Sona Nair

Venous thromboembolism in young patients from western India: a study.

The goal of this article is to study the association of known markers of thrombophilia with venous thrombosis in young patients (< 45 years) from the Western part of India. A prospective study of 432 patients (252 males and 180 females, age 1-45 years) was conducted between 1994 and 2000 (6 years). The diagnosis was confirmed in all the patients by ultrasound with Doppler or by a computed tomograph (CT) scan of the brain with or without contrast depending on the case. Detailed clinical examination, and family history was taken to establish recurrent thrombosis and familial occurrence of thrombosis. The markers studied were protein C, protein S, antithrombin (AT) III, factor V Leiden mutation, prothrombin gene G20210A polymorphism, and the thermolabile MTHFR variant C677T polymorphism, using appropriate techniques. Lupus inhibitor was tested in the first 72 patients using Dilute Russel Viper Venom Time (DRVVT) test, and anticardiolipin antibodies were tested by enzyme-linked immunosorbent assay. Protein C, protein S, and AT III deficiency was detected in 9.5%, 6.5%, and 2.6%, respectively, among the patients. Anticardiolipin antibody was present in 9.9% of the patients, whereas lupus anticoagulant was present in 8.3% of patients; factor V Leiden mutation was detected in 3% of patients; thermolabile variant of MTHFR C677T polymorphism was present in 14.9% of patients with 1.2% homozygotes. Prothrombin G20210A polymorphism was not detected in any sample in this population. One hundred and four patients of 432 (24.9%) had recurrent attacks of thrombosis without any proximate precipitating cause, whereas 7.5 % of the patients had another close member of the family with a history of deep venous thrombosis. Eighty-six members from 28 families (out of 32 families giving family history of thrombosis) were investigated and found to have protein C and protein S deficiency in seven each; factor V Leiden was present in 6, and MTHFR C677T polymorphism was present in 5 cases. Hence, 25 of 86 members (28%) from the family of patients with familial history deep venous thrombosis had positive markers for thrombophilia. Thus, we could show that in young patients presenting with thrombosis, at least 34% of them had a demonstrable cause for thrombophilia. Prothrombin gene polymorphism G20210A seems to be nonexistent in our population and AT III deficiency also appears to be low compared to other markers of thrombophilia. There is a high prevalence of variant MTHFR C677T in our series, but the incidence of MTHFR C677T in our general population is also high. Hence, the significance of this finding in our cases of deep venous thrombosis remains to be seen, but we did not see any homozygotes when we tested 70 randomly selected asymptomatic persons, whereas in the present series, 1.8% of the patients had homozygosity for the MTHFR C677T polymorphism.

Glanzmann's thrombasthenia: updated

Glanzmanns thrombasthenia is an autosomal recessive disorder, rare in a global context, but a relatively more common platelet function defect in communities where consanguineous marriages are more frequent. On clinical grounds alone, it cannot be distinguished from other congenital platelet function defects. Epistaxis, gum bleeding, menorrhagia are the common clinical manifestations, whereas large muscle hematoma or hemarthrosis seldom occur in these patients. Essential diagnostic features are a normal platelet count and morphology, a greatly prolonged bleeding time, absence of platelet aggregation in response to ADP, collagen, epinephrine, thrombin and to all aggregating agents which ultimately depend on fibrinogen binding to platelets for this effect, flow cytometry, studies of GPIIb-IIIa receptors on the platelet membrane surface using monoclonal antibodies. The present review describes some of the uncommon features of the disorders and the currently available options which the treating physicians should be aware of during the management of these patients. Although by definition all patients with Glanzmanns thrombasthenia have a virtually complete failure of platelet aggregation, a number of variant forms of GT have been described in which the glycoproteins are present in normal or near normal amounts but are functionally defective. Understanding the pathophysiology of the disorder by the treating physicians is of utmost importance. Presence of high affinity platelet receptors resulting in thrombasthennia-like phenotype may require an antagonistic treatment atypical of classical GT management. It has now been established that different genetic mutations of either GPIIb or IIIa genes results in such a heterogeneity of thrombasthenia phenotype. Glanzmanns thrombasthenia is a paradigm for treating coronary artery disease patients with GPIIb-IIIa antibody and inhibitors. By using these medicines we create a temporary GT-like situation. Hence, understanding this disease is of utmost importance to the practicing cardiologist. As mutations for different variant forms of GT become known, our understanding of how GPIIb-IIIa molecules can be activated to act as a receptor for fibrinogen molecules will be increased. Such understanding undoubtedly will help us to devise better drugs with GPIIb-IIIa inhibitors. Molecular biology techniques have enabled us to equivocally detect heterozygote carriers who are clinically asymptomatic. However, there may be several laboratories in the developing world, which have no access to molecular biology techniques. Development of more robust techniques of quantitation of platelet receptors has enabled an accurate diagnosis of heterozygote carriers or an unborn fetus in the second trimester. The importance of the GPIIb-IIIa polymorphisms in carrier and prenatal diagnosis has not been properly studied. Nowadays the less direct method of PLA1 typing (determination of the levels of platelet antigen) of the foetal platelets as early as 16 weeks of intrauterine life can be used for prenatal diagnosis of GT.

