Factor VIII Assays Research Articles

The Use of Chromogenic Factor VIII Assay Changes Treatment Approach in a Portion of Mild Hemophilia A Patients with Factor VIII Assay Discrepancy

Introduction Hemophilia A is an X-linked inherited bleeding disorder characterized by a deficiency of coagulation factor VIII (FVIII). FVIII assays used for diagnosis and classification of hemophilia are the widely used one-stage assay (OSA) and the chromogenic assay (CSA), a more costly and less commonly used test. The CSA has the advantages of improved precision and insensitivity to pre-activation effects of FVIII that can lead to erroneous results. In approximately one-third of mild hemophilia A cases, reliance on OSA can lead to overestimation of factor level in comparison to CSA, and this 'discrepancy' phenomenon is closely associated with the FVIII molecular abnormality. In mild or moderate hemophilia A, assay discrepancy can lead to misclassification or inappropriate treatment. The objectives of this retrospective study were to determine the proportion of patients with OSA/CSA discrepancies defined by OSA to CSA ratio of >1.5 or <0.5, to identify previously reported and new F8 gene mutations and to classify the impact of assay discrepancy on clinical treatment approach. Also, the study aimed to re-evaluate published discrepant OSA/CSA ratios to identify a ratio with the highest sensitivity to indicate that a change of treatment approach or intensity may be required. Methods We reviewed the charts of adult (>18 years old) patients with mild or moderate hemophilia A followed at the Adult British Columbia Hemophilia clinic with concomitant OSA and CSA results between January 2013 and March 2019. Data collected included patient age, disease severity classification, baseline OSA FVIII:C, and F8 gene mutation. Bleeding phenotype and impact of discrepancy on treatment approach was evaluated and determined independently by two individuals and was correlated with concomitant OSA and CSA. Gene mutation data was collected to determine whether OSA/CSA discrepancy has been previously reported in the international hemophilia A mutation database (www.factorviii-db.org). Descriptive data was reported as medians and ranges. Statistical analyses using Spearman's correlation, logistic regression and receiver operating characteristics (ROC) curve were performed using IBM SPSS v. 22.0. Results 98 patients were included in the study with median age of 53 years (18-88). 75 patients were classified by OSA at baseline as mild and 23 patients as moderate severity. Median FVIII:C by OSA was 15% (6%-40%) in the mild group and 3% (1%-5%) in the moderate severity group. Median FVIII:C using CSA was 13% (2%-66%) in the mild group and 4% (1%-7%) in the moderate group. Based on ratio alone, OSA/CSA discrepancy was detected in 51 (52%) patients out of which 3 (3%) were reverse discrepancies (CSA higher than OSA). Treatment approach was changed as a result of CSA introduction in 14 (27%) patients with OSA/CSA discrepancy (14% of the entire study population). OSA had overall good correlation with CSA (r = 0.84) and as expected, there was no correlation between age and OSA to CSA ratio (r = 0.03). In the patients with OSA/CSA discrepancies, neither age nor OSA/CSA predicted a change of treatment approach (OR 1.02 for age; 95% CI 0.99-1.06; p value 0.213 and OR 1.48 for OSA/CSA; 95% CI 0.96-2.28; p value 0.08). The OSA to CSA ratio of 1.8 to 3.5 demonstrated the highest area under the ROC curve and sensitivity for identification of requirement for treatment change in patients with OSA/CSA discrepancy (AUC 0.75; sensitivity 71%, negative predictive value 75%). Six F8 gene mutations with OSA/CSA discrepancies previously not reported in the database were identified. Conclusions OSA/CSA discrepancies were detected in 52% of the patients with mild or moderate hemophilia A and resulted in change of treatment approach in 27% of discrepant patients. Use of an adjusted OSA/CSA ratio between 1.8 to 3.5 was more likely to detect cases with a requirement for change of treatment approach and use of this ratio to indicate discrepancy may be more clinically meaningful. Six gene mutations associated with OSA/CSA discrepancies previously not described in the international hemophilia A mutation database were identified in the study population, highlighting the need for genotyping discrepant cases. Disclosures No relevant conflicts of interest to declare.

