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POD21PAS _Podiatric Assessment_

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					POD21PAS (Podiatric Assessment)

LABORATORY TESTING MANUAL – ANSWER BOOK

Matthew Oates Department of Podiatry, 2004

INDEX
Introduction to Unit What is „normal‟? Specificity, Sensitivity & Predictive values Factors affecting laboratory test results Units of Measurement in Laboratory Tests Haematology Review of blood formation, composition & function Blood examination Collection of blood specimen Common blood tests Review of haemostasis Common haemostatic tests Other blood tests Urinalysis Review of urine formation & composition Urine examination Collection of urine specimens Macroscopic analysis Microscopic analysis Microchemical analysis Diagnostic microbiology Microscopy Culture Wound culture Clinical mycology 3 4 4 7 7 8

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Synovial fluid analysis 29 Indications & Contraindications Synovial fluid examination Collection of synovial fluid specimen Macroscopic analysis, Microscopic analysis, Microbiologic analysis & Chemical analysis Biopsy 31 Incisional biopsy Excisional biopsy Specimen preparation
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Introduction
Throughout your POD12PAS Podiatric Assessment lectures you have been gathering vital information about the ways in which you can begin to assess various structures within the lower limb. For example, by now you should be able to assess the vascular supply to the feet by manual palpation of pulses, taking ankle-brachial pressure measurements or by making very simple observations which might also serve to provide relevant and important information about your patient‟s vascular integrity. Laboratory tests, often referred to as „pathology tests‟, are another means of obtaining important information, which may serve to enhance your ability to diagnose a patient‟s presenting complaint. These tests usually require obtaining a specimen of the body tissue you wish to examine, e.g the patient‟s blood, and then having this sample examined in a pathology laboratory in order to report findings of various tests which may provide you with information to suggest a particular pathology or condition or alternatively, to rule out a potential diagnosis. Until recently, Podiatrists in Victoria were unable to refer patients directly to pathology services. Instead, the patient required a referral from their General Practitioner or other medical specialist in order to receive pathology services and the associated Medicare rebate. However, the Australian Podiatry Association of Victoria has recently formed a commercial agreement with Mayne Health Dorevitch Pathology, which offers patients of Victorian podiatrists (who are members of the ApodA (Vic.)) the option of being directly referred to pathology for a reduced out-of-pocket fee. It is anticipated that this new avenue of referral may lead to increased referral by podiatrists to pathology. As a result of this, it has been necessary to educate undergraduate students in the understanding of common laboratory tests. This manual and activity book is designed to provide you with relevant lecture notes and additional reading material as well as offering you the opportunity to develop an understanding of this area of assessment through simple learning activities. I hope that you find this resource useful. Matthew Oates Associate Lecturer, Department of Podiatry

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What is ‘normal’?
One of the obvious questions when we begin to look at laboratory tests and begin to interpret their results is, “What is considered a normal test result?” To discuss this very question, let‟s use the example of cholesterol levels. That is, what is a normal cholesterol level? Murphy (1967, 1971) identified seven different ways of defining the term „normal‟. „Normal‟ may be considered as:        a bell shaped probability distribution of cholesterol values the most representative cholesterol level as defined by a mean or average the most commonly encountered cholesterol values defined by a range cholesterol levels most suited for reproduction and survival cholesterol levels unlikely to cause harm a committee‟s consensus (i.e approved cholesterol levels) the „ideal‟ cholesterol value

While the above definitions may serve to define normal in certain areas of study, e.g statistics, politics, sociology, etc., we are aware of the diverse nature of the human population. For example, there are age, gender and racial differences that exist which may influence the so called „normal range‟ of various laboratory tests. So instead, we refer to the term „Reference Values‟. This reference range identifies the most commonly encountered values for a specific test as specified for various groups.

Specificity, sensitivity & predictive values
If we are going to use a laboratory test, it is vital that the test is accurate and does actually test what it is intended to test. Otherwise, this could have devastating effects on people having the test. Imagine being told that a test result has indicated that you are positive for HIV when in fact you are not or vice versa.

Sensitivity refers to the percentage of positive results obtained by a test in persons with
a given disease.

Specificity refers to the percentage of negative results obtained by a test among people
who do not have a given disease. Let‟s explore these terms a little further. Examine the table below.

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No. with disease No. without disease TOTALS

No. with result TP FP TP+FP

+

test No. with – test result FN TN FN+TN

TOTALS TP+FN FP+TN TP+FP+TN+FN

TP: True positive, FP: False positive, TN: True negative, FN: False negative The table above provides an „algebraic‟ consideration of the accuracy of a particular laboratory test. If we look at the sensitivity of this test, that is, its ability to identify those who are positive for a disease, we can see that sensitivity = TP/(TP+FN) For example, if we know there are 10 people with the disease and the test returns 4 positive results and 6 negative results, then sensitivity will be Sens. = 4/(4+6) = 0.4. The sensitivity of this test is considered to be low. Ideally, sensitivity would be 1.0 where the test produces 100% true positive results. However, this is not always the case with some laboratory tests. Now, looking at the specificity of the test or its ability to correctly identify those who don‟t have the disease, specificity = TN/(TN+FP) For example, if we know that there are 10 people without the disease and the test returns 2 positive results and 8 negative results then, Spec. = 8/(8+2) = 0.8. This test has high specificity for this disease. The Predictive Value of a test, then, is its ability to predict that someone who has a disease does have the disease and someone who doesn‟t have the disease doesn‟t have the disease. Therefore, the predictive value of a positive test (i.e the percentage of patients with a positive test who are diseased), = TP/(TP+FP)

