Search

The diagnosis of plasma cell leukaemia, a rare disorder with an aggressive clinical course and poor prognosis, is not always straightforward and may be dependent on the results of immunophenotyping. Samples from two cases of plasma cell leukaemia have been issued by the UK NEQAS for Leucocyte Immunophenotyping Scheme during the last 5 years and on each occasion a significant number of laboratories failed to make the correct diagnosis. The details of the two samples issued and the results of both surveys are presented. The data highlights the need to adhere to guidelines for immunophenotyping, with respect to using the correct antibody panels, the importance of data interpretation in conjunction with morphological appearance as well as the need to participate in external quality assurance schemes.

Leucocyte counts of < 5 x 106 per blood transfusion product are currently recommended in the UK in order to reduce transfusion-related infections and febrile reactions. Routine leucocyte depletion, however, requires the development of reliable internal and external quality assurance (EQA) programmes. We report preliminary findings from the UK NEQAS for Low-Level Leucocyte Counting from 18 UK Transfusion Centres over a four month period. Data analysis showed that the IMAGN 2000 had the lowest CVs (range 7.5-36%, mean 16.7) for samples with counts of 5-30 cells/microl when compared to the flow cytometric (range 13.8-88%, mean 29.5) and Nageotte methods (range 20.6-117%, mean 61.8). In addition, laboratories using commercial nuclear stains (LeucoCOUNTTM) had consistently lower CVs than those using 'in-house' propidium iodide staining methods. Important differences in flow cytometric gating strategies were also identified. This study highlights the current variability in low level leucocyte counting, especially within the critical range of 5-30 cells/microl (equating to < 5 x 106/l). The acceptance of consensus protocols, including gating strategies and nuclear staining techniques, is required to reduce the observed interlaboratory variation. Finally, we demonstrate that stabilized blood preparations can be successfully used to provide a national/international low-level leucocyte EQA scheme.

We report the development of painful scrotal ulceration in two patients during treatment with all-trans-retinoic acid (ATRA) for acute promyelocytic leukaemia (APL). ATRA 45 mg/m2 was administered orally for 8 days prior to the addition of standard induction chemotherapy. Painful scrotal ulceration developed in both cases within 2 weeks of therapy (9 and 13 days) and responded slowly to drug withdrawal and systemic, or topical, corticosteroids. A total of 17 APL patients have been treated with ATRA at our institution during the last 10 years, giving an incidence of approximately 12%. The present report, together with a review of literature, suggests that scrotal ulceration is a specific adverse effect of ATRA therapy and that this complication may be more common than previously documented.

We describe two cases of autoimmune thrombocytopenia precipitated by fludarabine therapy in patients with chronic lymphatic leukaemia. Both were treated with high dose steroids and initially responded with recovery of normal platelet counts. One patient developed recurrent autoimmune thrombocytopenia on two occasions following re-exposure to the drug when his disease had become refractory to all other treatments. A retrospective review of the case notes of 45 patients with lymphoproliferative disorders treated with fludarabine over the past 6 years indicated the development of autoimmune thrombocytopenia in 4.5% (two out of 45) and autoimmune haemolytic anaemia in 6.7% (three of the 45).

As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.

The flow cytometric determination of antigen density, or cellular antibody binding capacity, is now an accepted technique for the characterization of cells in health and disease. In HIV infection, for example, antigen density changes in CD38 expression may be an important indicator of disease progression. Our experience of using one such method, Quantum Simply Cellular, which measures antibody binding capacity (ABC), has highlighted several technical factors which can affect the results. We report the influence of pH, incubation temperature and time, antibody fluorochrome and titre, as well as lysing reagent (FACS Lysing Solution v. Ortho-mune Lysing Reagent) on the ABC of anti-CD3, CD4 and CD8 of normal lymphocytes. In addition, the effect of single, double or triple-staining was assessed. The results indicate that the ABC values are influenced by all the variables studied. The pH range tested (6.0-9.0) demonstrated that pH 7.4 gave maximal binding. Furthermore, temperature also influenced the pH of the two lysing solutions, and thus potentially the ABC. Antibody concentration, fluorochrome and staining technique are also important factors with an observed difference of up to 458,855 ABC between the various fluorochromes. In addition a maximal difference of 130,119 ABC was observed between single and triple staining techniques. In conclusion, if antigen quantification is to be used in the clinical setting, an internationally standardized method is required to ensure the reproducibility of results from centre to centre. Our data suggests that single staining, using fluorescein isothiocynate (FITC) conjugated antibodies with all reagents at pH 7.4 + 0.1, with incubation and lysing carried out at 20 + 1 degrees C, could be used as a 'benchmark' method for ABC determination using the QSC system.

