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CLINICAL CHEMISTRY -SAMPLES

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CLINICAL CHEMISTRY -SAMPLES Powered By Docstoc
					                     MANUAL ON
STANDARD OPERATION PROCEDURES, SAMPLE COLLECTION AND
               REFERENCE RANGES FOR

        CLINICAL CHEMISTRY




               WORLD HEALTH ORGANISATION,
                   MINISTRY OF HEALTH
                           AND

THE DEPARTMENT OF BIOCHEMISTRY, MEDICAL RESEARCH INSTITUTE

                        SRI LANKA
                   MANUAL ON
STANDARD OPERATION PROCEDURES, SAMPLE COLLECTION
            AND REFERENCE RANGES FOR
                  CLINICAL CHEMISTRY




                     Dr. Meliyanthi M. Gunatillaka
   Consultant Chemical Pathologist and Head, Department of Biochemistry
                   Medical Research Institute, Colombo


                         Ms. D. K. Daya Silva
          Superintendent Grade Medical Laboratory Technologist
                   Medical Research Institute, Colombo


                     Mr. M. Muhammed Hunais
                    Medical Laboratory Technologist
                   Medical Research Institute, Colombo
                          This document is NOT for sale.
The document may, however, be freely reviewed, abstracted, reproduced or translated, in
                  part or in whole for non commercial purposes.
                                                                                                                                                             i
CONTENTS
Contents ..................................................................................................................................................i
Acknowledgements ..............................................................................................................................ii
Preface ...................................................................................................................................................iii
General introduction...........................................................................................................................iv
1. Format of a technical procedure manual................................................................................1
2. Albumin ........................................................................................................................................3
3. Amylase.........................................................................................................................................7
4. Alkaline phosphatase................................................................................................................10
5. Aspartate amino transferase....................................................................................................13
6. Alanine amino transferase .......................................................................................................18
7. Bilirubin ......................................................................................................................................21
8. Calcium .......................................................................................................................................27
     CALCIUM IN URINE ...................................................................................................................31
9. Creatinine....................................................................................................................................32
10.     Urine creatinine ....................................................................................................................35
11.     Cholesterol.............................................................................................................................37
12.     Glucose ..................................................................................................................................40
13.     Inorganic phosphate ............................................................................................................46
14.     Inorganic phosphate in urine .............................................................................................49
15.     Total protein..........................................................................................................................50
16.     Urea.........................................................................................................................................55
17.     Uric acid .................................................................................................................................59
18.     Urine uric acid.......................................................................................................................62
19.     Electrolytes ............................................................................................................................63
20.     Urine sodium and potassium .............................................................................................68
21.     Appendix 1 - Sample Collection and Transportation ..................................................69
22.     Appendix 2 - Diabetes mellitus ........................................................................................73
23      Appendix 3 - Reference ranges ........................................................................................75
Reference:.............................................................................................................................................96
ii



ACKNOWLEDGEMENTS
     We would like to acknowledge the WHO representative to Sri Lanka, Dr. Kan
     Tun, for identifying the need for quality assurance in local laboratories and offering
     us the opportunity to publish this handbook.

     We thank the Director General of Health Services Dr. Athula Kahandaliyanage,
     the Deputy Director General (Planning) Dr. T. S. B. Tennekoon and the Deputy
     Director General (Education, Training and Research) Dr. Stanley De Silva, Deputy
     Director General (Laboratory Services) Dr. Ajith Mendis and Director Laboratory
     Services Dr. Jayasundara Bandara, for approval and facilitation of this project.

     We are grateful to the Director of the MRI Dr. G. S. S. K. Colombage and the
     Deputy Director of MRI Dr. Lulu Raschid, for all their support and
     encouragement in bringing this project to fruition.

     We appreciate the assistance of administrative staff of World Health Organisation
     and colleagues, resource persons, administrative staff of the Medical Research
     Institute and staff of the Department of Biochemistry.
                                                                                         iii




PREFACE
    Clinical laboratory services have become an important component of modern
    medicine. Clinical laboratories play a major role in the diagnosis, treatment,
    prognosis and monitoring of diseases.
    Quality assurance in laboratory services, aimed at improving reliability, efficiency
    and facilitating inter – laboratory comparability in testing is the backbone of quality
    health care delivery. The use of standard operating procedure in laboratory testing
    is one of the major factors in achieving quality.
    This manual provides guidelines on standard operating procedure, sample
    collection and reference ranges.
    The publication aims at the use of World Health Organization recommended
    methods which may be adapted to local needs, based on the experience of the
    authors.
    It is hoped that this publication will be useful in achieving its objective of
    improving the quality of laboratory results using the available resources.
      .
iv

GENERAL INTRODUCTION
     Standard Operating Procedures (SOP) Manuals are required in the laboratory and
     are a key element in internal Quality Control within the laboratory. Hence, assuring
     quality health care, method manuals should include properly authenticated
     methods, or methods recommended by relevant professional organisation shall be
     used. A methods manual containing all methods and procedures authorised for
     using the laboratory shall be available in the appropriate work areas as bench copy
     and it is recommended that a master copy be maintained by the head of the unit.
     These manuals should be review at least annually, by the competent member of the
     technical staff and the head of the unit.

     The first consideration is the selection of the test methods best suited to full fill the
     function of the laboratory. Each test performed in the laboratory (for e.g.
     screening, routine clinical testing, reference laboratory service) must be evaluated
     and appropriate method adopted. Each method should be evaluated in terms of
     sensitivity, specificity, accuracy, simplicity, speed reliability and economy. It should
     be the compromise which best full fills the role of the laboratory, using the
     available resources.

     The methods that are included in this manual are based on World Health
     Organization recommended biochemical tests, adapted and evaluated by the
     Authors taking into consideration the clinical and technical experience, Quality
     Control measures and the available resources. This standard operating procedure
     manual includes the routine biochemical tests based on manual spectrophotometric
     methods. The clinical significance of each analyte has been described in detail for
     the benefit of the users. The precautions to be followed are included for each
     analyte to maximize the performance. This is based largely on the experience of the
     Authors.

     The manual contains the collection procedures of blood and urine samples. It
     includes the method of collection, selection of containers and preservatives,
     storage, transport and stability. There must be vigorous control of the procedures
     involved in the collection and identification of specimens to ensure the quality of
     the specimen to be examined in the laboratory. Copies of collection procedures
     should be available at all collection areas. The collection manual should be updated
     regularly at for laboratory procedure manual by the competent member of the
     technical staff.

     Specimen which do not conform to the requirements or are of inadequate quality
     for the test involved may be rejected with the consultation of the head of the unit
     and the sender informed adequate techniques shall be used for the continue correct
     identification, specimens and results. Specimen shall be retained by the laboratory
     for a time appropriate to the nature and origin of the specimen.

     The manual includes a section for reference ranges. The reference ranges are given
     for many biochemical analytes considering the age and the sex. Standard adult and
     paediatric text books along with the information available on the internet were
     taken into consideration in accepting the relevant reference ranges.
                                                                                                1


1.   FORMAT OF A TECHNICAL PROCEDURE MANUAL
     The following format for technical procedures should be adhered to as closely as possible
     and double spaced typing is recommended and a table of contents required of each test of
     procedure

     TITLE
     Name of test or procedure

     PRINCIPLE
     A short but informative statement which describe the basis of the methods

     References(s)
     List primary reference(s) as well as modifications.

     SPECIMEN
     List the types of specimens which are appropriate and indicate stability limits if known

     PROCEDURE
     Present a concise stepwise description of the method.

     CALCULATIONS
     These explain use of formulae and indicate, briefly, derivation of factors.

     REAGENTS
     State concentration or use descriptive name where appropriate (e.g. Biuret reagent) and
     give detailed stepwise instructions for preparing each reagent. Specify chemical brand and
     grade where it is known to be critical. Unusual reagents/components should have source
     or company stated. Expiry of reagents should be specified.

     UNITS
     Units used and abbreviations.

     REFERENCE RANGE
     Adult reference ranges for males and females and where applicable paediatric ranges

     CLINICAL SIGNIFICANCE
     Give reasons as to why the test is usually requested and indicates the significance of low or
     high results using the reference

     Notes
     Elaborate on any particular points which may require further explanation

     QUALITY CONTROL
     Quality control procedures, including documentation and methods of statistical analysis

     APPROVAL
     Each procedure to be dated and signed at the bottom of the final page by the appropriate
     senior authorised qualified staff
                                                                                      2

METHOD HISTORY
This to be a separate sheet and include date notations of method changes and review

REPORTING
Format and procedure of reporting, including procedures for urgent and especially
clinically significant results, must be described.

COPIES
Any copies of method for bench use are to be made in full from the master copy and
should include method history and approval sections

SUPPLEMENTARY INFORMATION
Certain information to be used at the bench to implement procedures may be extracted
from the procedure manual. This information may include flow diagrams, index cards, and
manufacturer product literature. Such supplementary information must be current and be
referenced to the procedure manual by date, procedure, reviewer’s initials, etc.
                                                                                                 3


2.   ALBUMIN

     2.1     INTRODUCTION

     Albumin is the most abundant protein in human plasma from 20 weeks of gestation,
     representing 40-60% of the total protein. It is synthesized exclusively in the liver. The rate
     of synthesis is depended on protein intake and subject to feed back regulation by the
     plasma albumin level. The half life of albumin is estimated at 15-19 days. Traces of
     albumin can be found in almost all extra vascular body fluids. The loss of albumin via the
     glomerular filtrate is very small as almost all the albumin is reabsorbed by the proximal
     tubular cells. Albumin is catabolised in various tissues where it is taken up by cells by
     pinocytosis. Its constituent amino acids are released by intracellular proteolysis and
     returned to the body pool. Albumin has a molecular weight of approximately 66,000.
     Albumin is an anion at pH 7.4 with more than 200 negative charges/molecule. The chief
     biological functions of albumin are to transport and store a wide variety of ligands to
     maintain the plasma osmotic pressure and to serve as a source of endogenous amino acid.
     The capacity of albumin to act as a binding protein is due to the large numbers of charges
     of each molecule as well as very large no of molecules available. Albumin binds nonpolar
     compounds such as Bilirubin & long chain fatty acids. Albumin binds hormones such as
     thyroxin, Triiodothyronin, cortisol and aldosteron, thus act as a reservoir in which these
     compounds are stored in inactive form but from which they are readily mobilised. Some
     40% of serum calcium is bound to albumin. Many drug such as phenyl glutazone, warfferin
     and salicylates are also strongly bounded to albumin. Albumin concentration is the major
     determinant of plasma oncotic pressure one of the factors that regulate partition water
     between intra and extra vascular compartments.

     2.2     CLINICAL SIGNIFICANCE

         Hypoalbuminaemia is very common and may result due to the following factors
         o Impaired synthesis: Diminished protein intake or liver disease
         o Increased catabolism: Due to tissue damage on inflammation
         o Reduce absorption of amino acid: caused by malabsorption syndrome or malnutrition.
         o Protein loss in urine: Due to Nephrotic syndrome chronic glomerular nephritis,
             Diabetes,or systemic lupus erythromatosis
         o Protein loss in faeces: Due to protein losing enteropathy
         o Protein loss through the skin: Burns
         o Altered distribution: as for instance in ascites
     Most severe hypoalbuminaemia is caused by protein loss by way of urine or faeces. When
     plasma albumin levels are less 2.0 g/l oedema is usually present.
         Hyper albumin: is of little diagnostic signification except in dehydration.

     Albumin has more than 20 genetic variants, which are not associated with disease but
     which cause two bands or single band in the albumin region on electrophoresis. The
     condition is called bisalbuminaemia. Congenital absence of albumin or analbuminaemia is
     asymptomatic except for occasional slight oedema.

     2.3     PRINCIPLE OF THE METHOD

     The requirements of a dye binding method for albumin include specific binding of the dye
     to albumin in the presence of other plasma or serum protein, high binding affinity between
     dye and albumin so that small changes in ionic strength and pH or presence of competing
     ligands do not break the dye-protein complex; a substantial shift in the absorption
                                                                                          4
wavelength of the dye in the bound form so that it remains spectrally distinct from the
free form present in excess, and absorption maximum for the bound form at a wavelength
distinct from those at which Bilirubin and haemoglobin can interfere. Serum albumin and
buffered BCG (Bromocresol green) are allowed to bind at pH 4.2, and absorption of the
BCG/Albumin complex is determined spectrometrically at 632 nm (filter No 607)
Albumin act as a cation to bind the anionic dye. Absorption reading taken within 30
seconds of mixing the serum and BCG avoids the problem of non specific reaction of
BCG with globulin. The manual method described here for albumin by BCG is adaptable
to automated analysis.

2.4     SPECIMEN TYPE, COLLECTION AND STORAGE

Serum is preferred. Fasting specimen is not an absolute requirement but it may be desirable
because marked lipaemia interferes in the assay. Avoid, apply a tourniquet in specimen
collection because haemoconcentration due to venous stasis increases the apparent
concentration of albumin and other plasma proteins. Storage: Separate the serum within 2
hours of collection, separated serum in a tightly stoppered container is stable for 24 hours
at room temperature 25-30 0 C, for 1 week at 2-8 0C, for 3 months at -20 0C.

APPARATUS AND CHEMICALS
APPARATUS:
Visible spectrophotometer, wavelength 632 nm or Colorimeter, orange filter, Ilford 607
(600nm)
pH meter
GLASSWARE:
Volumetric flasks (1 litre and 100 ml volumes)
Micro pipette (20 µl)
Graduated pipettes (10 ml in 0.1 ml)
Beakers (5ml, 50ml, 500 ml and 1 litre)
Amber colour reagent bottles (1 litre)
Test tubes (125 mm x 16 mm)
Measuring cylinders (1 litre)
CHEMICALS:
(All chemicals must be analytical grade)
Bromocresol green sodium salt, also called BCG, water soluble
Sodium azide caution: handle with care
Sodium chloride
Succinic acid
Sodium hydroxide pellets
Brij-35 (polyoxy7ethylene (23) lauryl ether) solid or solution 30% w/v
Standard buffers for pH meter
Bovine albumin or other available calibrator(fraction v powder)

REAGENTS
1. Succinic acid solution 50 g/l: weigh out 1.0 g of Succinic acid, dissolve and make up to
   about 20 ml with distilled water. Prepare as required, discard after use.
2. Sodium hydroxide solution 10 g/l: Weigh out 1.0 g of sodium hydroxide in a glass beaker,
   dissolve and make up to 100 ml with distilled water. This solution is stable for several
   months at 20-25 C. Store in polypropylene bottle.
3. Brij-35 solution 250 g/l: Warm 25 g solid Brij-35 in a small volume of distilled water to
   dissolve and make up to 100 ml with distilled water or use the 30 % w/v Brij-35
   solution.
4. Working dye solution: Dissolve 5.6 g of Succinic acid, 58 mg of Bromocresol green
   (Sodium salt) and 100 mg of sodium azide in about 900 ml of distilled water in a clean
   beaker. Add 1.0 g of sodium hydroxide and dissolve, and then add 2.5 ml of Brij-35
                                                                                           5
    solution. (If 30% Brij solution is used add 2.1 ml)Adjust to pH 4.2 using small
    volumes of sodium hydroxide solution or Succinic acid solution if necessary. Transfer
    slowly (avoid frothing) to 1 litre volumetric flask and make up to volume with distilled
    water, mix gently and transfer into a clean brown bottle. This solution is stable for
    several months at 2-8 0C.

NOTE: The BCG working dye solution requires careful preparation. Some laboratories
may find it economical to purchase this solution. There are several different BCG reagents
available. Make sure you select a BCG reagent in Succinate buffer at pH 4.2.
In 10 mm light path cuvette (cell) the working dye solution should have the following
absorbance readings (zero the instrument on distilled water).

Spectrometer                                   Colorimeter
Wavelength             Absorbance              Filter                 Absorbance
430 nm                 About 1.4               601                    About 1.1
615 nm                 About 0.25              607                    About 0.2

5. Succinate buffer solution: Prepare in exactly the same way as the working dye solution but
   do not add any Bromocresol green. This solution is stable for several months at 2-8 0C
6. Albumin standard 40 g/l: Using a 5ml volumetric pipette dilute 5.0 ml of the bovine
   albumin standard (80 g/l) with 5.0 ml of sodium chloride/sodium azide solution to
   prepare an albumin standard containing 40 g/l. This standard is stable for 6 months at
   2-8 0C. (Bovine albumin standard 80 g/l provide by the Department of Biochemistry,
   MRI)
7. Sodium chloride /Sodium azide solution: Weigh out 9.0 g of sodium chloride and 1.0 g of
   sodium azide, dissolve and make up to 1 litre with distilled water. This solution is
   stable indefinitely at 20-25 0C (room temperature)

2.5     PROCEDURE
1. Pipette 4.0 ml of working dye solution into test tubes.
2. Add 20 µl of standard or control or test sample, mix and measure the absorbance
    immediately (within 30 seconds).
3. Read the absorbance at 632 nm or filter No 607 after setting the instrument to zero
    absorbance with the working dye solution.

PREPARATION OF CALIBRATION GRAPH
Preparation of working albumin standard solutions: They are prepared by dilution of the
albumin standard (40 g/l) in sodium chloride/sodium azide solution as follows:
Working standard No                           (01)      (02)        (03)       (04)
Albumin standard 40 g/l (ml)                  0.5       )
                                                        1.0         1.5        2.0
Sodium chloride/Sodium azide solution (ml)      1.5          1.0       0.5        0.0
Concentration of working standards (g/l)        10           20        30         40

Working dye solution (ml)                      4.0    4.0          4.0        4.0
Working standard No (20 µl)                    (01)   (02)         (03)       (04)
Mix each tube thoroughly                              )
Read the absorbance of each tube immediately at 632 nm (or filter No 607) after setting
the instrument to zero with working dye solution.
                                                                                            6
Plot the absorbance of each tube against the concentrations of working standards
solution on the graph. The calibration graph should be linear up to 40 g/l. If the graph is
linear then a single standard (40g/l) may be used for routine analysis but linearity should be
confirmed for each new batch of working dye solution and at least once a month.
The recommended method proposes the use of a bovine albumin solution as a calibrator.
Alternative commercial bovine albumin solutions are available.

2.6    CALCULATION
Albumin concentration = T/S x 40 g/l

Where T=the absorbance of the test
      S=the absorbance of the standard

NOTE: Always include a standard (40 g/l) in each and every batch of samples.

QUALITY CONTROL
OPTIMAL CONDITIONS VARIANCE                       : A coefficient of variation of around 3%
should be attainable.
ROUTINE CONDITIONS VARIANCE                       : The value obtained for the RCV should not
exceed 6%

REFERENCE VALUES
New born                            : 25-50 g/l
1 Year                              : 35-50 g/l
2-3 Year                            : 36-50 g/l
4th Year and after                  : 37-50 g/l
Adult                               : 30-45 g/l

2.7        LIMITATIONS
If a serum sample is extremely lipaemic a serum blank should be used. (Moderate lipaemia
does not affect the results) The blank is prepared by adding 20 µl of sample to 4.0 ml of
Succinate buffer solution. The absorbance of this blank, with distilled water as reference is
subtracted from the test. Grossly haemolysed specimens are unsuitable for albumin
determination.


REFERENCES
Ann.clin.Biochem.14 (1977)105-115
Tietz text book of clinical chemistry
                                                                                                 7


3.   AMYLASE

     3.1       INTRODUCTION
     Amylases are a group of hydrolases that split complex carbohydrates constituted of α-D-
     glucose units linked through carbon atoms 1 and 4 located on adjacent glucose residues.
     Both straight-chain (linear) polyglucans, such as amylose, and branched polyglucans, such
     as amylopectin and glycogen, are hydrolyzed, but at different rates. In the case of amylose,
     the enzyme splits the chains at alternate α-1, 4-hemiacetal (-C-O-C-) links, forming maltose
     and some residual glucose; maltose, glucose, and a residue of limit dextrins are formed if
     branched –chain polyglucans are used as substrate. The α-1, 6- linkages at the branch
     points are not attacked by the enzyme. Two types of amylases are recognized. Beta-amylase
     (e.g. plant and bacterial exoamylase) acts only at the terminal-reducing end of polyglucan
     chain; it splits off two glucose units (maltose) at a time. Animal amylases, including those
     present in human tissues, are α-amylases. They are also called endoamylases because they
     attack α-1, 4-linkages in a random manner anywhere along the polyglucan chain. Linear
     starch chains in helical form react with molecular iodine to form the well-known deep blue
     starch-iodine complex. The enzyme present in normal serum and urine is predominantly of
     pancreatic (p-type) and salivary gland (S-type) origin. These Isoenzymes are products of
     two closely linked loci on chromosomes1. Each gene is allelic; thus there are 12 distinct
     phenotypes for the salivary Isoenzyme and 6 for the pancreatic Isoenzyme.

     3.2      CLINICAL SIGNIFICANCE
     Assays of Amylase activity in serum and urine are largely of use in the diagnosis of disease
     of the pancreas and in the investigations of pancreatic function in acute pancreatitis. A
     transient rise in serum amylase activity occurs in 2- 12 hours of onset; levels return to
     normal by the 3 rd or 4th day. A 4-6 fold elevation of amylase activity above reference limit
     is usually with maximum level attained in 12-72 hours. The magnitude of elevation of
     serum enzyme activity is not related to the severity of pancreas involvement. However the
     greater the rise, grater the probability of acute pancreatitis. A significant amount of serum
     amylase is excreted in urine and therefore elevation of serum activity is reflected in rise of
     urine amylase activity. Urine amylase as compared on serum amylase appears to be more
     frequently elevated, reaches higher level and pursuit for longer periods. In quiescent
     chronic pancreatitis both serum and urine activity are usually subnormal. Acute pancreatitis
     is sometimes difficult to diagnose because it must be differentiate from other acute intra
     abdominal disorders and because an increase serum amylase activity may not necessary due
     to acute pancreatitis.

     3.3     PRINCIPLE OF THE METHOD
     In solution iodine reacts with starch to give an intense blue – violet complex. Amylase
     hydrolyses starch, forming maltose and other fragments which do not react with iodine.
     After incubation of serum with buffered starch solution, the amount of starch remaining is
     assayed by measuring the absorbance at 660 nm after the addition of iodine

     3.4     SPECIMEN TYPE, COLLECTION AND STORAGE
     3-5 ml clotted blood in a clean dry bottle, avoid haemolysis. Separate serum as early as
     possible. Enzyme activity loss is negligible in sterile serum store at 2-8 0C for a week (free
     of bacterial contamination).

     3.5     APPARATUS AND CHEMICALS
     APPARATUS:
     Hot plate or Bunsen burner
                                                                                            8
Water bath at 37 0C
pH meter
Visible Spectrometer wavelength at 660nm or Colorimeter with red filter Ilford No: 608
(680 nm)

GLASSWARE:                                             CHEMICALS:
Volumetric flasks (1litre volume)                      Soluble starch pharmaceutical grade
Automatic micro pipette 20 µl                          Potassium iodide AR
Graduated pipettes (1ml, 5ml, 10 ml                    Potassium iodate AR
and 0.1 ml)                                            Disodium        hydrogen      ortho-
Beakers (50ml, 1 litre)                                phosphate (anhydrous) AR
Graduated cylinders (100 ml and 1                      Sodium chloride AR
litre)                                                 Benzoic acid AR
Amber colour reagents bottles (100                     Hydrochloric acid (concentrated (37
ml and 1 litre)                                        % w/v) caution: highly corrosive)
                                                       Buffers for pH meter
REAGENTS
1. Buffered starch substrate: Dissolve 26.6 g of anhydrous disodium hydrogen phosphate,
   1.75 g of sodium chloride and 8.6 g of benzoic acid in about 500 ml of distilled water
   in a large beaker. Heat to boiling. In 50 ml of beaker, mix separately 0.4 g of soluble
   starch in 10 ml of cold distilled water to form a paste. Add the paste with stirring to
   the boiling mixture, rinsing the beaker with distilled water. Continue to boil for one
   minute. Cool to room temperature, and transfer to volumetric flask and dilute to 1 litre
   with distilled water. This solution is stable for at least one year at 20- 25 0C and should
   have a pH of 6.9-7.1; the stability is monitored by noting the absorbance of the reagent
   blank with each set of tests. We recommend that the solution is stored at 4-8 0C in the
   refrigerator. Aliquot the required amount for daily use. If 1 litre substrate is excess
   then prepare 500 ml for use.
2. Stock iodine solution 50 mmol/l: Dissolve 3.57 g of potassium iodate and 45 g of
   potassium iodide in about 800 ml of water in a volumetric flask. Slowly and with
   mixing add 9.0 ml of concentrated hydrochloric acid. Dilute to 1 litre with distilled
   water. This solution should be stored in a dark bottle and is stable for a year at 4 -8 0C.
3. Working iodine solution: Dilute 10 ml of stock iodine solution with 90 ml distilled water
   in a graduated 100 ml volumetric flask. This solution should be stored in a dark bottle
   and is stable for 2 months at 2-8 0C.

3.6     PROCEDURE
1. Pipette 1.0 ml of buffered starch substrate into 150 x 16 mm test tubes. You will need
    1 tube for each patient and control sample and 1 tube for a reagent blank.
2. Place all of the tubes in a water bath at 37 0C for 5 minutes to warm the contents.
3. Pipette 20 µl of patient’s or control serum into the bottom of the test tubes, mix and
    incubate at 37 0C for exactly 7 minutes and 30 seconds. (No serum is added to the
    reagent blank).
4. After 7 minutes and 30 seconds remove the test tubes from the water bath
    immediately add 1.0 ml of working iodine solution to each tube (samples and reagent
    blank) then add 8 ml of distilled water.
5. Mix the contents of each tube well then measure the absorbance without delay at 660
    nm (red filter Ilford No. 608) setting the spectrometer to zero with distilled water.

NOTE: Avoid contamination of the pipette with saliva

3.7     CALCULATION
Amylase activity U/L      = B-T x 1470
                            B
B                         = absorbance of reagent blank
                                                                                            9
T                         = absorbance of test
1470                      = factor to express values in U/L

If the result is greater than 735 U/L (i.e. there is no blue colour in tube T) then the sample
must be diluted with saline (20 µl serum + 100 µl saline) and the analysis repeated using 20
µl of the diluted sample. The measured value must be multiplied by 6 to calculate the
amylase activity of the sample to take into account the dilution factor.

QUALITY CONTROL
OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 6% should be
attainable.
ROUTINE CONDITIONS VARIANCE: This value should not exceed 12 %

A quality control sample with a value in the range 200 – 700 U/L should be analysed with
each batch of specimens. If single specimens are analysed a control specimen should
always be included.

REFERENCES VALUES: Approximate reference values: 70 – 340 U/L


REFERENCES
WHO Manual LAB/86.3
                                                                                                  10
4.   ALKALINE PHOSPHATASE

     4.1      INTRODUCTION
     The alkaline phosphatases (ALP) are a group of glycoprotein enzymes that act as
     phosphotransferases by hydrolysing various types of monophosphate bond at alkaline pH.
     ALP activity is found in virtually all tissues, particularly bone, liver, kidney, intestine,
     adrenal, placenta. The protein moieties comprise about 510 amino acid residues, to which
     is attached various amounts of carbohydrate and sialic acid. Tissue-specific
     posttranslational modifications occur to the carbohydrate content, leading to the formation
     of isoforms, e.g. bone, liver and kidney ALP, each of which contains the same tissue non-
     specific protein. ALP is found attached to the outer lipid bilayer of cell membranes by a
     glycosyl-phosphatidylinositol group, in the case of liver ALP possibly as a tetramer of
     identical subunits; if released from cell membranes, ALP is dimeric. Liver ALP is located in
     the cell membranes of the hepatcoyte, and particularly in the outer layer of the cells
     adjacent to the bile canaliculi and also in the cells lining the sinusoids. Adult intestinal ALP
     lacks sialic acid and is found in the epithelial cells of the intestinal brush border. The
     placental enzyme is formed by the syncitiotrophoblast cells lining the microvilli that
     interface the placental and fetal blood circulations but the placental ALP does not cross
     into the fetal circulation.

     4.2      CLINICAL SIGNIFICANCE
     Serum ALP measurements are of particular interest in the investigation of two groups of
     conditions: hepatobiliary and bone disease associated with increased osteoblastic activity.
     The response of the liver to any form of biliary tree obstruction is to synthesize more ALP.
     The main site of new enzyme synthesis is the hepatocytes adjacent to the biliary canaliculi.
     Some of the newly formed enzyme enters the circulation to raise the enzyme level in
     serum. The elevation tends to be more marked (more than three-fold) in extrahepatic
     obstruction (e.g. by stone or by cancer of the head of the pancreas) than in intrahepatic
     obstruction and is greater the more complete the obstruction. Serum enzyme activities may
     reach 10 to 12 times the upper limit of normal, returning to normal on surgical removal of
     the obstruction.

     4.3      PRINCIPLE OF THE METHOD
     4 - Nitophenylphosphate is hydrolysed by alkaline phosphatase at pH 10.3 at 37 C and 4 -
     Nitrophenol is liberated. Alkali is added to stop the enzyme activity at the end of the timed
     incubation period and the increase in absorbance due to the 4-Nitrophenol released is
     measured at 410 nm.

     4.4      SPECIMEN TYPE, COLLECTION AND STORAGE
     Collect about 2-3 ml blood, separate serum as soon as possible, avoid haemolysis. Freshly
     collected serum sample should be kept at room temperature and assayed as soon as
     possible. If there is a delay in the assay store the serum at -20 C. Sample should be
     completely thawed before the assay. Refrigeration of the serum at 4 C will increase the
     ALP activity.

