Control de la calidad y ensayos de rendimiento de CromoCen® SALM según la norma ISO 11133:2014/Amd. 1: 2018 / Quality control and performance testing of CromoCen® SALM according to ISO 11133: 2014 / Amd.1: 2018

Introducción: En Cuba, se desarrolló un medio de cultivo cromogénico y fluorogénico, para la detección, aislamiento y diferenciación de Salmonella de otras bacterias Gram negativas. El método que emplea el medio fue validado y su uso se adoptó en una norma cubana. El aseguramiento de la calidad y el control del rendimiento de los medios garantizan la confiabilidad de los resultados analíticos. La norma ISO 11133 establece criterios mínimos y métodos para evaluarlos. Objetivo: Evaluar los criterios de control de la calidad y de rendimiento de CromoCen® SALM, establecidos en la ISO 11133:2014/Amd.1:2018, para demostrar su fiabilidad para el análisis microbiológico de los alimentos de consumo humano. Métodos: Se evaluaron los indicadores de calidad físico-químicos de tres lotes y se definió un conjunto de ellos que caracteriza la calidad del medio antes y después de terminado, así como la consistencia entre lotes. Para el ensayo de rendimiento se seleccionaron 10 cepas de diferentes géneros. Se determinó la relación de productividad, el factor de selectividad y la electividad de CromoCen® SALM, según la ISO 11133. Resultados: La evaluación físico-química mostró una consistencia entre lotes en color, homogeneidad, apariencia del polvo y del medio preparado. Los valores de contenido de humedad y pH se encontraron dentro de los valores establecidos para este producto. La relación de productividad de CromoCen® SALM con respecto al agar triptona soya, fue superior al 50 por ciento, mientras que el factor de selectividad resultó de 4. Se demostró que en el medio de cultivo se puede diferenciar un grupo representativo de géneros microbianos de Salmonella. Conclusiones: CromoCen® SALM cumple con los requisitos de calidad establecidos para este tipo de productos, según la ISO 11133 vigente. La correcta formulación de los lotes, así como el cumplimiento de los requisitos de calidad aseguran el funcionamiento adecuado para lo que fue diseñado(AU)

Introduction: In Cuba, a new chromogenic and fluorogenic culture medium was developed for the detection, isolation and differentiation of Salmonella from other Gram negative bacteria. The method and medium were validated and their use was adopted as a Cuban standard. Quality assurance and control of media is essential and mandatory to ensure the reliability of the results of the analysis in which they are used. ISO 11133 establishes minimum criteria and methods to evaluate them. Objective: To evaluate the quality and performance criteria of CromoCen® SALM, as recommended in ISO 11133:2014/Amd.1:2018 to demonstrate its reliability for the microbiological analysis of food for human consumption. Methods: The physical-chemical quality indicators of three batches were evaluated and a group of them was defined to characterize its quality before and after finishing, as well to evaluate the consistency between batches. For the performance test, 12 strains of different genera were selected. The productivity ratio, the selectivity factor and the electivity of CromoCen® SALM were determined. Results: The physico-chemical evaluation showed a consistency between batches in color, homogeneity, appearance of the powder and of the prepared medium. The moisture content and pH values ranged within the established values for this product. The productivity ratio of CromoCen® SALM with respect to tryptone soy agar was greater than 50 percent, while the selectivity factor was 4. It was shown that in the culture medium a representative group of Salmonella microbial genera can be differentiated. Conclusions: CromoCen® SALM meets the quality requirements established for this type of products, according to the current ISO 11133 standard. The correct formulation of the batches, as well as the fulfillment of the quality requirements ensure the proper functionality and match the design purpose(AU)

Comparative analysis of chromogenic vs clot.based one stage APTT assay for determination of factor VIII level.

