S. C. Nair

Tests of global haemostasis and their applications in bleeding disorders

Summary.  There is a potential for significant paradigm shift in the assessment of haemostasis from the conventional plasma recalcification times, such as prothrombin time (PT) and activated partial thromboplastin time (APTT), which correspond to artificially created compartments of haemostasis to tests that assess the entire process in a more physiological and holistic manner. These include the thrombin generation test, thromboelastogram and the clot wave form analysis. While these tests have been described many years ago, there is renewed interest in their use with modified technology for assessing normal haemostasis and its disorders. Although early data suggest that they can provide much greater information regarding the overall haemostasis process and its disorders, many challenges remain. Some of them are possible only on instruments that are proprietary technology, expensive and are not widely available. Furthermore, these tests need to be standardized with regard to their reagents, methodology and interpretation, and finally, much more data need to be collected regarding clinical correlations with the parameters measured.

Role of minimal residual disease monitoring in acute promyelocytic leukemia treated with arsenic trioxide in frontline therapy

Data on minimal residual disease (MRD) monitoring in acute promyelocytic leukemia (APL) are available only in the context of conventional all-trans retinoic acid plus chemotherapy regimens. It is recognized that the kinetics of leukemia clearance is different with the use of arsenic trioxide (ATO) in the treatment of APL. We undertook a prospective peripheral blood RT-PCR-based MRD monitoring study on patients with APL treated with a single agent ATO regimen. A total of 151 patients were enrolled in this study. A positive RT-PCR reading at the end of induction therapy was significantly associated on a multivariate analysis with an increased risk of relapse (relative risk = 4.9; P = .034). None of the good risk patients who were RT-PCR negative at the end of induction relapsed. The majority of the relapses (91%) happened within 3 years of completion of treatment. After achievement of molecular remission, the current MRD monitoring strategy was able to predict relapse in 60% of cases with an overall sensitivity and specificity of 60% and 93.2%, respectively. High-risk group patients and those that remain RT-PCR positive at the end of induction are likely to benefit from serial MRD monitoring by RT-PCR for a period of 3 years from completion of therapy.

Diversity of Glanzmann thrombasthenia in southern India: 10 novel mutations identified among 15 unrelated patients

Summary.  Background: Glanzmann thrombasthenia (GT) is a congenital bleeding disorder caused by either a lack or dysfunction of the platelet integrin αIIbβ3. Objectives: To determine the molecular basis of GT in patients from southern India. Patients: Fifteen unrelated patients whose diagnosis was consistent with GT were evaluated. Results: Platelet surface expression of αIIbβ3 was < 10%, 10%–50%, and > 50% of controls in five, nine, and one patient(s), respectively. Immunoblotting of the platelet lysates showed no αIIb in 14 patients, and no β3 in 10 patients, although severely reduced in four patients. Platelet fibrinogen was undetectable in 13 patients, and severely reduced in one patient. One patient showed normal surface αIIbβ3 expression, and normal αIIb, β3 and fibrinogen levels in the lysate. Ten novel candidate disease‐causing mutations were identified in 11 patients. The missense mutations included Gly128Ser, Ser287Leu, Gly357Ser, Arg520Trp, Leu799Arg in αIIb, and Cys575Gly in β3. We have already shown that Gly128Ser, Ser287Leu, and Gly357Ser mutations variably affect αIIbβ3 surface expression. The Cys575Gly mutation may disrupt the disulphide link with Cys586 to cause the GT phenotype. The molecular pathology of the other missense mutations is not clear. Two nonsense mutations, Trp‐16Stop and Glu715Stop in αIIb, and a 7‐bp deletion (330‐336TCCCCAG) in β3 are predicted to result in truncated proteins. An IVS15(−1)G → A mutation in αIIb induced a cryptic splice site as confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR) analysis. Thirteen polymorphisms were also identified (five in αIIb and eight in β3), among which five were novel. Conclusions: While identifying a significant number of novel mutations causing GT, this study confirms the genetic heterogeneity of the disorder in southern India.

Six novel mutations including triple heterozygosity for Phe31Ser, 514delT and 516T-->G factor X gene mutations are responsible for congenital factor X deficiency in patients of Nepali and Indian origin.