     4.5      APPARATUS AND CHEMICALS

     APPARATUS:
     Water bath at 37 0C
     pH meter
     Spectrometer wavelength at 410 nm or Colorimeter with violet filter, Ilford 600 (410 nm)
                                                                                            11

GLASSWARE:                                             Automatic pipette (50µl and 100µl)
Volumetric flasks (100 ml and 1
litre volumes)                                         CHEMICALS:
Beakers (1 litre)                                      All chemicals must be analar grade
Measuring cylinders (100 ml and 1                      Standard buffer solutions for pH
litre)                                                 meter
Test tubes (125 mm x 16 mm or                          Hydrochloric acid (Concentrated
150 mm x 16 mm)                                        (37%     w/v);   caution:   highly
Volumetric pipettes (5 ml in 0.1                       corrosive)
ml)                                                    2-Amino-2-methyl-1-propanol
Polyethylene reagent bottles (1                        Magnesium chloride hexahydrate-
litre)                                                 Disodium     4   –     Nitrophenyl
Amber colour bottles                                   phosphate hexahydrate
Graduated pipettes (1ml, 5ml, 10ml)                    Sodium hydroxide pellets
                                                       4 - Nitrophenol

REAGENTS
1. AMP Buffer pH 10.3: Dissolve 78.5 g of 2-amino-2-methyl-1-propanol (CH3)2 C (NH2)
   CH2 OH in about 900 ml of distilled water. Adjust to pH 10.3 with concentrated
   hydrochloric acid (about 18 ml) and make up to 1 litre with distilled water. Store in an
   Amber colour reagent bottle. This solution is stable for 1 month at 20-25 0C.
   Suggestive procedure method done at MRI: Use the chemical with the
   specification 95 % (CH3)2 C(NH2) CH2OH with MW of 89.14 and specific gravity of
   0.93 g/ml (BDH). Add 84.4 ml of the above liquid to 800 ml of distilled water.
2. Magnesium chloride solution 1.5mmol/l: Dissolve 300 mg of magnesium chloride
   hexahydrate in water and make up to 1 litre. This solution is stable indefinitely at 20 -
   250C. MgCl2.6H2O chemical and the solution (1.5mmol/l) is best stored in
   refrigerator.
3. Substrate solution 225 mmol/l in the magnesium chloride solution: Dissolve 83.5 mg of
   disodium 4 -Nitrophenyl phosphate hexahydrate (store the chemical in freezing
   compartment) in 1.0 ml of magnesium chloride solution as required. This solution is
   stable for one working day. It’s best to keep the solution in refrigerator since at room
   temperature colour development may occur.
4. Sodium hydroxide solution 250 mmol/l: Dissolve 10 g of sodium hydroxide in distilled
   water and make up to 1 litre. Store in a tightly Stopperd polyethylene bottle. This
   solution is stable indefinitely at 20-25 0C. We have observed that this solution is stable
   even at room temperature at 20-30 0C.
5. 4 – Nitrophenol stock solution 10.8 mmol/l: Weigh out 150 mg of 4 – Nitrophenol
   accurately in a beaker & transfer the chemical from beaker to a 100 ml volumetric flask
   using a funnel and wash any chemical remaining in the container into the volumetric
   flask with distilled water. Make up to the mark with distilled water. This solution is
   stable for about 6 months in an amber colour bottle at 4 0C.
6. 4 – Nitrophenol working solution 54 µmol/l: Pipette 0.5 ml of the 4 – Nitrophenol stock
   solution into a 100 ml volumetric flask, makes up to 100 ml with sodium hydroxide
   solution (250mmol/l) Prepare this solution freshly before use.

Note: The quality of the disodium 4 – Nitrophenyl phosphate should be checked to ensure
that it does not contain excessive amounts of free 4 – Nitrophenol. Prepare sodium
hydroxide solution (10mmol/l) by diluting 4.0 ml of sodium hydroxide solution
(250mmol/l) to 100 ml with distilled water. Add 200 µl of substrate solution (reagent 3
above) to 3.8 ml of sodium hydroxide solution (10 mmol/l) Mix and measure the
absorbance at 410 nm after setting the spectrometer to zero with sodium hydroxide
solution (10 mmol/l). The quality of the substrate is satisfactory if its absorbance after
dilution in sodium hydroxide solution is less than 0.25 (10 mm light path cuvette at room
                                                                                        12
temperature and 410 nm) In a colorimetric the absorbance should be less than 0.12
(10mm light path and Ilford filter No 600) This procedure should be carried out when a
new batch of chemical/bottle is introduced to the bench.

4.6         PROCEDURE
1. Pipette 1.4 ml of AMP buffer into sufficient test tubes for patients’ samples, controls
   and reagent blank and preincubate in the water bath at 37 0C for about 5 minutes.
2. To each tube add 50 µl of serum, to the blank; add 50 µl of distilled water .Mix well.
3. To each tube in sequence add 100 µl of substrate solution at timed intervals. Mix well.
4. Incubate for exactly 15 minutes at 37 0C then add 4.0 ml of sodium hydroxide solution
   (250mmol/l) to each tube in sequence, maintaining timed intervals. Mix each tube and
   allow them to cool to room temperature.
5. Measure the absorbance of each test solution at 410 nm ( violet filter ,Ilford No :600)
   setting the spectrometer to zero with the blank
6. If the absorbance is greater than the absorbance of a 400 U/L standard, then repeat
   the procedures, but at step 4, incubate for exactly 5 minutes (instead of 15 minutes)
   then add 4.0 ml sodium hydroxide solution (250mmol/l) and complete the procedure
   described in steps 4 and 5. Do not forget to multiply the activity by 3 before reporting
   the result.

PREPARATION OF CALIBRATION GRAPH
Tube No                                     (1)     (2)      (3)    (4)      (5)   (6)
4 – Nitrophenol solution 54 µmol/l (ml)      1       2        4      6        8    10
Sodium hydroxide 250 mmol/l (ml)             9       8        6      4        2     0
Activity (U/L)                              40      80       160    240     320    400
Mix well and measure the absorbance of each tube at 410 nm (violet filter Ilford No 600)
setting the spectrometer to zero with sodium hydroxide solution (250mmol/l) Plot the
absorbance of each tube on the graph. Prepare a new calibration graph every 3 months.

4.7         CALCULATION
Read off the activities of alkaline phosphatase in the unknown and control samples from
the calibration graph. Remember to multiply by 3 if you used 5 minute incubation instead
of 15 minutes incubation.

QUALITY CONTROL
At least two serum control specimens, having stated values in the range 20-350 U/L, one
of which is unknown to the operator should be included with each batch of specimens. If
single specimens are analysed a control specimen should always be included.

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 10% should be
ROUTINE CONDITIONS VARIANCE: The value should be less than 20%

APPROXIMATE REFERENCE VALUES
Males (age 20 -60 years) : 20- 90 U/L
Females (age 15-60 years) : 20-90 U/L
Children (age 1-12 years) : up to 350 U/L
During the growth spurt of puberty: up to 500 U/L


REFERENCES
LAB/86.3
A guide to diagnostic clinical chemistry By R.N. Walmsley and G.H. White -page 312
TIETZ TEXTBOOK OF Clinical chemistry page 831-832
                                                                                                  13


5.   ASPARTATE AMINO TRANSFERASE

     5.1      INTRODUCTION
     Aspartate Amino Transferase & Alanine Amino Transferase
     The aminotransferases constitute a group of enzymes that catalyze the interconvertion of
     amino acids and α- oxo-acids by transfer of amino groups. Aspartate amino transferase
     (AST) and Alanine amino transferase (ALT) are the two enzymes that are of clinical
     significance. Distinct isoenzymes of AST are present respectively in cytoplasm and
     mytochondria of cells. The α- oxoglutarate/glutamate couple serves as one amino group
     acceptor and donor pair in all amino-transfer reactions; the specificity of the individual
     enzymes derives from the particular aminoacid that serves as the other donor of an amino
     group.

     L-Aspartate + 2- Oxaglutarate                            Oxaloacetate + L –Glutamate

     L-Alanine + 2- Oxaglutarate                              Pyruate      + L –Glutamate

     The reactions are reversible, but the equilibria of the AST and ALT reactions favour
     formation of aspartate and alanine, respectively. Pyridoxal -5’ phosphate and its amino
     analogue, pyridoxamine-5’ –phosphate function as coenzymes in the amino transfer
     reactions. Transaminases are widely distributed in tissues. Both AST and ALT are normally
     present in human plasma, bile, cerebrospinal fluid and saliva, but none is found in urine
     unless a kidney lesion is present.

     5.2      CLINICAL SIGNIFICANCE
     In viral hepatitis and other forms of liver disease associated with hepatic necrosis, serum
     AST and ALT levels are elevated even before the clinical signs and symptoms of the
     disease (such has jaundice) appear. Levels for both enzymes may reach values as high as
     100 times the upper reference limit, although 20- to 50- fold elevations are most frequently
     encountered. Peak values of transaminase activity occur between the 7th and 12th days;
     activities then gradually decrease, reaching normal levels by the 3rd to 5th week if recovery is
     uneventful. Alcoholic hepatitis has more modest elevations. In infectious hepatitis and
     other inflammatory conditions affecting the liver, ALT is characteristically as high as or
     higher than AST, and the ALT/AST ratio, which normally and in other conditions is less
     than 1, becomes greater than unity. The use of different assay methods for the two
     enzymes may alter the activities of the two enzymes relative to each other, and hence the
     numerical values of the ratio observed may differ between laboratories. Nevertheless, the
     principle that hepatitis is associated with comparable elevations of the two activities
     remains valid. The relatively similar elevations of AST and ALT in hepatitis have been
     attributed to the release of only the cytoplasmic isoenzyme of AST into the circulation
     from reversibly damaged parenchymal cells. When necrosis of the cells occurs,
     considerable amounts of mitochondrial AST are also released, depressing the ALT/AST
     ratio. The picture in toxic hepatitis is similar to that in infectious hepatitis, with very high
     ALT and AST activity being observed in severe cases. Elevations up to 20 times the upper
     reference limit may be encountered in infectious mononucleosis with liver involvement and
     somewhat lover values in intrahepatic cholestasis. Increased levels may also be observed in
     extrahepatic cholestatis, with levels tending to be higher the more chronic the obstruction. The
     aminotransferase levels observed in cirrhosis vary with the status of the cirrhotic process;
     they range from upper normal to some four to five times normal, with the level of AST
     activity higher than that of ALT activity. Elevations probably indicate continuing cellular
     necrosis. Five- to 10- fold elevations of both enzymes occur in patients with primary or
     metastatic carcinoma of the liver, with AST usually being higher than ALT, but levels are
                                                                                            14
often normal in the early stages of malignant infiltration of the liver. Slight or moderate
elevations of both AST and ALT activities may be observed after the intake of alcohol, in
delirium tremens, and after administration of various drugs, such as opiates, salicylates, or
ampicillin. Although serum levels of both AST and ALT become elevated whenever
disease processes affect liver cell integrity. ALT is the more liver-specific enzyme. Serum
elevations of ALT activity are rarely observed in conditions other than parenchymal liver
disease. Moreover, elevations of ALT activity persist longer than do those of AST activity.
Measurement of both AST and ALT has some value in distinguishing hepatitis from other
parenchymal lesions. After myocardial infarction, increased AST activity appears in serum, as
might be expected from the relatively high AST concentration in heart muscle. On average,
serum levels do not become abnormal, however, until 6 to 8 h has elapsed after the onset
of the chest pain. Abnormal AST levels are observed in more than 97% of cases of
myocardial infarction when correctly timed blood specimens are analyzed. Peak values of
AST activity are reached after 18 to 24 h, and the activity values fall within the normal
range by the fourth of fifth day, provided no new infarct has occurred. The peak values of
AST activity are roughly proportional to the extent of cardiac damage. Average increases
are of the order of four to five times the upper limit of normal; levels of 10 to 15 times
normal are frequently associated with fatal infarct. However, small elevations in serum
levels do not necessarily indicate a favorable prognosis. ALT levels are within normal
limits or are only marginally increased in uncomplicated myocardial infraction, because the
concentration of ALT activity in heart muscle is only a fraction of that of AST activity.
AST (and occasionally ALT) activity levels are increase in progressive muscular dystrophy
and dermatonyositis, reaching levels up to 8 times normal; they are usually normal in other
types of muscle diseases, especially in those of neurogenic origin. Pulmonary emboli can
raise AST levels to two to thee times normal, and slight to moderate elevations (two to five
times normal) are noted in acute pancreatitis, crushed muscle injuries, gangrene, and hemolytic
disease.

5.3      PRINCIPLE OF THE METHOD
Aspartate aminotransferase (AST or SGOT) effects the conversion of alpha keto -
glutarate and Aspartate to glutamate and oxaloacetate respectively, by amino group
transfer. The oxaloacetate thus formed is coupled with 2, 4 - dinitrophenylhydrazine to
produce a coloured complex whose absorbance in alkaline solution is measured at 505 nm

5.4      SPECIMEN TYPE, COLLECTION AND STORAGE
3-5 ml clotted blood, avoid haemolysis. Separate serum as soon as possible and performed
the assay or keep in the refrigerator, it is stable up to 24 hours at 4 C. Minimal loss of
activity occurs at 0-4 0C over 1-3 days. Specimen is best stored frozen if they are to be kept
more than 3-4 days

5.5      APPARATUS AND CHEMICALS

APPARATUS:
Water bath at 37 0C
pH meter
Visible spectrometer wavelength at 505 nm or Colorimeter with green filter Ilford No 604
(520 nm)
                                                                                           15

GLASSWARE:
Volumetric flasks (100 ml and 1 litre volumes)
Measuring cylinders (1 litre)
Automatic micro pipette (100 µl)
Graduated pipettes (1ml, 2 ml, 5ml, 10 ml in 0.1 ml)
Test tubes (150 x16 mm), Beakers (5ml, 10ml, 100ml and 1 litre)
Reagents bottles clear and amber coloured (250 ml and 1 litre)
Polypropylene bottle

CHEMICALS:
Disodium hydrogen ortho-phosphate-anhydrous, analytical grade
Potassium dihydrogen phosphate-anhydrous, analytical grade
Alpha – ketoglutaric acid -analar
DL- aspartic acid -analar, Sodium hydroxide pellets analar
2, 4- dinitrophenylhydrazine-AR; caution: may explode violently when dry,
Hydrochloric acid concentrated-AR (37% w/v), caution: highly corrosive
Sodium pyruvate (analytical grade)

REAGENTS
1. Phosphate buffer pH 7.4: Dissolve 11.9 g of disodium hydrogen phosphate (anhydrous)
   and 2.2 g of potassium dihydrogen phosphate (anhydrous) in distilled water and make
   up to 1 litre. Check the pH adjust to pH 7.4 if necessary using small amounts of the
   appropriate phosphate (e.g. if the pH is more than 7.4 add potassium dihydrogen
   phosphate). This solution is stable for about 2 months at 2-8 0C.
2. Sodium hydroxide solution 1 mol/l: Weigh 40 g of sodium hydroxide in beaker, slowly
   dissolve in distilled water, transfer into a volumetric flask and make up to 1 litre. Store
   in a tightly Stopperd polypropylene bottle. This solution is stable indefinitely at 20 -
   250C, which can be achieved by air condition system. However we have observed that
   this solution is stable in our room temperature (20-30 0C) for about 2 months.
3. Buffered substrate reagent: Weigh 29.2 mg of alpha- ketoglutaric acid 2.66 g of DL-aspartic
   acid into a small beaker. Dissolve in 20 ml of sodium hydroxide solution (1 mol/l)
   then adjust to pH 7.4 with more sodium hydroxide solution. Transfer to a 100 ml
   volumetric flask and make up to 100 ml with phosphate buffer and mix well. Store the
   reagent frozen in screw capped bottles; the volume should be the amount need for a
   day analysis.
4. Hydrochloric acid 1 mol/l: Dilute 9 ml of hydrochloric acid (concentrated (37% w/v)) to
   100 ml with distilled water.
5. Colour reagent 2,4 –dinitrophenylhydrazine 1 mmol/l: Dissolve the equivalent of 19.8 mg of
   dry 2,4-dinitrophenylhydrazine in 100 ml of hydrochloric acid ( 1mol/l)and transfer
   into a amber colour bottle; Note that the weight of 2, 4 – dinitrophenylehydrazine
   must be adjusted to take into account the water content. e.g. If the label reads as 33%
   by weight of water is added to ensure safety in transit the calculation is as
   follows.100/67 x 19.8 = 29.55 mg. The prepared solution is stable for 2 months at 2 -
   8 0C.
6. Sodium hydroxide solution 400 mmol/l: Dissolve 16.0 g of sodium hydroxide in distilled
   water in a beaker and make up to 1 litre. Store in a tightly stoppered polypropylene
   reagent bottle. This solution is stable indefinitely at 20- 25 0C. We have observed that
   this solution is stable at our room temperature at 20-30 0 C for 2 months.
7. Pyruvate standard solution 4 mmol/l: Weigh out 44 mg of sodium pyruvate in a beaker,
   transfer into a 100 ml volumetric flask and make up to the mark with phosphate
   buffer. Mix well, divide into small portions (about 1ml) and store in the freezer
   compartment of the refrigerator. The standard solution is stable for 6 months in the
   freezer.
                                                                                      16
5.6     PROCEDURE
1. Two test tubes are required for each serum or control sample( one for the “Test” and
   one for a sample blank) one for a reagent blank and one for the standard
2. Transfer 0.5 ml buffered substrate to each tube and pre-incubate in the water bath
   (37 0 C) for 5 minutes
3. Add 100 µl of patient’s or control serum to the “Test” tubes or 100 µl distilled
   water(reagent blank) or 100 µl pyruvate standard (standard) mix and incubate at 37 0C.
4. After exactly 60 minutes add 0.5 ml colour reagent to each tube, mix and remove from
   the water bath.
5. Add 100 µl of patient’s or control serum to the sample blank tubes and mix well.
6. Leave for 20 minutes at room temperature and then add 5.0 ml of sodium hydroxide
   solution (400mmol/l) and mix thoroughly.
7. Leave the tubes at room temperature for at least 5 minutes, but not longer than 30
   minutes, and then read the absorbance at 505 nm. Set the spectrometer to zero with
   the reagent blank.

CALIBRATION
In this method the amount of pyruvate formed is calculated by comparing the absorbance
of the samples (Test- Blank absorbance) with that of the pyruvate standard (4mmol/l).
However alpha - ketoglutarate also contributes to the absorbance and the change in
absorbance is not linearly related to enzyme activity expressed in U/L. The table must
therefore be used to convert the amount of pyruvate formed into U/L.

5.7     CALCULATION

Amount of pyruvate formed        = (Abs of Test-Abs of Sample Blank) x 4x1x1000
(µmol/min/litre)                         Abs of Standard                  60
                                 = Abs of Test- Abs of Sample Blank x 66.7
                                         Abs of Standard

With the reagent blank set to zero the spectrophotometer at 505 nm
Abs = absorbance

Use the table below to convert the amount of pyruvate into U/L (expressed as
µmol/minute/litre at 37 0C)

Calculated pyruvate   AST result (U/L at Calculated pyruvate         AST result (U/L at
(µmol/min/litre)      37 0C)             (µmol/min/litre)            37 0C)
2                     4                  28                          52
4                     6                  30                          56
6                     10                 32                          60
8                     12                 34                          64
10                    15                 36                          69
12                    19                 38                          73
14                    23                 40                          77
16                    27                 42                          81
18                    31                 44                          85
20                    35                 46                          92
22                    40                 48                          98
23                    42                 50                          106
24                    44                 52                          114
26                    48                 54                          125
                                                                                          17
NOTE: When the activity of a sample exceeds 125 U/L the measurement should be
repeated with 10 minute incubation (instead of 60 minutes) and the results multiplied by 6.
When the activity is greater than 750 U/L, the serum should be diluted 1 in 10 with sodium
chloride solution (150mmol/l), an incubation time of 10 minutes should be used and the result
multiplied by 60.
QUALITY CONTROL
At least two serum control specimens, having stated values in the range 20-125 U/L, one
of which is unknown to the operator, should be included with each batch of specimens.
Even if single specimens are analysed a control specimen should always be included.

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 8% should be
attainable
ROUTINE CONDITIONS VARIANCE: The value should not exceed 16%
REFERENCE VALUES
Approximate reference values: 4 - 42 U/L.

REFERENCES
Reitman, .S. & Frankel. S. (1957) Am. J. Clin.Pathol., 28, 56-63
                                                                                                18


6.   ALANINE AMINOTRANSFERASE

     6.1     PRINCIPLE OF THE METHOD
     Alanine aminotransferase (ALT or SGPT) effects the conversion of alpha-ketoglutarate
     and Alanine to pyruvate. Pyruvate formed is coupled with 2, 4-dinitrophenylhydrazine to
     produce a coloured complex. The absorbance in alkaline solution is measured at 505 nm.

     6.2     SPECIMEN TYPE, COLLECTION AND STORAGE
     3-5 ml clotted blood, avoid haemolysis. Separate serum as soon as possible and performed
     the assay or keep in the refrigerator, it is stable up to 24 hours at 4 0C. Minimal loss of
     activity occurs at 0-4 0C over 1-3 days. Specimen is best stored frozen if they are to be kept
     more than 3-4 days

     6.3     APPARATUS AND CHEMICALS
     APPARATUS:
     Water bath at 37 0C
     pH meter
     Visible spectrometer wavelength at 505 nm or Colorimeter with green filter Ilford No
     604 (520 nm)
     GLASSWARE:
     Volumetric flasks (100 ml and 1 litre volumes)
     Automatic micro pipette (100 µl)
     Graduated pipettes (1ml, 2ml, 5ml and 10 ml in 0.1 ml)
     Test tubes (150 x16 mm), Beakers (5ml, 50ml, 100ml and 1 litre)
     Reagents bottles amber coloured (250 ml and 1 litre)
     Polypropylene bottle
     CHEMICALS:
     Disodium hydrogen phosphate-Anhydrous analytical grade
     Potassium dihydrogen phosphate-Anhydrous analytical grade
     Alpha ketoglutaric acid AR
     DL Alanine AR
     Sodium hydroxide pellets analytical grade
     2, 4-dinitrophenyl hydrazine AR
     Hydrochloric acid AR
     Sodium pyruvate analytical grade

     REAGENTS
     1. Phosphate buffer pH 7.4: Dissolve 11.9 g of disodium hydrogen phosphate (anhydrous)
        and 2.2 g of potassium dihydrogen phosphate (anhydrous) in distilled water and make
        up to 1 litre. Check the p H and adjust to pH 7.4 if necessary using small amounts of
        the appropriate phosphate (e.g. if the pH is more than 7.4 add potassium dihydrogen
        phosphate). This solution is stable for about 2 months at 2-8 0C.
     2. Sodium hydroxide solution 1 mol/l: Slowly dissolve 40.0 g of sodium hydroxide in distilled
        water in a beaker and make up to 1 litre. Store in a tightly stopperd reagent bottle. This
        solution is stable indefinitely at 20-25 0C. we have observed this solution is even stable
        at our room temperature 20-30 0C
     3. Buffered substrate reagent: Weigh out 3.56 g of DL Alanine in a beaker and weigh 30 mg
        of alpha ketoglutaric acid accurately in another small beaker. Transfer it to same
        beaker by dissolving in the phosphate buffer. Add 0.5 ml of sodium hydroxide
        (1mol/l) solution. Check the pH. The pH should be at 7.4.Make up to 100 ml with
        phosphate buffer and mix. Divide the prepared substrate into small volumes and store
                                                                                             19
      in the freezing compartment or in the freezer. Discard the remaining substrate after
      use. Do not freeze the remaining substrate again.
4.    Hydrochloric acid 1 mol/l: Dilute 9 ml of hydrochloric acid (concentrated (37% w/v)) to
      100 ml with distilled water.
5.    Colour reagent 2,4 –dinitrophenylhydrazine 1 mmol/l: Dissolve the equivalent of 19.8 mg of
      dry 2,4-dinitrophenylhydrazine in 100 ml of hydrochloric acid ( 1mol/l); Note that the
      weight of 2, 4 – dinitrophenylehydrazine must be adjusted to take into account the
      water content. e.g. If the label reads as 33% by weight of water is added to ensure
      safety in transit the calculation is as follows.100/67 x 19.8 = 29.55 mg. The prepared
      solution is stable for 2 months at 2-8 0C.
6.    Sodium hydroxide solution 400 mmol/l: Dissolve 16.0 g of sodium hydroxide in distilled
      water in a beaker and make up to 1 litre. Store in a tightly stoppered polypropylene
      reagent bottle. This solution is stable indefinitely at 20- 25 0C. We have observed that
      this solution is stable at our room temperature (20-30 0C) for 2 months.
7.    Pyruvate standard solution 4 mmol/l: Weigh out 44 mg of sodium pyruvate in a beaker,
      transfer into 100 ml volumetric flask and make up to the mark with phosphate buffer.
      Mix well, divide into small portions (about 1ml) and store in the freezer compartment
      of the refrigerator. The standard solution is stable for 6 months in the freezer.

6.4       PROCEDURE
1. Two test tubes are required for each serum or control sample( one for the “Test” and
   one for a sample blank) one for a reagent blank and one for the standard
2. Transfer 0.5 ml buffered substrate to each tube and pre-incubate in the water bath (37
   0C) for 5 minutes

3. Add 100 µl of patient’s or control serum to the “Test” tubes or 100 µl water(reagent
   blank) or 100 µl pyruvate standard (standard) mix and incubate at 37 0C
4. After exactly 30 minutes add 0.5 ml colour reagent to each tube, mix and remove from
   the water bath.
5. Add 100 µl of patient’s or control serum to the sample blank tubes.
6. Leave for 20 minutes at room temperature and then add 5.0 ml of sodium hydroxide
   solution (400mmol/l) and mix thoroughly.
7. Leave the tubes at room temperature for at least 5 minutes, but not longer than 30
   minutes, and then read the absorbance at 505 nm. Set the spectrometer to zero with
   the reagent blank.

PREPARATION OF CALIBRATION GRAPH
In this method the amount of pyruvate formed is calculated by comparing the absorbance
of the samples (Test- Blank absorbance) with that of the pyruvate standard (4mmol/l).
However alpha - ketoglutarate also contributes to the absorbance and the change in
absorbance is not linearly related to enzyme activity expressed in U/L. The table must
therefore be used to convert the amount of pyruvate formed into U/L.

6.5       CALCULATION
Amount of pyruvate formed          = (Abs of Test-Abs of Sample Blank) x 4x1x1000
(µmol/min/litre)                            Abs of Standard                 30
                                   = Abs of Test- Abs of Sample Blank x 133
                                            Abs of Standard
With the reagent blank set to zero the spectrophotometer at 505 nm
Abs=absorbance
                                                                                        20
Use the table below to convert the amount of pyruvate into U/L (expressed as
µmol/minute/litre at 37 0C)

Calculated pyruvate           ALT result (U/L at Calculated pyruvate   ALT result (U/L at
(µmol/min/litre)              37 0C)             (µmol/min/litre)      37 0C)
2                             2                  54                    42
4                             4                  56                    44
6                             4                  58                    46
8                             5                  60                    47
10                            7                  62                    49
12                            7                  64                    53
14                            9                  66                    55
16                            11                 68                    56
18                            13                 70                    60
20                            13                 72                    62
22                            15                 74                    64
23                            15                 76                    66
24                            16                 78                    67
26                            16                 80                    69
28                            18                 82                    71
30                            20                 84                    73
32                            22                 86                    76
34                            24                 88                    80
36                            25                 90                    84
38                            27                 92                    87
40                            29                 94                    91
42                            31                 96                    95
44                            33                 98                    98
46                            35                 100                   102
48                            36                 102                   109
50                            38
52                            40

QUALITY CONTROL
At least two serum control specimens, having stated values in the range 20-109 U/L, one
of which is unknown to the operator, should be included with each batch of specimens.
Even if single specimens are analysed a control specimen should always be included

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 8 % should be
attainable
ROUTINE CONDITIONS VARIANCE : The value should not exceed 16 %

REFERENCE VALUES Approximate reference values: up to 2-27 U/L.

REFERENCES
King and wooton- enzymology
                                                                                                 21
7.   BILIRUBIN

     7.1      INTRODUCTION

     Haem, released from aged red cells and maturing cells during erythropoiesis, or from
     degraded haemoproteins is converted to biliverdin in the reticuloendothelial system.
     Biliverdin is reduced to bilirubin which is secreted into the plasma where it is transported
     to the liver reversibly bound to albumin. The hepatocytes take the bilirubin up from the
     plasma, conjugate it to glucuronic acid and excrete it in the bile. There are six steps in this
     process namely; production, transport to the liver, hepatocyte uptake, conjugation, biliary
     secretion, gut degradation and excretion.
     The total bilirubin produced is around 250-350 mg/day (4 – 6 mmol/day). 15% to 20% is
     derived from immature red cell and haemoproteins (early labelled fraction) whilst the
     remainder comes from senescent red cells ( 1g of haemoglobin produces 620µmol of
     bilirubin ) Haem is converted to bilirubin in the reticuloendothelial system by two
     enzymes, haem oxygenase and biliverdin reductase. Haem oxygenase breakes the alpha –
     CH bridge of protoporphyrin IX to produce biliverdin IX alpha, which is then reduced to
     bilirubin IX alpha by biliverdin reductase. Some cleavage occurs at the ß, gamma and delta
     bridges, but insignificant amounts of these isomers are produced. Although bilirubin IX
     alpha has two polar propionic acid side chains it is poorly soluble in water because of
     intramolecular hydrogen bonding between the propionic acid residues and other parts of
     the molecule. This bonding may also account for the necessity for conjugation with
     glucuronic acid prior to biliary excretion. Bilirubin is transported to the liver reversibly
     bound to albumin. This protein has one high – affinity binding site and an additional low –
     affinity site which is activated at high concentrations of bilirubin. The amount of unbound
     bilirubin is low, =4 nmol/l at a total plasma concentration of 20 µmol/L. Compounds
     such as free fatty acids, sulphonamides, salicylate and ampicillin will displace bilirubin from
     its binding sites. This species of bilirubin is called unconjugated bilirubin or indirect
     reacting bilirubin. The bilirubin – albumin complex dissociates in the liver and the bilirubin
     is transported across the hepatocyte membrane into the cell where it is reversibly bound to
     cytosolic proteins; one such protein being ligandin. The function of these proteins appears
     to be prevention of efflux of bilirubin from the cell. Bilirubin is conjugated in the
     endoplasmic reticulum with glucuronic acid and to a lesser extent with glucose and xylose.
     The enzyme responsible is UDP – glucuronosyltransferase which esterifies one or both
     propionic acid side chains to produce di – and monoglucuronides. In man the product is
     predominantly bilirubin diglucuronide with lesser amounts of the monoglucuronide. The
     bilirubin conjugates are secreted into the biliary passages by an activ eprocess which is yet
     to be clarified. This transport mechanism differs from that responsible for the biliary
     excretion of bile acids. In the gut the bacterial flora reduce the bilirubin conjugates to
     urobilinogens which are excreted in the faeces as such. Some of the urobilinogens are
     reabsorbed from the gut to be re – excreted by the liver ( enterohepatic circulation ) ; as
     these compounds are water soluble they will also be excreted by the kidney, but this is an
     unimportant excretory route. Two points worthy of note are;
     conjugated bilirubin is more prone to reductive processes than unconjugated bilirubin.
     Unconjugated bilirubin can be reabsorbed from the gut. Thus, if bacterial flora are absent (
     eg: neonate, antibiotic therapy ) deconjugation of bilirubin glucuronides by intestinal
     mucosal ß – glucuronidase may occur and reabsorption of the unconjugated compound
     result in an increase in the plasma unconjugated bilirubin level. The total plasma bilirubin
     concentration in the normal subject is usually less than 20 µmol/L. The clinical sign of
     jaundice appears when the plasma total bilirubin rises beyond 40 µmol/L. When normal
     plasma is evaluated by high performance liquid chromatography techniques four bilirubin
     fractions alpha, beta, gamma and delta are obtained.
                                                                                             22
Alpha fraction
This fraction is unconjugated bilirubin which is water insoluble and bound to albumin. It is
also called indirect reacting bilirubin because the diazo reaction used to measure the plasma
level occurs only after the addition of accelerators. It is the major bilirubin fraction in
normal plasma (> 90%). It does not appear in the urine because of its attachment to
albumin and can only be cleared by the liver.