BACKGROUND: In the laboratory, factor VIII can be measured by three different methodologies, such as one-stage clotting assay, two-stage clotting assay, and chromogenic assay. These assays differ in ease of use, variety of reagents available, sensitivity to mild hemophilia A, and interference from lupus anticoagulants (LACs). Certain factor VIII gene mutations can cause discrepancy in results between one-stage activated partial thromboplastin time (APTT) and chromogenic assays. MATERIALS AND METHODS: The coagulometer for factor VIII assay is Sysmex CS-5100. All data were expressed as mean ± standard deviation (SD). RESULTS: A total of 135 cases were studied. Of these, 100 cases were of mild hemophilia A diagnosed by molecular genetics and, 15 cases were positive for LAC, which were confirmed by dilute Russell Viper venom test. Clot-based one-stage APTT assay showed 65% sensitivity and 80% specificity in diagnosing mild hemophilia A cases and out of 15 LAC cases, it showed false positivity in five cases. Chromogenic assay showed 85% sensitivity and 90% specificity in diagnosing mild hemophilia cases and was 100% specific in excluding LAC cases. CONCLUSIONS: One-stage APTT assay is the most commonly used test for determining factor VIII levels but chromogenic assay are considered as the gold standard and recommended as the reference method by European Pharmacopoeia and ISTH subcommittee. Mild hemophilia A patients with missense mutations show discrepancy between the one-stage clot-based APTT assay and chromogenic assays for determination of factor VIII level and this can lead to misdiagnosis or misclassification of mild hemophilia A. Therefore, it is recommended that both the assays should be used in the evaluation of mild hemophilia cases.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity.

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0-135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Comparison of clot-based and chromogenic assay for the determination of protein c activity.

: Activated protein C inactivates factor Va and VIIIa. Deficiency of this natural anticoagulant may result in recurrent venous thrombosis. Performance characteristics of clot-based and chromogenic protein C activity assays are different. The clot-based assay has limitations because of interference with coagulation inhibitors resulting in spuriously increased protein C levels or underestimation because of elevated levels of factor VIII and Factor V-Leiden mutation. The chromogenic assay is not influenced by such interferences but only detects functional defects of protein C that involve the active site rendering it insensitive to rare mutations. To compare two methods, we conducted a retrospective study from January 2015 to June 2017. Our results showed a good correlation between clot-based and chromogenic assay (R = 0.94 and r = 0.88). The study of agreement between the two methods by the Bland-Altman method showed that chromogenic method on an average measures 7.8% more protein C than that of clot-based. The results also showed that the bias between the two methods is significant. The positive trend noted was contributed by the values of less than 20% of protein C. Both clot-based and chromogenic assays had high sensitivity; however, the chromogenic assay showed better specificity (97%) as compared with the clot-based assay (93%). In conclusion, we recommend the chromogenic method as the assay of choice, which is also recommended by the College of American Pathologist Consensus Study over activated partial thromboplastin time-based assay. We have shown here that despite a good correlation between the two techniques, there is a difference as highlighted by the difference plots.

Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA® -Cephascreen® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. SUMMARY: Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective assay qualifications. Conclusion The one-stage clotting assay with the STA-Cephascreen APTT reagent, the ROX Factor IX chromogenic assay and the BIOPHEN Factor IX chromogenic assay are considered to be qualified for the measurement of N9-GP in 3.2% (0.109 m) citrated human plasma.

[The effectiveness of hand hygiene products on MRSA colonization of health care workers by using CHROMagar MRSA]. / El Hijyen Ürünlerinin Saglik Çalisanlarinda MRSA Kolonizasyonuna Etkilerinin CHROMagar MRSA ile Arastirilmasi

