Internal quality control for HIV testing of blood donors - Dried tube specimen as a cost-effective alternative.

BACKGROUND: An important aspect of ensuring blood safety is the performance of mandatory serological testing for transfusion transmissible infections. The practice of internal quality control (IQC) in blood banks in India is nonuniform, especially the use of third-party materials. Cited reasons are cost, lack of access to control materials, and need for deep-freezers for storage, if prepared in-house. OBJECTIVE: Validation of dried tube specimen (DTS) from HIV-positive plasma as a low-cost, stable material for use as IQC material in blood banks. METHODS: Fresh-frozen plasma (FFP) prepared from four HIV-positive blood-donors were pooled. Equal numbers of seronegative FFPs were pooled. Twenty microlitre aliquots of plasma were made in micro-centrifuge tubes and air-dried overnight at room-temperature. These were stored in 2-8°C refrigerators and tested once weekly for 6 months on multiple platforms with different detection principles: Rapid tests, second-generation enzyme-linked immunosorbent assay (ELISA), fourth-generation ELISA, and fourth-generation Chemiluminescence immunoassay. The protocol was sustained over the next 6 months with decreased testing frequency to study the extended stability of DTS. RESULTS: A total of 139 positive-DTS and 139 negative-DTS were tested with 100% samples showing consistent results on all platforms over 1 year. There was mild deterioration in reaction strengths, which did not interfere in result interpretations. CONCLUSION: Plasma in form of DTS maintained stability when stored at 2-8°C for 1 year. This provides evidence that DTS can be a modality for the production of cost-effective, stable, in-house control material for resource-restricted countries.

Pre-administration of a carboxypeptidase inhibitor enhances tPA-induced thrombolysis in mouse microthrombi: Evidence from intravital imaging analysis.

INTRODUCTION: Thrombolysis using recombinant tissue-type plasminogen activator (rt-PA) is the pharmacological treatment of choice in acute thrombotic events. However, a narrow therapeutic window and bleeding complications limit its use. We describe the role of carboxypeptidase inhibitor from potato tuber (PTCI), an inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), on Glu-plasminogen accumulation and microthrombus dynamics in vivo and demonstrate its influence on rt-PA-mediated thrombolysis. MATERIALS AND METHODS: In conjunction with real-time intravital two-photon excitation fluorescence microscopy, we produced and imaged laser-induced microthrombi in the mesenteric venules of Green Fluorescent Protein (GFP)-expressing mice. We examined microthrombus dynamics and thrombolysis patterns in vivo by measuring the changes in the fluorescence intensity of labeled Glu-plasminogen following administration of epsilon aminocaproic acid (EACA), PTCI, and rt-PA. RESULTS: PTCI enhanced Glu-plasminogen accumulation at the core of the thrombus by inhibiting TAFIa, while EACA inhibited this process. Exogenous rt-PA effectively triggered Glu-plasminogen activation within the thrombus and promoted thrombolysis. Administration of PTCI and rt-PA together showed no significant benefit on thrombolysis compared to rt-PA administration alone. However, early-phase systemic administration of PTCI before thrombolytic therapy by rt-PA expedited clot lysis as evidenced by significantly faster time to reach peak Glu-plasminogen fluorescence intensity and shorter time to achieve near-complete clot lysis (P = 0.014 and P = 0.003, respectively). CONCLUSIONS: PTCI potentiates rt-PA-mediated thrombolysis when administered early in acute thrombotic events. Further studies are warranted to explore the potential of TAFI inhibitors as adjunct agents in thrombolysis or thromboprophylaxis.

Rituximab therapy for neuro - inflammatory disorders - Real world experience from South India.

