Objectives of this study were to adapt a commercial human protein C (PC) colorimetric assay for use in dogs and to investigate effects of various storage conditions. The human assay was modified by using pooled canine plasma for calibration and by increasing the activation time. PC activity was measured in fresh canine plasma and in plasma stored under various conditions. PC activity of some stored samples was significantly different from that of fresh plasma; however, differences were small. No difference was detected in samples stored under similar conditions but analyzed in different laboratories using similar methodology. Results of this study indicate that the human colorimetric assay is suitable for canine samples if pooled canine plasma is used for calibration, that Clinical and Laboratory Standards Institute sample storage guidelines developed for testing in humans are appropriate for dogs, and that comparisons of results from laboratories using similar methodology are legitimate.
Protein C is a vitamin K-dependent glycoprotein that is primarily synthesized in the liver [
A canine-specific protein C assay is not commercially available; therefore, protein C activity in dogs is measured with human assays that employ colorimetric or clotting principles. Several reports cite the use of pooled canine plasma for assay calibration, instead of a commercial human standard; these studies have been performed using various combinations of instruments and assay reagents, including clotting-based and colorimetric (chromogenic substrate-based) assays [
Since few veterinary laboratories currently offer protein C assays, most veterinarians must ship samples in order to obtain results. According to Clinical and Laboratory Standards Institute (CLSI) guidelines [
The objectives of this study were to adapt a commercial human protein C colorimetric assay for use in dogs, and then to determine the effects of various storage conditions on results. Our hypothesis was that different storage conditions would significantly affect results. Secondary objectives were to compare results from our laboratory with those from a laboratory in another state, and to establish preliminary canine protein C reference values for our laboratory.
The project involved the use of 28 clinically normal adult dogs owned by faculty, staff, or students. All live animal work was approved in advance by an institutional animal care and use committee (University of Tennessee, protocol #1498). Pooled plasma was made using samples from 15 dogs (median age = 7 years; 9 mixed breed, 6 different pure breeds; 9 spayed females, 6 neutered males). Experimental phases 1 and 2 were done using samples from 13 dogs of varying age (phase 1, range = 3–9 years, median = 8 years; phase 2, range = 3–9 years, median = 6.5 years), breed, and sex; samples from 2 dogs were included in both phases.
Samples were analyzed using an automated instrument (STA Compact), and commercially available reagents (STA-Stachrom protein C colorimetric assay, STA-Owren-Koller buffer, STA-System Controls) from the same manufacturer (Diagnostica Stago, Asnieres, France). The assay involves a 2-step reaction in which one reagent (purified
Standard curves were established using either a commercial human standard (STA-Unicalibrator, Diagnostica Stago, Asniers, France) according to manufacturer’s instructions, or pooled canine plasma as described below. In the regular assay procedure, the 110% human standard calibrator is diluted 1/3, and all test samples are automatically diluted 1/3 (personal communication, Diagnostica Stago technical support); another veterinary laboratory using the same assay for canine samples uses a 1/3 dilution of pooled canine plasma as the 100% standard, and an activation time of 10 minutes instead of the default 5 minutes (personal communication, Cornell Animal Health Diagnostic Center). Based on this information, and on our own experience using the human standard calibrator, we established a canine-specific standard curve using the following dilutions of pooled canine plasma, and a 10-minute activation time (dilution = protein C activity): 1/3 = 100%, 1/4 = 75%, 1/6 = 50%, 1/10 = 30%, 1/20 = 15%, and pure buffer = 0%. To assess precision, two separate curves were generated in immediate succession on the same day, both run in duplicate. Using the second of these curves, a plasma sample from one normal dog was chosen at random and 6 aliquots were measured as part of a single run to determine intra-assay precision. A new canine-specific curve generated one week later (Curve 2) was used for all subsequent assays.
Blood samples (6 mL) were collected by jugular venipuncture from 15 dogs into 3.2% sodium citrate anticoagulant (BD Vacutainer, Becton, Dickenson, and Co., Franklin Lakes NJ). Samples were centrifuged twice for 120 s at 13,700 g (StatSpin, StatSpin, Inc., Norwood MA), then plasma was harvested within 30 minutes of collection, placed in 2 mL screw-cap tubes (Sarstedt, Newton NC), frozen at −80°C within 60 minutes of harvesting, and kept frozen for 12 to 28 d. Samples were thawed by letting the tubes stand at room temperature, and occasionally rolling them between open palms. Pooled plasma was created from 250
Blood samples were collected from 9 dogs, and plasma was harvested as described for making pooled plasma. Each plasma sample was analyzed within 60 minutes and the remainder was immediately frozen at −80°C. After 4 days (
Plasma storage conditions for Phase 1 (a) and Phase 2 (b).
