Under certain conditions, use of a different anticoagulant or no anticoagulant may be helpful. Some patients’ blood undergoes an in vitro phenomenon called platelet satellitosis³ when anticoagulated with EDTA. The platelets surround or adhere to neutrophils, which potentially causes pseudothrombocytopenia when counting is done by automated methods (Figure 15-1).^3,4 In addition, spuriously low platelet counts and falsely increased WBC counts (pseudoleukocytosis) can result from EDTA-induced platelet clumping.⁵ Pseudoleukocytosis occurs when platelet agglutinates are similar in size to WBCs and automated analyzers cannot distinguish the two. The platelet clumps are counted as WBCs instead of platelets. Platelet-specific autoantibodies that react best at room temperature are one of the mechanisms known to cause this phenomenon.⁶ In these circumstances, the examination of a blood film becomes an important quality control strategy, identifying these phenomena so that they can be corrected before the results are reported to the patient’s chart.

Figure 15-1 Photomicrograph of platelet satellitosis.

Problems such as these can be eliminated by re-collecting specimens in sodium citrate tubes (light blue top) and ensuring that the proper ratio of nine parts blood to one part anticoagulant is observed (a properly filled tube). These new specimens can be analyzed in the usual way by automated instruments. Platelet counts and WBC counts from sodium citrate specimens must be corrected for the dilution of blood with the anticoagulant, however. In a full-draw tube, the blood is nine tenths of the total tube volume (2.7 mL of blood and 0.3 mL of sodium citrate). The “dilution factor” is the reciprocal of the dilution (i.e., 10/9 or 1.1). The WBC and platelet counts are multiplied by 1.1 to obtain accurate counts. All other CBC parameters should be reported for the original EDTA tube specimen and slide.

Another source of blood for films is from finger and heel punctures. In general, the films are made immediately at the patient’s side. There are, however, a few limitations to this procedure. First, some platelet clumping must be expected if films are made directly from a drop of finger-stick or heel-stick blood or if blood is collected in heparinized microhematocrit tubes. Generally, this clumping is not enough to interfere with platelet estimates if the films are made promptly before clotting begins in earnest. Second, only a few films can be made directly from blood from a skin puncture before the site stops bleeding. If slides are made quickly and correctly, however, cell distribution and morphology should be adequate. These problems with finger and heel sticks can be eliminated with the use of EDTA microcollection tubes, such as Microtainers (Becton, Dickinson and Company, Franklin Lakes, N.J.) (see Chapter 3).

Peripheral Film Preparation

Types of Films

Manual Wedge Technique

The wedge film technique is probably the easiest to master. It is the most convenient and commonly used method for making peripheral blood films. This technique requires at least two 3-inch × 1-inch (75-mm × 25-mm) clean glass slides. High-quality, beveled-edge microscopic slides with chamfered (beveled) corners for good lateral borders are recommended. A few more slides may be kept handy in case a good-quality film is not made immediately. One slide serves as the film slide, and the other is the pusher or spreader slide. They can then be reversed. It is also possible to make good wedge films by using a hemacytometer coverslip attached to a handle (pinch clip or tongue depressor) as the spreader.

A drop of blood (about 2 to 3 mm in diameter) from a finger, heel, or microhematocrit tube (nonheparinized for EDTA-anticoagulated blood or heparinized for capillary blood) is placed at one end of the slide. The drop also may be delivered using a Diff-Safe dispenser (Alpha Scientific Corporation, Malvern, Penn.). The Diff-Safe dispenser is inserted through the rubber stopper of the EDTA tube, which eliminates the need to remove the stopper.⁷ The size of the drop of blood is important: too large a drop creates a long or thick film, and too small a drop often makes a short or thin blood film. The pusher slide, held securely in the dominant hand at about a 30- to 45-degree angle (Figure 15-2, A), is drawn back into the drop of blood, and the blood is allowed to spread across the width of the slide (Figure 15-2, B). It is then quickly and smoothly pushed forward to the end of the slide to create a wedge film (Figure 15-2, C). It is important that the whole drop be 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 film. Maintaining an even, gentle pressure on the slide is essential. It is also crucial to keep the same angle all the way to the end of the film. When the hematocrit is higher than normal, as is found in patients with polycythemia or in newborns, the angle should be lowered (i.e., 25 degrees), so the film is not too short and thick. For extremely low hematocrit, the angle may need to be raised. If two or three films are made, the best one is chosen for staining, and the others are disposed of properly. Some laboratories make two good films and save one unstained in case another slide is required.

Figure 15-2 Wedge technique of making a peripheral blood film.

