M. B. Van't Veer

Cyclosporin A for the treatment of patients with chronic idiopathic thrombocytopenic purpura refractory to corticosteroids or splenectomy

Patients with chronic immune thrombocytopenic purpura (ITP) who are unresponsive to corticosteroids require splenectomy, but if this fails, treatment is difficult. We tried to induce durable remissions in ITP patients refractory to corticosteroids before or after splenectomy by applying strong immunosuppression with the combination of cyclosporin A (CyA 5 mg/kg/d) and prednisone (0·4 mg/kg/d). Patients were assigned to one of two groups. Group 1, 10 patients refractory to prednisone; and group 2, 10 patients refractory to at least prednisone and splenectomy. Overall response rate was 55% (50% in group 1 and 60% in group 2 patients). Nine of the 10 patients in group 1 finally had a splenectomy because of relapse, insufficient response or toxicity of CyA. Thirty percent of the patients discontinued CyA because of side‐effects; hypertension, severe headache and muscle pain being the most frequent encountered. It is concluded that CyA treatment does not avoid, but only postpones, splenectomy in chronic ITP patients who are refractory to corticosteroids. However, CyA can be useful in a subgroup of patients with corticosteroid‐ and splenectomy‐refractory ITP, but treatment toxicity is high.

Intensive postremission chemotherapy without maintenance therapy in adults with acute lymphoblastic leukemia. Dutch Hemato-Oncology Research Group.

PURPOSE To investigate the value of intensive consolidation chemotherapy not followed by maintenance therapy in adult acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS A multicenter phase II trial was conducted in 130 adult patients with ALL between 16 and 60 years of age. After standard induction therapy, postinduction chemotherapy was given: three courses of high-dose cytarabine (2,000 mg/m2 every 12 hours for four doses) in combination with amsacrine (course one), mitoxantrone (course two), and etoposide (course three). CNS prophylaxis consisted of 10 injections of intrathecal methotrexate (IT MTX). Patients younger than 50 years with an HLA-identical sibling were eligible to receive allogeneic bone marrow transplantation (BMT). RESULTS Ninety-five patients (73%) achieved complete remission (CR); 82% were younger than 50 years and 41% were older than 50 years. Seventeen patients (13%) were resistant to chemotherapy, and 18 (14%) died during induction treatment. Only age and performance status were significantly associated with response (P<.001 and .03, respectively). Death during consolidation occurred in four patients. The estimated 5-year overall survival (OS) was 22% for the entire group and 26% for patients younger than 35 years. Disease-free survival (DFS) at 5 years was 28% +/- 6 for patients younger than 35 years, 25% +/- 9 for patients between 35 and 50 years, and 0% for patients older than 50 years. Increasing age (P<.01) and expression of CD34 (P<.01) were adverse factors. Only three patients (3%) developed an isolated CNS relapse. CONCLUSION Intensive consolidation including high-dose cytarabine not followed by maintenance therapy provides an outcome for adult patients with ALL that may be worse or even inferior compared with studies using long-term maintenance therapy. High-dose cytarabine in combination with IT MTX was effective for CNS prophylaxis.

Phenotypic and karyotypic properties of hyperdiploid acute lymphoblastic leukaemia of childhood

Summary. The DNA/cell content was measured by flow cytometry in samples obtained from 98 unselected children with acute lymphocytic leukaemia (ALL) at diagnosis. The frequency of anomalies in modal DNA content was compared to that encountered in acute childhood non‐lymphocytic leukaemia (ANLL) and disseminated non‐Hodgkins lymphoma (NHL). In ALL the most frequent (35%) aberration in DNA content was an increase by 20% relative to the modal value of normal white blood cells. This subcategory, referred to as hyperdiploid ALL (HD‐ALL), was characterized by a close association with the expression of the c‐ALL surface marker (20/20 patients) and characteristic numerical chromosome changes, including tri‐ or tetrasomy of chromosome 21. Moreover, patients with hyperdiploid ALL had a much lower peripheral leucocyte count (P= 0.001) than those with diploid disease and a varying proportion of their leukaemic cells existed in the peripheral blood as morphologically normal lymphocytes expressing the c‐ALL antigen. Within the standard risk category, patients with HD‐ALL had a longer disease‐free survival than those with diploid disease (P= 0.058). It is concluded that routine analysis by flow cytometry can conveniently and consistently detect ALL patients with hyperdiploid chromosome numbers. Hyperdiploid ALL constitutes a fairly large subtype of childhood ALL with specific biological and karyotypic properties, possibly associated with favourable prognosis.