Bleeding in Mild Hemophilia A Due to a Splice-Site F8 Mutation May be Fully Abrogated By Prothrombotic Gene Variants

Introduction: While Factor VIII (FVIII) activity correlates with phenotype in most Hemophilia A (HA) males, the study of rare individuals with discrepant bleeding phenotypes can provide insight into F8 genotype-bleeding phenotype relationships, refine HA classification and improve clinical management. We present an adult male subject who carries a F8 missense mutation, c.5999G>C (p.Gly2000Ala), but remarkably has had no bleeding despite numerous challenges, including multiple surgeries. To resolve genotype-phenotype discrepancy, we evaluated F8 transcripts and screened for additional gene variants by exome sequencing. Methods: We performed clinical laboratory assays including one-stage FVIII:C and chromogenic FVIII assays, FVIII antigen ELISA, VWF antigen and activity assays, and von Willebrand Disease (VWD) Type 2 Normandy binding activity assay. The F8 c.5999G>C mutation is proposed to affect F8 splicing, and result in a transcript that deletes exon 19. To evaluate F8 splice variants, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using primers that distinguish FVIII isoforms. Samples were normalized relative to a housekeeping gene, GAPDH. Normalized relative fold expression for each primer pair was calculated by the 2−ΔΔCT Livak method. Whole exome sequencing was performed at 80X coverage. Sequencing reads were mapped to the GRCh37/hg19 reference genome with BWA-MEM, and high-quality variant calls were made using GATK4.0 with annotation based on refSeq 1.9, employing best practice recommendations. Results: Clinical assays confirmed reduced FVIII activity levels consistent with mild HA. By qRT-PCR, the F8 mutation results in a partial splicing defect. Exon 19 deleted transcripts predominate, but are likely not secreted. Indeed, the reduced levels of full-length F8 isoform closely mirror measured FVIII protein and activity levels. These findings explain FVIII levels, but to do not explain the absent phenotype. Exome sequencing analysis first confirmed the F8 mutation and excluded other genetic causes of FVIII deficiency. Additionally, we excluded thrombophilic mutations proposed to attenuate bleeding severity in HA, including Factor V Leiden (c.1601G>A, p. R534Q) and the c.*97G>A (p.G20210A) mutation in the F2 gene encoding prothrombin. To identify additional variants that may modify hemostasis, we then screened for non-pathogenic variants in 96 genes that are known to be mutated in bleeding disorders or that alter FVIII clearance or half-life. Of 352 single nucleotide variants (SNVs) in these genes, we prioritized 10 non-synonymous variants with annotated effects on hemostasis. Of these, four are associated with functional effects that would potentiate bleeding phenotype in coagulation factor deficiency. In contrast, five variants are associated with prothrombotic effects and are strong candidates for ameliorating bleeding. Of these, two non-pathogenic VWF variants, rs1063856 and rs7962217, have been shown to increase FVIII. Three other variants in F5 reside within the R2 haplotype. R2 has effects in the absence of Factor V Leiden and can increase thrombin generation and FVIII levels. Conclusion: The identification of multiple variants that are expected to attenuate bleeding suggests that a single gene variant is unlikely to ameliorate bleeding phenotype in this subject, particularly in the presence of additional variants that promote bleeding. These results suggest evaluation of non-pathogenic variants in non-F8 genes may further explain other cases of discrepant HA not resolved by clinical assays. While these new modulators of HA phenotype require functional confirmation, they provide new avenues for therapeutic development. Disclosures Eyster: Bayer: Other: research support; Baxalta: Other: research support; Shire: Other: research support; NovoNordisk: Other: research support; SPARK: Other: research support.

The Discordance between Offsite to Onsite Testing for Von Willebrand Disease in Post-Menarchal Females: A Multi-Center Study

The Discordance between Offsite to Onsite Testing for Von Willebrand Disease in Post-Menarchal Females: A Multi-Center Study

Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves.

The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs. The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels. We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism. Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25IU/dL in spiked samples and in detection of FVIII activity (≥1IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results. Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments.

Application of Turbidity Curve Analysis to New Coagulation Factor VIII and IX Assays with Superior Analytical Resolution in Low-Level Range