Beta and gamma fractions
The beta fraction is composed of bilirubin monoglucuronide whilst diglucuronide
constitutes the gamma fraction. These bilirubins (< 10% in normal plasma) are water
soluble and will appear in the urine if present in the blood in excess. They are called direct
reacting bilirubins as they react with the diazo reagent without the addition of
accelerations.

Delta fraction
The delta fraction, usually referred to as ‘delta’ bilirubin – not to be confused with bilirubin
IX delta –is an interesting compound with the following characteristics:
     − it is direct reacting
     − it is tightly bound to albumin
     − its plasma level is increased in diseases associated with high plasma levels of
         conjugated Bilirubin
It appears to be derived from conjugated Bilirubin as it can be produced by incubating
Bilirubin glucuronides with plasma albumin. It is thought that the Bilirubin migrates from
the glucuronic acid residues and becomes covelantly bound to albumin. As noted above
this fraction increases in conditions associated with chronic conjugated
hyperbilirubinaemia (eg: cholestasis) and, as it is tightly bound to albumin, it has a t1/2
comparable to that of protein (=20 days) .Thus, after formation delta Bilirubin ‘hangs
around ‘as it is not cleared by the liver or the kidney. The bile pigments that may be found
in the urine are Urobilinogen and conjugated Bilirubin. Unconjugated Bilirubin is water
insoluble and bound to albumin, and is thus not available for urinary excretion. The
normal subject excretes 1 – 4 mg (2 – 7 µmol) of Urobilinogen daily (derived from the
enterohepatic circulation) Increased values occur in liver disease (inability to excrete the
small amounts reabsorbed from the gut) and in haemolytic disease (increased Bilirubin
production). Decreased excretion occurs in bile duct obstruction. (cholestasis) Bilirubin
appears in the urine when the plasma level of conjugated Bilirubin rises as in hepatocellular
disease and cholestasis.

7.2      CLINICAL SIGNIFICANCE
The earliest clinical manifestation of hepatobiliary disease is often jaundice, but jaundice
need not necessarily indicate liver pathology (eg: haemolysis) and liver pathology can
present without jaundice (e.g.: space-occupying lesions). However, it is convenient to
classify liver disease in terms of jaundice and to this end it is helpful to divide
hyperbilirubinaemia into three categories: Prehepatic: liver disease not present, Hepatic:
hepatocellular disease, Post hepatic: cholestasis (obstruction)

7.3      PRINCIPLE OF THE METHOD
Sulfanilic acid is diazotized by the nitrous acid produced from the reaction between
sodium nitrite and hydrochloric acid. Both conjugated and unconjugated Bilirubin reacts
with diazotized sulfanilic acid (Diazo reagent) to produce azobilirubin. Caffeine is an
accelerator by splitting the unconjugated Bilirubin protein complex and gives a rapid and
complete conversion to azobilirubin. The pink acid azobilirubin is converted to blue
azobilirubin by an alkaline tartrate reagent and the absorbance of the blue green solution is
measured at 600 nm. Measurement of the azobilirubin in an alkaline medium removes
turbidity and increases specificity. There is very little interference by other pigments at 600
                                                                                              23
nm wavelength. Conjugated Bilirubin is determined by diazotization at an acidic pH,
only the conjugated forms of Bilirubin react with the diazo reagent in the absence of the
accelerator caffeine benzoate. The reaction is stopped by the addition of ascorbic acid
minimizes the effect of haemolysis.

7.4      SPECIMEN TYPE, COLLECTION AND STORAGE
Clear non haemolysed serum or collect 3-5 ml of blood in a clear dry container. A fasting
specimen is preferred to avoid lipaemia. Haemolysis should be avoided because it produces
falsely low values. Specimens should be protected from direct exposure to either artificial
light or sunlight as soon as they drawn because conjugated and unconjugated Bilirubin is
photosensitive. The sensitivity to light is temperature dependent for optimal stability.
Serum separation should be done as early as possible and assay should be carried out
within 2 hours of sample collection if not storing the serum in the dark and at low
temperature (2-8 0C) is essential.

APPARATUS AND CHEMICALS
APPARATUS: Spectrophotometer, wavelength at 600 nm or Colorimeter, orange filter,
Ilford 607

GLASSWARE:
Volumetric flasks (100 ml, 500 ml and 1 litre volumes)
Beakers (5ml, 100 ml and 1 litre)
Automatic micro pipettes (50 and 100 µl)
Graduated pipettes (1ml and 10 ml in 0.1 ml)
Test tubes (100 x 13mm) & Rubber bulb
Reagent bottle, clear and amber coloured
CHEMICALS: (ANALYTICAL GRADE)
Bilirubin powder (or commercial Bilirubin standards)
Caffeine
Sodium benzoate
Sodium acetate trihydrate
Sulphanilic acid
Hydrochloric acid, concentrated (37% w/v) caution: highly corrosive
Sodium nitirite
Sodium hydroxide pellets
Potassium sodium tartrate tetra hydrate
Ethylenediamine tetra – acetic acid disodium salt dehydrate
Ascorbic acid
Sodium carbonate anhydrous

REAGENTS
1. Caffeine- benzoate reagent: Dissolve 93 g of sodium acetate trihydrate, 56 g of sodium
   benzoate and 1 g of disodium EDTA in approximately 500ml of distilled water. Add
   38 g of caffeine. Dissolve and dilute to 1 litre in a volumetric flask. Mix well and filter.
   This solution is stable for at least 6 months at 20-25 0C (Room temperature)
2. Sulfaninlic acid reagent: Add 2.5 g of sulfanilic acid to about 200 ml of distilled water in a
   500 ml volumetric flask. Using a rubber bulb carefully pipette 7.5 ml of concentrated
   hydrochloric acid into the flask. Dissolve and make up to 500 ml. This solution is
   stable for up to 6 months at 20-25 0C (Room temperature)
3. Sodium nitrite solution: Dissolve 500 mg of sodium nitrite in distilled water and make up
   to 100 ml. This solution should be stored at 2-8 0C in an amber coloured bottle and
   must be renewed every month. Prepare about 25ml or 50 ml.
4. Diazo reagent: Mix 4 ml of sulfanilic acid reagent with 0.1 ml of sodium nitrite solution,
   leave for 2 minutes, then use within 5 hours. Keep at 2-8 0C in an amber coloured
   bottle.
                                                                                            24
5. Alkaline tartrate reagent: Dissolve 75 g of sodium hydroxide and 350 g of potassium
    sodium tartrate in about 800 ml of distilled water. Transfer to a volumetric flask and
    make up to 1 litre. This reagent is stable for at least 6 months at 20- 25 0C. Store in a
    polypropylene bottle.
6. Hydrochloric acid 50 mmol/l: Using a rubber bulb, carefully pipette 4.2 ml of hydrochloric
    acid (concentrated) and dilute to 1 litre with distilled water, require only for conjugated
    Bilirubin method.
7. Ascorbic acid 40 g/l: Dissolve 0.2 g in 5 ml of water, this solution must be prepared
    daily. Required only for the conjugated Bilirubin method.
8. Sodium carbonate 100mmol/l: Weigh out 1.06 g of sodium carbonate anhydrous very
    accurately and transfer quantitatively to a 100 ml volumetric flask, containing about 50
    ml distilled water. Mix well and make up to 100 ml with distilled water.(Prepare about
    50 ml for use)
9. Sodium hydroxide 100 mmol/l: Weigh out rapidly 2.0 g of sodium hydroxide in a beaker,
    dissolve and make up to 500 ml with distilled water. ( prepare about 100 ml for use)
10. Tris buffer(0.1 mol/l) pH 7.4 : Tris(hydoxymethyl) amino methane 1.21 g, distilled water
    80 ml, adjust pH 7.4 ±0.05, with hydrochloric acid 2 N, make up to 100 ml with
    distilled water stable for about 1 month at 4 0C
11. Bovine serum albumin (BSA) diluent 40 g/l: Weigh out 4 g of Bovine albumin powder in a
    beaker. Dissolve in the Tris buffer and transfer into 100 ml volumetric flask and make
    up to the mark with Tris buffer. This solution is stable for one week at 4 0C
12. Bilirubin standard 342µmol/l: Weigh out 20 mg of Bilirubin powder accurately in a small
    beaker or in a weighing bottle. Cover the beaker with aluminium foil or black paper to
    protect from sunlight. Add 2 ml of sodium carbonate solution and 1.5 ml of sodium
    hydroxide solution and dissolve it .This solution must be red and clear. This procedure
    must not be carried out in strong sunlight. Transfer the solution quantitatively to a 100
    ml volumetric flask. (This should be protecting form direct sunlight by wrapping
    around with an aluminium foil or with a black carbon paper). Make up to 100 ml with
    BSA diluent mix the solution carefully without forming froth. Divide into small
    volumes in clean bottles keep in a box to protect from light. Kept store deep frozen.
    Do not reuse the standard once it has been used.

7.6      PROCEDURE (TOTAL BILIRUBIN)
1. For the standard, patient specimen, control and their blanks (SB, TB, and CB) pipette
   1.0 ml of caffeine benzoate reagent into each of two test tubes.
2. Add 100 µl of standard or patient or control serum to each pair of tubes
3. Add 0.5 ml of diazo reagent to the test, standard, control and 0.5 ml of sulfanilic acid
   to the entire blank.
4. Mix well and let stand for 10 minutes at room temperature.
5. Add 1.0 ml of alkaline tartrate reagent to each tube and mix thoroughly
6. Read the absorbance at 600 nm (Ilford filter No: 607) immediately, setting the
   spectrometer to zero with distilled water.

7.61     PROCEDURE (CONJUGATED BILIRUBIN)
1. For each patient or control specimen label two tubes: one test one blank.
2. Add 100 µl of serum to each tube.
3. Add 1.0 ml of hydrochloric acid (50mmol/l) to each tube.
4. Add 0.5 ml of diazo reagent to the tube marked test and 0.5 ml of sulfanilic acid to the
   tube marked blank, mix well.
5. After 5 minutes add 50 µl of ascorbic acid solution to each tube.
6. Add 1.0 ml of alkaline tartrate reagent to each tube. Mix well and read the absorbance
   of each solution at 600 nm immediately, setting the spectrometer to zero absorbance
   with distilled water.
                                                                                                                           25
   PREPARATION OF CALIBRATION GRAPH
   Prepare Bilirubin working standard solutions by diluting the Bilirubin standard solution
   (342µmol/l) with BSA solution as shown in the table below. The working standard
   solution must be freshly prepared each time a calibration graph is made.
   Bilirubin working
                                          (1)     (2)      (3)     (4)      (5)  (6)      (7)
   standard
   Bilirubin standard
                                                   0     0.5         1           1           2               3              4
   Solution 342µmol/l(ml)
   Bovine serum Albumin (ml)
                                                   4     9.5         9           3           2               1              0
   (BSA solution)
   Concentration of Bilirubin
   Working standard solution                       0     17.1      34.2      85.5            171         256.5             342
   (µmol/l)
   A calibration graph is prepared from the Bilirubin working standards using the volumes of
   standard and reagents described in the table below:

Tube No               1 (Blank)                2                             3           4         5             6         7
Caffeine
benzoate              1.0                      1.0                           1.0         1.0       1.0           1.0       1.0
Reagent (ml)
Bilirubin
working               100                      100                           100         100       100           100       100
Standard (µl)
                                 Mix well, protect the tubes from light then add
Diazo reagent
                      -                        0.5                            0.5         0.5       0.5           0.5       0.5
(ml)
   Mix well and allow the solutions to stand at room temperature for 10 minutes. Protect the tubes from light.
Sulfanilic acid
                      0.5                      -                                     -         -         -             -         -
Reagent(ml)
                                                   Mix well
Alkaline tartrate
                     1.0                       1.0                            1.0 1.0 1.0 1.0                               1.0
Reagent (ml)
Mix well. Read the absorbance of each tube at 600 nm after setting the spectrometer to zero with the blank
(Tube No 1)

   Plot the absorbance of the each tube on the vertical axis against the concentrations in
   µmol/l of working standards on the horizontal axis
   o The calibration graph should be prepared whenever the new batch of reagents are
       prepared or any changes made in the spectrophotometers
   o Freshly prepare all reagents and use clean glassware
   o Measure the standards (each concentration) in duplicate
   o Check the calibration graph by measuring a quality control serum

   NOTE:
     All the tubes or racks used for the assay should be covered with black papers to
     protect from direct light
        Icteric serum can be diluted 1 in 2 or highly icteric serum can be diluted 1 in 4 with
        normal saline and multiply the result by diluting factor
                                                                                         26
    Carryover is minimized by measuring the absorbance of all the serum blank
    solutions first, followed by all the test solutions. The colour of the azobilirubin is
    stable for about 30 minutes. After 30 minutes turbidity may occur and the absorbance
    of the serum blank increases

7.7       CALCULATION
If the calibration graph is linear, calculate the results using the following formula:
Concentration of Bilirubin (µmol/l) = T – TB x 342
                                           S – SB
Where:
T         =Absorbance reading of sample or control
TB        =Absorbance reading of control or patient sample blank
S         =Absorbance reading of Bilirubin standard (342 µml/l)
SB        =Absorbance reading of standard blank

If the calibration graph is not linear, then results should be read from a calibration curve
prepared using working standards 1, 2, 3, 5 and 6.


QUALITY CONTROL
At least two serum control specimens having stated values in the range 20 – 200 µmol/l,
one of which should be unknown to the operator, should be included with each batch of
specimens. If single specimens are analysed a control specimen should always be included.

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 6% should be
attainable.
ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed
12%

REFERENCE VALUES
For adults: 3 – 21 µmol /l
Conversion from SI units into ‘old’ units: µmol/l x 0.0585=mg/dl

REFERENCES
WHO manual LAB /86.3
                                                                                                27
8.   CALCIUM

     8.1       INTRODUCTION
     Calcium is the fifth most common element and the most prevalent cation found in the
     body. For example, an average human body contains approximately 1kg (24.95 mol) of
     calcium. Calcium is found in three main compartments: the skeleton, soft tissues, and the
     extra cellular fluid. The skeleton contains 99% of the body’s calcium, predominantly as
     extra cellular crystals of unknown structure with a composition approaching that of
     hydroxyapatite. Soft tissues and extra cellular fluid contain about 1% of the body’s calcium.
     In blood, virtually all of the calcium is in serum or plasma, which has a mean normal
     calcium concentration of approximately 9.5mg/dL (2.37 mmol/L). Calcium exists in three
     physiochemical states in plasma, of which approximately 50% is free or ionised. Another
     40%is bound to plasma proteins, chiefly albumin. Because calcium binds to negatively
     charged or anionic sties on albumin, it’s binding in pH-dependant. Alkalosis leads to an
     increase in binding and a decrease in free calcium; conversely, acidosis leads to a decrease
     in binding and an increase in free calcium. For each 0.1-unit change in pH, approximately
     0.2-mg/dL (0.05mmol/L) of inverse change occurs in the serum free calcium
     concentration. Approximately 20% of protein-bound calcium in serum is associated with
     globulins. In some patients with multiple myeloma, the high concentrations of serum
     globulin may bind sufficient calcium, about 10%, is complexed with small diffusible anions
     including bicarbonate, lactate, phosphate and citrate. Calcium can be redistributed among
     the three plasma pools, acutely of chronically, independently affecting the quantities of free
     calcium and total calcium in the serum. The skeleton is a major storehouse for providing
     calcium for the extra cellular and intracellular pool. Approximately 5g of calcium is rapidly
     available from the skeletal exchangeable pool, which is accessible for maintaining normal
     physiological functions. Intracellular calcium has many important physiological functions
     within the cells, including muscle contraction, hormone secretion, glycogen metabolism
     and cell division. Extra cellular calcium provides a source for maintenance of intracellular
     calcium. In addition, it has an important role in providing calcium ions for bone
     mineralization, coagulation cascade and maintaining plasma membrane potential. Calcium
     stabilises the plasma membranes and influences permeability and excitability. A decrease in
     serum free calcium concentration increases neuromuscular excitability and tetany. An
     elevated free calcium concentration results in reduced neuromuscular excitability.

     8.2     CLINICAL SIGNIFICANCE
     Hypercalcaemia is found commonly in clinical practice. It may be uncovered as a
     biochemical abnormality in an otherwise asymptomatic patient or in association with
     severe illness. Hypercalcaemia occurs when the flux of calcium into the extra cellular fluid
     is greater than the efflux of calcium out of this compartment. For example, when excessive
     resporption of bone mineral occurs in malignancy, hypercalciuria develops. When the
     capacity of the kidneys to excrete filtered calcium is exceeded, Hypercalcaemia develops.
     Hypercalcaemia can be due to increased intestinal absorption of calcium (vitamin D
     intoxication), enhanced renal retention of calcium (thiazide diuretics), increased skeletal
     resorption (immobilization), or a combination of these mechanisms (primary
     parathyroidism). The pathogenesis, clinical presentation, and differential diagnosis
     therefore vary widely. Primary hyperthyroidism is the most common cause of
     Hypercalcaemia in outpatients, and malignancy is the most common cause in hospitalized
     patients. Together these two account for 90 to 95% of all cases if Hypercalcaemia.
     Hypoalbuminemia is probably the most common cause of reduction in the concentration
     of total serum calcium. Common clinical conditions associated with low serum albumin
     concentrations include chronic liver disease, nephritic syndrome, congestive heart failure,
     and malnutrition. Chronic renal failure is also frequently associated with hypocalcaemia.
     Contributing reasons for the low calcium values are hyperphosphatemia, impaired
     synthesis of 1, 25(OH)2D due to inadequate renal mass, and skeletal resistance to the
                                                                                          28
action of Parathyroid hormone..(PTH) Magnesium deficiency is the other common
clinical cause of hypocalcemia. Magnesium deficiency impairs PTH secretion as well as the
action of PTH of bone and kidneys. Acute symptomatic hypocalcaemia may be noted in
hospitalized patients for various reasons. Patients undergo surgical treatment for
hyperthyroidism or primary hyperparathyroidism of receiving therapy for haematological
malignancies may have rapid remineralisation of bone (hungry bone syndrome) causing a
drop in serum calcium. Acute hemorrhagic and edematous pancreaitis is frequently
complicated by hypocalcaemia. Ostomalacia of rickets secondary to vitamin D deficiency
may also be associated with hypocalcaemia. The hypocalcaemia may be due in part to
impaired intestinal absorption of calcium. In addition, vitamin D deficiency renders the
skeleton resistant to PTH and thereby limits calcium resorption from bone.

8.3     PRINCIPLE OF THE METHOD
Serum or plasma calcium is measured with o-cresolphthalein complexone reagent
containing ethanediol which maintains a clear solution in the presence of proteins and
suppresses the ionization of o-cresolphthalein complexone in the reagent. Interference by
magnesium is eliminated by the inclusion of 8-hydroxyquiline.

8.4     SPECIMEN TYPE, COLLECTION AND STORAGE
2-3 ml clotted blood, fasting specimen without tourniquet and haemolysed free sample is
collected into acid washed bottles. Separate the serum from red cells as early as possible.
Pyrex vials with Teflon lined screw caps are recommended for specimen storage. Specimen
may stored at 4 0C for several weeks or months

8.5     APPARATUS AND CHEMICALS
APPARATUS: Spectrometer with wavelength 575 nm or colorimeter with yellow filter-
Ilford 606(580nm), Safety bulb

GLASSWARE:
Volumetric flask class A (100ml, 500ml and 1 litre volumes)
Automatic micro pipettes (50 µl200 µl 5ml and 10ml)
Graduated pipettes (1 ml, 2 ml, 5ml and 10 ml in 0.1ml)
Beaker (5ml, 250 ml)
Measuring cylinder (50ml), Test tubes (125 x 16 mm)
CHEMICALS:
Hydrochloric acid concentrated (37% w/v); caution: highly corrosive
O-cresolphthalein complexone-AR
Ethanediol-AR
2-amino-2-methyl-1-propanol-AR
8-hydroxyquinoline-AR
Calcium carbonate-AR

NOTES: All glassware must be thoroughly cleaned then soaked overnight in hydrochloric
acid (0.5mol/l) to remove traces of calcium then thoroughly rinsed with distilled or
deionised water and finally dried before use.

REAGENTS
1. Hydrochloric acid 0.5 mol/l: Adding the acid to the distilled water, dilute about 45 ml of
   hydrochloric acid (concentrated) to 1 litre with distilled water. Use this solution for
   soaking glassware as described above.
2. Stock CPC reagent: Add 38 ml ethanediol and 13 ml of 2- amino-2-methyl-1-propanol to
   a 500 ml volumetic flask containing about 400 ml of distilled water. Weigh out 15 mg
   of o-cresolphthalien complexone and add it to the volumetric flask, mix until the all
   the chemicals are completely dissolved, make up to the mark with distilled water and
                                                                                             29
   transfer the reagent into a clean brown bottle. This solution is stable for 3 weeks at 4
   -6 0C
3. Working CPC reagent: Weigh out 100 mg of 8-hydroxyquinoline and transfer into a 100
   ml volumetric flask using small volumes of stock CPC solution. Add about 80 ml of
   stock CPC solution mix until the chemical is completely dissolved and make up the
   volume to 100ml of stock CPC reagent. The 8-hydroxyquinoline dissolves quite
   slowly. This solution is stable for 1 week at 4-6 0C and should have and absorbance at
   575 nm of about 0.2 when measured with the spectrometer set to zero with distilled
   water. An absorbance higher than 0.2 indicates either that the reagent has deteriorated
   or that it is contaminated with calcium
4. Calcium stock standard 25mmol/l: Dry about 300 mg of calcium carbonate in a dry
   container in an oven for 4 hours at 80-100 0C. After heating remove it from the oven
   and immediately close the container with a lid. When it has cooled to room
   temperature weigh out exactly 250 mg and transfer to a 100 ml volumetric flask.
   Dissolve the calcium carbonate in a minimum volume of hydrochloric acid
   (concentrated) approximately 0.5 ml is require, then make up to100 ml with distilled
   water.
5. Calcium working standard 2.5mmol/l: Using a volumetric pipette transfer 10.0 ml of the
   stock standard to a 100 ml volumetric flask. Make up to 100 ml with distilled water.

8.6      PROCEDURE
1. The analysis must be performed in duplicate. Transfer 50 µl of serum or plasma to
    each of two clean tubes, add 5.0 ml working CPC reagent to each tube, mix well.
2. Transfer 50 µl of working calcium standard (2.5mmol/l) to each of 2 clean tubes; add
    5.0 ml working CPC reagent to each tube, mix well.
3. Transfer 50 µl of distilled water to a clean tube, add 5.0 µl working CPC reagent, Mix
    well. This is the reagent blank.
4. Set the spectrometer to zero at 575 nm with distilled water and measure the
    absorbance of the reagent blank which should be about 0.2.
5. Measure the absorbance of the standards (2.5mmol/l) and serum sample
If the absorbance of duplicate readings varies by more than 0.015 then precision is
unsatisfactory. Check that the test tubes and pipettes are clean. Check the precision of the
50 µl volumetric pipette. Use a 5.0 ml volumetric pipette for the working CPC reagent.
Check that the spectrometer cuvettes (cells) are clean.

PREPARATION OF CALIBRATION GRAPH
The calibration graph must be prepared in order to confirm the linearity of the method
and should be checked monthly. The calibration graph should not be used for calculating
patients’ results. Prepare the calibration graph standards from the calcium stock standard
as described in the table below in clean test tubes as accurately as possible using 1, 2 and 10
ml graduated pipettes


Tube Number                                        1       2         3       4        5

Calcium stock standard
                                                   0.2     0.7       1.0     1.2      1.5
( 25mmol/l) ml

Distilled water (ml)                               9.8     9.3       9.0     8.8      8.5

Calcium concentrated (mmol/l)                      0.5     1.75      2.5     3.0      3.75
                                                                                           30
Transfer 50 µl of distilled water into a clean test tube for the reagent blank and transfer
50 µl of each calibration graph standard to 5 other test tubes. Add 5.0 ml of working CPC
reagent to each tube using a 5 ml volumetric pipette and safety bulb. Mix and measure the
absorbance of each tube at 575 nm setting the spectrometer to zero with distilled water.
Plot the absorbance of each tube on the vertical axis against the calcium concentration of
the calibration graph standards in mmol/l on the horizontal axis.
The purpose of preparing the calibration graph is to confirm the linearity of the method. If
this is not linear beyond 3.0 mmol/l, then patients’ samples with calcium concentrations
greater than 3.0 mmol/l should be diluted two-fold with distilled water before analyse. If
the graph is linear up to 3.75 mmol/l then samples with calcium concentrations greater
than 3.75 mmol/l should be diluted two-fold with distilled water before analysis.

8.7     CALCULATION
Calculate the results using the following formula:
Concentration of calcium (mmol/l)            = T-B x 2.5
                                                S-B
Where:
T        = Absorbance reading of sample or control
S        = Absorbance reading of working calcium standard (2.5 mmol/l)
B        = Absorbance reading of reagent blank
If the sample or control result is above the linearity of the method then repeat the analysis
after accurately diluting 200 µl of sample with 200 µl of distilled water in a clean tube. Use
50 µl of the diluted sample for the analysis. Remember to multiply the result by 2 to obtain
the calcium concentration of the sample.

QUALITY CONTROL
At least two serum control specimens, having stated values in the range 2.00-2.90 mmol/l
one of which is unknown to the operator, should be included with each batch of
specimens. If single specimens are analysed a control specimen should always be included.

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 1.5% should be
attainable.
ROUTINE CONDITIONS VARIANCE      : The value obtained for the RCV should not
exceed 4%


REFERENCE VALUES
The reference interval for healthy ambulant adults is 2.25-2.60 mmol/l. Conversion of SI
units into “old” units: mmol/l x 4 = mg/dl.

The reference values are only appropriate if the patient has a normal serum albumin
concentration. If the patient has a low serum albumin then it may be helpful to report the
albumin corrected calcium as well as the measured calcium. The albumin corrected calcium
is calculated in this way:
(40 – Patient’s albumin) + measured calcium = albumin corrected calcium
  40
For example if the patient has an albumin of 20 g/l and a measured calcium of
1.90mmol/l, then the albumin corrected calcium is 2.40mol/l

(40-20) + 1.90 = 2.40 mmol/l
   40
The albumin corrected calcium will be approximately 2.20-2.60 mmol/l if low measured
calcium is a consequence of a low serum albumin.
                                                                                        31
Note: The estimation of calcium is difficult, particularly because of the possibility of
contamination by calcium. It is essential that high quality chemicals are used and that the
recommendations regarding cleaning of glassware are strictly followed. It may be found
helpful to keep separate test tubes, pipettes etc, only for the analysis of calcium
When preparing the reagents, be careful not to contaminate other chemicals or glassware
with calcium carbonate.

PRECAUTIONS
   Use dry glass container which must be chemically cleaned and acid washed
   Avoid use of plastic containers and use of rubber stoppers
   Avoid use of acid etched glassware. It may lead either to calcium loss because
   adsorption of calcium on to the damage surface or to contamination with calcium
   because the etched areas cannot be cleaned thoroughly.

REFERENCES
LAB/86.3




CALCIUM IN URINE
SPECIMENS: a 24 hour urine collection in an acid washed 2.5 L bottle; 10 ml of 1 N HCl
is added as the preservative. Acid washed beaker and a funnel should be issued from the
laboratory along with collection bottle.