Changes in platelet glycoprotein receptors after smoking--a flow cytometric study.

Cigarette smoking is accepted to be one of the major factors which increase the risk of coronary artery disease, peripheral vascular disease and stroke. A number of studies have been carried out on the acute and chronic effects of tobacco smoking on platelet activation. An enhancing effect of high nicotine cigarette smoking on platelet aggregation has been reported. Since platelet receptors are involved in the final stage of platelet aggregation, the intention of this study was to investigate platelet receptors in acute and chronic smokers before and after smoking. Nineteen chronic smokers, 18 acute smokers and 18 healthy non-smoking controls were included in the present study. Platelet aggregation was carried out using ristocetin, adenosine diphosphate (ADP) and collagen both before and after smoking in acute and chronic smokers. Flow cytometric studies of platelets were carried out utilizing fluorescein isothiocyanate (FITC)-labelled anti-human fibrinogen antibody in unstimulated and ADP-stimulated platelets, FITC-labelled anti-GP IIb/IIIa antibody, FITC-labelled anti-GP Ib/IX antibody and FITC-labelled P-selectin antibody. The intensity of fluorescence was graded into three groups and expressed in arbitrary units. The interesting data generated in the present work is that in vivo platelet activation occurs immediately after smoking a cigarette which is detected by using FITC-labelled anti-human fibrinogen antibody binding to platelet and by P-selectin expression. It is also quite evident from the present study that a significant number of circulating platelets are in the activated state in chronic smokers. Therefore this study suggests that smoking-induced platelet activation may be an important contributory mechanism for acute coronary events in smokers.

Response to hydroxyurea in β thalassemia major and intermedia: Experience in western India

BACKGROUND The clinical and hematological response to hydroxyurea was evaluated in beta thalassemia patients in western India with variable clinical severity and correlated with genetic factors. MATERIALS AND METHODS Seventy-nine patients-[38-beta thalassemia intermedia-(group I), 41-beta thalassemia major-(group II)] on hydroxyurea therapy were followed-up for 20-24months. RESULTS Among the frequently transfused patients in group I, 58% became transfusion independent and 16% showed a 50% reduction in transfusions after therapy which correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. Forty-one percent of patients in group I had associated alpha-thalassemia and 72.7% were XmnI (+/+). beta thalassemia chromosomes among the responders of group I (41%) were linked to haplotype (- + + - + + - - +) as against haplotype (+ - - - - - - - +) being more common among the non-responders. Response was not linked to the beta thalassemia mutations. Thirty-two percent of group II patients showed a 50% reduction in their transfusion requirements after therapy which also correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. A significant decrease in serum ferritin was seen in both groups. 19% of patients could not tolerate the drug. CONCLUSIONS In group I, clinical response to hydroxyurea was better in patients with alpha-thalassemia, XmnI (+/+) and a higher mean fold increase in gamma mRNA expression. In group II, only one-third of patients showed a partial response.

Hydroxyurea in sickle cell disease--a study of clinico-pharmacological efficacy in the Indian haplotype.