Background: The needs for sensitive coagulation factor assays able to measure factor VIII (FVIII) and factor IX (FIX) in the range of 0.0 to 1.0 %, are continuously growing with diversification of hemophilia management. However, practical methods with sufficient analytical sensitivity available in clinical laboratory have not yet been introduced. We developed new coagulation factor assays applying various parameters derived from a turbidity based coagulometer and examined their ability to measure low-level FVIII and FIX and analytical resolution in that range.Method: We prepared 12 spiked samples with FVIII and FIX levels from 0.0 to 2.4 % and conducted conventional one-stage coagulation factor assays in repeat. We collected measured values of APTT, velocity and acceleration peaks of coagulation (peak 1 and peak 2) from each measurement. We also calculated values of peak 1 and peak 2 from the mathematical model of turbidity curves. From the measured values of these parameters we derived calibration formulae for coagulation factor assays, FVIIICT, FVIIIpeak1, FVIIIpeak2, FVIIIcalc1, FVIIIcalc2, FIXCT, FIXpeak1, FIXpeak2, FIXcalc1, and FIXcalc2.Results: The reliability interval (range of FVIII levels producing unequivocal results) of FVIIICT (the conventional FVIII assay) covered only 9 % of 0.0 to 1.0 % range. For new assays, the coverages were 54, 31, 55, and 65 % for FVIIIpeak1, FVIIIpeak2, FVIIIcalc1, and FVIIIcalc2 respectively. The resolution between immediate levels of spiked samples could be determined from modeled distributions or be checked simply by inspecting the actual assay result distributions. For FVIIIpeak1, 0.2 % and 0.6 % results stood apart from each other. For FVIIIcalc1 and FVIIIcalc2, 0.2, 0.4, and 0.6 % were distinguished from each other. When we measured recombinant human (rh) FVIII, the coverages were 7, 64, 52, 73, and 79 % for rhFVIIICT, rhFVIIIpeak1, rhFVIIIpeak2, rhFVIIIcalc1, and rhFVIIIcalc2 respectively. (rh)FVIIIpeak1, (rh)FVIIIcalc1, and (rh)FVIIIcalc2 particularly showed wide measurable ranges of guarantee. For FVIIIpeak1, 0.2 % and 0.6 % results stood apart from each other. For FVIIIcalc1 and FVIIIcalc2, 0.2, 0.4, and 0.6 % were distinguished from each other. rhFVIIIpeak1 and rhFVIIIcalc1 showed slightly better resolution than the former. rhFVIIIcalc2 was notable in that every 0.1, 0.2, 0.4, 0.6, 0.8 % result stood apart from each immediate level result. We could not determine certainty interval (the range of unequivocal values) of FIXCT and FIXpeak2 because the 0.0 % and 1.0 % ranges overlapped. Thus, the conventional FIX assay cannot measure between 0.0 and 1.0 %. FIXpeak1, FIXcalc1, and FIXcalc2 worked better and the certainty interval of unequivocal results could be determined between 0.0 and 1.0 %. The reliability interval was not available for any FIX assay. Results from rhFIX measurements were similar those of plasma FIX assays.Conclusion: We introduce new FVIII and FIX assays with superior analytical resolution in the range of 0.0 to 1.0 % in comparison to the conventional assays. DisclosuresNo relevant conflicts of interest to declare.

A Practical, One Clinic Visit, Population Pharmacokinetic (PK) Protocol for Generation of PK Profiles in Subjects with Severe Hemophilia a

IntroductionClearance of infused factor VIII (FVIII) varies approximately 2-fold between persons with severe hemophilia A. This results in significant interpatient differences in factor levels following an infusion of FVIII and contributes to potentially significant differences in protection against spontaneous musculoskeletal bleeding in patients on fixed dose prophylaxis regimens.AimThe aim of this study is to compare two PK protocols: 1) a 6-point PK protocol with a 72 hour washout; and 2) a 2-point, one clinic visit PK protocol with no washout using the following pharmacokinetic (PK) parameters: clearance (Cl) and time to FVIII:C of 1% above baseline (tt1%) in persons with severe hemophilia A.MethodsInhibitor negative males with severe hemophilia A (FVIII<2%) receiving a standard half-life recombinant FVIII (rFVIII) concentrate (ADVATE®) were consented into a research ethics board approved study. In the 6-point PK protocol, participants were infused with approximately 50 IU/kg rFVIII after a minimum washout of 72 hours and FVIII levels were measured pre-infusion and at 1, 3, 9, 24 and 48 hours post-infusion. The 2-point PK protocol consisted of a blood sample taken in clinic approximately 24 hours after the participant infused their prophylactic dose at home (15-50 IU/kg), followed by a 25 IU/kg dose given in clinic and a 3 hour post-infusion sample. Frozen plasma samples were sent to a central laboratory in Kingston, Ontario where one-stage and chromogenic FVIII assays were performed. PK parameters (Cl and tt1%) were estimated using the 2 compartmental models of PK programs Phoenix WinNonlin 7.0 (Certara USA Inc.) and myPKFiT version 3.0 (Baxalta US Inc). Intra-class correlations (ICCs) were used to compare the PK parameters derived from the two PK protocols using WinNonlin and myPKFiT.Results28 males (median age: 12 years, range: 2-69 years) participated. The frequency distribution of clearance and the median half-life (t1/2) generated using myPKFiT is presented in Figure 1. There was a substantial to almost perfect agreement observed when comparing the PK parameters derived from the 6-point PK protocol with washout using the two PK programs (Table 1). There was a moderate to almost perfect agreement observed when comparing the PK parameters derived from the 6-point PK protocol with washout to the 2-point PK protocol with no washout using the myPKFiT program (Table 2).ConclusionThe 2-point, one clinic visit, PK protocol (24 and 3 hrs) with no washout offers a convenient and practical approach to generating clinically relevant PK parameters in persons with severe hemophilia A. It can provide information relevant to selection of personalized prophylaxis regimens that aim to reduce to a minimum/eliminate spontaneous joint bleeding. [Display omitted] DisclosuresBlanchette:Shire: Other: Investigator-initiated research funding; Novo Nordisk: Other: Speaker's fees; Shire: Other: Speaker's fees; Bayer: Other: speaker's fees; Bioverativ: Other: Investigator-initiated research funding; Pfizer: Other: Speaker's fees. Jackson:Pfizer: Honoraria; Roche: Honoraria; Bayer: Honoraria; Novo Nordisk: Honoraria; Shire: Honoraria; Bioverativ: Other: Investigator initiated grant funding. Carcao:Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; LFB: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL-Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotest: Honoraria, Membership on an entity's Board of Directors or advisory committees; Grifols: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bioverativ/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Khoo:Shire: Research Funding; Biogen Idec: Research Funding. Blatny:Shire, Pfizer, Roche: Consultancy, Speakers Bureau.

