Show Preparing Blood SmearsIf you are using venous blood, blood smears should be prepared as soon as possible after collection (delay can result in changes in parasite morphology and staining characteristics). Thick smearsThick smears consist of a thick layer of dehemoglobinized (lysed) red blood cells (RBCs). The blood elements (including parasites, if any) are more concentrated (app. 30×) than in an equal area of a thin smear. Thus, thick smears allow a more efficient detection of parasites (increased sensitivity). However, they do not permit an optimal review of parasite morphology. For example, they are often not adequate for species identification of malaria parasites: if the thick smear is positive for malaria parasites, the thin smear should be used for species identification. Prepare at least 2 smears per patient!
Scratch Method for Thick smearsThe scratch method is an alternate method for making thick films that allows for improved adherence and faster turnaround times. The process is similar to making a normal thick film, but instead of using a stick to spread the blood, the edge of a glass microscope slide is used, while applying firm pressure to create small scratches in the underlying slide. The scratches allow for improved adherence of the blood film to the slide without affecting the smear morphology. The smear can then be stained as soon as it is dry, generally within 20-30 minutes of smear preparation. Reference: Norgan AP, Arguello HE, Sloan LM, Fernholz EC, Pritt BS. Malaria Journal 2013; 12: 231. Thin smearsThin smears consist of blood spread in a layer such that the thickness decreases progressively toward the feathered edge. In the feathered edge, the cells should be in a monolayer, not touching one another. Prepare at least 2 smears per patient!
Note: Under field conditions, where slides are scarce, national malaria programs (and CDC staff) prepare both a thick and a thin smear on the same slide. This works adequately if one makes sure that of the two smears, only the thin smear is fixed. Special Procedures for Detecting MicrofilariaeBlood microfilariae:
For additional information on making blood smears, call the Division of Parasitic Diseases at (404) 718-4110. Reference:Eberhard ML, Lammie PJ. Laboratory diagnosis of filariasis. Clin Lab Med 1991;11:4.
Blood for hematologic testing must be collected into an anticoagulant, preferably EDTA (purple top tube). Blood smears should be made at the time of collection to minimize storage-associated changes, which invariably occur. Venipuncture should be minimally traumatic to minimize platelet activation and should be done using a minimum of a 23G needle (21-22G is ideal for small animals and 18-20G is ideal for large animals) and 3-5 mL syringe (depending on the desired volume). Vacutainers can be used for sample collection, as can short butterfly catheters. For very small or pediatric patients, samples can be placed into microtainer tubes. Regardless of the tube used for sample collection, the blood must be adequately mixed with the anticoagulant during and after collection to prevent clotting. This is done by gentle inversion of the tube (tubes should never be shaken). View other sample collection pages specific for hemostasis, chemistry, urinalysis and cytology. Sample collection and handling guidelinesAll samples should be kept cool (at refrigerated temperatures, but not frozen) during storage and shipping to minimize changes in cells that can occur with storage. The sample should be wrapped in paper towels to prevent direct contact of the tube with ice, which will result in freezing and lysis of red and white blood cells within the tube. Also, blood smears should be made from freshly collected blood and submitted along with the tubes to help facilitate blood smear examination and ensure provision of the most accurate results. The blood in the tube should be kept cold, however slides should be stored and shipped at room temperature. Both tubes and slides should be placed in break-proof containers for shipping. The following anticoagulants can be used for hematologic testing, however EDTA (purple top) is the preferred anticoagulant.
ArtifactsArtifacts in blood samples for hematologic testing stem mainly from either sample collection, aging of sample, or poor maintenance of the staining solutions (stain precipitate and water artifact). See also a summary of some of these artifacts. Collection artifactsThese are associated with problems with sample collection. The most common problems are:
Storage artifactsStorage of blood can result in many false changes in hematology results. These changes are minimized but not eliminated by cold storage (refrigerated, shipping on ice packs). Ensure that the slides are maintained at room temperature and are not stored cold or placed in direct contact with ‘”cool packs”, as the slide can freeze or become moist, thus rupturing the cells. Artifacts of specimen handling and preparation can significantly impair examination of blood cells, but are easily avoided. If a delay in analysis is anticipated (e.g., lab closed or when sending a sample to a reference lab), smears should be made and sent along with the EDTA tube. Air-dried unfixed smears hold up very well unless subjected to flies or moisture. In vitro aging of blood cells in the specimen tube causes changes in the appearance of the cells on stained blood films. Eventually, cells will break down altogether, rendering the specimen useless for analysis. Shown on the right is a smear from a sample which had been in EDTA for 48 hours at room temperature. Extreme crenation of the erythrocytes is evident, which could easily mask or render suspect significant pathologic shape abnormalities. The leukocyte nucleus has undergone pyknosis and karyorrhexis, making certain identification impossible. Age-related changes that occur are:
Stain artifactsDiff-Quik®, Hemacolor®, and other commonly used quick stains for hematology and cytology can provide good staining quality if properly used and maintained:
Common stain-related artifacts are water artifact and stain precipitate.
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