Introduction
Ferrets (Mustela putorius furo) are commonly used as research animals but are also popular as pets, which engenders an increased willingness by owners to pursue advanced medical care.1,2 These animals are subject to several diseases that may affect coagulation, including hepatic disorders, rodenticide poisoning, disseminated intravascular coagulation, ferret hemorrhagic syndrome, and hereditary coagulopathies.3,4 Thus, reference intervals for coagulation times in ferrets represent an essential tool in diagnosis and management of coagulopathies, as well as the evaluation of potential risk prior to surgery. As clotting time may be analyzer and reagent dependent, it is of great importance that the veterinarian interprets values according to the analyzer used. Similar collection techniques are also necessary as they can influence the results. Only a few reports provide coagulation values in ferrets.5,6 In all of them, the numbers of individuals were limited to < 20 ferrets, which did not permit the establishment of statistical reference values for this species.7,8
Prothrombin time (PT) and activated partial PT (aPTT), evaluated in this study, are the most common screening tests for coagulopathies in mammals. PT evaluates the integrity of the extrinsic and common pathways of coagulation (fibrinogen, factors II, V, VII, and X), and aPTT evaluates the integrity of the intrinsic and common pathways (fibrinogen, factors II, V, VIII, IX, X, XI, and XII).
The aim of this study was to establish reference intervals for PT and aPTT in healthy ferrets, using 2 different point-of-care analyzers.
Materials and Methods
Animals
Eighty-seven adult ferrets, 48 females (55.2%) and 39 males (44.8%) were included in this study. All animals were under 3 years of age (12 months to 36 months old; median, 24 months) and originated from private owners from 2 private practices and 4 distinct small-scale pet ferret breeders (5 to 15 ferrets/breeder), in which 3 to 5 animals sampled/breeder were from the same lineage. All ferrets had mixed coat color and pattern, and 10 were angora.
Ferrets were considered healthy on the basis of a thorough individual history and complete physical examination (determination of body condition, heart and respiratory rates, abdominal palpation, peripheral lymph node palpation, auscultation, and examination of the teeth, eyes, ears, and external genitals). All ferrets presenting with a disease that could potentially influence blood coagulation values or a history of chronic disease were excluded from the study. Ferrets were either entire, surgically neutered, or subcutaneously implanted with GnRH agonist implants (deslorelin acetate; Suprelorin; 4.7 mg). A 2-month washout medication period was respected prior to blood sampling. All owners signed an informed consent form prior to blood collection for the purposes of this study.
Blood collection
Venipuncture was performed on all ferrets by 2 trained veterinarians without anesthesia. Animals were restrained in dorsal recumbency with the forelegs pulled caudally, hind legs restrained just cranial to the pelvis by 1 assistant, and head and neck extended by the person performing the blood collection (Figure 1).
Blood collection from the cranial vena cava in a ferret without anesthesia. The animal is restrained in dorsal recumbency with the forelegs pulled caudally, the hind legs restrained just cranial to the pelvis by 1 assistant, and the head and neck extended by the person performing the blood collection.
Citation: Journal of the American Veterinary Medical Association 261, 5; 10.2460/javma.22.12.0595
Blood samples were collected from the cranial vena cava, allowing easy venipuncture and quick collection of sufficient quantities of blood. Eighty-seven samples were collected with a 23-G needle and 2.5-mL plastic syringe. The needle was inserted between the first rib and manubrium at a 30° to 45° angle to the body and directed toward the opposite rear leg. The blood volume collected was 1 mL. The blood was placed in trisodium citrated plastic tubes provided by the laboratories. The ratio of blood to anticoagulant was 9:1. The tubes were then gently inverted 3 times. Blood samples with signs of clotting were rejected and not included in the study.
Ethics approval
This study was approved by the ethics committee of the Veterinary School VetAgroSup in Lyon, France (proposal No. 2268). Legal and ethical requirements were met with regard to the humane treatment of animals described in the study.
