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Blood smear examination

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Blood smear examination Powered By Docstoc
					        Introduction
    In Medical Technology



         Lecture 11


        Making Blood smear

1
Preparation of blood smear
   There are three types of blood smears:
    1. The cover glass smear.
    2. The wedge smear .

    3. The spun smear.

   The are two additional types of blood smear used for
    specific purposes
    1. Buffy coat smear for WBCs < 1.0×109/L

    2. Thick blood smears for blood parasites .




                                                           2
Wedge blood smear
   Specimen : EDTA blood within 2 to 3 hours & collected
    to the mark on tube.

Not's : May change RBCs morphology such as Spiculated
      (crenated) cells if :
  1. Excessive amount of anticoagulant to specimen

  2. Old blood - long standing.

  3. Warm environment (room temperature) may hasten
     changes.




                                                            3
Procedure
1. Placing a drop of blood from mixed sample on a clean
   glass slide.
2. Spreader slide using another clean glass slide at 30-40
   degree angle.
3. Control thickness of the smear by changing the angle of
   spreader slide
4. Allow the blood film to air-dry completely before
   staining. (Do not blow to dry. The moisture from your
   breath will cause RBC artifact.)




                                                         4
STEPS FOR BLOOD FILM




                       5
6
The thickness of the spread
1. If the hematocrit is increased, the angle of the
  spreader slide should be decreased.
2. If the hematocrit is decreased, the angle of the
  spreader slide should be increased.




                                                      7
Characteristics of a Good Smear
1.   Thick at one end, thinning out to a smooth rounded
     feather edge.
2.   Should occupy 2/3 of the total slide area.
3.   Should not touch any edge of the slide.
4.   Should be margin free, except for point of application.

Note: As soon as the drop of blood is placed on the glass slide, the smear
       should be made without delay. Any delay results in an abnormal
       distribution of the white blood cells, with many of the large white
       cells accumulating at the thin edge of the smear.



                                                                        8
Common causes of a poor blood smear
   Drop of blood too large or too small.
   Spreader slide pushed across the slide in a jerky manner.
   Failure to keep the entire edge of the spreader slide
    against the slide while making the smear.
   Failure to keep the spreader slide at a 30° angle with the
    slide.
   Failure to push the spreader slide completely across the
    slide.
   Irregular spread with ridges and long tail: Edge of
    spreader dirty or chipped; dusty slide
   Holes in film: Slide contaminated with fat or grease
   Cellular degenerative changes: delay in fixing, inadequate
    fixing time or methanol contaminated with water.
                                                            9
Examples of unacceptable smears
A: Blood film with jagged tail made from a spreader with achipped
   end.
B: Film which is too thick
C: Film which is too long, too wide, uneven thickness and made on
   a greasy slide.
D: A well-made blood film.




                                                                    10
Examples of unacceptable smears




                                  11
Biologic causes of a poor smear
1.   Cold agglutinin - RBCs will clump together. Warm the
     blood at 37° C for 5 minutes, and then remake the smear.
2.   Lipemia - holes will appear in the smear. There is nothing
     you can do to correct this.
3.   Rouleaux - RBC’s will form into stacks resembling coins.
     There is nothing you can do to correct this

Notes:
1. The WBCs are unevenly distributed and RBC distortion is seen at the
   edges Smaller WBCs such as lymphocytes tend to reside in the middle
   of the feathered edge.
2. Large cells such as monocytes, immature cells and abnormal cells can be
   found in the outer limits of this area.
3. Spun smears produce the most uniform distribution of blood cells.


                                                                       12
SLIDE FIXATION & STAINING

    LEISHMAN'S STAIN




                            13
Fixing the films
   To preserve the morphology of the cells, films must be fixed
    as soon as possible after they have dried.
   It is important to prevent contact with water before fixation is
    complete.
   Methyl alcohol (methanol) is the choice, although ethyl
    alcohol ("absolute alcohol") can be used.
   Methylated spirit (95% ethanol) must not be used as it
    contains water.
   To fix the films, place them in a covered staining jar or tray
    containing the alcohol for 2-3 minutes. In humid climates it
    might be necessary to replace the methanol 2-3 times per day;
    the old portions can be used for storing clean slides.

                                                                  14
Staining the film
Romanowsky staining: Romanowsky stains are universally
employed for staining blood films and are generally very
satisfactory.
 There are a number of different combinations of these dyes,
  which vary, in their staining characteristics.
  1. May-Grunwald-Giemsa is a good method for routine work.
     Giemsa stain is thought to produce more delicate staining
     characteristics.
  2. Wright's stain is a simpler method.
  3. Leishman's is also a simple method, which is especially
     suitable when a stained blood film is required urgently or the
     routine stain is not available (e.g. at night).
  4. Field's stain is a rapid stain used primarily on thin films for
     malarial parasites.

                                                                15
Principle
   The main components of a Romanowsky stain are:
     A cationic or basic dye (methylene blue or its oxidation
      products such as azure B), which binds to anionic sites and
      gives a blue-grey color to nucleic acids (DNA or RNA),
      nucleoproteins, granules of basophils and weakly to granules
      of neutrophils
     An anionic or acidic dye such as eosin Y or eosin B, which
      binds to cationic sites on proteins and gives an orange-red
      color to hemoglobin and eosinophil granules.

       pH value of phosphate buffer is very important.




