Introduction

In Medical Technology

Lecture 11

Making Blood smear

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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 purposes1. Buffy coat smear for WBCs < 1.0×109/L 2. Thick blood smears for blood parasites .

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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 specimen2. Old blood - long standing. 3. Warm environment (room temperature) may

hasten changes.

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Procedure1. 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 slide4. Allow the blood film to air-dry completely before

staining. (Do not blow to dry. The moisture from your breath will cause RBC artifact.)

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STEPS FOR BLOOD FILM

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The thickness of the spread1. 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.

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Characteristics of a Good Smear1. 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.

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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 smearsA: Blood film with jagged tail made from a spreader with a chipped 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.

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Examples of unacceptable smears

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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.

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SLIDE FIXATION & SLIDE FIXATION & STAININGSTAINING

LEISHMAN'S STAINLEISHMAN'S STAIN

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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.

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Staining the filmRomanowsky 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.

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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.

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Staining procedure 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.

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too acidic too acidic suitable suitable too basic too basic

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Causes & correction Too Acid Stain:1. insufficient staining time2. prolonged buffering or

washing3. old stain Correction:1) lengthen staining time2) check stain and buffer pH3) shorten buffering or wash

time

Too Alkaline Stain:1. thick blood smear2. prolonged staining3. insufficient washing4. alkaline pH of stain

components  Correction :1) check pH2) shorten stain time3) prolong buffering time

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Performing A Manual differential Performing A Manual differential And assessing RBC Morphology And assessing RBC Morphology

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PurposesPurposes 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.

Platelets. 1. Estimate number present. 2.  Examine for morphologic

abnormalities.

Red Blood Cells, 1. Size and shape.

2. Relative hemoglobin content.

3. Polychromatophilia.

4. Inclusions.

5. Rouleaux formation or agglutination

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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.

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Observations Under ×40 : WBC estimation1. 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.

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Observations Under × 100: Platelet Estimates1. 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 = decreased2) 8 to 20 platelets/OIF = adequate3) >20 platelets/OIF = increased

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PLATELETS26

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 countedThen 5,000× 100/110 = 4545.50The corrected white count is 4545.50.

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Manual Differential CountsManual 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 resultsAbsolute number of cells/µl = % of cell type in

differential x white cell count

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Observing Observing direction:direction:

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

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Normal peripheral blood Normal peripheral blood smearsmear

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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 leukocytosisMonocytosis : Monocytic leukocytosisBasophilia: Basophilic leukocytosis

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Leukocytosis

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Stab (Band) neutrophil Diameter:12-16 Cytoplasm : pink Granules: primary

secondary Nucleus: dark purple blue dense chromatin

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Segmented neutrophil

Diameter: 12-16 Cytoplasm : pink Granules: primary,

secondary Nucleus: dark purple

blue dense chromatin, 2-5 lobes

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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.

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

Decreased neutrophil count (neutropenia)1. Typhoid fever2. Brucellosis 3. Viral diseases, including hepatitis, influenza, rubella, and

mumps.

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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%

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Segmented neutrophile Band neutrophil

Shift to left Increased bands mean acute infection, usually bacterial.

Shift to right Increased hypersegmented neutrophile.

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Eosinophil Diameter: 14-16 Cytoplasm : full of granules Granules: large refractile,

orange-red Nucleus: blue dense

chromatin 2 lobes like a pair of glass

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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

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Basophil Diameter: 14-16 Cytoplasm : pink Granules: dark blue –

black obscure nucleus

Nucleus: blue

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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.

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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

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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

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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.

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Monocyte Diameter: 14-20 Cytoplasm : grey blue Granules: dust-like lilac

color granules Nucleus: blue large irregularly shaped

and folded

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

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NotesNotes1. 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

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2- Abnormal differentials1. 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.

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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..

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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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