Sample | Storage conditions |
---|---|
Control | NA (fresh plasma) |
Aliquot 1.1 | 4 d (7 dogs) or 7 d (2 dogs) at −80°C |
Aliquot 1.2 | 4 d (7 dogs) or 7 d (2 dogs) at −80°C, then thawed, refrozen at −20°C, and shipped with a cold pack the next day overnight to an independent laboratoryA; assay performed 2 d after initial thaw from −80°C |
Aliquot 1.3 | 4 d (7 dogs) or 7 d (2 dogs) at −80°C, then thawed, refrozen at −20°C, and packaged the next day identically to Aliquot 1.2 (package kept at room temperature); assay performed 2 d after initial thaw from −80°C |
Aliquot 1.4 | 4 d (7 dogs) or 7 d (2 dogs) at −80°C, then thawed and refrozen at −80°C for another 4 d |
Aliquot 1.5 | 4 d (7 dogs) or 7 d (2 dogs) at −80°C, then thawed and refrozen at −80°C for another 7 d |
ACornell University, Animal Health Diagnostic Center.
Sample | Storage conditions |
---|---|
Control | NA (fresh plasma) |
Aliquot 2.1 | 7 d at −80°C |
Aliquot 2.2 | 7 d at −20°C |
Aliquot 2.3 | 28 d at −80°C |
Aliquot 2.4 | 28 d at −20°C |
Blood samples were collected from 6 dogs, and plasma was harvested as described above. Each plasma sample was divided into 5 aliquots (0.5 mL per aliquot). One of the aliquots was analyzed immediately, and the others were immediately frozen under different conditions for later testing (Table
Precision of the protein C assay was determined by calculating coefficients of variation (CV, defined as standard deviation/mean). Results obtained using different types of samples (fresh v. various storage conditions) were compared using commercial software (SAS Version 9.2 of the SAS System for Windows, Cary, NC). Analyses included: Pearson’s correlation, Student’s
Based on 4 data points per dilution (2 standard curves, both run in duplicate), the CVs of OD measurements were < 5% (low CV = 2.0%, high CV = 4.8%) for each dilution except pure buffer; for pure buffer, 3 readings were 0.000 and 1 was 0.001. Based on 6 separate measurements of the same sample as part of a single assay run, the CV was 1.4%. Standard curves generated 1 week apart were very similar (Table
Comparison of standard curves generated 1 week apart, using pooled canine plasma (Curves 1 and 2), and a curve generated using the manufacturer’s standard protocol (Mfr Std). Results for protein C (PC) activity shown here are hypothetical, calculated by inserting arbitrary optical density (OD) values into the regression equations for the curves (Curve 1: c = [1278.276 × d] − 12.397; Curve 2: c = [1257.324 × d] − 9.60; Mfr Std curve: c = [515.734 × d] − 0.608; where c = PC activity and d = OD). Actual instrument output for Curve 2 is shown in Figure
Protein C activity (%) | |||
OD | Curve 1 | Curve 2 | Mfr Std curve |
0.02 | 13 | 16 | 10 |
0.04 | 39 | 41 | 20 |
0.06 | 64 | 66 | 30 |
0.08 | 90 | 91 | 41 |
0.10 | 115 | 116 | 51 |
0.12 | 141 | 141 | 61 |
Instrument output for Curve 2. The standard curve was established using the following dilutions of pooled canine plasma (dilution = protein C activity): 1/3 = 100%, 1/4 = 75%, 1/6 = 50%, 1/10 = 30%, 1/20 = 15%, and pure buffer = 0%.
Phase 1 results are summarized in Table
Protein C activity results of fresh (control) and stored plasma for Phase 1 (a) and Phase 2 (b). Cross symbols indicate results of comparisons of control and treatment groups, and alphabetical symbols indicate results of comparisons of different treatment groups; results for groups including the same symbols are not significantly different.
Protein C Activity (%, mean ± SD).