Laboratory methods
The same inclusion criteria, method of blood sampling, and procedure were used in all practices and breeding farms. Blood samples were kept at room temperature and analyzed as whole blood within an hour after sampling. In total, 87 samples were analyzed in 2 different practices, using one of the analyzers. Sixty-six blood samples from the 4 ferret breeding farms (42 samples) and 1 private practice (24 samples) were analyzed using the citrate PT/PTT cartridges from Idexx Coag DX (Idexx Laboratories Inc) and 21 from the other private practice using the MS QuickVet Coag Combo (Melet Schloesing Laboratoires). Both analyzers use a photo-optical clot detection system.
Statistical evaluation
Statistical analysis was performed using R software (version 3.6.2; R foundation for Statistical Computing). The samples available in the study were fewer than the minimum of 120 recommended for the establishment of de novo reference intervals in animal species. Alternative methods were used and adapted to the number of samples available.7 Reference intervals and their 90% reference limits were calculated with the ‘referenceIntervals’ package (R foundation for Statistical Computing) based on the robust method as recommended by the American Society for Veterinary Clinical Pathology reference interval guidelines7 for a sample size of 20 < N < 120.
Normality was tested by the Shapiro-Wilk normality test, and Q-Q plots were reviewed for deviation from normality. To determine whether different groups of animals required different reference intervals, the effect of sex, reproductive status (neutered, implanted, or entire), angora status, age, and analyzer type on the values of aPTT and PT variables were evaluated using a Student t test, Mann-Whitney-Wilcoxon test, or regression analysis. A Student t test was used with variables that did not significantly diverge from normality, and a Mann-Whitney-Wilcoxon test was used for variables that were significantly not normal. Statistical significance was evaluated by the Benjamini-Hochberg procedure applied to P values with a false discovery rate set at .05.9
Results
Evaluation of aPTT distribution revealed the presence of a statistical outlier (aPTT > 300 seconds). Although the reason for this unexpectedly high value was not identified, either an illness or error in the processing of the sample was suspected, and this individual was excluded from the study. The resulting aPTT distribution from the 86 remaining animals did not significantly differ from normality (Shapiro-Wilk, P = .11). The PT variable distribution was not normal (Shapiro-Wilk, P < .001).
Reference intervals were established as follows for the Idexx Coag DX: aPTT (n = 65) reference interval, 69.84 to 105.99 seconds; CI 90% lower, 65.83 to 73.71 seconds; CI 90% upper, 102.26 to 110.48 seconds; PT (65) reference interval, 14.44 to 21.98 seconds; CI 90% lower, 9.88 to 15.24 seconds; CI 90% upper, 21.22 to 24.96 seconds.
Reference intervals were established as follows for the MS QuickVet Coag Combo: aPTT (n = 21) reference interval, 74.90 to 115.50 seconds; CI 90% lower, 68.27 to 80.46 seconds; CI 90% upper, 109.73 to 122.58 seconds; PT (21) reference interval, 18.31 to 23.05 seconds; CI 90% lower, 17.47 to 18.97 seconds; CI 90% upper, 22.25 to 23.70 seconds (Figures 2 and 3; Table 1). With both types of analyzers, there was no significant difference between age of ferret and aPTT and PT (linear regression analysis, P > .05).
Prothrombin time (PT) distribution using Idexx Coag DX and MS QuickVet Coag Combo in healthy ferrets (Mustela putorius furo). Bold horizontal lines represent the medians. Boxes encompass the second and third quartiles. The whiskers represent the highest and lowest data included in 1.5 times the 25th to 75th percentile. The circles are data points outside the whiskers.
Citation: Journal of the American Veterinary Medical Association 261, 5; 10.2460/javma.22.12.0595
Activated partial prothrombin time (aPTT) distribution using Idexx Coag DX and MS QuickVet Coag Combo in healthy ferrets (M putorius furo). Bold horizontal lines represent the medians. Boxes encompass the second and third quartiles. The whiskers represent the highest and lowest data included in 1.5 times the 25th to 75th percentile. The circles are data points outside the whiskers.