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For example, in a population of 20 people there are 10 people with the disease and 10 people without the disease but the test returns 15 positive results. Therefore, PV = 10/(10+5) = 0.67 Here, the dilemma is that 5 people have been told that they are positive for a disease when in fact they don‟t have the disease at all. Alternatively, the predictive value of a negative test (i.e the percentage of patients with a negative test who are not diseased), = TN/(TN+FN) For example, in a population of 20 people there are 10 with the disease and 10 without the disease but the test returns 18 negative results. Therefore, PV = 10/(10+8) = 0.56 So, here the problem is that the test is a poor predictor of negativity. Eight patients have been told they don‟t have the disease when in fact they do. This obviously has great implications for the health and safety of these patients. In screening for disease, practitioners are most interested in the predictive value of the positive result. We don‟t want our test to identify those who have a disease as not having it. There is a strong relationship which exists between the predictive value and the prevalence of disease. Prevalence refers to the proportion of individuals in a population having a disease. The higher the prevalence of a specific disease, the greater the predictive value of a positive test. Consider the following table. LOW Clinic prevalence Test + MI 48 Non-MI 47 Total 95 HIGH CCU prevalence Test + MI 475 Non-MI 25 Total 500

2 903 905

Total
50 950 1000

25 475 500

Total
500 500 1000

This table considers the predictive value of an enzyme test in patients with myocardial infarction (MI) in a general medical clinic (Clinic) and in a coronary care unit (CCU). We would expect there to be a higher prevalence of patients with MI in the CCU compared to the Clinic. Now, let‟s see how specificity, sensitivity and predictive value of the enzyme test differ with prevalence.

Clinic

prevalence of MI = 50/1000 = 5% Sensitivity = 48/50 = 0.96 Specificity = 903/905 = 0.95 Predictive value = 48/95 x 100 = 50% 6

CCU

prevalence of MI = 500/1000 = 50% Sensitivity = 475/500 = 0.95 Specificity = 475/500 = 0.95 Predictive value = 475/500 x 100 = 95% This example demonstrates that in populations with a higher prevalence of a disease, the test may demonstrate a higher predictive value by sheer fact that the number of people with this disease is greater.

Factors affecting laboratory test results
There are a variety of factors which may influence the results obtained by laboratory tests. Some to consider are:       Genetic (or interindividual) factors: racial, ethnic Physiologic (or intraindividual) factors: age, sex, build Sampling variables: diet, time of day, season, fasting states, physical states, geographical location, stress Drug influences: physiologic or direct chemical interference with test methodology Specimen collection variables: torniquet, anticoagulant Specimen handling

Units of Measurement in Laboratory Tests
The units of measurement used by laboratories in reporting their reference ranges may vary from laboratory to laboratory. In the United States, there is a move away from conventional units of measurement to the Systeme International (SI) values. In Australia, laboratories have tended to adopt SI values. E.g Normal serum cholesterol for person <29 years  <200 mg/dl (conventional)  <5.15 mmol/L (SI) Nonetheless, the main point is that the laboratory will always report their reference range beside your patient‟s results so that you can identify whether the patient is below or above the reference range. Often laboratories will print the values obtained in the units they are not using also.

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Learning activity
1) Go to the following website: http://gim.unmc.edu/dxtests/howto.htm You are provided with what is known as a 2-by-2 table. It is similar to the tables I have presented to you in this section. Read the instructions provided then click on the link „click here for practice problems‟. Work your way through as many problems as you like until you feel comfortable with calculating predictive values, etc.

Haematology
The study of the morphology of the blood and blood forming tissues.”
The study of the morphology of the blood and blood forming tissues

Review of blood formation, composition & function
Formation of blood cells occurs via the process of haemopoiesis and this begins in the first two months of life. In infants blood cells are produced in the bone marrow of all bones while in adults the vertebrae, sternum, skull, ribs, pelvis, sacrum and the head of the femur become the primary centres for cell formation. Erythropoiesis refers to the formation of red blood cells (RBCs) or erythrocytes. This process requires the presence of metals, vitamins, amino acids and hormones in order to produce RBCs. Deficiency of any of these materials can lead to a disorder known as anaemia but we will discuss this later. As well as producing new RBCs the body also removes old RBCs. This occurs in what is known as the reticuloendothelial system (bone marrow, liver and the spleen). The average person circulates ~5 litres of blood. Approximately 3 litres of this is plasma while the remaining 2 litres consist of blood cells. Plasma is derived from the intestines and other organs and serves as the vehicle for the measurement of cells within the blood in laboratory medicine. Blood is made up of white blood cells (WBCs) also known as leukocytes, RBCs and platelets or thrombocytes. Blood is primarily responsible for the transport of oxygen from the lungs to the body tissues and to return carbon dioxide from the tissues back to the lungs. Other functions include maintenance of haemostasis, thermoregulation, regulation of pH and fluid balances, movement of nutrients and hormones to body tissues and disposal of metabolic waste.

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Blood examination
Collection of blood specimen
Blood may be collected from the patient using various techniques.  Capillary puncture, skin puncture  at the fingertips, earlobe (adults), hallux or heel (infants)  this methods is commonly used by people with diabetes who regularly check the blood glucose levels. Venipuncture  this is the most commonly used method of specimen collection  allows for obtainment of large volumes of blood  the antecubital veins are usually used Bone marrow aspiration  this technique uses a needle biopsy procedure  it is important in the evaluation of several haematologic disorders and infectious diseases





Common blood tests
Learning about various laboratory tests can be a tedious process, especially when the information is presented in a very didactic way. In this section and in those to follow where specific laboratory tests are mentioned, it is hoped that by getting you to source your own information about these tests will enhance your learning experience in this subject. Blood is a wonderful body tissue in terms of being able to test blood for many substances, chemicals, drugs, cells, etc. and provide the clinician with a wealth of information related to a patient‟s health status. The blood tests that we will look at are:  The Full Blood Examination (FBE) or as it is referred to by our American colleagues, the Complete Blood Count (CBC)  The Stained Cell Examination  The Erythrocyte Sedimentation Rate (ESR)

Full Blood Examination
As the name suggests the FBE is a conglomerate of tests which together serve to evaluate the cellular composition of blood.

