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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5015-5015
Author(s):  
Jaewoo Song ◽  
Juwon Kim ◽  
Jungwoo Han ◽  
Jin Seok Kim ◽  
Jihye Ha
Keyword(s):  
Factor Viii ◽  
Clinical Laboratory ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Analytical Sensitivity ◽  
Low Level ◽  
Assay Result ◽  
Viii And Ix ◽  
Analytical Resolution

Abstract 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. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Transcriptional Analysis Of a Tissue-Specific Factor VIII Knockout Model Demonstrates The Importance Of Endothelial Factor VIII Synthesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 27-27 ◽  
Author(s):  
Scot A. Fahs ◽  
Matthew T. Hille ◽  
Robert R. Montgomery
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Coagulation Factor ◽  
The Body ◽  
Transcriptional Analysis ◽  
Specific Cell ◽  
Tissue Specific ◽  
Low Level

Abstract Definitively identifying the cells responsible for synthesis of coagulation factor VIII (F8) has proven to be a challenge. Transplantation studies demonstrate that as an organ liver is the major, but not exclusive source of plasma F8. Within the liver F8 expression has been variously attributed to hepatocytes, and/or liver sinusoidal endothelial cells, and/or Kupffer cells. Extrahepatic transcription of F8 mRNA appears to be nearly ubiquitous at a low level throughout the body. Previous studies have relied upon retrospective post-expression detection of F8 protein or mRNA using a variety of immunochemical, in situ, and cell isolation techniques, but continuing controversy speaks to the difficulties in localizing expression of a trace protein such as F8. We used a rather different, pre-emptive approach to address the question of F8 synthesis. We developed a conditional F8 knockout (KO) mouse model that allows inactivation of the F8 gene, thus preventing expression, in specific cell types. Exons 17/18 of the F8 gene were flanked by LoxP sites (floxed) resulting in their excision in cells expressing Cre recombinase. Tissue-specific Cre-expressing mouse strains were cross-bred with floxed (F8F) mice to generate tissue-specific F8-KO models. Embryonic Cre expression resulted in a new F8KOstrain displaying a severe hemophilia A phenotype. A hepatocyte-specific F8-KO has completely normal plasma F8 levels, while each of 3 endothelial cell (EC)-Cre models displays a reduced-F8 phenotype that correlates in severity with endothelial Cre efficiency. Presumably due to a shared hemangioblast progenitor, Cre is expressed with similar efficiency in both EC and hematopoietic cells in these models. Plasma F8 is undetectable in the most efficient EC-KO model. In contrast, a highly efficient hematopoietic F8-KO model presents with only modestly reduced F8 levels, likely due to off-target effects. RNA analysis revealed that the F8KO allele produces 2 alternatively spliced transcripts in roughly equivalent amounts. The 1st transcript represents the predicted exon 16/19 splicing event. In the 2nd transcript, 46bp at the 5’ end of intron 16 are retained due to the same cryptic splice site observed in the Kazazian exon 17-disrupted F8null model. Combined, the 2 F8KO allele transcripts are present at ∼1/8 to 1/5 of normal levels in the F8KO strain. No normal F8 transcripts are present. In the phenotypically normal hepatocyte-KO model ∼70% of total liver gDNA is converted to the F8KO allele, indicative of very efficient hepatocyte Cre activity, yet almost exclusively normal F8 mRNA is present, with only traces of F8KO message. This is consistent with endothelial synthesis as our further results indicate. For the 3 EC-KO models, plasma F8 levels were correlated with hepatic levels of normal F8 mRNA, and inversely correlated with F8KO transcripts. Excessive F8F to F8KOconversion in the hematopoietic-Cre model suggests variable loss of tissue-specificity. In the most efficient, functionally hemophilic EC-KO model, ∼20% of liver gDNA is converted to the F8KO allele, in good agreement with the expected number of hepatic EC, and F8KO mRNA is present at ∼10% of normal liver levels. With undetectable plasma F8, the continued production of normal F8 mRNA at a similar low level (∼10%) by the remaining 80% of Cre-negative, presumably non-endothelial hepatic cells, was unexpected. In addition to liver we found both normal and F8KO message only in kidney and perhaps brain. As expected, only F8KOmRNA was found in spleen and bone marrow, but the presence of exclusively normal mRNA in heart, intestine, testis, lung, and thymus, at relatively normal (low) levels, was surprising. The persistence of widespread transcriptional “expression” of F8, albeit in a functionally hemophilic mouse, is reminiscent of the near-ubiquitous presence of low level F8 transcription in normal mice. This low level transcription apparently does not support functional plasma F8 production, at least not in these EC-KO mice. In summary, our results support the hypothesis that synthesis of F8 is a function of endothelial cells, both in the liver and presumably elsewhere. Neither hepatocytes nor hematopoietic cells appear to contribute significantly to steady-state plasma F8 levels. Transcriptional analysis of normal and F8KO-specific transcripts provides further support for the localization of F8 expression to endothelial cells. Disclosures: No relevant conflicts of interest to declare.