PROCEDURE:
    Mix the 24 hour urine collection and measure the total urine volume
    Follow the procedure as for serum calcium analysis
Calcium samples with calcium concentration greater than 3.75 mmol/l should be diluted
with distilled water before analysis. Multiply the results by the dilution factor
                                                                                            32
9.       CREATININE

9.1      INTRODUCTION
Creatine is synthesized in the kidneys, liver and pancreas by two enzymatically mediated
reactions. In the first, transamidination of arginine and glycine forms guanidiniacetic acid:
the second methelation of guanidinoacetic acid occurs with S- adenosylmethionine as
methyl donor. Creatine is then transported in blood to other organs such as muscle and
brain, where it is phosphorylated to phosphocreatine a high energy compound.
Interconversion of phosphocreatine and creatine is a particular feature of metabolic
processes of muscle contration; some of the free creatine in muscle spontaneously
converts to creatinine, its anhydride. Between 1 and 2% of muscle creatine is converted to
creatinine daily. Because the amount of endogenous creatinine produced is propotional to
muscle mass, the production varies with age and sex; non obese men excrete about 1.5
g/day, women 1.2 g/day. Daily excretion of creatinine can be 10% to 30% greater as a
result of dietary intake of creatine and creatinine in meats. On the whole however, dietary
fluctuations of creatinine intake cause only minor variation in daily creatinine excretion on
the same individual. The excretion rate in one individual, in the absence of renal disease, is
relatively constant and parallels endogenous production. Most of the interindividual
variations of creatinine excretion in healthy persons are attributable in the main to age, sex,
and lean body mass. The interindividual variation tends to be less than 15% from day to
day.
9.2      CLINICAL SIGNIFICANCE
Because creatinine is endogenously produced and released into body fluids at a constant
rate and its plasma levels maintained within narrow limits, its clearance may be measured as
an indicator of GFR. However, a small quantity of creatinine is reabsorbed by the tubules
and as a result; creatinine clearance (if creatinine is measured with an accurate method) is
approximately 7 & greater than inulin clearance. Some methods for creatinine used in
clinical laboratories are nonspecific, however, and thus this difference is often smaller. The
creatinine clearance is performed by obtaining a 4-, 12- or 24-h urine specimen and also a
blood specimen sometime within the period of urine collection. The volume of the urine is
measured, urine flow rate is calculated (millimetres per minute), and the assay for creatinine
is performed on plasma and urine to obtain the concentration in milligrams per decilitre or
per millilitre. Two factors influence measurement of creatinine clearance and thus its
interpretation. First, the most common methods for measuring creatinine use the
nonspecific alkaline picarate reaction, and thus noncreatinine chromogens in plasma
increase the apparent plasma concentration by as much as 30% if serum values are less
than 1.0mg/dL, and by approximately 10% is values exceed 1.0mg/dL. The percent
increase is progressively less with higher creatinine concentrations. (Urine contains
considerably fewer noncreatinine chromogens.) this overestimation of plasma creatinine
concentration result in underestimation of creatinine clearance and partially offsets the
apparent high clearance of creatinine that is due to tubular secretion. As a result, the
endogenous creatinine clearance agrees closely with the inulin clearance throughout a
substantial range of clearances. However, if accurate emethods are used for assay of plasma
creatinine, the GFR estimated by creatinine clearance may not correlate with the GFR
estimated by inulin clearance. Secondly, GFR measured by creatinine clearance and GFR
measured by inulin clearance in the same patient progressively diverge as renal failure
progresses and plasma creatinine level rises. The greater apparent GFR found by creatinine
clearance may be due to an increase in tubular secretory activity for creatinine when plasma
levels increase much above normal and to the relatively smaller contribution of
noncreatinine chromogens in the nonspecific assay of plasma creatinine. In clinical
practice, it is now accepted that, by the time patients have lose one half to two thirds theirs
normal renal function, as demonstrated by creatinine clearance, it is more reliable and
prudent to monitor their subseqnet renal function and response to therapeutic initiatives
by using radioisotopic markers of glomerular filtration and renal plasma flow.
                                                                                            33
9.3      PRINCIPLE OF THE METHOD
Protein free filtrate is mixed with an alkaline picrate solution which forms a yellow –red
complex with Creatinine. The absorbance of the complex is measured at 500 nm.
9.4      SPECIMEN TYPE, COLLECTION AND STORAGE
Non haemolysed serum; collect about 4 - 5 ml blood into a clean dry bottle. Avoid
haemolysis. Separate the serum as early as possible from the cells within 12 hours of
collection. Serum is stable at 2-8 0C up to 24 hours
Referral: Send about 1.0 ml serum kept cool to reach destinations within 18 hours
9.5      APPARATUS AND CHEMICALS
APPARATUS:
pH meter
Spectrophotometer at wavelength 490 nm or Colorimeter with blue green filter, Ilford
603(490 nm)
GLASSWARE:
Volumetric flask (100 ml and 500 ml volumes)
Automatic micro pipettes (50, 200 and 500 µl)
Graduated pipettes (10 ml in 0.1 ml)
Measuring cylinders (50 ml and 100 ml)
Beakers (100ml, 500 ml)
Test tubes (125 mm x 13mm)
Reagent bottles, clear and amber coloured (500 ml), Rubber bulb
Conical centrifuge tubes 15 ml
CHEMICALS:
Sodium hydroxide pellets
Picric acid; Note: water is added to picric acid to ensure safety in transit
Standard buffers for pH meter
Creatinine anhydrous (pure)
Hydrochloric acid concentrated (37% w/v) caution: highly corrosive
Benzoic acid
Sodium tungstate dihydrate
Polyvinyl alcohol

REAGENTS
1. Saturated picric acid solution: Picric acid is supplied as a moist chemical. Weigh out the
   equivalent of 7 g of picric acid i.e. mix well the bottle and weigh out about 10.5 – 11 g
   if your picric acid container states that 50 % by weight of water has been added. Add
   11 g of moist picric acid to 500 ml distilled water, stir for several hours to ensure that a
   saturated solution is produced. Transfer in to a brown bottle. This solution is stable
   indefinitely at room temperature.
   Note: the amount of picric acid weighed out will depend on the water content of the
   chemical
2. Picric acid 0.036 mol/l: Measure 705 ml of saturated picric acid solution in to volumetric
   flask and make up to 1 litre with distilled water store in a brown bottle. This solution is
   stable indefinitely
3. Acid tungstate solution: Weigh out 11.1 g of sodium tungstate dihydrate (9.8 g anhydrous
   salt) and dissolve in about 300 ml of distilled water in a 1 litre volumetric flask.
   Dissolve 1 g of polyvinyl alcohol in about 100 ml of dish water with heating (do not
   boil) Allow to cool to room temperature then transfer into the volumetric flask
   containing sodium tungstate. Measure 2.1 ml of concentrated sulphuric acid in to 300
   ml of distilled water in a beaker mix well. Add this solution also-in to the same
   volumetric flask containing tungstate solution. Dilute to 1 litre with distilled water
   when the solution is cool to room temperature. Store in a brown bottle.
4. Sodium hydroxide solution 1.4mmol/l: Dissolve 56 g of sodium hydroxide in distilled water
   and dilute to 1 litre, store in a polypropylene bottle.
                                                                                           34
5. Hydrochloric acid 0.1mol/l: Carefully pipette 9.0 ml hydrochloric acid concentrated in
   to a volumetric flask containing distilled water, dilute to one litre with distilled water.
6. Creatinine standard 1.32mmol/l: Keep about 200 mg of Creatinine for overnight in a
   dessicator. Weigh out 149 g of Creatinine anhydrous and dissolve in a small volume of
   hydrochloric acid (0.1mmol/l) in a small beaker, transfer quantitatively to a one litre
   volumetric flask and make to 1 litre with hydrochloric acid. Store in a brown bottle
9.6     PROCEDURE
1. Label two conical centrifuge tubes one for the quality control serum and one for the
   patient’s sample. Pipette 4.0 ml of Acid tungstate reagent into each tube.
2. Pipette 500 µl of quality control serum or patients’ serum to the appropriate tubes.
   Mix vigorously for about 10 seconds then centrifuge for about 10 minutes to obtain a
   clear supernatant.
3. Transfer 3.0 ml of clear supernatant in to test tubes for each quality control serum or
   patient’s serum. For blank 3.0 ml of distilled water, for the standard pipette 3.0 ml of
   distilled water and 50 µl of Creatinine standard solution.
4. To each tube add 1.0 ml of picric acid solution (0.036 mol/l) mix well
5. Add 0.5 ml sodium hydroxide solutions (1.4mol/l) to the first tube, mix well at 30
   second intervals add sodium hydroxide solution to remaining tubes.
6. Exactly 15 minutes after addition of sodium hydroxide read the absorbance against the
   reagent blank at 500 nm. Read absorbance of the tubes in sequence maintaining 30
   seconds intervals between readings
9.7     CALCULATION
Dilution of standard in the assay           : 0.05ml STD + 3.0 ml of distilled water
                                            = 3/0.05 = 60
Concentration of the stock standard         = 1.32 mmol/l (1320 µmol/l)
In the assay condition                      =1.32/60 =0.022 mmol/l
Dilution of serum in the assay              =1 in 9
∴ Serum Creatinine concentration            =Abs of Test x Con of Std x Dilution of serum
                                              Abs of Std
                                            =T/S x 0.022 x 9
                                            =T/S x 0.198
                                            =T/S x 200 µmol/l

QUALITY CONTROL
OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 4% should be
attainable.
ROUTINE CONDITIONS VARIANCE: This value should not exceed 8%

9.8     LIMITATIONS
Proteins give positive Jaffe reaction therefore
    The supernatant fluid should be clear
    Pipetting of 3 ml of supernatant should be done carefully without disturbing the
    precipitate.
    Sodium hydroxide solution should be added in timed intervals
    Absorbance reading should be measured exactly 15 minutes after addition of Sodium
    hydroxide
    Interfering substances reacting like Creatinine are acetone, acetoacetate, pyruvate and
    some cephalosporin antibiotics contribute to total colour production
    Blood constituents such as glucose, ascorbate histidine and adrenaline may cause
    fading or enhancement of the colour.
                                                                                                 35
10.   URINE CREATININE

      10.1       SPECIMEN TYPE, COLLECTION AND STORAGE
      24 hours collection of urine, empty the bladder first and note the time, from that time
      onward collect the sample into amber colour preservative added 2 litre bottle till 24th hour
      and keep the bottle in the refrigerator during collection period, after completion of 24
      hours collection, bring the bottle to the laboratory
      10.2       PRINCIPLE OF THE METHOD
      After dilution, the urine is mixed with picric acid and sodium hydroxide solution which
      forms a yellow red complex with Creatinine. The absorbance of the complex is measured
      at 500 nm.
      10.3       APPARATUS AND CHEMICALS
      Same as for serum Creatinine estimation.

      PRESERVATIVE
      One Normal hydrochloric acid- 10ml or
      Few crystals of thymol or 5 ml of a 100g/l solution in isopropanol
      10.4       PROCEDURES
      1. Mix the 24 hours urine collection. Measure the total urine volume.
      2. Pipette 1 ml of urine into a 100 ml volumetric flask. Make up to 100 ml with distilled
         water. Mix well (1:100 dilution)
      3. Three test tubes are required. One for the Test, one for a reagent blank, and one for
         the standard
      4. Transfer 3.0 ml of diluted urine into the Test. 3.0 ml of distilled water into the reagent
         blank. 3ml of distilled water and 0.1 ml of standard solution into the standard.
      5. To each tube add 1.0 ml picric acid solution (0.036mol/l). Mix well.
      6. Add 0.5 ml sodium hydroxide solution (1.4mol/l) solution to the first tube, mix well at
         30 seconds intervals, and add sodium hydroxide solution to the remaining tubes.
      7. Exactly 15 minutes after adding sodium hydroxide read the absorbance against the
         reagent blank at 500 nm. Read the tubes in sequence maintaining 30 seconds intervals
         between readings.
      10.5       CALCULATION
      Same method as serum Creatinine but 3 ml of diluted urine (1:100 with distilled water) is
      used for the filtrate
      No protein precipitation is needed

             Concentration of standard            = 1.32 mmol/l (1320 µmol/l)
             Standard dilution is                 = 1 in 30 (i.e. 0.1 ml standard diluted with 3.0
                                                           ml of distilled water)
             ∴standard concentration              = 1.32 mmol/l = 0.044 mmol/l
                                                           30
             urine dilution is                    = 1: 100
             ∴ urine Creatinine                   = Test x 0.044 x 100 mmol/l
                                                    Standard
             24 hour Urine Creatinine             = Test(T) x 4.4 x Total volume in litre(TV)
             (mmol/24 hour)                         Standard(S)

      REFERENCES VALUES: 8.84 to 17.6 mmol/24 hours

      CHILDREN
         Urine Creatinine                         = T/S x 4.4 x Total volume x 1000/B.W
                                                  = x µmol/kg/24 hours
                                                                                                36
10.6        CREATININE CLEARANCE
      The following details of the patients are required for calculation of Creatinine
      Clearance
          Height
          Body weight(BW)
          Age
          Total volume of urine collection over timed period (over 24 hours)
      Blood sample should be collected during the 24 hours urine collection
      Serum Creatinine and 24 hours urine Creatinine should be done for calculation of
      Creatinine clearance
      Creatinine Clearance =UV/P
      U-Urine Creatinine in mmol/l
      V-Rate of urine flow in ml/minute
      P-Serum Creatinine in mmol/l
      V= 24 hours urine volume in ml
                   24 x 60

Creatinine Clearance=Concentration of urine Creatinine in mmol/l x 24 hour urine volume in ml
                     Concentration of serum Creatinine in mmol/l         24 x 60

Clearance varies with body size and is proportional to the body area (A) where this varies
much from the normal in adults and in all cases in all cases in children.
The determined clearance is corrected to a standard surface area of 1.73 m2 by multiplying
by 1.73/A. The A (Body surface area can be calculated using a nomogram. From height
and weight)

Corrected Creatinine Clearance= Creatinine clearance x 1.73
                                                            A

REFERENCES VALUES: Refer the appendix 2

REFERENCES
WHO manual
Varley’s practical clinical Biochemistry sixth edition
                                                                                           37
11.     CHOLESTEROL

11.1    INTRODUCTION
Although every living organism examined has been found to contain sterols, cholesterol is
found almost exclusively in animals and humans, in which it is also the main
sterol.Virtually all cells and body fluids contain some cholesterol.Like other
sterols,cholesterol is a solid alcohol of high molecular weight and possesses the tetracyclic
perhydrocyclopentanophenanthrene skeleton. The molecule contains 27 carbon
atoms.Cholesterol is the initial starting point in many metabolic pathways. These include
vitamin D synthesis, steroid hormone synthesis, and bile acid metabolism.
       Cholesterol is presented to the intestinal wall from three sources: the diet, bile and
intestinal secretions and cells. Animal products, especially meat, egg yolk, seafood, and
whole-fat dairy products, provide the bulk of dietary cholesterol. Cholesterol intake varies
considerably according to the dietary intake of animal products. A similar amount of
cholesterol is present in the gut from biliary secretion and the turnover of mucosal cells.
Practically all cholesterol in the intestine is present in the unesterified (free) form.
Esterified cholesterol in the diet is rapidly hydrolyzed in the intestine to free cholesterol
and free fatty acids (FFA) by cholesterol esterase in pancreatic and small intestinal
secretions.
         Although portion of the body’s cholesterol is derived from dietary intake, most
tissue and plasma cholesterol is synthesized endogenously by the liver and other tissues
from simpler molecules, particularly acetate.
       Once synthesized cholesterol is released into the circulation for transport in
combination with specific apoproteins, the apolipoproteins, in complexes known as
lipoproteins. Minimal cholesterol esterification occurs within the liver before its release,
and cholesterol is mainly esterified within the vascular compartment. Esterification is
important because it serves to enhance the lipid carrying capacity of the lipoproteins. The
reaction is catalyzed by the enzymes lecithin-cholesterol acyltransferase (LCAT) in the
plasma and acyl-cholesterol acyltransferase (ACAT) intracellularly.The intracellular ACAT
pathway is the major pathway in the liver, intestine, adrenal cortex, and probably in the
arterial wall.
       Once cholesterol enters the cell, the esters are hydrolyzed by the action of specific
esterases and enters into specific metabolic pathways.
      Cholesterol reaching the liver is either secreted unchanged into bile or metabolized to
bile acids. Approximately one third of the daily production of cholesterol is catabolized
into bile acids. The first step in the bile acid synthesis involves the rate limiting step, 7
alpha hydroxylation. Two bile acids, cholic and chenodeoxycholic, constitute the primary
bile acids.They are conjugated with either glycine or taurine and enter the bile canaliculi.
Some of the bile acids are deconjugated and converted by bacteria in the intestine to
secondary bile acids. Cholic acid is converted to deoxycholic acid, and chenodeoxycholic
acid is metabolized to lithocholic acid.Virtually all bile acids except lithocholic are
reabsorbed in the lower third of the ileum and returned to the liver via the portal vein, thus
completing the enterohepatic circulation.

11.2    CLINICAL SIGNIFICANCE
High prevalence of atherosclerosis and ischaemic heart disease is seen where dietary fat
intake is relatively high. High plasma levels of LDL, IDL and possibly VLDL are
associated with an increased risk of premature atherosclerosis and ischaemic heart disease.
This relationship appears to be a continuous (curvilinear) one, i.e. there is no threshold
above which risk abruptly appears. Plasma HDL cholesterol concentration is a negative
risk factor, so that high levels appear to protect against ischaemic heart disease and low
levels are associated with an increased risk of ischaemic heart disease.
                                                                                        38
11.3       PRINCIPLE OF THE METHOD
The cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator
quinoneimine is formed from hydrogen peroxide and 4 – aminophenazone in the presence
of phenol and peroxidase.

11.4       PATIENT PREPARATION
       No change in dietary habits for at least 3 weeks
       To analyze cholesterol alone 12 hours fasting is preferred.

BLOOD DRAWING TECHNIQUE
   Patient should be seated.
   Blood is drawn from antecubital vein.
   Patient should be subjected to minimum amount of stress during blood drawing.

11.5       SPECIMEN TYPE, COLLECTION AND STORAGE
12 hours fasting, 2-3 ml clotted blood or 2 ml EDTA blood without tourniquet avoid
haemolysis. Serum should be separated from cells as early as possible. Specimen preferably
be analysed on the day of collection. Serum is stable for 4 days at 4 0C, for 3 months at
-20 0C and for many years at -70 0C. As referral sample send about 0.5 ml clear serum, kept
cool, to reach destination within 24 hours.

11.6       APPARATUS AND CHEMICALS

APPARATUS:
Spectrophotometer at wavelength 500 nm
Water bath at 37 0C
Vortex mixer
GLASSWARE:
Test tubes 100mm x 13mm
Automatic micropipette 10 µl
Automatic pipette 1000µl
Semi micro cuvette (capacity =1ml)

REAGENTS
There are various reagents and standard are commercially available for cholesterol
estimation , evaluate the kits using Quality Control sample with low, high, normal value
and consider following to choose commercially available kits
a. Reagent stability
b. Expiry date
c. Test procedure
d. Interfering substances
e. Other related factors

11.7       PROCEDURE
1. label tubes for Blank, Standard, Quality control and test
2. Add 10 µl of distilled water to Blank , 10 µl of standard solution to Standard, 10µl of
   QC sample to Quality control and 10µl of patients serum to test
3. Add 1000 µl of cholesterol reagent to all the tubes
4. Mix well and incubate all the tubes at 37 0C for 5 minutes in water bath
5. Mix well, zero the spectrophotometer with reagent blank and read the absorbance at
   500 nm
   Strict to the test procedure available with the reagent kits that is being used
                                                                                          39
11.8       CALCULATION
Concentration = Absorbance of test x Standard concentration (mmol/l)
                Absorbance of standard

QUALITY CONTROL
Include Quality Control sample for every batch of tests.

OPTIMAL CONDITIONS VARIANCE                  : A coefficient of variation of around 7%
should be attainable.
ROUTINE CONDITIONS VARIANCE                  : This value should not exceed 14%

11.9       LIMITATION
       The test is linear up to a cholesterol concentration of 750 mg/dl (19.3mmol/l). Dilute
       samples with a higher cholesterol concentration 1+2 with physiological saline (0.9 %)
       and repeat the determination. Multiply the result by 3.
       Haemoglobin up to 200 mg/dl does not interfere with the test
       Bilirubin >5mg/dl and ascorbic acid>10 mg/dl interfere the test
       This limitation varies with kits to kits.

NOTE: Do not report results from specimens with suspected interference, inform the
physician, of the problem.

PRECAUTIONS
   Allow the samples, standard, QC and reagents to attain room temperature
   Mixed well the thawed sample
   Take not more than 20 samples for a batch to maintain good quality
   Verify the temperature of the water bath (at 37 C) before start the test.
   Since final volume would be 1 ml , do not use long tube it makes difficult in pipetting
   Wipe outside of the pipette tip using a piece of gauze
   Add serum to the bottom of the tube
   Mix well in each step
   Any colour change of the blank should be compare with previous day blank reading
                                                                                                40
12.   GLUCOSE

      12.1    INTRODUCTION
      Glucose is the primary energy source for the human body. It is derived from the
      breakdown of carbohydrates in the diet (grains, starchy vegetables and legumes) and in the
      body stores (glycogen), as well as by endogenous synthesis from protein or the glycerol
      moiety of triglycerides. When energy intake exceeds expenditure, the excess is converted to
      fat and glycogen for storage in adipose tissue and liver or muscle respectively. When energy
      expenditure exceeds caloric intake, endogenous glucose formation occurs from the
      breakdown of carbohydrate stores and from non carbohydrate sources.(amino acids,
      lactate, and glycerol)

      The glucose level in blood is maintained within a fairly narrow range under diverse
      conditions (feeding, prolonged fasting or severe exercise) by regulatory hormones. These
      include insulin, which decreases blood glucose, and the counter regulatory hormones
      (glucagon, cortisol, noradrenalin and growth hormone) which increase blood glucose
      levels.

      12.2    CLINICAL SIGNIFICANCE
      Diabetes mellitus is a group of metabolic disorders of carbohydrate metabolism in which
      glucose is underutilized, producing hyperglycemia. Some patients may develop acute life
      threatening hyperglycemic episodes, such as ketoacidosis or hyperosmolar coma. As the
      disease progresses the patients are at increased risk of developing specific complications
      including retinopathy leading to blindness, renal failure, neuropathy(nerve damage),and
      atherosclerosis.The last may result in stroke, gangrene or coronary disease.
      (Please refer the article in annexure for current World Health Organization recommended
      criteria for diagnosis of Diabetes Mellitus.)

      Hypoglycemia is a blood glucose concentration below the fasting range, but it is difficult to
      define specific limits. No symptoms are specific for hypoglycemia. A rapid decrease in
      plasma glucose to hypoglycemic levels usually triggers a sympathetic response, with the
      release of nor adrenaline, which produces classical signs and symptoms of hypoglycemia:
      weakness. Sweating, nausea, rapid pulse, lightheadedness and hunger. The brain is totally
      dependent on blood glucose, and very low levels of plasma glucose(less than 20 or 30 mg
      /dl cause severe central nervous system dysfunction.

      Neonatal blood glucose concentrations are much lower than adults and decline shortly
      after birth when live glycogen stores are depleted. Glucose levels as low as 30 mg/dl in a
      term infant and 20mg/dl in a premature infant may occur without any clinical evidence of
      hypoglycemia. The more common causes in the neonatal period include prematurity ,
      maternal diabetes and maternal toxemia. These are usually transient. Hypoglycemia with
      onset in early infancy is usually less transitory and may be due to inborn errors of
      metabolism or ketotic hypoglycemia, which usually develop after fasting or febrile illness.

      12.3    PRINCIPLE OF THE METHOD
      The aldehyde group of β - D – Glucose present in the plasma is oxidized by the enzyme
      Glucose oxidase to gluconic acid with liberation of hydrogen peroxide. The hydrogen
      peroxide is converted to water and molecular oxygen by the enzyme peroxidase. In the
      presence of an oxygen acceptor or 4 - aminophenazone together with phenol, a pink
      colour is formed which is measured at 510 nm.

      SPECIMEN CONTAINERS
         Blood containers should be leak proof and be easy to close and open without
         contaminating the fingers.
                                                                                      41
    Screw capped bottle with a rubber liner (Bijou bottles) is satisfactory.
    Bottles should be washed with a detergent, rinsed in several changes of clean water,
    rinsed in distilled water and dried well.

ANTICOAGULANT AND PRESERVATIVE
   4 mg of a mixture of potassium oxalate and sodium fluoride in the ratio of 3:1 is
   sufficient to collect 1 ml of blood.
   A solution can be prepared so that 0.1 ml contains 3 mg of potassium oxalate and 1mg
   of sodium fluoride.

PREPARATION BLOOD SUGAR BOTTLES
   Weigh out 3 g of potassium oxalate and 1 g of sodium fluoride separately into beakers
   Dissolve the chemicals well and transfer into a 100 ml volumetric flask, mix well and
   make up to 100ml with distilled water.
   Store the solution in a bottle at room temperature
   To collect 1 ml of blood add 0.1 ml of the prepared solution into bijou bottle and dry
   in the oven at 60 0C
   Allow to cool; stopper and label the bottles, the amount of blood is to be collected
   should be mentioned

12.4    SPECIMEN TYPE, COLLECTION AND STORAGE
    1 ml blood collected into blood sugar bottle
    Haemolysis free plasma

FASTING SPECIMEN
   For adults the fasting time is usually 10-12 hours.
   For children the fasting time is 6 hours unless longer time is indicated.

POST PRANDIAL SPECIMEN
   Blood collected 2 hours after a meal

RANDOM SPECIMEN
   Blood sample collected at any time regardless of food intake

STABILITY
   Glucose stabilized up to 24 hour at room temperature when collected in an oxalate
   and fluoride mixture.
   Plasma should be separated soon after collection preferably within 1 hour
   Separated plasma should not contain RBC or Leucocytes

BLOOD COLLECTION
VENOUS BLOOD
   Avoid an intravenous (IV) infusion arm
   Do not shake the blood but gently mix with the anticoagulant.(to prevent haemolysis)
   Exact amount of an anticoagulant and blood should be mixed since sodium fluoride
   inhibits the action of glucose oxidase and peroxidase in the assay.

12.5    APPARATUS AND CHEMICALS
APPARATUS:
Analytical balance accurately calibrated
Oven, temperature at 100 0C
Water bath, temperature at 37 0C
Spectrophotometer with 510 nm
                                                                                            42


GLASSWARE:                                              CHEMICALS:
Volumetric flask (100 ml A grade                        Benzoic acid
and 500 ml volumes)                                     4 - amino phenazone/4 - amino anti
Automatic micro pipettes (100 µl)                       pyrine-AR
Graduated pipettes (2 ml in 0.1 ml)                     Glucose oxidase
Petri dish, watch glass or beaker                       Peroxidase lyophilized powder
Pasteur pipette                                         Phenol crystals-AR
Test tubes (100 mm x 13mm)                              Tween 20
Reagent bottles, clean and amber                        D-Glucose anhydrous-AR
coloured                                                Disodium hydrogen phosphate
Rubber bulb                                             dihydrate – AR (Na2HPO4.2H2O)
                                                        Potassium dihydrogen phosphate-
                                                        AR      (KH2PO4)
                                                        Sodium azide AR

REAGENTS
1. Benzoic acid solution 1g/l: Weigh 1g of benzoic acid and transfer it to a 1 litre volumetric
   flask. Add about 800 ml of distilled water and mix to dissolve the chemical completely.
   Make up to 1 litre mark with distilled water and mix well. Transfer to a clean bottle,
   label the bottle and store at room temperature. The solution is stable indefinitely.
   Benzoic acid will take some time to dissolve (Distilled water at 50 – 70 0C can be used
   –Monica)
2. Stock glucose standard solution 1 g %( 55.55 mmol/l)
        Use dry and clean glassware
        Weigh 1.3 g of D-Glucose anhydrous (analytical grade) into a watch glass or Petri
        dish or into a beaker. Spread the chemical over the bottom of the container and
        keep in an oven at 60- 80 0C for 4 hours.
        Allow to cool in a desiccator and weigh out 1 g of dried glucose accurately
        Transfer the chemical from the weighing container to a volumetric flask using a
        funnel. Wash any chemical remaining in the container into the volumetric flask
        with the benzoic acid solution (1 g/l). Always use a funnel to transfer the chemical
        or solutions from any container to a flask
        Half fill the volumetric flask with benzoic acid solution and mix until the chemical
        is completely dissolved. Make the solution up to 100 ml with benzoic acid
        solution. Make sure the bottom of the meniscus of the fluid is on the graduation
        mark when viewed at eye level
        Use a Pasteur pipette to add the final volume of the benzoic acid solution to the
        flask.
        Mix the solution well by inverting the flask for several times. Rinse the bottle with
        small quantity of the standard solution, transfer in to the bottle and put the date of
        the preparation on the label
        The standard is stable for three months at 2- 8 0C
   Note: The glucose standard solution should be kept at room temperature for 24 hours
   to α - β forms to reach in equilibrium after preparation (H&W- 6ht edition)
3. Working glucose standard 5.55 mmol/l:
        Allow the stock glucose solution to attain room temperature.
        Pipette accurately 10 ml of stock glucose solution using a volumetric pipette A
        grade ( bulb pipette)
        Carefully dispense into a 100 ml volumetric flask A grade
        Make up to the 100 ml mark with benzoic acid solution (1 g/l) use a Pasteur
        pipette to add the final volume of the benzoic acid solution to the flask. Make sure
        the bottom of the meniscus of the fluid is on the graduation mark when viewed at
        eye level. Stopper and mix the solution well by inverting the flask several times.
                                                                                           43
       Rinse a clean dry bottle with small quantity of standard solution and transfer the
       solution into the bottle. Store in the refrigerator at 2- 08 C. This solution is stable
       for three months at 2- 08 C
4. Phosphate buffer 100 mmol/l pH 7.0 :
   Disodium hydrogen phosphate dihydrate [Na2HPO4.2H2O]                12.95 g
   Anhydrous Potassium dihydrogen phosphate [KH2PO4 ]                  4.95 g
   Sodium azide [NaN3 ]                                                0.50 g
   Distilled water to                                                  1 litre
       Measure about 800 ml of distilled water into a 1 litre volumetric flask
       Weigh out chemicals and add one by one in the order into the flask. Mix to
       dissolve the chemicals
       Check that the pH is 7.0 ± 0.05 with a pH meter, make up to 1 litre mark with
       distilled water and mix well.
       Transfer the reagent to a clean bottle and label. The reagent is stable for 3-4
       months at 2-8 0C
5. Colour reagent(100ml)
   4 – Amino phenazone                                                 16 mg
   Glucose oxidase                                                     1800 units
   Peroxidase                                                          100 units
   Phenol                                                              105 mg
   Tween 20                                                            50 µl
   Phosphate buffer to                                                 100 ml

To prepare 500 ml of colour reagent:
    i.   Glucose oxidase (GOD): Available as lyophilised powder and as liquid form.
         Different products are found in different definitions for units of activity of
         glucose oxidase. Read the label for the activity. E.g. 250 units/mg
         Therefore the amount of GOD powder to be weighed to contain 1800 units of
         GOD is 1800/250=7.2 mg
         To prepare 500 ml of colour reagent 7.2 x 5 =36 mg is required, weigh out 36 mg
         of GOD lyophilized powder accurately in a small beaker and dissolve in 10 ml of
         phosphate buffer carefully.
   ii.   Peroxidase (POD): Read the label for the activity. E.g. 63 units/mg
         Therefore the amount of peroxidase powder to be weighed to contain 100 units
         of POD is 100/63 = 1.58 mg
         To prepare 500 ml colour reagent 1.58 x 5 = 7.9 mg is required; weigh out 7.9 mg
         of POD in a small beaker and dissolve in about 10 ml of phosphate buffer.
  iii.   Transfer about 400 ml of phosphate buffer into a 500 ml volumetric flask
  iv.    Add the glucose oxidase solution into the flask using a funnel. Rinse out the
         beaker into the flask with a little of the phosphate buffer to make sure all the
         GOD is transferred to the flask
   v.    Add the POD solution into the flask described above ( iv)
  vi.    Weigh out 80 mg of 4 – aminophenazone in a small beaker and transfer into the
         flask with rinsing with the phosphate buffer.
 vii.    Weigh rapidly 525 mg of crystalline phenol in a beaker. Transfer into the flask
         carefully using the funnel. Rinse the beaker into the flask with phosphate buffer
         and mix well.
         Note: Phenol is highly corrosive, toxic and hygroscopic chemical. Therefore
         handle it with great care. To avoid damaging the balance pan, always remove the
         beaker when adding or subtracting the chemical. Make sure the stock bottle of
         phenol is tightly stopperd after use.
 viii.   Measure 250 µl (0.25 ml) of Tween 20 and add into the flask. Make up to the 500
         ml of mark with phosphate buffer, stopper and mix well.
  ix.    Transfer to a clean brown bottle, label and store at 2-8 C. The reagent is stable for
         about one month.
                                                                                            44
12.6    PROCEDURE
1. Label sufficient test tubes for the batch including standard (S) Quality controls (C1,C2)
    and patients samples (1,2,3, etc)
2. pipette into the appropriately labelled 13 x 100 mm tubes as follows
                                                       S1     S2      C1, C2        1, 2, 3
Distilled water (ml)                                   1.9    1.8     1.9           1.9
Glucose standard 5.5 mmol/l (µl)                       100    200     -             -
Quality control/Patient’s plasma (µl)                  -      -       100           100
Mix well
3. Label a second set of tubes including reagent blank (B), standard (S1, S2), Quality
    controls (C1, C2 ) and patient’s samples (1, 2, 3 )
4. Pipette into the tubes as follows.
                                           Blank         S1     S2      C1, C2        1, 2, 3
Distilled water (µl)                        100           -      -         -             -
Diluted standards (µl)                       -        100      100         -            -
Diluted patient’s sample(µl)                 -          -       -         100         100
Colour reagents(ml)                         1.2       1.2      1.2        1.2         1.2

5. Mix all tubes well, incubate at 37 0C in a water bath for 15 minutes.
6. Shake tubes two or three times during this period to ensure adequate aeration
7. Remove from the water bath, cool to room temperature and read the absorbance in a
   spectrophotometer at 510 nm. Set the instrument to zero with the reagent blank (B).
8. Perform the standards in duplicate for greater accuracy and precision.
9. Calculate the results in mmol/l and check the quality control results.

