key: cord-0049927-j0ihw7c8 authors: Norman, Elizabeth J.; Barron, Ronnie C. J.; Nash, Andrew S.; Clampitt, Roger B. title: Prevalence of Low Automated Platelet Counts in Cats: Comparison with Prevalence of Thrombocytopenia Based on Blood Smear Estimation date: 2008-03-05 journal: Vet Clin Pathol DOI: 10.1111/j.1939-165x.2001.tb00422.x sha: dd359a7338e28ba38b6e1ede1d90a1a3cb531558 doc_id: 49927 cord_uid: j0ihw7c8 Abstract: True thrombocytopenia is uncommon in cats; however, low platelet counts frequently are found using automated cell counters. Although this discrepancy is a well known problem, the prevalence of low automated platelet counts in feline blood samples has not been documented. We retrospectively compared the prevalence of low automated platelet counts with low blood smear‐estimated platelet counts in feline blood samples. Results of blood sample analysis from 359 cats during a 1‐year period at the University of Glasgow Veterinary Haematology Laboratory were examined. Smear estimates of platelet number were done in those cases in which records did not indicate adequate platelet numbers. Platelet counts obtained with an impedance counter (Minos Vet, Abx Hematologie) were <200×10(9) cells/L in 256 samples (71%) and <50×10(9) cells/L in 43 samples (12%). However, based on estimation of platelet numbers from blood smears, only 11 samples (3.1%) had platelet counts of <200×10(9) cells/L and 9 samples (2.5%) had counts of <50×10(9) cells/L. Four cats with thrombocytopenia estimated by blood smear evaluation had clinical signs of a bleeding disorder. Disorders associated with thrombocytopenia included neoplasia, cytotoxic chemotherapy, and infectious diseases. There was no evidence that delay due to mailing of samples was associated with lower automated platelet counts than would have been obtained on the day of sampling. The high prevalence of apparent thrombocytopenia in automated platelet counts was attributed to a combination of platelet aggregation and the impedance method of cell differentiation by size. Vigilance and careful examination of blood smears is required to identify the few cats with true thrombocytopenia. Records of automated hematology counts for all feline blood samples on which complete blood counts were done at the University of Glasgow Veterinary Haematology Laboratory were retrospectively examined for the period from April 1, 1997, to March 31, 1998. Data were collected from the records and, if necessary, by examination of stored blood films. When an individual cat was sampled on more than 1 occasion, only the results from the first blood sample were included in the study. Blood was submitted in EDTA for hematologic analysis using any one of a variety of commercially available EDTA tubes. Samples collected from patients hospitalized at the University of Glasgow Veterinary Hospital (internal samples) were stored at room temperature until analyzed within 24 hours (in most cases within 8 hours). Samples also were received from veterinarians elsewhere by first-class mail (external samples) and were held at room temperature until analyzed on the day of receipt. Notation was made if clots were seen or if the amount of sample in the tube was grossly inadequate such that a disproportionately high concentration of EDTA would be present; these samples were excluded from the study. After thorough mixing of each blood sample on an automated mixer for 10 minutes, a complete automated blood count was performed using an impedance cell counter (Minos Vet, Abx Hematologie, Montpellier, France), which was maintained and calibrated as recommended by the manufacturer. A WBC differential count and smear cytologic analysis also was performed on each sample. Thin air-dried blood smears made after thorough mixing of the sample were stained with a modified May-Grünwald-Giemsa stain and examined under light microscopy. Platelet numbers were reported to be adequate when aggregates were seen or, subjectively, based on the experience of the laboratory technicians. Where the record did not note the results of smear evaluation for platelets, the slides were re-examined by one of us for platelet aggregates. If no aggregates were found, platelet count was estimated by averaging the number of platelets in 5 oil-immersion fields in the monolayer of the smear. An Olympus BX50 microscope was used with a ϫ100 oil-immersion lens and an ocular field number of 22. Mean platelet number per oilimmersion field was multiplied by a factor of 15.8 to give an approximate count ϫ10 9 cells/L. 5,6 Thrombocytopenia was defined as a platelet count of <200ϫ10 9 cells/L. Statistical analysis was performed using Minitab for Windows software (release 10.2, 1994, Minitab, State College, Penn). All counts were log transformed, and comparisons were made using an unpaired t-test. A P value of <.05 was