"In December 2003, we took delivery of a LaserCyte® and have since completed all our haematology monitoring in-house. This has been of particular benefit whilst monitoring chemotherapy patients as well as being speedy and reliable support for the general veterinary surgery. I find the LaserCyte exceedingly useful and would feel our practice was losing a good friend should we no longer have it on the bench."

Roger Baker, BSc, BvetMed, MRCVS
Roger Baker's Veterinary Surgery and
Cancer Treatment Unit
Kent, UK

Visit IDEXX representatives at AAHA to learn about two new products:
IDEXX VetStat® Electrolyte and Blood Gas Analyzer IDEXX Urine P:C Ratio
Event:	Amercian Animal Hospital Association Conference
When:	Exhibition is open March 20–22, 2005
Where:	Baltimore, Maryland
Booth:	623

PEER-REVIEWED
Three-minute peripheral blood film evaluation: Preparing the film
No single hematology procedure produces more valuable information yet requires so little time and expense than a peripheral blood film evaluation. Proper preparation and staining of the film are critical.

A PERIPHERAL BLOOD FILM evaluation should be part of all complete blood counts (CBCs), regardless of whether hematology is performed in-house or at a reference laboratory. Proper sample collection, slide preparation, and staining are essential to accurately evaluate a blood film, as is the correct use of a high-quality microscope. This article describes the steps in preparing a blood film and the equipment you'll need.

Components of an indication for a CBC
Important components of a CBC include evaluating the erythron (hematocrit, total red blood cell [RBC] count, hemoglobin concentration, absolute reticulocyte count, and RBC indices), the leukon (total white blood cell [WBC] count, five-part differential count including immature neutrophils), the thrombon (platelet count and platelet indices), and the total protein concentration. As mentioned earlier, always include a peripheral blood film evaluation in the CBC. A CBC should be included in evaluations of every sick patient, every patient with vague signs of disease, and every patient receiving long-term medications. In addition, a CBC should be performed as part of every preanesthetic workup, for adult wellness and geriatric profiles, and as a recheck test for patients in which RBC, WBC or platelet abnormalities were previously diagnosed.

Collecting a sample for the blood film
Artifacts must be avoided for proper hematologic interpretation. Causes of artifacts include poor blood collection techniques, inadequate sample volumes, prolonged sample storage, and delayed sample analysis. Proper blood collection is vital to prevent erroneous results from sample clotting and cellular lysis. Obtain hematology samples from the largest blood vessel possible to minimize cellular trauma and to prevent the activation of clotting mechanisms. For accurate results, discard clotted samples and collect fresh samples. Common venipuncture sites in dogs and cats include the jugular, cephalic, and lateral and medial saphenous veins. Anticoagulants include EDTA, heparin and citrate. EDTA is the preferred anticoagulant for blood film preparation because it preserves cellular detail better than other anticoagulants do and does not interfere with Romanowsky staining of WBCs.

Inadequate sample volume is a common cause of inaccurate hematologic results. Properly fill anticoagulated blood collection tubes to avoid falsely decreased hematocrits and cell counts and to prevent RBC shrinkage. Hematologic samples must be analyzed as soon as possible to prevent artifacts created by exposure to anticoagulants and cell deterioration due to storage and shipment. Analyze samples within three hours or refrigerate them at 39.2°F (4°C) to avoid an artificially increased hematocrit, increased mean corpuscular volume, and decreased mean corpuscular hemoglobin concentration.¹ Prepare blood films within one hour of collection to avoid morphologic artifacts. RBC crenation, neutrophil hypersegmentation, lymphocytic nuclear distortion and general WBC degeneration, including vacuolization in neutrophils, may occur in aged samples. In addition, monocyte vacuolization, monocyte cytoplasmic pseudopod formation and platelet agglutination can be encountered in stored samples.² If you use a reference laboratory for primary hematologic analyses, we recommend submitting freshly prepared blood films along with the anticoagulated blood.

Clearly, gauging the appropriateness of the marrow's response to an anemia requires both an accurate history and sequential CBCs. For example, during the first 2–3 days of any anemia, regenerative or nonregenerative, the erythrogram would be classified as nonregenerative simply because there had been insufficient time for reticulocytosis to develop.