There is clinical variability in the presentation of sickle cell disease among Indians. Vaso-occlusive crisis is common among non-tribal patients. Hydroxyurea, induces fetal hemoglobin (HbF) synthesis and reduces the clinical severity of sickle cell disease but individual patients have a variable response. This study was undertaken to investigate the efficacy and safety of hydroxyurea in Indians with severe manifestations where the beta(s) gene is linked to the Arab-Indian haplotype and is associated with higher HbF levels. Seventy-seven patients (29 adult sickle homozygous, 25 pediatric sickle homozygous, 23 adult sickle beta-thalassemia) selected for hydroxyurea therapy were evaluated for clinical, hematological, biochemical and genetic parameters and were followed for 24 months. Ninety-eight point seven percent of the sickle chromosomes were linked to the Arab-Indian haplotype, 27% of patients had associated alpha thalassemia and 65% were Xmn I +/+. Seventy-eight percent of the patients had no further crises after starting hydroxyurea. This effect was accompanied by a significant increase in HbF (p<0.001), but this increase was variable in individual cases. There was also an increase in gamma gene mRNA expression in the few cases so studied. Hemoglobin levels increased significantly (p<0.001) resulting in the cessation of blood transfusions. Leucopoenia was observed in one patient. Hydroxyurea was effective in reducing the clinical severity in Indian patients who initially had higher HbF levels and the presence of ameliorating factors, such as alpha-thalassemia and the Xmn I polymorphism. Hydroxyurea therapy with careful monitoring can thus change the quality of life of Indians with sickle cell disease.

Development of inhibitors in patients with haemophilia from India

Four hundred and seven patients (352 haemophilia A and 55 haemophilia B) were investigated for the presence of factor VIII and IX inhibitors. Twenty‐four out of 292 severe and two out of 36 moderate haemophilia A patients showed the presence of inhibitors. The mean age at development of inhibitors was 17.7 years (range 6–52 years). In 12 patients the inhibitors were detected due to suboptimal response to factor replacement therapy (symptomatic) and in the remaining 14 patients the inhibitors were detected during the routine screening of the patients’ samples for inhibitors. They had, however, responded well to the usual doses of factor concentrates and there was no suspicion in these patients that they had developed an inhibitor (asymptomatic). There were two families in which the inhibitors were detected in more than one family member. The level of inhibitors in symptomatic patients ranged from 2.2 Bethesda units (BU) mL–1 to 460.6 BU mL–1, and in asymptomatic patients it ranged from 0.8 BU mL–1 to 3.2 BU mL–1. The inhibitors persisted in all patients except one, who developed an inhibitor postoperatively for a brief period of 3 months. All these patients were followed up from first factor exposure and were tested for inhibitors at least twice a year. The mean number of exposure days before they developed inhibitors was 47.5 exposure days (range 17–98 exposure days). No inhibitors appeared after more than 100 exposure days in any of the patients. When 50 consecutive patients were investigated for intron 22 inversions of the factor VIII gene, 17 patients were found to be positive for inversions (10 proximal inversion; seven distal inversion) out of whom four patients developed inhibitors, three patients belonging to the same family. Out of 35 haemophilia B patients, only one patient developed an inhibitor. The overall prevalence of inhibitors was thus 8.2%, which is similar to the reports from western countries, prior to the introduction of highly purified factor concentrate therapy.

Effect of hydroxyurea on the transfusion requirements in patients with severe HbE-β-thalassaemia: a genotypic and phenotypic study

Background Haemoglobin E (HbE)-β-thalassaemia has a very variable clinical presentation. The management of severe cases that are often transfusion dependent is similar to that of cases of β-thalassaemia major; however, this is often not possible in India because of its high cost and the lack of availability of safe blood at many places. Thus there was a need for a drug such as hydroxyurea, which is known to reduce the transfusion requirements of patients with thalassaemia intermedia. This study was undertaken to evaluate the response of Indian patients with HbE-β-thalassaemia to hydroxyurea. Materials and methods 11 patients with HbE-β-thalassaemia receiving regular transfusion plus two less frequently transfused patients were selected for hydroxyurea therapy. Clinical and haematological evaluation was performed before and after treatment for 2 years. Molecular studies included β-globin genotype, β-globin gene haplotype, Xmn I polymorphism and α-genotyping. Results Four clinically severe patients became transfusion independent (responders) after hydroxyurea therapy, four patients showed a reduction in their transfusion requirements (partial responders), and three patients were non-responders. Responders showed a statistically significant increase in Hb, mean corpuscular volume, mean cell Hb, fetal Hb and F cells with a reduction in their transfusion requirements. A reduction in serum ferritin concentration was also seen in responders and non-responders. Conclusions Genetic markers such as Xmn I polymorphism and α-gene deletions were not always beneficial for the response to hydroxyurea therapy. Thus many more markers and a larger cohort need to be studied to predict the response in these patients.

