Making the peripheral blood smear

Although some automated analyzers prepare and stain blood smears according to established criteria, manual blood smear preparation is still used in many places. The wedge smear is a convenient and commonly used technique for making peripheral blood smears. This technique requires at least two 3 × 1-inch (75 × 25-mm) clean glass slides. High-quality, beveled-edge microscope slides are recommended. One slide serves as the blood smear slide and the other as the spreader slide. These can then be reversed to prepare a second smear. A drop of ethylenediaminetetraacetic acid (EDTA) anticoagulated blood about 3 mm in diameter is placed at one end of the slide. Alternatively, a similar size drop of blood directly from a finger or heel puncture is acceptable. The size of the drop of blood is important. Too large a drop creates a long or thick smear, and too small a drop often makes a short or thin smear. In preparing the smear, the technician holds the pusher slide securely in front of the drop of blood at a 30- to 45-degree angle to the smear slide (Figure 1-1, A). The pusher slide is pulled back into the drop of blood and held in that position until the blood spreads across the width of the slide (Figure 1-1, B). It is then quickly and smoothly pushed forward to the end of the smear slide, creating a wedge smear (Figure 1-1, C). It is important that the whole drop of blood is picked up and spread. Moving the pusher slide forward too slowly accentuates poor leukocyte distribution by pushing larger cells, such as monocytes and granulocytes, to the very end and sides of the smear. Maintaining a consistent angle between the slides and an even, gentle pressure is essential. It is frequently necessary to adjust the angle between the slides to produce a satisfactory smear. For higher than normal hematocrit, the angle between the slides must be lowered so that the smear is not too short and thick. For extremely low hematocrit, the angle must be raised. A well-made peripheral blood smear (Figure 1-2) has the following characteristics:

Figure 1–1 Wedge technique of making a peripheral blood smear. A, Correct angle to hold spreader slide. B, Blood spread across width of slide. C, Completed wedge smear.

(From Rodak BF, Fritsma GA, Keohane EM: Hematology: clinical principles and applications, ed 4, St. Louis, 2012, Saunders.)

Figure 1–2 Well-made peripheral blood smear.

(From Rodak BF, Fritsma GA, Keohane EM: Hematology: clinical principles and applications, ed 4, St. Louis, 2012, Saunders.)

Figure 1-3 shows examples of unacceptable smears.

Figure 1–3 Unacceptable peripheral blood films. Slide appearances associated with the most common errors are shown, but note that a combination of causes may be responsible for unacceptable films. A, Chipped or rough edge on spreader slide. B, Hesitation in forward motion of spreader slide. C, Spreader slide pushed too quickly. D, Drop of blood too small. E, Drop of blood not allowed to spread across the width of the slide. F, Dirt or grease on the slide; may also be caused by elevated lipids in the blood specimen. G, Uneven pressure on the spreader slide. H, Time delay; drop of blood began to dry.

(From Rodak BF, Fritsma GA, Keohane EM: Hematology: clinical principles and applications, ed 4, St. Louis, 2012, Saunders.)

Staining of peripheral blood smears

The purpose of staining blood smears is to identify cells and recognize morphology easily through the microscope. Wright or Wright-Giemsa stain is the most commonly used stain for peripheral blood and bone marrow smears. These stains contain both eosin and methylene blue, and are therefore termed polychrome stains. The colors vary slightly from laboratory to laboratory, depending on the method of staining.

The cells are fixed to the glass slide by the methanol in the stain. Staining reactions are pH dependent, and the actual staining of the cellular components occurs when a buffer (pH 6.4) is added to the stain. Free methylene blue is basic and stains acidic cellular components, such as RNA, blue. Free eosin is acidic and stains basic components, such as hemoglobin or eosinophilic granules, red. Neutrophils have cytoplasmic granules that have a neutral pH and accept some characteristics from both stains. Details for specific methods of staining peripheral blood and bone marrow smears, including automated methods, may be found in a standard textbook of hematology.

An optimally stained smear (Figure 1-4) has the following characteristics:

Figure 1–4 Optimally stained peripheral blood smear demonstrating the appropriate area in which to perform the white blood cell differential and morphology assessment and the platelet estimate. Only the center of the field is shown; an entire field would contain 200 to 250 red blood cells (×1000).

A well-stained slide is necessary for accurate interpretation of cellular morphology. The best staining results are obtained from freshly made slides that have been prepared within 2 to 3 hours of blood collection. Slides must be allowed to dry thoroughly before staining. Box 1-1 lists common reasons for poorly stained slides and may be used as a guide when troubleshooting.