Preparing the blood film
Producing high-quality blood films begins by using clean, new slides. Used slides frequently have imperfections such as scratches and other physical defects. Slides must be free 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 cm from the end of the slide. Next, draw a spreader slide back into the blood drop at about a 30-degree angle until the spreader slide edge contacts the sample drop and capillary action disperses the sample along the edge. Then, using a smooth steady motion, push the spreader slide away from the blood drop, creating a uniform film that covers nearly the entire length of the slide (Figure 1). Changing the angle of the spreader slide will change the length and thickness of the blood film. For blood samples with low hematocrits (severe anemia), you may need to increase the angle of the spreader slide to make a good-quality slide. In contrast, for samples with high hematocrits (severe dehydration and polycythemia due to a variety of conditions), you may need to decrease the angle of the spreader slide. Drying is an important step in the production of good-quality blood films. Allow the blood film to thoroughly air-dry before applying stain, or use a heat block (at the low setting) or a hair dryer to hasten the drying time, thus minimizing refractile markings that can distort erythrocyte morphology. In addition, keep formalin and formalin-containing containers away from all blood and cytology smears to prevent staining artifacts such as increased cytoplasmic granularity and basophilia.

Staining the blood film
Most veterinary practices use a modified Wright's stain (Romanowsky stain) for both hematology and cytology. Modified Wright's stains are available as either three- or two-solution kits. We prefer kits with three separate solutions: an alcohol fixative, an eosinophilic staining solution and a dark-blue staining solution. Veterinary practices should have two separate Coplin stain jar sets—one for hematology and cytology samples and one for contaminated samples such as those collected for ear and fecal cytology. For optimal results, replace staining solutions regularly. Recommended blood-staining protocol includes dipping the air-dried slide five to 10 times in each solution while blotting one edge briefly in between each solution. Rinse the slide with distilled water after the final staining step, and allow the blood film to air-dry before examination. Good staining technique is critical to identifying polychromatophils on a peripheral blood film. If the staining is done improperly, many of the quick Romanowsky-type stains will not give the needed tinctorial differences between mature RBCs (orange-red) and polychromatophils (bluish-pink). Typically, poor staining with the various quick stains results in all RBCs having a bluish or muddy tint.

Evaluating the blood film
The microscope
A high-quality microscope is essential for hematology. We recommend a binocular microscope with a minimum of 10X, 20X and 100X (oil-immersion) objectives and widefield 10X oculars. When evaluating the slide, make sure maximum light reaches the blood film. This means positioning the substage condenser as close as possible to the stage with the iris wide open. The light source should be equipped with a variable rheostat to allow maximal control of light intensity. To ensure optimal focused light for the microscopic evaluation, the microscope should be in Köhler illumination. Contact your microscope vendor, or review the microscope's manual to ensure proper illumination.

The three zones
A good-quality blood film has three zones: the body (near the point of blood application), the monolayer (the zone between the body and feathered edge), and the feathered edge (the area most distant from the point of application) (Figure 2). The definition of the monolayer varies from institution to institution, but the definition used to ensure consistent semiquantification of various morphologic abnormalities is the area where about half of the RBCs are touching one another without overlapping. The monolayer is the area of the blood film where WBCs and platelet numbers are estimated and cell morphology is examined. Although the monolayer is the only zone where morphologic evaluation of individual cells is performed at oil-immersion magnification, systematic blood film evaluation includes assessing of all three zones.

First, examine the blood film at low magnification (10X or 20X), and scan the entire slide to evaluate overall film thickness, cell distribution and differentiation of the three different zones. Evaluate the feathered edge (Figure 3) for microfilaria, 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), estimate the total WBC count (see the third article in this symposium), and predict the expected WBC differential count. Finally, use the oil-immersion objective (100X) to examine RBC, WBC and platelet morphology in the monolayer. Evaluate RBCs for evidence of anisocytosis, poikilocytosis, polychromasia, hemoglobin concentration and RBC parasites. Important RBC morphologic abnormalities include spherocytes, schistocytes, acanthocytes and leptocytes (see the second article in this symposium). Evaluate neutrophils for toxicity and the presence or absence of a left shift (increased numbers of band neutrophils). Evaluate lymphocytes for reactivity and monocytes for phagocytized organisms (see the third article in this symposium). With relatively little practice, you can identify most important morphologic abnormalities in the RBCs, WBCs and platelets with the 20X objective field of view; these can be quickly validated with the 100X oil-immersion view.

Summary
Examining a properly prepared peripheral blood film offers invaluable information about cellular morphologic changes not provided by automated instruments and provides a quality assurance confirmation of CBC data generated by in-clinic or reference laboratory hematology analyzers. We recommend taking about three minutes to view the blood film and, using the questions discussed in the next two articles of this symposium, to systematically evaluate the RBC, platelet and WBC components of the peripheral blood film.

ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance and images provided by Dennis DeNicola, DVM, PhD, DACVP.

© 2004 Veterinary Medicine Magazine, Volume 99, Number 12. All rights reserved. Originally published as, Three-minute peripheral blood film evaluation: Preparing the film. Used by IDEXX Laboratories with permission.

Watch for the second and third articles in this symposium in upcoming issues of Diagnostic Edge...

Three-minute peripheral blood film evaluation: Preparing the film
Fred L. Metzger, Jr. and Alan Rebar
When examining the red blood cells and platelets in a blood film, ask yourself these questions to quickly identify and further characterize conditions such as anemia and thrombocytopenia.

Three-minute peripheral blood film evaluation: The leukon
Fred L. Metzger, Jr. and Alan Rebar
Taking a moment to briefly examine white blood cells will help you identify conditions such as inflammation or stress that may indicate serious disease.

The recommendations contained in Diagnostic Edge educational materials are intended to provide general guidance only. As with any diagnosis or treatment, you should use clinical discretion with each patient based on a complete evaluation of the patient, including physical presentation and complete laboratory data. With respect to any drug therapy or monitoring program, you should refer to product inserts for a complete description of dosages, indications, interactions and cautions.

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SNAP® Giardia Test for dogs and cats

Remember learning the Giardia life cycle?

Want a refresher? See it come to life in an informative animation that will also show you how the new SNAP® Giardia assay takes advantage of an excreted cyst wall protein to enhance your ability to accurately detect this pervasive pathogen.

To find out more information on the SNAP Giardia Test for dogs and cats, visit www.idexx.com/snap.

Celebration greets 2,500^th IDEXX LaserCyte® Hematology Analyzer customer

The celebration was on in Martinez, California, as IDEXXers joined Martinez Animal Hospital staff to celebrate the 2,500^th installation of the IDEXX LaserCyte® Hematology Analyzer.

The IDEXX team brought champagne, flowers and balloons to the clinic, where they toasted the happy occasion. "They were ecstatic," said Brad Brazell, Senior Marketing Program Manager, who flew to California for the event. "They were as happy to celebrate the 2,500^th placement as we were."

Photographed from left to right are Shelley Lagomarsino, Dona Hoffman, Teri Nunes, Elana Easter, Dr. René Butler (holding Goldie), Pam Soberanes (holding Stanley), Dr. Stacy Enke (holding Dixie), and Brad Brazell, Senior Marketing Program Manager at IDEXX Laboratories.

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Education and Events
We offer a variety of seminars and teleconferences about emerging trends and best practices in veterinary diagnostics—in a forum designed to involve, educate and motivate you and your staff.

• Visit the seminar and teleconference calendar and click the date to view the details.
• Fill out and submit the form to register.

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As part of the LaserCyte® version 1.30 software upgrade, interpretive guides for many of the LaserCyte hematology parameters and VetTest analytes are now available.

The interpretive guides offer valuable background information regarding the individual parameters of the test results. This information can be particularly helpful in explaining diagnostic results to your clients. For example, the diagnostic implications of a low hematocrit level can be explored by clicking the HCT link on the LaserCyte touch screen. All parameters having interpretive guides are underlined on the Test Results screen on the LaserCyte touch screen. The diagram shows an example of some of the hematocrit information available in the interpretive guides.

To access the interpretive guides:

1. Tap Records on the main screen, select the desired patient and tap OK.
2. On the Select Results screen, select the desired test results and tap OK.
3. On the Test Results screen, tap an underlined parameter to view its interpretive guide.
4. Tap Print Page if you want to print the information.
5. Tap Close Window to close the interpretive guide and return to the Test Results screen.

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Calling All Qualified Urine Protein:Creatinine/Renal Disease Case Studies

Do you have a case study in which a urine protein:creatinine ratio helped you detect renal disease? If so, you could win a copy of Renal Disease in Dogs and Cats by Dr. Jonathan Elliott and Dr. Scott Brown, just for sending us a qualified submission!

The case that best exemplifies how clinics can "practice what's possible" will be featured in a special edition case study booklet on renal disease and proteinuria.

Qualified submissions must include:

• The patient's name, signalment, history, physical examination, bloodwork and a complete urinalysis (including an IDEXX Urine P:C Ratio result)
• A diagnosis of renal disease (either primary or secondary)
• The name, address and telephone number of your clinic; and the names of your veterinarians and veterinary technicians
• Pictures, if possible

Limit one case per practice.

If you have any questions, please contact your IDEXX representative or call Dr. Michelle Kahn at 1-207-556-8589.

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