Mutations in GPIIIa molecule as a cause for Glanzmann thrombasthenia in Indian patients

Summary.  Background: Glanzmann thrombasthenia (GT) results from a quantitative or qualitative defect of GPIIb–IIIa complex, the fibrinogen receptor on platelets, which plays a very important role in platelet aggregation. In this report we describe the molecular studies on 22 patients with Glanzmann Thrombasthenia at our institute. Objectives: The main objective was to identify the mutations present in our GT population in order to establish a strategy for genetic counseling and antenatal diagnosis. Methods: Twenty‐two patients with GT were included in the present study. Complete blood count (CBC), platelet aggregation, flow cytometry, Western blot, single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) were performed in all the patients. The patients showing an abnormal migration pattern in SSCP or DGGE were sequenced further on an automated sequencer. Results: Of the 22 patients studied, mutations were detected in 12 individuals. Of these, 11 were novel mutations and one mutation Y115C was reported earlier. Flow cytometric analysis showed the absence of receptors in type I GT, highly reduced levels in type II GT and normal levels in type III GT. The DGGE analysis and SSCP analysis of the patients showed different migration patterns. Sequencing was performed in all patients showing an abnormal migration pattern. Of the 22 cases studied mutations could be detected in 12 cases of GT. We could detect six patients with point mutations, four patients with insertions and five patients with deletion mutations. Exon 4 has been found to be the most common site for mutations in our patients. Conclusion: This study has shown a wide array of mutations present in our GT patients which would be extremely useful in genetic counseling and prenatal diagnosis, essential in preventing these disorders in succeeding generations.

Human platelet alloantigen polymorphism in Glanzmann's thrombasthenia and its impact on the severity of the disease.

Summary. Glanzmanns thrombasthenia (GT) is an autosomal recessive disorder of platelets caused by the deficiency or abnormality of platelet receptors. Several platelet alloantigen systems reside on glycoprotein (GP) IIb and GPIIIa, of which the human platelet antigen 1 (HPA‐1) system is important. Studies have shown that, in the normal population, the HPA‐1b phenotype results in increased platelet aggregation and increased fibrinogen binding, increasing the risk of myocardial infarction. GT produces severe bleeding, but in a subset of patients has a relatively milder course. Forty‐one GT patients and 100 healthy control subjects were genotyped for platelet alloantigens HPA‐1 to HPA‐6, using PCR–ASA (polymerase chain reaction–allele‐specific amplification), and for GPIIb‐IIIa expression and fibrinogen binding using flow cytometric techniques. Platelet alloantigen distributions were similar in the patient and control groups. With the exception of the two HPA‐1b/1b homozygous patients (> 10%), 25 GT patients had less than 5% aggregation to 6 µmol/l ADP, and 16 patients showed between 5% and 10% aggregation to 6 µmol/l ADP. Seven out of 37 patients with HPA‐1a/1a phenotype showed 1–5% fibrinogen binding and GPIIb–IIIa receptors. The two HPA‐1b/1b patients showed 34·6% and 32% fibrinogen binding and > 10% GPIIb–IIIa receptors. This study determined the platelet alloantigen distribution in a large cohort of unrelated GT patients from western India. GT patients homozygous for HPA‐1b/1b had higher levels of platelet aggregation and fibrinogen binding as well as a milder course, as evidenced by infrequent epistaxis and no transfusion requirement to date.

First case of Hb Fontainebleau with sickle haemoglobin and other non-deletional α gene variants identified in neonates during newborn screening for sickle cell disorders

Objective To evaluate the significance of non-deletional α gene variants identified in neonates during newborn screening for sickle cell disorders. Methods 1534 newborn babies were screened in the last 2 years for sickle cell disease using a targeted screening approach. Investigations included a complete blood count, high performance liquid chromatography analysis, cellulose acetate electrophoresis (pH 8.9), heat stability test, restriction digestion and Amplified Refractory Mutation System for confirmation of sickle haemoglobin (Hb S), α genotyping by multiplex PCR and DNA sequencing. Results Three non-deletional α gene variants, Hb Fontainebleau, Hb O Indonesia and Hb Koya Dora, were identified in heterozygous condition in newborns. This is the first report of Hb Fontainebleau in association with Hb S. The baby had anaemia at birth (Hb 11.4 g/dl) with no cyanosis, icterus or need for transfusion. She had occipital encephalocoele and was operated on day 24 to remove the mass. The baby diagnosed with Hb O Indonesia in combination with Hb S also had a low haemoglobin level of 12.7 g/dl. Conclusion Newborn screening for sickle cell disorders also enabled us to identify three α globin chain variants. Two babies who inherited Hb Fontainebleau and Hb O Indonesia along with Hb S had reduced Hb levels at birth and need to be followed up.