This document provides instructions for making and examining a blood smear. It describes three types of blood smears - the cover glass smear, wedge smear, and spun smear. The wedge smear technique is explained in detail, including steps for placing a blood drop and spreading it across the slide at a 30-40 degree angle while controlling thickness. Characteristics of a good smear and common causes of a poor smear are also outlined. The document also covers fixing slides, staining techniques including Leishman's stain, and manually performing a differential white blood cell count and examining red blood cell morphology.
7. The thickness of the spread
Notes:
1. If the hematocrit is increased, the angle of
the s preader slide should be decreased.
2. If the hematocrit is decreased, the angle of
the spreader slide should be increased.
9. CHARACTERISTICS OF A GOOD SMEAR
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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 anabnormal
distribution of the white blood cells, with many of the large white
cells accumulating at the thin edge of the smear.
11. 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
14. Notes:
1. Although this is the easiest and most popular methods for
producing a blood smear, it does not produce a quality smear.
2. 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.
3. 2. Large cells such as monocytes, immature cells and abnormal
cells can be found in the outer limits of this area.
4. 3. Spun smears produce the most uniform distribution of blood
cells.
16. II- 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.
17. III. 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.
2. Giemsa stain is thought to produce more delicate staining
characteristics.
Wright's stain is a simpler method.
4. 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).
5. Field's stain is a rapid stain used primarily on thin films for malarial
parasites.
18. 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.
21. 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.
37. 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.
1. Neutrophilic leukocytosis = neutrophilia
2. Lymphocytic leukocytosis = lymphocytosis
3. Eosinophilic leukocytosis = eosinophilia
4.Monocytic leukocytosis =monocytosis
5.Basophilic leukocytosis = basophilia
43. NEUTROPHILS.1
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.
45. 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".
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46. Segmented neutrophile Band neutrophil
Shift to left Increased bands mean acute infection, usually bacterial.
Shift to right Increased hypersegmented neutrophile.
49. 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
52. 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.
55. LYMPHOCYTES.4
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
56. 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.
61. 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.
62. 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..
64. 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.