Advantages, disadvantages and optimization of one-stage and chromogenic factor activity assays in haemophilia A and B.

Haemophilia A and B diagnosis and disease severity classification are determined on the basis of results from factor VIII (FVIII) and factor FIX (FIX) activity assays, respectively. These assays are also used for potency labelling, postinfusion monitoring of factor replacement products and testing for FVIII/FIX inhibitors. This review focuses on activated partial thromboplastin time (APTT)-based one-stage assays (OSAs) and two-stage chromogenic substrate assays (CSAs). Currently, there is considerable inter-laboratory variability in the results obtained using OSAs, which can be intensified in a reagent-specific manner by the presence of the new modified recombinant factor replacement products that are entering the market. Furthermore, the use of CSAs, which tend to show less variability, especially with the new modified products, is recommended in a number of clinical scenarios. Clinical laboratories may, therefore, need to establish CSAs for routine use. In this review, we aim to improve understanding and help establish best practices by describing the methodology behind OSAs and CSAs and highlighting assay advantages and limitations. We argue that there can be value in offering both assay methodologies in clinical laboratories that contribute to the care of patients with haemophilia A or B. Educating both laboratory scientists and clinicians about the strengths and weaknesses of each type of assay will help to establish the necessary dialogue that is key to ensuring not only that the appropriate assays are used in the right clinical situations, but also that the results are interpreted correctly.

Laboratory testing issues for protein C, protein S, and antithrombin.

Thrombophilia is a complex disease process, which clinically expresses as venous thrombosis. The presence of a genetic defect in one of the major contributing components (protein C [PC], protein S [PS], and antithrombin [AT]) to thrombophilia can be determined by clinical laboratory assays. However, understanding the limitations and problems associated with assays is paramount to an accurate analysis of the genetic status. This review will discuss the major analytical issues and provide recommendations for assaying PC, PS, and AT in plasma. Recommendations are also made about pre-analytical and postanalytical issues clinically affecting these assays.

The effect of instrumentation and laboratory site on the accuracy of the APTT-based heparin therapeutic range.

BACKGROUND: Monitoring of unfractionated heparin therapy by activated partial thromboplastin time using the ex vivo method for determining the heparin therapeutic range (HTR) is the standard of practice. Many extrinsic and intrinsic factors influence accuracy of the HTR. This study investigates the affect of instrumentation and laboratory site on the accuracy of the ex vivo HTR method. METHODS: Patients on unfractionated heparin are used to determine the HTR by published guidelines. Various instruments and different laboratories are compared to investigate the affect of these variables on the HTR. RESULTS: The HTR is the same when the same model of instrument or even different models (same methodology) are used in the same laboratory. However, a significant clinical difference is observed when different hospital laboratories using same lot and instrument model are compared. CONCLUSIONS: The ex vivo method for HTR is the best protocol currently available. The HTR should be determined and averaged on all of the same method instruments used in the laboratory. However, determination of the HTR in different laboratories should not be shared.

Inhibition of thrombin by iopromide in vitro.

Iopromide is a nonionic, iodinated, monomeric, radiographic contrast agent used in various indications, including coronary angiography and visceral and peripheral arteriography. Nonionic contrast media have been postulated to increase thrombogenicity when compared with ionic contrast media. The goal of this study was to characterize the interaction of iopromide with thrombin, specifically to determine the rate, extent, specificity, and reversibility of the thrombin inhibition by iopromide, the integrity of the thrombin-iopromide complex, and the inhibitory potency of iopromide using a validated assay methodology. Iopromide was mixed with purified thrombin or pooled serum from healthy male and female donors. The final concentrations of iopromide in the presence of estimated physiologic concentrations of thrombin (1 nmol/L) were 0-184 mmol/L. After incubation for defined time intervals, the activity of thrombin was determined by adding substrate and measuring the absorbance of the generated chromophores at 405 nm. The possible inhibition of the protease trypsin by iopromide was investigated to evaluate the specificity of thrombin inhibition by iopromide. Iopromide was compared with Thromstop, a known thrombin inhibitor, to assess the relative potency of iopromide. The inhibition of thrombin by iopromide was immediate, rapidly reversible, and proportionate to the iopromide concentrations. The minimum inhibitory concentration of iopromide was 50 mmol/L. At the highest iopromide concentration tested, 184 mmol/L, the mean inhibition of thrombin activity was 44.5%. The mean concentration of iopromide associated with a 50% inhibition was 206 mmol/L. The inhibitory potency of iopromide was 4 x 10(6) times smaller than that of Thromstop. The inhibition of thrombin by iopromide is specific, because trypsin was not inhibited by iopromide. The results indicate that in vitro iopromide at clinically relevant concentrations partially inhibits thrombin activity. However, the in vitro model used does not consider other factors that may be relevant for the overall coagulation response in vivo.

