Effects of in vitro hemodilution of canine blood on platelet function analysis using the PFA-100

Authors

  • Noel Clancey,

    1. Departments of 1Pathology and Microbiology and 2Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada
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  • 1 Shelley Burton,

    1. Departments of 1Pathology and Microbiology and 2Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada
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  • 1 Barbara Horney,

    1. Departments of 1Pathology and Microbiology and 2Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada
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  • 1 Allan MacKenzie,

    1. Departments of 1Pathology and Microbiology and 2Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada
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  • 1 Andrea Nicastro,

    1. Departments of 1Pathology and Microbiology and 2Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada
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  • and 2 Etienne Côté 2

    1. Departments of 1Pathology and Microbiology and 2Companion Animals, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada
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Correspondence
Noel Clancey, Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, Canada C1A 4P3
E-mail: nclancey@groupwise.upei.ca

Abstract

Background: The platelet function analyzer (PFA)-100 is a point-of-care instrument previously evaluated in humans and dogs. In both species, artificially prolonged platelet closure time (CT) occurs with anemia. Reliability of the analyzer in dogs becomes a concern when the HCT is between 0.25 and 0.35 L/L.

Objective: The objective of this study was to further define the level of HCT at which CT is prolonged, using in vitro diluted canine blood.

Methods: Citrated whole blood samples were collected from 22 healthy dogs. Initial HCT was determined and autologous platelet-rich plasma was added to samples to achieve HCTs of 0.33, 0.30, and 0.27 L/L. CT was determined in duplicate on the PFA-100 using collagen/adenosine-5′-diphosphate cartridges.

Results: Compared with the initial CT in samples with HCT 0.39–0.54 L/L (CT mean±SD=57.8±5.75 seconds), significantly prolonged CTs were found in hemodiluted samples with HCT 0.33 L/L (61.1±4.64 seconds), 0.30 L/L (64.3±6.79 seconds), and 0.27 L/L (70.8±7.90 seconds) (P=0.029; repeated measures ANOVA).

Conclusion: Although statistical differences were found, further studies are needed to determine the clinical significance of the mild prolongation in CT associated with mild anemia. Until then, dogs with HCTs slightly <0.35 L/L should be evaluated cautiously for platelet dysfunction using the PFA-100.

Platelets play a vital role in hemostasis. Dogs experience a variety of serious conditions potentially involving increased or decreased platelet function.1–6 Assessment of platelet hypofunction and hyperfunction involves sophisticated testing including flow cytometry and platelet aggregation. These techniques are not routinely available in diagnostic laboratories, are labor intensive, and require specialized training. The development of a simple point-of-care instrument, the platelet function analyzer (PFA-100),7–9 has allowed identification of human platelet hyperfunction10,11 and hypofunction.12,13 The PFA-100 is easy to use and has been investigated and validated for use in dogs.14–16 Other than unreliable results when thrombocytopenia is present, the main drawback of using the PFA-100 is that anemia prolongs the time to formation of a platelet plug, the closure time (CT). This has been demonstrated in humans17,18 and in dogs.16,19 Anemia is believed to result in decreased CT due to rheological alterations in the sample20 and because the lower number of RBCs provides less physical stimulus for platelet arachidonic acid release.21 Manufacturer guidelines for human samples indicate that an increased CT may be seen in samples with HCT<0.35 L/L.22 This effect has been only minimally investigated in dogs, using small sample sizes16,19 and at specific in vitro HCTs of 0.15, 0.25, and 0.35 L/L, without evaluation of values in-between these levels.23 In the latter study, CTs in samples from 10 dogs were significantly prolonged at a HCT of 0.25 L/L, but not 0.35 L/L, compared with an initial mean HCT of 0.58 L/L.23 As the range between 0.25 and 0.35 L/L is broad, more precise information is needed regarding the threshold HCT at which CT is significantly prolonged. Specifically, it would be helpful to know whether mildly anemic dogs with HCTs between 0.25 and 0.35 L/L can be assessed reliably for platelet function. The goal of this study was to further define the level of anemia in dogs at which CT is significantly prolonged using in vitro hemodiluted samples.

All experimental protocols were designed and performed in accordance with the standards of the Canadian Council on Animal Care and were approved by the University of Prince Edward Island Animal Care Committee. Written owner consent was obtained for all dogs used in the study. The population consisted of 22 healthy dogs owned by hospital staff or veterinary students. Dogs were deemed healthy and appropriate for the study if they had no history of a bleeding tendency, had not received any medications known or suspected to interfere with platelet function or number within 7 days before blood collection, had no abnormalities on physical examination, had no abnormalities in a CBC or serum clinical biochemistry profile, had a platelet count >150 × 109/L, had no evidence of platelet clumps on blood smears, and had a plasma von Willebrand factor:antigen (vWF:Ag) concentration >50%.

