Determination of Plasma Fibrinogen Concentrations in Beagle Dogs, Cynomolgus Monkeys, New Zealand White Rabbits, and Sprague–Dawley Rats by Using Clauss and Prothrombin-Time–Derived Assays

Abstract

The most widely used technique for determination of fibrinogen concentration is the Clauss fibrinogen (FIB_Clauss) assay, which measures the clotting time of plasma after addition of excess thrombin. More recently, the PT-derived fibrinogen (FIB_PT) assay has been developed, based on the relationship between fibrinogen concentration and the kinetics of clot formation during the prothrombin time. The objective of this study was to compare the fibrinogen concentration determined by the FIB_Clauss and FIB_PT assays in citrated plasma samples from healthy dogs (n = 40), monkeys (n = 40), rabbits (n = 26), and rats (n = 58) by using an automated coagulation analyzer. Results of a t test analysis indicated that the mean plasma fibrinogen concentrations measured by the 2 assays for all 4 species were significantly different. According to Pearson correlation coefficients, the FIB_PT assay displayed a high correlation (0.93 to 0.98) with the FIB_Clauss assay for all species. When the FIB_PT and FIB_Clauss assays were compared by using Deming regression, positive or negative constant and proportional biases emerged for all species. Intra- and interassay coefficients of variation for the FIB_PT and FIB_Clauss assays were 0.8% to 2.3% and 1.8% to 7.4%, respectively. In conclusion, the FIB_PT assay is a rapid and economical method for estimating fibrinogen concentration in plasma samples from dogs, monkeys, rabbits, and rats. However, it should not be used without restriction. Further studies are required to investigate the performance of this assay in animals with various pathologic states, including coagulopathy, dysfibrinogenemia, and hypo- or hyperfibrinogenemia.

Fibrinogen, a plasma glycoprotein synthesized in the liver, is the soluble precursor of insoluble fibrin, a major component of blood clots.¹⁰ In addition, fibrinogen plays an important role in platelet aggregation by linking activated platelets. Activated platelets express the integrin αIIb β3, which binds plasma fibrinogen as well as that secreted from activated platelet granules.⁶ The function and quantity of plasma fibrinogen can be altered by both inherited and acquired disorders.¹⁸ Some common causes of decreased fibrinogen concentration are increased consumption during localized or disseminated intravascular coagulation, severe hepatic dysfunction, and hemodilution. Low plasma fibrinogen concentration is associated with an increased risk of bleeding, due to impaired primary or secondary hemostasis. Furthermore, fibrinogen is a positive acute-phase protein and, therefore, is increased in response to inflammatory processes.^3,17,18

Because of its clinical relevance, fibrinogen concentration must be determined accurately. The concentration of fibrinogen is measured routinely as part of a CBC analysis in various species. Various laboratory methods are available for the measurement of fibrinogen concentration. The most widely used technique for determination of fibrinogen concentration is Clauss (FIB_Clauss) assay, which is a modification of the assay first described by Clauss.^3,18 In this method, dilutions of a plasma standard of known fibrinogen concentration are clotted by addition of a high concentration of thrombin, and a standard curve is prepared. Because the clotting time is inversely proportional to the fibrinogen concentration, the clotting time of diluted patient plasma is used to read the fibrinogen concentration from the standard curve. This method is reliable, accurate, and precise, and easily adapted to automated coagulation analyzers.¹³ However, the FIB_Clauss assay is time-consuming, with requirements of dilution buffer and special thrombin reagent, which may cause carryover problems in subsequent tests on some fully automated coagulation analyzers and false shortening of clotting times.⁴ Furthermore, performance of the FIB_Clauss assay by automated coagulation analyzers may decrease the throughput for samples that require only analysis of prothrombin time or activated partial thromboplastin time. Therefore, many laboratories only perform the FIB_Clauss assay when there is specific indication.⁷ In addition, fibrinogen degradation products that are formed in excess of fibrin split products during thrombolytic therapy interfere with the FIB_Clauss assay, resulting in an artifactually increased fibrinogen concentration level.¹¹

Quantification of fibrinogen concentration based on the relationship between fibrinogen level and the kinetics of clot formation during the PT has recently been developed.² Several photo-optical coagulometers use the change in optical properties during the prothrombin time reaction to determine fibrinogen concentration, by comparing the response of a test plasma with that of a standard of known fibrinogen concentration.^4,14 This method provides substantial cost savings and is known as the prothrombin-time–derived fibrinogen (FIB_PT) assay.¹⁶

Although fibrinogen estimation derived from the FIB_PT assay is widely used in human medicine,⁷ to our knowledge, comparison between the FIB_Clauss and FIB_PT in domestic animals has never been reported. The objective of the current study was to compare the fibrinogen concentrations determined by the FIB_Clauss and FIB_PT assays in citrated plasma samples from clinically healthy dogs, monkeys, rabbits, and rats.