Biochemical evidence of cannibalism at a prehistoric Puebloan site in southwestern Colorado.

The existence of cannibalism is one of the most controversial issues in the archaeology of the American Southwest. Disarticulated, cut-marked and heat-altered human remains from non-burial contexts at prehistoric Puebloan (Anasazi) archaeological sites in the Four Corners region of the American Southwest have been interpreted by some scholars as evidence of cannibalism. Osteological studies indicate that many of the disarticulated bodies found at these sites were processed in a manner consistent with food preparation. Opponents of this interpretation point out that non-cannibalistic practices such as secondary interment, corpse mutilation and ritualized witch executions might account for the assemblages. Osteological evidence alone does not document the actual ingestion of human flesh. Here we show consumption of human flesh did occur as demonstrated in preserved human waste containing identifiable human tissue remains from a site with osteological evidence of cannibalism.

Vitreoretinal findings similar to retinopathy of prematurity in infants with compound heterozygous protein S deficiency.

OBJECTIVE: To present previously undescribed vitreoretinal findings similar to severe retinopathy of prematurity (ROP) in two siblings (daughter and son) with a thrombophilic disorder, compound heterozygous protein S (PS) deficiency. DESIGN: Family genotype study and literature review. PARTICIPANTS: Two unrelated heterozygous PS-deficient parents and their two children with compound heterozygous PS deficiency were studied. The gestational age and birth weight of the daughter were 40 weeks and 3200 g, respectively, and those of the son were 34 weeks and 2150 g, respectively. Three other neonates with homozygous or compound heterozygous PS deficiency and ophthalmologic findings were identified in the literature. INTERVENTION: The daughter underwent lensectomy-vitrectomy at 48 weeks adjusted age bilaterally. The son underwent therapy developed for severe ROP: laser therapy of the peripheral avascular retina at 39 weeks adjusted age, and bilateral lensectomy-vitrectomy with membrane peel of intravitreous proliferation from the optic disc at 42 weeks adjusted age. MAIN OUTCOME MEASURES: The main clinical outcome measures were retinal appearance and functional vision. Genotypes of the family members were determined. RESULTS: One of the four eyes retained functional vision. A normal-appearing posterior retina, normal scotopic and photopic flash electroretinograms, and a normal flash visual-evoked response were documented from the left eye of the son at 62 weeks adjusted age. The other three eyes had inoperable retinal detachments and no functional vision. The mother had type I PS deficiency and the father had type II PS deficiency. Compound heterozygous PS deficiency was confirmed in both children. CONCLUSION: In both children, normal vasculogenesis was interrupted. At 39 weeks adjusted age, the retinal examination of the son revealed extraretinal fibrovascular proliferation at the optic disc (reactivation of the hyaloid system) and in the peripheral retina (interruption of inner retinal vascularization). Patients with homozygous or compound heterozygous PS deficiency may present as infants with severe ROP. The authors' experience suggests that appropriately timed surgical procedures, which are efficacious for ROP, can preserve vision in infants with thrombophilic disorders.

Thrombomodulin gene defects in families with thromboembolic disease--a report on four families.

It has been suggested that an impaired thrombomodulin (TM) function could constitute an abnormality leading to thromboembolic disease (TED). The TM gene from 51 unrelated American patients with TED and 100 American blood donors was screened for mutations. Four heterozygous point mutations in the TM gene were detected. The mutations are distributed throughout the TM gene and predict amino acid changes 1) Pro483 to Leu, 2) Gly61 to Ala, 3) Asp468 to Tyr (earlier described) and 4) a silent mutation not predicting any amino acid change at Glu163. Family studies reveal that the occurrence of the different TM mutations is associated with a history of TED, but there are indications of multiple risk factors and no perfect co-segregation of the TM defects and TED. Among the controls. three individuals carried heterozygous TM variants predicting either a Pro477-Ser mutation (two cases) or an Asp468-Tyr mutation. Our results thus demonstrate that a previously undocumented abnormality in the protein C anticoagulant pathway, a defect in the TM gene, to a certain extent co-segregates with familial thrombophilia. Further studies are needed to prove the causality of these TM mutations.

