Cation Exchange High Performance Liquid Chromatography for Diagnosis of Haemoglobinopathies.

BACKGROUND: Cation exchange high performance liquid chromatography (HPLC) is emerging as the method of choice for initial screening and diagnosis of haemoglobinopathies. The use of alkaline and acid gel electrophoresis in the developing countries may result in incorrect diagnosis of haemoglobinopathies. The aim of the study is to assess the accuracy and precision of diagnosis of haemoglobinopathies by HPLC and its possible advantage over conventional techniques. METHODS: Over a two year period, 955 patients presenting with anaemia were evaluated by HPLC for diagnosis of haemoglobinopathies. All cases showing 'unknown peaks' and other rare haemoglobin variants on HPLC were further analyzed by agar gel electrophoresis at alkaline pH (8.6) and at acid pH (6.0). RESULT: A total of 137 (14.3%) patients showed different abnormal haemoglobins variants. Of these 91 (66.4%) were diagnosed to have beta - heterozygous thalassaemia based on high level of HbA2 (>3.9%), five (3.7%) as beta - homozygous thalassaemia (HbF 25 - 91%), 15 (10.9%) as sickle cell trait, two (1.5%) as compound heterozygous state of sickle - ß(+) thalassaemia and three (2.2%) patients as homozygous sickle cell anaemia (HbSS). One (0.7%) patient had unknown peak on HPLC with retention time of 4.78 minutes, constituting 16.8% of total haemoglobin. Sickling test was negative. He was diagnosed as HbQ - India heterozygous. Thirteen (9.5%) patients were diagnosed as HbE syndrome and were further sub classified as HbE trait (five cases) and HbE disease (eight cases). Seven (5.1%) patients were diagnosed as Hb - D Punjab heterozygous. CONCLUSION: The simplicity of the sample preparation, superior resolution of the method and accurate quantitation of haemoglobin concentration, combined with complete automation, makes this an ideal methodology for diagnosis of haemoglobinopathies.

The Role of anti-HBc IgM in Screening of Blood Donors.

BACKGROUND: Transfusion transmitted hepatitis B has always been a dreaded disease, with incidence of increased transmission through donated blood. The screening test for hepatitis B infection is detection of HBsAg that does not rule out the risk of transmission of hepatitis B as the donor may be in the 'window period'. During this period, detection of the antibody to the hepatitis B core antigen (anti-HBc) IgM type serves as a useful serological marker. The aim of this study was to screen blood donors for anti-HBc type IgM and anti - HBc Ag total for detection and to find their incidence amongst blood donors. METHODS: 2552 voluntary blood donors were screened by the ELISA method for HBsAg and anti - HBc IgM and other mandatory screening markers. 704 of the test blood samples were also screened for anti-HBc total. RESULT: Of the 2552 donor, 47 (1.84 %) cases were HBsAg positive. A total of 11 (0.43 %) blood units were reactive for HBcAg IgM and of these, 10 (0.39 %) were HBsAg negative and reactive for anti-HBcAg IgM. Of the 704 samples tested for anti - HBcAg total, 112 (15.9%) samples were reactive. CONCLUSION: Screening of blood for anti-HBc total is practical in the western world as the incidence of HBsAg and anti-HBc is low in these countries and these positive blood units for anti - HBcAg total can be discarded. This may not be practical in India as the incidence of anti- HBcAg total is high in our population. It is recommended that all blood units should be tested for anti - HBc IgM for infectivity status of the blood donors in the window period and to discard blood if positive.

Role of gel based technique for Coomb's test.

The direct (DAT) and indirect antiglobulin tests (IAT) are one of the most important diagnostic tools used in the investigation of immune mediated disorders. Recently, transfusion laboratories have seen the introduction of column technology in the form of the gel technology (GT). Aim of this study is to compare the conventional tube tests (CTT) and the GT for Coomb's test and to evaluate their sensitivity and specificity. 1656 samples were included in this study, in which 1054 samples were subjected to IAT and 602 samples were subjected to DAT Of the 602 samples tested for DAT, 587 (97.5%) showed concordant DAT results. DAT by the GT could detect 8.6% positivity as compared to 6.1% by CTT. The sensitivity and specificity of the GT was 100% and 97.3% respectively and its negative predictive value was 100%. Among the 1054 samples for IAT, 1041 (98. 8%) showed concordant results. The IAT by the GT showed 6.6% positivity as compared to 5.4% positivity by CTT The sensitivity, specificity and the positive and negative predictive value were 100%, 97.7%, 81.4% and 100% respectively. In conclusion, the GT is a better alternative to the CTT for both DAT and IAT. The GT is highly recommended to be implemented as a routine method of testing in all zonal / regional blood transfusion centers.