Materials and Methods

Animals.

The animals included in the study were 40 beagle dogs (Canis familiaris; 20 male; 20 female; age, 5 to 10 mo), 40 cynomolgus monkeys (Macaca fascicularis; 20 male; 20 female; age, 2 to 6 y), 26 New Zealand white rabbits (Oryctolagus cuniculus; 13 male; 13 female; age, 5 to 6 mo), and 58 Sprague–Dawley rats (Rattus norvegicus; 29 male; 29 female; age, 7 to 58 wk). The animals were healthy based on physical examination and hematology, coagulation, serum clinical chemistry, and urinalysis profiles. The animals had not been included in any previous toxicology study and were housed under an IACUC-approved protocol. Blood samples were withdrawn from the jugular vein of dogs and rats, cephalic vein of monkeys, and marginal ear vein of rabbits, collected in vacuum phlebotomy tubes containing 3.2% sodium citrate (0.105 M; Becton Dickinson, Franklin Lakes, NJ) with a blood:anticoagulant ratio of 9:1 (v/v), and immediately transported to the clinical pathology laboratory for analysis.

Fibrinogen assays.

Plasma was prepared by centrifugation of citrated blood at 1500 × g for 10 min at room temperature; plasma was analyzed within 2 h of preparation. The FIB_Clauss and FIB_PT assays both were performed on an automated coagulation analyzer (ACL TOP, Beckman Coulter, Fullerton, CA) according to the manufacturer instructions. This instrument is a fully automated, benchtop analyzer designed specifically for in vitro coagulation and fibrinolysis diagnostic testing in the assessment of thrombosis and hemostasis. Both assays used the same manufacturer's reagents (RecombiPlasTin; Instrumentation Laboratories, Orangeburg, NY). The FIB_Clauss and FIB_PT assays were calibrated by using the HemosIL Calibration Plasma kit (Instrumentation Laboratories), which contains a standard reference material specific to this particular coagulation analyzer and its reagents. All samples were analyzed in triplicate.

Standardization of the FIB_PT and FIB_Clauss assays requires the development of species-specific fibrinogen standards and careful evaluation of their commutability between assays. Because such species-specific fibrinogen standards are currently unavailable, we evaluated the accuracy of the assays by analyzing a calibration standard and human-based normal and low abnormal fibrinogen control materials (HemosIL; Instrumentation Laboratories). For intraassay (within-run) and interassay (between-run) precision studies, species-specific pooled plasma samples were prepared by mixing equal volumes of specimens from male or female animals of each species.

The FIB_Clauss assay uses thrombin reagent (HemosIL Fibrinogen-C; Instrumentation Laboratories) composed of lyophilized bovine thrombin with bovine albumin, calcium chloride buffer, and stabilizers. In the FIB_Clauss assay, excess thrombin is used to convert fibrinogen to fibrin in diluted plasma. At high thrombin and low fibrinogen concentrations, the rate of reaction is a function of the fibrinogen concentration.⁴ The analyzer uses sample absorbance at 405 nm to determine the reaction end-point and measures the reaction time in seconds. Fibrinogen concentration then is reported in milligrams per deciliter after interpolation of the reaction time against a fibrinogen standard curve.

In the FIB_PT assay the reagent for prothrombin testing (HemosIL RecombiPlasTin; Instrumentation Laboratories) is used. According to the package insert, this reagent contains thromboplastin that is a liposomal preparation of recombinant human tissue factor in a synthetic phospholipid blend, calcium chloride buffer, and a preservative. The addition of this reagent to the test plasma initiates the activation of the extrinsic coagulation pathway, which ultimately results in the conversion of fibrinogen to fibrin. To perform the FIB_PT assay, the analyzer measures the total change in sample absorbance at 671 nm from the endpoint of the prothrombin reaction. The total change in optical density is directly proportional to the fibrinogen content of the sample.⁴

Statistical analysis.

