Objective.Serum 14-3-3η is a novel joint-derived proinflammatory mediator implicated in the pathogenesis of rheumatoid arthritis (RA). In our study, we assessed the diagnostic utility of 14-3-3η and its association with standard clinical and serological measures.Methods.A quantitative ELISA was used to assess 14-3-3η levels. Early (n = 99) and established patients with RA (n = 135) were compared to all controls (n = 385), including healthy subjects (n = 189). The sensitivity, specificity, positive and negative predictive values of 14-3-3η, and the likelihood ratios (LR) for RA were determined through receiver-operator curve analysis. The incremental value of adding 14-3-3η to anticitrullinated protein antibody (ACPA) and rheumatoid factor (RF) in diagnosing early and established RA was assessed.Results.Serum 14-3-3η differentiated established patients with RA from healthy individuals and all controls (p < 0.0001). A serum 14-3-3η cutoff of ≥ 0.19 ng/ml delivered a sensitivity and specificity of 77% and 93%, respectively, with corresponding LR positivity of 10.4. At this cutoff in early RA, 64% of patients with early RA were positive for 14-3-3η, with a corresponding specificity of 93% (LR+ of 8.6), while 59% and 57% were positive for ACPA or RF, respectively. When ACPA, RF, and 14-3-3η positivity were used in combination, 77 of the 99 patients (78%) with early RA were positive for any 1 of the 3 markers. Serum 14-3-3η did not correlate with C-reactive protein, erythrocyte sedimentation rate, or Disease Activity Score, but patients who were 14-3-3η-positive had significantly worse disease.Conclusion.Serum 14-3-3η is a novel RA mechanistic marker that is highly specific, associated with worse disease, and complements current markers, enabling a more accurate diagnosis of RA.
Thrombopoietin (TPO) is the major regulator of megakaryopoiesis. Measurement of serum TPO levels may help distinguish between various causes of thrombocytopenia and predict treatment response to TPO receptor agonists. Serum TPO levels from 118 healthy volunteers and 88 patients with abnormal platelet counts were measured using a quantitative ELISA assay. The mean (range) TPO level in healthy volunteers was 39 (7-99) pg/mL. TPO values were correlated with the patient's diagnosis, platelet count, and response to TPO receptor agonists. 88 patients with history of consumptive thrombocytopenia (39) or hypoproliferative thrombocytopenia (49) were analyzed. Median (interquartile range) TPO level for consumptive thrombocytopenia patients was 63 (48-98) pg/mL with a corresponding median (interquartile range) platelet count of 73 (28-146) 3 10 9 /L. In contrast, hypoproliferative thrombocytopenia patients had platelet counts [59 (30-117) 3 10 9 /L] comparable with consumptive thrombocytopenia patients, but significantly higher serum TPO levels [706 (358-1546) pg/mL, P < 0.0001]. Analysis of 21 ITP patients treated with TPO receptor agonists demonstrated that a TPO level >95 pg/mL was associated with lack of clinical response (P < 0.002). TPO levels may have diagnostic utility in discriminating between patients with hypoproliferative and consumptive thrombocytopenia. Elevated TPO levels in ITP patients may predict a poor clinical response to treatment with TPO receptor agonists. Am. J. Heamtol. 88:1041Heamtol. 88: -1044Heamtol. 88: , 2013. V C 2013 Wiley Periodicals, Inc. IntroductionThrombopoietin (TPO) is the major regulator of platelet production. Prior studies in animal models [1-3] and humans [4] have demonstrated that TPO levels vary inversely with circulating platelet mass. Additional clinical studies have suggested that TPO levels also vary inversely with the rate of megakaryopoiesis [5][6][7][8][9][10]. Thus, TPO levels may help distinguish between the various disorders of thrombocytopenia, with elevated TPO levels associated with hypoproliferative thrombocytopenia (e.g., aplastic anemia) but normal TPO levels associated with consumptive thrombocytopenia (e.g., immune thrombocytopenia) [5][6][7][8]10]. In addition, the introduction of TPO receptor agonists for the treatment of thrombocytopenia has created an interest in predicting the response of patients to these agents [11]; TPO levels may help predict treatment responses.To assess the performance characteristics of a TPO assay in a clinical setting, we measured the serum TPO concentration in a panel of healthy humans as well as patients with diverse hematologic diseases. We then correlated the TPO level with platelet count, diagnosis, and treatment response. Our goals were (1) to determine the normal range of TPO levels, (2) to determine whether TPO levels could help discriminate between different disorders of platelet production, and (3) determine whether TPO levels might help predict the platelet response in those patients treated with a TPO receptor agonist ...