Summary.  Factor X (FX) deficiency is a rare (1 : 100000) autosomal recessive disorder caused by heterogeneous mutations in FX gene. We have studied the molecular basis this disease in six Indian and one Nepali patients. Diagnosis was confirmed by measuring the FX coagulant activity (FX: C) using a PT based assay. Six of them had a FX: C of < 1% and one patient had 24% coagulant activity. Mutations were identified in all the seven patients. These included eight (88.8%) missense and one frame‐shift (11.2%) mutations of which six were novel. Three of the novel mutations, a Phe31Ser affecting ‘Gla’ domain and 514delT and 516T→G mutations affecting Cys132 in ‘connecting region’ were identified in a triple compound heterozygous state in a Nepali patient presenting with a severe phenotype. Two other novel mutations, Gly133Arg, may affect the disulphide bridge between Cys132‐Cys302 in the connecting region while Gly223Arg may perturb the catalytic triad (His236, Asp282 and Ser379). The other novel mutation, Ser354Arg, involves the replacement of a small‐buried residue by a large basic aminoacid and is likely to have steric or electrostatic effects in the pocket involving Lys351‐Arg347‐Lys414 that contributes to the core epitope of FXa for binding to FVa. Three previously reported mutations, Thr318Met; Gly323Ser; Gly366Ser were also identified. This is the first report of the molecular basis of FX deficiency in patients from the Indian subcontinent.

Correlating clinical manifestations with factor levels in rare bleeding disorders: a report from Southern India.

Summary.  Data on the clinical manifestations of patients with clotting factor defects other than Haemophilia A, B and von Willebrand disease are limited because of their rarity. Due to their autosomal recessive nature of inheritance, these diseases are more common in areas where there is higher prevalence of consanguinity. There is no previous large series reported from southern India where consanguinity is common. Our aim was to analyze clinical manifestations of patients with rare bleeding disorders and correlate their bleeding symptoms with corresponding factor level. Data were collected in a standardized format from our centre over three decades on 281 patients who were diagnosed with rare bleeding disorders (fibrinogen, prothrombin, factor V (FV), FVII, FX, FXI, FXIII and combined FV or FVIII deficiency). Patients with liver dysfunction or those on medications which can affect factor level were excluded. All patients with <50% factor levels were included in this analysis. Patients were analysed for their salient clinical manifestations and it was correlated with their factor levels. The data shows that FXIII deficiency is the commonest and FXI deficiency is the rarest in Southern India. There was no significant difference in bleeding symptoms among those who were < or >1% factor coagulant activities among all disorders, except for few symptoms in FVII and FX deficiency. An international collaborative study is essential to find out the best way of classifying severity in patients with rare bleeding disorders.

Towards standardization of clot waveform analysis and recommendations for its clinical applications

*Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan; †Department of Haematology, Manchester Royal Infirmary,Manchester, UK; ‡Department of Transfusion Medicine and Immunohaematology, Christian Medical College; and §Department ofHaematology, Christian Medical College, Vellore, IndiaTo cite this article: Shima M, Thachil J, Nair SC, Srivastava A. Towards standardization of clot waveform analysis and recommendations for itsclinical applications. J Thromb Haemost 2013; 11: 1417–20.

Polymorphism in factor VII gene modifies phenotype of severe haemophilia

Summary.  The basis for 10–15% of patients with severe haemophilia having clinically mild disease is not fully understood. We hypothesized that polymorphisms in various coagulant factors may affect frequency of bleeding while functionally significant polymorphisms in inflammatory and immunoregulatory genes may also contribute to variations in the extent of joint damage. These variables were studied in patients with severe haemophilia, who were categorized as ‘mild’ (<5 bleeds in the preceding year, <10 World Federation of Haemophilia clinical and <10 Pettersson scores, n = 14) or ‘severe’ (all others, n = 100). A total of 53 parameters were studied in each individual for their association with the clinical severity. Age, F8:c activity and the incidence of thrombotic markers were comparable between the groups while the median number of bleeds, number of affected joints, clinical, radiological and functional joint scores (P ≤ 0.001) and life‐time clotting factor use (P ≤ 0.007) were different. Patients with severe molecular defects had a 4.1‐fold increased risk for a severe phenotype (95% CI: 1.18–14.42, P = 0.026) compared with other mutations. Of the polymorphisms studied, the FVII353Q (RR = 3.5, 95% CI: 1.04–12.05, P = 0.044) allele was associated with a severe phenotype. This data shows that apart from the F8/F9 genotype, functional polymorphisms in FVII gene affect the phenotype of patients with severe haemophilia.