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Go to the following website address: http://www.labtestsonline.org/understanding/analytes/cbc/glance.html The labtestsonline website is a fantastic resource and will serve as the basis for learning about several laboratory tests. Complete the following tasks using this website:   Click on the “The Test” icon. Answer the following using the information provided:

Q.1 What individual tests form the FBE (CBC)?

WBC count, WBC differential, RBC count, Haemoglobin (Hb), Haematocrit (Hct), Platelet count, Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), Red Cell Distribution Width (RCDW)

Q.2 When is the FBE ordered by a clinician?

Determine general health status, Screen for anaemia, infection, nutritional status, exposure to toxic substances Part of routine medical examination

You are now asked to click on the links to each of the following tests and answer the questions about each test as indicated:

Red Blood Cell Count
 What does this test evaluate?

Determines actual numbers of RBCs per unit volume of blood.
 What is anaemia? You may also want to click on „ANAEMIA‟ under the „CONDITIONS/DISEASES‟ drop down box.

Anaemia = less than normal levels of haemoglobin. Different types of anaemia but most common include: iron deficiency anaemia, Vitamin B12 deficiency anaemia, Folic acid deficiency anaemia, aplastic anaemias, haemolytic anaemias (eg. sickle cell anaemia, thalasaemia)
 What is polycythemia?

Increase in number of RBCs.
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

What may a raised RBC count indicate?

Obstructive lung disease Congenital heart disease Dehydration Bone marrow over-production
 What may a low RBC count indicate?

Anaemia Malnutrition Haemorrhage (bleeding) Kidney disease Haemoglobin
 What does this test evaluate?

Bone marrow failure (radiation, tumor) ? Iron, folate, B12, B6 deficiencies

Measures amount of Hb in blood.
 When might this test be ordered?

Measures severity of anaemia or polycythemia if present; monitors response to treatment, assists in the decision-making about blood transfusions. Pre-operatively when blood transfusion is anticipated.
 What might an above-normal result indicate?

Dehydration, excess RBC production in bone marrow, severe lung disease
 What might a below-normal result indicate?

Anaemia (iron deficiency), inherited Hb defects, liver cirrhosis, excess bleeding, vitamin/mineral deficiencies, kidney disease, cancer of bone marrow, chronic disease.
 How might, pregnancy, time of day, smoking, altitude and age effect haemoglobin test results?

Pregnancy – natural slight decrease in Hb levels is normal Time of day – Hb level peaks around 8am and lowest at 8pm Heavy smokers – higher Hb levels than non-smokers Altitude – increased Hb levels because of reduced O2 levels at altitude Older men & women, children – slightly lower Hb levels
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Haematocrit
 What does this test evaluate?

Also known as Packed Cell Volume (PCV), Measures proportion of blood that is made up of RBCs. Value is expressed as a % or fraction of cells in blood. Eg. Hct of 40% = 40mL of RBCs in 100mL of blood.
 When might this test be ordered?

As part of CBC; used in diagnosis and monitoring treatment of anaemia; evaluate recovery from dehydration, monitor ongoing bleeding & its severity. Also used in transfusion decision making and to assess transfusion effectiveness. Diagnosis of polycythemia.
 What might an above-normal result indicate?

Normally seen in those living at high altitudes. Most common cause is dehydration Polycythemia vera – increase in RBCs due to bone marrow problem or as compensation for inadequate lung function.
 What might a below-normal result indicate?

Normally seen during pregnancy because of increased fluid in blood. Indicates anaemia (but further testing required to determine type) Other causes include: vitamin/mineral deficiencies, recent bleeding, liver cirrhosis, malignancies.
 What is hemachromatosis?

“Iron overload” – most commonly cause by inherited disorder of iron metabolism but can be caused by other medical conditions. Iron is normally absorbed via small intestines & amount absorbed is dependent on body‟s requirements. In hamachromatosis, more iron than is needed is absorbed resulting in excessive build up of iron in the body tissues and organs. Net effect is organ dysfunction and failure. Early symptoms include fatigue, loss of well-being. Progressively, complications develop & may include arthritis, diabetes, liver cirrhosis, hear arrhythmias & failure, widespread skin pigmentation due to haemosiderin deposition.
Now return to the following web address: http://www.labtestsonline.org/understanding/analytes/cbc/test.html Scroll down the page and you should see the following tests mentioned:  Mean Corpuscular Volume (MCV)  Mean Corpuscular Haemoglobin (MCH) 12

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Mean Corpuscular Haemoglobin Concentration (MCHC)

These tests are know as the Red Cell Indices. The value of these tests rests in their ability to differentiate between different types of anaemia by providing information about the size of red blood cells and their oxygen-carrying capabilities. This information can assist the clinician in determining the type of anaemia present (eg. Iron deficiency anaemia, vitamin B-12 deficiency anaemia) and in doing so assist in the appropriate management of the patient (e.g Iron supplements to increase serum Iron levels or Vitamin B-12 injections to reduce deficiency). For each of these tests complete the information below.

Mean Corpuscular Volume (MCV)
 What does this test measure?

The average size of RBCs.
 What does an elevated result indicate?