The aims of this study were; to investigate the hand hygiene compliance of the health care workers (HCWs) during their routine patient care, to determine the methicillin-resistant Staphylococcus aureus (MRSA) hand colonization of the HCWs, to investigate the effect of different hand hygiene products on MRSA colonization and to evaluate the effectiveness of chromogenic agar for detecting MRSA. HCWs were investigated during their routine patient care and hand cultures were taken before and after hand wash/hygiene. Two different techniques were used to obtain the hand cultures: fingertip method (CHROMagar MRSA containing HygiSlide); and direct swab method and then inoculation to CHROMagar MRSA media. MRSA strains grown on those cultures were confirmed with conventional methods. A total of 100 HCWs (of them 61 were female; mean age: 32.7 ± 5.2 years; age range: 25-51 years) involving physicians (n= 33), nurses (n= 38) and health care assistants (n= 29), were included in the study. MRSA was detected in 39% and 11% before hand hygiene and in 13% and 6% after hand hygiene, with HygiSlide CHROMagar media and with CHROMagar in plate media, respectively. No difference were found regarding clinics, occupations, or the type of patient handling in those HCWs who were positive (n= 13) for MRSA colonization following hand hygiene, and those who were negative (n= 26). However, the type of the hand hygiene product used exhibited a statistical difference. None of the seven HCWs who used alcohol based hand rub revealed growth in the second culture while 10 of 19 (53%) HCWs who used soap and three of 13 (23%) HCWs who used chlorhexidine were still colonized with MRSA. In terms of reduction in the MRSA counts, the most effective one was the alcohol based hand rub while the soap was the least, since seven of 19 (37%) HCWs who used soap showed no reduction at all in the MRSA counts. A high ratio of hand colonization with MRSA was detected in our hospital staff (39%). It was shown that the colonization could be reduced significantly (with a rate of 66%) with hand hygiene. Alcohol based hand rub was found to be the most effective method in hand hygiene. The fingertip technique was found to be superior to inoculation to plate media for obtaining hand cultures and CHROMagar MRSA media was found to be rapid, effective and practical for detecting the MRSA hand colonization.

Calibration of human coagulation factor VII concentrate Ph. Eur. BRP batch 2.

The potency assay of human coagulation factor VII concentrate preparations as described in the European Pharmacopoeia (Ph. Eur.) requires a reference preparation calibrated in International Units (IU). The current Ph. Eur. Biological Reference Preparation (BRP) batch 1 was established in 2005 during an international collaborative study. It has an assigned potency of 8.2 IU/vial for the chromogenic assay method. Stocks of this BRP are dwindling and a replacement batch needs to be established. A candidate material was produced by a manufacturer from a plasma-derived concentrate preparation, with the same formulation and approximately the same potency, in the interest of continuity. The candidate material fulfilled the requirements of a BRP with regard to precision and homogeneity of fill, residual water content and stability. The potency of the candidate BRP (cBRP) was determined using chromogenic assays as required by the Ph. Eur. and in-house clotting assays in an attempt to assign a potency for both methods, as is the case for the current batch. The statistical model used for most laboratories was the maximum likelihood of the parallel line model using a logarithmic transformation of the responses. In the chromogenic assay, a potency of 9.9 IU/vial (+/- 1.8 %) was obtained for the cBRP with a very good consistency between laboratories. The results from the clotting assay, however, were less homogenous and yielded consistently higher results (13 IU/vial +/- 12 %), probably due to a higher activated factor VII (FVIIa) content than in the current BRP (3 % as compared to 0.3 %). Due to the large difference between the values obtained with the 2 different methods, it was not possible to reconcile the outcomes with each other. On the other hand, the uncertainty observed with the clotting assay method was quite large and seemed questionable for a reference preparation. Therefore the use of BRP batch 2 as a reference for the clotting assay method is not recommended. Nevertheless, the results of the study showed that the candidate BRP (cBRP) is suitable as a reference standard for the chromogenic assay according to the Ph. Eur. general chapter 2.7.10 Assay of human coagulation factor VII. It was adopted by the Ph. Eur. Commission in December 2009 as an official Ph. Eur. BRP for human coagulation factor VII concentrate with an assigned potency of 9.9 IU/vial.

Establishment of the human coagulation factor VII concentrate European Pharmacopoeia biological reference preparation batch 1.