considered significant. A total of 583 feline blood samples were submitted during the study period. Of these, records were incomplete in 14 cases for various reasons, including cancellation of the request by the submitting veterinarian. In another 4 samples, substantial underfilling of the EDTA tube was noted, and in 26 samples gross clotting of the sample had occurred, making the sample unsuitable for further analysis. These samples were excluded from the study, leaving a total of 539 samples from 359 cats, comprising 325 internal samples from 227 cats and 214 external samples from 132 cats. In 256 of the 359 cats sampled (71%), automated platelet counts indicated thrombocytopenia (<200ϫ10 9 cells/L) ( Figure 1 ). In 43 of these cats (12%), platelet counts were severely decreased (<50ϫ10 9 cells/L), and in 7 cats (1.9%) counts were <20ϫ10 9 cells/L. Based on evaluation of smears, 11 of 359 cats (3.1%) had platelet counts of <200ϫ10 9 cells/L. Platelet counts were markedly decreased (<50ϫ10 9 cells/L) in 9 cats (2.5%), and in 8 cats (2.2%) counts were <20ϫ10 9 cells/L. In all samples with blood smear-estimated thrombocytopenia, the automated platelet count was <200ϫ10 9 cells/L. In only 4 of the 11 samples was thrombocytopenia mentioned in the final hematology report to the submitting veterinarian. Of the 11 samples with blood smear-estimated thrombocytopenia, 4 cats had histories that suggested a hemostatic defect. In all 4 cats, both the automated and estimated platelet counts were <20ϫ10 9 cells/L. One of the cats had pemphigus foliaceus and was being treated with myelosuppressive drugs. One cat had feline immunodeficiency virus-related disease. In 2 cats, an underlying disease was not reported. Of the remaining 7 thrombocytopenic samples, 1 cat was receiving chemotherapy for lymphosarcoma, 1 cat had a positive feline coronavirus titer and intracranial disease of suspected nutritional origin, and 1 cat each had renal neoplasia, haemobartonellosis, and hepatic disease. In 2 cats an As has been previously reported, neoplasia and infectious diseases are the most common disorders in cats with thrombocytopenia. 1 In some cats, thrombocytopenia may be a component of disseminated intravascular coagulation (DIC); 38% of cats undergoing coagulation testing have been found to meet some or all of the diagnostic criteria for DIC. 7 No cases of immune-mediated thrombocytopenia were diagnosed during this 1year survey, in keeping with the low prevalence of this disease in cats. 1, 7 Automated platelet counts performed by an impedance counter were low in the majority of cats sampled (71%), whereas the prevalence of thrombocytopenia based on blood smear estimation was only 3.1%. Thus, apparent thrombocytopenia was a significant problem in automated counts using an impedance cell counter. Although automated counts were low in all cats with blood smear-estimated thrombocytopenia, the frequent occurrence of falsely low automated platelet counts meant that thrombocytopenia commonly was ignored. The impedance counting method, in which platelets and RBCs are differentiated by size alone, contributes to falsely low automated platelet counts in cats. With the Minos Vet analyzer, RBCs and platelets are analyzed concurrently in a single channel with a 50-µm-diameter aperture. The impedance generated by each particle passing through the sensing zone is plotted against the number of impulses (particles) for analysis. A fixed upper platelet and lower RBC threshold of 17.5 fL for cats has been determined by the manufacturer. In comparison, a threshold of 27.0 fL is used for dogs. In most cases, this threshold cuts the histogram at the trough between platelets and RBCs. However, the platelet and RBC histograms commonly overlap in cats, such that the threshold may exclude larger platelets from the platelet count and smaller RBCs from the RBC count. Feline platelets are larger than those of other species, with a mean volume of 11.0-18.1 fL. 8 Mean platelet volume (MPV) for dogs, pigs, and human beings is 7.6-8.3 fL. 4 The magnitude of error in counting platelets is much greater than for counting RBCs because of the difference in relative numbers of platelets and RBCs. Aggregation of platelets increases this error because clumped platelets appear to the cell counter as a single larger cell. Although other factors such as RBC microcytosis and schistocytosis also contribute to the problem, platelet aggregation is a far more frequent occurrence, affecting at least 50% of the feline blood samples analyzed during this 1-year period. Platelet aggregation occurred in 66.6% of blood samples collected from 48 healthy, anesthetized cats, 2 and in another study, aggregation-induced interference was found in 56% of 41 feline blood samples undergoing automated cell counting. 