PREPARATION OF CALIBRATION GRAPH
The calibration graph must be prepared in order to confirm the linearity of the method
and should be checked whenever a new batch of reagents are introduced or any change in
the spectrophotometer is being made.
Prepare the calibration graph standards from the Glucose working standard 5.5 mmol/l as
described in the table below in clean tubes (13 x 100 mm) as accurately as possible.
                                                   S1    S2     S3      S4     S5    S6
Glucose working standard 5.5mmol/l (µl)            50    100 200 300 400 500
Distilled water (ml)                               1.95 1.9     1.8     1.7    1.6   1.5
Glucose concentration mmol/l                       50    100 200 300 400 500
                                         Mix well
Draw a calibration graph by plotting the absorbance values of standards against the
concentration of standards.
The points should be linear and the graph should pass through the origin

12.7    CALCULATION
When the calibration graph is linear one of the standards used to prepare the calibration
graph should be included in each batch of tests.
The Beer & Lambert formula can be used to calculate the concentration of unknown
samples.
Concentration of Glucose in mmol/l = Test /Standard x Concentration

QUALITY CONTROL
Include QC sample for each batch of tests

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 3 % should be
attainable.
                                                                                          45
ROUTINE CONDITIONS VARIANCE: This value should not exceed 6 %

REFERENCE VALUES
Random plasma Glucose level                          ≤7.8 mmol/l
Fasting plasma Glucose level                         3.3-6.1 mmol/l
Post prandial plasma Glucose level                   ≤11.1mol/l

12.8        LIMITATION
      Any sample that gives a glucose value >450 mg/dl should be diluted 1:2 with 0.9 %
      Sodium chloride solution and the correct value obtained by multiplying the result by 3.
      At high plasma levels of uric acid, glutathione and Bilirubin may interfere with the
      assay by causing a decrease in glucose values. Ascorbic acid will decrease glucose
      values by retarding colour development. Do not report results from specimens with
      suspected interference. Inform the requesting physician of the problem.

PROBLEMS AND PRECAUTIONS
   In preparation of sugar bottle add correct volume of anticoagulant since NaF inhibit
   enzyme activity , excess NaF may lead to falsely low glucose level
   As the glucose oxidase is enzymatic method the pH of buffer should be 7.0 ,
   temperature should be 37 0C and the period of incubation also be accurate as
   mentioned in the technique
   No colour development or low colour development may be due to
   o Expired colour reagent
   o Unsuitable or expired glucose oxidase or any other chemicals
   o Check incubation time and temperature of water bath
   o Pipetting errors
   o If the problem is with glucose oxidase chemical then increase the amount of
       glucose oxidase in colour reagent preparation and compare the previous standard
       reading
   End product volume is 1.3 ml , ∴ use semi micro cuvette , do not use macro cuvette

12.9        OTHER METHODS
COPPER REDUCTION
       1. Phosphomolybdate (Folin wu)
       2. Arsenomolybdate (Somogyi-Nelson)
       3. Neocuproine
       4. Alkaline fericyanide method
       5. O-Toluidine method

ENZYMATIC
      6. Hexo kinase (HK)
      7. Glucose oxidase (GOD)-oxygen consumption
      8. Glucose dehydrogenase
      9. Radiation energy attenuation

OTHER
             1. Isotope dilution mass fragmentography

REFERENCES
Trinder,p. (1969).Annals of Clin.Biochem.6:24-27
Barham D and Trinder P. (1972). Analyst 97:142-145
                                                                                                 46




13.   INORGANIC PHOSPHATE

      13.1    INTRODUCTION
      Phosphorus in the form of inorganic or organic phosphate is an important and widely
      distributed element in the human body. An adult human has approximately 600g (19.4
      mol) of phosphate expressed as phosphorus, of which about 85% is in the skeleton and
      the rest principally in soft tissues. In the soft tissues most phosphate is cellular. Although
      both inorganic and organic phosphate are present in cells, most is organic and
      incorporated into nucleic acids, phospholipids and high energy compounds involved in
      cellular integrity and metabolism.
      Serum phosphate has a diurnal variation. It is higher in the afternoon and evening. The
      serum phosphate level is dependent on meals and variation in the secretion of hormones
      such as parathyroid hormone. Serum calcium and phosphate levels are regulated by the
      kidneys.
      The most important intracellular function of phosphate is the high energy bond in
      adenosine triphosphate. These energy sources maintain many physiological functions such
      as muscle contractility, neurological functions and electrolyte transport. Phosphate is a
      constituent of cyclic adenine and guanine nucleotides as well as nicotinamide adenine
      dinucleotide phosphate, which is important in many enzyme systems. It is an element in
      phospholipid cell membranes, nucleic acids and phosphoproteins. It is also involved in the
      regulation of intermediary metabolism of proteins, fats and carbohydrates, as well as in
      gene transcription and cell growth.
      Extra cellular phosphate maintains the critical intracellular concentration and provides
      substrate for bone mineralization.
      The skeleton serves as a store house for phosphate. The cellular demands for metabolic
      function in bone cells are similar to those in other cells.

      13.2    CLINICAL SIGNIFICANCE
      Hypophosphataemia is defined as the concentration of inorganic phosphate in the serum
      below the normal reference interval. Hypophosphatemia does not necessarily imply
      intracellular phosphate depletion. Hypophosphataemia may be present when cellular levels
      are normal, and cellular phosphate depletion may exist when serum concentrations are
      normal or even high. Phosphate depletion may have four general causes

      Intra cellular shift (A high carbohydrate diet stimulates insulin secretion, increasing the
      transport of glucose and phosphate into the cell.)
      Renal Phosphate wasting (Any cause of excessive parathyroid hormone secretion may
      result in hypophosphataemia due to phosphaturia
      Decreased intestinal phosphate absorption (Increased loss due to vomiting, diarrhoea and
      phosphate binding antacids: Decreased absorption in malabsorption syndromes.)
      Cellular phosphate loss (Acidosis results in catabolism of organic compounds within the
      cell so that inorganic phosphate shifts into the plasma and excreted in the urine.

      Hyperphosphatemia is usually due to acute or chronic renal failure because the kidneys fail
      to excrete the amount taken in the diet. Lack of Parathyroid hormone and increased
      growth hormone causes increased tubular reabsorption of phosphate results in increased
      phosphate levels in blood. Increased phosphate intake, increased extra cellular phosphate
      load in acidosis and any cause leading to cell lysis causes hyperphostaemia
                                                                                        47
13.3     PRINCIPLE OF THE METHOD
The filtrate obtained after removing proteins by means of trichloro acetic acid is treated
with an acid molybdate reagent which reacts with inorganic phosphate to form
phosphomolybdic acid. The molybdenum of the phosphomolybdic acid is reduced by
means of 1, 2, 4 amino naphtholsulphonic acid to give a blue compound which is
measured colorimetrically at 700 nm.

13.4    SPECIMEN TYPE, COLLECTION AND STORAGE
Clear, non haemolysed serum is suitable, collect about 4-5 ml blood without haemolysis
into a clean container. Ideally specimens should be obtained without the tourniquet from a
recumbent fasting patient. Serum should be separated from erythrocytes as soon as
possible within 1 hour of collection, as organic phosphate present in erythrocytes are
hydrolysed with formation of inorganic phosphate causing the serum concentration to rise.
Hydrolysis proceeds more rapidly at room temperature and at 37 0C. Haemolysed samples
are unsuitable because erythrocyte contain high concentration of organic phosphates which
can be hydrolysed to inorganic phosphate during storage.

13.5    APPARATUS AND CHEMICALS
APPARATUS:
Centrifuge
Spectrophotometer
GLASSWARE:
Volumetric flasks (100 ml, 200ml, 500 ml)
Beakers (5ml, 200 ml, 500ml)
Automatic pipette (500 µl, 1000 µl)
Graduated pipette (0.5 ml, 1ml, 5ml, 10 ml)
Centrifuge tubes 15 ml
Test tubes
CHEMICALS:
Ammonium molybdate-AR
1, 2,4Aminonapthol sulphonic acid
Perchloric acid AR
Sodium metabisulphite-AR
Sodium sulphite-AR
Potassium dihydrogen phosphate KH2 PO4 - AR
Trichloro acetic acid - AR

REAGENTS
1. Reducing agent: Dissolve 12 g of sodium Meta bisulphite and 2.4 g of sodium sulphite in
   about 80 ml of distilled water. Add 0.2 g of 1.2.4 amino naphthol sulphonic acid.
   Dissolve and dilute to 100 ml in volumetric flask. Store in the refrigerator in a brown
   bottle. The solution keeps up to 4 weeks. It is better prepare fresh reagent ( about 10
   ml)
2. Trichloroacetic acid 10 %: Dissolve 10 g of Trichloroacetic acid in water and make up to
   100 ml with distilled water. Store in the refrigerator.
3. Perchloric acid AR
4. Ammonium Molybdate 5%: Dissolve 5 g of Ammonium molybdate in distilled water and
   make up to 100 ml with distilled water. Store at room temperature. Discard the
   solution when a precipitate appears.
5. Stock phosphate standard solution 32mmol/l: Keep about 2.5 g of pure potassium
   dihydrogen phosphate in a dessicator to dry, weigh exactly 2.194 g of potassium
   dihydrogen phosphate and transfer into a 500 ml flask. Dissolve in distilled water and
   make up to 500 ml with distilled water. Store at room temperature.
6. Working phosphate standard solution 0.128 mmol/l: Dilute 2 ml of stock phosphate
   standard to 500 ml with distilled water. Store at room temperature
                                                                                               48
13.6    PROCEDURE
1. Label sufficient centrifuge tubes for quality control (C) and patient’s samples (1, 2, 3)
2. Pipette into tubes as follows

                                                                         C           1, 2, 3
Trichloroacetic acid 10 % (ml)                                           9.0          9.0
Quality control serum (ml)                                               1.0            -
Patient’s sample (ml)                                                     -           1.0

3. Mix well and leave for about 5 minutes. And centrifuge for about 10 minutes
4. Label a set of tubes including reagent blank (B) Standard (S) Quality control (C1, C2)
   and patient’s samples (1, 2, 3 …)
5. Pipette into the tubes as follows

                                                          B         S          C       1, 2, 3
Trichloro acetic acid 10% (ml)                           5.0        -           -         -
Working standard solution (ml)                            -        5.0          -         -
Supernatants from tubes above(ml)                         -         -          5.0       5.0
Perchloric acid (ml)                                     0.4       0.4         0.4       0.4
Ammonium Molybdate 5% (ml)                               0.4       0.4         0.4       0.4
Reducing reagent (ml)                                    0.2       0.2         0.2       0.2

6. Mix the tubes after each addition of reagents.
7. Leave at room temperature for 10 minutes
8. Read the absorbance at 700 nm , set the instrument to zero with tube B

13.7    CALCULATION
Concentration of standard is 0.128 mmol/l, serum dilution is 1:10
∴Concentration of phosphorus =           T x 0.128 x 10
                                         S
                                  =      T x 1.28 mmol/l
                                         S

13.8    LIMITATION
Glucose phosphate, CPK and other organic phosphates may also be hydrolyzed by assay
conditions, resulting in overestimation of inorganic specimens.

PRECAUTIONS
   Glass ware should be properly cleaned and rinsed because phosphate is a common
   component of many detergents
   Discard Ammonium Molybdate solution when a precipitation formed as the bottom
   of the container
   Prepare about 10 ml of reducing agent as it last only for about 1 month
                                                                                                 49
14.   INORGANIC PHOSPHATE IN URINE

      14.1    INTRODUCTION
      Urinary phosphate excretion is more influenced by diet, because a higher proportion of
      phosphate intake is absorbed from the gut, Over 96 % of the total phosphorus
      compounds excreted is inorganic, present mostly as a mixture of HPO42- , the proportion
      varying with the urinary pH. Dihydrogen salts are acidic and monohydrogen alkaline.
      Dihydrogen salts of calcium and magnesium are more soluble than monohydrogen forms
      so that the latter precipitate more readily if the urine becomes alkaline.

      14.2    PRINCIPLE OF THE METHOD
      Urine is diluted with distilled water and mixed with ammonium molybdate in acid solution
      to form ammonium phosphomolybdate a reducing agent containing 1-2-4 amino naphthol
      salphonic acid, sodium bisulphite and sodium sulphate is then added to reduce the
      molybdenum phosphate complex to the blue coloured complex. The intensity of the blue
      colour is measured spectrophotometrically.

      14.3    SPECIMEN TYPE, COLLECTION AND STORAGE
      A 24 hours collection of urine is need. 10 ml of 1 N HCl is added as the preservative
      APPARATUS, CHEMICALS, GLASSWARE AND REAGENTS ARE AS FOR SERUM
      PHOSPHATE ESTIMATION

      14.4    PROCEDURE
      1. Mix the 24 hours urine collection
      2. Measure the total volume of urine
      3. Pipette 1 ml of urine into a 100 ml volumetric flask. Make up to 100 ml with distilled
         water.
      4. Label 3 test tubes for Blank (B), Standard (S) and Test (T)
      5. Pipette 5 ml of distilled water into the tube B, 5 ml of standard into the tube S and 5
         ml of diluted urine into the tube T
      6. Add 0.4 ml of Perchloric acid into each tube, Mix well
      7. Then add 0.4 ml of Ammonium Molybdate 5% solution to each tube and mix well
      8. Add 0.2 ml of reducing agent to each tube, Mix well and leave for 10 minutes at room
         temperature
      9. Read the absorbance at 700 nm, setting the spectrophotometer to zero with blank
         solution

      14.5    CALCULATION
      Concentration of the Standard     = 0.128 mmol/l
      Urine phosphate                   = T x 0.128 x 100 mmol/l
                                          S
                                        = T x 12.8 mmol/l
                                          S
      24 hour urine phosphate           = T x 12.8 x V (24 hour urine volume in litre) mmol/24 hrs
                                           S
                                                                                                 50
15.   TOTAL PROTEIN

      15.1     INTRODUCTION
      The term ‘ plasma proteins’ describes the very large number of complex molecules that
      share a common primary structure ; but have an enormous diversity of function .Many of
      the plasma proteins are classified according to function, e.g. enzymes, clotting factors,
      acute phase proteins, immunoglobulins complement components, protease inhibitors,
      apolipoproteins, transport proteins , etc. In addition to special functions the plasma
      proteins contribute general properties such as buffer capacity and oncotic pressure. Apart
      from those groups of proteins that have formed the diagnosis and research base for
      separate disciplines of laboratory medicine. E.g. immunology, relatively few plasma
      proteins are routinely measured by the diagnostic clinical chemistry laboratory. Some
      proteins are also measured in ot
      her body fluids , such as cerebrospinal fluid [CSF], ,and pleural and ascitic fluids.

      Acute Phase Proteins
      The term ‘acute phase proteins’ describes a groups of 20 or more apparently unrelated
      plasma proteins [excluding immunoglobulins] whose concentrations significantly alter in a
      characteristic fashion following cell injury, e.g. infection, surgery, trauma, tumor growth,
      tissue necrosis, etc. The acute phase proteins represent part of the complex physiological
      and metabolic responses by the body to limit further tissue damage [e.g. by proteases free
      radicals] and to initiate and maintain repair.

      Most of the proteins rise in plasma concentrations by up to twofold in the days following
      significant cell injury. Plasma C-reactive protein [CRP] is normally barely detectable and is
      not stored in the liver, but plasma levels can rise within 5-6h of injury to many hundredfold
      within 1-2 days [t1/2 2 h] CRP] is able to bind to nuclear material and other debris from
      damaged cells ,and activates the complement system that leads to inflammation and
      phagocytosis. The concentration of some plasma proteins often fall during the acute phase
      response (e.g. albumin by up to 20%, transport proteins: transferrin, thyroxin-binding
      globulin (TBG), pre albumin) reflecting an increased capillary wall permeability, the shift by
      hepatocytes to synthesizing acute phase protein and the mobilization of repair
      mechanisms.

       ALBUMIN
      Some 10-15 g/day of albumin [=66.5 kDa] is synthesized by the adult liver, but production
      can be doubled if severe loss occurs: the liver stores very little albumin .Albumin synthesis
      is reduced if the plasma concentration of other plasma proteins raises, e.g. acute phase
      proteins, myeloma. Albumin is distributed mainly in the extra cellular fluid [ECF], with
      about 40% located in the intravascular compartment. Vascular permeability allows up to
      150 g/day to be lost into the ECF, which is then returned via the lymphatic system; losses
      are more severe in inflammatory conditions. Albumin is mainly catabolized by endothelial
      tissue, with small losses into the gut [=1g/day] and urine [less than =20 mg /day. Water
      content of the intravascular compartment affects albumin concentration, e.g. plasma
      volume expansion in pregnancy, congestive cardiac failure, and liver failure posture. In the
      latter case plasma albumin concentration can raise 10-15% on standing as water in the
      lower extremities is lost from vessels due increased hydrostaic pressure; separate reference
      intervals are advisable for inpatients and ambulant patients. Aprolonged application of a
      tourniquet for venepuncrure can cause blood stasis and water loss from the distal vein and
      a rise in albumin concentration of 5-10g/L.
       GLOBULINS
      Electrophoresis of normal serum performed on a carrier such as cellulose acetate reveals
      four major non-albumin band s staining for protein/lipoprotein, designated by mobility as
      α1,β1, γ
                                                                                            51
Band                Concentration (g/L)            Major components
α1-globulin               2-5                       α1- antitrypsin, Apolipoprotein
α2 –globulins             4-10                      Caeruloplasmin, α2macroglobulins,
                                                    Haptoglobins
β-globulin                6-12                     Transferrin β-lipoproteins
γ-globulins8-16                                     Immunoglobulins
α1-Antitripsin        1.5 –3.5 g/L
A protease inhibitor [54kDa], a positive acute phase reactant also being increased in liver
disease ,pregnancy and by anabolic steroids ,The major diagnostic use is the detection of
hereditary deficiency ,low plasma concentrations [homozygotes are 10-15 % of normal
heterozygotes are 50-60 %] predisposing individuals to neonatal jaundice ,childhood
hepatities , hepatoma. Adult emphysema.
Caeruloplasmin
Caeruloplasmin [copper oxidase, 135k binds up to eight copper atoms which are necessary
for the hepatic release of the protein and for oxidase activity]
Apolipoprotein A
α2-Macroglobulin [1.5 –4.0.g/L]
Haptoglobulins [0.5 –3.0 g/L]
Transferrin
Apolipoprotein B
Immunoglobulin
Class    MW               Concentration (g/L)       Antibody function
Ig G     150000           8 -15                     Viruses, bacteria, Protects body
                                                    spaces, crosses placenta
IgA      160000           1-5                       Protects tissue surfaces: gut,
                                                    Respiratory tract; in
                                                    tears, sweat, saliva
IgM      950000           0.5-2.0                   First Ig class to respond to antigens
                                                    bacterial, viral
IgD      175000           <150 mg                   Ag recepter on B Lymphocytes,
                                                    antibodies
IgE      190000           =0.3 mg                   Allergic hypersensitivity



15.2     CLINICAL SIGNIFICANCE

15.3     PRINCIPLE OF THE METHOD
Serum proteins form a violet- blue complex with copper ions in alkaline solution. The
absorbance of the complex is measured at 540 nm. A method using a sample blank is
recommended in order to avoid errors due to turbidity.
                                                                                           52
15.4    SPECIMEN TYPE, COLLECTION AND STORAGE
3 ml clotted blood collected into dry clean glass bottle without applying tourniquet, avoid
haemolysis, fasting specimen is desired to decrease lipaemia. Separate the serum from cells
as early as possible. Serum is stable at 4 0C

15.5    APPARATUS AND CHEMICALS
APPARATUS:
Spectrophotometer at wavelength 540 nm or colorimeter with yellow-green filter, Ilford
605(550 nm)
GLASSWARE:
Volumetric flasks (500 ml and 1 litre volumes)
Automatic micro pipettes (50 µl)
Graduated pipette (5 ml in 0.1 ml)
Measuring cylinders (100ml and 500 ml)
Test tubes (150 mm x16 mm)
Beaker (250 ml)
Polyethylene reagent bottles (I litre)
CHEMICALS:
Sodium chloride-AR
Sodium azide-AR caution: handle with care
Sodium hydroxide pellets-AR
Copper sulphate pentahydrate-AR
Potassium sodium tartrate tetrahydrate-AR
Potassium iodide-AR
Albumin bovine, a fraction v powder is suitable

REAGENTS
1. Sodium hydroxide solution 6 mol/: Dissolve 120 g of sodium hydroxide little by little in
   about 400 ml of distilled water. After cooling dilute to 500 ml. Store in a tightly- closed
   polyethylene bottle. This solution is stable indefinitely at 20 -25 0C(room temperature)
2. Biuret reagent: Dissolve 3.0 g of copper sulphate in about 500 ml of distilled water. Add
   9.0 g of potassium sodium tartrate and 5.0 g of potassium iodide. When they have
   completely dissolved add 100 ml of sodium hydroxide solution ( 6 mol/l) and make up
   to solution is stable indefinitely at 20 -25 0C(room temperature) store in a tightly closed
   polyethylene bottle
3. Blank Biuret reagent: Dissolve 9.0 g of potassium sodium tartrate and 5.0 g of potassium
   iodide in distilled water. Add 100 ml of sodium hydroxide solution (6mol/l) and make
   up to 1 litre with distilled water. When kept in a tightly-stoppered polyethylene bottle
   this solution is stable indefinitely at 20 -25 0C(room temperature)
4. Sodium chloride/Sodium azide solution: Weigh out 9.0 g of sodium chloride and 1.0 g of
   sodium azide, dissolve and make up to 1 litre with distilled water. This solution is
   stable indefinitely at 20 -25 0C (room temperature)
5. Protein standard 80 g/l: Weigh out about 4.3 g of bovine albumin powder and dry it
   overnight in the oven at about 60 C. After drying weigh out exactly 4.0 g of dry bovine
   albumin powder. Float the powder on the surface of about 30 ml of sodium
   chloride/sodium azide solution in a small beaker. When the albumin has dissolved
   transfer the solution into a 50 ml volumetric flask (pour slowly down the side of the
   flask to avoid frothing). Rinse the beaker with small volumes of sodium
   chloride/sodium azide solution. This solution is stable for 6 months at 2-8 0C. Store in
   a clean sterile bottle.

15.6    PROCEDURE
1. For the standard and each patient or control sample, pipette into test tubes 2.5 ml of
   Biuret reagent (standard and test) and 2.5 ml of blank Biuret reagent (standard and
   sample blanks)
                                                                                              53
2. Add 50 µl of standard (80 g/l) or samples to each pair of tubes.
3. A reagent blank is set up for each batch and contains 2.5 ml of Biuret reagent and 50
   µl of water.
4. Mix each tube and allow them to stand at room temperature for 30 minutes or at
   370 C for 10 minutes. Note: the same temperature must be used for standards and
   samples.
5. Measure the absorbance at 540 nm (yellow-green filter, Ilford No 605) setting the
   spectrometer to zero with blank Biuret reagent. First read the absorbance of the
   sample blanks, then the reagent blank, and then the tests.
6. If results are greater than 120 g/l then repeat the analysis using 20 µl of sample.
   Multiply the result by 2.5 to obtain the protein concentration.

CALIBRATION
Although the measurement of total protein is simple, results of external quality assessment
programmes indicate that many laboratories have difficulty in producing accurate without
sample blank correction.
The recommended method proposes the use of a bovine albumin solution as a calibrator.
One alternative is to use lyophyilised serum calibrators. However many of these show
significant turbidity when they are reconstituted and sometimes it is difficult to know
whether the contribution that the turbidity makes to the absorbance at 540 nm has been
subtracted in the calculation of the total protein value. When possible use a calibrator with
a value assigned by a total protein method that incorporates a sample blank. A sample
blank for the standard (SB) is not necessary if a solution of bovine albumin is used.
A second alternative is to use an out – of-date of human serum albumin from a blood
transfusion or pharmacy department. Use the stated concentration of albumin for the total
protein value. One bottle will last for many months at 4-6 0Cc if small volumes (5-10 ml)
are withdrawn as required using a sterile syringe.
A calibration curve must be prepared as described below to check the linearity of the
spectrometer. If it is linear, then a single standard can be used routinely as described under
“Technique” .The calibration curve should be repeated at least once a month.

PREPARATION OF CALIBRATION GRAPH USING BOVINE ALBUMIN
Prepare a calibration graph to confirm that the method is linear with your spectrometer to
at least 80g/l. Provided that it is linear, a single standard (80 g/l) can then be used with
each batch of patients’ samples.

Working standard No                                               1       2      3       4

Protein standard 80 g/l (ml)                                    0.25    0.50    0.75    1.0

Sodium chloride/sodium azide solution (ml)                      0.75    0.50    0.25     0

Concentration of working standard solution (g/l)                 20      40      60      80

Label four test tubes as follows: reagent blank (RB),
Working standard: No.1 (20 g/l);
No.2 (40 g/l);
No.3 (60g/l);
No.4 (80g/l);
Pipette 2.5 ml of Biuret reagent into each tube; add 50 µl of distilled water to the reagent
blank, and 50 µl of each working standard solution into the corresponding tubes. Mix and
leave to stand at room temperature for 30 minutes or at 37 0C for 10 minutes.
Read the absorbance at 540 nm after setting the instrument to zero with the reagent blank.
                                                                                        54
Prepare the calibration graph by plotting the absorbance against the protein
concentration for each tube.

15.7       CALCULATION
S= A standard - A standard blank – A reagent blank
T= A sample - A sample blank- A reagent blank

The serum protein concentration of sample=T/S x 80 g/l
Where 80 is the concentration of the standard (g/l)
Conversion from SI to “old” units g/l x 0.1=g/100 ml

QUALITY CONTROL
At least two quality control specimens, having stated values in the range 40-80 g/l, one of
which is unknown to the operator, should be included with each batch of specimens. If
single specimens are analysed a control specimen should always be included.

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 2% should be
attainable.
ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed
4%
REFERENCE VALUES
Approximate reference values are 60-80 g/l

REFERENCES
Doumas, B.T. (1975) Clin. Chem.., 1159-1166.
                                                                                                 55
16.   UREA
      DIACETYLE MONOXIME METHOD

      16.1    INTRODUCTION
      Urea is the major nitrogen containing metabolic product of protein catabolism in humans,
      accounting for more than 75% of the non protein nitrogen eventually excreted. The
      biosynthesis of urea from amino nitrogen-derived ammonia is carried out exclusively by
      hepatic enzymes of the urea cycle.
      More than 90% of urea is excreted through the kidneys, with losses through the
      gastrointestinal tract and skin accounting for most of the remaining minor fraction. Urea is
      neither actively reabsorbed nor secreted by the tubules but is filtered freely by the
      glomeruli. In a normal kidney, 40 to 70% of the highly diffusible urea moves passively out
      of the renal tubule and into the interstitium, ultimately to re-enter plasma. The back
      diffusion of urea is also dependent on urine flow rate; with more entering the interstitium
      in slow-flow states. Urea production is dependent on several non renal variables such as
      diet and hepatic synthesis.

      16.2    CLINICAL SIGNIFICANCE
      A wide variety of renal diseases with different permutations of glomerular, tubular,
      interstitial or vascular damage can cause an increase in plasma urea concentration. The
      usefulness of urea as an independent indicator of renal function is limited by the variability
      of its blood levels as a result of non renal factors. Mild dehydration, high protein diet, the
      increased protein catabolism, muscle wasting as in starvation, reabsorption of blood
      proteins after a gastrointestinal haemorrhage, treatment with cortisol, and decreased
      perfusion of kidneys may cause increased blood urea that is called pre renal ureamia.
      Impaired perfusion may be due to decreased cardiac output or shock secondary to blood
      loss or other causes.

      16.3    PRINCIPLE OF THE METHOD
      Proteins in whole blood, plasma or serum are precipitated with trichloroacetic acid. The
      urea in the supernatant reacts with diacetyle monoxime in the presence of
      thiosemicarbazide and cadmium ions under acid conditions. The absorbance of the
      resulting rose-purple solution is measured at 530 nm.

      16.4    SPECIMEN TYPE, COLLECTION AND STORAGE
      3 ml of clotted blood collected into clean dry bottle avoid haemolysis. Separate serum from
      cells. Urea is stable for 24 hours at room temperature (250C), for 7 days at 2-60C, for longer
      duration (2-3 months) when frozen. As referral sample send about 0.5 ml clear serum, kept
      cool, to reach correct destination within 24 hours.