10× examination

Examination of the blood smear is a multistep process. Begin the smear examination with a scan of the slide using the 10× or low-power objective (total magnification = 100×). This step is necessary to assess the overall quality of the smear, including abnormal distribution of RBCs, suggesting the presence of rouleaux or autoagglutination and/or the presence of a disproportionate number of large nucleated cells, such as monocytes or neutrophils, at the edges of the smear. If the latter exists, another smear should be prepared. In addition, the 10× smear examination allows for the rapid detection of large abnormal cells such as blasts, reactive lymphocytes, and parasites.

40× or 50× examination

Using the 40× (high dry) objective or the 50× oil objective (400× and 500× total magnification, respectively), find an area of the smear in which the RBCs are evenly distributed and barely touching one another (two or three cells may overlap; Figure 1-5). Scan 8 to 10 fields in this area of the smear and determine the average number of white blood cells (WBCs) per field. Although an exact factor varies with the make and model of microscope, in general, an approximate WBC count per cubic millimeter can be determined by multiplying the average number of   WBCs per high-power field by 2000 (if 40× is used) or 2500 (if 50× is used). This estimate is a useful quality-control tool for validating WBC counts from hematology analyzers. Any discrepancy between the instrument WBC count and the slide estimate must be resolved. Some reasons for discrepancy include the presence of   WBC or platelet clumps, fibrin strands, severe RBC agglutination, cryoprecipitate, giant platelets, as well as a mislabeled smear, a smear made from the wrong patient’s sample, and an instrument malfunction.

Figure 1–5 Correct area of blood smear in which to evaluate cellular distribution and perform white blood cell estimate (×400).

100× examination

The next step in smear evaluation is to perform the WBC differential. This is done in the same area of the smear as the WBC estimate but using the 100× oil immersion objective (1000× total magnification). When the correct area of the smear from a patient with a normal RBC count is viewed, about 200 to 250 RBCs per oil immersion field are seen (see Figure 1-4). Characteristically, the differential count includes counting and classifying 100 consecutive WBCs and reporting these classes as percentages. The differential count is performed in a systematic manner using the “battlement” track (Figure 1-6), which minimizes WBC distribution errors. The results are reported as percentages of each type of WBC seen during the count. An example of a WBC differential count is 3% bands, 55% segmented neutrophils, 30% lymphocytes, 6% monocytes, 4% eosinophils, and 2% basophils (Table 1-1). Any WBC abnormalities, such as toxic changes, Döhle bodies, reactive lymphocytes, and Aüer rods, are also reported. When present, nucleated red blood cells (NRBCs) are counted and reported as number of NRBCs per 100 WBCs. The RBC, WBC, platelet morphology evaluation, and platelet estimates are also performed under the 100× oil immersion objective. RBC inclusions, such as Howell-Jolly bodies, and WBC inclusions, such as Döhle bodies, can be seen at this magnification. Each laboratory should have established protocols for standardized reporting of abnormalities.

Figure 1–6 “Battlement” pattern for performing a white blood cell differential.

(From Rodak BF, Fritsma GA, Keohane EM: Hematology: clinical principles and applications, ed 4, St. Louis, 2012, Saunders.)

Evaluation of the RBC morphology is an important aspect of the smear evaluation and is used in conjunction with the RBC indices to describe cells as normal or abnormal in size, shape, and color. Each laboratory should establish a standard reporting protocol. Most laboratories use concise statements describing overall RBC morphology that is consistent with the RBC indices. The microscopic evaluation of RBC morphology must be congruent with the information given by the automated hematology analyzer. If not, discrepancies must be resolved before reporting patient results.

The final step in the performance of the differential count is the estimation of the platelet number. This is done under the 100× oil immersion objective. In an area of the smear where RBCs barely touch, the number of platelets in 5 to 10 oil immersion fields is counted. The average number of platelets is multiplied by 20,000 to provide an estimate of the total number of platelets per cubic millimeter. This estimate is reported as adequate if the estimate is consistent with a normal platelet count, decreased if below the lower limit of normal for that laboratory, and increased if above the upper limit of normal. A general reference range is 150,000 to 450,000/mm³ (150–450 × 10⁹/L). When a patient is extremely anemic or has erythrocytosis, a more involved formula for platelet estimates may be used:

The estimate can be compared with an automated platelet count as an additional quality-control measure. If the estimate and the instrument platelet count do not agree, discrepancies must be resolved. Some causes for discrepancy include the presence of giant platelets, many schistocytes, and platelet satellitism. Notably, high-quality 40× or 50× oil immersion objectives can be used by the experienced technologist to perform the differential analysis of the blood smear. However, all abnormal findings must be verified under the 100× objective.