Quality assessment of immunological marker analysis and the immunological diagnosis in leukaemia and lymphoma: a multi-centre study

Summary In order to standardize and assess the quality of immunophenotyping of leukaemias and lymphomas for diagnostic purposes, a cooperative study group in the Netherlands, SIHON, has formulated guidelines for the composition of antibody panels to be applied and guidelines for the interpretation of the marker analysis. To assess the value of these guidelines frozen cell samples of three patients with different haematological malignancies were sent to the 26 participating laboratories twice a year. Here we present the results with respect to the marker analysis and to the immunological diagnosis on 387 samples from 18 patients. A large inter‐laboratory variation was seen in the percentage of positive cells for each marker, which influenced the valuation of a marker to be discordant positive in up to 23% and discordant negative in up to 40%. No single major factor could be traced to explain the large variation in the results. However, probably due to the balanced composition of the antibody panel and to the application of the guidelines for interpretation, this variation did not much influence the agreement in immunological diagnosis. In only 13/387 samples (3.3%) differences in the percentage of positive cells caused disagreement in the final diagnosis. In 23 samples (5.9%) the disagreement was due to an incorrect application of the guidelines. Quantitative data of single observations obtained from different laboratories, in which the materials and methods are not standardized, cannot be compared; but standardization of guidelines for marker sets and for interpretation contributes to a high grade of agreement in immunological diagnosis.

A Negative Direct Antiglobulin Test with Strong IgG Red Cell Autoantibodies Present in the Serum of a Patient with Autoimmune Haemolytic Anaemia

Summary. The case is presented of a boy with an autoimmune haemolytic anaemia of 10 years duration. He had a positive direct antiglobulin test with IgG and complement detected on the red cells and with IgG autoantibodies in the serum. During a recent episode of severe haemolysis, the Hb level fell to 3.8 g/dl and the direct antiglobulin test became negative although his autoantibodies still reacted with all the red cells in a panel. The serum reacted more strongly with C‐ and e‐positive cells. The Rh phenotype of the patient was CcDee as it had always been. Possible explanations of the unexpected findings are discussed.

Pitfalls in the immunophenotyping of leukaemia and leukaemic lymphomas: survey of 9 years of quality control in The Netherlands

During the last decade, biannual quality controls were performed in The Netherlands focusing on the immunophenotyping of leukaemic haematological malignancies. All results on 48 specimens obtained by 18–34 laboratories were analysed. The interlaboratory variability and percentages of discordant results from 30 markers were measured by assessing false positive or negative (cut‐off 10%) results in comparison with median results of the group. The quality of the immunophenotypic diagnoses obtained from the interpretation of these markers in relation to clinical data was evaluated by scoring them as ‘correct’, ‘minor fault’, ‘major fault’, ‘not based upon the markers used’, and ‘no diagnosis’. CD3, CD8, CD19, CD61 and Smλ had the lowest percentage discordancy (sum of total negative and positive discordant values 5–7.5% of assays); CD13, CD15, cyCD22, CD33 and TdT scored worst with 14–20% cumulative discordancy. The analysis of each diagnosis yielded 78% acceptable immunophenotypic conclusions (correct 54% and minor fault 24%). It appeared that the major faults in immunophenotyping were caused by suboptimal antibody selection and erroneous interpretation of the results obtained, rather than by technical errors. Large differences per diagnostic category were observed, with the best scores for mature B‐cell leukaemias, AMLs and common‐ALL, and the poorest scores for T‐cell malignancies which were correctly diagnosed in only 24–60% of specimens. Mature T‐NHL and T‐PLL were mistakenly diagnosed as T‐ALL by 40% of the centres. Misinterpretation of TdT immunofluorescence or omitting this marker contributed significantly to these wrong diagnoses. A median of 4% of immunophenotypic diagnoses were not based on a correct panel of antibodies, but upon the morphology of the accompanying blood smear, and was often flawed by overinterpretation.