For the potency assay of human coagulation factor VII concentrate preparations according to the European Pharmacopoeia (Ph. Eur.) a reference preparation calibrated in International Units (IU) is needed. Currently, the 1st International Standard (97/592, potency: 6.3 IU/ampoule) but no Ph. Eur. reference preparation is available. A collaborative study was run to calibrate a candidate Ph. Eur. Biological Reference Preparation (BRP) for human coagulation factor VII concentrate against the 1st International Standard; the BRP is intended to be used as working standard. A candidate BRP batch 1 was produced from a plasma-derived human factor VII concentrate preparation available on the European market. It fulfilled the requirements of a BRP with regard to precision and homogeneity of fill, residual water content and stability. In addition, the content of activated factor VII was low. Sixteen laboratories from 9 countries participated in the collaborative study. The potency of the candidate BRP was determined using the participants' chromogenic assay based on the Ph. Eur. and their in-house clotting assay, if available. The statistical model used for analysis of the results from most laboratories was the maximum likelihood of the parallel line model following a logarithmic transformation of the responses. In the chromogenic assay, a potency estimate of 8.2 IU/vial (+/-3.7%) was obtained for the candidate BRP. Results from the clotting assay were lower and less homogenous (6.7 IU/vial+/-11.6%). The results from the collaborative study showed that the candidate BRP is suitable as a reference standard for the chromogenic assay according to the Ph. Eur. It was adopted by the Ph. Eur. Commission in March 2006 as official Ph. Eur. BRP for this purpose.

Factor VIII test in reference preparations: compensation for different dilutions.

An approach is described that aims to prevent the problems associated with factor VIII (FVIII) assays. This approach takes a number of factors into consideration: 2 different assay types are currently being performed (clotting and chromogenic), different standards coexist (for plasma and for concentrates) and not only are reference standards being diluted for test purposes, but likewise plasmas are diluted by adding reference standards which are significantly less concentrated than commercial concentrates. Moreover, artificially FVIII-depleted plasmas are diluted as compared to normal or haemophilic plasma with respect to non-FVIII proteins as well. Taken together, these observations have led us to re-examine FVIII assays and to establish an algorithm for FVIII measurements with plasma as the natural reference.

Chromogenic assay of human coagulation factor VIII: statistical comparison of 2 working dilution procedures.

The effect of 2 different practices for preparation of working dilutions in the chromogenic substrate method for potency assay of factor VIII was evaluated. In this study the potency of several concentrate materials was shown to be statistically equivalent, whether performing the assay with independent or serial working dilutions.

A multicentre assessment of the endogenous thrombin potential using a continuous monitoring amidolytic technique.

This study assessed the inter-laboratory imprecision associated with the measurement of the endogenous thrombin potential (ETP). The initial studies used techniques that had evolved in each of the participating laboratories. Samples from normal healthy subjects (n = 10), two patients receiving coumarin therapy [International Normalized Ratio approximately 2.0 and approximately 4.0] and a further two subjects receiving treatment with unfractionated heparin (anti-Xa 0.07 i.u./ml and 0.31 i.u./ml) were assayed relative to a lyophilized normal plasma that had arbitrarily been assigned a potency of 100%. Considerable variation in potency estimates was observed between the centres, although individual laboratories using fully automated techniques achieved acceptable levels of imprecision as assessed by the coefficient of variation (CV) (intra-assay CV < 9.5%, inter-assay CV < 12.5%). A second study to assess a similar range of samples, using a standardized assay protocol and incorporating appraisal of two chromogenic substrates, CBS.0068 or Pefachrom TG, demonstrated markedly improved agreement in potency estimates between centres and good correlation (r > 0.96) between the chromogenic substrates. Our data demonstrates that an automated ETP method can be standardized between laboratories and suitable levels of imprecision achieved, using different analysers (COBAS Mira at two centres and an ACL-300R) and two thrombin substrates. This indicates that more widespread use of ETP measurements in clinical laboratories is feasible.

Calibration of the Ph. Eur. BRP Batch 3/Mega 2 (US/FDA) standard for human coagulation factor VIII concentrate for use in the potency assay.

