BLOOD SMEAR EXAMINATION Making Blood smear

I- 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 .

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.

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.

large angle

low HCT

small angle

high HCT

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 slide.

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

8. Cellular degenerative changes: delay in fixing, inadequate

fixing time or methanol contaminated with water.

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.

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 blood cells.

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 slides.

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.

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).

5. Field's stain is a rapid stain used primarily on thin films for malarial parasites.

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.

Eosinophilic granules

Basophilic granules Blue nucleus

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

Too Acid Stain:

1. insufficient staining time

2. prolonged buffering or washing

3. old stain

Correction:

1) lengthen staining time

2) check stain and buffer pH

3) shorten buffering or wash time

Too Alkaline Stain:

1. thick blood smear

2. prolonged staining

3. insufficient washing

4. alkaline pH of stain components

Correction :

1) check pH

2) shorten stain time

3) prolong buffering time

PERFORMING A MANUAL DIFFERENTIAL AND ASSESSING RBC

MORPHOLOGY

PRINCIPLE

White Blood Cells.

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

Red Blood Cells, Examine for :

1. Size and shape.

2. Relative hemoglobin content.

3. Polychromatophilia.

4. Inclusions.

5. Rouleaux formation or agglutination

Platelets.

1. Estimate number present.

2. Examine for morphologic abnormalities.

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.

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)

1) <8 platelets/OIF = decreased

2) 8 to 20 platelets/OIF = adequate

3) >20 platelets/OIF = increased

PLATELETS

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)

Example : If WBC = 5000 and 10 NRBCs have been counted

Then 5,000× 100/110 = 4545.50

The corrected white count is 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.

Reporting results

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

*avoid repeat or miss some cells

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".

Left-shift: non-segmented neutrophil > 5%

Right-shift: hypersegmented neutrophil >3%

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

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

Diameter: small 7-9

large 12-16

Cytoplasm: medium blue

Granules: small agranular

large a few primary granules

Nucleus: 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

Diameter: 14-20

Cytoplasm : grey blue

Granules: dust-like lilac color granules

Nucleus: blue

large irregularly shaped and folded

MONOCYTE

Diseases that cause a monocytosis include:

•Tuberculosis

•Brucellosis

•Malaria

•Monocytic leukemia

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

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..

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 differentials.