                                                               16
17
Staining procedure (Leishman’s stain)
   Thin smear are air dried.
   Flood the smear with stain.
   Stain for 1-5 min. Experience will indicate the
    optimum time.
   Add an equal amount of buffer solution and mix the
    stain by blowing an eddy in the fluid.
   Leave the mixture on the slide for 10-15 min.
   Wash off by running water directly to the centre of
    the slide to prevent a residue of precipitated stain.
   Stand slide on end, and let dry in air.


                                                        18
too acidic   suitable   too basic



                                    19
Causes & correction
   Too Acid Stain:                Too Alkaline Stain:
    1. insufficient staining        1. thick blood smear
       time                         2. prolonged staining
    2. prolonged buffering or
                                    3. insufficient washing
       washing
                                    4. alkaline pH of stain
    3. old stain
                                       components
   Correction:
                                    Correction :
    1) lengthen staining time
                                    1) check pH
    2) check stain and buffer
                                    2) shorten stain time
        pH
                                    3) prolong buffering time
    3) shorten buffering or
        wash time


                                                           20
Performing A Manual differential
And assessing RBC Morphology




                                   21
Porposes
   White Blood Cells.                        Red Blood Cells,
    1.   Check for even distribution and       1. Size and shape.
         estimate the number present           2. Relative hemoglobin
         (also, look for any gross                content.
         abnormalities present on the
         smear).                               3. Polychromatophilia.

    2.   Perform the differential count.       4. Inclusions.
                                               5. Rouleaux formation or
   Platelets.                                    agglutination
    1. Estimate number present.
    2. Examine for morphologic
       abnormalities.




                                                                          22
Procedures
   Observations Under ×10
    1. Check to see if there are good counting areas
       available free of ragged edges and cell
       clumps.
    2. Check the WBC distribution over the smear.
    3. Check that the slide is properly stained.
    4. Check for the presence of large platelets,
       platelet clumps, and fibrin strands.




                                                       23
   Observations Under ×40 : WBC estimation
    1. Using the × 40 high dry with no oil.
    2. Choose a portion of the peripheral smear where
       there is only slight overlapping of the RBCs.
    3. Count 10 fields, take the total number of white
       cells and divide by 10.
    4. To do a WBC estimate by taking the average
       number of white cells and multiplying by 2000.




                                                   24
   Observations Under × 100: Platelet Estimates
    1. Use the oil immersion lens estimate the
       number of platelets per field.
    2. Look at 5-6 fields and take an average.

    3. Multiply the average by 20,000.

    4. Note any macroplatelets.



   Platelets per oil immersion field (OIF)
    1)   <8 platelets/OIF = decreased
    2)   8 to 20 platelets/OIF = adequate
    3)   >20 platelets/OIF = increased


                                                   25
PLATELETS
            26
Observing and Recording Nucleated Red
Blood Cells (nRBCs)
   If 10 or more nucleated RBC's (NRBC) are seen,
    correct the White Count using this formula:




Example : If WBC = 5000 and 10 NRBCs have been
           counted
            Then 5,000× 100/110 = 4545.50
            The corrected white count is 4545.50.


                                                     27
Manual Differential Counts
   These counts are done in the same area as WBC and
    platelet estimates with the red cells barely touching.
   This takes place under × 100 (oil) using the zigzag
    method.
   Count 100 WBCs including all cell lines from
    immature to mature.

   Reporting results
    Absolute number of cells/µl = % of cell type in
    differential x white cell count




                                                             28
Observing direction:




Observe one field and record the number of WBC according
to the different type then turn to another field in the snake-
liked direction
* avoid repeat or miss some cells

                                                            29
Normal peripheral blood smear




                                30
Leukocytosis
Leukocytosis, a WBC above 10,000 is usually due to an increase
in one of the five types of white blood cells and is given the name
of the cell that shows the primary increase.

Neutrophilia :         Neutrophilic Leucocytosis
Lymphocytosis :        Lymphocytic leukocytosis
Eosinophilia :         Eosinophilic leukocytosis
Monocytosis :          Monocytic leukocytosis
Basophilia:            Basophilic leukocytosis




                                                                  31
32
Stab (Band) neutrophil

                 Diameter:12-16
                 Cytoplasm : pink
                 Granules: primary
                  secondary
                 Nucleus: dark purple
                  blue
                 dense chromatin




                                         33
Segmented neutrophil

               Diameter: 12-16
               Cytoplasm : pink
               Granules: primary,
                secondary
               Nucleus: dark purple
                blue dense chromatin,
                2-5 lobes




                                        34
Segmented neutrophil
   Neutrophils are so named because they are not well
    stained by either eosin, a red acidic stain, or by
    methylene blue, a basic or alkaline stain.
   Neutrophils are also known as "segs", "PMNs" or
    "polys" (polymorphonuclear).
   They are the body's primary defense against bacterial
    infection.