Urate oxidase (uricozyme) is an enzyme of non-human origin capable of oxidizing human uric acid to allantoin, a highly soluble product at renal tubule pH. We report its efficacy in three patients with acute urate nephropathy due to tumour lysis in chronic lymphatic leukaemia and high grade lymphoma. Two patients had an additional obstructive nephropathy due to ureteric urate crystals. An intravenous infusion (100 units/kg in 50 ml saline over 30 min) was given for between two and five consecutive days. All patients showed a rapid fall in serum urate levels with associated diuresis, correction of metabolic disturbance and full resolution of uraemia within a week. The treatment was well tolerated and caused a rapid resolution of clinical symptoms in all cases. We review the literature relating to the use of this agent both in the treatment of hyperuricaemic acute renal failure and gouty arthritis.

We have characterized the first case of Rhnull phenotype to be identified in England. The red cells were serologically negative for C, c, Cw, D, E, e, f, hr B, Rh17, Lw a, Lw ab and Duclos, while the patient's serum contained anti-Rh29, which was subsequently boosted by transfusion. The Rh phenotype of the patient's son (R1r) confirmed that this was a regulator type of Rhnull in the patient. Follow up studies confirmed the presence of a mild chronic anaemia with stomatocytes and spherocytes; electron microscopy revealed the presence of cells with deep central indentations. Osmotic fragility was increased to a level intermediate between normal and hereditary spherocytic controls. The presence of ongoing haemolysis was indicated by a mild reticulocytosis and splenomegaly. The potent anti-Rh29 has made the provision of compatible blood difficult and autologous units have been frozen. The case illustrates the rare phenomenon of the Rhnull phenotype which not only causes problems for the transfusionist but should also be recognized as a cause of haemolytic anaemia secondary to a membrane defect. Blood film and Rh phenotyping are useful preliminary investigations in suspected cases.

The diagnosis of haematopoietic disorders by immunological methods, termed 'immunophenotyping', or 'cell marker analysis', is now a routine investigation in many haematology laboratories. A wide range of antibodies and fluorochromes are commercially available, in addition to antigen detection systems: immunocytochemistry, direct or indirect immunofluorescence, incorporating either light microscopy or flow cytometry. In view of this variety in methodology, it is important that all antibodies are used appropriately. The following review highlights a variety of issues, including antibody selection and dilution, negative controls, storage, fixatives as well as quality control, that require consideration prior to the routine use of antibody reagents.

The CD20 antigen has been regarded as a B lineage specific, 35 kDa, non-glycosylated membrane phosphoprotein, which functions as either a Ca2+ ion channel or as a regulatory protein of such a channel. Weak expression of CD20 (CD20dim), however, has recently been reported on a sub-population of T lymphocytes. We present results which confirm the existence of a CD20dim T lymphocyte population and show that such cells have a reduced antibody-binding capacity, when compared to CD20bright B-cells (10337 +/- 642 and 346311 +/- 24264 respectively). In addition, CD20dim cell counts vary with age, with the highest levels occurring in octogenarians: cord blood 0.3 +/- 0.1% (n = 13), 20-60 year-old group 2.1 +/- 1.1% (n = 18) and individuals > or = 61 years of age 6.9 +/- 3.2% (n = 10) (P < 0.001). Further characterization of CD20dim T cells, using three colour flow cytometry, demonstrated a predominantly memory cytotoxic phenotype, in that the cells were CD8+CD28+CD45RO+T-CR alpha beta +CD38-HLA-DR-.

The distribution of collagen type VI and tenascin has been determined in both normal and myelofibrotic bone marrow by immunohistological techniques. In normal sections positivity was demonstrated in the periosteum (collagen type VI and tenascin) and in the walls of small blood vessels (tenascin). In contrast, myelofibrotic bone marrow showed an increased deposition of both proteins, especially collagen type VI, although this increase was restricted to the later fibrotic stages of the disease. Serum concentrations of collagen type I (PICP), collagen type III (PIIIP) and laminin (laminin P1) related polypeptides were determined in a further 26 patients. PIIIP levels were significantly raised, in contrast to PICP and laminin P1 concentrations. All three markers, however, were significantly elevated in patients with active/transforming disease. Laminin P1 and PICP levels showed a strong correlation, indicating a relationship between basement membrane and interstitial collagen metabolism, although they do not appear to offer any advantage over PIIIP for the monitoring of disease activity.