That RBCs are of larger size than normal (ie. macrocytic). This is characteristic of Vitamin B12 deficiency anaemia (pernicious anaemia) where the body stops production of intrinsic factor needed to absorb Vitamin B12 from diet. This results in nerve problems such as neuropathy, tingling in hands and feet.
 What does a low result indicate?

RBCs are of smaller size than normal (ie. microcytic). Eg. as seen in iron deficiency anaemia.
 What is iron deficiency anaemia?

Most common type of anaemia and usually due to bleeding. In women, iron deficiency can occur secondary to heavy menstrual bleeding. In older men and women, intestinal bleeding/disease is a common cause. Reduced dietary iron may also be an issue as is often the case in pregnancy. Mean Corpuscular Haemoglobin (MCH)
 What does this test measure?

Calculates the amount of oxygen carrying haemoglobin inside RBCs.
 What does an elevated result indicate?

Macrocytic RBCs (see notes on MCV above)
 What does a low result indicate?

Microcytic RBCs (see notes on MCV above)
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Mean Corpuscular Haemoglobin Concentration (MCHC)
 What does this test measure?

Calculation of the percentage of Hb in RBCs
 What does an elevated result indicate?

As per MCH and MCV when elevated (see above)
 What does a low result indicate?

Hypochromasia – decreased oxygen-carrying capacity of RBCs because of reduced Hb inside the cell. Seen in iron deficiency anaemia and in thalassemia.
 What is thalassemia?

It is a haemolytic-type anaemia. A hereditary abnormality of Hb production, frequently observed in those of Mediterranean, African & Asian heritage. It causes microcytic RBCs that resemble those seen in iron-deficiency anaemia. There are 2 types/forms: the major type in which all thalassemia genes have been inherited and the minor type in which half of the thalassemia genes and half normal genes have been inherited. White Blood Cell Count (WBC count)
 What does this test measure?

Number of WBCs in blood.
 When might it be of value?

Monitor recovery from illness, to test for infection, allergy, leukaemia.
 What might a raised WBC count indicate?

Referred to as „leukocytosis‟ – may result due to bacterial infection, inflammation, leukaemia, trauma, stress.
 What might a lowered WBC count indicate?

Referred to as „leukopenia‟ – seen with chemotherapy, radiation therapy, immune system disease (eg. HIV).
 What is leukaemia?

Cancer of WBCs - cause is unknown although associated with exposure to radiation, benzene, anticancer drugs, genetic disorders, viral infections. Bone marrow disorder when 1 abnormal, immature WBC begins to replicate itself (clone) continuously. These cells are unable to perform normal functions of WBCs, they
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don‟t die at the same rate as other WBCs so they begin to accumulate, inhibiting the production of other necessary blood cells in bone marrow. This results in anaemia, bleeding, recurrent infections. These „leukaemia cells‟ can enter the blood stream and divide to form tumours and damage organs, eg. brain & liver.

White Blood Cell Differential (Differential white cell count)
 What is the purpose of this test?

To diagnose infection and other immune system disorders.
 What is the function of: (a) (b) a neutrophil?

granular leukocyte, phagocyte that destroys bacteria & cellular debris
an eosinophil?

1-3% of WBC count – functions in allergic responses and in preventing some infections.
a basophil?

(c) (d) (e)

1% or less of total WBC count
a monocyte?

granular leukocyte, phagocytic of bacteria and other foreign particles.
a lymphocyte?

two types – T-cells (cell-mediated immune system) B-cells (antigen-antibody immune response) account for 25% of WBC count and levels are raised in presence of infection.



What may increase or decrease neutrophil numbers?

Inc. = acute bacterial infections, certain malignant neoplastic diseases, inflammation Dec. = severe infection, medication response.
 What may increase or decrease eosinophil numbers?

Inc. = allergic disorders, inflammation of skin, parasitic infections, bone marrow malignancies Dec. = infection
 What may increase or decrease basophil numbers?

Inc. = leukaemia, chronic inflammation, allergy to food, radiation therapy Dec. = acute infection.
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What may increase or decrease monocyte numbers?

Inc. = infection of all kinds, inflammatory disorders Dec. = bone marrow injury or failure, some forms of leukaemia
 What may increase or decrease lymphocyte numbers?

Inc. = bacterial or viral infection, leukaemia, cancer of bone marrow, radiation therapy Dec. = diseases of immune system (eg. lupus, later stages of HIV)

The Stained Cell Examination
As the name suggests, this laboratory tests involves the visual inspection of a smear of blood on a microscope slide which is then examined under a microscope. Similar to the red cell indices, this test can assist in the determination of different types of anaemia as blood cell size can be examined.

The stained cell examination can provide information regarding size, colour, shape and structure of red blood cells.

The diagram above shows the normal structure of healthy RBCs. You can see that they are of uniform colour, shape and size and there is a central nucleate region within each cell. These features may change in various disease states, such as anaemia. For example, the photograph below, shows the RBCs of a person with sickle cell anaemia.

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Observe how the shape, structure, size and colour of these cells differs to those of normal RBCs.

Erythrocyte Sedimentation Rate (ESR)
In various disease states, for example inflammation, RBCs will aggregate due to the presence of proteins such as fibrinogen. The ESR measures the rate at which RBCs settle out of plasma in a column or tube. As RBCs clump together they become heavier and fall out of plasma at a faster rate. The ESR test is often used as an indicator of inflammation as elevated levels of proteins like fibrinogen cause an increased sedimentation of RBCs.  Why is the ESR considered non-specific?

To recap, Specificity = % of negative results obtained by a test among people who do not have the disease. Therefore, the ESR is non-specific because it must obtain a lower percentage of negative results in those without the disease, ie. a high rate of false positive results. Additionally, while the ESR is an indirect marker of inflammation, it cannot pinpoint the exact cause of inflammation and must therefore be used in conjunction with other tests and the clinical history.
 When might the ESR be requested?