Control | 4–7 d, −80°C | 4–7 d, −80°C; thawed, 2 d, −20°C, send-out | 4–7 d, −80°C; thawed, 2 d, −20°C, in-house | 4–7 d, −80°C; thawed, 4 d, −80°C | 4–7 d, −80°C; thawed, 7 d, −80°C |
---|---|---|---|---|---|
95 ± 10† | 100 ± 15†, a, b, c | 97 ± 14†, b | 102 ± 11†, a, b, c | 111 ± 16‡, c | 105 ± 15‡, a, b, c |
Protein C Activity (%, mean ± SD).
Control | 7 d, −80°C | 7 d, −20°C | 28 d, −80°C | 28 d, −20°C |
---|---|---|---|---|
94 ± 12† | 94 ± 11†, a | 97 ± 11‡, a | 88 ± 10‡, b | 87 ± 10‡, b |
Correlation for comparisons of fresh samples with samples stored under the 5 different conditions ranged from
Samples stored for 4 to 7 d at −80°C, then thawed and refrozen at −80°C for either an additional 4 d or 7 d, had significantly higher protein C activity than control samples. Samples frozen for 4 to 7 d at −80°C, then thawed and refrozen at −20°C for an additional 2 d and measured in-house, had higher protein C activity than control samples; this difference was marginally significant (
There was no significant difference in results determined by two different laboratories using samples stored under the same conditions (4 to 7 d at −80°C, then thawed and refrozen at −20°C for an additional 2 d). Samples stored for 4 to 7 d at −80°C, then thawed and refrozen at −20°C for an additional 2 d and measured by a laboratory in another state, had significantly lower protein C activity than samples stored for 4 to 7 d at −80°C, then thawed and refrozen at −80°C for an additional 4 d. Samples stored for 4 to 7 d at −80°C had lower protein C activity than samples stored for 4 to 7 d at −80°C, then thawed and refrozen at −80°C for an additional 4 d; this difference was marginally significant (
Phase 2 results are summarized in Table
Correlation was high (
Samples stored for 7 d at −20°C had significantly higher protein C activity, and samples stored for 28 d at either temperature (−20°C or −80°C) had significantly lower protein C activity, than control samples.
Samples stored for 7 d had significantly higher protein C activity than samples stored for 28 d, irrespective of storage temperature.
Based on the combined control sample data sets of Phase 1 and Phase 2, the minimum and maximum values for protein C activity were 80% and 115%, respectively. We are using these as preliminary lower and upper reference limits, respectively, in our laboratory.
This study was motivated by our desire to measure canine protein C activity in our laboratory, and to learn more about effects of different storage conditions on test results. Limitations of the study included relatively small sample sizes and fragmentation into two experimental phases to investigate storage effects. Despite these limitations, we expect this report to be useful to anyone interested in establishing a canine protein C assay in their own laboratory or seeking more information about effects of different storage conditions (including shipping samples to another institution for analysis).
In our laboratory, the human standard for assay calibration had unacceptably low results (Table
We investigated effects of storage conditions different from those previously reported. In one previous study, plasma samples were stored at −20°C for 2 months or −60°C for 10 months before analysis [
Based on these findings, we interpret the differences in results after the experimental storage conditions to be of little to no clinical significance. This interpretation is consistent with the findings of investigators who have studied the effects of different storage conditions on human or canine protein C activity [
Protein C activity was not significantly different in samples stored under similar conditions but analyzed in different laboratories. The two laboratories in this study used the same instruments and assays but different batches of pooled canine plasma to calibrate the assay. The lack of significant difference suggests that data generated from laboratories using essentially the same methodology can be legitimately compared. Variability in protein C results within and between laboratories has been the subject of study in human laboratories but, to our knowledge, not in veterinary laboratories [
In summary, this report describes how we adapted a commercial human colorimetric protein C assay for use in dogs. Our preliminary reference values are consistent with those of other investigators using colorimetric or clotting-based assays, and the precision of the assay compared favorably with other reports and with performance goals established for human laboratories. In addition, we report the effects of previously unreported storage conditions on test results. We believe that the differences between fresh samples and those stored under the study conditions were of little to no clinical significance, that CLSI guidelines developed for testing in humans are also appropriate for dogs, and that comparisons of results from laboratories using essentially the same methodology are likely to be valid.
This work was supported in part by the UTCVM Center of Excellence Summer Student Research Program, and the UTCVM Angel Fund for Research and Treatment of Congenital Portosystemic Shunts in Dogs.