evaluating blood films evaluating blood films - Hungarovet

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FIGURE 1Good staining technique is criticalto identifying polychromatophilson a peripheral blood film.1. The steps in preparing a blood film. A small dropof blood is placed near the end of a slide (top). Aspreader slide is drawn back into the blood dropat a 30-degree angle (middle). Then the spreaderslide is pushed away from the blood drop,creating a uniform film across the slide (bottom).Producing high-quality blood films begins byusing clean, new slides. Used slides frequentlyhave imperfections such as scratchesand other physical defects. Slides must befree of fingerprints, dust, alcohol, detergents,and debris. Using a microhematocrit tube,place a small drop (2 to 3 mm in diameter)of well-mixed EDTA blood about 1 to 1.5 cmfrom the end of the slide. Next, draw aspreader slide back into the blood drop atabout a 30-degree angle until the spreaderslide edge contacts the sample drop and capillaryaction disperses the sample along theedge. Then, using a smooth steady motion,push the spreader slide away from the blooddrop, creating a uniform film that coversnearly the entire length of the slide (Figure1). Changing the angle of the spreader slidewill change the length and thickness of theblood film. For blood samples with lowhematocrits (severe anemia), you may needto decrease the angle of the spreader slide tomake a good-quality slide. In contrast, forsamples with high hematocrits (severe dehydrationand polycythemia due to a variety ofconditions), you may need to increase theangle of the spreader slide.Drying is an important step in the productionof good-quality blood films. Allow theblood film to thoroughly air-dry before applyingstain, or use a heat block (at the low setting)or a hair dryer to hasten the drying time,thus minimizing refractile markings that candistort erythrocyte morphology. In addition,keep formalin and formalin-containing containersaway from all blood and cytologysmears to prevent staining artifacts such as increasedcytoplasmic granularity and basophilia.Staining the blood filmMost veterinary practices use a modifiedWright’s stain (Romanowsky stain) for bothhematology and cytology. Modified Wright’sstains are available as either three- or two-solutionkits. We prefer kits with three separatesolutions: an alcohol fixative, an eosinophilicstaining solution, and a dark-blue stainingsolution. Veterinary practices should havetwo separate Coplin stain jar sets—one forhematology and cytology samples and onefor contaminated samples such as those collectedfor ear and fecal cytology. For optimalresults, replace staining solutions regularly.Recommended blood staining protocolincludes dipping the air-dried slide five to10 times in each solution while blotting oneedge briefly in between each solution. Rinsethe slide with distilled water after the finalstaining step, and allow the blood film to airdrybefore examination. Good staining techniqueis critical to identifying polychromatophilson a peripheral blood film. If thestaining is done improperly, many of thequick Romanowsky-type stains will not givethe needed tinctorial differences between matureRBCs (orange-red) and polychromatophils(bluish-pink). Typically, poor staining withthe various quick stains results in all RBCs havinga bluish or muddy tint.Evaluating the blood filmThe microscopeA high-quality microscope is essential forhematology. We recommend a binocular microscopewith a minimum of 10, 20, and100 (oil-immersion) objectives and widefield10 oculars. When evaluating the slide,make sure maximum light reaches the bloodfilm. This means positioning the substage condenseras close as possible to the stage withthe iris wide open. The light source should beequipped with a variable rheostat to allowmaximal control of light intensity. To ensureoptimal focused light for the microscopic evaluation,the microscope should be in Köhler il-Feathered edgeMonolayer MonolayerBody of blood filmApplication point2. The components of the peripheral blood film slide.FIGURE 2Label4
FIGURE 3 FIGURE 4 FIGURE 5Patient IDPatient IDPatient IDlumination. Contact your microscope vendor,or review the microscope manual to ensureproper illumination.The three zonesA good-quality blood film has threezones: the body (near the point of blood application),the monolayer (the zone betweenthe body and feathered edge), and the featherededge (the area most distant from thepoint of application) (Figure 2). The definitionof the monolayer varies from institutionto institution, but the definition used to ensureconsistent semiquantification of variousmorphologic abnormalities is the area whereabout half of the RBCs are touching one anotherwithout overlapping. The monolayer isthe area of the blood film where WBCs andplatelet numbers are estimated and cell morphologyis examined. Although the monolayeris the only zone where morphologicevaluation of individual cells is performed atoil-immersion magnification, systematicblood film evaluation includes assessing ofall three zones.First, examine the blood film at low magnification(10 or 20), and scan the entireslide to evaluate overall film thickness, celldistribution, and differentiation of the threedifferent zones. Evaluate the feathered edge(Figure 3) for microfilariae, phagocytized organisms,atypical cells, and platelet clumping.Next, examine the body of the blood film(Figure 4 ) for rouleau formation or RBC agglutination.Then, still using low magnification,evaluate the monolayer (Figure 5), estimatethe total WBC count (see the third article in thissymposium), and predict the expected WBCdifferential count. Finally, use the oil-immersionobjective (100) to examine RBC, WBC,and platelet morphology in the monolayer.Evaluate RBCs for evidence of anisocytosis,poikilocytosis, polychromasia, hemoglobinconcentration, and RBC parasites. ImportantRBC morphologic abnormalities include spherocytes,schistocytes, acanthocytes, and leptocytes(see the second article in thissymposium).Evaluate neutrophils for toxicity and thepresence or absence of a left shift (increasednumbers of band neutrophils). Evaluate lymphocytesfor reactivity and monocytes forphagocytized organisms (see the third articlein this symposium).With relatively little practice, you canidentify most important morphologic abnormalitiesin the RBCs, WBCs, and platelets withthe 20 objective field of view; these can bequickly validated with the 100 oil-immersionview.SummaryExamining a properly prepared peripheralblood film offers invaluable informationabout cellular morphologic changes not providedby automated instruments and providesa quality assurance confirmation of CBCdata generated by in-clinic or reference laboratoryhematology analyzers. We recommendtaking about three minutes to view the bloodfilm and, using the questions discussed in thenext two articles of this symposium, to systematicallyevaluate the RBC, platelet, and WBCcomponents of the peripheral blood film.ACKNOWLEDGMENTThe authors gratefully acknowledge the technicalassistance and images provided by Dennis DeNicola,DVM, PhD, DACVP.3. Look for platelet clumps, microfilariae, and large cellsin the feathered edge of the film ( purple section).4. Look for rouleau formation and RBC agglutination inthe body of the film ( purple section).5. Estimate platelet and WBC counts and examine cellmorphology in the monolayer zone ( purple section).REFERENCES1. Willard, M. et al.: The complete blood countand bone marrow examination. Textbook ofSmall Animal Clinical Diagnosis by LaboratoryMethods, 3rd Ed. W.B. Saunders, Philadelphia,Pa., 1999; pp 11-30.2. Rebar, A.; Metzger, F.: The Veterinary CE Advisor:Interpreting Hemograms in Cats and Dogs.Vet. Med. (suppl.) 96 (12):1-12; 2001.5
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