High B-cell activating factor levels in multi-transfused thalassemia patients.

OBJECTIVES: To assess the associations between B-cell activating factor (BAFF) and alloimmunisation in multi-transfused thalassemia. BACKGROUND: Red blood cell (RBC) alloimmunisation is a complication of multi-transfused thalassemia. BAFF is promoting B cells that produce alloantibodies. METHODS/MATERIALS: Multi-transfused thalassemia, 15 years or older, were recruited in the cohort study. Alloantibodies and BAFF levels were analysed. RESULTS: Of 114 patients, the overall prevalence of RBC alloimmunisation was 29.8%. The most common alloantibodies were anti-E, anti-Mia and anti-c. BAFF levels were different among the three groups; the patients with baseline alloantibodies (median ± interquartile range 1251 ± 474 pg/ml), without alloantibodies (1098 ± 453) and healthy controls (719 ± 306), p < 0.001. The BAFF level was elevated in the >25 years old patients (vs. the <25, p = 0.011) and the buffy-coat-reduced blood recipients (vs. the pre-storage leukocyte-depletion, p = 0.005). Absolute lymphocyte count was higher in the patients without baseline alloantibodies (vs. with baseline alloantibodies, p = 0.049) and the splenectomised patients (vs. the non-splenectomised patients, p < 0.001). Of the 72 patients without baseline antibodies, four who developed new antibodies showed no statistically different BAFF levels compared with those without new antibodies after 40-month follow-up (1296 ± 734 vs. 1062 ± 460, p = 0.491). In multivariate analysis, BAFF to absolute lymphocyte ratio was independently associated with RBC alloimmunisation (odds ratio 3.07, 95% confidence interval 1.124-8.369, p = 0.029). CONCLUSION: B-cell activating factor (BAFF) levels were elevated in multi-transfused thalassemia and the BAFF to absolute lymphocyte ratio was associated with red blood cell (RBC) alloimmunisation.

Antiplatelet activity of deferiprone through cyclooxygenase-1 inhibition.

Thalassemia patients are susceptible to both iron overload and thromboembolism. Deferiprone is an iron chelator that shows an antiplatelet activity and thus may alleviate platelet hyperactivation in thalassemia. Therefore, this study aimed to characterize the inhibitory effects and mechanisms of deferiprone on normal human platelets. The results illustrated that deferiprone inhibited platelet aggregation at the iron chelating concentrations (0.08-0.25 mmol/l). Deferiprone inhibited human platelet aggregation stimulated by arachidonic acid and ADP more potently than epinephrine and collagen, with the IC50 of 0.24 mmol/l and 0.25 mmol/l vs. 3.36 mmol/l and 3.73 mmol/l, respectively. Interestingly, deferiprone significantly inhibited COX-1 activity, with the IC50 of 0.33 mmol/l, and slightly increased cAMP level at the high concentration of 4 mmol/l. Moreover, the results from molecular docking showed that deferiprone interacted closely with key residues in the peroxidase active site of COX-1. These results suggested that deferiprone possessed antiplatelet activity mainly through the inhibition of COX-1 activity.

Comparison of original and generic clopidogrel 600 mg loading dose in the patients who planned undergoing coronary angiography.

OBJECTIVE: To compare the efficacy and safety of original (Plavix) and generic (Apolets) clopidogrel 600 mg loading in patients planning to undergo coronary angiography. MATERIAL AND METHOD: This is an experimental design, parallel, randomized-controlled study. Coronary artery disease patients planned for cardiac catheterization were recruited Patients were randomized to receive either original or generic clopidogrel 600 mg loading dose. Platelet aggregation induced by 5 micromol/L and 20 micromol/L adenosine diphosphate (ADP) was measured by light transmission aggregometry (LTA) at baseline and 6 hours after clopidogrel 600 mg administration. RESULTS: Forty-nine patients were enrolled, 24 patients received original clopidogrel, and 25 patients received generic clopidogrel. After six hours of loading, there was significantly reduction in platelet aggregation induced by adenosine 5 micromol/L from 41.08 +/- 3.04% to 19.50 +/- 1.68% (p < 0.001) in original group compared to 36.76 +/- 2.66% to 21.32 +/- 2.60% (p < 0.001) in generic group. When induced by 20 micromol/L, the platelet aggregation was reduced from 58.50 +/- 2.09% to 32.25 +/- 2.30% (p < 0.001) in original group and from 61.12 +/- 2.54% to 30.04 +/- 3.14% (p < 0.001) in generic group. There was no significant difference between original and generic clopidogrel in reducing platelet aggregation induced by both adenosine 5 and 20 micromol/L. Groin hematoma was found in one case (4.2%) in the original clopidogrel group. CONCLUSION: Generic clopidogrel (Apolets) 600 mg loading dose is as effective as original clopidogrel (Plavix) in term of platelet aggregation inhibition.