The effect of time and temperature variables on routine coagulation tests.

This study evaluates the effects of time and temperature variables on routine coagulation assays [Prothrombin Time test and Activated Partial Thromboplastin Time (APTT) test]. Four different groups were studied: healthy volunteers, hospitalized patients not receiving anticoagulants, patients receiving oral anticoagulant therapy and patients receiving unfractionated heparin therapy. Samples were subjected to one of four conditions: (1) centrifuged immediately and stored at room temperature (20-22 degrees C); (2) centrifuged immediately and stored on ice (4 degrees C); (3) stored as whole blood without centrifugation, at room temperature and (4) stored without centrifugation, on ice. Coagulation tests were performed as soon as possible after phlebotomy and at specified times up to 24 h. Our data demonstrate that prothrombin time results are stable for up to 24 h, remaining constant regardless of storage conditions. APTT assays are stable for up to 8 h, except for patients receiving unfractionated heparin therapy. Heparinized samples, when stored uncentrifuged at room temperature, demonstrate a clinically significant shortening of the APTT and individual samples demonstrate a greater than 50% decrease in ex-vivo heparin levels at 4 h.

Minimum specimen volume requirements for routine coagulation testing: dependence on citrate concentration.

We evaluated the effect of sample volume and citrate concentration on results of routine coagulation assays (prothrombin time [PT] and activated partial thromboplastin time [APTT]). The study was performed on samples obtained from healthy persons and patients receiving oral anticoagulant therapy. Standard evacuated tubes (3.2% and 3.8% sodium citrate) were filled to varying total sample volumes ranging from 3.0 to 5.0 mL, and results of routine coagulation tests were compared. Underfilling may significantly affect the APTT and PT, resulting in artifactual prolongation of results. This effect is most pronounced in samples drawn into 3.8% citrate. By using 3.8% citrate, there is a statistically significant difference in the results of PT assays in the samples less than 80% filled compared with those that are 100% filled. For APTT assays performed on samples drawn into 3.8% citrate, a statistical difference occurred at less than 90% filled. This effect was less pronounced when samples were drawn into 3.2% sodium citrate. We found no statistically significant difference in PT results from a 3.2% citrate tube between fill volumes of 60% and 100% and none for APTT results between fill volumes of 70% and 100%. This study further supports the recommendation to use 3.2% sodium citrate concentration, because 60% of the optimum filled volume for PT and 70% of the optimum filled volume for APTT are acceptable.

A laboratory approach to the evaluation of hereditary hypercoagulability.

The concept of hypercoagulability and especially its evaluation in the clinical laboratory has changed dramatically during the last few years. The genetic basis and the mechanisms of the various factors responsible for hypercoagulability are briefly reviewed with emphasis on the most common genetic deficiencies. The major thrust of this review centers on the cost-effective approach to examining patients with a personal or family history of venous thrombosis. Several new concepts dealing with thrombotic risk are presented with a focus on the theory that multiple factors cause thrombosis in affected patients. A proposal for a cost-effective sequential testing scheme for the accurate diagnosis of hereditary hypercoagulability is discussed. The knowledge of thrombotic risk factors is evolving rapidly, requiring the clinical laboratory to remain flexible. Ultimately, the clinical laboratory must take a leading role in the diagnosis of hereditary thrombotic disease by serving as the consultant to the primary caregiver by providing an up-to-date and cost-effective evaluation.

Thrombomodulin gene variations and thromboembolic disease.

Thrombomodulin (TM) is the endothelial cell cofactor for protein C activation. Since deficiencies of other protein C system proteins are known to cause thrombotic disease, then defects in the gene coding for TM could be responsible for inherited thrombophilia. We have searched for mutations in the TM gene among healthy controls as well as patients with thrombophilia and identified eight patients heterozygous for TM mutations that are distributed throughout the TM gene. We have shown that the respective TM mutation co-segregates with thromboembolic disease (TED) in four families. Moreover, we have demonstrated that the C allele in a common C/T dimorphism in the TM gene is significantly more frequent among survivors of premature myocardial infarction (MI) than in matched controls. We suggest that TM defects should be added to the list of risk factors in TED, and after further evaluation possibly be included in a routine laboratory evaluation of thrombophilia.