Hypercoagulable State.

BACKGROUND: The hypercoagulable state results from a complex interplay of blood coagulation factors, coagulation-inhibitory factors, platelets and the vascular endothelium. Imbalance of the complex interplay between these factors results in thrombosis often complicated by embolism. The causes of thrombosis are varied and maybe congenital or acquired. The current interest is centered on the congenital deficiency of coagulation inhibitors as there is an increasing awareness of their involvement in thrombosis, especially in the young. METHODS: A total of 42 patients with thrombosis were studied. The most common clinical presentation was deep vein thrombosis. All the cases were evaluated for coagulation inhibitors Antithrombin, resistance to activated protein C, Protein C and Protein S using standard assay kits. RESULTS: Resistance to activated protein C (n=10) was seen to be the commonest cause of thrombophilia. This was followed by deficiency of Antithrombin (n-4), Protein C (n=3) and Protein S (n=2). Majority of our cases were in the third decade of life. CONCLUSION: The identification of the underlying aetiology is important for instituting specific therapy and patient management.

Leucodepletion and Blood Products.

BACKGROUND: Leucoreduction of blood products is increasingly being employed to produce blood products with residual WBCs < 5 × 10(6) per unit (99.9 percent or a log 3 leucoreduction). Clinical data suggests that non-haemolytic febrile transfusion reactions can be prevented by leucodepletion. The procedure also prevents alloimmunisation to HLA antigens in patients who will repeatedly require transfusion of blood/blood products. METHOD: Of the methods available to reduce the number of WBC in blood products washing of red cells, freezing and deglycerolisation are effective and yield a product with only a 24 hour shelf life. Other methods such as leucodepletion filters are relatively inexpensive, simple and the final product has a normal shelf life. Modern generation of leucoreduction filters and apheresis machines can provide greater than 4 log reduction of WBC. RESULTS: After the introduction of leucodepletion of blood for Thalassemics at our center in 2003, the incidence of non haemolytic febrile transfusion reactions (NHFTR) fell from 4% in 2002 to 1% in 2003. CONCLUSION: In patients undergoing long-term blood transfusion therapy e.g. Thalassemics, alloimmunisation against the HLA antigens on donor white cells is prevented by leucodepletion and prevents NHFTRs.

Transfusion Transmitted Infections in Armed Forces: Prevalence and Trends.

BACKGROUND: This study presents data on the prevalence rate of infectious markers among voluntary and replacement donors in the blood transfusion service in Armed Forces from 2000 to 2004. METHODS: 39,646 units of blood were collected from donors during the period from 2000 to 2004. All the samples were screened for hepatitis B surface antigen (HBsAg), human immunodeficiency virus (HIV) 1&2, hepatitis C virus (HCV), and by venereal disease research laboratory test (VDRL). RESULTS: 24,527 (61.9%) were voluntary donations and 15,119 (38.1%) replacement donations. Prevalence of HBsAg had decreased, amongst voluntary donors from 1.67% to 0.77% but the positivity rate has not showed significant change. Seropositivity of HIV had decreased both in voluntary and replacement donors to 0.22% and 0.86% respectively. The seropositivity for anti-HCV showed steady decrease amongst voluntary donors from 0.46% to 0.20% in 2004, but in replacement donors, there was an increase in reactivity rate from 0.43% to 0.65%. CONCLUSION: The increased seropositivity for HCV, HIV and HBsAg could be decreased by introduction of nucleic acid amplification testing (NAT) in minipools for HCV and HIV and introduction of anti-HBcAg (IgM) for hepatitis B virus (HBV) infection. But this may not be possible in near future in developing countries due to financial constraints. At present implementation of strict donor criteria and with use of sensitive laboratory screening tests it is possible to reduce the incidence of transfusion transmitted infections (TTI) in Indian scenario.