Prevalence of thrombocytopenia, anti-platelet factor 4 antibodies and D-dimer elevation in Thai people After ChAdOx1 nCoV-19 vaccination.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare distinctive syndrome characterized by unusual site thrombosis accompanied by thrombocytopenia after ChAdOx1 nCoV-19 vaccination. Platelet-activating anti-platelet factor 4-dependent antibodies (anti-PF4 Abs) were detected in most cases of VITT. To date, data from Asian countries are lacking. OBJECTIVES: To determine the prevalence of thrombocytopenia, anti-PF4 Abs, and D-dimer elevation in Thai people administered the ChAdOx1 vaccine. PATIENTS/METHODS: A total of 521 vaccinated and 146 nonvaccinated subjects were enrolled. Blood samples were collected to determine platelet counts, anti-PF4 Abs using ELISA and D-dimer levels 5 to 30 days after the first vaccination. RESULTS: None of the participants developed thrombocytopenia or had significantly decreased platelet counts from baseline after ChAdOx1 vaccination. The frequencies of anti-PF4 Abs between vaccinated (16/521; 3.1%; 95% confidence interval [CI], 1.8-4.9) and nonvaccinated Thai people (6/146; 4.1%; 95% CI, 1.5-8.7) were similar. None of the detectable anti-PF4 Abs activated platelets in vitro. The average D-dimer levels between vaccinated and control groups were similar (282.2 ± 286.3 vs 267.8 ± 219.3 ng/mL; P = 0.58). Four vaccinated and one nonvaccinated participants had markedly elevated D-dimer levels >2000 ng/mL without detectable anti-PF4 Abs. Imaging studies of these asymptomatic subjects revealed incidental pulmonary embolism in a vaccinated elderly woman. CONCLUSIONS: This study demonstrated a low prevalence of thrombocytopenia and pathogenic anti-PF4 Abs after ChAdOx1 vaccination. D-dimer testing revealed no significant coagulation activation. Routine tests for platelet counts, anti-PF4 Abs, and D-dimer levels are not recommended for VITT screening without clinical suspicion.

Whole exome sequencing for diagnosis of hereditary thrombocytopenia.

Hereditary thrombocytopenia comprises extremely diverse diseases that are difficult to diagnose by phenotypes alone. Definite diagnoses are helpful for patient (Pt) management.To evaluate the role of whole exome sequencing (WES) in these Pts.Cases with unexplained long-standing thrombocytopenia and/or suggestive features were enrolled to the observational study. Bleeding scores and blood smear were evaluated. The variant pathogenicity from WES was determined by bioinformatics combined with all other information including platelet aggregometry, flow cytometry, and electron microscopy (EM).Seven unrelated Pts were recruited. All were female with macrothrombocytopenia. Clinical bleeding was presented in four Pts; extra-hematological features were minimal and family history was negative in every Pt. WES successfully identified all the 11 responsible mutant alleles; of these, four have never been previously reported. Pt 1 with GNE-related thrombocytopenia showed reduced lectin binding by flow cytometry, increased glycogen granules by EM and a novel homozygous mutation in GNE. Pts 2 and 3 had phenotypic diagnoses of Bernard Soulier syndrome and novel homozygous mutations in GP1BB and GP1BA, respectively. Pt 4 had impaired microtubule structures, concomitant delta storage pool disease by EM and a novel heterozygous TUBB1 mutation. Pt 5 had sitosterolemia showing platelets with reduced ristocetin responses and a dilated membrane system on EM with compound heterozygous ABCG5 mutations. Pts 6 and 7 had MYH9 disorders with heterozygous mutations in MYH9.This study substantiates the benefits of WES in identifying underlying mutations of macrothrombocytopenia, expands mutational spectra of four genes, and provides detailed clinical features for further phenotype-genotype correlations.

Vascular and hemostatic alterations associated with pulmonary hypertension in ß-thalassemia hemoglobin E patients receiving regular transfusion and iron chelation.

