Lecture #13: Morphological Examination of Blood Films

  

BLOOD FILM OR SMEAR EXAMINATION

Making and staining blood films or smear

Examination of the blood film is an important part of the hematologic evaluation. The reliability of the information obtained depends heavily on well–made and well stained films which are systematically examined. So, the production of a good stained film is an absolute requirement if one hopes to obtain all information possible from the morphology of blood cells.

Blood smears or films can be made on glass slides or coverslips. The latter have the single possible advantage of a more even distribution of the leukocytes, but in every other respect slides are to preferred. Unlike coverglasses, slides are not easily broken; they are simple to label and when large number of films are to be dealt with, slides will be found much easier to handle.

Methods:

A. The two–slide or wedge method

This is the simplest and most widely used method. It uses two slides, one for the smear and the other serves as spreader or pusher.

Criteria of a good blood smear using two–slide or wedge method

1. The thick area makes a gradual transition to the this area (feathery like edge)

2. The blood on the thin area does not extend to the end of the slide – the smear may cover ¾ of the slide’s length

3. Must have smooth even surface, free from ridges, waves and holes

4. Leukocytes must not be bunched at the edge or at the end of the smear.

B. Ehrlich’s two–coverglass method

No.1 or 1 ½ coverglasses 22 mm square are recommended. In this method, the coverglass with blood side down is placed crosswise on another coverglass so that the corners appear on an eight pointed star. When the blood has spread evenly between the two surfaces, pull the coveglasses quickly but firmly apart on a plane parallel to their surface.

 

C. Beacom’s coverglass and slide method

THE THIN AND THICK SMEAR

1. Thin smears are prepared by any of the three methods previously mentioned. These smears are used for

a.     Differential leukocyte count

b.     Stained red cell examination

c.      Platelet count (indirect method)

d.     Reticulocyte count

e.     Siderocyte count

f.       Malarial parasite (blood parasite) examination

g.      Thorough study of the morphology of blood cells

2. Thick smears are prepared as follows:

a. Place a small drop of blood in the center of a slide

b. Spread it out with a corner of another slide to cover an area about 4 times its original area

c. The correct thickness for a satisfactory film will be achieved if, with the slide placed on a piece of newspaper, small print is just visible

Thick smears are used in:

a.     Diagnosis of malaria

b.     Diagnosis of filarial

c.      Diagnosis of trypanosomes

d.     Diagnosis of spironemas

Factors which affect the thickness of blood films:

a.     Variation in the angle of the spreader

b.     Variation in the pressure of the spreader against the slides

c.      Variation in the size of the drop of blood

Requirements to be able to produce proper blood films:

a.     Use of chemically clean slides and coverglasses

b.     Use of not too large nor too small drop of blood

c.      Work is done quickly before coagulation of blood

d.     Proper angle and pressure of the spreader

Methods of drying the film

a.     Air drying

b.     Heating in the oven or at low flame

c.     Chemical drying in ethyl alcohol

Chemicals used to fix blood smears

a.     Pure methyl alcohol

b.     Absolute ethyl alcohol

c.      Absolute alcohol and ether

d.     1% solution of HgCl₂

e.     1% formalin

STAINING OF BLOOD SMEARS

The microscopic study of stained, peripheral blood smear constitutes the most important part of the routine hematological examination. Cytochemical stains are essential for the identification of hemopoeitic cells. The most commonly used stains are polychrome stains; those belonging to the Romanowsky group.

A polychrome stain is a stain of many colors and the original polychrome stain was discovered by Romanowsky. Polychrome methylene blue and eosin stains are the outgrowth of the original time–consuming Romanowsky method and are widely used. They stain differently most normally and abnormal structures in the blood.

All Romanowsky stains are composed of mixture of thiazine eosinate produced by the interaction of polychrome methylene blue with eosin. The neutral dye precipitated is redissolved in methyl alcohol and used as stain; the difference among the various stains in the proportion of the reagent and in the method of preparation.

Methylene blue on oxidation produces colored compounds, termed “azures” which have the ability to combine with eosin. Oxidation of methylene blue is known as “polychomasia” or “polychroming,” it may be formed by heating for 12 hours at 65^oC and allowing it to be exposed to atmospheric oxygen for 10 days or by accelerating the oxidizing action by allowing it to be exposed to free steam. When the Romanowsky stains color the cellular constituents blue or purple, they are termed basophilic. They stain red, pink or orange, they are said to be acidophilic or oxyphilic or eosinophilic. Those cellular constituents staining between the two extremes are termed neutrophilic.