Citation: Journal of the American Veterinary Medical Association 261, 5; 10.2460/javma.22.12.0595
Reference intervals for prothrombin time (PT) and activated partial PT (aPTT) in healthy ferrets (Mustela putorius furo) using 2 point-of-care analyzers (Idexx Coag DX and MS QuickVet Coag Combo).
| Parameter | PT Idexx Coag DX | PT MS QuickVet Coag Combo | aPTT Idexx Coag DX | aPTT MS QuickVet Coag Combo |
|---|---|---|---|---|
| N | 65 | 21 | 65 | 21 |
| Reference interval (seconds) | 14.44–21.98 | 18.31–23.05 | 69.84–105.99 | 74.90–115.50 |
| 90% CI for lower limit (seconds) | 9.88–15.24 | 17.47–18.97 | 65.83–73.71 | 68.27–80.46 |
| 90% CI for upper limit (seconds) | 21.22–24.96 | 22.25–23.70 | 102.26–110.48 | 109.73–122.58 |
With the Idexx Coag DX, the mean aPTT in males (91.74 seconds; n = 27) was significantly higher than that of females (86.2 seconds; 38; Student t test, P = .01). Median of PT in males was identical to females (19 seconds).
Reference intervals were established as follows for the Idexx Coag DX in females: aPTT (n = 38) reference interval, 69.62 to 103.00 seconds; CI 90% lower, 64.97 to 74.09 seconds; CI 90% upper, 98.71 to 107.87 seconds; PT (38) reference interval, 14.95 to 21.09 seconds; CI 90% lower, 12.90 to 15.98 seconds; CI 90% upper, 20.13 to 25.18 seconds. Reference intervals were established as follows for the Idexx Coag DX in males: aPTT (n = 27) reference interval, 72.04 to 109.63 seconds; CI 90% lower, 66.63 to 78.92 seconds; CI 90% upper, 103.56 to 116.52 seconds; PT (27) reference interval, 19.00 to 19.00 seconds; CI 90% lower, 19.00 to 25.31 seconds; CI 90% upper, 14.05 to 19.00 seconds.
Because there were < 20 neutered individuals, specific reference intervals could not be established for aPTT or PT.
As for the variable angora coat, < 20 individuals were included and specific reference intervals could not be calculated. Furthermore, variation related to sex and those related to the phenotype angora could not be distinguished due to an important bias of sex in angora ferrets in our population (11 males and 0 females).
Discussion
The aim of our study was to establish reference blood coagulation values for PT and aPTT in healthy ferrets. Several point-of-care coagulation analyzers are currently found in veterinary practices. Those used in our study are dedicated to coagulation testing and are 2 of the most commonly used by veterinarians in France (MS QuickVet Coag Combo and Idexx Coag DX).10 Reference intervals for coagulation times with these 2 analyzers have been established in cats and dogs11 as well as in rabbits,10 but no reference values have been determined for ferrets.
Information on reference values for coagulation times in ferrets is scarce. Two studies5,6 provide coagulation times in anesthetized ferrets using a fibrometer (Becton, Dickinson and Co) and a photo-optical clot detection system (ACL 3000 coagulation analyzer; Beckman Coulter Inc) in one study5 and a Start4 analyzer/reagent combination (Diagnostica Stago Inc) in the other,6 both on plasma samples. Results obtained in these reports for PTT and aPTT were different from those in our study.
Multiple parameters could contribute to these differences. Clotting time assays are known to be sensitive to changes depending on the type of analyzer.5,10 Point-of-care analyzers were used in our study, and the procedures and techniques were different from those used in previous reports. Particularly, poor agreement has been documented between point-of-care analyzers and reference laboratory determinations of aPTT, which may reflect the different activators as well as differences between plasma and whole blood values.11
Normal values in both previous reports were based on a low number of individuals (4 and 18 ferrets), and reference intervals could not be established. According to Dodds8 and Friedrichs et al,7 reference limits can only be established with a minimum of 20 individuals. By default, mean values in these reports were defined by minimum and maximum. In our study, a much larger population (86 ferrets) was included, with 65 samples analyzed using the Idexx Coag DX and 21 using the MS QuickVet Coag Combo. A minimum of 120 individuals is recommended to establish reference limits by nonparametric methods with 90% CIs. But as collection of sufficient samples can be challenging in veterinary medicine, recommended procedures for establishing reference intervals based on reference sample size and distribution have been determined.7 Reference intervals could be determined for the Idexx Coag DX (sample size of 40 < X < 120)7 and for the MS QuickVet Coag Combo (sample size of 20 < X < 40),7 using the robust method.