INTRODUCTION: Pulmonary hypertension (PH) is the commonest cardiac complication in ß-thalassemia intermedia, including ß-thalassemia/hemoglobin E (ß-thal/HbE), and is strongly associated with splenectomy. We aimed to define the prevalence and comprehensively explore mechanisms of PH in ß-thal/HbE patients receiving regular transfusion and iron chelation, which were reported to alleviate PH. MATERIALS AND METHODS: ß-Thal/HbE patients receiving regular transfusion and iron chelation over one year were enrolled. Patients at risk for PH were defined by tricuspid-regurgitant-jet-velocity (TRV) ≥ 2.5 m/s. Laboratory and echocardiographic variables were compared with healthy controls. RESULTS: There were 68 ß-thal/HbE, including 31 (45.6%) splenectomized patients, and 38 controls included for analysis. PH was detected in 29 ß-thal/HbE (42.6%). ß-Thal/HbE with PH had a significant reduction in nitric oxide metabolites (NOx) but elevations in thrombin-antithrombin (TAT) complex, soluble thrombomodulin (sTM), endothelin-1 (ET-1) and flow-mediated dilation (FMD) values compared to those without PH (all, p < 0.05). TRV was significantly correlated with NOx, TAT, sTM, ET-1 and FMD values (r = -0.514, r = 0.281, r = 0.313, r = 0.245 and r = -0.474; all p < 0.05). Erythropoietic activity, serum ferritin, circulating total tissue factor (TF) antigen, microparticle-associated TF activity, microparticle's procoagulant activity and soluble p-selectin levels were not different between PH and non-PH subgroups. Notably, there were no significant associations between splenectomy and PH. CONCLUSIONS: PH remains prevalent in ß-thal/HbE patients receiving long-term transfusion and iron chelation. PH is not associated with splenectomy status but correlated with NO depletion, TF-independent hypercoagulability and endothelial perturbation.

Natural anticoagulant deficiencies in Thais: A population-based study.

INTRODUCTION: In contrast to Caucasians, hereditary anticoagulant deficiencies are more common in Asians and mutations are heterogeneous among different countries. This study aimed to determine the prevalence and genetic basis of protein C and protein S deficiencies in Thai population. METHODS: Healthy volunteers were tested for protein C activity and free protein S antigen. Subjects with low values were requested for repeated testing and exclusion of acquired causes. Cases with persistently low levels were assayed for respective gene mutations using direct sequencing and multiplex ligation-dependent probe amplification (MPLA) when PROS1 point mutation was undetectable. RESULTS: For protein C activities (N = 5234), the values of men were lower than those of post-menopausal women (P < 0.001). In 17 of 18 subjects, there were 7 types of PROC mutations, 64.7% of which were p.R189W and 2 were previously unreported. Protein S levels (N = 5242) were highest in men, followed by post-menopausal and pre-menopausal women, respectively (P < 0.001). The repeatedly low protein S was mostly in female (88.2%). Among 29 subjects with protein S below the sex-specific 2.5 percentile cut-points, 4 types of mutations were found in 5 subjects (17.2%) with one previously unreported mutation. Free protein S levels were below 30 U/ml in all cases with mutations. The estimated prevalence of PROC and PROS1 mutations in Thai population was 0.69% and 0.22%, respectively. CONCLUSIONS: PROC mutations, especially p.R189W, are common in Thais. However, mildly depressed protein S levels were frequently found in women with undetectable PROS1 mutation.

Bleeding Symptoms and von Willebrand Factor Levels: 30-Year Experience in a Tertiary Care Center.

Correlations between bleeding symptoms and von Willebrand factor (VWF) levels may help to predict hemorrhagic severity in the Westerners with von Willebrand disease (VWD), but data in Asians are lacking. In this study, Thai patients with VWF levels <50 IU/dL without any secondary causes were enrolled from 1988 to 2018 to determine the relationship between VWF levels and hemorrhagic manifestations. According to the current concept, we reclassified VWD and low VWF by VWF levels ≤30 and 30 to 50 IU/dL, respectively. Type 2 VWD was diagnosed if VWF activity to antigen ratio was ≤0.6. Bleeding severity was determined by the condensed MCMDM-1VWD bleeding score (BS). Among 83 patients, VWF activities showed negative correlations with BS (P = .001), which were higher in type 2 (median: 7, interquartile range [IQR]: 5-11) compared with type 1 VWD (median: 3, IQR: 2-4) and low VWF (median: 4, IQR: 2-8). Bleeding symptoms were indistinguishable between type 1 VWD and low VWF using the 30 IU/dL cutoff point. However, VWF ristocetin cofactor activity or gain-of-function mutant glycoprotein Ib binding activity <36.5 IU/dL and VWF collagen binding activity <34.5 IU/dL could predict increased bleeding risk (BS ≥3) by 92.3% specificity and 70.0% sensitivity (P < .0001).