BACKGROUND AND OBJECTIVES: To determine the real-world data regarding the use of Rituximab (RTX) in neuroinflammatory disorders (NIDS) and assess the outcomes following RTX treatment. METHODS: A cohort of consecutive patients with NIDS started on RTX (2018-2020) was included. The outcomes assessed were the proportion with favourable clinical response (FCR) as defined by clinical remission/ improvement using disease specific disability scores, comparative efficacy based on timing of initiation and B cell kinetics. RESULTS: A total of 97 patients with NIDS were included. The mean age was 36.43 (±14.4) years and median duration of follow - up being 15 months (IQR 12-16). Forty patients (41.2%) were initiated on RTX "early" in disease course. Favourable clinical response at last follow-up was seen in 94.9% (n = 92). The mean change in disability score (mRS) was 1.89 (SD 1.30) (p < 0.001). RTX appeared more effective when initiated "early" with higher remission rates (75% vs. 42%, p 0.015). B cell kinetics varied across NIDS, with 73% having adequate depletion at 6 months. Minor adverse events including infusion related reactions were reported in 9%. CONCLUSIONS: RTX has a favourable efficacy and safety profile. Future prospective studies are needed to establish the optimal timing of initiation and need for disease-based dosage regimens.

Laboratory characterization of obligate carriers of type 3 von Willebrand disease with a potential role for Platelet Function Analyzer (PFA-200).

INTRODUCTION: Type 3 von Willebrand disease (VWD) is a rare autosomal recessive disorder characterized by undetectable von Willebrand Antigen (VWF:Ag). Carriers of type 3 VWD carry one null allele and have von Willebrand factor (VWF) at about 50% of normal. The aim of this study was to characterize type 3 VWD carriers and to study the role of Platelet Function Analyzer (PFA-200) in this cohort. METHODS: This was a cross-sectional study where data were collected from carriers (parents/offspring) of type 3 VWD patients and evaluated with activated partial thromboplastin time, factor VIII, blood group, ristocetin cofactor assay (VWF:RCo), VWF:Ag, and closure time on PFA-200 with collagen/epinephrine (COL/EPI), and collagen/ADP (COL/ADP). RESULTS: One hundred carriers were included in the study of which 85 were included for PFA-200 analysis. The mean (SD) of VWF:Ag (IU/ml) and VWF:RCo (IU/ml) was 0.63 (0.24) and 0.61 (0.26), respectively. Among the 100 carriers, based on VWF levels (VWF:Ag and/or VWF:RCo) and bleeding history, there were 7 type 1 VWD, 10 type 2 VWD, 25 borderline VWF (0.30-0.50 IU/ml and no bleeding), and 58 normal VWF (>0.50 IU/ml). PFA-200 was prolonged in 71% of the carriers, all carriers with type 1 and type 2 VWD phenotype, 80% carriers with borderline VWF, and 59% with normal VWF. COL/EPI was more sensitive than COL/ADP and showed better correlation with VWF parameters than COL/ADP. CONCLUSION: Carriers of type 3 VWD can have a variable laboratory phenotype. PFA-200 showed good sensitivity among the carriers at VWF levels <0.50 IU/ml.

A study to compare Hematopoietic Progenitor Cell count determined on a next-generation automated cell counter with flow cytometric CD34 count in peripheral blood and the harvested peripheral blood stem cell graft from autologous and allogenic donors.

INTRODUCTION: A successful bone marrow transplant requires a minimum of 2-4 × 106 cells/kg patient body weight of CD 34+ cells to be transfused, where peripheral blood CD34+ cell count being and ideal predictor. We compared the correlation and predictive capacity of both hematopoietic progenitor cell count (HPC) determined on the Sysmex XN-9000 and flow cytometric CD34 in autologous and allogenic donors. METHODS: Autologous and allogenic donors were taken as per criteria. TLC (Total Leukocyte Count), MNC (Mononuclear cell count), HPC, and CD34 assay were done in both the peripheral blood prior to apheresis, and the harvest product postapheresis. Sysmex XN-9000 was used for TLC, MNC, and HPC tests, and a modified ISH-AGE protocol was used to enumerate CD34 by flow cytometry. Statistical analysis was done using SPSS 16.0. RESULTS: Sixty-seven allogenic and 35 autologous donors were enrolled. 45% were females, and 55% were males. Correlation between HPC and CD34 was found to be 0.887 with P value < .01 in peripheral blood and 0.847 with P value < .01 in the harvested product. On the other hand, TLC had a correlation of 0.424 and 0.520 in peripheral blood and harvested, respectively. MNC had a weak association. The cutoff value for a target dose of 2 × 106 CD34 cells/kg was 37 × 106 /L for pre-HPC. For a target of 4 × 106 CD34 cells/kg, the cutoff value calculated to be 54 × 106 /L (Sensitivity: 85%, Specificity: 89%) for peripheral blood HPC. CONCLUSION: We conclude that HPC is comparable to CD34 in predicting harvest product's adequacy.