Assessment Levels of Factor VIII and IX Inhibitors in Patients with Hemophilia in North West of Iran: What Is the Main Reason of Low Inhibitor Titer and Rate in Our Patients?

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4662-4662
Author(s):  
Roya Dolatkhah ◽  
Ali Akbar Movasaghpour Akbari ◽  
Iraj Asvadi Kermani ◽  
Zohreh Sanaat ◽  
Azim Rezamand ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Factor Ix ◽  
Haemophilia A ◽  
Inhibitor Development ◽  
Haemophilia B ◽  
On Demand ◽  
Viii And Ix

Abstract Abstract 4662 Introduction Today, the development of inhibitors against Factor VIII (FVIII) and Factor IX (FIX) is seen as the most serious complication of haemophilia A and B therapy. Recent studies on the role of the causative haemophilic mutation, race and ethnicity, family history of inhibitors and the possible influence of HLA genotype in inhibitor formation have revealed new and exciting insights. That is challenging conventional thinking about inhibitor development risk and type of factor products, recombinant or plasma-derived. The use of recombinant factor concentrates has revolutionized the treatment of severe factor VIII and IX deficiency. One of the most important complications is the development of antibodies (Inhibitors). Material and Methods Ninety two patients with haemophilia A and 12 patients with Haemophilia B have been studied. Confirmatory tests including one stage FVIII and FIX assay has been performed using STA Deficient FVIII and FIX, an immune-depleted plasma intended in plasma by analyzers of the STA line suitable with these reagents (Diagnostica Stago,France).Presence of Factor VIII and IX inhibitors have been tested by Bethesda Assay. All of the patients use mostly plasma-derived factor products, and on-demand treatment. Results Among 92 haemophilia A patients, FVIII levels were between 0.14–14.40 IU/dl (mean 2.91 ± 2.62), FIX levels were between 0.17 to 4.37 IU/dl (mean 1.53 ± 1.38) in12 haemophilia B patients. PT activity was 68.7–134 (mean 101.05 ± 15.13), APTT was 28.90 – 102 (mean 60.66 ± 13.50). FVIII inhibitor levels were between 0–1.14 BU (mean 0.04 ± 0.20) in 5 severe Hemophilia A patients (5.45%) and FIX Inhibitor levels were between 0–0.65 BU (mean 0.10 ± 0.23) in 2 Hemophilia B patients. Discussion Alloantibodies (inhibitors) against FVIII or FIX represent a major complication in patient care because they render classical substitution therapy ineffective. Inhibitors occur at a frequency of 20–30% in severe and 3–13% with mild or moderate haemophilia A, and 3% in haemophilia B, respectively. An alternative pathomechanism may underlie inhibitor development in patients with mild hemophilia A. Although it has been reported that inhibitors in patients with mild haemophilia are related to periods of intensive treatment or surgery, this has never been properly studied in children with severe haemophilia. The low inhibitor rate with Low Titers in our patients may be demonstrate the role of type of factor products, recombinant or plasma-derived, which in this study was mostly use of plasma-derived factor products, and on-demand treatment. Also detailed evaluation of major risk factors of development of Factor VIII and IX inhibitors in our patients is required. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gene Therapy in Hemophilia: Recent Advances

2021 ◽  
Vol 22 (14) ◽  
pp. 7647
Author(s):  
E. Carlos Rodríguez-Merchán ◽  
Juan Andres De Pablo-Moreno ◽  
Antonio Liras
Keyword(s):  
Gene Therapy ◽  
Adverse Events ◽  
Factor Viii ◽  
Coagulation Factor ◽  
Cost Savings ◽  
Factor Ix ◽  
Clotting Factors ◽  
Advanced Therapies ◽  
Potential Benefits