The importance of commutability in material used for quality control purposes.

External Quality Assessment (EQA) is an important part of laboratory quality assurance. Spiking of normal plasma is sometimes employed to mimic clinical samples. It is important that spiked material gives similar results to clinical samples (ie, is commutable) to ensure appropriate conclusions can be drawn from EQA exercises. We describe here data from UK National External Quality Assessment Scheme for Blood Coagulation (NEQAS BC) exercises where spiked samples were tested alongside samples from patients to explore commutability of artificial material. Normal plasma was spiked with unfractionated heparin (UFH), low molecular weight heparin (LMWH), Dabigatran or Rivaroxaban. Factor VIII (FVIII) deficient plasma was spiked with FVIII concentrate. Spiked samples and ex vivo patient material were sent to laboratories for testing. For LMWH, good agreement was seen between results for samples from patient plasma and plasma spiked with heparin. For UFH, APTT ratios differed between spiked and patient samples for the same drug concentration, with no correlation in ranking of reagents. Precision for patient and spiked material for Rivaroxaban and Dabigatran assays was comparable. However, the pattern of results for some Dabigatran assay kits differed between spiked and patient samples. For FVIII assays, results obtained with spiked and postinfusion samples gave comparable results. Spiked material is suitable for EQA if commutability is demonstrated. Our data show commutability for plasma spiked with Rivaroxaban, LWMH and some FVIII concentrates. For some tests, notably APTT for UFH, marked differences between patient and spiked samples indicate not all tests can be evaluated using artificial samples.

The effect of unmeasurable endogenous plasma factor activity levels on factor VIII dosing in patients with severe hemophilia A

BackgroundPatients with hemophilia A are defined as “severe” if they present <0.01 IU mL−1 of clotting factor VIII (FVIII) activity endogenously (i.e. in absence of FVIII concentrate administration). However, an exact measurement of baseline FVIII is often impossible in such patients as the lower limit of quantification for most FVIII assays is around 0.01 IU mL−1, forcing assumptions to be made regarding endogenous circulating level. ObjectiveThis work aims to assess the consequences of different assumptions when selecting a prophylaxis regimen for the treatment of hemophilia A using a pharmacokinetics (PK)-driven approach. MethodsUsing a validated population PK model for conventional FVIII, we simulated the dose and frequency required to maintain various target troughs as a function of different postulated baseline levels. ResultsPatients with a true baseline of 0 IU mL−1 would require an additional 40 IU kg−1 to achieve any target trough below 0.05 IU mL−1, as compared to those with a true baseline close to 0.01 IU mL−1. When tailoring individual treatment regimens using a PK-based approach, baseline assumptions were highly influential and the assumption producing the most conservative dosing regimen was not consistent; rather, it depended on the particular scenario (e.g. increase vs. decrease of an observed trough). ConclusionsTargeting a trough close to the assay sensitivity creates a unique problem in forecasting the dose needed for optimal treatment of hemophilia that, while still without solution, deserves consideration when making dosing decisions.

Immune tolerance induced by platelet‐targeted factor VIII gene therapy in hemophilia A mice is CD4 T cell mediated

Background Immune responses are a major concern in gene therapy. Our previous studies demonstrated that platelet-targeted factor VIII (FVIII) (2bF8) gene therapy together with in vivo drug selection of transduced cells can rescue the bleeding diathesis and induce immune tolerance in FVIIInull mice. Objective To investigate whether non-selectable 2bF8 lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and how immune tolerance is induced after 2bF8LV gene therapy. Methods Platelet-FVIII expression was introduced by 2bF8LV transduction and transplantation. FVIII assays and tail bleeding tests were used to confirm the success of platelet gene therapy. Animals were challenged with rhF8 to explore if immune tolerance was induced after gene therapy. Treg cell analysis, T-cell proliferation assay and memory B-cell-mediated ELISPOT assay were used to investigate the potential mechanisms of immune tolerance. Results We showed that platelet-FVIII expression was sustained and the bleeding diathesis was restored in FVIIInull mice after 2bF8LV gene therapy. None of the transduced recipients developed anti-FVIII inhibitory antibodies in the groups preconditioned with 660 cGy irradiation or busulfan plus ATG treatment even after rhF8 challenge. Treg cells significantly increased in 2bF8LV-transduced recipients and the immune tolerance developed was transferable. CD4+ T cells from treated animals failed to proliferate in response to rhF8 re-stimulation, but memory B cells could differentiate into antibody secreting cells in 2bF8LV-transduced recipients. Conclusion 2bF8LV gene transfer without in vivo selection of manipulated cells can introduce immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell mediated.