To detect and monitor the activity of inflammation and as an aid in diagnosis of the underlying cause. helpful in diagnosing 2 inflammatory disorders (temporal arteritis – inflammation of large arteries of the head and face; polymyalgia rheumatica - A disease that causes pain and weakness in the shoulder muscles and pelvis, and morning stiffness. It mainly affects women over 50 years of age.)
 What may cause an elevated ESR?

Inflammation, anaemia, infection, pregnancy, old age.
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

What may cause a lowered ESR?

Polycythemia, extreme leukocytosis, protein abnormalities.
 What factors may affect ESR results?

Females tend to have higher ESR; menstruation & pregnancy can cause temporary elevations. Drugs such as dextran, methyldopa (Aldomet), oral contraceptives, penicillamine procainamide, theophylline, and vitamin A can increase ESR, while aspirin, cortisone, and quinine may decrease it.

Review of haemostasis
To review haemostasis go to the following website:

http://www.uq.edu.au/vdu/HDUHaemostasis.htm

Common haemostatic tests
Bleeding disorders occur when the body is unable to initiate haemostasis in order to arrest bleeding. There are four broad categories of bleeding disorders: 1) Vascular disorders These are mild disorders which cause spontaneous small vessel haemorrhage. They may be acquired or hereditary. An example of these are Purpura and Scurvy 2) Thrombocytopaenia This a state in which there is a reduced circulating level of platelets in the blood. This may be due to a reduction in platelet synthesis due to bone marrow illness or failure or viral infection. Alternatively, the rate at which platelets are destroyed may be increased. This may be caused by autoimmune disease, following blood transfusion or as a result of chemotherapy. This results in increased bleeding time following vessel injury. 3) Platelet Function Disorders In these disorders, the patient will have a normal platelet count but a prolonged bleeding time will be observed. These disorders may be of the hereditary type, e.g Von Willibrands disease or acquired, e.g as a result of aspirin therapy. 18

4) Coagulation Disorders Coagulation disorders may occur as a result of an hereditary deficiency of a coagulation factor. These hereditary deficiencies are of numerous types with some common examples including Haemophilia A, Christmas disease (Haemophilia B). Other factors which may lead to the development of a coagulation disorder include Vitamin K deficiency and liver disease (which may lead to reduced Vitamin K, fibrinogen and factor V levels). Haemostatic tests do one of two things:   Measure platelet activity or Measure the effectiveness of coagulation

Platelet Activity Tests

Bleeding Time
This test measure the duration of bleeding after a standardised skin incision has been made in the patient and can detect abnormal platelet function. The duration of bleeding will vary depending on the elasticity of the vessel wall and on the number and functional capacity of the platelets themselves. A patient with normal platelet levels and function will bleed for between 3 to 8 minutes.

Platelet Count
Go to the following website address and answer the following questions concerning the Platelet Count test: http://www.labtestsonline.org/understanding/analytes/platelet/test.html What is this test measuring?



Number of platelets in blood.
 When is it ordered?

To assist in diagnosis of bleeding disorders or bone marrow disease, in cases of unexplained bruising or prolonged bleeding time from a small cut, wound.
 What is a normal adult healthy platelet count?

150,00 – 450,000 platelets/microlitre of blood

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

What might the results of this test indicate?

thrombocytopenia – reduced number of platelets – seen in bone marrow disease (eg. leukaemia), autoimmune disorder (eg. lupus), chemotherapy. thrombocytosis – increased number of platelets – no significant medical condition, myeloproliferative disorder.
Coagulation Tests

Prothrombin Time (PT time)

Many patients visiting the podiatry clinic will be taking some form of anticoagulant therapy, e.g Warfarin. This has implications for podiatric management as any procedure which may be potentially invasive needs to be approached with care as the patient will exhibit a prolonged bleeding time and a poor ability of their blood to coagulate. The Prothrombin Time test is an important test used in determining someone‟s response to anticoagulant therapy. Again, go to the following website address and answer the questions that follow: http://www.labtestsonline.org/understanding/analytes/pt/glance.html  What is this test measuring?

Time taken for blood to begin forming clots; measures the effectives of clotting factor in converting prothrombin to thrombin.
 How is this test used?

To assess effectiveness of anticoagulant therapy by determining how much it prolongs the prothrombin-to-thrombin conversion time.
 When is it ordered?

Check/monitor anticoagulant therapy. May be used preoperatively to ensure clotting capabilities and to check for bleeding disorder.
 What is the International Normalised Ratio or INR?

Developed by the World Health Organisation (WHO) & International Committee on Thrombosis, Haemostasis. It is an adjusted PT result which aims to standardise PT results internationally taking into consideration differences between instruments and reagents used. This allows people on anticoagulant therapy to travel and still receive comparable test results.

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What are the implications of a prolonged or increased PT time?

Blood is taking too long to form a clot – adjustments to anticoagulant therapy needed potentially. May indicate liver disease if you have increased PT result and not on anticoagulant therapy.

Other blood tests
As you are probably beginning to understand, the most wonderful thing about blood (apart from its physiological importance) is its ability to allow clinicians a way of testing and measuring an enormous range of components including, cells, chemicals, and physiological function. There are many substances which can be measured via a blood sample and it is worth mentioning some of the other more common blood tests that you should be aware of. These include:       Blood glucose tests (especially for people with diabetes) Blood gases & electrolytes Enzyme analysis Hormones Proteins, such as albumin. Lipids (inc. triglycerides an cholesterol)

Blood glucose tests Go to the following website address. Here you will find specific information on tests related to diabetes. At the bottom of the webpage you will see a number of links directing you to tests related to diabetes. You should click on the following links: Glucose and A1C. Make some notes on these tests for your own reference and revision purposes.