Green pit viper (Trimeresurus albolabris and T. macrops) venom antigenaemia and kinetics in humans.

Green pit viper bite is a common public health problem in Southeast Asia. Although most patients experience only local swelling, some may suffer from severe systemic bleeding that can be delayed. Venom antigenaemia was measured by enzyme-linked immunosorbent assay and correlated with clinical findings in 42 patients. Initial venom antigenaemia was not predictive enough for clinical uses. A kinetic study (n = 27) showed highest levels at presentation and, then, progressive decline. The average half-life was 27.5 h during the first three days and over 50 h on days 5-7 after bite. Two small subsets (7.4% each) showed persistently detectable venom on day 14 and a subsequent rise in venom antigenaemia. They were associated with prolonged thrombocytopaenia and coagulopathy, respectively. These data demonstrated the long half-life of the venom, suggesting that waiting for spontaneous resolution of coagulopathy is not preferable. In addition, the delayed venom disappearance, not the initial values, was correlated with haemostatic disorders.

Thrombophilia profile in Thai patients with arterial thrombotic disorders.

BACKGROUND: Arterial thrombosis is attributed mainly to atherosclerosis and the roles of hypercoagulability remain unclear In addition, there are ethnic variations in thrombophilia profiles. OBJECTIVE: The authors performed a survey of the thrombophilia profile in Thai patients with arterial thrombosis MATERIAL AND METHOD: The authors analyzed 103 consecutive cases of proven arterial thrombosis and requested thrombophilia profile in Chulalongkorn Hospital during 2003-2004. The mean age was 42.5 years. The proportions of stroke, peripheral arteries, and other sites were 70.9%, 22.3% and 6.8%, respectively. RESULTS: Abnormal profile was found in 35.0% with the prevalence of hyperhomocysteinemia, low protein S, antiphospholipid antibody and low protein C was 15.5%, 12.6%, 9.7%, and 5.8%, respectively. There was no difference in clinical characteristics between cases with or without detectable abnormalities. However, the authors found significant associations of low protein S with poor outcome and HIV seropositivity with antiphospholipid. CONCLUSION: The present study found that the defective protein C pathway may be the most common thrombophilia found in Thais with arterial thrombosis. Future study is required to prove the cause-effect relationship and its clinical significance.

Von Willebrand factor (vWF) antigen levels and function in healthy Thais.

Type I von Willebrand disease (vWD) is very common in caucasians. Its genetic basis is possibly heterogenous, lying both within and out of the vWF gene locus. We sought to investigate vWF levels in the Thai population, to compare with those of western countries. The vWF antigen and activity were measured using ELISA and Collagen Binding Assay (CBA), respectively, in 311 healthy Thai volunteers. The mean age was 32.3, ranging from 18 to 75 years. Fifty-four percent were female. Low vWF antigen and activity (below 50 U/dl) were found in 3.5% and 10.2%, respectively. Around 75% and 20% of these cases had O and A blood groups, respectively. Three (0.96%) had definitely low levels of vWF (vWF antigen level below 35 U/dl), suggesting the diagnosis of vWD. Similar to previous studies, vWF levels were lowest in subjects with group O blood. We found that subjects with blood group A had higher vWF levels than group O subjects, but significantly lower vWF levels than those with group B. The average ratio between the vWF activity and antigen was 0.96, ranging from 0.66 to 1.66. These ratios were inversely correlated with age (p=0.047), suggesting a decline in vWF activity per vWF protein with advancing age. Low levels of vWF are common in healthy Thais. Clinicians should be aware of vWD in bleeding patients and beware low levels of vWF in therapeutic plasma products, especially from blood groups O and A.

Protein S deficiency is common in a healthy Thai population.

Currently, venous thromboembolism is a growing menace in Asians, approaching to that of Western countries. The most common genetic mutations causing thrombosis in Caucasians are factor V Leiden and prothrombin mutation. However both are very rare in Asians. On the other hand, natural anticoagulant protein (protein S, protein C and antithrombin) deficiencies are more common in Asian than in Western thrombotic patients. The prevalence of these deficiencies is very low in healthy Caucasians (0.02-0.3%). It is possible that the prevalence is higher in an Asian general population. However there have been very few prevalence studies to prove this hypothesis. Protein S deficiency was found in 3.7% (13/352, 95% confident interval 1.72-5.66) healthy Thais. Seven of them were type III deficiency. Similar to previous studies, total and free protein S levels were lower in females, but positively and negatively correlated with age, respectively. In contrast, one protein C deficiency (0.27%, 1/370) and no antithrombin deficiency (0/206) were detectable in our population. Furthermore, the authors found that antithrombin was significantly lower in women and there was a positive correlation between protein C activity and age. In conclusion, protein S deficiency is more common in Thais than in Caucasians. This result remains to be confirmed by a large population-based study.