Prevalence of HLA-B*27 subtypes in the Tamil population of India with Ankylosing spondylitis and its correlation with clinical features.

INTRODUCTION: HLA-B*27 is strongly associated with Ankylosing spondylitis (AS). Its subtypes show considerable geographic and ethnic difference. The main aim of this study was to assess the frequency of subtypes of HLA-B*27 in the Indian Tamil AS patients. METHODS AND MATERIALS: Adult AS patients positive for HLA-B*27 were considered for the study. The high-resolution typing to define HLA-B*27 subtypes were done using Invitrogen B kits from One Lambda (SeCore® Sequencing Kits, Thermo Fisher, United States). RESULTS AND CONCLUSION: Prevalence of subtypes identified were HLA-B*27:04 (52.2%), HLA-B*27:05 (41.6%), HLA-B*27:07 (3.5%) and HLA-B*27:02 (2.7%). All subtypes showed disease predisposition for males. The most common extra articular manifestation seen was enthesitis in HLA-B*27:04 and HLA-B*27:05. Uveitis was mainly associated with HLA-B*27:05 and dactylitis with HLA-B*27:04. A significant peripheral joints involvement for female and axial joint involvement for males was seen in HLA-B*27:04. Our study establishes the prevalence of HLA-B*27 subtypes and the associated clinical phenotypes among the Indian Tamil population. Considering the variability of presentation, organ involvement, and disease course in different subtypes and across ethnicities it is critical to define these associations in the ethnic populations we treat for their appropriate care considering the significant negative health and socioeconomic effects of AS.

Autoantibodies Among HIV-1 Infected Individuals and the Effect of Anti-Retroviral Therapy (ART) on It.

BACKGROUND: Antiretroviral therapy (ART) has led to a decline in autoimmune diseases but lacks studies on its effect on autoantibodies. METHODS: It is a cross-sectional study with archived samples from 100 paired HIV-1 infected ART naïve and experienced individuals and 100 prospectively collected matched blood-donor controls. Antinuclear antibody, IgG anticardiolipin antibody, IgM and IgG ß2 glycoprotein-1 antibodies, and total IgG levels were detected. Results are expressed as mean with standard deviation (SD), median, percentage positivity, and a p<0.05 is considered significant. The study was approved by the Institutional Review Board. RESULTS: The median viral load of the treatment naïve samples was 4.34 Log copies/mL, while all were virally suppressed post ART with a median duration of treatment for 12 months (range: 3-36 months). The percentage of antinuclear antibody positivity was 5% among ART naïve and controls, with a decrease of 2% post ART (p= 0.441). The positivity for anti-cardiolipin antibody was 15% among ART naïve while none of the ART experienced or controls were positive (p<0.05). IgM ß2 glycoprotein-1 were 4%, 1% and 3% among ART naïve, treated and controls, respectively (p<0.05). IgG ß2 glycoprotein-1 was 2% among ART naïve while none of the treated and controls were positive (p<0.05). The mean total IgG level among ART naïve, experienced, and controls were 21.82 (SD 6.67), 16.91 (SD 3.38), 13.70 (SD 2.24) grams/Litre, respectively (p<0.05). CONCLUSION: ART has a significant effect on IgG anti-cardiolipin antibody and total IgG but only a marginal effect on ANA, IgM, and IgG ß2 glycoprotein-1 antibodies.

Microsatellite instability in young patients with mucinous colorectal cancers - characterization using molecular testing, immunohistochemistry, and histological features.

