A novel ELISA for diagnosis of Glanzmann’s thrombasthenia and the heterozygote carriers

A sensitive and specific sandwich ELISA was developed for the diagnosis of Glanzmann’s thrombasthenia (GT) and the heterozygote carriers of the disease using whole blood platelets. The assay used anti-CD36 antibody to capture platelets from platelet-rich plasma which was subsequently treated with a bioengineered disintegrin/alkaline phosphatase hybrid protein specific for GP IIb/IIIa. The test allows large number of samples to be typed and can also be used on stored samples. The assay correctly diagnosed 40 normal healthy individuals, 10 GT cases, 10 heterozygotes, 3 Bernard–Soulier syndrome cases and 2 type 3 GT cases. ELISA plates were stable at room temperature up to 3 weeks without any loss of activity. This novel and simple test can be widely used for heterozygote detection besides diagnosing GT cases without using a sophisticated flow cytometer or a platelet aggregometer and has wide applicability in countries like India where many of these cases remain undiagnosed due to the lack of diagnostic facilities.     

Comparison of three newer generation freely available intraocular lens power calculation formulae across all axial lengths

Purpose:The aim of this study is to evaluate the accuracy of three newer generation formulae (Barrett Universal II, EVO, Hill-RBF 2.0) for calculation of power of two standard IOLs—the Acrysof IQ and Tecnis ZCB00 across all axial lengths.Methods:In this retrospective series, 206 eyes of 206 patients, operated for cataract surgery with above two IOLs over the last 6 months, were included in the study. Preoperative biometry measurements were obtained from LenstarLS900. By using recommended lens constants, the mean error for each formula was calculated and compared. Then, the optimized IOL constants were calculated to reduce the mean error to zero. Mean and median absolute errors were calculated for all eyes and separately for short (AL<22.5 mm), medium (22.5–24.5 mm), and long eyes (>24.5 mm). Absolute errors and percentages of eyes within prediction errors of ±0.25 D, ±0.50 D, ±0.75 D, and ±1.00 D were compared.Results:Prediction error with using recommended lens constants was significantly lower in the Barrett Universal II formula as compared to the other two formulae. However, after optimizing lens constants, there were no significant differences in the absolute errors between the three formulae. The formulae ranked by mean absolute error were as follows: Barrett Universal II (0.304 D), EVO (0.317 D), and Hill-RBF (0.322) D. There were no significant differences between absolute errors in the three formulae in each of the short-, medium-, and long-eye subgroups.Conclusion:With proper lens constant optimization, the Barrett Universal II, EVO, and Hill-RBF 2.0 formulae were equally accurate in predicting IOL power across the entire range of axial lengths.     

Plasma Cytokine Analysis in Patients with Advanced Extremity Melanoma Undergoing Isolated Limb Infusion

Background

Preprocedure clinical and pathologic factors have failed to consistently differentiate complete response (CR) from progressive disease (PD) in patients after isolated limb infusion (ILI) with melphalan for unresectable in-transit extremity melanoma.

Methods

Multiplex immunobead assay technology (Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel, Millipore Corp., Billerica, MA; and Magpix analytical test instrument, Luminex Corp., Austin, TX) was performed on pre-ILI plasma to determine concentrations of selected cytokines (MIP-1α, IL-1Rα, IP-10, IL-1β, IL-1α, MCP-1, IL-6, IL-17, EGF, IL-12p40, VEGF, GM-CSF, and MIP-1β) on a subset of patients (n = 180) who experienced CR (n = 23) or PD (n = 24) after ILI. Plasma from normal donors (n = 12) was also evaluated.

Results

Of 180 ILIs performed, 28 % (95 % confidence interval 22–35, n = 50) experienced a CR, 14 % (n = 25) experienced a partial response, 11 % (n = 21) had stable disease, 34 % (n = 61) had PD, and 13 % (n = 23) were not evaluable for response. Tumor characteristics and pharmacokinetics appeared similar between CR (n = 23) and PD (n = 24) patients who underwent cytokine analysis. Although there were no differences in cytokine levels between CR and PD patients, there were differences between the melanoma patients and controls. MIP-1α, IL-1Rα, IL-1β, IL-1α, IL-17, EGF, IL-12p40, VEGF, GM-CSF, and MIP-1β were significantly higher in normal controls compared to melanoma patients, while IP-10 was lower (p < 0.001) in controls compared to melanoma patients.

Conclusions

Patients with unresectable in-transit melanoma appear to have markedly decreased levels of immune activating cytokines compared to normal healthy controls. This further supports a potential role for immune-targeted therapies and immune monitoring in patients with regionally advanced melanoma.     

ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor–Induced Thrombotic Microangiopathy

Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor–related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor–related TMA. ADAMTS13^−/− mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor–related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13^−/− mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.