Genotype and phenotype correlation in intracranial hemorrhage in neonatal factor VII deficiency among Thai children.

Congenital factor VII (FVII) deficiency is a rare inherited coagulopathy. The clinical manifestations and clinical findings vary widely, ranging from asymptomatic to life-threatening bleeding, including intracranial hemorrhage (ICH), with prolonged prothrombin time, normal partial thromboplastin time and normal platelet counts, which are confirmed by the low level of FVII assay. Treatment consists of fresh frozen plasma (FFP), prothrombin complex concentrates (PCCs), and recombinant activated FVII to treat bleeding and prophylactic therapy. Here, we report four patients with FVII levels <5% (severe type) who presented ICH during the neonatal period. The IVS6+1G>T was the most common (50%) mutation identified in our study, followed by the K376X nonsense mutation (37.5%). In our study, we found that genetic information affected the severity of congenital FVII deficiency with ICH.

Factor Activity Assays for Monitoring Extended Half-Life FVIII and Factor IX Replacement Therapies.

The advent of modified factor VIII (FVIII) and factor IX (FIX) molecules with extended half-lives (EHLs) compared with native FVIII and FIX represents a major advance in the field of hemophilia care, with the potential to reduce the frequency of prophylactic injections and/or to increase the trough level prior to subsequent injections. Monitoring treatment through laboratory assays will be an important part of ensuring patient safety, including any tailoring of prophylaxis. Several approaches have been used to extend half-lives, including PEGylation, and fusion to albumin or immunoglobulin. Some of these modifications affect factor assays as routinely performed in hemophilia centers; so, laboratories will need to use FVIII and FIX assays which have been shown to be suitable on a product-by-product basis. For some products, there are marked differences between results obtained using one-stage or chromogenic assays and results obtained using different reagents in the one-stage assay. The laboratory should use an assay in which the recovery of the product closely aligns with the assay used by the pharmaceutical company to assign potency to the product, so that the units reported by the laboratory agree with those used to demonstrate efficacy of the product during clinical trials. Reported assay differences in relation to several of the EHL FVIII and FIX molecules will be reviewed in this article.

The Use of Activated Partial Thromboplastin Time As a Surrogate to Predict Residual FVIII Activity in the Bethesda Assay

Introduction: Bethesda assays, used in the quantification of factor VIII inhibitors, are time and labor intensive. They are performed by mixing one part of normal pooled plasma (NPP) with an equal amount of serial dilutions of patient plasma in imadizole buffered saline (IBS), and incubating at 37°C for 2 hours. After the incubation time, most laboratories perform factor VIII (FVIII) activities on each tube. These results are divided by the activity resulted on the control (NPP+IBS) to obtain the percent residual activity. The dilution with a percent residual FVIII activity between 25-75% is then converted to a Bethesda Unit using the Bethesda graph. This value is multiplied by the dilution to calculate final Bethesda units/mL.We propose an alternative method, involving performing partial thromboplastin times (PTTs) on all dilution tubes and only performing FVIII activity testing on a limited number of tubes e.g. 2 tubes rather than all 10 tubes. The study below describes the methodology and provides data to show how the PTT can be used as a surrogate. Using PTTs may reduce cost per test by reducing the amount of FVIII deficient plasma used for FVIII activity testing, especially in the case of very high titer inhibitors.Methods: Serial dilutions of patient plasma with IBS are prepared to the 512 dilution (10 tubes) and incubated with an equal amount of NPP along with a control consisting of IBS and NPP. PTTs are run using Siemens Actin FSL reagent on the BCS coagulometer (Siemens) on all 10 tubes and the control. The tube with a resultant PTT within 5 seconds above the control is identified and then that tube, along with the next higher dilution, are tested for residual factor VIII activity (ACL TOP, Instrumentation Laboratory). These results are used to calculate the percent residual FVIII activity and Bethesda units/mL as described above. To support our hypothesis, we performed PTT and FVIII activities on all dilution tubes.Results: We performed 14 inhibitor assays on 9 patients. 44% (4/9) had congenital hemophilia A with an inhibitor and 56% had acquired hemophilia A. Table 1 shows results from dilution tubes that resulted in residual FVIII activities between 25-75%. The dilutions with residual activity closest to 50% were used in the final interpretation and are bolded. Using the 5 seconds above control PTT to identify dilutions to limit FVIII assays correlated with residual FVIII activities in 12 of the 14 samples tested (86%). Furthermore, this method identified the dilution with the residual FVIII activities closest to 50% in 10 of the 12 samples.Two samples demonstrated no correlation between PTT and FVIII activities. These samples had low titer inhibitors (<5 BU), which could have been predicted by the low PTTs in the first two dilutions. In these cases, serial FVIII activities were used in calculations. More data is needed to definitively say whether this methodology could not be used for low titer inhibitors.Conclusion: An alternative method, using PTT testing to perform targeted FVIII activity testing, may be used in the Bethesda Assay. [Display omitted] DisclosuresSarode:CSL Behring: Consultancy, Honoraria.

Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.

The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half-lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long-acting therapeutic concentrates contain molecular modifications of Fc fusion, site-specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half-life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored.

Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.

Although the variability in factor VIII (FVIII):C measurement is well recognized, this has not been widely reported for post-FVIII infusion samples. Three samples from haemophilia A patients were distributed in a UK National External Quality Assessment Scheme survey, each after treatment with either ReFacto AF, Kogenate FS or Advate. Fifty-two UK haemophilia centres performed FVIII assays using one-stage (n = 46) and chromogenic (n = 10) assays. Centres calibrated assays with the local plasma standard and with ReFacto AF laboratory standard for the ReFacto AF sample. Chromogenic assays gave significantly higher results than one-stage assays (P < 0.0001, 32% difference) in the post-Kogenate sample but not in the post-ReFacto AF (11% higher by chromogenic assay, ns) or post-Advate samples (3% lower by chromogenic, ns) when assays were calibrated with plasma standards. Twenty centres used all Instrumentation Laboratory (IL)-activated partial thromboplastin time reagents (Synthasil)/IL deficient plasma/reference plasma) in the one-stage assay and 15 used all Siemens reagents (Actin FS/Siemens deficient plasma/reference plasma); this made a significant difference to results post-ReFacto AF (41% higher by IL reagents, P < 0.0001) and Advate (39% higher by IL reagents, P < 0.0001), but not Kogenate (7% higher by IL, ns) when calibrated with plasma standards. Differences between results obtained with different one-stage assay reagents for monitoring Advate have implications for dosing patients. Furthermore, there was considerable inter-laboratory variation as indicated by CVs in the range 15-26% for chromogenic assay and 12-19% for one-stage assay results. This study suggests that external quality assessment schemes should offer participation in post-FVIII infusion schemes where haemophilic patients are monitored.

Commutability of reference materials for coagulation factor VIII and factor IX activity on three measurement systems

To evaluate the comparability of measurement results for coagulation factor VIII (FVIII)and factor IX (FIX) activity and the commutability of reference materials on different measurement systems. The study was performed according to CLSI guideline EP30 and China health standard WS/T 356-2011. Clinical samples with different levels of FVIII and FIX which covered over the clinical analytical range, five lots of homemade reference materials (F20140601-F20140605) and a coagulation reference material (SSCLOT4) provided by NIBSC were detected for FVIII and FIX activity on three popular measurement systems in China, which including Stago STA-R Evolution, IL ACL TOP700 and Sysmex CA7000 automatic coagulation analyzers using supplementary reagents. The results between measurement systems were analyzed pairwise. To evaluate the comparability, the linear regression and the biases between the results of clinical samples from two measurement systems were calculated. The comparability was evaluated by the regression coefficient and the biases inside the acceptable range. After eliminated outliers from the results, linear regressions were run again and the 95% confidence intervals were calculated. The commutability of the homemade reference materials and NIBSC reference material were evaluated by comparing the results with the limits of the intervals. The ranges of FVIII and FIX level of clinical samples were 0.5%-218.0% and 1.6%-156.5%, which covered the sample levels in routine work and fit the requirements for commutability evaluation. The square of correlation coefficients (R²) of measurement results of clinical samples for FVIII and FIX activity assays were 0.89-0.94 and 0.81-0.93. The proportions of outliers were all less than 10%. The comparability of measurement results of FVIII and FIX in different measurement systems was acceptable.According to the acceptable criteria for bias, the measurement results of 42, 41 and 45 clinical samples for FVIII and 44, 42 and 41 clinical samples for FIX were used in the commutability evaluation for homemade reference materials and NIBSC reference material after deleting the outliers. The five lots homemade reference materials and NIBSC reference material were all within the 95% prediction intervals from the linear regression of clinical samples' results. Good correlation and comparability are found for FVIII assay and those for FIX assay are acceptable. All homemade reference materials and NIBSC reference material are commutable for the three measurement systems.