      16.5    APPARATUS AND CHEMICALS
      APPARATUS:
      Water bath or heating block (temperature range 10- 110 0C)
      Spectrophotometer wavelength 530 nm
      Colorimeter green filter, Ilford 604 (520nm)
      Rack for boiling tubes
      GLASSWARE:
      Amber coloured reagent bottles (500 ml volume)
      Graduated pipettes (5 and 10 ml in 0.1 ml)
      Graduated cylinders (50 ml, 100 ml and 500 ml)
      Volumetric flasks (50 and 100 ml and 500 ml volumes)
      Automatic micro pipettes (50 and 100 µl)
      Glass stoppered boiling tubes (160 x 18 mm)
                                                                                           56
Conical centrifuge tubes 15ml
CHEMICALS:
Benzoic acid-AR
Cadmium sulphate (as 3CdSO4.8H2O)-AR
Diacetyle monoxime-AR
Orthophosphoric acid (85% w/v)-AR caution: corrosive, handle with care
Sulfuric acid, concentrated (95 – 97 % w/v)-AR caution: corrosive, handle with care
Thiosemicarbazide-AR
Trichloroacetic acid-AR caution: corrosive, handle with care
Urea (pure)

REAGENTS
1. Acid reagent : Add about 200 ml of distilled water to a 500 ml volumetric flask. Keep
   the flask in basin containing water and then add slowly 44 ml of sulphuric
   (concentrated) acid and 66 ml of orthophosphoric acid. Cool the solution to room
   temperature but do not use ice water as a cooling bath. Add 50 mg thiosemicarbazide
   and dissolve, then add 1.6 g cadmium sulphate and dissolve, add 1.5 ml of the urea
   working standard solution (2.5mmol/l). Make up to 500 ml with distilled water.
   Transfer to an amber coloured bottle. This reagent is stable for at least six months at
   2- 8 0C.
   NOTE          : The presence of a small amount of urea in the reagent improves the
   linearity of the calibration curve. The cadmium sulphate improves the stability of the
   final coloured product.
2. Diacetyl monoxime reagent: Weigh out 2.0 g of diacetyl monoxime, dissolve in
   distilled water and dilute to 500 ml with distilled water in a volumetric flask. This
   solution is stable for at least six months at 2-8 0C
3. Colour reagent: Use a graduated cylinder and mix 50 ml of acid reagent with 50 ml of
   diacetyl monoxime reagent in a small bottle. This amount is sufficient for 33 reactions.
   This reagent must be prepared daily, therefore, the volume made up should depend on
   the number of reactions anticipated; 3 ml is required for each reaction.
4. Benzoic acid solution 1 g/l: Weigh out 0.5 g of benzoic acid and transfer to a 500 ml
   volumetric flask. Add distilled water, mix well to dissolve and make up to 500 ml with
   distilled water. This solution is stable for several months at 20-250C (room
   temperature)
5. Trichloroacetic acid solution 50 g/l: Weigh out 25 g of trichloroacetic acid in a beaker,
   dissolve, transfer into 500 ml volumetric flask and make up to 500 ml with distilled
   water. This solution is stable for several months at 20-25 0C. we recommend to store
    the solution at refrigerator.
6. Stock urea solution 125 mmol/l: Weigh out 1g of urea –AR in a beaker and keep in
    the dessicator overnight. Weigh out 750 mg urea from the dessicator and transfer into
    a 100 ml volumetric flask. Add 50 ml of benzoic acid solution; dissolve and dilute to
    100 ml with benzoic acid solution. This reagent is stable for several months at 2-80 C
7. Working urea standards: Prepare working urea standards in 50 ml volumetric flasks
    according to the table below;
Working standard No                      1        2        3         4      5        6
Stock urea standard (ml)                 1        2        3         4      6        8
Benzoic acid solution                                 Up to 50 ml for each
Concentration of working standard         2.5     5.0   7.5     10       15           20
Solution (mmol/l)
These standards are stable for several months at 2-80 C
                                                                                            57
PREPARATION OF CALIBRATION GRAPH
In this method the formation of the coloured product depends on the composition of the
colour reagent and the period of heating at 100 0C. Small variations may occur from day to
day and it is therefore essential to check the calibration each time that patients’ samples are
analysed. When you are familiar with the shape of the calibration graph on your
spectrometer you may find that you can omit some of the standards, e.g. prepare your daily
calibration using for example the 5, 10 and 20 mmol/l standards, or use the 10 mmol/l
standards should be prepared when the acid reagent and diacetyl monoxime reagent are
renewed to check that the reagents are correct.
Follow the procedure described under “Procedure”. Plot the absorbance of each tube on
the vertical axis against the concentration of the working urea standard solutions in mmol
on the horizontal axis.

16.6     PROCEDURE
1. Pipette 0.5 ml of trichloroacetic acid solution using a rubber bulb into centrifuge tubes
   for each standard, control serum and patient’s sample. Add 50 µl of standard, control
   serum or patient’s sample to the appropriate tube, mix and leave at room temperature
   for 5 minutes, then centrifuge to obtain a clear supernatant.
2. Label another set of tubes (18 x 160mm) and pipette 3.0 ml of colour reagent into test
   tubes for each standard, blank, control serum and sample.
3. Add 100 µl of trichloroacetic acid solution to the blank tube, and 100 µl of
   supernatants form the standards, controls or samples to the appropriate tube.
4. Mix well and heat at 100 0C for 15 minutes exactly.
5. Cool the tubes to room temperature in a bowl of water (about 5 min) mix and read the
   absorbance at 530 nm (green filter, Ilford No 604) first read the absorbance of the
   blank against distilled water and note down the reading, then set to zero with the blank
   and read the standards and unknowns. The absorbance measurements should be made
   as soon as possible and not more than 30 minutes after the end of step 4.

16.7     CALCULATION
Read the results from the calibration curve or use the following formula if your calibration
graph is linear.
Concentration of urea in mmol/l = T x C
                                        S
Where:
T        = Absorbance reading of patient’s test
S        = Absorbance reading working standard (10 mmol/l)
C        = Concentration of working standard (10 mmol/l)

If the result is greater than 20mmol/l, dilute 50 µl of supernatant from that sample with
100 µl of trichloroacetic acid solution. Mix well. Repeat the analysis using 3 ml of colour
reagent and 100 µl of the diluted supernatant. Remember to multiply the result by 3 to
obtain the urea concentration in the patient’s sample.

QUALITY CONTROL
At least two serum control specimens, having stated values in the range 3-20 mmol/l, one
of which is unknown to the operator, should be included with each batch of specimens. If
single specimens are analysed, a control specimen should always be included.

OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 3% should be
attainable.
ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed
6%
                                                                                        58
REFERENCE VALUES
Approximate reference intervals for “healthy” ambulant adults: 2.1-7.1 mmol/l
Conversion of SI units into “old” units: mmol/l x 6 = mg/100 ml

NOTE: The method recommended uses the reagents described in the above
references. However a protein precipitant has been included because the direct method
(No protein precipitation) gave results on patients’ samples which were significantly
higher than other methods.

16.8     LIMITATION
Cadmium sulphate has been included as recommended by Wybenga et al. In the absence
of cadmium ions, the absorbance decreased by about 7 % in 30 minutes; when the colour
reagent is prepared as described, then the absorbance decreased by about 4% in 30
minutes. Provided that the batch size is kept small, so that the absorbance readings can be
made over a period of a few minutes, the presence or absence of cadmium ions has little, if
any effect on the results.


REFERENCES
WHO Manual LAB/86.3
                                                                                                59
17.   URIC ACID

      PHOSPHOTUNGSTATE REDUCTION –CARAWAY METHOD

      17.1    INTRODUCTION
      Uric acid is the end product of purine metabolism (adenine,guanine) in the human. On a
      normal diet some 5-6 mmol of urate is produced daily.Of this amount about 3-4 mmol is
      produced from purines synthesized in the body (de novo synthesis), whilst the remaining
      1-2 mmol are contributed by dietary purines.
       The purine base (adenine,guanine,hypoxanthine,etc.) is a double ring structure assembled
      from a variety of precursors: glutamine, glycine, aspartate, tetrahydrofolate and
      bicarbonate. A base attached to a pentose (ribose) is a nucleoside (e.g. adenosine), while a
      phosphate ester of a nucleoside is termed a nucleotide (e.g. adenosine monophosphate,
      AMP).
      Purine ring is assembled on ribose phosphate; the committed step being the formation of 5
      – phosphoribosylamine from 5- phosphoribosylpyrophosphate(PRPP) and glutamine by
      the enzyme PRPP amidotransferase(PRPP-AT) –the PRPP being previously synthesized
      from ribose 5- phosphate by PRPP synthetase. The enzyme PRPP-AT is subjected to
      negative feedback          control by inosine monophosphate(IMP), guanosine
      monophosphate(GMP) and AMP, and is stimulated by PRPP(IMP,AMPand GMP also
      inhibit PRPPsynthetase). After a number of intermediate reactions, which utilize adenosine
      triphosphate (ATP), AMP is formed. GMP and AMP are formed from IMP.
      In addition to this de novo synthesis, free purine bases are also formed by hydrolytic
      degradation of nucleic acids and nucleotides; and purine nucleotides can be synthesized
      from these bases by a salvage pathway involving two enzymes: hypoxanthine-guanine
      phosphoribosyl transferase(HGPRT) and adenine phosphoribosyl transferase (APRT).
      HGPRT synthesizes IMP and GMP from guanine and hypoxanthine and APRT
      synthesize AMP from adenine- the other sbstrate in both cases is PRPP. These two
      pathways do not require ATP and thus there is an energy saving compared with de novo
      synthesis (PRPP to IMP utilizes four molecules of ATP)

      Urate, the end product of purine base degradation, is formed from xanthine by the enzyme
      xanthine oxidase.Xanthine is in turn formed from hypoxanthine(purine base of inosine) by
      xanthine oxidase and from guanine by guanase.Adenosine enters the degradation cycle by
      convertion to inosine by adenosine deaminase(ADA). Xanthine oxidase also acts on
      adinine to form 2, 8-dioxyadenine.

      Around 25-30% of the 5-6mmol of urate produced daily is eliminated via the
      gastrointestinal tract, where it is degraded by bacterial uricases. The remainder is excreted
      by the kidney. In renal failure the intestinal excretion can be markedly increased.

      The renal clearance of urate is of the order of 5-10% of inulin clearance. Some 98% of the
      filtered urate is reabsorbed in the proximal tubule. The 2% that escapes reabsorption
      contributes around 20% of the total excreted, proximal tubular secretion accounting for
      the remainder. Minor reabsorption also occurs in the distal nephron Renal clearance is
      influenced by various drugs and metabolic products.

      17.2    CLINICAL SIGNIFICANCE
      Hyperuricemia is most commonly defined by serum or plasma uric acid concentrations
      greater than 7.0 mg/dL (0.42 mmol/L) in men or greater than 6.0 mg/dl (0.36 mmol/l) in
      women (if specific methods are used to measure the uric acid). Asymptomatic
      hyperuricemia is frequently detected through biochemical screening; Long term follow up
      of asymptomatic hyperuricemic patients is undertaken because many are at risk for renal
                                                                                            60
disease that may develop as a result of hyperuricemia or hyperuricosuria; few of these
patients ever develop the clinical syndrome of gout.
Gout occurs when monosodium urate precipitates from supersaturated body fluids;the
deposits of urate are responsible for the clinical signs and symptoms.Gouty arthritis may
be associated with urate crystals in joint fluid as well as with deposits of crystals(tophi) in
tissues surrounding the joint. The deposits may occur in soft tissues as well , and wherever
they occur they elicit an intense inflammatory response consisting of polymorphonuclear
leukocytes and macrophages.Renal disease associated with hyperuricemia may take one or
more of several forms: (1)gouty nephropathy with urate deposition in renal
parenchyma,(2)acute intratubular deposition of urate crystals, and (3) urate nephrolithiasis
Hyperuricemia is also attributable to primary defects of enzymes in the pathways of purine
metabolism.

17.3     PRINCIPLE OF THE METHOD
Serum proteins are precipitated with acid tungstate and a clear supernatant is obtained after
centrifugation. A portion of the supernatant is added to alkaline phosphotungstate.
Phosphotungstate reagent oxidizes the urate to allantoin and it self reduced to tungsten
blue which is measured by its absorbance at 700 nm

17.4     SPECIMEN TYPE, COLLECTION AND STORAGE
4ml of clotted blood, collected into clean dry bottle, avoid haemolysis. Separate the serum
from cells as early as possible. Uric acid in serum is stable for 48-72 hours at room
temperature (250C), for 3-7 days at 4-6 0C and for 6-7 months when frozen. As referral
sample send about 1 ml of clear serum, kept cool, to reach the correct destination within
24 hours

17.5     APPARATUS AND CHEMICALS
APPARATUS:
Water bath at 25 0C or Basin with water temperature maintained at 250C
GLASSWARE:
Conical centrifuge tubes (15ml)
Test tubes 16 x 150 mm
Quick fit complete assembly (round bottom flask and condenser) for refluxing
Volumetric flask 100 ml, 200 ml and 1 L
Graduated pipettes 1 ml, 5 ml and 10 ml
Automatic micro pipette 500 µl
CHEMICALS:
Molybdate free sodium tungstate dihydrate-AR
Orthophosphoric acid-AR
Sodium carbonate-AR
Sulphuric acid-AR
Uric acid –AR

REAGENTS
1. Stock Phosphotungstic acid reagent: Weigh out 50 g of molybdate free sodium tungstate
   dihydrate or 44.5 g anhydrous salt and dissolve in about 400 ml of distilled water in
   refluxing flask. Add 40 ml of Orthophosphoric acid (concentrated). Mix well and
   reflux gently for 2 hours. Allow the solution to cool. Then transfer with rinsing to a
   500 ml volumetric flask. Make up to 500 ml with distilled water. Mix well. Transfer this
   solution into a clean dry brown bottle. This solution is stable in the refrigerator for
   about one year.
2. Working Phosphotungstic acid reagent: Dilute 10 ml of stock phosphotungstic acid reagent
   to 100 ml with distilled water. Transfer in to a clean, dry brown bottle. Stable in the
   Refrigerator for two weeks.
                                                                                            61

3. Sodium carbonate 10g/dl: Weigh 10 g of Sodium carbonate anhydrous, dissolve in distilled
   water and make up to 100 ml with distilled water. Store in a poly propylene bottle.
4. Sodium tungstate 10g/dl: Weigh out 10 g Sodium tungstate dihydrate, transfer into a
   100ml volumetric flask, dissolve and make up to 100 ml with distilled water. Store in a
   brown bottle.
5. Sulphuric acid (2/3 N) 0.33mol/l: Add slowly 3.7 ml of sulphuric acid concentrated in to
   150 ml distilled water in a beaker. Cool and stir well. Transfer in to a 200 ml
   volumetric flask and make up to 200 ml distilled water. Transfer in to a brown bottle.
6. Acid tungstate reagent: To 80 ml of distilled water while mixing add 5 ml of sodium
   tungstate solution, 0.05 ml of phosphoric acid (concentrated), 5 ml of 2/3 N sulphuric
   acid solution. Transfer in to a brown bottle. Keep at room temperature.
7. Stock Uric acid standard solution 1.5mmol/l: Keep about 300 mg of uric acid analytical grade
   chemical in a desiccator overnight. Weigh out 252.2 mg of uric acid and transfer in to a
   one litre volumetric flask. Weigh out 150 mg of Lithium carbonate; dissolve in about
   50 ml of distilled water. Filter, heat the filtrate to 60 0C. Add this warm solution to the
   volumetric flask containing uric acid. Mix until the uric acid completely dissolved.
   Allow the flask to cool. Then add 20 ml formaldehyde 40 % solution. Add 500 ml of
   distilled water .Then add slowly 25 ml of sulphuric acid solution. (Prepared by adding
   1 ml of sulphuric acid (concentrated) to 35 ml of water) Make up to one litre with
   distilled water. Mix and store in a brown bottle. This solution is stable for about one
   year at 4- 6 0C.
8. Working uric acid standard: Allow the stock uric acid standard solution to attain the room
   temperature. Pipette 2 ml of stock standard solution in to 100 ml volumetric flask.
   Make up to 100 ml with distilled water. This standard is equivalent to 300 µmol/l
   under the assay conditions employed. Solution is stable for one week at 4-6 0C.
9. Working Uric acid standard series for calibration: Prepare working standard series by
   quantitative transfer of 1ml, 2ml, 3ml and 4 ml of stock solution to each of four 100
   ml volumetric flasks. Dilute to 100 ml with distilled water. Mix well. These standards
   are equivalent to 150µmol/l, 300µmol/l, 450µmol/l and 600µmol/l under the
   employed assay condition.

17.6     PROCEDURE
1. Pipette 4.5 ml of acid tungstate regent in to centrifuge tubes for each quality control
    sample and patient’s sample.
 2. Pipette 0.5 ml of quality control serum or patients sample to the appropriate tube with
     mixing.
 3. Leave at room temperature for 10 minutes and centrifuge for 10 minutes at 3000 rpm
     to obtain a clear supernatant.
 4. Label a set of test tubes for each blank, standard, quality control sample and patient’s
     samples.
 5. Pipette 2.5ml of distilled water to the tube marked blank, 2.5ml of working standard to
     the tube marked Standard, and 2.5 ml supernatants from the quality control sample or
     patients’ samples to the appropriate tube.
 6. Add 0.5 ml of Sodium carbonate solution to all tubes, mix well and allow standing for
     10 min at 25 0C.
 7. Then add 0.5 ml of working phosphotungstic acid reagent to all tubes. Mix
     immediately. Allow to stand at 250C for 30 min.
8. Measure the absorbance at 700 nm, setting the spectrophotometer to zero with blank
    solution.

PREPARATION OF CALIBRATION GRAPH
Run the uric acid working standards together with the regent blank exactly in the same
manner as describe under technique. Plot the absorbance values of standards against the
                                                                                                62
      concentration of standards. The points should be linear and the graph should pass
      through the origin.

      17.7    CALCULATION
      Calculate the uric acid concentration in the patient’s specimens from the absorbance of the
      standard as follows

      Concentration of Uric acid in µmol/l= T/S x C=T/S x 30 x 10=T/S x 300 µmol/l
      Where
      T= Absorbance of patient’s test
      S=Absorbance of working standard
      C=Concentration of working standard (30µmol/l)
      Serum dilution 1:10

      QUALITY CONTROL
      Include Quality control sera for each batch of test

      17.8    LIMITATION
      Haemolysed sera interfere with the measurement of uric acid.
      Supernatants should be clear as turbidity may interfere with colour development

18.   URINE URIC ACID
      Method similar to used for serum uric acid can be applied to urine uric acid estimation

      18.1    SPECIMEN TYPE, COLLECTION AND STORAGE
              Collect 24 hour urine into a clean, dry, sterile 2.5 L bottle and keep in the
              refrigerator during the collection.

      18.2    PROCEDURE
              1. Mix the urine collection well. Measure the total volume. Warm an aliquot of
                 urine for a few minutes to 600C to dissolve any urates in the deposit. Add 1ml
                 of urine in to a 100ml volumetric flask. Make up to 100ml with distilled water.
                 Mix well. (Dilution 1in 100)
              2. Pipette 2.5 ml of distilled water to the tube marked B for blank , 2.5 ml of
                 working standard to the tube marked S as standard and pipette 2.5 ml of
                 diluted urine in to the tube marked T for test.
              3. Carry out the same procedure as for serum uric acid estimation.

      18.3    CALCULATION
              Calculate urine uric acid concentration in the sample using the absorbance of the
              standard as follows.

              Urine uric acid concentration µmol/l = T/S x C
              Where             T = Absorbance of patients test
                                S = Absorbance of working standard
                                C = Concentration of working standard
                                Urine dilution 1:100

              Urine Uric acid µmol/l             = T/S x 30 x 100
              Urine Uric acid (mmol/l)           = T/S x 30 x 100
                                                    1000
              24 hour urine uric acid            =T/S x 30 x100 x 24 hour urine volume in litre
              (mmol/24 hour)                      1000
                                                                                                   63
19.   ELECTROLYTES

      DETERMINATION OF SERUM SODIUM AND POTASSIUM BY FLAME PHOTOMETRY
      [FLAME EMISSION SPECTROSCOPY]

      19.1     INTRODUCTION
      SODIUM
      In a normal adult the total body sodium is about 55mmol/kg of body weight; about 30% is
      tightly bound in the crystalline structure of bone and thus is nonexchangeable. Thus only
      40 mEq/kg is exchangeable among the various compartments and accessible to
      measurements. The exchangeable sodium is distributed primarily in the extra cellular space.
      About 97% to 98% of the exchangeable sodium is found in the extra cellular water space
      and only 2% to 3% in the intracellular water space. Approximately 16% of exchangeable
      sodium is in plasma, 41% is in interstitial fluid (ISF) that is readily accessible to the plasma
      compartment, 17% is in ISF of dense connective tissue and cartilage, 20% is in ISF of
      bone and 3% to 4% in the transcellular water compartment. Total bone sodium
      (exchangeable and nonexchangeable) accounts for the 40% to 45% of the total body
      sodium.
      The amount of sodium in the body is a reflection of the balance between sodium intake
      and output. Sodium intake depends on the quantity and type of food intake. Under normal
      conditions, the average adults takes in about 50 – 200 mmol of sodium/ day. Sodium
      output occurs through three primary routes; the gastrointestinal tract, the skin and the
      urine.
      Under normal circumstances loss of sodium through the GIT is very small. Faecal water
      excretion is only 100- 200 ml/day for a normal adult and faecal sodium excretion only 1 to
      2 mmol/ day. However, one should bear in mind that although losses of water and
      electrolytes are normally small, the total volume of GIT fluid secreted is large, averaging
      about 8 L/day. Almost all this volume is normally reabsorbed. However, with impaired
      GIT reabsorption, loss of water and electrolytes are large. Thus with severe diarrhoea or
      with gastric or intestinal drainage tubes, sodium loss via the GIT may exceed 100mmol/
      day.
      The sodium content of sweat averages about 50 mmol/ L but is somewhat variable. The
      sweat sodium concentration is decreased by aldosterone and increased in cystic fibrosis.
      The rate of sweat production is highly variable, increasing in hot environments, during
      exercise, and with fever. Under extreme conditions sweat production can exceed 5 L/ day,
      accounting for a loss of more than 250 mmol of sodium .Under normal conditions, in a
      cool environment; sodium losses from the skin are small. With extensive burns or
      exudative skin lesions there is great loss of sodium and water.
      Major route of sodium excretion is through the kidney. Furthermore, urinary excretion of
      sodium is carefully regulated to maintain body sodium homeostasis, which in turn is critical
      to control of extra cellular volume.
      Sodium is freely filtered by the glomerulus. Approximately 70% of the filtered sodium is
      reabsorbed by the proximal tubule, about 15% by the loop of henle, 5% by the distal
      tubule, 5% by the cortical collecting tubule, and another 5% by the medullary collecting
      duct; thus normally less than 1% of the filtered sodium is excreted.

      POTASSIUM
      Approximately 98% of the total body potassium is found in the intracellular water space
      (ICW),reaching a concentration there of about 150 to 160 mmol/ l .In the ECW space
      ,the concentration of potassium is only 3.5 to 5 mmol/l . Total body potassium in an
      adult male is about 50 mmol/kg of body weight and is influenced by age, sex, and very
      importantly muscle mass, since most of the body’s potassium is contained in muscle.
      The difference in potassium concentration between plasma and ISF is attributable to the
      Gibbs-Donnan equilibrium. The difference in potassium concentration in ISF and ICF is
                                                                                           64
the result of active transport potassium into the cell in exchange for sodium. Factors that
enhance potassium transport into the cell and there by increase the ratio of IC to EC
potassium are insulin, aldosterone, alkalosis, and beta – adrenergic stimulation. Factors that
decrease potassium transport into the cell or enhance leakage out of the cell include
acidosis, alfa- adrenergic stimulation and, tissue hypoxia.
The amount of potassium in the body is a reflection of the balance between potassium
intake and output. Potassium intake depends on the quantity and type of food intake.
Under normal conditions the average adult takes in about 50 to 100 mmol/ day, about the
same amount as sodium. Potassium output occurs through three primary routes; the
gastrointestinal tract, the skin and the urine.
 Under normal circumstances loss of potassium through the GIT is very small, amounting
to less than 5mmol/day for an adult. The concentration of potassium in the sweat is less
than that of sodium, and so potassium losses via the skin are usually small.
The major means of potassium excretion is by the kidney. The kidney is capable of
regulating the excretion of potassium to maintain body potassium homeostasis.

19.2    CLINICAL SIGNIFICANCE
SODIUM
Hyponatremia occurs when there is a greater excess of extra cellular water than of sodium
or a greater deficit of sodium than water.
The symptoms of hyponatremia depend on the cause, magnitude, and rate of fall in serum
sodium. With acute, pronounced hyponatremia caused by water intoxication,nausea,
vomiting, seizures, and coma occur. Symptoms are less fulminant with chronic
hyponatremia caused by salt depletion. With progressively severe degrees of chronic
hyponatremia, constant thirst, muscle cramps, nausea, vomiting, weakness, lethargy, and
finally delirium and impaired consciousness occur.

Hypernatremia occurs when there is greater deficit of extra cellular water than of sodium.
Greater excess of sodium than of water rarely occurs.
Hypernatremia usually occurs as a chronic process secondary to loss of water in excess of
sodium. Symptoms are therefore those of dehydration.

POTASSIUM
POTASSIUM EXCESS
Potassium accumulates in the body when the intake of potassium exceeds output because
of some abnormality of the potassium homeostatic mechanism. It should be noted that
under most conditions the normal kidney is capable of excreting a great deal of potassium,
and a high potassium intake leads to potassium retention only when kidney function is
compromised.

POTASSIUM DEPLETION
This occurs when potassium output exceeds intake. Only small amount of potassium is
loss in the faeces under normal conditions. GIT loss of potassium during diarrhoea and
drainage of GIT secretions can be large.
Alkalosis results in the total body potassium depletion. With alkalosis, potassium moves
from the EC to the IC space. In the cells of the distal nephrone of the kidney, this increase
in IC potassium stimulates potassium secretion and therefore increases renal excretion of
potassium.

19.3    PRINCIPLE OF THE METHOD
Using compressed air diluted serum is sprayed as a fine mist of droplets in to a non
luminous flame. In the flame the elements in the compound are converted in to the atomic
state. As the temperature rises due to the thermal energy of the flame, a small portion of
these atoms excited and the electrons moves to higher energy level. When atoms excited
they emit light in the form of a fixed wavelength to produce a spectrum. Light emitted
                                                                                          65
from the thermally excited ions is directed to photo sensitive detector system. The
amount of light emitted depends on the concentration of metallic ions present. The
response compared with those obtained from standards.

19.4    SPECIMEN TYPE, COLLECTION AND STORAGE
Sample suitable is Haemolysis free serum. Blood collected into Clean, dry bottle or
commercially available evacuated tubes or capillary blood collected in either micro tube or
capillary tubes.
Blood should not be collected from the arm receiving an electrolyte or intravenous
infusion. Avoid muscle activity (clenching the fist) when collecting the blood sample as this
can artificially increase the potassium values. Blood specimens should not chilled before
separation of serum, false increase in potassium level occurs as a result of K+ leakage from
erythrocytes and other cells.
Serum should be separated from cells immediately within 1 h of collection at room
temperature. Blood samples should not be centrifuge for longer time. Grossly lipaemic
serum samples are unsuitable for electrolyte estimation and should be ultra centrifuged.
Serum samples should be stored at 20C to 40C or frozen for delayed analysis.

19.5    APPARATUS AND CHEMICALS
APPARATUS:
Analytical balance
Flame photometer
Automatic micro pipette 100µl
GLASSWARE:
Conical flasks 50 ml
Graduated pipette 25 ml
Disposable sample cups (plastic)
CHEMICALS:
Sodium chloride (Analytical grade)
Potassium chloride (Analytical grade)

REQUIREMENTS (ENVIRONMENTAL CONDITIONS)
TEMPERATURE : Operating 100C to 35 0C
HUMIDITY           : Operating 85% maximum at 350 C
FUEL               : High grade propane should be free of heavy hydrocarbon deposits and
                   regulated at the cylinder to approximately 2.1 kg/cm2 (30 psig)
  AIR              : A supply of clean air at a minimum pressure of 1 kg/cm2 (14 psig) at 6
                   litres /minute, as supplied by a Corning 850 Air Compressor. Maximum
                   inlet pressure 2.1 kg/cm2 (30 psig).If condensation problems arises a
                   ‘Corning 856 Air Compressor’ should be used, which has a water
                   separator fitted.
VOLTAGE            : 90V to 127V or 198V to 264 V, 50/60Hz
POWER              : 20VA, 410 only
REAGENTS
 All glassware used to prepare standard solutions should be chemically cleaned
 and finally rinsed with distilled water.
Weigh out separately in to two watch glasses or in to two Petri dishes about 15 g of
analytical grade Sodium chloride and about 1 g of Potassium chloride.
Dry for 6 hours at 100 0C in an oven and allowed to cool to room temperature in a
desiccator.
1. Stock Sodium solution 1000 mmol/l: To prepare 200ml, weigh accurately 11.7 g of
    dried Sodium chloride in a weighing bottle or in a beaker. Transfer it in to an ‘A grade’
    200 ml volumetric flask using a funnel. Wash in any chemical remaining in the
    weighing bottle or in the beaker in to the flask with a little amount of distilled water
    (glass distilled water) or deionised water. Dissolve in about 150 ml of distilled water
                                                                                           66
     and make up to 200 ml with distilled water. Use a pasture pipette or a wash bottle to
     add the final volume of distilled water to the flask. Mix the solution well by inverting
     the flask for several times.
2.   Stock Potassium solution 100 mmol/l: Weigh accurately 0.746 g of dried Potassium
     chloride in a weighing bottle or beaker. Transfer it in to an ‘A grade’ 100 ml
     volumetric flask using a funnel. Wash any remaining in the weighing container in to
     the flask with distilled water. Dissolve in about 80 ml of distilled water. Make up to
     100 ml with distilled water. Use a Pasture pipette or a wash bottle to add the final
     volume of distilled water to the flask. Mix the solution well by inverting the flask for
     several times.
3.   Flame photometry deproteinising solution: The pack of deproteinising solution
     contains Deproteinising base solutions and Sacher of catalyst. For use add the catalyst
     into base solution and mix thoroughly. This solution is stable for 4 weeks at 2-80 C.
4.   Working Standard solution (Sodium-140 mmol/l, Potassium-5.0 mmol/l): Into
     a clean 500 ml ‘A grade’ volumetric flask, add 70 ml of stock solution-1(Stock sodium
     solution) and 25 ml of stock solution-2 (Stock potassium solution) Make up to 500 ml
     with good quality distilled water or deionised water. Use a pasture pipette or a wash
     bottle to add the final volume of distilled water to the flask. Mix the solution well by
     inverting the flask for several times. Rinse a prolypropylene bottle with little of the
     prepared solution. Then transfer the standard solution into the bottle. Label and keep
     at room temperature. (Contamination with sodium may occur if a glass bottle is used)
     Stopper the bottle tightly to avoid evaporation.
5.   Working diluent concentrate: Diluent concentrate recommended by the
     manufacturer should be used. For ‘Corning 410 C’ Corning diluent concentrate is
     used. Pipette 0.5 ml diluents concentrate in to clean 500 ml volumetric flask. Dilute up
     to 500 ml with good quality distilled water or deionised water. Store the working
     diluent concentrate in a polypropylene bottle. This solution is stable for 5 days at room
     temperature.

