paper into a brown bottle.
4. Label and store protected from light; the shelf life is 36 months, provided that results are within quality control guidelines.
The staining procedure requires phosphate buffer solutions (see directions in the preceding discussion of Giemsa stain); the pH required for Wright’s stain is 6.6 to 6.8.
Procedure for Staining Thin Films
Since Wright’s stain contains alcohol, the slides do not require fixation before staining.
1. Place a slide on a rack in a horizontal, level position, and cover the surface with stain.
2. Count the number of drops of stain needed to cover the surface. Let stand for 1 to 3 min (the optimal staining time varies with each batch of stain).
3. Add an equal number of drops of phosphate-buffered water to the slide; mix the stain and buffer by blowing on the surface of the fluid.
4. After 4 to 8 min, flood the stain from the slide with phosphate buffer. Do not pour the stain off before washing. If you do, a precipitate will be deposited on the slide.
5. Wipe the bottom of the slide to remove excess stain.
6. Allow the slide to drain and air dry.
Procedure for Staining Thick Films
Thick films stained with Wright’s stain are usually inferior to those stained with Giemsa solution. Great care should also be taken to avoid excess stain precipitate on the slide during the final rinse. Before being stained, thick films must be laked in distilled water (to rupture and remove RBCs) and air dried. The staining procedure is the same as for thin films, but the staining time is usually somewhat longer and must be determined for each batch of stain.
Results
Wright’s stain colors blood components as follows: RBCs, light tan, reddish, or buff; nuclei of WBCs, bright blue with contrasting light cytoplasm; eosinophilic granules, bright red; and neutrophilic granules, pink or light purple.
If malaria parasites are present, the cytoplasm stains pale blue and the nuclear material stains red. Schüffner’s dots and other inclusions in the RBCs usually do not stain or stain very pale with Wright’s stain. Nuclear and cytoplasmic staining characteristics of the other blood parasites such as Babesia spp., trypanosomes, and leishmaniae are like those of the malaria parasites. While the sheath of microfilariae may not always stain with Wright’s stain, the nuclei within the microfilaria itself stain pale to dark blue.
General Notes on Staining Procedures
1. When large numbers of slides are being processed, remember that dry films should be stored in dust-free containers before staining, to protect fresh smears from insects.
2. If the slides cannot be stained within 48 h, thin films should be fixed in methyl alcohol and thick films should be laked in distilled water before storage (some users prefer buffered water at pH 6.8 to 7.2). Rapid stains can also be used. Parasites will stain like the WBCs.
3. Slides should be stored at reasonably low temperatures (below 80°F [ca. 27°C]) before being stained. If the slides are exposed to high temperatures, thick films that have not been laked will become heat fixed; hemoglobin will remain fixed in the RBCs, and heavy stain retention will then prevent parasite identification. Thin films also stain poorly after exposure to high temperatures.
4. Fresh working stain should be prepared just before use. If a large number of thick films are being laked during staining, the stain should be changed after 50 slides because of the accumulation of excess hemoglobin.
Proper Examination of Thin and Thick Blood Films
In any examination of thin blood films for parasitic organisms, the initial screen should be carried out with the low-power objective of a microscope. Microfilariae may be missed if the entire thin film is not examined. Microfilariae are rarely present in large numbers, and frequently only a few organisms occur in each thin-film preparation. Microfilariae are commonly found at the edges of the thin film or at the feathered end of the film, because they are carried to these sites during the process of spreading the blood. The feathered end of the film where the RBCs are drawn out into one single, distinctive layer of cells should be examined for the presence of malaria parasites and trypanosomes. In these areas, the morphology and size of the infected RBCs are most clearly seen.
Depending on the training and experience of the microscopist, examination of the thin film usually takes 5 to 10 min or less (200 to 300 oil immersion fields) at a magnification of ×1,000. Although some people use a 50× or 60× oil immersion objective to screen stained blood films, there is some concern that small parasites such as plasmodia, Babesia spp., or L. donovani may be missed at this smaller total magnification (×500 or ×600) compared with the ×1,000 total magnification obtained with the more traditional 100× oil immersion objective. Because people tend to scan blood films at different rates, it is important to examine a minimum number of fields, regardless of the time it takes to perform this procedure. If something suspicious has been seen in the thick film, the number of fields examined on the thin film is often considerably more than 200 to 300. The request for blood film examination should always be considered a STAT procedure, with all reports (negative as well as positive) being reported immediately to the physician as soon as possible. Appropriate governmental agencies (local, state, and federal) should be notified within a reasonable time frame in accordance with guidelines and laws.
Diagnostic problems with the use of automated differential instruments have been reported (13, 14). Both malaria and Babesia light infections (like those seen with travelers with no prior exposure to malaria) were missed with these instruments, and therapy was delayed. Although these instruments are not designed to detect intracellular blood parasites, the inability of the automated systems to discriminate between uninfected RBCs and those infected with parasites may pose serious diagnostic problems.
In the preparation of a thick blood film, the greatest concentration of blood cells will be in the center of the film. A search for parasitic organisms should be carried out initially at low magnification to detect microfilariae more readily. Examination of a thick film usually requires 3 to 5 min (approximately 100 oil immersion fields). Search for malarial organisms and trypanosomes is best done under oil immersion (total magnification, ×1,000). Intact RBCs are frequently seen at the very periphery of the thick film; such cells, if infected, may prove useful in malaria diagnosis, since they may demonstrate the characteristic morphology necessary to identify the organisms to the species level.
It is important to report the level of parasitemia when blood films are reviewed and found to be positive for malaria parasites. Because of the potential for drug resistance in some of the Plasmodium species, particularly P. falciparum and P. vivax, it is important that every positive smear be assessed and the parasitemia reported exactly the same way on follow-up specimens as on the initial specimen. This allows the parasitemia to be monitored after therapy
