Comparative analysis of different enzyme immunoassays for assessment of phosphatidylserine-dependent antiprothrombin antibodies

Phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) were strongly correlated with the presence of lupus anticoagulant showing a high specificity for the diagnosis of antiphospholipid syndrome. However, the main criticism for the clinical applicability of aPS/PT testing is the lack of reproducibility of the results among laboratories. In this study, we measured IgG and IgM aPS/PT using our original in-house enzyme-linked immunosorbent assays (ELISA) and commercial ELISA kits to assess the assay performance and to evaluate the accuracy of aPS/PT results. The study included 111 plasma samples collected from patients and stored at our laboratory for aPS/PT assessment. Sixty-one samples were tested for IgG aPS/PT using two assays: (1) aPS/PT in-house ELISA and (2) QUANTA Lite? aPS/PT IgG ELISA kit (INOVA Diagnostics, Inc., USA). Fifty samples were evaluated for IgM aPS/PT using two assays: (1) aPS/PT in-house ELISA and (2) QUANTA Lite? aPS/PT IgM ELISA kit (INOVA Diagnostics). Ninety-eight percent of samples yielded concordant results for IgG aPS/PT and 82 % for IgM aPS/PT. There was an excellent agreement between the IgG aPS/PT assays (Cohen κ = 0.962) and moderate agreement between the IgM aPS/PT assays (κ = 0.597). Statistically significant correlations in the aPS/PT results were obtained from both IgG and IgM aPS/PT assays (r = 0.749, r = 0.622, p < 0.001, respectively). In conclusion, IgG and IgM detection by ELISA is accurate. The performance of aPS/PT is reliable, and concordant results can be obtained using different ELISA methods.     

Fecal calprotectin and lactoferrin as predictors of relapse in patients with quiescent ulcerative colitis during maintenance therapy

Purpose

This prospective study was to evaluate the significance of fecal calprotectin and lactoferrin for the prediction of ulcerative colitis (UC) relapse.

Methods

Eighty UC patients in remission for ≥3 months on mesalamine as maintenance therapy were included. At entry, stool samples were collected for the measurement of calprotectin and lactoferrin. All patients were followed up for the following 12 months. To identify predictive factors for relapse, time-dependent analyses using the Kaplan-Meier graphs and Cox's proportional hazard model were applied.

Results

During the 12 months, 21 patients relapsed. Mean calprotectin and lactoferrin levels were significantly higher in patients with relapse than those in remission (calprotectin—173.7 vs 135.5 μg/g, P?=?0.02; lactoferrin—165.1 vs 130.7 μg/g, P?=?0.03). A cutoff value of 170 μg/g for calprotectin had a sensitivity of 76 % and a specificity of 76 % to predict relapse, while a cutoff value of 140 μg/g for lactoferrin had a sensitivity of 67 % and a specificity of 68 %. In a multivariate analysis, calprotectin (≥170 μg/g) was a predictor of relapse (hazard ratio, 7.23; P?=?0.002). None of the following parameters were significantly associated with relapse: age, gender, duration of UC, number of UC episode, severity of the previous episode, extent of UC, extraintestinal manifestation, and lactoferrin level.

Conclusions

Fecal calprotectin showed a higher sensitivity and specificity than fecal lactoferrin for predicting UC relapse. Fecal calprotectin level appeared to be a significant predictor of relapse in patients with quiescent UC on mesalamine as maintenance therapy.     