Molecular genetics of hereditary prothrombin deficiency in Indian patients: identification of a novel Ala362→Thr (Prothrombin Vellore 1) mutation

Summary.  Prothrombin deficiency is a rare (1:200 000) autosomal recessive disorder caused by diverse mutations in prothrombin gene. We have studied the molecular basis of this disorder in four unrelated Indian patients. The diagnosis was based on prolonged prothrombin (PT) and activated partial thromboplastin times and low factor II coagulant activity (FII: C) measured using a PT based assay. FII: C levels ranged between 4.7% and 17.5%. Mutations were identified in all the four patients. Five different causative mutations including four (80%) missense and an in‐frame deletion (20%) were identified. One of them was a novel, Ala362→Thr aminoacid change affecting ‘B’ chain of α‐thrombin. This mutation was present in a compound heterozygous state with a previously reported Arg‐1→Gln missense change affecting pro‐peptide cleavage site. Ala362→Thr occurred at a codon, evolutionarily conserved in all the 24 different prothrombins or its related serine proteases studied. Molecular modeling of this mutation was found to cause a conformational change around the region involving a catalytic triad residue His363 and a cysteine residue at codon 364. The FII: C level in this patient was 17.5%. Three other previously reported mutations were also detected in the homozygous state: Arg271→Cys in Kringle‐2 region, a Glu309→Lys in ‘A’ chain of α‐thrombin and an in‐frame deletion of 3 bp (AAG) leading to Del Lys301/302 in ‘A’ chain of α‐thrombin. This is the first report of the molecular basis of prothrombin deficiency in Indian patients and we suggest the eponym ‘Prothrombin Vellore 1’ for Ala362→Thr mutation.

Molecular basis of Bernard-Soulier syndrome in 27 patients from India.

To cite this article: Sumitha E, Jayandharan GR, David S, Jacob RR, Sankari Devi G, Bargavi B, Shenbagapriya S, Nair SC, Abraham A, George B, Viswabandya A, Mathews V, Chandy M, Srivastava A. Molecular basis of Bernard–Soulier syndrome in 27 patients from India. J Thromb Haemost 2011; 9: 1590–8.

Successful surgical haemostasis in patients with von Willebrand disease with Koate®DVI

Summary.  This report describes our experience with Koate®DVI, a factor VIII (FVIII) concentrate containing von Willebrand factor (VWF) for surgery in patients with von Willebrand’s disease (VWD). Twenty‐one patients underwent 26 procedures, 10 of which were major and 16 were minor. The median age was 27 years (3–55) and the mean weight was 52 kg (16–88). Among the ten patients (type 2–5; type 3–5) who underwent major procedures, the pre‐operative dose was 35 IU kg−1 of FVIII followed by 10–20 IU kg−1 once daily depending on FVIII:C levels. The mean total dose of FVIII used per procedures was 106 IU kg−1 (30–190) over a mean duration of 7 days (3–11). In this group, pre‐infusion FVIII:C, VWF:Ag and VWF: ristocetin cofactor (RCoF) level that were 19.5% (1–64), 20 U dL−1 (0–96) and 12% (0–66) increased to 72% (54–198), 131 U dL−1 (68–206) and 68% (27–108) postinfusion, respectively. Sixteen minor procedures were performed in 11 patients (type 1–3, type 2–6, type 3–2). The preparative dose of FVIII was 10–20 IU kg−1. The average duration of factor support was 2 days (1–3) for a mean total dose of 23 IU kg−1 (9–60). The pre‐infusion levels of FVIII:C, VWF:Ag and VWF:ristocetin cofactor (RCo) which were 31% (22–64), 25.5 U dL−1 (0–63) and 21% (0–76), respectively, increased to 76% (27–111), 73 U dL−1 (30–137) and 45% (2–106) postinfusion. Whereas surgical haemostasis was achieved in all patients, minor postoperative bleeding occurred after one procedure in each group. Both were controlled with additional doses of factor replacement. We conclude that Koate®DVI in modest doses provide adequate haemostasis for surgery in patients with VWD.