Inhibitors of FVIII are usually IgG polyclonal antibodies that develop as alloimmune responses in patients with congenital haemophilia A or as autoimmune responses resulting in acquired haemophilia. Their recognition can be difficult, especially when the titre is low. Furthermore, results from a Bethesda assay often require several days as samples are referred to a specialty laboratory. The aim of this study is to assess the utility of an ELISA system for detecting immune responses to FVIII. A total of 246 plasma samples submitted from 176 individuals with immune responses to FVIII, as verified with the Bethesda assay, and samples from 50 control subjects were tested for the presence of FVIII-specific IgG using an ELISA-based assay. Paired sera from 18 of the patients were also tested by the ELISA. Of the 246 samples that were positive for a FVIII inhibitor by the Bethesda assay, 235 (95.5%) were also positive by ELISA. The regression coefficient, using Log BU was r = 0.82. The correlation data were strengthened when 27 inhibitor samples were diluted further. There was a strong correlation between ELISA results for the 18-paired serum and plasma samples (r = 0.99). There is a strong correlation between the ELISA and Bethesda methods in detecting immune responses to FVIII. The ELISA provides rapid screening that could be available well in advance of confirmation by the Bethesda assay.
A 30-year-old female with severe factor XI deficiency of 0-2% acquired factor XI inhibitor following many infusions for fresh frozen plasma (FFP) for surgical procedures starting at 4 years of age. Seven months before this inhibitor was diagnosed, surgery was complicated by prolonged bleeding resistant to FFP, requiring epsilon aminocaproic acid (EACA) and surgical packing. The inhibitor was measured at 2.2 Bethesda units, 7 months since the last FFP. The inhibitor was confirmed as specific anti-XI and anti-XIa binding by patient's IgG to immobilized factor XI and factor XIa from whole plasma and purified IgG. For repair of a painful anterior cruciate ligament (ACL) defect she was given recombinant factor VIIa (rVIIa) at 90 mug kg(-1), starting one-half hour preoperatively and continued every 2 h for 8 h when haemostasis was complete. Thereafter the rVIIa was given every 3 h for two doses, and then every 4 h for four doses at which time she was discharged on EACA which was continued for 6 days. There was excellent haemostasis during and following the surgery. There was no evidence of consumptive coagulopathy, with no change in the fibrinogen, platelet count, or D-D dimer; and no increase of platelet factor 4, beta-thromboglobulin, or prothrombin fragment F 1.2. The thrombin-antithrombin complex increased over baseline after 24 h. There was no postoperative deep vein thrombosis or pulmonary embolus. In this patient with a factor XI inhibitor, the recombinant factor VIIa was effective and safe, ensuring adequate haemostasis with no thrombotic complications. This product which was designed for patients with inhibitors to factor VIII or factor IX, and factor VII deficiency, has now been given successfully to four patients with factor XI inhibitors.
Laboratory diagnosis of von Willebrand disease type 2N (VWD2N) is based on costly mutation analysis or in vitro measurement of the ability of plasma von Willebrand factor (VWF) to bind exogenous factor VIII (FVIII); however, the VWF-FVIII binding activity assay is complex and not widely used. Our aim was to assess the utility of the in-house VWF-FVIII binding assay in the investigation of patients with suspected VWD2N. A previously described ELISA-based FVIII binding method was simplified and adapted for the clinical laboratory use by optimizing incubation time, reagent concentrations and assay standardization. The assay was validated using samples from eight individuals with known homozygous or heterozygous VWD2N mutations, and 100 healthy adults. An additional 392 patient samples were tested, including 314 with FVIII activity <50% of normal and 78 received for routine VWF-FVIII binding activity testing. Intra- and inter-assay variations were less than 10% and 17%, respectively, and the limit of quantification was estimated as 0.12. The reference range for healthy adults was 0.73-1.42. VWF:FVIII binding activity was consistent with the genotype in subjects with available genetic data, being low in three individuals with homozygous mutation (<0.12) and intermediate in five heterozygous individuals (0.44-0.61). Screening of the 392 clinical samples identified reduced VWF:FVIII binding in 19 subjects. This assay provides accurate measurement of VWF:FVIII binding activity and successfully identifies homozygous VWD2N patients and heterozygous carriers. Use of this ELISA-based assay may help avoid the need for mutation analysis in patients with unexplained low FVIII activity.
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