Current laboratory practices in the diagnosis and management of haemophilia: a global assessment.

Haemophilia management is complicated by the extreme variability in laboratory practices. Lack of consistency or comparability in testing makes it difficult to establish diagnostic criteria or disease severity, and complicates response assessment. A global survey was conducted to document current practices. A 35-min survey was completed by 30 laboratory scientists in each of seven countries (France, Germany, Italy, Japan, Spain, UK, USA; 210 in total); results were weighted by average country testing volume in haemophilia. Eighty-three per cent of participants reported participation in a Quality Assurance scheme. Ninety per cent reported using clotting tests in haemophilia A and 88% in haemophilia B (55% and 53% frequent use respectively). Sixty-eight per cent reported chromogenic assays were used in haemophilia A, with only 23% reporting frequent use, compared to only 11% reporting any use in haemophilia B. Twenty-nine separate activated partial thromboplastin time (aPTT) reagents were reported for haemophilia A and 27 aPTT reagents were reported for haemophilia B, with one-quarter or less obtaining reagents or kits from any single manufacturer. Fifty-four per cent run a calibration curve with every factor VIII (FVIII) assay. The mean number of plasma dilutions varied from 2 to 4 for FVIII assays and from 1 to 3 for FIX assays. Results indicate very low consistency in materials and practices used to test for factor activity in haemophilia. A number of responses suggest that some laboratory scientists' understanding of best practices or guidelines in haemophilia could be improved. More education and broader understanding is recommended regarding assay types, assay components, test material and instrument features and capabilities.

EVALUATION OF A CHROMOGENIC FACTOR VIII ASSAY ON THE SYSMEX CS 2100I INSTRUMENT

Background: The one-stage factor VIII (FVIII) assay is the most widely used assay for the quantification of FVIII. Several disadvantages of the one-stage assay have been described. The two-stage- and chromogenic FVIII assays purportedly overcame these disadvantages. The chromogenic method is easier to automate and the reagents are readily available. In this evaluation we evaluated the chromogenic FVIII assay on the Sysmex CS 2100i instrument. Methods: We compared the FVIII levels of healthy individuals and haemophilia A patients, as determined by the one-stage and chromogenic FVIII assays, respectively. Results: FVIII levels differed significantly between assays. The lower FVIII detection limit of both assays did not allow the quantification of FVIII for the haemophilia A patients. The addition of heparin led to a decrease in FVIII levels detected by the one-stage FVIII assay, but did not affect the chromogenic assay results. Conclusion: The chromogenic FVIII assay can be introduced into the diagnostic laboratory as part of haemophilia A diagnosis and monitoring. However, the one-stage FVIII assay is still recommended as the first-line screening test as it had a lower FVIII detection limit and was more cost effective.

Is some better than none: are TEG and TGA profiles different in severe FVIII-deficient patients with inhibitors?

Severe factor VIII (FVIII)-deficient patients with and without FVIII inhibitors cannot be distinguished using FVIII levels. The FVIII assay is sensitive to detect factor levels below 1%. While severe FVIII-deficient, non-inhibitor patients have FVIII<1%, they may retain unmeasurable residual factor activity. In contrast, inhibitor patients have a FVIII antibody that presumably fully eliminates FVIII activity. It is unknown whether thromboelastography (TEG) and thrombin generation assay (TGA) can differentiate between patients with FVIII<1% with and without the presence of FVIII inhibitors. The primary objective was to discern whether TEG and TGA could differentiate between severe FVIII-deficient patients with and without the presence of FVIII inhibitors. A secondary objective was to correlate TEG and TGA to annualized bleeding rates. This observational study performed TEG and TGA in healthy volunteers (N=15), severe FVIII-deficient (N=15) and severe FVIII-deficient patients with inhibitors (N=15). Kaolin-activated TEG was better at differentiating reaction time (31.3 vs. 120min respectively, P=0.004) and kinetics time (6.1 vs. 23.1min respectively, P=0.028) between the non-inhibitor and inhibitor patients. TEG activated by tissue factor in plasma-containing corn trypsin inhibitor failed to differentiate groups. The TGA failed to differentiate peak thrombin, endogenous thrombin potential and lag time between groups. There was no correlation between TEG and TGA with annualized bleeding rates. Kaolin-activated TEG, but not TGA, differentiated between severe FVIII-deficient patients with and without inhibitors. These assays did not find a correlation to annualized bleeding rate.