Gene conversions are a common cause of von Willebrand disease.

von Willebrand disease (VWD), the most common inherited bleeding disorder, is very heterogeneous, both in its phenotype and genotype. One particular molecular mechanism of VWD is due to recombination events between the true gene and its pseudogene on chromosome 22. We assessed the frequency and extension of such events in 50 multi-ethnic index patients with severe VWD type 3 and in five index patients with VWD type 2M Vicenza. One additional unclassified patient had been diagnosed with possible VWD in Russia solely on a clinical basis. Gene conversions, previously thought to be rare events, were identified in >10% of our study population: in six multi-ethnic patients with severe VWD type 3, in one patient with VWD type 2M Vicenza and the Russian patient was finally diagnosed with VWD type 2B New York/Malmoe. Our results suggest a significant contribution of this particular molecular mechanism to the manifestation of VWD. The location of the gene conversions, their extension and their occurrence as homozygous, compound heterozygous or heterozygous mutations determines the resulting phenotype.

Difficulties in Immunohaematology : The Weak D Antigen.

BACKGROUND: The Rh blood system is one of the most polymorphic and immunogenic systems known to humans. The expression of Rh blood group antigen is complex. The Rh D antigen is the most important of the antigens that constitute the Rh antigen system. In most cases, D antigen can easily be detected. However, due to variability of expression, weak forms antigen are encountered. The reactivity of weak D with antisera is variable and presents as a problem in blood banking. METHODS: A retrospective analysis for a five-year period was done. Blood samples that were negative for Rh D by immediate spin tube method were tested for weak D antigen by additional lab tests. RESULT: Of 34932 serial Rh grouping tests done in our Blood Bank, the incidence of weak D Rh antigen was 0.189%. All these were confirmed by the antiglobulin test. CONCLUSION: These patients present as a problem for the blood banker and a curiosity to the clinician. Although uncommon, all health care workers should be aware of this entity to avoid anti D alloimmunisation.

Cytokines : as useful Prognostic Markers in Lymphoma Cases.

Cytokines are believed to be involved in the pathogenesis and enhanced expression in patients with Hodgkin's and Non-Hodgkins lymphoma. Based on this phenomenon, a multicentric study was carried out in various lymphoma cases. The diagnosis of lymphoma was made on tissue biopsies and fine needle aspiration cytology (FNAC). Out of a total of 72 cases studied, 45 were of Hodgkin's lymphoma (62.5%) and 27 cases were of Non-Hodgkin's lymphoma (37.5%). Maximum cases of Hodgkin's disease occurred in the age group of 30-40 years and males outnumbered females. Hodgkin's lymphoma cases were predominantly of mixed cellularity histologic type (46.66%) whereas majority cases of Non-Hodgkin's lymphoma were of high grade histologic type (48.14%) with predominance in the age group 51-60 years. In both these type of lymphomas, the IL-2R and IL-6 levels were found to be increased more than four fold (as compared to healthy controls) (p<0.05). The cytokine levels decreased after chemotherapy in patients showing response to therapy. However, there were few conflicting and unreliable trends in the IL-6 levels after chemotherapy where elevated IL-6 levels persisted in patients in clinical remission. Overall, it was seen that both IL-2R and IL-6 can be used as an indicator for assessing prognosis and drug therapy in lymphoma cases. IL-2R was found to be a better prognostic marker than IL-6 in assessing the response of lymphoma patient to chemotherapy, more so in Hodgkin's disease.

A rare germ-cell tumor site: vaginal endodermal sinus tumor.

Malignant germ-cell tumors (MGCT) are rare tumors of childhood accounting for less than 3% of pediatric malignancies. Endodermal sinus tumor (EST) forms the most common histologic subtype of MGCT. The vagina is an extremely rare site for GCTs. A 9-month-old female was admitted with a short history of vaginal bleeding, a mass protruding from the vagina, and difficulty in passing urine. She was pale, the bladder was full, and a mass was palpable anteriorly on rectal examination. Ultrasound showed an ovoid, hyperechoic mass posterior to the bladder. A biopsy revealed a vaginal EST. The serum alpha-fetoprotein (AFP) was elevated partial vaginectomy was done and the tumor was excised in toto. The patient was subsequently given six courses of cis-platinum, etoposide, and bleomycin and on follow-up continues to be free from disease. EST is the most common GCT in children. In females, it is usually encountered in the ovary. EST of the vagina is a rare, highly malignant GCT that exclusively involves children less than 3 years of age. The diagnosis is based on histology and raised AFP. Vaginal EST is both locally aggressive and capable of metastasis. Untreated patients have died within 2 to 4 months of presentation. Radical surgery leads to a loss of sexual and reproductive function. Long-term irradiation has secondary effects of sterility, aseptic necrosis of the femoral head, and abnormal growth of the pelvic bones. Partial vaginectomy with combination chemotherapy is the most recommended line of treatment. The surgery eradicates local tumor cells and makes subsequent chemotherapy more effective. Simple tumor excision is not sufficient, as residual tumor cells induce early recurrence and make chemotherapy ineffective. The serum AFP level is a useful marker for diagnosis and monitoring the recurrence of vaginal EST in infants.