19.6     PROCEDURE
The details of the operation vary from one instrument to another. Following steps are
related to ‘Corning 410’ clinical model flame photometer.
1. Sample dilution: Dilute each serum, quality control sample and working standard
     solution 1:200 with working diluent concentrate. Into 50 ml conical flasks pipette 19.9
     ml of working diluent concentrate and 0.1 ml of working standard solution or quality
     control sample or patient’s serum and mix well.
2. Turn on the fuel supply at source
3. Depress the ‘power’ switch to switch on the instrument 410. The ‘power on’ LED will
     be illuminated, the air compressor will start an ignition cycle will commence.
4. If the flame on LED is not illuminated at the end of the ignition cycle, (Refer the
     operator’s manual available with the instrument) check that the air pressure gauge
     indicates a reading between 11 and 13 psig, if it does not, lower the air regulator
     locking ring and adjust the regulator for a reading of 12 psig on the air pressure gauge.
     Raise the locking ring to lock the air regulator adjuster.
5. Set the filter selector to the required position. Non luminous blue flame with distinct
     cones can be seen, if does not; adjust the fuel to get distinct blue cone flame.
6. Insert the nebulizer inlet tube in a beaker containing approximately 100 ml of diluent
     and allow 15 minutes for the operating temperature to stabilize. This will ensure a
     stable burner temperature when solutions are aspirated, after the warm up period.
7. While aspirating the diluent, adjust the ‘blank’ control so that the display reads zero
8. Aspirate a pre diluted standard solution
9. Allow 20 seconds for a stable reading and then adjust ‘coarse’ and ‘fine’ controls for a
     convenient reading (if a 140 mmol/l Sodium standard is aspirated, set the display to
     140)
                                                                                          67
10. Carefully adjust the ‘fuel’ control for a maximum reading on the display, ensuring
    that only small adjustments are made, with a pause of several seconds between
    adjustments.
11. Remove the standard solution, wait 10 seconds, then aspirate a blank solution of
    diluent for 20 seconds. Adjust the ‘blank’ control for a zero reading. Remove the blank
    solution and wait 10 seconds.
12. Repeat paragraphs 8, 9 and 11 until the blank reading is zero (within ± 0.2) and the
    calibration reading is within ± 1%.
13. Aspirate each of the unknown solutions for 20 seconds, then note the reading in
    mmol/l
14. Check the calibration frequently
15. When analyzing large batches of samples, recheck instrument calibration every 10
    samples with a single standard solution.

NOTE:
        Always use same batch of diluent for the blank, dilution of samples and standard,
        alternatively the corning dilutor can be used.
        In any difficulty of obtaining a maximum sodium reading proceed as follows:
        Open the inspection flap and adjust the ‘fuel’ control until the flame just starts to
        lift off the burner. Then turn the ‘fuel’ control back, counter clockwise, until the
        cones of the flame are on the burner. Close the flap and proceed with paragraph
        11.

PRECAUTIONS
A diluent recommended by the manufacturer of instrument should be used. Deionised or
high quality distilled water should be used to prepare the diluent. Deionised or distilled
water must be free from contaminative elements (bacteria or moulds can cause
inaccuracies by interrupting or blocking the flow of sample through the neubuliser always
use the same batch of diluent for the blank and the dilution of samples and standards.
1. Anticoagulants containing sodium or potassium salts must not be used. Use serum for
     measurement of sodium and potassium
2. Dilute the sera with care. Good quality calibrated pipette or a sensitive diluter must be
     used. Use the same pipette or dilution equipment for both standard and sample
3. Accuracy of the results depends on the accuracy and purity of the calibration standard.
     Always use accurately prepared standards.
4. Both the accuracy and precision of results depends on maintenance and adherence to
     operating instructions provided by the manufacturer. Careful cleaning of the atomizer-
     burner, cleanliness of sample containers, the aspirating systems, proper adjustment of
     flame size, (blue flame with distinct cone) aspiration rate, and geometry of the flame
     and uniform entry of atomized, diluted sample into the flame are also critical for
     accuracy and precision. Thermal equilibrium must be established before analysis of
     unknown samples. Warm up period is necessary because the initial evaporation of
     water in the flame decreases the temperature of the burner and the entire burner
     chamber.
5. Safety: Propane is highly inflammable and potentially explosive and commonly
     supplied as a liquid under pressure in a cylinder for use with the 410. Cylinder should
     never be subjected to heat or mechanical shock. Leakage of propane from tank,
     instrument fittings or from values may be detected with the aid of soap solutions.

6. Site conditions:
        Never install the flame photometer beneath overhanging cupboards. There must
        be at least 1 metre of clear space above the 410 chimney
        The environment must be clean and free from dust
        The instrument must be placed on a strong, level work stop, free from vibration
        Avoid the instrument to direct sunlight or draughts
                                                                                                  68
      QUALITY CONTROL
      At least two serum control specimens, having stated values in the range 120-150 mmol/l
      for sodium, 3.0-6.0 mmol/l for potassium, and one of which is unknown to the operator
      should be included with each batch of specimens. If single specimens are analysed a
      control specimen should always be included
      OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 1% for sodium
      and 1.5% potassium should be attainable
      ROUTINE CONDITIONS VARIANCE: The value should not exceed 2% for sodium and
      3% for potassium

      REFERENCE VALUES
      Serum Sodium : 136mmol/l - 146mmol/l
      Serum Potassium: 3.5mmol/l - 5.6mmol/l

      19.7       LIMITATION
      Haemolysed sera interfere with the measurement of Electrolytes which causes liberation of
      potassium from the red blood cells.

      REFERENCES
      Operator’s Manual Corning 410
      LAB/86.3




20.   URINE SODIUM AND POTASSIUM

      20.1       PRINCIPLE OF THE METHOD
      Same as for serum sodium and potassium

      20.2       SPECIMEN TYPE, COLLECTION AND STORAGE
      Collect 24 hours urine (timed collection) into a dry, sterile, 2.5 litres brown bottle without
      addition of any preservatives

      20.1       PROCEDURE
                    Mix the 24 hour urine collection and measure the total volume using a clean
                    measuring cylinder
                    Perform the test as for serum electrolytes
                    If the sodium level is low then dilute 1:50 or 1:100 and multiply the result by 4
                    or 2 respectively; dilution is depend on the level of the sodium
                    If the potassium level is higher than 10 mmol/l then dilute the urine 1:500 or
                    1:1000 and multiply the result by 2.5 or 5 respectively. Dilution depends on
                    the level of sodium

      20.3       CALCULATION
                 Sodium (mmol/24 hour)              = mmol/l x 24 hour urine volume in litre
                 Potassium (mmol/24 hour)           = mmol/l x 24 hour urine volume in litre

                 REFERENCE VALUES
                 Urine Sodium : 40-220 mmol/24 hour
                 Urine Potassium : 25-150 mmol/24 hour

                  REFERENCES
                  Operator’s Manual Corning 410
                  LAB/86.3
                                                                                          69

21.   APPENDIX 1 - SAMPLE COLLECTION AND TRANSPORTATION

      21.1        COLLECTION OF A BLOOD SAMPLE
             Confirm the identification of the patient
             Pre-labelling of containers
             Venepuncture:
             Performed only by an experienced phlebotomist (Injury to the median nerve)
             Standardization of the position
             Labelled containers – determine the volume
             Site- median cubital vein of the antecubital fossa
             Non dominant arm: Phlebotomist- gloves
             Patient with infectious disease – gloves, masks, goggles
             Selection of the vein by palpation
             Clean the site with 70% isopropanol swab
             Allow to dry in air (Haemolysis)
             (Alcohol assays – dilute benzylchonium chloride)
             Tourniquet (4-6 inches above the site)
             (Velcro bands: width 2.5 cms)
             Correct gauge of the needle
             Sterile, sharp and without barbs
             The larger the gauge size smaller the bore
             (Adults 22 gauge)
             (Trace metal analysis –stain steel needle)
             Draw required amount of blood
             Release the tourniquet, gauze pad on the site
             Separate the needle from the syringe
             Needle should be placed in a sharps bin
             Put required volumes in to the containers
             Mix gently where necessary- by circular motion
             Do not squeeze the blood through the needle.

      21.2     VENOUS OCCLUSION
      Tourniquet – obstruct the return of blood to the heart
      Time - < 1 min
      Venous occlusion
              ↓
      Increased filtration pressure across the capillary walls
              ↓
      Fluid and low molecular wt compounds leave the vascular compartment
              ↓
      Haemoconcentration
               ↓
      Protein and protein bound compounds increase
      (Total protein, Calcium, Iron, total lipids)

      Increase in CPK and SGOT (stasis of blood in tissues)

      Pumping of the fist → breakdown of RBC (↑K, PO4, LDH)
        LDH → acidosis →calcium released from bone →free Ca↑

      First drawn sample has least changes
      Stress associated with venepuncture- ↑GH, cortisol, glucose
      Struggling - ↑enzymes CPK, SGOT
                                                                                          70
21.3     BLOOD COLLECTION WITH SYRINGE
Avoid vigorous suction and forceful transfer to the container
(Haemolysis)

21.4     BLOOD COLLECTION WITH AN EVACUATED TUBE
PROCEDURE:
Needle screwed into the holder
Tap the tube gently to dislodge any additive from the stopper
Gently insert the tube into the holder
Clean the skin and apply the tourniquet
Guide the needle into the vein and once the needle is in place, press the tube forwards
 into the holder to puncture the stopper and release the vacuum.
When blood is drawn into the vein release the tourniquet.
The tube is filled until the vacuum is exhausted
Withdraw the tube from the holder
Replace another tube if necessary
Evacuated tube with or without serum separating material
Different additives
Stoppers with out glycerin coat –lipid analysis
Gel (serum separating material) inert
Allow the sample to clot/ separation of plasma
Centrifuge
Gel will place between the serum and blood cells
Primary tube –directly into the analyzers
Gel might aspirate into the ion selective electrodes (avoid)
Glass or plastic
Special tubes with metal contamination –Trace metals
Expiry date

21.5     SKIN PUNCTURE
Adults and grown up children
Pre warm if necessary with a slightly warm cloth
Third or fourth finger of the non dominant hand
Clean the centre of the palmer side of the tip (distal phalanx)
Allow it to air dry (avoid Haemolysis)
Quickly puncture with a sharp stab with a lancet
Depth should be < 2.5 mm to avoid contact with bone
Hold the hand downwards to collect the blood by gravity
First drop should be wiped off
Transfer to appropriate capillary tube/card
Do not massage the finger

Infant < 1 year- lateral or medial planter surface of the foot
Patients who are ambulatory (diabetic) foot puncture should be avoided

Neonatal screening –Special filter paper with circles
Touch the paper against a blood drop to fill the circle
Airs dry the paper

21.6     ARTERIAL PUNCTURE
Only performed by an experienced medical officer who had training on the technique.
Sites: femoral artery of the leg, radial artery of the wrist
                                                                                            71
     Blood gas analysis:
Needle and syringe flushed with heparin solution to ensure adequate anticoagulation
and to eliminate trapped air in the needle and in the dead space of the nozzle.
Excess heparin causes reduction of p CO2
Glass syringes
Evacuated tubes should not be used (residual air in the tube will give erroneous results)
Apply pressure after the sample is drawn.
The nozzle should be sealed; syringe placed in a plastic bag and placed in melting ice
(to inhibit the metabolic activity of white cells; prevent change in p H) for
immediate transportation.Analysis should be performed immediately.

Children – arterialized capillary blood –ear lobe
Young child, Infant – heel
Heparinised capillary tubes

    Types –Whole blood- blood gas, ammonia, trace metals
          Serum – preferred specimen
          Plasma – Obtain by including an anticoagulant
                    Repeated thawing and freezing –fibrin clots
                   (Block the probes of analyzers.)

    Anticoagulants and preservatives
    Heparin- least interference with tests
            Available in Na, K, NH4, Li
            Prevent prothrombin into thrombin
            20 units /1ml of blood
            Na and K salts avoided in electrolyte estimation
            Lithium salt is preferred
            (Insignificant effect on flame photometers with
            Lithium internal standard)
            Heparin inhibit - acid phosphatase and LDH activity,
            Binding of thyroid hormones to globulins
            (↑free hormone)
     EDTA -Chelating agent: binds Ca- prevent clotting
            Preserves cellular components of blood
            1-2 mg/ml
            Potassium salt more soluble than sodium
            Unsuitable for ALP, CPK, Ca and Iron
    Sodium fluoride –
            Preservative for blood glucose
            Weak anticoagulant
             Enzyme inhibitor of the glycolytic pathway
             In high concentrations inhibit glucose oxidase and
             urease
             2mg/ml

    Oxalate –Na, K, Li, NH4 oxalate inhibit coagulation by forming
             Insoluble complexes with Ca ions
             Potassium oxalate 1-2mg/ml blood
             Drying temperature should not exceed 100 C
             (Avoid decomposition of oxalate to carbonate which
             Has no anticoagulant activity)
                   3mg/ml oxalate – Haemolysis
                   Inhibit –acid and alkaline; phosphate, amylase and LDH
                                                                                       72
           Influence of the site of collection
           Capillary value> venous (Glucose, Potassium)
           Capillary < Venous        (Bilirubin, calcium, chloride, Sodium, T.P)

21.7   COLLECTION OF BLOOD FROM IV LINES
          Direct from IV line shows increased analytes conc.; of the infusion
          Draw the sample from the arm below the IV line.

          Haemolysis
        ↑aldolase, acid phosphatase, LDH, potassium, phosphate, SGOT
        Other tests- ↑absorbance; Blanking required

           Sample preservation in transit
           Transport without delay
           Blood gas-melting ice
           Hormones – PTH, ACTH –Prechilled tubes (plastic –ACTH)
                         Melting ice, refrigerated centrifuge, freeze-20C
                         Other hormones – plaintubes, separate after
                         2 hours, freeze -20C
           Bilirubin- protect from light and heat
           Na/k – plain bottle – Do not refrigerate
           Calcium – acid washed tubes, no tourniquet
           Enzymes – heat labile
                       ALP – Do not refrigerate

           Separation and storage
           Plasma or serum separation from cells should be carried out 2 hours after
           collection unless specified
           Immediate analysis
           Refrigerate at 4C for the analytes that are stable for 24 hours
           Freeze -20C or -70C
           DO NOT CONTAMINATE THE SAMPLES
           Multiple aliquots –Prevent freezing and thawing
           Method to maintain the identity of samples
           Physiological factors that affect results
           Posture –upright - ↑↑protein and protein bound compounds
           Physical training - ↑ Enzymes of skeletal muscle
           Circadian Variation- cortisol - ↑8.00 am ↓mid night
           Age, gender, race
           Childhood to puberty
           Adult, Elderly Adult, Menopause
           Food ingestion- lipaemia –interference in assays
                                                                                              73


22.   APPENDIX 2 – DIABETES MELLITUS
      Definition: A group of diseases characterized by elevated blood glucose level
      (hyperglycaemia) resulting from defects in insulin secretion, in insulin action or both.
      Classification
          Type 1 Diabetes mellitus
          (Insulin dependent, juvenile D.M.)
          Immune mediated
          Autoimmune destruction of β cells of the pancreas
          Age of onset: childhood and adolescence; any age
          Antibodies against islet cells
          Idiopathic
          Asian /African: permanent low insulin

          Type 2 Diabetes mellitus
          Maturity onset; non insulin dependent
          Due to insulin insensitivity          hyper secretion of insulin
          Relative insulin deficiency

      characteristics                 Type 1                         Type 2
      Age of onset                    <35 years                      >35years
      Genetic predisposition          low                            high
      Antibodies to β cells           yes                            No
      Body habitus                    Normal/wasted                  Obese
      Plasma insulin                  Low /absent                    High
      C -peptide
      Metabolic feature               Insulin deficiency             Insulin insensitivity

          Specific types of Diabetes
          Genetic defects of islet cell function
          Endocrinopathies
          Drug induced

          Gestational D.M
          Any degree of clinical glucose intolerance with onset or first recognition during
          pregnancy.

      Symptoms
      Polyuria
      Polydypsia
      Blurring of vision
      Weight loss

      Diagnostic Strategy for Diabetes (Refer annexure 1)W.H.O 2002 – pg 17
      Corrections – Fasting plasma glucose and random plasma glucose

      Fasting plasma glucose without symptoms

      Fasting plasma glucose on 2 occasions
      Normal fasting plasma glucose 3.3 - 6.1 mmol/L
      Impaired fasting glycaemia        > 6.1 <6.9 mmol/L
      Diabetes                          >7.0 mmol/L
      FPG = 7.0 mmol/l →repeat →7.0 mmol/L→DM
                                                                                      74

Oral Glucose Tolerance Test
Provide information on latent DM
Is more sensitive than FPG

Preparation of the patient
Thee days carbohydrate rich diet and activity
No medication on the day of the test
12 hour fast
No smoking
Glucose load: adults              75g in 300-400 ml of water
               Children           1.75 g/Kg up to 75g of glucose
Plasma glucose sampling           10 min before glucose load
                                  120 min (2 hours) post glucose
Urine glucose corresponding to the samples
Evolution;

               Fasting plasma               120min glucose
                Glucose
IFG                                         6.1 -6.9 mmol/L
                                            (110 – 125mg/dl)

IGT              < 7.0 mmol/L               7.8 – 11.1m.mol/L
                 (< 126mg/dl)               (140- 199mg/dl)

Diabetes         >7.0m.mol/L               >11.1 mmol/L
                 (>126 mg/dl)              > 200mg/dl

OGTT is affected by
  Metabolic stress( ↑ glucose secretion)
  Major surgery, M.I, drugs (steroids)
  Malabsorption
  Vomiting

Gestational Diabetes
Diagnosis;
Fasting plasma glucose >7.0 mmol/L (126mg/dl)
Random plasma glucose >11.1 mmol/L (200mg/dl)

Lab diagnosis
One step approach 75g glucose-OGTT

Two step approach
   First OGTT with 50g glucose; cut off value after 1 hour plasma glucose > 7.8
   mmol/L (140 mg/dl)
   Second OGTT with 75g of glucose load and evaluation as the standard OGTT

Monitoring of Disease
Maintain Plasma glucose level as close as possible to normal levels during the day.

    Use of the glucometer – only for monitoring not for the diagnosis
    CALIBRATED glucometer
                                                                                                                                             75


23       APPENDIX 3 - REFERENCE RANGES
                                                                     Reference Ranges
Analytes                          Sample                          Reference range                           Units   Some common indication
ACE                               5 ml clotted blood /            Adult                          30-100     U/L     Sarcoid
(Angiotensin converting enzyme)   Lithium Heparin
                                  1 ml Blood                      Children 6m-4y                  35-75
                                                                  Children 4-9y                   42-90
                                                                  Children 9-13y                 49-105
                                                                  Boys 13-18y                     45-98
                                                                  Girls 13-18y                    35-75

ACTH                              4 - 5 ml of blood into EDTA     Cord                           50-570     ng/L    Pituitary function
(Adrenocorticotropic Hormone )    bottle ( Separated and frozen   Newborn                        10-185             Adrenal function
                                  within 30 min) Avoid glass      Adult
                                  tubes as they adsorb ACTH       8 h unrestricted activity       8-79
                                  Use pre-chilled polystyrene     16 h supine                     7-30
                                  tubes

AFP Maternal                      3-5 ml clotted blood            14 wks                            46      µg/l    Neural tube defects
                                                                  15 wks                            58              Downs syndrome
                                                                  16 wks                            64
                                                                  17 wks                            72
                                                                  18 wks                            84
                                                                  19 wks                            94
                                                                  20 wks                           108
                                                                  21 wks                           118
                                                                  22 wks                           120

AFP                               3-5 ml Clotted blood            Adult                           < 10      µg/L    Hepatoma
(Alpha Feto protein)                                              Newborns                         <55000   U/ml    Testicular teratoma
                                                                  (may be higher if premature)
                                                                  Infants at           8 wk      <3100      U/ml
                                                                  Infants at          20 wk        <40      U/ml
                                                                  Children                         <15      U/ml
                                                                                                                                                                                76



Albumin                                  2 ml of Clotted Blood       New borne                                    25-50     g/l                  Malabsorption                 and
                                                                     1 Year                                       35-50                          malnutrition
                                                                     2-3 Year                                     36-50                          Protein losing states
                                                                     4th Year and after                           37-50                          Chronic liver disease
                                                                     Adult                                        30-45

Aldosterone                              4 -5 ml EDTA / Heparin or   Newborn                                0.14-1.66       nmol/L               Adreno cortical function
                                         Clotted Blood               1wk -1 y                              0.03-4.43
                                                                     1–3y                                  0.14-1.66
                                                                     3-5 y                                <0.14-2.22
                                                                     5-7 y                                <0.14-1.39
                                                                     7-11 y                                0.14-1.94
                                                                     11-15 y                              <0.14-1.39

                                                                     Adult, average Sodium diet (100-200 nmol/day)
                                                                     Supine                               0.08-0.27

                                                                     Upright (At least 2 hours)
                                                                     Female                                 0.14-0.83
                                                                     Male                                0.166-0.609

Alkaline Phosphatase Isoenzymes          5 ml clotted blood          Reference range available in text books                Qualitative     by   Differentiation of increased
(Only analysed if patient has alkaline                                                                                      electrophoresis      ALP
phosphatase >250 U/L)                                                                                                                            (Liver , Bone disorders)



ALP                                      3 ml clotted blood          Males (age 20-60 Years)               20-90            IU/L 37C             Bone and Liver diseases
( Alkaline Phosphatase)                                              Females (age 15-60 Years)             20-90
                                                                     Children (age 1-12 Years)          up to 350
                                                                     During the growth spurt of puberty up to 500

Alpha-1 Antitrypsin                      4 -5ml Lithium Heparin or   Newborn                                     1.45-2.7   g/L                  α 1- antitrypsin deficiency
(Phenotyping performed if <1.4           5 ml clotted blood          Adult                                     0.78-2.00
g/L)                                                                 >60 years                                 1.15-2.00
                                                                                                                                                                     77


ALT                            3 ml clotted blood               Adults                                       2-27          IU/L 37C   Liver disease
(Alanin Aminotransferase)                                       Infants                                     10-80
                                                                Children                                   10-40
Amino Acids (Plasma)           4.ml Lithium Heparin or          Reference ranges are available in text books               µmol/L     Metabolic diseases
                               5 ml clotted blood
Amino Acids (Urine)            Early morning first urine        This is a special test, contact the reference laboratory              Metabolic diseases
                               sample. Sample should be
                               frozen and a control sample
                               should be provided
Ammonia                        4 ml EDTA in pre chilled         Adult                                          <40         µmol/L     Acute hepatic failure
                               tubes. Must reach the lab        Newborn                                      53-88                    Urea cycle defects
                               within 20 minutes. A control     Infants and older children                   21-47
                               sample should be provided.
                               Contamination            from
                               environment,          smoking,
                               contamination of the glass
                               ware should be avoided. For
                               specific instructions contact
                               the Reference laboratory
Amylase                        3 ml Clotted blood                                                        70 – 340          IU/L 37C   Acute Pancreatitis
Androstenedione (plasma)       3 ml clotted blood               Pre pubertal                               0.3-1.7         nmol/L     Congenital adrenal       cortical
                                                                Puberty (10-17 y)                         0.3-8.4                     disease

                                                                Adult
                                                                Male                                       2.6-7.2
                                                                Female                                       3-9.6

Apo- Lipoprotein B             3 ml clotted blood               Male                                    0.63-1.33          g/L        Investigation of lipid disorders
                                                                Female                                  0.60-1.26
Apo-Lipoprotein A-1            3ml clotted blood                Male                                    0.94-1.78          g/L        Investigation of lipid disorders
                                                                Female                                  1.01-1.99

AST                            3 ml Clotted blood, avoid        Newborn                                      10-75         IU/L 37C   Hepatocellular disease
(Aspartate aminotransferase)   haemolysis                       Children                                     10-45                    Cardiac disease

                                                                Adult                                         4-42
                                                                                                                                                 78



Bicarbonate         5 ml clotted blood              Newborn                              18-23     mmol/l             Acid base disorders
                                                    Adult                                23-31

Bilirubin (Urine)   Random Urine                    Not normally detectable                        Qualitative        Liver disease

Bilirubin (Total)   3 ml clotted blood              Cord blood                             < 50    µmol/l             Liver disease
                    Protect from light              Cord blood premature infants           < 58
                                                    First 24 h                            <103
                                                    2-5 days                              <205
                                                    >1 month                              1.7-26
                                                    Adult                                  3-21

Bilirubin-Direct    3 ml clotted blood              Adults and Children                  0-3.4     µmol/l             Neonatal jaundice
                    Protect from light                                                                                Liver disease
CA 125              5 ml clotted blood                                                     < 35    kU/L               Ovarian cancer
                                                    (Refer the analytical method)
CA 15-3             5 ml clotted blood              Non pregnant                           < 28    kU/L               Raised in cirrhosis
                                                    Pregnant                                                          Breast cancer
                                                    1 & 2 Trimester                       < 50

CA 19-9             5 ml clotted blood                                                     < 37    kU/L               Pancreatic cancers



Calcium (Urine)     24 hours Urine Collected into   New Born                             0-17.5    µmol/kg/24 hours   Disorders       of    calcium
                    acid washed bottle              Infants                          up to 1000    µmol/kg/24 hours   metabolism
                                                    Older Children                     up to100    µmol/kg/24 hours
                                                                                    Or 750-3750    µmol/24 hours

                                                    Adults
                                                    Calcium in diet
                                                    Calcium free                       0.13-1.0    mmol/24 hours
                                                    Low –average                      1.25-3.75    mmol/24 hours
                                                    Average( 800mg/day)                  2.5-7.5   mmol/24 hours
                                                                                                                                                          79


Calcium ( Ionised)              3 ml Heparinised blood            Cord blood                         1.25-1.5    mmol/l     Disorders         of     calcium
ISE (Ion Selective Electrode)                                     Newborn 3-24 h                    1.07-1.27               metabolism
                                                                          24-48 h                   1.00-1.17
                                                                          Thereafter                1.12-1.23

Calcium ( Total)                3 ml clotted blood Collected      Cord Blood                        2.33-3.05    mmol/l     Disorders         of     calcium
                                into acid washed bottle without                                                             metabolism
                                tourniquet                        New Born ( 1st week)
                                                                  Bottle fed                        1.85-2.75
                                                                  Breast fed                        2.05-3.05
                                                                  Thereafter up to 12 years         2.20-2.75
                                                                  Adults                            2.25-2.60

CEA                             5 ml clotted blood                Non smokers                            <2.5    µg/L       Tumour marker, especially of
(Cacino Embryonic Antigen)                                        Smokers                             2.6-5.0               colorectal, lung, breast and
                                                                                                                            pancreas
Ceruloplasmin                   5 ml clotted blood                1 day- 3 months                         5-18   mg/L       Wilson’s disease
                                                                  6/12 months-1 year                     33-43
                                                                  1 year-7 years                       24-56
                                                                  >7 years , Adult                      18-45

Chloride                        5 ml clotted blood                Cord blood                          96-104     mmol/l     Acid base disturbances
                                                                  Newborn 0-30 days                   98-113
                                                                  After 1 month                       98-107

                                CSF                               Infant                             110-130     mmol/l
                                                                  Adult                              118-132

                                Urine (24 hours sample)           Infant                                2-10     mmol/day
                                                                  Child                                15-40
                                                                  Adult                              110-250
                                                                  (Varies greatly with Cl intake)

                                Sweat                             Normal                                 5-35    mmol/l     Cystic fibrosis
                                                                  Marginal                             30-70
                                                                  Cystic fibrosis                     60-200
                                                                                                                                                80



Cholesterol                3 ml Clotted blood             Cord Blood                        0.60-3.50    mmol/l         Lipid disorders
                                                          1-6 week                          2.40-5.60
                                                          up to 1 year                      3.50-6.80
                                                          1-3 years                         1.15-4.70
                                                          4-6 years                         2.80-4.80

                                                                                 Male        Female
                                                          6 - 9 years           3.26-4.94   3.16-5.41    mmol/l
                                                          10-14 years           3.36-5.28   3.21-5.61
                                                          15-19 years           2.95-5.12   3.23-5.48
                                                          22-24 years           3.21-5.64   3.16-5.59
                                                          25-29 years           3.44-6.32   3.33-5.75
                                                          30-34 years           3.57-6.58   3.37-5.96
                                                          35-39 years           3.78-6.99   3.63-6.27
                                                          40-44 years           3.91-6.94   3.81-6.53
                                                          45-49 years           4.09-7.15   3.94-6.86

                                                          50-54 years           4.09-7.17   4.20-7.38
                                                          55-59 years          4.04-7.15    4.45-7.77
                                                          60-64 years          4.12-7.15    4.45-7.69
                                                          65-69 years          4.09-7.10    4.43-7.85
                                                          > 70 years           3.73-6.86    4.48-7.25

Cholinesterase phenotype   5 ml clotted blood of family   Dibucaine                             77-83    %              Scoline apnoea
(Pseudo cholinesterase)    members of patient and         Fluoride                              56-64                   Organophosphorus pesticide
                           control sample                                                                               exposure
Cholinesterase screen      5 ml clotted blood of family                                        0.6-1.4   kU/l at 25 C   Scoline apnoea
(Pseudo cholinesterase)    members of patient and                                             1.08-2.4   kU/l at 37 C   Organophosphorus pesticide
                           control sample                                                                               exposure
CK (Total)                 3 ml clotted blood             New Born                               <300    IU/L 37 C      MI
(Creatine kinase)                                         Children                              <200                    Skeletal muscle disease
                                                          Male                                38-174
                                                          Female                              26-140