Improved Sensitivity and Discriminative Power of Factor FVIII Assay By Applying Turbidimetric Clotting Curve Analysis

Background: The need for sensitive detection of coagulation factor between the levels of 0.0 and1.0% is now growing continuously. The most popular method of measuring factor VIII (FVIII) activity is one stage clotting time (activated partial thromboplastin time, APTT)-based assay. From the first and the second derivatives of the original turbidimetric curve, the velocity and acceleration of clot formation can be followed and parameters like maximum velocity (peak1) and acceleration (peak2) of clot formation can also be derived. We examined the limit of detection of FVIII activities measured based on clotting time, peak1 and peak2, following the recommendations from Clinical and Laboratory Standards Institute guideline.Method: We performed APTT with sample/reagent volume and incubation time modified to be identical to those adopted for factor assay on factor deficient plasma as blank and plasmas prepared to have variable FVIII activities from 0.1 to 1.6%. Peak1 and Peak2 were also measured to determine the limit of blank (LoB) and limit of detection (LoD) corresponding to each parameter. Also, by modifying the method of LoB and LoD determination, we determined lower limit of 1.0% (lower Lo1) corresponding to each parameter.Results: The mean clotting time of blank sample (FVIII 0.0%) was 131.4 ± 1.90 seconds (mean ± SD). Thus, 95% of clotting times measured from a blank sample (FVIII 0.0%) are longer than 128.3 seconds, which was determined to be the LoB for clotting time. The pooled standard deviation (SD) of clotting times measured from low FVIII samples was 2.29 seconds and 124.49 seconds which was the theoretically minimum value that was statistically different from LoB was set as the clotting time corresponding to LoD of FVIII. The LoD clotting time was between the clotting times of FVIII 0.2% and FVIII 0.4% sample and 0.4% was safely determined to be LoD of FVIII. This implied that plasma sample with at least 0.4% FVIII level was guaranteed to be measured higher than blank (FVIII 0.0%) sample. The mean peak1 height for blank sample was 21.4 ± 0.68 and LoB peak height was determined to be 22.53. The pooled SD for peak1 height was 0.68 and peak1 height of LoD was calculated to be 23.63. Because the mean peak1 height for 0.2% sample was 24.27, the LoD FVIII activity could be safely determined to be 0.2%. Thus, by applying peak1 as primary measure to estimate FVIII activity, the sensitivity of FVIII assay was increased with lower LoD of 0.2% compared with clotting time based assay. For peak2 height, LoB and LoD peak2 height were 14.86 and 18.59 respectively and The LoD could be set at FVIII 0.4%. Next, we determined lower Lo1, which meant FVIII level that was guaranteed to be measured significantly lower than 1.0% sample. For clotting time, lower Lo1 was FVIII 0.2% and for peak1 and 2 FVIII, was 0.4%. These results implied that by conventional clotting time based FVIII assay FVIII activity between 0.0 and 1.0% could not be measured credibly. FVIII should be at least 0.4% to be ever detected but ironically FVIII should be less than 0.2% to be assuredly measured lower than 1.0%. However, with peak1 there was an interval of FVIII value that could be assured to be measured higher than 0.0% but lower than 1.0%.Conclusion: We concluded that the maximum clotting velocity derived from turbidimetric curve analysis can be applied to measure FVIII activity between 0.0 and 1.0% credibly. DisclosuresNo relevant conflicts of interest to declare.

Assay discrepancy in mild haemophilia A.

There are three main methods used to assay factor VIII (FVIII) activity: the one-stage and two-stage clotting assays and the two-stage chromogenic method. The most commonly used assay for the diagnosis of haemophilia A is the automated one-stage FVIII assay. The classical two-stage FVIII assays are less frequently used. The chromogenic FVIII:C assay is a variant of the two-stage assay. It is easier to use and therefore used more commonly. Recently significant assay discrepancy has been recognised in the FVIII:C measurements in approximately one-third of mild haemophilia A patients. This so-called discrepant mild haemophilia A is characterised by a high ratio of one-stage/two-stage assay with one-stage FVIII levels that are typically more than double those of the two-stage coagulation assay. There are several mutations that destabilise the FVIIIa structure that can explain this result of a more pronounced decrease of the chromogenic FVIII:C activity compared with the one-stage activity. These mutations are clustered at the interfaces of the A1, A2 and A3 domains of the FVIII protein. The inverse discrepancy, where the one-stage assay gives lower FVIII:C results than the chromogenic assay, seems to be associated with mutations found close to important sites for thrombin cleavage or FIX binding. We are carrying out a study of mild haemophilia A samples from the Malmö Haemophilia Centre of families with a unique F8 genotype. The activity of FVIII will be measured using a chromogenic assay and two different one-stage assays. We hope to estimate the true size of assay discrepancy. This project will review assay discrepancy in mild/moderate haemophilia A and the risk of misdiagnosis. The overall aim is to estimate the size of the problem and to learn from the literature and experiences from our centre as well as to suggest recommendations on how to avoid misdiagnosis.