Effect of 3.2% vs 3.8% sodium citrate concentration on routine coagulation testing.

The effects of 3.2% and 3.8% sodium citrate concentration on the results of routine coagulation assays (prothrombin time [PT] and activated partial thromboplastin time [aPTT]) were evaluated by means of two sets of reagents, one responsive and the other nonresponsive. Five groups were entered in the study: healthy volunteers; outpatients receiving stable oral anticoagulant therapy; and hospitalized patients receiving intravenous (i.v.) heparin therapy, both i.v. heparin and oral anticoagulant therapy, or no anticoagulant therapy. With use of nonresponsive PT and aPTT reagents, varying the citrate concentration has little clinical significance except in patients receiving i.v. heparin therapy. In contrast, when responsive PT and aPTT reagents are used, the concentration of sodium citrate anticoagulant has a significant effect on assay results. Eighteen percent of samples from patients receiving stable oral anticoagulant therapy demonstrated a change of less than 0.7 INR (International Normalized Ratio) units between citrate concentrations. Nineteen percent of patients receiving i.v. heparin therapy had a greater than 7-second difference when aPTT results were compared. These data demonstrate that citrate concentration affects the results of coagulation tests. On the basis of these data, it is recommended that 3.2% citrate be used for all coagulation tests.

Coagulation and fibrinolytic profiles in patients with severe pulmonary hypertension.

STUDY OBJECTIVES: Although in situ thrombosis is a prominent finding in lung vessels from patients with primary and secondary pulmonary hypertension, to our knowledge, plasma coagulation factors that might contribute to a hypercoagulable state have not been fully investigated. We hypothesized that the local coagulation environment in the lung vasculature is important to progression if not initiation of pulmonary hypertension. DESIGN: Quasi-experimental cross-sectional design with concurrent controls. SETTING: Referral clinics and inpatient services of a University Hospital and a Veterans Administration Medical Center. PARTICIPANTS: To investigate the role of plasma coagulation factors in severe pulmonary hypertension, we sampled plasma from patients with primary pulmonary hypertension, patients with pulmonary hypertension secondary to a discernible etiology, and normal adult control subjects. RESULTS: We detected abnormalities of the thrombomodulin/protein C anticoagulant system, evidenced by a decrease in soluble thrombomodulin, in patients with primary pulmonary hypertension. In the patients with primary pulmonary hypertension, we found impaired fibrinolytic activity, with a rise in the fibrinolytic inhibitor plasminogen activator 1 and elevated euglobulin lysis time. Lower fibrinolytic activity correlated with high mean pulmonary artery pressure. In contrast, in patients with secondary pulmonary hypertension, von Willebrand factor antigen and fibrinogen levels were increased, and fibrinolytic activity decreased. CONCLUSIONS: Different patterns of coagulation and fibrinolytic abnormalities are apparent in plasma from patients with primary and secondary pulmonary hypertension. Although we are unable to address causality with this study, we speculate that abnormalities of these coagulation mechanisms may initiate or play a role in perpetuation of pulmonary hypertension.

Heparin levels to guide thromboembolism prophylaxis during pregnancy.

OBJECTIVE: Our purpose was to determine the dose of heparin required in pregnant women to achieve the same heparin levels as standard doses of 5000 units given subcutaneously every 12 hours in the nonpregnant population. STUDY DESIGN: Fourteen pregnant women placed on heparin prophylaxis for a history of thromboembolism had blood drawn for 64 anti-Xa level determinations in the second and third trimesters. Heparin doses were adjusted in an attempt to achieve a midinterval or peak level of 0.05 to 0.25 U/ml, which corresponds to the range seen in nonpregnant patients given standard doses of 5000 units subcutaneously every 12 hours. RESULTS: A standard heparin dose of 5000 units given subcutaneously every 12 hours was inadequate to achieve the desired range in this pregnant population. In five of nine second-trimester pregnancies 7500 units given subcutaneously every 12 hours was inadequate to attain this range. In six of 13 third-trimester pregnancies, > 10,000 units subcutaneously every 12 hours was needed. CONCLUSIONS: Heparin requirements may increase and are highly variable in patients during pregnancy. Until appropriate clinical outcomes trials can determine optimal dosing, measuring anti-Xa activity may be useful to guide therapy.