The procedure just described is for a push-type wedge preparation. It is called push because the spreader slide is pulled into the drop of blood and the film is made by pushing the blood along the slide. The same procedure can be modified to produce a pulled film. In this procedure, the spreader slide is pushed into the drop of blood and pulled along the length of the slide to make the film. Although this method is less commonly used, it also provides a satisfactory wedge preparation and is easier for some individuals to perform. Other variations on the wedge technique include using the 3-inch side of the slide as the spreader slide or balancing the spreader slide on the fingers to avoid placing too much pressure on it. Learning to make consistently good blood films takes a lot of practice but can be accomplished if one is patient and persistent.

Features of a well-made wedge peripheral blood film

1. The film is two thirds to three fourths the length of the slide (Figure 15-3).

Figure 15-3 Well-made peripheral blood film.

2. The film is finger shaped, very slightly rounded at the feather edge, not bullet shaped; this provides the widest area for examination.

3. The lateral edges of the film are visible.

4. The film is smooth without irregularities, holes, or streaks.

5. When the slide is held up to the light, the thin portion (feather edge) of the film has a “rainbow” appearance.

6. The whole drop of blood is picked up and spread.

Figure 15-4 shows unacceptable films.

Figure 15-4 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 due to elevated lipids in the blood specimen. G, Uneven pressure on the spreader slide. H, Time delay; drop of blood began to dry.

Coverslip Technique

The coverslip method of film preparation is an older technique that is now used only rarely for peripheral blood films, but it is sometimes still used for making bone marrow aspirate smears. The only advantage of this method is that it produces an excellent leukocyte distribution, which lends itself to more accurate determination of differential counts. For routine morphologic evaluation, however, this technique is neither convenient nor practical. Impeccably clean glass coverslips must be used.² Labeling, transport, staining, and storage of these small, breakable smears present many problems.

This technique requires that a small drop of blood or bone marrow be placed on one clean coverslip (22 × 22 mm) and another coverslip be placed on top, with the blood allowed to spread across the two coverslips. One is then pulled across the other to create two thin films, one film on each coverslip (Figure 15-5). The two films can be stained and mounted on a 1-inch × 3-inch glass slide. When bone marrow smears are made using this technique, very slight pressure is applied to the coverslips between the index finger and thumb to help spread the bone marrow spicule before the two smears are pulled apart (this is sometimes called a crush preparation). Refining the skills required to make high-quality crush preparation bone marrow smears takes practice. Too much pressure on the coverslips causes cell rupture, which makes morphologic evaluation impossible. Inadequate pressure prevents the spicule from spreading to a satisfactory monolayer. Bone marrow crush preparations similar to this can be made using regular glass slides instead of coverslips, and the smears are of equal quality.

Figure 15-5 Coverslip method of making a peripheral blood film.

Automated Slide Making and Staining

The Sysmex SP-1000i (Sysmex Corporation of America, Mundelein, Ill.) is an automated slide making and staining system. After the instrument has performed a CBC for a specimen, a conveyor moves the racked tube to the SP-1000i, where the barcode is read. The computer already has determined from the automated results whether a slide is required. Criteria for a manual slide review are determined by each laboratory based on its patient population. Dependent on the hematocrit reading, the system adjusts the size of the drop of blood used and the angle and speed of the spreader slide in making a wedge preparation. After each blood film is prepared, the spreader slide is automatically cleaned and is ready for the next blood film to be made. Films can be produced approximately every 30 seconds. Information such as name, number, and date for the specimen is printed on the slide. The slide is dried, loaded into a cassette, and moved to the staining position, where stain and then buffer and rinse are added at designated times. When staining is complete, the slide is moved to a dry position, then to a collection area where it can be picked up for microscopic evaluation. Smears made off-line, such as bone marrow smears and cytospin preparations, may be stained using this system as well. Other blood analyzer manufacturers, such as Beckman Coulter (Brea, Calif.), also have automated slide making and staining instruments (Figure 15-6) (see Chapter 39).

Figure 15-6 COULTER LH slide maker and stainer. (Courtesy Beckman Coulter, Inc., Brea, Calif.)

Drying of Films

Regardless of film preparation method, before staining, all blood films and bone marrow smears should be dried as quickly as possible to avoid drying artifact. In some laboratories, a small fan is used to facilitate drying. Blowing breath on a slide is counterproductive, because the moisture in breath causes RBCs to become echinocytic (crenated) or to develop water artifact (also called drying artifact) (see later).

Staining of Peripheral Blood Films

Pure Wright stain or a Wright-Giemsa stain (Romanowsky stain)⁸ is used for staining peripheral blood films and bone marrow smears. These are considered polychrome stains because they contain both eosin and methylene blue. Giemsa stains also contain methylene blue azure. The purpose of staining blood films is simply to make the cells more visible and to allow their morphology to be evaluated.