                                                            35
Increased neutrophils count (neutrophilia)
   1.   Acute bacterial infection.
   2.   Granulocytic leukemia.

Decreased neutrophil count (neutropenia)
   1.   Typhoid fever
   2.   Brucellosis
   3.   Viral diseases, including hepatitis, influenza,
        rubella, and mumps.




                                                    36
LEFT-SHIFT AND RIGHT-SHIFT OF NEUTROPHIL
   Normally, most of the neutrophils circulating in the
    bloodstream are in a mature form, with the nucleus of the cell
    being divided or segmented. Because of the segmented
    appearance of the nucleus, neutrophils are sometimes referred
    to as "segs.”
   The nucleus of less mature neutrophils is not segmented, but
    has a band or rod-like shape. Less mature neutrophils - those
    that have recently been released from the bone marrow into
    the bloodstream - are known as "bands" or "stabs".
   Left-shift: non-segmented neutrophil > 5%
   Right-shift: hypersegmented neutrophil >3%

                                                                37
Segmented neutrophile                       Band neutrophil


 Shift to left  Increased bands mean acute infection, usually bacterial.
 Shift to right  Increased hypersegmented neutrophile.




                                                                            38
Eosinophil

                Diameter: 14-16
                Cytoplasm : full of granules
                Granules: large refractile, orange-
                 red
                Nucleus: blue dense chromatin 2
                 lobes like a pair of glass




                                                   39
   The most common reasons for an increase in the
    eosinophil count are
    1.   Allergic reactions such as hay fever, asthma, or drug
         hypersensitivity.
    2.   Parasitic infection
    3.   Eosinophilic leukemia




                                                            40
Basophil


              Diameter: 14-16
              Cytoplasm : pink
              Granules: dark blue –black
               obscure nucleus
              Nucleus: blue




                                            41
Basophils
    The purpose of basophils is not completely
     understood.

    Basophile counts are used to analyze allergic
     reactions.

    An alteration in bone marrow function such as
     leukemia may cause an increase in basophils.




                                                     42
Lymphocyte
                Diameter:
                  small 7-9

                  large 12-16

                Cytoplasm: medium blue
                Granules:
                  small agranular

                  large a few primary
                   granules
                Nucleus: dark blue \round
                 dense chromatin




                                             43
Lymphocytes
   Lymphocytes are the primary components of the body's
    immune system. They are the source of serum
    immunoglobulins and of cellular immune response.
   Two types of lymphocytes:
     1. B lymphocyte : Humoral immunity
     2. T lymphocyte : Cellular immunity




                                                       44
   Lymphocytes increase (lymphocytosis) in:
     1. Many viral infections
     2. Tuberculosis.
     3. Typhoid fever
     4. Lymphocytic leukemia.

   A decreased lymphocyte (lymphopenia) count of
    less than 500 places a patient at very high risk of
    infection, particularly viral infections.




                                                          45
Monocyte

              Diameter: 14-20
              Cytoplasm : grey blue
              Granules: dust-like lilac
               color granules
              Nucleus: blue
                large irregularly shaped
               and folded



                                           46
Diseases that cause a monocytosis include:
  • Tuberculosis
  • Brucellosis
  • Malaria
  • Monocytic leukemia




                                             47
Notes
1. Do not count cells that are disintegrating
    Eosinophil with no cytoplasmic membrane and with
     scattered granules
    Pyknotic cell (nucleus extremely condensed and
     degenerated, lobes condensed into small, round clumps
     with no filaments interconnecting).
    smudge cells

    Basket cells




                                                        48
2- Abnormal differentials
   1. 200 Cell diff:
       A. WBC > 15.0 (>20.0 for babies under 1 month and labor unit)
       B. Three or more basophils seen.
  2.   If more than five immature WBC's are seen (or any
       blasts) let someone else diff slide and average results.
  3.   Correct WBC for NRBC's if you seen ten or more
       NRBCs/100 WBC.
  4.   Always indicate number of cells counted on diff.
  5.   If any cell type is extremely elevated (such as bands,
       monos, or eos > 20) indicate that you are aware of the
       abnormality by circling or checking on the card next to
       the results.


                                                                   49
3- Morphologic Changes Due To Area Of Smear
    Thin area- Spherocytes which are really
     "spheroidocytes" or flattened red cells. True
     spherocytes will be found in other (Good) areas of
     smear.
    Thick area - Rouleaux, which is normal in such areas.
     Confirm by examining thin areas. If true rouleaux,
     two-three RBC's will stick together in a "stack of coins"
     fashion..




                                                            50
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4. A well-made and well-stained smear is essential to the accuracy
   of the differential count. The knowledge and ability of the cell
   morphologist is critical to high-quality results.
5. Before reporting significant abnormalities such as blasts,
   malaria or other significant finding on a patient’s differential,
   ask a more experienced tech to review the smear for
   confirmation. In clinical settings where a pathologist or
   hematologist is present, the smear is set aside for Pathologist
   Review.
6. Never hesitate to ask questions concerning morphology or the
   identification of cells. The differential is one of the most
   difficult laboratory tests to learn. In fact, learning about cells
   and their morphology is a process that continues for as long as
   you perform differentials.


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