CONTEXT: The incidence of colorectal cancers (CRCs) in young Indian patients is higher than the international average. CRCs in young patients are commonly of mucinous type and show microsatellite instability (MSI). AIMS: To ascertain the MSI status of mucinous CRCs in patients ≤40 years of age by molecular testing and to correlate this with immunohistochemical (IHC) analysis and tumor histology. SUBJECTS AND METHODS: Archived formalin-fixed paraffin embedded tissue blocks of 30 young mucinous CRC patients were retrieved. MSI testing was done using two mononucleotide markers - BAT26 and NR24. IHC analysis was done using MLH1, MSH2, and MSH6. Histological features of all cases were studied. Data were analyzed using the SPSS software and the Pearson's chi-square test and Fisher's exact test. RESULTS: Eight out of 30 cases (26.7%) showed MSI by molecular testing. IHC identified seven of these cases. Histological features showing a statistically significant association with MSI were the presence of a well-differentiated adenocarcinoma component (P = 0.003), peritumoral lymphocytes (P = 0.002) and tumor budding (P = 0.021). CONCLUSION: The detection of defective mismatch repair (MMR) proteins using IHC for MLH1, MSH2, and MSH6 and molecular testing using BAT26 and NR24 appears to be a good protocol to detect CRCs with MSI. Histology could be useful in identifying cases that require screening for presence of MMR protein defects.

Evaluation of nonneutralizing antibodies against factor VIII in severe haemophilia A patients from India.

: Haemophilia A is treated by replacement therapy with factor VIII (FVIII) concentrate. This strategy of treatment is ineffective in some patients due to the development of neutralizing antibodies (NNAs) against FVIII. The inhibitors have been identified to act against the functional domains of FVIII. The presence of NNAs against FVIII has also been identified. There is limited data on the prevalence and significance of NNA in haemophilia. To identify the presence of NNA in severe haemophilia A in our population, patients who were recruited from community-based camps were evaluated for FVIII activity. The patient's samples were further analysed for inhibitor activity with Nijmegen-Bethesda Assay and for NNAs using an in-house ELISA. 312 severe haemophilia patients were analysed for inhibitors and NNA. In-house ELISA picked up antibodies in 56 patients (17.9%). Of these 42 (13.7%) had inhibitory antibodies and in 14 patients (4.5%) there was no evidence of FVIII inhibitory activity. A substantial number of patients with severe haemophilia A have NNA. Continuous long-term follow-up is required in this cohort to evaluate the significance of this observation.

Rodenticidal hepatotoxicity: Raised plasma Von Willebrand factor levels predict in-hospital survival and preliminary report of the outcome of Von Willebrand factor reducing management protocol.

BACKGROUND: High Von Willebrand factor (VWF) levels may predispose to multi-organ failure in acute liver failure (ALF). In rodenticide-induced hepatotoxicity patients, we analyzed if plasma VWF levels predicted survival and also the outcome of VWF lowering by N-acetyl cysteine (NAC), fresh frozen plasma (FFP) infusions, and plasma exchange (PLEX). METHODS: We retrospectively analyzed prospectively collected data. Hepatotoxicity was classified as uncomplicated acute hepatitis (UAH), acute liver injury (ALI), and ALF. ALF patients, if not opting for liver transplantation, had PLEX and NAC; ALI patients received NAC ± FFP (PLEX, if worsening); UAH patients had NAC. Plasma VWF antigen was measured (normal, 50% to 150%). In-hospital survival was analyzed as discharged alive or died/discharged in a terminal condition (poor outcome). RESULTS: Twenty-four consecutive rodenticide-induced hepatotoxicity patients (UAH in 1, ALI in 20, ALF in 3) from December 2017 to January 2019 were studied. Baseline VWF levels were 153%, 423 (146-890)% median (range), and 448 (414-555)% in UAH, ALI, ALF patients; model for end-stage liver disease (MELD) scores were 11, 24 (12-38), 36 (32-37) and in-hospital survival rates were 100%, 85%, 67%, respectively. VWF levels were higher in patients with poor outcome (555 [512-890]%) than in those discharged alive (414 [146-617]%) (p-value = 0.04). The area under the receiver operating curve of the VWF level, MELD score, and sequential organ failure assessment score to predict survival was 0.92, 0.84, and 0.66, respectively. Of 4 patients meeting criteria for liver transplantation (none had transplantation), 3 (75%) survived. CONCLUSIONS: High VWF levels predict poor outcome in rodenticide-induced hepatotoxicity. VWF reduction may be useful in such patients.