Longitudinal measurement of anticardiolipin antibodies during normal pregnancy: a prospective study.

We have previously shown that elevation of anticardiolipin antibodies (aCL) at the first prenatal visit is associated with increased fetal loss in normal pregnancy. The variation in aCL levels during normal pregnancy has not been established. To examine this question we measured IgG, IgM and IgA aCL levels five times during pregnancy at weeks 5-15, 16-25, 26-35, 36-37 and at delivery. Data were analyzed to determine: (a) the within and between subject variability of aCL during pregnancy; (2) the temporal trend of aCL; and (3) the relation of serial measures of aCL with maternal complications of pregnancy. We divided our cohort of 354 subjects into two groups. Group A included those subjects with consistently normal levels of aCL and group B those subjects with at least one elevated level of aCL. In group A the within subject variability was relatively low (28-34%). In group B we found wide fluctuations in aCL levels and a within subject variability of 88-91%. Subjects in group B had no increase in maternal complications of pregnancy. The present data suggest that aCL may fluctuate significantly during normal pregnancy and there is little clinical value in measuring aCL on a serial basis during pregnancy.

Developmental expression of protein C and protein S in the rat.

In order to better understand the expression of the Protein C/Protein S anticoagulant system, we have isolated and characterized cDNAs coding for rat Protein C and Protein S. These cDNAs were used in Northern analysis to determine tissue-specificity and developmental expression patterns for mRNAs coding for Proteins C and S. In rats, Protein C mRNA is expressed almost exclusively in liver with a small amount of expression in kidney, diaphragm, stomach, intestine, uterus and placenta. Protein C mRNA was not expressed in brain, heart, lung, spleen, small intestine, large intestine, ovary, or urinary bladder. In liver, Protein C mRNA is expressed at very low levels at prenatal day 18 and these levels increased to maximal levels by postnatal day 13. The size of the mRNA coding for rat Protein C is approximately 1.9 kb. Rat Protein S mRNA was expressed in all tissues examined: brain, heart, lung, diaphragm, liver, spleen, stomach, small intestine, large intestine, kidney, adrenal ovary, uterus, placenta, and urinary bladder. Interestingly, there were 4 bands hybridizing with the rat protein S cDNA that were evident in many of the tissues examined, corresponding to mRNA sizes of approximately 3.5, 2.6, 1.8, and 0.3 kb. There was a difference in tissue-specificity of each mRNA. The 1.8 kb band is generally the most prominent autoradiographic band in any tissue. From these results, it is evident that the expression of Protein C mRNA is similar to that of other vitamin K-dependent proteins. The expression of Protein S mRNA, however, is surprisingly complex and may include alternative splicing of mRNA to generate the various sizes evident on Northern analysis.

The first mutation identified in the thrombomodulin gene in a 45-year-old man presenting with thromboembolic disease.

Thrombomodulin (TM) is the anticoagulant endothelial cell membrane-bound protein cofactor in the thrombin-mediated activation of protein C (PC). It has been clearly demonstrated that the anticoagulant and profibrinolytic functions of the PC system are important for the prevention of a thromboembolic disease. Patients with PC, protein S, or PC "'cofactor"' deficiency and/or dysfunction develop thromboembolic diseases. However, the molecular abnormality in at least 20% to 30% of thrombophilic patients cannot be identified by hitherto recognized defects. A putative pathologic lesion in the TM gene could be one of several candidates for these prothrombotic mutations. A directed search strategy for deletions, insertions, or point mutations in the TM gene has not been performed. Therefore, in the present study, we have analyzed the entire TM gene, including the promoter region, by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) in normal healthy volunteers and in patients presenting with a thromboembolic disease. We have identified a patient with a thromboembolic disease and a TM point mutation. In a 45-year-old Hispanic man with a documented pulmonary embolism, PCR-SSCP showed an aberrant band pattern and subsequent DNA sequence analysis showed a heterozygous substitution for G1456 to T. This substitution predicts an Asp468 to a Tyr change in the amino acid sequence that is located between the transmembrane domain and the sixth epidermal growth factor-like domain. The Asp468 to Tyr change would probably lead to significant structural changes not allowing the expression of the TM protein or to a conformational change that is not functional.