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Background: Cold agglutinins are IgM autoantibodies, which are often detected incidentally. They are rarely pathogenic. Natural cold autoantibodies occur in titers less than 1:64 measured at 4°C and have no activity at temperature higher than 22°C, However pathogenic cold agglutinins causing haemolysis typically have titers well over 1:1000 and may react at 28-31°C or even upto 37°C (wide thermal amplitude). Case Report: We encountered a case of 62-year-old male presenting to Urology department of our hospital in January 2007 with the complains of recurrent haematuria, symptoms of obstruction in the lower urinary tract and progressive weakness. He was subsequently diagnosed as a case of cold agglutinin causing haemolysis. The case came to notice because of the difficulty in carrying out routine investigations in the laboratory due to repeated agglutination and haemolysis. Investigations: • Complete haemogram, peripheral smear examination and blood grouping was done. All the investigations were done while keeping the blood at 37°C. • Direct and indirect Coombs test. Routine urine microscopy. • Biochemical investigations include total and direct bilirubin AST/ALT, Urea, creatinine and LDH. Results: • Macroscopic agglutination was visible in the blood collection tubes. • Hb was 5.6 g% with normal WBCs and platelets count. Corrected reticulocyte count was raised (5%). • Biochemistry indirect hyper bilirubinaemia, increased LDH, normal AST/ALT and creatinine. • Urine was positive for Hb and haemosiderin and showed no RBC in sediments. • X-ray chest was normal. • The serum of the patient could agglutinate all allo-RBC.s in a titer >1:1000 and at a temperature range from 4 to 37°C. • Coombs test was reported as positive for C3d and agglutinins showed anti-I reactivity. Discussion and Conclusion: • Patient was diagnosed to have primary cold agglutinin disease on the basis of the positive Coombs test against C3d and anti-I reactivity. There was no clinical evidence of any other underlying disease. • Patient was refractory to corticosteroids and subsequently managed by Cyclophosphamide with a good response. The case high lights the need for laboratory personnel to be aware of this condition so the clinician can be alerted about this possibility and timely diagnosis made. [ABSTRACT FROM AUTHOR]

Background & Objectives: Although the outcome of children with acute lymphoblastic leukaemia (ALL) has improved dramatically over the last decade, some children still fare poorly and relapses are seen. The sensitivity of leukaemic cells to corticosteroids has emerged as an important prognostic factor in ALL. The t(9,22) translocation, resulting in the bcr-abl fusion gene, is a non-random translocation found in B-lineage acute lymphoblastic leukaemia. It is also known to be an independent poor prognostic factor for long-term disease free survival. We studied the association between the presence of bcr-abl fusion gene and in vitro prednisolone resistance in children with B-lineage acute lymphoblastic leukaemia at diagnosis., Methods: A total of 23 children (aged 1-16 yr, median age: 12 yr) with B-lineage acute lymphoblastic leukaemia at diagnosis were included in the study. The presence of bcr-abl fusion gene was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and the in-vitro resistance to prednisolone was measured by short term colorimetric methyl thiazol tetrazolium (MTT) assay., Results: A median LD50 (lethal dose for 50% cells) for prednisolone in bcr-abl positive children (n=7) was 1.6 mg/ml (range: 0.25-5.0 mg/ml) and that of bcr-abl negative children (n=16) was 0.35 mg/ml (range 0.62-1.0 mg/ml). The median LD50 for prednisolone differed significantly between the bcr-abl positive and negative groups of children with acute lymphoblastic leukaemia (P<0.005)., Interpretation & Conclusion: This is probably the first report to show that leukaemic blasts of bcr-abl positive children with ALL are about four-fold resistant to prednisolone as compared to blasts from bcr-abl negative children. This suggests that one of the reasons for the poor prognosis of bcr-abl positive ALL could be a lower steroid sensitivity.

Background & Objectives: Drug sensitivity assays are useful in oncology practice for evaluating the sensitivity of malignant cells to anti-cancer drugs. The usefulness of such assays for the prediction of clinical response to therapy has also been demonstrated. The existing methods used for this purpose are time consuming and labour intensive. Here we report a simplified flow cytometry based assay for evaluating the in vitro drug sensitivity of leukaemic cells., Methods: The chemo-sensitivity of three human leukaemic cell lines (a lymphoblastoid cell line, Jurkat; an erythroleukaemic cell line, K 562 and a myelomonocytic cell line HL-60) was investigated by flow cytometry. Flow cytometry was used to determine LD50 (50% inhibitory concentration) for prednisolone on Jurkat and daunorubicin on HL 60 and K 562 cell lines respectively. Per cent cell death could directly be assessed on a flow cytometer by measuring the fluorescence after staining with propidium iodide (PI). For comparison MTT assay was also performed using prednisolone on Jurkat and daunorubicin on HL-60., Results: Cytotoxic effect of drugs was found to be dose dependent. Mean LD50 of prednisolone for Jurkat cells by flow cytometry was 0.805 +/- 0.058 mg/ml and by MTT assay 0.866 +/- 0.115 mg/ml. Mean LD50 of daunorubicin for HL-60 was 1.96 +/- 0.05 micrograms/ml by flow cytometry and 1.90 +/- 0.282 micrograms/ml by MTT assay. The mean LD50 of daunorubicin to K 562 was 0.49 +/- 0.049 mg/ml by the flow cytometry method. The inter-assay variation for the LD50 by flow cytometry based assay was found to be 6, 14 and 10 per cent for Jurkat, HL-60 and K 562 respectively., Interpretation & Conclusion: We report a flow cytometry based drug-sensitivity assay for leukaemic cells, which uses a single dye staining and is rapid, technically simple and reproducible. The results compare well with the more commonly used MTT assay, which is labour intensive and time consuming. The limitation of our method is that it can only be used for studying cells in suspension and is therefore not suitable for adherent cell lines.