PRINCIPLES IN STAINING

Physical theories of staining assume that the dye either precipitates the porous cellular wall or is consequently absorbed into the cell or that the stain is precipitated within the cell after such penetration. The proponents of the chemical theory of staining postulate that because of the nuclear material is composed primarily of DNA and consequently acidic in nature, it has an affinity for basic dyes and that the cytoplasm is normally basic in pH and has an affinity for acidic dyes. This theory also assumes that the tissue acids and bases are amphoteric and act as electrolytes dissolved in any solution in which they are immersed. Stains for blood are used at a pH of either 6.4 to 6.8, depending on the stain used; at those pH levels the nuclei takes basic stains and the cytoplasm, acidic dyes. Bone marrow are usually better stained at a pH of 6.4, while malarial parasites at a pH of 7.2.

 Basically, the procedure of staining with Romanowsky stains consists of flooding the smear with stain, which allows the cells to be fixed by the alcohol vehicle, staining the cell by the addition of water to the stain and finally removing the excess stain from the cells by differentiating with water. The staining period must be rigidly monitored otherwise the smear may appear understained, overstained, too acidic or too alkaline.

Note:   Romanowsky stains are dissolved in methyl alcohol and combine fixation with staining

1. Wright stain

In its preparation, the methylene blue is polychromed by heating with sodium bicarbonate

Formula:         Wright stain – 0.1 grams

                        Methyl alcohol – 60 ml

Preparation of buffer (pH 6.4)

            Primary potassium phosphate (monobasic) – 6.63 grams

            Secondary sodium phosphate (dibasic) – 2.56 grams

            Distilled water – 1 liter

2. May–Grunwald’s stain

May – Grunwald’s powdered dye – 0.3 grams

Methyl alcohol – 100 ml

3. Giemsa stain

Giemsa powdered dye – 1 gram

Glycerol – 66 ml

Methyl alcohol – 66 ml

4. Leishman’s stain

Leishman’s powdered dye – 0.2 grams

Methyl alcohol – 100 ml

5. Jenner’s stain

Jenner’s powdered dye – 0.5 grams

Methyl alcohol – 1 liter

6. May–Grunwald – Giemsa stain

Both May – Grunwald and Giemsa stain are covered on the blood films one after the other

Panoptic stain – a combination of a Romanowsky  stain and another stain to improve cytoplasmic granules. Examples: (a) Jenner–Giemsa stain (b) May–Grunwald – Giemsa stain

Intravital stain – it is used to stain the tissue by a dye which is introduced into a living organism and which, by virtue of selective attraction to certain tissues, will stain these tissues.

Supravital stain – a supravital stain is used to stain and inspect living cells which have been removed from the body. The stain enables the cells to remain alive and mobile. It does not stain the nucleus or cytoplasm but is stain significant structures in the cytoplasm. Example is the reticulocyte stain.

CRITERIA OF A GOOD STAIN

1. The film will appear pink to the naked eye.

2. Microscopically, the erythrocyte are pink

3. The neutrophilic granules are lilac

4. The eosinophilic granules are red

5. The nuclei of WBC are purplish blue

6. The oxychromatin of the nuclei are clearly differentiated

7. The areas between the cells are clear with no film or precipitated stain visible

Causes of overstained smears

1.     Too thick smears

2.     Insufficient washing

3.     Too prolonged staining time

4.     Excessive alkalinity of the stain, buffer or water

Appearance of cells in overstained smears

1.     Erythrocyte stains blue or green

2.     Cytoplasm of the lymphocytes become gray or lavender

3.     Granules of neutrophils are intensely overstained

4.     Eosinophilic granules become deep gray or blue

Causes of under stained smears

1.     Too thin smears

2.     Excessive washing of the smear   

3.     Excess acidity of the stain, buffer or water

Appearance of cells in understained smears

1.     Nuclear chromatin is stained pale blue rather than vivid blue

2.     Erythrocyte stain bright red or orange rather than pink

3.     Eosinophilic granules stain brilliant red

Causes of scum or precipitated stain between the cells

1. Unclean slide or coverglass

2. Faulty washing because of failure to hold the slide horizontally and to float off the scum

3. Permitting dust to settle on the film

Causes of poor staining

1.     Alkaline slides and alkaline distilled water

2.     Acid slides and acid distilled water

3.     Unclean slides

4.     Evaporation of the stain

5.     Incorrect buffer pH

6.     Imperfect polychroming of the stain

7.     Incomplete reaction of the staining fluid

8.     Errors of the operator

**** DIFFERENTIAL LEUKOCYTE COUNT ****

Differential leukocyte count is the linear representation of the percentage of the various types of leukocytes in the peripheral or venous blood. It is called “hemogram.”