The European Pharmacopoeia Biological Reference Preparation Batch 3/Mega 2 (United States/Food and Drug Administration) (Ph. Eur. BRP Batch 3/Mega 2 (US/FDA)) was developed as an internationally available, common working standard to replace the dwindling stocks of Mega 1 (the current US standard) and Ph. Eur. BRP Batch 2 (the current European standard). The potency was assigned in an international collaborative study with reference to four currently established standards, Ph. Eur. BRP batch 2, WHO 5th and 6th International Standard and Mega 1. Thirty-eight laboratories participated in the collaborative study. Each laboratory was asked to perform four independent assays. Participants used either the one stage clotting assay or the chromogenic assay or both. This publication reports the results obtained with both assays. The summary and conclusion, however highlight the results mainly with respect to the chromogenic assay, which is the assay prescribed in the European Pharmacopoeia. Data were analysed for both assays separately. A consensus potency value was calculated as the unweighted average of mean potencies determined against the four standards. A potency of 8.6 IU/vial as determined in the chromogenic substrate method was assigned to the candidate standard. Inter-laboratory agreement as assessed by calculation of the geometric coefficient of variation was below 10% for mean potencies against all four calibrators for the chromogenic assay. Ph. Eur. BRP Batch 3/Mega 2 (US/FDA) is a freeze-dried, plasma derived, high-purity concentrate. The material was filled into approximately 100,000 vials and lyophilised to a final residual moisture of < or = 2%. Approximately 90,000 vials of the standard are available, equally shared between the two co-ordinating centers. Based on the stability studies, the predicted mean percentage loss per year at -20 degrees C is 0.000% and thus the candidate standard appears to be stable. The Ph. Eur. BRP batch 3 was adopted by the European Pharmacopoeia Commission in November 2001.

Assaying the circulating factor VIII activity in hemophilia A patients treated with recombinant factor VIII products.

Large discrepancies between factor VIII assay methods have been reported from pharmacokinetic studies of recombinant factor VIII concentrates. In the assay of postinfusion patient plasma samples, traditional activated partial thromboplastin time (aPTT)-based one-stage clotting methods usually give results that are 20 to 50% lower than those obtained by chromogenic substrate assays. Investigations into the cause of these discrepancies have shown that the choice of phospholipid in the one-stage assay is crucial. The use of platelets or liposomes resembling platelet factor 3 instead of traditional aPTT reagents results in an increase in the apparent one-stage activity and a fairly good correlation with the chromogenic results. These and other functional test results, antigen measurements as well as clinical data, support the view that the chromogenic assay most accurately reflects the therapeutic effect. In addition to the differences among assay methods, there is also a discrepancy between the World Health Organization (WHO) standards for concentrates and plasma. The use of product-specific standards, prepared by diluting the factor VIII concentrate into hemophilic plasma, when assaying postinfusion plasma samples seems to be a feasible approach to overcome the problems encountered in pharmacokinetic studies.

Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.

Factor VIII (FVIII) is assayed by one-stage and two-stage clotting methods and by chromogenic methods, although the chromogenic method has largely replaced the two-stage clotting assay. Clinical plasma samples are assayed mostly by one-stage assays, but most manufacturers of concentrates use the chromogenic method, which is more precise and is the reference method of the European Pharmacopoeia and the International Society on Thrombosis and Haemostasis (ISTH). For most plasma-derived concentrates, assays against the World Health Organization (WHO) concentrate standard give similar results with the one-stage and chromogenic methods, but for products produced by the "method M" monoclonal antibody process, the one-stage potency is 25 to 30% higher than the chromogenic potency. For full-length recombinant products assayed against a plasma-derived concentrate standard, one-stage potencies are about 10% lower than chromogenic potencies, but for the B-domain deleted recombinant product ReFacto, the discrepancy is larger-from 20 to 50%. These discrepancies emphasize the need for an international methodology for labeling of concentrates. In ex vivo assays of hemophilic plasmas after infusion of concentrates, large discrepancies are found among laboratories and with different assay methods when a plasma standard is used. In most studies, the chromogenic potencies are higher than the one-stage potencies, and the discrepancy is highest for recombinant products. This discrepancy can be largely eliminated by the use of concentrate standards, diluted in FVIII-deficient plasma, to assay postinfusion plasma samples.

Measurement of factor VIII activity of B-domain deleted recombinant factor VIII.