Hemophilia is a monogenic mutational disease affecting coagulation factor VIII or factor IX genes. The palliative treatment of choice is based on the use of safe and effective recombinant clotting factors. Advanced therapies will be curative, ensuring stable and durable concentrations of the defective circulating factor. Results have so far been encouraging in terms of levels and times of expression using mainly adeno-associated vectors. However, these therapies are associated with immunogenicity and hepatotoxicity. Optimizing the vector serotypes and the transgene (variants) will boost clotting efficacy, thus increasing the viability of these protocols. It is essential that both physicians and patients be informed about the potential benefits and risks of the new therapies, and a register of gene therapy patients be kept with information of the efficacy and long-term adverse events associated with the treatments administered. In the context of hemophilia, gene therapy may result in (particularly indirect) cost savings and in a more equitable allocation of treatments. In the case of hemophilia A, further research is needed into how to effectively package the large factor VIII gene into the vector; and in the case of hemophilia B, the priority should be to optimize both the vector serotype, reducing its immunogenicity and hepatotoxicity, and the transgene, boosting its clotting efficacy so as to minimize the amount of vector administered and decrease the incidence of adverse events without compromising the efficacy of the protein expressed.

THE MODE OF ACTION OF PENTOSAN POLYSULPHATE IN PLASMA

1987 ◽  
Author(s):  
S Béguin ◽  
H C Hemker
Keyword(s):  
Factor Viii ◽  
Linear Increase ◽  
Factor Ix ◽  
Factor X ◽  
Heparin Cofactor Ii ◽  
Normal Plasma ◽  
Viii And Ix ◽  
Pentosan Polysulphate ◽  
Lag Times ◽  
Prothrombinase Activity

We developed a method which enables as to compute the course of prothrombinase activity in clotting plasma (H.C. Hemker, G.M. Willems, S. Béguin: Thromb. Haemostas. 56, 9-17, 1986) and used this for a study of the effect of pentosan polysulphate (PPS) on thrombin generation.When added to normal plasma in the concentration range of 0-8 μg/ml PPS induces a linear increase of the pseudo first order decay constant of endogenous thrombin like heparin does, 1 ug of PPS being equivalent to 0.045 Aig of heparin. Contrary to heparin this action is partly (∼ 65%) dependent upon AT III and partly (∼ 35%) upon heparin cofactor II.In normal plasma PPS causes an inhibition of both extrinsic and intrinsic prothrombinase formation. Only in the intrinsic system an increase of the lag time of prothrombinase appearance is observed. Unlike heparin, PPS does not inhibit factor IXa induced thrombin formation neither does it inhibit prothrombinase formation in the presence of preactivated factor VIII. The prolongation of the lag times must therefore be ascribed to inhibition by PPS of the activation of factor VIII.The inhibition of extrinsic prothrombinase formation by PPS increases with progressive dilution of thromboplastin and is not seen in haemophilia A or B plasma. This demonstrates the existance of a factor VIII and IX dependent process in extrinsic coagulation that gains in importance when the potency of factgr VII-tissue factor complex decreases, i.e. the Josso pathway.PPS, but also heparin causes an unexplained increase of prothrombinase action in haemophIIic plasma. The same phenomenon may be expected to exist in normal plasma, be it obscured by a concomitant inhibition. This, together with the incomplete inhibition of factor VIII activation by PPS makes that we cannot use this inhibitor as a means to quantitate the Josso pathway. The best estimate that we can obtain is that, in the presence of 2% thromboplastin, the factor IX dependent activation of factor X contributes more then 20% to prothrombinase generation.

Extravascular Clotting Factor Activity within Joint Tissues Protects Against Progression of Hemophilic Arthropathy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 496-496 ◽  
Author(s):  
Junjiang Sun ◽  
Narine Hakobyan ◽  
Leonard A. Valentino ◽  
Paul E. Monahan
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Joint Capsule ◽  
Factor Ix ◽  
Clotting Factor ◽  
Needle Puncture ◽  
Factor Activity ◽  
Clotting Factors ◽  
Hemophilic Arthropathy ◽  
Human Factor Viii