The analyzers used in our study did not give similar intervals. Comparisons between the 2 analyzers would have been possible if all measurements were performed using both machines and with greater numbers of samples for the MS QuickVet Coag Combo. In addition, a potential bias inherent to differences between animals issued from the ferret breeders versus owners is to be considered. The genetic link between some ferrets originating from breeders could influence coagulation parameters, although these results did not seem significantly different from those of private owners in the present study. Further research is needed to determine the impact of genetically linked animals on coagulation parameters.
In the present study, samples were analyzed as whole blood unlike previous studies in which plasma was used for coagulation times. Although no study on the impact of the use of plasma versus whole blood on coagulation times is reported in ferrets in the literature, the study conducted by Amukele et al12 on human beings demonstrated an average of 40% longer PT using whole blood versus plasma. It can be assumed that coagulation times differ depending on the type of sample used and that it could be part of the differences observed between our results and those of previous studies.
All samples in our study were collected without anesthesia unlike previous reports. The cranial vena cava in ferrets represents one of the most common sites of venipuncture in this species. This technique has been widely described and is safe and relatively easy to perform.13 The effect of anesthesia on coagulation time results was not assessed in our cases.
Data on the effect of anesthesia on PT and aPTT are scarce and controversial in human beings and different companion animals14–19 and have not been published in ferrets. Further research is needed and could be of interest, as venipuncture is not always feasible in uncooperative, conscious ferrets.
Reliable results are essential for the determination of de novo reference intervals in veterinary species; hence, procedures must be standardized for repeatability and avoid significant differences between samples. In our study, blood samples were taken from the cranial vena cava only. As in-house coagulation analysis requires quick and simple collection of large amounts of blood to avoid clot formation, the cranial vena cava remains the best large vessel sampling site and is the most used in ferrets. Plastic syringes and plastic or silicone-coated tubes should be used for sample collection and storage, which was the case in our study. Blood was then transferred into a trisodium citrate tube, the anticoagulant of choice for coagulation studies, as indicated by laboratories and coagulation studies.
Results of the present study suggested that the age of the ferrets might not have a significant effect on aPTT and PT. However, this should be verified with ferrets from a wider age range.
Because < 20 neutered and angora individuals were included in our study, specific reference intervals for these variables could not be established.7,8 Further research with greater numbers of ferrets would be relevant to evaluate the effects of these variables.
Reference intervals were established depending on sex for the Idexx Coag DX, as follows: mean of aPTT was significantly higher in males than in females, and the reference interval for PT was difficult to interpret due to the low number of males (n = 27). As with the MS QuickVet Coag Combo, greater numbers of samples would be necessary to evaluate the influence of the reproductive status on PT and aPTT. The unbalanced distribution of angora individuals between sex groups (overrepresentation of males) precluded any conclusion on coat pattern influence on coagulation times. The number of angora ferrets in the group did not allow for a reliable comparison of the male angora group with the male group.
Moreover, results of reference intervals in subgroups for the Idexx Coag DX raised the question of the relevance of these results in comparison with the overall reference intervals. As there was no significant difference, subgroups should not be considered separately and use of overall reference intervals can be combined.
The present study provided reference ranges of PT and aPTT for 2 commonly used point-of-care analyzers and on a much larger population than previous studies. Reference coagulation parameters for PT and aPTT using these 2 analyzers will help veterinarians diagnose coagulopathies in ferrets and evaluate particular risks in patients presenting with hepatic disease, for example. This report also supported the facts that results are analyzer dependent and it is essential to provide reference values for each point-of-care analyzer for accurate interpretation. Furthermore, procedures for sampling and methods have to be standardized and reference intervals should be established by testing at least 20 normal individuals.7,8 Further research is needed to confirm the influence of general anesthesia, pattern types (including angora coat), and reproductive status on PT and aPTT in-house assay.
Acknowledgments
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript. The authors declare that there were no conflicts of interest.
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