Glucose testing “Snapshot test” – only tells us what BGLs were at time of sample collection tests for hyper/hypoglycaemia Different types of glucose tests o fasting blood glucose test o oral glucose tolerance test (OGTT)  Used in diabetes diagnosis in conjunction with other tests
  

A1c or HbA1C
 

Checks the amount of Hb that carries glucose molecules. As glucose circulates in blood, some spontaneously combines with Hb, forming HbA1c. The amount of HbA1c formed is directly related to the amount of glucose in blood.

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If someone has diabetes that is not well controlled, their BGLs are high so there is more circulating glucose that can combine with Hb, therefore more HbA1c will be formed, causing increased HbA1c levels.  As blood cells last for 2-3 months, the HbA1c can give an indication of BGLs over the past 3 months.
 Blood gases Again, go http://www.labtestsonline.org/understanding/analytes/blood_gases/glance.html  When might these tests be conducted? to

O2/CO2 imbalance, acid-base imbalance which may indicate a respiratory, metabolic, kidney disorder.
 What can be tested?

pH, PO2, PCO2, O2 saturation, HCO3- (bicarbonate)
 Where is the test sample taken from?

Radial, femoral, brachial arteries (remember, difference between arterial & venous blood) In newborn – umbilical artery & vein
 What is respiratory acidosis and what might cause it?

lower pH, increased PCO2 (not enough O2 in and CO2 out) – pneumonia, COPD
 What is respiratory alkalosis and what might cause it?

raised pH, decreased PCO2 (too much O2 in and CO2 out) - hyperventilation
 What is metabolic acidosis and what might cause it?

lower pH, decreased HCO3- - net affect = blood too acidic – diabetes, shock, renal failure
 What is metabolic alkalosis and what might cause it?

raised pH, increased HCO3- - hypokalaemia, chronic vomiting, sodium bicarbonate overdose.
Proteins One of the more common proteins which is of relevance to podiatry is that of albumin. 22

Go to the following website address: http://www.labtestsonline.org/understanding/analytes/albumin/glance.html  What is albumin?

It is the most abundant protein in blood plasma, prevents leakage of fluid from blood vessels, nourishes tissues, transports hormones, vitamins and drugs.
 Why might you test someone for their albumin levels?

Chronic, non-healing wounds, screen for liver disease, kidney disease, evaluate nutritional status.
 What may cause a low albumin level?

liver disease, kidney disease (where albumin leaks out of blood into urine), inflammation, shock, malnutrition.
 What may cause a raised albumin level?

Dehydration.
Lipids Patients may have had a „Lipid Profile‟ conducted to assess various lipid components of their blood. The lipid profile includes measures of total cholesterol, triglycerides, High density lipoproteins (HDLs) and the Low density lipoproteins (LDLs). Go to http://www.labtestsonline.org/understanding/analytes/lipid/glance.html Again, make some notes for your own reference and revision under the following headings. (Click on the links to each of the individual tests)

Total cholesterol
 

Screens for risk of developing heart disease 3 categories of results: o Desirable: < 5.18mmol/L = low risk o Borderline high: 5.18-6.22mmol/L = moderate risk o High risk: > 6.22mmol/L = high risk

Triglycerides
 

Screen for risk of developing heart disease Normal Fasting triglyceride = <1.70 mmol/L
23

HDLs


If high cholesterol is due to high HDLs, person is probably at low risk of heart disease and no further testing usually required.

LDLs If high cholesterol is due to high LDLs, person is probably at risk of developing heart disease and further testing, treatment important.  Must be done following a 12 hour fast.


24

Urinalysis
Review of urine formation & composition
It may be worth reviewing your anatomy and physiology related to the genitourinary system. But, here are some of the main points related to urine formation and composition.

Urine formation
Blood enters the kidney via the renal artery and eventually ends up in the glomerulus via afferent glomerular arterioles. Each of these arterioles subdivide into a network of capilliaries enclosed by Bowman‟s capsule. Blood leaves the glomerulus via efferent glomerular arterioles and travels to a network of peritubular capiliaries which encircle the renal tubules. Glomerular filtrate leaves Bowman‟s capsule and enters the proximal convoluted tubule where 65% is selectively reabsorbed by the capilliaries, including water, glucose, some proteins, amino acids, vitamins and hormones. The remaining 35% proceeds to the loop of Henle and distal convoluted tubules where sodium and water are reabsorbed as needed. During secretion, fluid and solutes such as potassium, uric acid, exogenous waste (eg. drugs) move from the peritubular capilliaries back to the glomerular filtrate. As this filtrate evolves it is constantly modified by filtration, reabsorption and secretion. The end product of course, is urine.

Urine composition
The composition of urine changes with things such as diet, physical activity and emotional stress. However, urine will always contain water, urea, uric acid and sodium chloride. Urine usually includes the following components: non-protein nitrogen compounds, citric acid, other organic acids, phosphate, potassium, calcium, magnesium, vitamins, hormones, mucoproteins, catecholamines, sulphur-containing compounds.

Urine examination
Collection of urine specimens
There are various methods available for obtaining urine specimens for analysis. The methods chosen will depend on the patient type and what is being tested. With all urine specimens it is important to remember that urine will change following collection and so samples need to be sent to the pathology laboratory as soon as possible. 25

Random – specimen is taken at any time of the day Early morning – specimen taken early in the morning before the ingestion of any fluid Clean-catch midstream – the patient cleans the external urethral meatus. The first half
of the bladder urine is discarded then the collection vessel is introduced midstream to collect the last half of the bladder urine patient) where a catheter is inserted to collect urine sample.