9 The use of optical cell counters in which platelets and RBCs are differentiated by their light scattering pattern would be expected to avoid errors associated with impedance counters. However, the light scattering pattern of a platelet aggregate is not the same as that of a single platelet, so aggregates are excluded from the platelet count by optical counters. 2 Manual counting of platelets also is affected by aggregation because individual platelets cannot be counted within aggregates either on smears or in a hemocytometer. Aggregation also is likely to lead to uneven distribution of platelets, with aggregates accumulating on the edges of smears and counting chambers. Hence, aggregation of feline platelets in vitro contributes to technical difficulties in platelet counting, and accurate counting of feline platelets depends on the absence of aggregates. Platelets are reactive cells that can be stimulated to aggregate by a variety of factors including substances released from activated platelets themselves such as adenosine diphosphate (ADP) and serotonin, circulating substances such as adrenalin and vasopressin, extravascular substances such as collagen, products of the coagulation cascade such as thrombin, physical factors such as shear stress and stirring, and many foreign substances. [10] [11] [12] [13] [14] [15] Certain features of feline platelets may cause them to be more reactive than platelets of other species, such as their larger size, 9 their higher concentration of serotonin, 9 and their response to serotonin by irreversible platelet aggregation with granule release, which is unique among domesticated species. 16 Irreversible aggregation occurs at lower concentrations of ADP in cat platelets than in platelets from other species. 17 The small size and imperfectly tractable nature of cats contribute to venipuncture difficulties, which also may increase the likelihood of in vitro platelet aggregation. Gentle handling of the sample, avoidance of small-bore needles for venipuncture and undue negative pressure on the syringe, 16 use of siliconized glassware or plastic sample containers, 18, 19 and discarding the first few drops of the sample 19 have been advocated to reduce aggregate formation. However, the problem appears to be unavoidable even under favorable conditions such as anesthesia. A simple method to consistently avoid platelet aggregation in cats would be valuable. In this study, no significant contribution to platelet aggregation could be attributed to delays and additional handling arising from sample mailing; there was no significant difference in automated platelet counts between external and internal samples. The findings of this survey demonstrate that although thrombocytopenia is an uncommon occurrence in cats, false indications of thrombocytopenia are common and result from the inability of an impedance counter to accurately quantify platelet counts in feline blood samples.This lack of reliability necessitates examination of individual blood smears for adequate platelet numbers, and vigilance on the part of laboratory workers to detect the few cats in which thrombocytopenia is actually present. In this study, as was previously reported, thrombocytopenia in cats was most commonly associated with neoplasia, chemotherapy, and infectious diseases.◊ Thrombocytopenia in cats: a retrospective study of 41 cases Automated analysis of feline platelets in whole blood, including platelet count, mean platelet volume, and activation state Laboratory Procedures for Veterinary Technicians Schalm's Veterinary Hematology Estimation of platelet counts on feline blood smears Ocular field width and platelet estimates Hemostatic disorders in cats: a retrospective study and review of the literature Platelet concentration and platelet volume distribution in healthy cats Canine and feline haematology analysis: comparative performance of Technicon H*1 and AVL MS8 VET analysers Aggregation of cat platelets in vitro The biochemistry of platelet activation The biochemical basis for the regulation of platelet responsiveness The platelet surface membrane: ultrastructure, receptor binding and function Shear stress-induced von Willebrand factor binding to glycoprotein Ib initiates calcium influx associated with aggregation Poller L, ed. Recent Advances in Blood Coagulation. Number 6 The study of platelet function in other species A comparative study of platelet aggregation in man and laboratory animals Platelet disorders in the dog and cat. Part II: Diagnosis and management Platelet preparation and estimation of functional responses The authors thank Professors Max Murray and David Bennett and the clinical staff of the Glasgow University Veterinary Hospital for their encouragement and assistance with this project. Thanks are also due to Mr K Williamson for technical support and Professor Stuart Reid for statistical advice.