All statistical analyses were performed by using Analyse-It software for Excel, version 2.22 (Analyse-It Software, Leeds, United Kingdom). Data were analyzed for normality by using the Kolmogorov–Smirnov test and were expressed as mean ± 1 SD. Paired t tests were used to compare the means of fibrinogen concentration measured by 2 assays for each species, and differences were considered significant at P values of 0.05 or lower. Pearson correlation coefficient tests were used to calculate the correlation (r) between fibrinogen concentrations measured by 2 assays. The agreement between 2 assays was assessed by using the Altman–Bland method. Deming regression analysis was performed for bias estimation, and the results were expressed as the 95% confidence interval for the constant and proportional biases. To determine the accuracy of each assay, 5 replicate analyses were carried out for each standard and control material, and the average fibrinogen concentration was calculated. The intra- and interassay precisions expressed as percentages of the coefficients of variation were determined from the mean of the fibrinogen concentration of 5 separate runs of the male and female pooled plasma samples from each species.

Results

The results of t test analysis indicated that the mean plasma fibrinogen concentration measured by 2 assays for all 4 species were significantly (P ≤ 0.05) different (Table 1). When the FIB_PT assay was compared with the FIB_Clauss assay by using Deming regression, there were positive constant and proportional biases for dog plasma fibrinogen and negative constant and positive proportional biases for monkey, rabbit, and rat plasma fibrinogen (Table 2 and Figure 1). Using Pearson correlation coefficient (r), we found that the FIB_PT assay displayed high correlation (range, 0.93 to 0.98) with the FIB_Clauss assay for all 4 species (Table 2). Using the calibration standard and normal and low abnormal controls revealed that the average recovery was 96.3% to 102.1% for the FIB_PT assay and 100.4% to 106.3% for the FIB_Clauss assay. The intra- and interassay coefficients of variation for the FIB_PT assay ranged from 0.8% to 2.0% and 1.0% to 2.3%, respectively, and for the FIB_Clauss assay ranged from 1.8% to 7.4% and 2.0% to 7.3%, respectively.

Table 1.

Fibrinogen concentration (mg/dL) in citrated plasma samples as measured by the FIB_PTand FIB_Claussassays

		FIBPT assay		FIBClauss assay
		Mean ± 1 SD	95% Confidence interval	Mean ± 1 SD	95% Confidence interval
Dog	Male	190.7 ± 21.4a	175.5–204.8	224.3 ± 24.1a	196.8–241.8
	Female	188.4 ± 26.6a	176.6–201.5	217.4 ± 17.3a	204.0–240.8
Monkey	Male	270.2 ± 59.2a	242.5–316.6	238.5 ± 39.3a	214.0–269.4
	Female	261.5 ± 49.3a	237.6–275.1	214.2 ± 21.9a	200.9–221.4
Rabbit	Male	318.8 ± 48.5a	289.4–348.1	258.7 ± 28.7a	241.3–276.1
	Female	288.2 ± 31.6a	263.1–313.4	238.6 ± 13.6a	224.4–252.9
Rat	Male	239.9 ± 32.8a	229.4–264.4	190.3 ± 15.2a	183.5–198.1
	Female	199.6 ± 26.5a	181.3–203.8	158.2 ± 10.3a	149.3–164.2

^aPlasma fibrinogen concentrations as measured by the 2 assays are significantly (P ≤ 0.05) different.

Table 2.

Correlation coefficient (r) and constant and proportional biases between the FIB_PTand FIB_Claussassays for determination of fibrinogen concentration from citrated plasma samples

	n	r (95% confidence interval)	Constant bias (95% confidence interval)	Proportional bias (95% confidence interval)
Dog	40	0.97 (0.94 to 0.98)	45.1 (33.4 to 56.8)	0.65 (0.6 to 0.7)
Monkey	40	0.98 (0.96 to 0.99)	−30.0 (–59.6 to –0.5)	1.30 (1.2 to 1.4)
Rabbit	26	0.93 (0.84 to 0.97)	−134.1 (–222.7 to –45.4)	1.80 (1.4 to 2.1)
Rat	58	0.94 (0.90 to 0.96)	−116.1 (–189.0 to –43.2)	1.90 (1.5 to 2.4)

Figure 1.