Steps in making a differential leukocyte count

1.     Making the blood smear

2.     Staining the blood smear

3.     Counting the cells

4.     Reporting the result

Methods or techniques employed in counting the cells:

1. Strip differential count

All the cells are counted in the longitudinal strip that is, from the head to the tail of the smear.

2. Exaggerated battlement method

The count starts at one edge of the smear and counting all the cells, advancing inward to 1/3 of the width of the smear, then on the line parallel to the edge, then out of the edge, then along the edge.

3. Two–field meander method

The count is made by dividing the smear into two fields and proceeds as in exaggerated battlement method

4. Four–field meander method

The count is made by dividing the smear into four fields and proceeds as in exaggerated battlement method

Methods of classification of cells in differential count

1. Schilling hemogram

In this method, all the leukocytes (granulocytes and non–granulocytes) are classified and grouped according to maturity of the cells into:

Granulocytes:             Neutrophils, Eosinophils, Basophils

Non–granulocytes:     Lymphocytes, Monocyte

The polymorphonuclear neutrophils are further classified according to maturity of the cells as:

a.     Myelocytes

b.     Metamyelocytes

c.      Bands or stabs

d.     Segmenters

The Schilling hemogram may be represented as:

Basophil                      0.25 – 0.5%

Eosinophil                  2 – 4%

Myelocyte                   0%

Metamyelocyte          0 – 1%

Stab                             2 – 6%

Segmenters                 55 – 65%

Lymphocytes              25 – 35%

Monocytes                  2 – 8%

2. Arneth’s classification

In this method, the polymorphonuclear neutrophils are classified according to the number of lobes which their nuclei possess. The more lobes, the older the cells:

Class I (with lobe or indented nucleus)        –          5%

Class II (with 2 lobes)                                                 –          35%

Class III (with 3 lobes)                                                –          41%

Class IV (with 4 lobes)                                    –          17%

Class V (oldest with 5 lobes)                                     –          2%

Under the traditional unit, the result in differential leukocyte count is reported in percentage, under the S.I. unit, the proportion of each type of cell is reported as a decimal fraction and is called the leukocyte type number fraction.

3. Haden’s classification

This method classifies the neutrophils according to the presence of filaments. These neutrophils whose lobes are connected by thin filaments are classified as filamented, while those that are not connected by filaments are grouped under non–filamented cells.

Filamented cells – 60%

Non–filamented cells – 7%

Eosinophils – 3%

Basophils – 1%

Lymphocytes – 21%

Monocytes – 8%

SHIFTING PROCESSES:

Shift to the left – if there is an increase in younger forms of leukocytes particularly Classes I and II. Seen in pyogenic infections.

Shift to the right – if there is an increase in older forms of leukocytes particularly Classes IV and V. seen in megaloblastic anemia and in convalescence

Regenerative shift to the left – if the predominating cells are younger forms, with the presence of myelocyte and metamyelocytes and increase in band cells; and it is accompanied by a high leukocyte count

Degenerative shift to the left – if the predominating cells are younger forms, with an increase in band cells but without myelocyte and metamyelocytes; and it is accompanied by a low WBC count.

NORMAL DIFFERENTIAL WBC COUNT VALUES

Segmented neutrophils           –         50 – 70%

Stabs or bands                         –         5 – 16%

Eosinophils                             –          2 – 4%

Basophils                                –          0 – 1%

Monocytes                              –          2 – 6%

Lymphocytes                          –          20 – 35%

AUTOMATED DIFFERENTIAL LEUKOCYTE COUNT

Two general principle have been employed in the automation of differential leukocyte count

1. Digital image processing – a uniformly made and stained blood film is placed on a microscope stage, which is driven by a motor. A computer controls the movement, scanning the slide and stopping it when leukocytes are in the field. The optical images are then recorded by television camera, analyzed by computer and converted to digital form.

2. Flow–through system – these systems analyze the cells suspended in a liquid. In photo– optical system, measurements of light scattering and of light absorption are made while the cells are being counted.