CK-MB                      3 ml clotted blood             < 12 or < 2.8 % of Total CK                    IU/L 37C       Myocardial infarction
                                                                                                                                                        81


                                                          Refer the procedure and reference ranges in the kit
Copper                  5 ml clotted blood collected      Birth – 6 month                      3.14-10.99       µmol/l     Wilson’s disease
                        into acid washed bottle           6y                                 14.13-29.83
                                                          12 y                               12.56-25.12

                                                          Adult
                                                          Male                               10.99-21.98
                                                          Female                             12.56-24.34

Cortisol                5 ml clotted blood                At Birth                                 94-610       nmol/L     Adrenocortical function
                                                          12 hrs                                      440
                                                          24 hrs                                      193

                                                          Older children 0800 hrs                200-720
                                                          2200 hrs                                  <205
                                                          Adult
                                                          0800 hrs                               138-635
                                                          1600 hrs                                83-441
                                                          2000 hrs fraction of         < 50% of 0800 hrs

Cortisol-free (Urine)   24 hours urine collected into a   Child                                  5.5-74.5       nmol/day   Adrenocortical function
                        container with 10 g of Boric      Adolescent                             13.8-152
                        acid. Sample should be            Adult                                  27.6-276
                        refrigerated    during      the
                        collection

C-Peptide (RIA)         3 ml clotted blood                                                  0.26-0.62           nmol/L     Insulinoma
                                                          Refer the procedure and reference range of the                   Pancreatic β-cell function
                                                          kit/method                                                       Insulin overdose
C-Reactive Protein      3 ml clotted blood                < 6 months                             < 3.6          mg/L       Acute phase protein
                                                          >12 months                                <6

Creatinine              4 ml clotted blood                Under 12 years                        20-80           µmol/l     Renal function
                                                          Adults                               71-133           µmol/l
                                                          Refer the procedure and reference range of the
                                                          kit/method
                                                                                                                                                               82



Creatinine (Urine)     24 hours urine                     Under 12 years                              44-354     µmol/kg/24 hours   Marker of renal function
                                                          Adults                                    8.84-17.6    mmol/24 hours

Creatinine Clearance   24 hour urine and blood            Newborn (up to 1 month)                       40-65    ml/min/1.732       Renal function
                       sample                                                               Male      Female
                       ( taken during the collection of   Under 12 years                   98-150     95-123     ml/min/1.732
                       24 hour urine sample)              20-30 years                      88-146     81-134
                                                          30-40 years                      82-140     75-128
                                                          40-50 years                      75-133     69-112
                                                          50-60 years                      68-126     64-116
                                                          60-70 years                      61-120     58-110
                                                          70-80 years                      55-113     52-105

DHEA-Sulphate          3ml clotted blood                  Pre pubertal                              0.25-1.00    µg/ml              Adrenocortical function

                                                          Tanner                   Age              Male
                                                          1                       <9.8 y             0.13-0.83
                                                          2                  9.8 -14.5 y            0.42-1.09
                                                          3                10.7- 15.4 y             0.48-2.00
                                                          4                 11.8-16.2 y             1.02-3.85
                                                          5                12.8-17.3 y              1.20-3.70
                                                                                  Adult             1.80-4.50

                                                          Tanner                   Age               Female
                                                          1                      <9.2 y              0.19-1.14
                                                          2                 9.2-13.7 y              0.34-1.29
                                                          3                  10-14.4 y              0.32-3.26
                                                          4                10.7-15.6 y              0.58-2.60
                                                          5                11.8-18.6 y              0.44-2.48
                                                                                 Adult              0.60-2.55

Ethanol                3 ml clotted blood. Avoid          Impairment                                    11-22    mmol/l             Ethanol level
                       alcohol swabs to clean the         Depression of CNS                             >21.7
                       venepuncture site aqueous          Fatalities reported                           >86.8
                       benzalkonium       chloride
                                                                                                                                                                83


                             preferred

Faecal Fat                   Minimum 3 day collection-      Infant breast fed                              <1    g/day              Gastrointestinal malabsorption
                             patient must be on a normal    0-6 years                                      <2
                             diet(Collected between two     Adult                                          <7
                             markers)                       Adult (fat free diet)                          <4

Faecal Reducing substances   Fresh faeces (send to lab      Undetectable                                         Qualitative test   Gut sugar malabsorption
                             within 20 minutes or freeze)

Ferritin                     5 ml clotted blood             Newborn                                    25-200    µg/L               Iron status
                                                            1 month                                   200-600
                                                            2- 5 month                                 50-200
                                                            6 month -15 years                           7-140

                                                            Adult
                                                            Male                                       20-250
                                                            Female                                     10-120

Folate                       5 ml clotted blood             2-16 y                                      11-48    nmol/L             Megaloblastic anaemia
                                                            >16                                           7-36

Fructosamine                 3 ml clotted blood             Adult                                    205-285     µmol/L             Glycaemic control
                                                            Child                         5% below adult level

FSH                          3 ml clotted blood             Pre pubertal                                                            Pituitary-Gonadal axis
                                                            0-6 months                                   <1-4    mIU/ml
                                                            6 months – 1 year                           <1-13
                                                            Children <10 years                           <1-3

                                                            Tanner                          Age       Male

                                                            1                              <9.8 y     0.26-3.0   mIU/ml
                                                            2                         9.8 -14.5 y      1.8-3.2
                                                            3                       10.7- 15.4 y       1.2-5.8
                                                            4                        11.8-16.2 y       2.0-9.2
                                                                                                                                                          84


                                                                5                      12.8-17.3 y   2.6-11.0
                                                                                             Adult     2.0-9.2


                                                                Tanner                    Age          Female
                                                                1                       <9.2 y         1.0-4.2
                                                                2                   9.2-13.7 y       1.0-10.8
                                                                3                    10-14.4 y       1.5-12.8
                                                                4                  10.7-15.6 y       1.5-11.7
                                                                5                  11.8-18.6 y        1.0-9.2

                                                                Adult
                                                                 Follicular                          1.8-11.2
                                                                 Mid cycle                           6.0-35.0
                                                                 Luteal                               1.8-11.2
                                                                 Post menopausal                       30-120

Gamma GT                       3 ml clotted blood               Newborn                                  <200    IU/L 37 C   Liver function
(Gamma Glutamyl Transferase)                                    Infants                                  <120                Alcohol abuse
                                                                Children                                  <35

                                                                Adult
                                                                Male                                     ≤50
                                                                Female                                   ≤30

Gastrin                        3 clotted blood. (12 hour        Child                                < 10-125    ng/L        Zollinger-Ellison syndrome
                               fasting) Serum should be         Adult 16-60 y Male                     <100
                               centrifuged,   separated    &                  Female                     <75
                               frozen at -20 C without delay.
                               Samples      must   not    be             >60 y                           <100
                               haemolysed and lipaemia
                               should be avoided


Glucose                        2 ml Blood collected into        Plasma Glucose level
                               sodium fluoride and potassium    Cord blood                             2.5-5.3   mmol/l
                                                                                                                                                               85


                              oxalate in 1:3 ratio (Refer the   Premature                               1.1-3.3
                              volume of blood to be             Neonate                                 1.7-3.3
                              included in the sugar bottle      Newborn
                              from the local laboratory)            1 day                            2.20-3.30
                                                                    >1 day                            2.80-5.00
                                                                Child                                3.30-5.50

Glucose                                                         Fasting Plasma Glucose level                           mmol/l   Diagnosis of Diabetes Mellitus

                                                                (10-12 hours fasting) Adult            3.3-6.1
                                                                Random Plasma Glucose level             ≤7.8
                                                                Post Prandial Plasma Glucose level     ≤11.1

                                                                Oral Glucose Tolerance Test
                                                                Fasting                                   < 6.1
                                                                After 2 hours                             < 7.8
                                                                (Refer recent WHO criteria for diagnosis of diabetes
                                                                mellitus)

Glucose (CSF)                 1 ml of CSF into Fluoride/        70 % of plasma glucose                                 mmol/l   Meningitis (Bacterial/Viral)
                              Oxalate bottle (accompanied
                              with the blood sample into
                              fluoride oxalate)

Growth Hormone (serum)        3 ml clotted blood                Newborn 1st day                             5-53       ng/ml    Pituitary function
                                                                1 week                                      5-27
                                                                1 month – 1 year                            2-10
                                                                Child fasting at rest                    0.7-6.0
                                                                Adult                                    0.7-6.0

HBA1c                         5 ml blood in EDTA bottle         1-5 years                            2.1-7.7           %        Glycaemic control
(Glycated haemoglobin A 1c)                                     5-16 years                           3.0-6.2
                                                                Adult
                                                                (Column chromatography, Cation exchange )
                                                                                                      4.5-8.5
HCG                           5 ml clotted blood                Male and non pregnant female              <5           IU/L     Pregnancy
                                                                                                                                                   86


(Human Chorionic Gonadotrohpin)                                    Female                                                      Germ cell tumours
                                                                   After                    After LMP
                                                                   fertilization
                                                                   2 wk                      4 wk             5-100
                                                                   3 wk                      5 wk         200-3000
                                                                   4 wk                      6 wk     10000-80000
                                                                   5-12 wk               7-14 wk 90000-500000
                                                                   13-24 wk            15-26 wk        5000-80000
                                                                   26-38 wk            27-40 wk        3000-15000
                                                                   Trophoblastic disease                   >100000
HDL Cholesterol                   4 ml of clotted blood colleted   Age                     Male           Female      mmol/l   Lipid disorders
                                  without tourniquet               Cord blood          0.16-1.37         0.34-1.45
                                                                   5-9 y              0.98-1.94          0.93-1.89
                                                                   10-14 y             0.96-1.91         0.96-1.81
                                                                   15-19 y             0.78-1.63         0.91-1.91
                                                                   20-24 y             0.78-1.63         0.85-2.04
                                                                   25-29 y             0.80-1.63         0.96-2.15
                                                                   30-34 y             0.72-1.63         0.93-1.99
                                                                   35-39 y             0.75-1.60         0.88-2.12
                                                                   40-44 y             0.70-1.73         0.88-2.28
                                                                   45-49 y             0.78-1.66        0.88- 2.25
                                                                   50-54 y             0.72-1.63         0.96-2.38
                                                                   55-59 y             0.72-1.84         0.96-2.35
                                                                   60-64 y              0.78-1.91        0.98-2.38
                                                                   65-69 y            0.78 -1.94         0.91-2.48
                                                                   >70 y               0.80-1.94         0.85-2.38

Homocystine                       Plasma                                                                   <15        mmol/l   Cardiac risk
                                                                   Refer the procedure and reference range of the
                                                                   kit/method
Hydroxy progesterone(17 OHP)      Serum 3 ml clotted Blood         Male, Puberty stage -1              0.1-2.7        nmol/L   Adrenal status
                                                                   Adult                                1.5-7.5
                                                                   Female, Puberty stage -1           0.1-2.5
                                                                   Follicular                          0.6-3.0
                                                                   Luteal                            3.0-15.5
                                                                   Postmenopausal                        ≤2.1
                                                                                                                                                           87


                                                                  Neonates                             <30                     Congenital            Adrenal
                                                                  >1 month                               6                     Hyperplasia

Hydroxyindoleacetic Acid        24 hours urine                    Adult                           10.4-31.2    µmol/24 hours   Carcinoid syndrome
(5-HIAA)                                                          Children                             7-70

Insulin (12 hour fasting )      5 ml clotted serum should be      Newborn                            21-139    pmol/L          Insulinoma
                                centrifuged , separated &         Adult                             13-174                     Insulin overdose
                                frozen at -20 C within 2 hours    >60 years                         42-243

Iron                            5 ml clotted blood collected      New Born up to 1 month          17.9-44.75   µmol/l          Iron status
                                into acid washed bottle           Infant (1month-1 year)          7.16-17.9
                                                                  Child (1 year-12 year)        8.95-21.48

                                                                  Adult
                                                                  Male                          11.64-30.43
                                                                  Female                         8.95-30.43
                                                                  (Strongly method dependent)

Iron Binding Capacity , Total   5 ml clotted blood collected      Infant                          17.9-71.6    µmol/l          Iron status
(TIBC)                          into acid washed bottle           Thereafter                     44.5-80.55    µmol/l

Lactate                         4 ml of blood .Patient should     Venous                              0.5-13   mmol/l          Investigation of     metabolic
                                be complete rest for 2 hour       Arterial                           0.5-1.6                   disorders
                                and fasting & preferably                                                                       Lactic acidosis
                                without tourniquet. (Should be
                                collected    into     container
                                containing 10 mg of NaF & 2
                                mg of potassium oxalate per 1
                                ml of blood. Specimen should
                                be cooled immediately & cells
                                separated within 15 minutes)
                                                                                                                                                 88


LDH (Lactate dehydrogenase)   5 ml clotted blood   0-4 days                            290-775    IU/L     37 C   Haematological abnormalities
Total(L          P)                                4-10 days                         545-2000                     Liver disease
                                                   10days -2 years                     180-430
                                                   2 years -12 years                   110-295
                                                   12 -60 years                        100-190
                                                   >60 years                           110-210
LDL Cholesterol                                                            Male        Female                     Lipid status
(Calculated)                                       Cord blood             0.5-1.45   0.54-1.50    mmol/l
                                                   5-9 y               1.63-3.34     1.76-3.63
                                                   10-14 y             1.66-3.44     1.76-3.52
                                                   15-19 y              1.61-3.37    1.53-3.55
                                                   20-24 y              1.71-3.81    1.48-4.12
                                                   25-29 y              1.81-4.27    1.84-4.25
                                                   30-34 y              2.02-4.79    1.81-4.04
                                                   35-39 y              2.10-4.90    1.94-4.45
                                                   40-44 y              2.25-4.82    1.92-4.51
                                                   45-49 y              2.51-5.23    2.05-4.82
                                                   50-54 y              2.31-5.10    2.28-5.21
                                                   55-59 y              2.28-5.26    2.31-5.44
                                                   60-64 y              2.15-5.44    2.59-5.80
                                                   65-69 y              2.54-5.44    2.38-5.72
                                                   >70 y                2.28-4.82    2.49-5.34
LH                            5 ml clotted blood   Pre pubertal                                   mIU/L           Pituitary- gonadal axis
(Luteinizing Hormone)                              0-6 month                             1-18
                                                   6 month -10 year                      <1-5

                                                   Tanner                    Age       Male
                                                   1                        <9.8 y    0.02-0.3
                                                   2                    9.8-14.5 y     0.2-4.9
                                                   3                   10.7-15.4 y     0.2-5.0
                                                   4-5                 11.8-17.3 y     0.4-7.0
                                                                             Adult      1.5-9.0

                                                   Tanner                    Age        Female
                                                   1                       <9.2 y    0.02-0.18
                                                   2                   9.2-13.7 y     0.02-4.7
                                                                                                                                                                           89


                                                                 3                   10-14.4 y            1.0-12.0
                                                                 4-5                10.7-15.6 y           0.4-11.7

                                                                 Adult
                                                                 Follicular                                      29
                                                                 Mid cycle                                    18-49
                                                                 Luteal                                        2-11

Lipoprotein (a)                3 ml clotted blood                                                            20-570    mg/L                  Lipid disorders
                                                                 (refer the method for reference range)
Lithium                        5 ml clotted blood                Therapeutic                                0.6-1.2    mmol/l                Therapeutic dose monitoring
                                                                 Toxic                                           >2                          Overdose

Magnesium                      3 ml clotted bottle collected     Newborn 2-4day                              0.6-0.9   mmol/l                Electrolyte status
                               into acid washed bottle           5 months – 6year                         0.70-0.95
                               (preferably without tourniquet)   6-12 years                               0.70-0.86
                                                                 12-20 years                              0.70-0.91
                                                                 Adult                                     0.66-1.07

Mucopolysaccharide screening   Random Urine, fresh               Negative                                              Qualitative           Mucopolysaccharidosis
Myoglobin                      Random Urine                      Any Myoglobin detected is clinically significant      Qualitative           Acute tubular necrosis
Myoglobin (Serum)              4 ml of clotted blood             Male                                        19-92     µg/l                  Acute myocardial infarction
                                                                 Female                                      12-76
                                                                 Increases slightly with age
Occult Blood                   Samples from 3 consecutive        Not normally detected                                 Qualitative           GI bleeding
                               days                                                                                                          Colonic cancer
Oestradiol (17-β Oestradiol)              5 ml Clotted blood     Children                                       <60                  pg/ml                Gonadal function
                                                                 Male                                           <40
                                                                 Female
                                                                 Follicular phase                           30-120
                                                                 Ovulatory peak                            150-400
                                                                 Luteal phase                               70-200
                                                                 Menopause                                     <60

Osmolality                     Random Urine                                                                50-1200     mOsmol/kg             Urine concentrating ability
                                                                 Depending on the fluid intake
                                                                                                                                                                   90


Oxalate                       24 urine collected into bottle   Male                                   0.23-0.68     mmol/24 hours    Hyperoxaluric stone forming
                              containing 10 ml of 1 N HCL      Female                                 0.23-0.63
                                                               Children                                0.10-3.0
                                                               Excessive Vitamin C in Urine affects assay results
Para Thyroid Hormone (PTH)    5 ml clotted blood               Cord blood                                ≤0.32      pmol/L           Parathyroid tumour
Intact (IRMA)                                                  2 y – Adult                             0.95-6.8                      Hypercalcaemic states
Phosphate -inorganic          5 ml Clotted blood               Cord Blood                             1.03-2.45     mmol/l           Bone function
                                                               (Lower values found in breast fed infants)                            Renal function
                                                               New Born ( 1st week)
                                                               1st week                              1.87-2.91
                                                               2nd week                              1.58-2.87
                                                               up to 1 year                          1.30-2.10
                                                               Thereafter up to 12 years             1.16-1.91
                                                               Adults                                0.80-1.44
Phosphate-inorganic (Urine)   24 hours Urine                   Older children                         0.49-0.65     mmol/kg/24       Parathyroid , renal and bone
                                                               Infants                                       6.5    hours            disorders
                                                               Adults                                     16-48     mmol/kg/24
                                                                                                                    hours
                                                                                                                    mmol/24 hours
Potassium                     3 ml clotted blood (avoid        <2 month                                      3-7    mmol/l           Electrolyte status
                              haemolysis   &   do   not        2-12 month                               3.5-6.0
                              refrigerate)                     > 12 month                                3.5-5.0
                                                               Adult                                    3.5-5.6
Pregnancy Test(Urine)         Random Urine                     Qualitative                                          Not applicable   Pregnancy

Progesterone                  5 ml clotted                     Pre pubertal child (1-10 y)             0.2-1.7      nmol/l           Ovulatory status
                                                               Puberty
                                                               Tanner                    Male          Female
                                                               1                      <0.3-1.0        <0.3-1.0
                                                               2                      <0.3-1.0        <0.3-1.7
                                                               3                      <0.3-1.5         .3-14.3
                                                               4                      <0.3-3.4       <0.3-41.3
                                                               5                       0.7-2.6       0.3-30.2

                                                               Adult
                                                               Male                                     0.4-3.1
                                                                                                                                                    91



                                                           Female
                                                           Follicular                              0.5-2.2
                                                           Luteal                                 6.4-79.5
                                                           Pregnancy            7-13 wk        32.6-139.9
                                                                                4-37 wk          62-262.4
                                                                               30-42 wk       206.7-728.2

Prolactin                         5 ml of clotted blood    Cord blood                              45-539     µg/L    Pituitary function
                                                           Newborn                    5-3 d        30-495



                                                           Children
                                                           Tanner               Male               Female
                                                               1                  <10                3.6-12
                                                               2-3               <6.1               2.6-18
                                                               4-5             2.8-11               3.2-20

                                                               Adult
                                                               Male                                3-14.7
                                                               Female                             3.8-23.2
                                                               Pregnancy 3rd trimester             95-473

Prostate Specific Antigen (PSA)   5 ml clotted blood                                                  <4      µg/L    Prostate cancer
Protein (CSF), Lumbar             CSF in plain container   Premature                               15-130     mg/dl   Inflammatory conditions of the
                                                           Full term newborn                       40-120             meninges
                                                           <1 month                                  20-80
                                                           Thereafter                               15-40

Protein(Total)                    3 ml clotted blood       New borne                                46-77     g/l     Malnutrition , liver disease and
                                                           1 Year                                   56-73             protein losing conditions
                                                           2-3 Year                                 58-76
                                                           4th Year and after                       60-80
                                                           Adult                                    60-80
                                                           Body Fluid (Transudates)                    <3
                                                                                                                                                         92


                                                                   (Exudates)                          >3

Renin                            5 ml blood taken into a bottle    0-3 y                           <16.6    µg/L/hr        Hypertensive states
                                 containing EDTA ,must be          3-6 y                            <6.7
                                 separated at room temperature     6-9 y                            <4.4
                                 and frozen at -20 C or            9-12 y                           <5.9
                                 lower(Contact the reference lab   12-15 y                          <4.2
                                 before collection of blood)       15-18 y                           4.3
                                                                   Normal sodium diet
                                                                    Supine                        0.2-1.6
                                                                    Upright (4 hours)            0.7-3.3
                                                                   Low sodium diet
                                                                   Supine                        1.0-5.4
                                                                    Upright (4 hours)            0-19.0

SHBG                             5 ml clotted blood                Male                            10-50    nmol/L         Gonadal function
(Sex Hormone Binding Globulin)                                     Female                          30-90
                                                                   Pre pubertal                   55-120
Sodium                           3 ml clotted blood                Newborn                       134-146    mmol/l         Electrolyte status
                                                                   Infant                       139-146
                                                                   Child                        138-145
                                                                   Thereafter                   136-146
Somatomedin C ( IGF-1)           4 ml blood collected into                                     400-2000     IU/L           Growth Hormone disorder
                                 EDTA bottle                                                                               investigation
Stone analysis                   Renal Calculus                    Not applicable                           Qualitative    Investigation of Nephrolithiasis
                                                                                                            Quantitative
Sugar Chromatography (Urine)     Random urine, Fresh early         Not detectable                           Qualitative    Gut sugar malabsorption
                                 morning sample preferable
T3 (Free)                        3 ml clotted blood                Child                         2.6-4.8    pg/ml          Thyroid function
                                                                   Adult                       2.08-6.74
T4(Free)                         3 ml clotted blood                Premature infant                         ng/dl          Thyroid function
                                                                   (Gestational age in week)
                                                                   25-27                         0.6-2.2
                                                                   28-30                         0.6-3.4
                                                                   31-33                         1.0-3.8
                                                                   34-36                         1.2-4.4
                                                                                                                      93



                                            Infants, Children and Adults
                                            1-4 days                            2.2-5.3
                                            2-20 weeks                          0.9-2.3
                                            5-24 months                          0.8-1.8
                                            2-7 years                           1.0-2.0
                                            8-20 years                          0.8-1.9
                                            21-45 years                         0.9-2.5
                                            Adults >45 years                    0.8-2.3
Testosterone (Free)    5 ml Clotted blood   Age                     Male        Female     pg/ml   Gonadal function
                                            Cord                     5-22          4-16
                                            Newborn
                                            1-15 d               1.5-31.0       0.5-2.5
                                            1-3 month            3.3-18.0       0.1-1.3
                                            3-5 month            0.7-14.0       0.3-1.1
                                            5-7 month              0.4-4.8      0.2-0.6
                                            Pre pubertal
                                            1-10 y              0.15-0.6      0.15- 0.6
                                            Adult                 52-280        1.6-6.3
Testosterone (Total)   5 ml Clotted blood   Pre pubertal Child 1-10 y                      ng/ml
                                            Male                             <0.03-0.1
                                            Female                           <0.03-0.1
                                            Puberty Male
                                            Tanner                   Age
                                                 1                  <9.8 y   <0.03-0.1
                                                 2             9.8-14.5 y    0.18-1.5
                                                 3            10.7-15.4 y     1.0-3.2
                                                 4            11.8-16.2 y     2.2-6.2
                                                 5            12.8-17.3 y     3.5-9.7
                                                                    Adult     3.5-103
                                            Puberty Female
                                            Tanner                   Age
                                                 1                  <9.2 y     0.03-0.1
                                                 2             9.2-13.7 y    0.07-0.28
                                                 3              10-14.4 y    0.15-0.35
                                                 4           10.7 -15.6 y    0.13-0.32
                                                                                                                                                                     94


                                                                            5            11.8 -18.6 y       0.20-0.38
                                                                                               Adult           0.1-0.5
Thyroglobulin                         5ml Clotted blood                                                          3.0- 42    µg/L         Medullary carcinoma of thyroid

Total Protein (Urine)                 24 hours urine                                                              20-150    mg/24hours   Renal function
                                      preservative:1ml    of     10%                                                                     Protein losing status
                                      Thymol       in     isopropanol
                                      solution
Transferrin saturation (calculated)                                     Male                                       20-55    %
                                                                        Female                                     15-54    %

Triglycerides                         3 ml of clotted blood. 14 hours                             Male         Female                    Lipid disorders
                                      fasting is necessary              Cord Blood            0.15-1.07     0.12-0.86       mmol/l
                                                                        0-9 years             0.34-1.13     0.40-1.24
                                                                        10-14 y               0.36-1.41     0.42-1.48
                                                                        15-19 y               0.42-1.67     0.44-1.40
                                                                        20-29 y               0.50-2.81     0.41-1.63
                                                                        30-39y                0.56-3.62     0.44-1.99
                                                                        40-49 y               0.62-3.70     0.51-2.42
                                                                        50-59 y               0.65-3.23     0.59-2.62

Troponin I                            2 ml lithium                      Normal                                      < 0.5   µg/L         Myocardial infarction
                                      Heparin or 5 ml clotted            MI                                          >3                  Unstable angina
                                                                        (Refer the method for reference range)
Troponin T                            2 ml lithium                      Refer the method for reference range                             Myocardial infarction
                                      Heparin or 5 ml clotted                                                                            Unstable angina
TSH                                   5m Clotted blood                  Premature infants                                   mIU/L        Thyroid function
(Thyroid Stimulating Hormone)                                           (Gestational age in weeks)
                                                                        25-27                                    0.2-30.3
                                                                        28-30                                    0.2-30.6
                                                                        31-33                                    0.7-27.9
                                                                        34-36                                    1.1-21.6


                                                                        Infants , children and adults
                                                                        1-4 days                                    1-39
                                                                                                                             95


                                                      2-20 wk             1.7-9.1
                                                      5-24 months          0.8-8.1
                                                      2-7 years           0.7-5.7
                                                      8-20 years          0.7-5.7
                                                      21-45 years         0.4-4.2
                                                      Adult >45 years     0.3-5.0

Urea                3 ml clotted blood                Cord blood          7.5-14.3    mmol/l         Renal function
                                                      Premature 1 wk       1.1-8.9                   Dehydration
                                                      Newborn              1.4-4.3
                                                      Infant/ Child        1.8-6.4
                                                      Adult                2.1-7.1
                                                      >60 y                2.9-8.2

Uric Acid           4 ml clotted blood                1-5 y               100-350     µmol/l         Gout
                                                      6-11 y              130-390                    Tumour lysis
                                                                                                     Pre eclampsia
                                                      12-19 y
                                                      Male               180-460
                                                      Female             160-340
                                                      Adult              120-360

Uric Acid (Urine)   24 hours urine collected into                       1.48-4.43     mmol/24hours
                    sterile 2.5L bottle (Should be
                    refrigerated    during     the
                    collection)
Urine Analysis      Random Urine                      Not applicable                  Qualitative    Renal disease
(with Microscopy)
Vitamin A           5ml Clotted blood                 1-6 y                 0.7-1.5   µmol/L         Nutritional status
                    (fasting)                         7-12 y             0.91-1.71
                    Protect from light separate the   13-19 y           0.91-2.51
                    serum immediately.                Adult               1.05-2.8
                    Store at -20 0 C

Vitamin B 12        5ml Clotted blood                 Newborn            125-590      pmol/L         Megaloblastic anaemia
                                                      Thereafter         103-157
                                                                                                                                                                96


                                                                   >60 years                            81-590

Vitamin D (25 OHD3)                 5ml Clotted blood              Children                                                               Vitamin D metabolism and
(25 Hydroxy cholecalciferol)                                       1-30 d                              1.9-33.4       ng/ml               Calcium metabolism
                                                                   31d - 1 y                           7.4-53.3

                                                                   Adult                                 14-60

Vitamin D(1,25 OHD3)                5ml Clotted blood                                                   43-154        pmol/L              Vitamin D metabolism and
(1,25-dihydroxy cholecalciferol)                                                                                                          Calcium metabolism
Vitamin E                           5ml Clotted blood                                                 11.6-46.4       µmol/l              Nutritional status




                                   REFERENCE:
                                        1.   Teitz text book of clinical chemistry by Carl A.Burtis, Edward R.Ashwood; 2nd and 3rd Edition
                                        2.   Clinical chemistry – theory , analysis, correlation by Lawrence A.Kaplan, Amadeo J. Pesce; 3rd Edition 1996
                                        3.   WHO Guidelines on standard operating procedures for clinical chemistry, Sep 2000
                                        4.   Nelson text book of paediatrics by Behrman, Kliegman, Jenson; 16th Edition 2000
                                        5.   Forfar and Arneil’s Text book of paediatrics
                                        6.   Biochemical basis of paediatric disease by Steven J Soldin,Nader Rifai,Jocelyn M. Hicks ;3rd Edition 1998
                                                                                      97



WE SINCERELY THANK THE FOLLOWING COLLEAGUES / MEMBERS OF MRI AND WHO COUNTRY OFFICE

                  Administrative staff
                                         Ms. Kumuduni Ragel
                                         Secretary WHO country office Sri Lanka

                                         Mrs. G. Subramanium
                                         Accountant MRI

                                         Mr. A. Ravichandran
                                         Financial staff

                                         Mrs. Margret Prera
                                         Planning unit Ministry of Health

                                         Miss. Nilakshi Devindi Gunatillaka

                  Members of staff and NEQAS team

                                         Ms. Manjula Subashini
                                         Mr. K. S. T. Karunapala
                                         Ms. E.A.N.S. Peiris
                                         Mr. B. D. Lankananda
                                         Ms. S.K. Nanayakkara
                                         Ms. N. D. Wijekoon

                  Support staff
                                         Mr. N. A. H. H. Nissanka
                                         Mr. J. M. Wijesinghe
                                         Mr. D. L. Upasena
                                         Mr. T. V. Anton
                                         Ms. W. Pushpika Perera

				
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