Agreement between fibrinogen concentration (mg/dL) as measured by the FIB_PT and FIB_Clauss assays in citrated plasma samples from clinically healthy dog (n = 40), monkey (n = 40), rabbit (n = 26), and rat (n = 58). The intercept and slope for regression lines for dog, monkey, rabbit, and rat were y = 45.11 + 0.65x, y = –30.03 + 1.31x, y = –134.06 + 1.76x, and y = –116.10 + 1.93x, respectively. The dashed line represents the line of identity.

Discussion

Fibrinogen, an acute phase reactant, is an essential component of the hemostatic process. Fibrinogen concentration in blood may be increased in response to inflammation or reduced due to consumption. Currently, the FIB_Clauss assay is the most commonly used method for measurement of fibrinogen concentration in animals. An alternative method for determination of fibrinogen concentration is the FIB_PT assay, which is a mathematical derivation of the sample prothrombin time that is performed automatically by several photo-optical coagulation analyzers. The attraction of the FIB_PT assay is that it rapidly provides a fibrinogen value at no extra cost.

The results of present study showed that fibrinogen concentration determined by the FIB_PT assay significantly differed from those obtained by the FIB_Clauss assay. The fibrinogen concentration measured by the FIB_Clauss assay was higher in dog plasma samples and was lower in monkey, rabbit, and rat plasma samples than that obtained by using the FIB_PT assay. In human medicine, several studies have shown that the FIB_PT assay gives higher values than the FIB_Clauss assay;^1,2,16 however, other studies concluded that the FIB_PT assay is accurate and precise for most routine purposes² and have found a good comparability between the FIB_Clauss and FIB_PT assays.^2,5,14,15 Deming regression analysis revealed positive or negative constant and proportional biases between the results obtained by 2 assays for all 4 species. Although the cause for these biases remains unclear, the bias may be due in part to the methods of calibration for these assays. It has been shown that the degree of discrepancy between results obtained during FIB_Clauss and FIB_PT assays of human plasma samples is related to the type of thromboplastin used within the FIB_PT procedure.^4,7 Additional studies need to be performed to evaluate the effect of thromboplastin type on the fibrinogen concentration in animal plasma samples measured by the FIB_PT assay.

In the present study, the FIB_Clauss and FIB_PT assays displayed high correlation for fibrinogen concentration in all 4 species. A similar result has been reported for human plasma specimens by using these methods.^9,13,14 The inter- and intraassay precision studies revealed lower between- and within-run coefficients of variation for the FIB_PT assay compared with the FIB_Clauss assay. Several investigators have reported lower between- and within-run coefficients of variation for the FIB_PT assay compared with the FIB_Clauss assay for human plasma specimens.^12-14

Because the FIB_PT assay is an indirect measurement of fibrinogen concentration, the choice of calibrant or standard is very important. It has been shown that the FIB_PT assay results vary according to the reagent and analyzer, even between different models from the same manufacturer.⁸ It has been recommended that the results from different instruments or thromboplastin reagents should not be mixed, and separate reference intervals are required.⁷ Furthermore, various substances and pathologic conditions, such as inherited or acquired deficiency of extrinsic pathway factors and the presence of exogenous or endogenous inhibitors that influence determination of prothrombin time, potentially can affect the accuracy of fibrinogen measurement by the FIB_PT assay.^2,14

In conclusion, the FIB_PT assay is a rapid and economical method for estimating fibrinogen concentration in the plasma samples of dog, monkey, rabbit, and rat and can be used as an alternative method when results of a FIB_Clauss assay are unavailable. However, the FIB_PT assay should be used with caution and is not recommended for general use in clinical pathology laboratories analyzing animal samples. The FIB_PT assay should be used only in well-characterized groups of animals and where a within-reference-interval fibrinogen concentration can be predicted. Further studies are required to investigate the performance of this assay in animals with various pathologic states, such as disseminated intravascular coagulation, sepsis, renal disease, hepatic disorder, dysfibrinogenemia, and hypo- or hyperfibrinogenemia and in patients receiving anticoagulants.