Blood samples were obtained by careful jugular or lateral saphenous venipuncture with collection directly into Vacutainer tubes using a 21- or 22-G needle vacuum system or a 21-G butterfly catheter system (Becton Dickinson, Franklin Lakes, NJ, USA). Blood was collected into tubes containing K-EDTA for CBC and into plain tubes for serum biochemical analysis. Tubes containing 105 nM (3.2%) trisodium citrate were filled to the appropriate ratio of 1 part anticoagulant to 9 parts blood for platelet function analysis and vWF:Ag concentration. Plasma for vWF:Ag concentration was obtained following centrifugation of the citrate tubes at approximately 1300g for 5 minutes at room temperature (∼23°C) within 30 minutes of collection. Plasma was immediately stored in cryovials at −20°C for no longer than 21 days, then transferred to −80°C storage until shipment. The concentration of vWF:Ag was determined at the Comparative Coagulation Section Diagnostic Laboratory at Cornell University, Ithaca, NY, USA. Auto-stained (Ames Hematek-1000, Bayer Corp., Elkhart, IN, USA) blood smears of K-EDTA and citrate anticoagulated blood were used for manual platelet estimates and examined for platelet clumping. Automated platelet counts were performed on K-EDTA anticoagulated blood samples using a CELL-DYN 3500CS analyzer (Abbott Diagnostics, Santa Clara, CA, USA).

The PFA-100 analyzer (Dade Behring, Deerfield, IL, USA) was used and maintained according to manufacturer instructions.17,22 Citrated whole blood samples for platelet function analysis were stored at room temperature (∼23°C) and analyzed between 30 and 240 minutes after collection. Blood samples from all dogs were run in duplicate using collagen/adenosine diphosphate (COL/ADP) cartridges; mean CT and coefficients of variation (CV) were calculated automatically by the PFA-100 as (SD÷mean) × 100. In the event of encountering a single cartridge failure, repeat analysis using a single test cartridge was performed. If the CV was >15% between 2 results, repeat duplicate analysis using new test cartridges was performed.22

Three hemodiluted samples were prepared from each patient sample using fresh autologous platelet-rich plasma (PRP) as previously described to achieve in vitro HCTs of 0.33, 0.30, and 0.27 L/L.16,23 An automated HCT and platelet count were obtained on each dilution (CELL-DYN 3500CS) to ensure the results matched the anticipated calculated dilution and to ensure platelet concentration was >150 × 109/L. Data on any sample were excluded if the CV was >15%.

Statistical analysis was performed using Minitab 14 software (Minitab Inc., State College, PA, USA) to assess differences (P<.05) between CT in the hemodilution groups using repeated measures ANOVA and Dunnett's multiple comparison.

The 22 healthy dogs included 1 intact female, 6 spayed females, 1 intact male, and 14 neutered males ranging from 7 months to 10 years in age and comprising 11 breeds. Mean CTs of the 3 hemodilution groups were significantly different from mean baseline CT (Figure 1), with the CT increasing as HCT decreased. One sample in the 0.30 L/L group and 4 samples in the 0.27 L/L group had CTs above the reference interval (48–77 seconds) for our laboratory.24 Data from 1 dog each in the 0.30 and 0.27 L/L hemodilution groups were excluded due to unacceptably high CVs.

Figure 1.

 Closure times (CT) using the PFA-100 analyzer on fresh whole blood samples from healthy dogs initially (HCT 0.39–0.54 L/L, mean 0.47 L/L) and following in vitro hemodilution to HCT 0.33 L/L (CT mean ± SD=61.1 ± 4.64 seconds), 0.30 L/L (64.3 ± 6.79 seconds), and 0.27 L/L (70.8 ± 7.90 seconds). Cross-haired symbols represent the mean CT. *Significantly different compared with the initial HCT group (P=.029; repeated measures ANOVA). Horizontal dashed lines indicate the lower and upper reference interval for our laboratory (48–78 seconds). PFA, platelet function analyzer.

The significant influence of HCT on CT in the present study supports previous work, which generally showed prolongation of the CT as the HCT approached and dropped below 0.30 L/L.16,19,23 Therefore, finding a significant difference in CT between the initial HCT, 0.30, and 0.27 L/L in the present study was not surprising. Finding a significant difference between the initial HCT and 0.33 L/L, however, suggested that the lower limit of HCT at which the PFA-100 analyzer will function reliably in dogs likely needs to remain at 0.35 L/L. This agrees with the limitation on using this analyzer for human samples. However, this point could be argued that statistical significance does not always denote clinical relevance. Although CT in the 0.33 L/L HCT group was significantly different from that in the initial HCT group, the mean difference was only 3.3 seconds. As well, none of the 22 samples in the 0.33 L/L HCT group had a CT outside of the reference interval of 48–77 seconds, with the highest CT recorded in this group being 68 seconds. Even the lowest HCT group (0.27 L/L) had a mean CT within the reference interval (71 seconds) and only 4 of 21 samples had CTs above the upper limit of the interval. Until more studies are performed to determine the clinical significance of these findings, assessment of mildly anemic dogs using the PFA-100 analyzer may have diagnostic value. A platelet function abnormality can likely be ruled out when a normal CT is reported in a mildly anemic dog. However, if an anemic dog has a prolonged CT it will not be possible to determine whether this is due to the anemia or to a platelet function abnormality. Further in vivo studies are needed before sound clinical conclusions can be drawn.

In conclusion, while statistically significant prolongations of CT were seen with even mildly hemodiluted samples in this in vitro study, the prolongations were mild and of equivocal clinical relevance. While dogs with a HCT below 0.35 L/L can be evaluated cautiously for platelet function using this analyzer, further clinical studies are needed.

Acknowledgments

This study was supported by a grant from the Sir James Dunn Animal Welfare Centre. The authors would like to thank Dr. Henrik Stryhn for his statistical expertise; Ms. Anne Dover, Ms. Linda Ruschkowski-Kerr, Ms. Karen Blackburn, and Mrs. Donna Barnes for excellent technical assistance; and all clinicians, staff, and students for their participation and generosity during this study.

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