Wintrobe's Clinical Hematology 14e SC

Chapter 1: Examination of the Blood and Bone Marrow 7

Routine Staining of Blood Smears Blood smears are usually stained with either Wright or May-Grünwald-Giemsa stains. Both stains are modifications of the Romanowsky procedure. 88

The stain may be purchased commercially or made in the laboratory. The

basic stain is formulated from methylene blue and eosin. Giemsa stains

use known quantities of acid bichromate to form the converted azure

compounds. The Wright stain formulation uses sodium bicarbonate to

convert methylene blue to methylene azure, which stains the cell. All

types of Romanowsky stains are water insoluble but can be dissolved in

methyl alcohol. The stain must be free of water to induce RBC artifacts.

Water artifacts may be avoided by fixation of slides or coverslips in anhydrous methanol before staining. 91

Optimal staining conditions must be established for each new batch of

stain. The methylene blue conversion to azure compounds continues to

occur while the stain is in the bottle, so staining conditions may change

over time. Methyl azures are basic dyes that impar a violet-blue color-

ation when binding to the acidic components of the cell, such as nucleic

A

B

acids and proteins. The eosin reacts with the basic cellular elements,

FIGURE 1.3 Preparation of blood smears. Blood smears may be prepared by the coverslip (A) or slide wedge method (B). Coverslip smears are prepared by placing

imparting a reddish hue to cytoplasmic components and Hb. Aproperly

stained slide has a pink tint. The red cells will have an orange to pink

coloration, and leukocytes have purplish-blue nuclei. The Romanowsky

a drop of blood in the center of a coverslip and spreading the blood by rotating a

second coverslip over it. Wedge smears are prepared by placing a drop of blood

stains differentially stain leukocyte granules, which aids in morphologic

on a slide and using a second slide to push the blood out along the length of the

analysis of the cells. Thus, neutrophil granules are slightly basic and

slide. (Adapted and redrawn from Bauer J. Clinical Laboratory Methods . 9th

stain weakly with the azurophilic component. The eosinophils contain

ed. St. Louis, MO: C.V. Mosby; 1982.)

a strongly basic spermine derivative and stain strongly with eosin. In

contrast, basophil granules contain predominately acidic proteins and

stain a deep blue violet. No precipitate should overlie the cells because

Coverslip smears ( Figure 1.3A ) are prepared using a good grade of flat, × 22 mm) coverslips that are free of lint, dust, and grease. Such coverslips allow optimal spreading of the blood no. 1, 0.5-in square (or 22 mm

this indicates the use of slides or coverslips that were not cleaned prop-

erly. Dust on slides may also induce artifacts. Staining solutions should be filtered or replaced weekly, if used heavily, to avoid precipitation. 88

over the surface and minimal artifact. Usually, high-quality coverslips do

Occasionally, an excessive blue coloration of the cells is seen. This

not require additional cleaning, although there may be some deterioration

may be caused by excessive staining times, improperly prepared or aged

with age. Plastic “nonwettable” coverslips are not satisfactory for these

buffer that is too alkaline, old blood smears, or blood smears that are too

preparations. The smear is prepared by holding the coverslip by two

thick. The quality of the staining may be improved by quick and vigorous

adjacent corners between the thumb and index finger. A small drop of

Laboratory Hematology

rinsing with distilled water. If the areas of the slide between cells are

fresh or anticoagulated blood is placed in the center of the coverslip. The

staining, it usually indicates inadequate washing of the slide, heparin

size of the drop of blood is critical. If the drop is too large, a thick smear

anticoagulation, or possible paraproteinemia. When the staining appears

results. If the drop of blood is too small, a very thin smear is obtained. A

too pink or red, the usual problem is buffer that is too acidic. This results

second coverslip is then grasped in a similar manner with the other hand,

in pale-stained leukocyte nuclei, excessively orange RBCs, and bright

placed across the first coverslip, and rotated 45° with a steady, rapid, and

red eosinophil granules. Other causes of excessive red or pink coloration

gentle motion. The two coverslips are then immediately pulled apart and

include inadequate staining times or excessive washing of the slide. Most

allowed to air dry. If done properly, this procedure produces two coverslips with even dispersion of blood without holes or excessively thick areas. 87

often, problems with staining are caused by problems with the pH of the solutions, and the use of new buffer solutions often corrects the problem. 91

Blood smears may also be prepared on clean glass slides by the wedge

method (Figure 1.3B). This often leads to irregular distribution of cells on

Examination of the Blood Smear

the slide, a distinct disadvantage over the coverslip procedure. However,

glass slides are less fragile, are easier to handle, and may be labeled more

easily than coverslips. To prepare a slide blood smear, a drop of blood is

The blood smear should be initially examined under an intermediate power × 20 objective) to assess the adequacy of cellular distribution and staining. An estimate of the WBC count may also be made at this power, ( × 10 to

placed in the middle of the slide approximately 1 to 2 cm from one end. A

second spreader slide is placed at a 30° to 45° angle and moved backward

and scanning for abnormal cellular elements, such as blasts or nucleated

to make contact with the blood drop. The blood drop will spread along

RBCs, can be performed. It is important to scan the entire blood smear

the slide edge and then the spreader slide is moved rapidly forward. This

to ensure that abnormal populations, which may be concentrated at the edges of the smear, are not missed. The use of an oil immersion lens ( × 50 or × 100) or high-power dry lens ( × 40 or × 60) is usually sufficient for performing leukocyte differential counts, although a × 100 oil lens may be necessary for identification of cellular inclusions or abnormal cytoplasmic

technique creates a film of blood that is 3 to 4 cm long. Artifact may be

introduced by irregular edges in the spreader and by the speed at which

the spreader is moved. Glass slide preparations have increased incidence

of accumulation of the larger white cells at the edges of the film, intro-

ducing cellular distribution errors. Fast movement of the spreader results in a more uniformly distributed population of cells. 87,88

granules. Systematic evaluation of the blood smear is essential so that

all cell types are examined and characterized. Each cell type should be evaluated for both quantitative and qualitative abnormalities. 92,93

Automated techniques for blood smear preparation have also been

developed, and some instruments have integrated automated blood smear

preparation technology, thus allowing smear preparation directly from

It is difficult to evaluate quantitative abnormalities of red cells on

the CBC tube. Two major types of approaches are used: centrifugation

a blood smear; however, the RBCs should be evaluated for variations

in size, shape, Hb content and distribution, and the presence of cellular

and mechanical spreaders. Centrifugation techniques are often most

Copyright © 2019 Wolters Kluwer, Inc. Unauthorized reproduction of the content is prohibited. useful when a small number of cells must be concentrated in a small inclusions. The red cells are usually unevenly distributed throughout the blood film. Optimal red cell morphology is seen in an area of the smear

area, as in preparing smears of cells in fluids such as cerebrospinal fluid. 89 Mechanical spreaders mimic the manual technique and are useful when large numbers of blood smears are prepared. 90 In general,

where the red cells are close together but do not overlap. Areas where the

red cells are spread too thinly or thickly have increased artifacts. In some

smears made by automated techniques are inferior to those made by an

blood smears, the red cells appear to stick together, forming what appear

experienced technician.

to be stacks of RBCs, termed rouleaux. This finding may be mimicked in

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