Methanol in the stain fixes the cells to the slide. Actual staining of cells or cellular components does not occur until the buffer is added. The oxidized methylene blue and eosin form a thiazine-eosinate complex, which stains neutral components. The buffer that is added to the stain should be 0.05 M sodium phosphate (pH 6.4) or aged distilled water (distilled water placed in a glass bottle for at least 24 hours; pH 6.4 to 6.8). Staining reactions are pH dependent. Free methylene blue is basic and stains acidic (and basophilic) cellular components, such as ribonucleic acid (RNA). Free eosin is acidic and stains basic (and eosinophilic) components, such as hemoglobin and eosinophilic granules. Neutrophils are so named because they have cytoplasmic granules that have a neutral pH and pick up some staining characteristics from both stains. The slides must be completely dry before staining, or the thick part of the blood film may come off of the slide in the staining process.

Water or drying artifact has been a long-lived nuisance to hematology laboratories. It has several appearances. It can give a moth-eaten look to the RBCs or it may appear as a heavily demarcated central pallor. It also may appear as refractive (shiny) blotches on the RBCs. Other times, it manifests simply as echinocytes (crenation) seen in the areas of the slide that dried most slowly.

Multiple factors contribute to this problem. Humidity in the air as the slide dries may add to the punched-out, moth-eaten, or echinocytic appearance of the RBCs. It is difficult to avoid drying artifact on films from extremely anemic patients because of the very high ratio of plasma to RBCs. Water absorbed from the air into the alcohol-based stain also can contribute. Drying the slide as quickly as possible helps, and keeping a stopper tightly on the stain bottle keeps moisture out. In some laboratories, slides are fixed in pure, anhydrous methanol before staining to help reduce water artifact. More recently, stain manufacturers have used 10% volume-to-volume methanol to minimize water or drying artifact.

Wright Staining Methods

Manual Technique

Traditionally, Wright staining has been performed over a sink or pan with a staining rack. Slides are placed on the rack, film side facing upward. The Wright stain may be filtered before use or poured directly from the bottle through a filter onto the slide. It is important to flood the slide completely. The stain should remain on the slide at least 1 to 3 minutes to fix the cells to the glass. Then an approximately equal amount of buffer is added to the slide. Surface tension allows very little of the buffer to run off. The laboratory professional can blow gently on the slide to mix the aqueous buffer and the alcohol stain. A metallic sheen (or green “scum”) should appear on the slide if mixing is correct (Figure 15-7). More buffer can be added if necessary. The mixture is allowed to remain on the slide for 3 minutes or more (bone marrow smears take longer to stain than peripheral blood films). The timing may be adjusted to produce the best staining characteristics. When staining is complete, the slide is rinsed with a steady but gentle stream of neutral-pH water, the back of the slide is wiped to remove any stain residue, and the slide is air-dried in a vertical position. Coverslip-type blood films must be stained by the manual method. These films are placed on evacuated test tube rubber stoppers on the staining rack over a sink (Figure 15-8).

Figure 15-7 Manual Wright staining of slides. Note metallic sheen of stain indicating proper mixing.

Use of the manual Wright staining technique is desirable for staining peripheral blood films containing very high WBC counts, such as the films from leukemic patients. As with bone marrow smears, the time can be lengthened easily to enhance the staining required by the increased numbers of cells. Understaining is common when a leukemia slide is placed on an automated slide stainer. The main disadvantages of the manual technique are the increased risk of spilling the stain and the longer time required to complete the procedure. This technique is best suited for low-volume laboratories.

Automated Slide Stainers

Numerous automated slide stainers are commercially available. For high-volume laboratories, these instruments are essential. Once they are set up and loaded with slides, staining proceeds without operator attention. In general, it takes about 5 to 10 minutes to stain a batch of slides. The processes of fixing/staining and buffering are similar in practice to those of the manual method. The slides may be automatically dipped in stain and then in buffer and a series of rinses (Midas II, EM Science, Gibbstown, N.J. [Figure 15-9]) or propelled along a platen surface by two conveyor spirals (Hema-Tek, Siemens Healthcare Diagnostics, Inc., Deerfield, Ill. [Figure 15-10]). In the Hema-Tek device, stain, buffer, and rinse are pumped through holes in the platen surface, flooding the slide at the appropriate time. Film quality and color consistency are usually good with any of these instruments. Some commercially prepared stain, buffer, and rinse packages do vary from lot to lot or manufacturer to manufacturer, so testing is recommended. Some disadvantages of the dip-type batch stainers are that (1) stat slides cannot be added to the batch once the staining process has begun, and (2) working or aqueous solutions of stain are stable for only 3 to 6 hours and need to be made often. Stat slides can be added at any time to the Hema-Tek stainer, and stain packages are stable for about 6 months.