Catheterisation of the bladder – used is special circumstances (eg. coma, confused

Macroscopic analysis
Macroscopic or physical examination of urine can provide easily obtainable clues to the presence of pathology. Go to the following website address and discover information related to physical examination of urine and make some notes for your reference and revision: http://www.labtestsonline.org/understanding/analytes/urinalysis/ui_exams.html COLOUR  most often shades of yellow – very pale/colourless (lots of H2O excreted) to dark

yellow/amber (little H2O excreted – eg. fever, 1st morning urine)  red colour – may indicate blood in urine – serious!! or ingestion of beetroot (not so serious)

CLARITY  Clear, slightly cloudy, cloudy, turbid  Normal = clear to cloudy  Depends on what is causing cloudiness o Healthy cloudiness = sperm & prostatic fluid, skin cells, normal urine

crystals, contaminants (eg. body lotions)

CONCENTRATION  Determined by measuring specific gravity  Measures amount of dissolved substance inn urine

Microscopic analysis
Again, visit the following website address and formulate notes for your own revision and reference under the headings provided below: http://www.labtestsonline.org/understanding/analytes/urinalysis/ui_exams-3.html RBCs  

Normally a few present High levels – inflammation, injury, kidney disease
26

WBCs  Normally low  Raised levels can indicate infection, inflammation somewhere in urinary tract EPITHELIAL CELLS  Can be found from the bladder, external urethra  Cells from kidney less common  Cells can indicate where condition is located depending on type of cell. MICROORGANISMS  Urinary tract normally sterile and free of microorganisms  UTIs caused by bacteria from surrounding skin CASTS  Found in kidney‟s tubules  Kidney secretes protein (hyaline) that can gel in the tubules taking the shape of the

tube (referred to as “hyaline cast”). If there is disease, the cast may capture other cells eg. RBCs,(red blood cell cast), WBCs (white blood cell cast).

CRYSTALS  Identified by shape, colour and urine pH  Soulte in urine can form crystals if conditions of urine pH, solute concentration and

urine temperature are all right.

Microchemical analysis
This form of analysis occurs using dipstick analysis. We will explore this form of analysis in the clinic tutorials. Go to the following website and make notes for your own reference under the headings provided: http://www.labtestsonline.org/understanding/analytes/urinalysis/ui_exams-2.html SPECIFIC GRAVITY  Measures concentration; ie a comparison of amount of solute dissolved in urine

water versus pure water.

pH



kidneys play major role in maintaining acid-base balance of body. changes in pH may be reflected in urine pH. Diet can also affect.

Therefore,

PROTEIN  Normally, amount of protein (albumin) in urine not detectable.

(increased protein in urine) may indicate early sign of kidney disease.

Proteinuria
27

GLUCOSE  Normally not present in urine  Glucosuria – diabetes, kidney problems where ability to absorb glucose is reduced

or absent

KETONES  These are the intermediate products of fat metabolism  Fat metabolism occurs when a person does not eat enough carbohydrates, body

metabolises fat to meet energy requirements.  Presence in urine may be early indication of insufficient insulin in person with diabetes.  Ketonuria may also occur with strenuous and severe exercise, exposure to cold, loss of carbohydrate.

BLOOD  Detects haemoglobin from RBCs  Small number of RBCs is normal  Small increase in RBCs in urine is significant – may indicate numerous diseases of

kidney and urinary tract, trauma, medications, stress, severe excercise.

NITRITE  Can identify many UTIs  Some (but not all) bacteria can covert nitrates to nitrites, therefore presence points

to UTI.

Diagnostic microbiology
There are some notes which have been prepared as background reading to this section on diagnostic microbiology. They can be found at the back of this manual.

Microscopy
Magnification of organisms in specimen in order to identify a specific organism. There are various types of microscopes available commercially. These include light microscopy, electron microscopy, fluorescence microscopy, dark-field microscopy. Each has different applications. We will concentrate on light miccroscopy. In order to view a specimen under a light microscope, the specimen must be stained in order to highlight or view the components we wish to examine. There are many, many staining techniques which can be employed. The Gram Stain is the principle stain used for the microscopic examination of bacteria. Nearly all clinically significant bacteria can be detected using this staining technique. 28

Gram Stain
This stain divides most bacteria into two groups:  Those that take up the dye (crystal violet) known as Gram Positive bacteria  Those that allow the crystal violet dye to wash out easily with the decoloriser alcholol or acetone known as Gram Negative bacteria This difference occurs on the basis of the difference in cell wall structure between gram positive and negative bacteria. Once the identity of a bacteria has been obtained, appropriate antibiotic therapy can be selected. This is important as come antibiotics will only target a particular type of bacteria.

Culture
Culture is the process of growing microorganisms by the taking the infecting organism from the infection site and growing it in an artificial environment. Nutrients are incorporated into a culture media. If this medium meets an organism‟s nutritional requirements then growth will be supported, the organism will multiply to sufficient numbers to allow visualisation. There are two types or phases of growth media: liquid (or broth) and solid (agar).

Broth media – Nutrients are dissolved in water and bacterial growth is indicated by
changes in the broth‟s appearance agar dish.