IMMUNOBIOLOGICAL MONITORING OF VARIOUS GASTROINTESTINAL AND PRIMARY HEPATIC MALIGNANCIES.

Carcinoembryonic antigen (CEA) and alpha fetoprotein levels (AFP) were assayed by enzyme-labelled immunoassay in 75 cases of gastrointestinal (GI) and primary hepatic malignancies. Mean CEA levels were found to be significantly higher (p<0.005) in metastatic non-operative group of GI malignancies (range 5.32 ng/mL to 175.2 ng/mL) as compared to early pre-operative cases (range 1.25 ng/mL to 33.2 ng/mL) and post-operative cases (range 1.41 ng/mL to 22.24 ng/mL). Variable levels of AFP were visualised in 12 cases of primary hepatic malignancies with eight cases having values less than 100 ng/mL. When both CEA and AFP were assayed simultaneously, the markers were helpful in differentiating cases of primary hepatic malignancies with low levels of CEA from 3 cases of secondaries in the liver where CEA levels were found to be highly raised (80.4 ng/mL to 146.4 ng/mL). To evaluate the variation of CEA and AFP levels in different patients having same stage of the disease, immunological monitoring for the functions of T and B cells was carried out by estimation of cytokine, i.e. interleukin-1 alpha (IL-1a), interleukin-2R (II-2R) and various immunoglobulins. IL-1a and 1L-2R levels were significantly higher (p<0.05) in the groups of patients having higher CEA and AFP. This indicates an important T cell (TH1 and TH2) function in the tumour antigen production.

FIBRIN AND FIBRINOGEN DEGRADATION PRODUCTS IN MALIGNANCY.

Sixty four patients of cancer, comprising of 39 leukaemia, 8 lymphomas and 17 cases of solid tumours were included in this study. Quantitative estimation of FDP, fibrinogen and platelets were done in all. Elevated levels of FDP (≥ 10 µgm/mL) were found in 29 patients. These patients were further categorised as decompensated, overcompensated and compensated intravascular coagulation and fibrinolytic syndrome on the basis of platelet counts and fibrinogen levels.

HISTAMINE RELEASE AND CHANDONIUM IODIDE.

Histamine releasing activity of chandonium iodide and d-tubocurarine was studied in guinea pig ileum (in vitro), guinea pig peritoneum (in vivo) and in human volunteers (in vivo) by intradermal testing at dilutions of 1:1,000 and 1:10,000 of the clinical dosage of drugs. d-tubocurarine revealed significant histamine releasing activity in in vivo animal experiments and high incidence of positive intradermal reaction, 53.5 per cent and 13.5 per cent, in human volunteers. Chandonium iodide neither showed histamine releasing activity in animals nor in man. In clinical terms, chandonium iodide appears to be a safe neuromuscular blocking agent.

A clinical study of adult leukaemias.

The incidence of adult leukaemias, their response to therapy and the complications of therapy were studied in 121 cases over seven years (1981-1987). All cases were followed up till recovery or death for periods ranging from seven days to seven years. Adult leukaemias accounted for 2.56% of all admissions due to malignancies. There were 21 cases of acute lymphoblastic leukaemia, 61 of acute myelogenous leukaemia, 36 of chronic myelocytic leukaemia and 3 chronic lymphocytic leukaemia. All received aggressive combination chemotherapy. Remission could be achieved in 57% to 60% of cases. Infection (34%), bleeding (34%), and central nervous system involvement (25%) were the complications during therapy. The cause of death was ascertained in 87 of 90 deaths by a detailed postmortem. Haemorrhage (34.5%), infection (31%) and uncontrolled leukaemia (22%) were the leading causes, either singly or in combination. Some of the uncommon causes of death were fulminant hepatic failure, coronary artery disease, gangrene of the colon and disseminated tuberculosis.

Aggressive angiofollicular lymph node hyperplasia (Castleman's disease) treated with high dose melphalan and autologous bone marrow transplantation.

A case of aggressive widespread angiofollicular lymph node hyperplasia in a 42-year-old male, treated with high dose melphalan is presented. The disease had failed to enter a durable remission after chemotherapy. High dose melphalan with autologous bone marrow transplantation achieved a complete remission which has lasted for 15 months to date. This approach can be considered when other measures fail.