Abstract Hemophilic arthropathy is the major morbidity of congenital factor VIII and IX deficiency. Therapies localized to hemophilic joints could provide adjunctive protection, in addition to that provided by systemic factor replacement. However, the ability of extravascular clotting factors to contribute to hemostatic protection within joint tissue is unknown. We hypothesized that replacing deficient factor VIII or IX within the injured joint capsule of mice with hemophilia A (FVIII −/ −) or hemophilia B (FIX −/ −), respectively, would decrease the progression of synovitis. We developed a bleeding model consisting of a unilateral knee joint capsule needle puncture to induce hemorrhage in hemophilic mice. Pathology of the joint at two weeks after the injury is graded 0 to 10 using a murine hemophilic synovitis grading system (Valentino, Hakobyan. Haemophilia, 2006). Hemostatically normal mice do not develop synovitis following this injury, but > 95% of FIX −/ − mice develop bleeding and synovitis with a mean grade of 3–4 or greater. Coincident with needle puncture, recombinant human coagulation factor doses ranging from 0 to 20 IU/kg body weight of factor IX or 0 to 25 IU/kg of factor VIII were instilled intraarticularly (I.A.). Comparison groups received the same injury and intravenous (I.V.) factor IX or VIII doses of 25 IU/kg to 100 IU/kg (n= 4–7 mice per study group). Joint bleeding phenotype of the two strains of mice was similar. Mice receiving only saline injection at the time of needle puncture developed mean synovitis scores of 5 ±0.5 in the FVIII −/ − mice and 6 ±0.5 in the FIX −/ − mice. Protection by human clotting factor in the mouse coagulation system was incomplete; mice receiving 100 IU/kg I.V. of factor VIII or factor IX developed synovitis scores of 2.6 ± 1.7 and 2.1 ± 0.2, respectively. In contrast, pathology grade of FVIII −/ − mice dosed with 25 IU/kg I.A. was 0.67 ± 0.3 (p = 0.05 for comparison of 25 IU/kg I.A. with 100 IU/kg IV); FIX−/ − mice receiving 20 IU/kg I.A. had synovitis scores of 0.45 ± 0.58 (p < 0.01 for comparison of 25 IU/kg I.A. with 100 IU/kg I.V.). We next ruled out the possibility that I.A. factor was entering the circulation, and via that route resulting in joint protection, either through technical error at the time of injection, or from a depot effect in the joint with late equilibration into the circulation. Additional groups of mice received factor VIII or IX intravenously at 100 IU/kg, or intraarticularly at 4 times the doses used in the hemarthrosis challenge (80 IU/kg FIX or 100 IU/kg FVIII), and factor activity assays were performed at 1, 4, 12, 24, and 48 hours. Expected circulation kinetics were seen following I.V. dosing; no increase in circulating factor VIII or IX activity were seen in the intraarticular dosing groups at any timepoint. In considering the potential immunogenicity of an intraarticular therapy approach for hemophilic joint therapy, factor VIII −/ − mice were treated with three doses of human factor VIII 100 IU/kg at five day intervals either I.V. or I.A. At two weeks after exposure, 5/5 I.V.-treated mice developed inhibitor antibodies with titers ranging 0.8–7.2 BU; 2/5 I.A.-treated mice had detectable low-titer antibodies (1.3 BU), indicating no greater immunogenicity in the I.A. model. Extravascular factor VIII and factor IX can contribute to protection against blood-induced joint deterioration; enhancing local tissue hemostasis with protein or gene therapy may prove a useful adjunct to systemic replacement.

Molecular Diagnosis Of One Chinese Patient With Hemophilia B

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4790-4790
Author(s):  
Rong-Fu Zhou ◽  
Bo Gao ◽  
Jian Ou-yang
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Coagulation Factor ◽  
Chinese Patient ◽  
Factor Ix ◽  
Hemophilia B ◽  
Coagulation Tests ◽  
Prolonged Aptt ◽  
Pcr Products

FTo investigate the molecular mutation leading to Hemophilia B in one patient. Methods FOne-stage method was used to detect APTT, PT, TT, Fibrinogen and FVIII:C, FIX:C. The genomic DNA was extracted from the peripheral blood of proband. All exons and their flanks of Factor IX gene were amplified by polymerase chain reaction (PCR). The PCR products were sequenced directly. Results FThe proband was a 20-month boy presenting with scalp hematoma after trauma. Regular coagulation tests showed that his APTT was 135.1s, PT 11.9s, TT 15.6s and Fibrinogen 2g/l. Normal mixed plasma could correct the prolonged APTT to 35s. His FIX:C was 6.4% and FVIII:C was normal. Direct sequencing of PCR products suggested that there was a 5085T>C mutation (NG_007994.1) in Exon1, and a 36060G>C mutation in Exon8 of F9 gene. The former mutation caused the substitution of Leu19 by Pro, which lies in -28th in signal peptide. The later mutation lead to the substituion of Gln370 by His. Conclusion FMutations of 5085T>C in Exon1 and 36060G>C in Exon8 might be the causes of coagulation factor IX defiency for the patient. These mutations are de novo ones according to the database presenting in http://www.factorix.org/. Disclosures: No relevant conflicts of interest to declare.

Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3787-3787
Author(s):  
Pete Lollar ◽  
Ernest T. Parker ◽  
John F. Healey ◽  
Christopher B. Doering
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Immune Complexes ◽  
Hemophilia A ◽  
Analytical Ultracentrifugation ◽  
Conflicts Of Interest ◽  
Sedimentation Coefficient ◽  
Coagulation Factor ◽  
Velocity Sedimentation ◽  
Molar Excess

Abstract Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Case Report: A 19-Years Old Patient with Haemophilia a with High Response Inhibitor Screening and Quantitation and the Bethesda Method

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4956-4956
Author(s):  
Marianela Trejos Herrera ◽  
Alicia Lopez Campos
Keyword(s):  
Factor Viii ◽  
Conflicts Of Interest ◽  
Current Knowledge ◽  
High Titer ◽  
Laboratory Diagnosis ◽  
Factor Ix ◽  
High Response ◽  
Patient Specific ◽  
Aptt Reagent ◽  
Low Sensitivity

Abstract A severe haemophiliac patient, high response inhibitors, 19 years, who was diagnosed at 8 months old and since then he begins to administer factor VIII concentrates. However, a year later after the start of treatment, they were detected inhibitors that behave as high response inhibitors from the start. He discontinues treatment with Factor VIII concentrate and instead he begins to administer Factor IX concentrate and prothrombin complex as an alternative treatment. Through his illness, the patient has made significant bleeding at the level of joint and other muscles such as the psoas. It is a bleeding patient at rest up for what is currently administered prophylactically Prothrombin Concentrate 3 times a week. This case, the comment will be directed towards laboratory diagnosis and its evolution since he was diagnosed in 1994 to date. From 2013, screening protocols and quantification of both factors and inhibitors were modified in the Specialized Hematology laboratory of Hospital México, due to problems in the sensitivity and specificity of the method and reagents we were using. The results from these patient specific inhibitors are described in the following paragraphs. Observations With the experience and current knowledge of the following it is concluded, according to a literature review that was performed (see Table 1): 1) This time period has persisted inhibitor high title, which is evidenced of the study of mixtures which do not clearly show a potentiation by incubating 2 hours at 37 ° C, since the values of the Control Mix and patient give very similar high values. 2) We were shown only on the date of 14.02.2013, there was a real interference Lupic Anticoagulant (LA), which is confirmed by the method of Russel viper venom. 3) The dates high titer inhibitors were reported against factor VIII (from 6 November 2013 to date), both screening and for the Bethesda, aPTT reagent was used with Kaolin activator which is low sensitivity to LA. 4) On November 20, 2015 there was an error in the interpretation and anti factor VIII inhibitors as negative were reported as potentiation at 37 ° C is not evidenced, but rather was interpreted as interference of an LA, which was communicated to the medical and preventive measures cited in paragraph corrections were made. 5) The last date that the sample was processed, on February 1, 2016, the inhibitor against factor VIII did not affect dilution of silica APTT, so the index ROSNER was not affected (<12) and it was not necessary to mount the LA test, according to the request in identifying protocols inhibitors. 6) Factor VIII deficient plasma is currently being used, which contains von Willebrand factor, as recommended by international guidelines quantization factors. Conclusions According to an experience as support center Reference Center in our country, we conclude and recommend the following: 1) Registration of haemophiliac patients with high antibody titer is essential as the description of the protocol to be followed in these patients. 2) The behavior of this inhibitor in the screening test of time and temperature dependence, it was decided to directly mount the Bethesda assay, following the recommendations of the literature on when the use of a reagent with low sensitivity to lupus inhibitor. 3) Within the protocol and as far as we can, we will process the purchase for quantification of factor VIII chromogenic by ELISA methodology, as a confirmatory method. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document