Agar media – A solidifying agent is added to the nutrients and the water and placed in an

Wound culture
Wound culture both microscopic and culture analyses of a specimen taken from a lesion in order to confirm infection. Infection may be aerobic (caused by oxygen-requiring bacteria) or anaerobic (caused by nonoxygen-requiring bacteria). Wound culture is indicated where the patient is experiencing fever, or where there is inflammation and drainage of damaged tissue. It involves taking a wound swab and then sending off for microscopic analysis and culture. Sterile cotton swabs and a sterile culture tube are used to collect the specimen. anaerobes the tube is filled with carbon dioxide or nitrogen. For 29

Some common aerobic pathogens are:  Staph. aureus, Group A beta-hemolytic streptococci, Proteus, Escherichia coli, some Pseudomonas species. Some common anaerobic pathogens are:  Some Clostridium and Bacteroides species

Clinical mycology
There are two groups of fungi: the yeasts and the molds. Yeasts are unicellular and produce asexually („budding‟) while molds consist of hyphae Podiatrists are primarily concerned with the dermatophytes ( group of molds) as these commonly involve the skin, hair, or nails of the feet. Hair, skin & nail specimens These are obtained by scraping all suspected infected material into a specimen container or envelope. There is no need for these to be refrigerated. The pathology lab will conduct by microscopic examination and culture tests on these specimens. Direct microscopic examination The most common microscopic test is the potassium hydroxide (KOH) test. A drop of 5% KOH solution is added to the sample on a microscope slide and gently heated to drive off the KOH solution. This dissolves unwanted keratinised tissues, exposing the fungal elements, namely hyphae. The tests takes about 5-10 minutes and allows for the rapid detection of fungal elements. However, some experience is required as background artefacts may be visible. Culture Mycosel agar is commonly used for the recovery of dermatophytes but there are various others. The sample is incubated for a minimum of 30 days at 30 oC before being reported as negative. It is not uncommon for negative results to be returned from the laboratory even though it is plainly obvious that a fungal infection is present. Read the following article found at the end of this manual for more information: Ellis, D H. How to take a fungal scraping.

30

Synovial fluid analysis (SFA)
Involves obtaining a sample of synovial fluid from a joint for analysis.

Indications & Contraindications
SFA can be used diagnostically as well as therapeutically (arthrocentesis). SFA is indicated in cases of joint effusions of unknown aetiology, arthritis of unknown aetiology, in all cases of suspected septic arthritis, to confirm a diagnosis strongly suspected on clinical grounds (e.g gout) and to monitor synovial fluid response to antibiotic therapy in cases of septic arthritis. Therapeutically, arthrocentesis, may be indicated in the following situations:  decompression of tense and painful joint effusions  evacuation of pus in septic arthritis  removal of inflammatory cells & crystals is established cases of gout/pseudogout  intra-articular injection of corticosteroid SFA/arthrocentesis are contraindicated in the following circumstances:  local infection along the proposed needle entry point (e.g cellulitis)  unco-operative patient as the joint needs to remain immobile during and following the procedure  difficulty in identifying osseous landmarks  poorly accessible joint space  inability to demonstrate joint effusion on physical examination (except where septic arthritis is suspected)

Synovial fluid examination
Collection of synovial fluid specimen The procedure for obtaining a synovial fluid sample is considered a minor surgical procedure. Therefore, strict aseptic conditions and procedures must be adhered to. The diagram below illustrates appropriate technique. The following technique is followed:  The patient is positioned appropriately for the joint to be examined Area over the puncture site is disinfected with an alcohol swab and povidone-iodine solution A local anaesthetic is administered to minimise pain 31





  

The needle is inserted into the joint space As much fluid as possible is aspirated (min. of 10-15mL) Withdraw the needle and apply pressure to puncture site then dress with a sterile dressing.

Macroscopic analysis, Microscopic analysis, Microbiologic analysis & Chemical analysis For this section please read the following article which can be found at the back of this handbook, Brook, J W & Cohen, R B. The analysis of synovial fluid: an interpretation guide for the non-rheumatologist. British Journal of Podiatric Medicine & Surgery. 9 (1), 1997.

Biopsy
The extraction of a living tissue specimen for histologic examination. There are two types of biopsy technique, incisional and excisional biopsy. Incisional biopsy This involves removal of a portion of tissue for examination and may involve the use of a scalpel, punch or via needle aspiration. An example of incisional biopsy would be the aspiration of the contents of a cystic lesion. Excisional biopsy This involves the removal of all abnormal tissue and is often the preferred method of biopsy as it combines both diagnosis and treatment. For example, the surgical excision of a malignant melanoma. Specimen preparation Body tissue decomposes following its removal from the body. Therefore, histological specimens require „fixation‟ so as to arrest cellular structures and to prevent decomposition. The specimen is usually held in a „fixing fluid‟, commonly, 10% neutral buffered formaldehyde. Common tissue biopsies The more common types of tissue biopsy are outlined below and include:  excision 32

  

shaving needle aspiration punch incision (biopsy)

Excision – involves the removal of an entire lesion from ANY tissue
A local anaesthetic is usually administered. This procedure combines both diagnosis and treatment. However, often major surgery is required as part of this procedure.

Shaving – tissue is shaved from a raised surface lesion on the skin
This is a relatively safe procedure, combines both diagnosis and treatment and maintains good cosmesis.

Needle aspiration – removal of core tissue or tissue sample from bone, bone marrow,
breast tissue, lung, lymph nodes, kidney, prostate. Needle aspiration allows the patient to avoid traumatic surgery while preserving cell architecture for examination.

Punch biopsy – removal of a specimen from the core of a lesion in the skin or cervix.
Podiatrists may consider this technique for verrucae which do not respond to conservative management and where the diagnosis is uncertain. This procedure involves the use of a „punch‟ which obtains a plug of tissue for examination.  Here, LA is injected beneath the lesion

o An appropriately sized punch is selected for the lesion o The punch is twisted back and forth in order to incise the border of the specimen sample o Simultaneously, the punch is advanced downwards



Using sterile forceps and tissue nippers the plug is elevated and snipped Care is taken not to damage the 33



specimen

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