Flow cytometric analysis of punctate and aggregate reticulocyte responses in phlebotomized cats

Paula C. Perkins From the Department of Microbiology, Pathology, and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

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Carol B. Grindem From the Department of Microbiology, Pathology, and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

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Laura D. Cullins From the Department of Microbiology, Pathology, and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

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SUMMARY

Five cats were made anemic by one-time phlebotomy, and their reticulocyte responses were monitored daily for 20 days, using manual enumeration and a standardized feline reticulocyte protocol developed and validated in our laboratory. The reticulocyte responses of 38 clinically normal client-owned cats also were analyzed manually and cytometrically to determine clinical reference ranges.

Increases in the percentage of aggregate reticulocytes over the reference range were detected in 5 of 5 phlebotomized cats, using the cytometric protocol. Only 4 of the 5 cats had an increase by results of manual enumeration. Manual aggregate counts had considerable daily variation and often fluctuated in and out of reference range, whereas cytometric aggregate counts remained consistently increased for distinct periods. Increased numbers of aggregate cells could also be detected for longer periods when evaluated by flow cytometry.

Increased numbers of punctate reticulocytes were detected in 4 of 5 cats, using the cytometric protocol. None of the cats had increased numbers of punctate cells when evaluated by use of the manual technique.

Aggregate reticulocytes in the 38 clinically normal cats ranged from 0.1 to 0.5%, which corresponded to 8,487 to 42,120 cells/μl. Punctate reticulocytes ranged from 2 to 17%, which corresponded to 225,400 to 1,268,584 cells/μl.

Flow cytometry, using a standardized analysis protocol, was a more reliable and sensitive technique for detection and evaluation of feline reticulocytosis than was manual enumeration. The sensitivity of the flow cytometer to small amounts of intracellular nucleoprotein makes this assay especially valuable for detection of punctate reticulocytosis and low degrees of aggregate reticulocytosis in cats.

SUMMARY

Five cats were made anemic by one-time phlebotomy, and their reticulocyte responses were monitored daily for 20 days, using manual enumeration and a standardized feline reticulocyte protocol developed and validated in our laboratory. The reticulocyte responses of 38 clinically normal client-owned cats also were analyzed manually and cytometrically to determine clinical reference ranges.

Increases in the percentage of aggregate reticulocytes over the reference range were detected in 5 of 5 phlebotomized cats, using the cytometric protocol. Only 4 of the 5 cats had an increase by results of manual enumeration. Manual aggregate counts had considerable daily variation and often fluctuated in and out of reference range, whereas cytometric aggregate counts remained consistently increased for distinct periods. Increased numbers of aggregate cells could also be detected for longer periods when evaluated by flow cytometry.

Increased numbers of punctate reticulocytes were detected in 4 of 5 cats, using the cytometric protocol. None of the cats had increased numbers of punctate cells when evaluated by use of the manual technique.

Aggregate reticulocytes in the 38 clinically normal cats ranged from 0.1 to 0.5%, which corresponded to 8,487 to 42,120 cells/μl. Punctate reticulocytes ranged from 2 to 17%, which corresponded to 225,400 to 1,268,584 cells/μl.

Flow cytometry, using a standardized analysis protocol, was a more reliable and sensitive technique for detection and evaluation of feline reticulocytosis than was manual enumeration. The sensitivity of the flow cytometer to small amounts of intracellular nucleoprotein makes this assay especially valuable for detection of punctate reticulocytosis and low degrees of aggregate reticulocytosis in cats.

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