Acute lymphoblastic leukaemia in adults: immunological subtypes and clinical features at presentation

SummaryIn 91 of 106 adult patients with acute lymphoblastic leukemia (ALL) enrolled in the treatment protocol ALL HOVON-5 between May 1988 and October 1991, the immunophenotype of the leukemia was determined and correlated with clinical characteristics at presentation. The immunological marker analysis was performed in ten laboratories, all members of the Dutch Study Group on Immunophenotyping of Leukemias and Lymphomas (SIHON). Undifferentiated blasts were found in four patients, 67 had B-lineage ALL, 18 had T-lineage ALL, and two had biphenotypic ALL. The age of T-lineage ALL patients was lower (mean 29.3) than that of B-lineage ALL patients (mean 35.5). Tumor mass, as expressed by leukocyte count, organomegaly, and LDH, was more pronounced in T-lineage ALL. Hemoglobin and platelet count was similar in all (sub)types. CD34 was expressed in 58% of the leukemias, but most frequently in the common B-ALL (70%). Thirteen percent of the leukemias expressed one or more markers not associated with their lineage. In this prospective study immunological data were not evaluable for 15 patients. On four of them data were not available because of dry tap, for six patients the typing was technically insufficient, and for four patients the results were unclassifiable; with one patient the marker analysis was not performed.

SIHONSCORE: a scoring system for external quality control of leukaemia/lymphoma immunophenotyping measuring all analytical phases of laboratory performance.

For the diagnosis of leukaemia and leukaemic lymphoma, clinicians frequently have to rely on the results of immunophenotyping. To improve the quality of these results, the Dutch Foundation for Immunophenotyping of Haematological Malignancies (SIHON) initiated external quality rounds in 1986. Over a period of more than 10 years, this has led to improvements in the interpretation of immunophenotyping results. However, the evaluation of results focused mainly on the correctness of the interpretation of the immunophenotypical data, leaving the preceding analytical phases unevaluated. Therefore, in 1996 SIHON developed a more comprehensive scoring system, called SIHONSCORE, covering all three phases of immunophenotyping, namely the pre‐analytical (i.e. choice of the staining panels), analytical (i.e. the technical part consisting of sample preparation, data acquisition and analysis) and the post‐analytical phase (i.e. the interpretation) of the laboratory process. Here, we report how SIHONSCORE was successfully applied to three consecutive external quality rounds consisting of a total of nine different cases tested. For laboratory certification, participation in external quality control programmes is required. Evidently, criteria are needed to define the minimum acceptable performance of a certified laboratory. With SIHONSCORE, a useful instrument is obtained evaluating all phases of the performance of laboratories in leukaemia and lymphoma immunophenotyping.

Red-Cell Auto-Antibodies Mimicking Anti-Fyb Specificity

Abstract. An antibody is described, produced by an Fy(a+b‐) woman that at first reacted with her own cells, preferentially with Fy(b+) cells but not with Fy(a‐b‐) cells. Later, pure anti‐Fyb specificity occurred. Elution and absorption studies revealed that the antibody recognized a determinant absent from Fy(a‐b‐) cells but present on Fy(a+b‐) and Fy(a‐b+) cells, much more strongly, however, on the latter, and that it was an autoantibody.

Murine Monoclonal Anti-P

Monoclonal antibodies (mAbs) reactive with human red cells may show specificity for the antigens of various human blood group systems, such as the blood group ABH, Le, MN system (1). Because of their strength, purity, and uniform quality they are excellent tools for serological studies as well as for more basic hematological and biochemical studies on blood group antigens.