Laboratory identification of lupus anticoagulants using the combination of activated partial thromboplastin time and Russell's viper venom at two phospholipid concentrations.

The most appropriate combination of tests for lupus anticoagulants (LA) is unknown. The standard double centrifugation method to prepare plasma was inadequate for platelet elimination, interfering with kaolin clotting time and mixing studies. In the present study, the percentage correction of activated partial thromboplastin time (APTT) and Russell's viper venom time (RVVT) by high compared with low concentrations of phospholipid was used for both screening and confirmation of LA. Abnormality in either one was reported as positive. The specificity of the tests in 122 individuals without LA was 100 per cent for RVVT and 96.7 per cent for APTT, which yielded false positive by heparin. The mixing study was omitted from the authors' strategy without decreasing the specificity. In 795 patients with thrombosis, LA was detectable in 6.03 per cent. The sensitivity of diluted activated partial thromboplastin time (dAPTT) and diluted Russell's viper venom time (dRVVT) alone, compared with the combination of the two was 83.3 per cent and 79.2 per cent respectively. Therefore, this new test scheme is a simple, inexpensive and efficient method for Thai patients.

Comparison of diagnostic performance of the heparin-induced thrombocytopenia expert probability and the 4Ts score in screening for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a serious immunological complication of heparin administration. Diagnosis of HIT is challenging, especially in critically ill patients. The clinical scoring model for predicting HIT is helpful for guiding clinical decision. We analysed data of patients who underwent the heparin-induced platelet aggregation (HPA) test from 2006 to 2010 and compared diagnostic performance of the novel model HIT expert probability ('HEP'), which has been validated in a population mainly comprising surgical patients first, by the previously published model '4Ts' score. Clinical courses of the patients were also reviewed to ensure that HPA test results were accurate. There were 47 suspected HIT patients. The majority was from medical (70.2%) and/or critical care (61.7%) units. Ten (21.3%) yielded positive HPA. Among positive HPA patients, eight were medical patients. The HEP score ranged from -3 to 13, whereas the 4Ts score ranged from 3 to 7 in positive HPA patients. Both HEP and 4Ts scores were significantly higher in positive HPA than in negative HPA patients (5.35 vs. 1.81, P=0.010 and 4.85 vs. 3.32, P=0.001, respectively). The HEP score did not display better diagnostic performance than the 4Ts score, with receiver operating characteristic (ROC) area under curve of 0.72 and 0.79 (P=0.31), respectively. The HEP score did not show better diagnostic performance than the 4Ts score for predicting HIT in our population. A large prospective validation in different sets of patients is warranted.

The role of prothrombin time (PT) in evaluating green pit viper (Cryptelytrops sp) bitten patients.

Viper bites cause consumptive coagulopathy resulting in hypofibrinogenaemia. Whole-blood clotting time is a standard test used to assess bleeding risk. Prothrombin time (PT) and activated partial thromboplastin time (APTT) are better standardised assays that are widely available, but their diagnostic accuracy in viper bites remains unknown. Adult patients presumed bitten by green pit vipers (Cryptelytops sp.) were enrolled. Conventional venous clotting time (VCT), 20min whole-blood clotting time (20WBCT), PT with international normalized ratio (INR) and APTT were determined. A fibrinogen level below 1.0g/litre was used as the gold standard. There were 97 patients. The average age was 46.1 years and 49.5% were men. VCT >30min, INR >1.2 and fibrinogen level <1.0g/litre were found in 9.3, 10.3 and 7.2%, respectively. The sensitivities of VCT >30min, 20WBCT (N=55), INR and APTT were 57.0%, 85.7%, 85.7% and 57.1%, respectively. The respective specificities were 94.4%, 95.8%, 95.6% and 72.4%. Three hypofibrinogenaemic patients who did not receive antivenom because of VCT <30min had persistently normal VCT and went home without clinical bleeding. In conclusion, PT with INR can be an alternative test for evaluation of coagulopathy in green pit viper bitten patients with potentially improved inter-laboratory standardisation.