The factor VIII activity of B-domain deleted recombinant factor VIII (BDDrFVIII) measured by activated partial thromboplastin time (APTT)-based one-stage assays is approximately 50% of the activity obtained by the chromogenic assay. Similar results have been reported for the two licensed full-length recombinant factor VIII products. In view of these findings, comprehensive studies have been undertaken to find the cause of the assay differences. Only the phospholipid reagent, used as a platelet substitute in the one-stage assay, proved to be crucial for explaining the assay difference. When platelet-rich plasma was used as the source of phospholipid in the one-stage assay, the factor VIII activity assay results correlated well with those measured by the chromogenic assay. Similar results were obtained when the platelets were replaced by liposomes prepared using platelet factor 3 (PF3) as a model that has a low content (5% to 10%) of phosphatidylserine (PS). In contrast, the use of liposomes with 20% to 30% PS, as in the crude lipid extracts used in ordinary APTT reagents, resulted in underestimation of the factor VIII activity. Antigen measurements using an enzyme-linked immunosorbent assay (ELISA) method demonstrated a good correlation between the antigen and chromogenic activity, but not always between antigen and one-stage activity results. Based on these findings and the clinical data, it can be concluded that the chromogenic assay most accurately measures the functional activity of BDDrFVIII. However, modifications of the one-stage assay, such as the use of a product-specific standard or development of a PF3-like phospholipid reagent, could address the observed assay discrepancies.

Determination of plasma aprotinin levels by functional and immunologic assays.

We compared a functional (amidolytic) and an enzyme-linked immunosorbent assay (ELISA) method for determining aprotinin concentration in 82 plasma samples obtained from patients undergoing cardiac surgery with aprotinin therapy. There was good correlation between methods (r = 0.87); however, aprotinin measurements by chromogenic assay were significantly higher than by ELISA [234 +/- 104 kallikrein inhibitory units (KIU)/ml versus 155 +/- 88 KIU/ml; P = 0.0001]. This appeared to be attributable to differences in the potency of the material used to standardize the assays. When results were corrected to allow for potency of the standard, there was no significant difference between chromogenic and ELISA methods (234 +/- 104 KIU/ml versus 240 +/- 137 KIU/ ml), although the ELISA results tended to be higher in some samples. These data suggest that aprotinin concentrations measured by these methods cannot be used interchangeably, and care must be taken when interpreting data from studies measuring aprotinin.

Measurement of antithrombin activity by thrombin-based and by factor Xa-based chromogenic substrate assays.

Functionally active antithrombin can be quantified by chromogenic substrate assays utilizing the heparin cofactor activity of antithrombin and the inhibition rates of thrombin or of activated factor X (FXa). Thrombin-based assays but not FXa-based assays may overestimate the antithrombin activity due to their sensitivity toward heparin cofactor II. We focused on the question whether an overestimation of antithrombin activity by thrombin-based assays involves the risk of misdiagnosing antithrombin-deficient individuals as being non-deficient. We determined antithrombin using two thrombin-based assays and one FXa-based assay in 27 plasma samples from patients with acquired antithrombin deficiency spiked with lepirudin, in antithrombin-deficient plasma and in mixtures of antithrombin-deficient plasma and normal plasma. We also measured antithrombin in healthy subjects, in patients with inherited and acquired antithrombin deficiency and in patients under high-dose heparin treatment. At therapeutic final concentrations of lepirudin, antithrombin activities were considerably overestimated by the thrombin-based assays but not by the FXa-based assay. The residual antithrombin activities in antithrombin-deficient plasma determined by the thrombin-based assays were markedly higher than the corresponding values obtained with the FXa-based assay. The thrombin-based assays also overestimated antithrombin activity in patients under high-dose heparin. However, the degree of overestimation in the range between 50 and 100 IU/dl was too low to misidentify individuals with inherited or acquired antithrombin deficiency as normal. We conclude that functionally active antithrombin can be reliably determined using FXa-based chromogenic substrate assays in all settings examined. Thrombin-based assays must not be used in patients under treatment with hirudin or other direct thrombin inhibitors.

Issues with the assay of factor VIII activity in plasma and factor VIII concentrates.

A review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VII concentrates such as Hemofil M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements. it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor VIIIa during the assay process.