A GCH1 haplotype confers sex‐specific susceptibility to pain crises and altered endothelial function in adults with sickle cell anemia

GTP cyclohydrolase (GCH1) is rate limiting for tetrahydrobiopterin (BH4) synthesis, where BH4 is a cofactor for nitric oxide (NO) synthases and aromatic hydroxylases. GCH1 polymorphisms are implicated in the pathophysiology of pain, but have not been investigated in African populations. We examined GCH1 and pain in sickle cell anemia where GCH1 rs8007267 was a risk factor for pain crises in discovery (n = 228; odds ratio [OR] 2.26; P = 0.009) and replication (n = 513; OR 2.23; P = 0.004) cohorts. In vitro, cells from sickle cell anemia subjects homozygous for the risk allele produced higher BH4. In vivo physiological studies of traits likely to be modulated by GCH1 showed rs8007267 is associated with altered endothelial dependent blood flow in females with SCA (8.42% of variation; P = 0.002). The GCH1 pain association is attributable to an African haplotype with where its sickle cell anemia pain association is limited to females (OR 2.69; 95% CI 1.21–5.94; P = 0.01) and has the opposite directional association described in Europeans independent of global admixture. The presence of a GCH1 haplotype with high BH4 in populations of African ancestry could explain the association of rs8007267 with sickle cell anemia pain crises. The vascular effects of GCH1 and BH4 may also have broader implications for cardiovascular disease in populations of African ancestry. Am. J. Hematol. 89:187–193, 2014. © 2013 Wiley Periodicals, Inc.     

Imaging flow cytometry for automated detection of hypoxia‐induced erythrocyte shape change in sickle cell disease

In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged “sickled” and “normal” erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = ?0.558, P = 0.027), negatively with pH (R = ?0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes‐103 (5‐hydroxymethyl‐2‐furfural) (R = ?0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator‐independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes‐103. SIFCA is a new method that may be useful in sickle cell drug development. Am. J. Hematol. 89:598–603, 2014. © 2014 Wiley Periodicals, Inc.     

First-line treatment for severe aplastic anemia in children: bone marrow transplantation from a matched family donor versus immunosuppressive therapy

The current treatment approach for severe aplastic anemia in children is based on studies performed in the 1980s, and updated evidence is required. We retrospectively compared the outcomes of children with acquired severe aplastic anemia who received immunosuppressive therapy within prospective trials conducted by the Japanese Childhood Aplastic Anemia Study Group or who underwent bone marrow transplantation from an HLA-matched family donor registered in the Japanese Society for Hematopoietic Cell Transplantation Registry. Between 1992 and 2009, 599 children (younger than 17 years) with severe aplastic anemia received a bone marrow transplant from an HLA-matched family donor (n=213) or immunosuppressive therapy (n=386) as first-line treatment. While the overall survival did not differ between patients treated with immunosuppressive therapy or bone marrow transplantation [88% (95% confidence interval: 86–90) versus 92% (90–94)], failure-free survival was significantly inferior in patients receiving immunosuppressive therapy than in those undergoing bone marrow transplantation [56% (54–59) versus 87% (85–90); P<0.0001]. There was no significant improvement in outcomes over the two time periods (1992–1999 versus 2000–2009). In multivariate analysis, age <10 years was identified as a favorable factor for overall survival (P=0.007), and choice of first-line immunosuppressive therapy was the only unfavorable factor for failure-free survival (P<0.0001). These support the current algorithm for treatment decisions, which recommends bone marrow transplantation when an HLA-matched family donor is available in pediatric severe aplastic anemia.     

Noninvasive flow evaluations of coronary artery bypass grafting using dynamic cardiac CT

We aimed to investigate the correlation of graft flow measurements between transit-time flow measurement (TTFM) during coronary artery bypass grafting (CABG) surgery and dynamic cardiac CT after the surgery.Fourteen patients underwent CABG with TTFM and postoperative dynamic cardiac CT; 11 internal thoracic artery (ITA) grafts and 15 saphenous venous grafts (SVGs) were included for analysis. Pearsons correlation analysis was performed for the comparisons of the TTFM and cardiac dynamic CT flow parameters.TTFM was not significantly correlated with the CT flow of the ITA grafts (r = −0.23, P = .49), but it had a very strong correlation with the CT flow of the SVGs (r = 0.83, P < .01).In patients who underwent CABG surgery, dynamic cardiac CT enabled quantitative evaluation of SVG flow, with good correlation with TTFM.