BLOOD SMEAR EXAMINATION Making Blood smear
I- Preparation of blood smear There are three types of blood
smears:
1. Th Thee cov cover er gl glas asss sme smear ar.. 2. The wedge smear . 3. The sp spun sm smear. The are two additional types of blood
smear used for specific purposes
1. Bu Buff ffy y co coat at sm smea earr fo forr WB WBCs Cs < 1. 1.0× 0×10 109/L 2. Th Thic ick k bl blood ood sm smea ears rs fo forr bl bloo ood d pa para rasi site tess .
I- Preparation of blood smear There are three types of blood
smears:
1. Th Thee cov cover er gl glas asss sme smear ar.. 2. The wedge smear . 3. The sp spun sm smear. The are two additional types of blood
smear used for specific purposes
1. Bu Buff ffy y co coat at sm smea earr fo forr WB WBCs Cs < 1. 1.0× 0×10 109/L 2. Th Thic ick k bl blood ood sm smea ears rs fo forr bl bloo ood d pa para rasi site tess .
WEDGE BLOOD BLOOD SMEAR
: EDTA blood within 2 to 3 hours & collected to the mark on tube.
May change RBCs morphology such as Spiculated (crenated) cells if :
1. Exce Excess ssiv ivee amou amount nt of of anti antico coag agul ulan antt to specimen 2. Ol Old d bl bloo ood d - lo long ng st stan andi ding ng.. 3. War arm m enviro environm nmen entt (room (room tempe tempera ratu ture re)) may hasten changes.
PROCEDURE placing a drop of blood from mixed sample on a clean glass slide. Spreader slide using another clean glass
slide at 30-40 degree angle. Control thickness of the smear by
changing the angle of spreader slide Allow the blood film to air-dry completely
before staining. (Do not blow to dry. The moisture from your breath will cause RBC artifact.)
STEPS FOR BLOOD FILM
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.
high HCT
small angle low HCT
large angle
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 anabnormal distribution of the white blood cells, with many of the large white cells accumulating at the thin edge of the smear.
COMMON CAUSES OF A POOR BLOOD SMEAR
1. Drop of blood too large or too small.
2. Spreader slide pushed across the slide in a jerky manner. 3. Failure to keep the entire edge of the spreader slide against the slide while making the smear. 4. Failure to keep the spreader slide at a 30° angle with the slid 5. Failure to push the spreader slide completely across the slide. 6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide 7. Holes in film: Slide contaminated with fat or grease
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
Examples of unacceptable smears
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.
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.
Large cells such as monocytes, immature cells and abnormal cells can be found in the outer limits of this area.
4.
Spun smears produce the most uniform distribution of cells.
blood
SLIDE FIXATION & STAINING LEISHMAN'S STAIN
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
III. Staining the film 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.
3. 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).
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.
Eosinophilic granules
Blue nucleus
Basophilic granules
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.
Staining procedure 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.
TOO ACIDIC
SUITABLE
TOO BASIC
CAUSES & CORRECTION
1. insufficient staining time 2. prolonged buffering or washing 3. old stain
1)
lengthen staining time
2)
check stain and buffer pH
3)
shorten buffering or wash time
1. 2. 3. 4.
thick blood smear prolonged staining insufficient washing alkaline pH of stain components
1) check pH 2) shorten stain time 3) prolong buffering time
PERFORMING A MANUAL DIFFERENTIAL AND ASSESSING RBC MORPHOLOGY
PRINCIPLE
1. Check for even distribution and estimate the number present (also, look for any gross abnormalities present on the smear). 2. Perform the differential count.
PRINCIPLE , Examine for :
1. Size and shape. 2. Relative hemoglobin content. 3. Polychromatophilia. 4. Inclusions. 5. Rouleaux formation or agglutination
1. Estimate number present.
PROCEDURES
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.
OBSERVATIONS UNDER× 40X : WBC ESTIMATES Using the × 40 high dry with no oil. Choose a portion of the peripheral smear where
there is only slight overlapping of the RBCs. Count 10 fields, take the total number of white
cells and divide by 10. To do a WBC estimate by taking the average
number of white cells and multiplying by 2000.
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)
platelets/OIF = platelets/OIF = platelets/OIF =
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:
Corrected WBC Count = WBC x 100/( NRBC + 100) counted
If WBC = 5000 and 10 NRBCs have been
5,000× 100/110 = 4545.50 4545.50.
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.
Absolute number of cells/µl = % of cell type in differential x white cell count
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
NORMAL PERIPHERAL BLOOD SMEAR
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
STAB NEUTROPHIL
Diameter:12-16
Cytoplasm : pink
Granules: primary secondary
Nucleus: dark purple blue
dense chromatin
BAND NEUTROPHIL
SEGMENTED NEUTROPHIL
Diameter: 12-16
Cytoplasm : pink
Granules: primary secondary
Nucleus: dark purple blue dense chromatin 2-5 lobes
SEGMENTED NEUTROPHIL
1.NEUTROPHILS 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.
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.
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".
: non-segmented neutrophil : hypersegmented neutrophil
Segmented neutrophile
Band neutrophil
Shift to left Increased bands mean acute infection, usually bacterial. Shift to right Increased hypersegmented neutrophile.
EOSINOPHIL
Diameter: 14-16
Cytoplasm : full of granules
Granules: large refractile, orange-red
Nucleus: blue dense chromatin 2 lobes like a pair of glass
EOSINOPHIL
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
BASOPHIL
Diameter: 14-16 Cytoplasm : pink Granules: dark blue – black black
obscure nucleus Nucleus: blue
BASOPHIL
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.
LYMPHOCYTE
: small 7-9 large 12-16 : medium blue small agranular large a few primary granules dark blue \round dense chromatin
LYMPHOCYTE
4.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
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.
MONOCYTE
: 14-20 : grey blue
dust-like lilac color granules
blue large irregularly shaped and folded
MONOCYTE
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
•
smudge cells
•
•
Basket cells
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.
3-Morphologic Changes Due To Area Of Smear
- Spherocytes which are really "spheroidocytes" or flattened red cells. True spherocytes will be found in other (Good) areas of smear.
- 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..
tail
body
head
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
