Dear Collegues, from our experience I want to comment on the ongoing discussion on PNH: The patients in question either present with a hemolytic anemia (primary PNH) or secondary to aplastic anemia (PNH syndrome). The non flow laboratory data should support one of these conditions (LDH, bili, urobili, haptoglobin, retics etc). Only about 25% of the primary PNH show nocturnal hemoglobinuria (PNH = paroxysmal nocturnal hemoglobinuria). Esp. young patients may show spontaneous remission (about 10%?). Most of the patients (esp. those who are not suspect) show a mosaic of normal and affected cells. In aplastic anemia the proportion may be very small in the beginning (5% or less). In preparation of a "consensus" protocol (what ever that may mean) I discussed with Dr. Schrezenmeier (who has seen the most patients in Germany) the following: As the acquired defect may be differently affect the blood cell lineages, red cells, platelets and leukocytes have to be checked separately. Reticulocytes are of special interest as patients might have received blood transfusions. CD55 and CD59 with phycoerythrin (orange fluorescence) versus thiazol orange (green fl.) is a good combination. Each lineage should be checked by two different CDs. Immunological gating is preferred. The problem of false negative cells is a major issue: CD14 and esp. CD16 are maturation dependent and not recommended. Scatter gates of these cells produce negative cells as they include lymphs in the mono gate or eosinophils, myelocytes or less mature granulocytes in the neutrophil gate (CD16 negative). In aplastic patients and those with infections this represents a significant proportion. CD16 on neutrophils in addition undergoes alternative RNA splicing which ends up in a polymorphism and you need a monoclonal antibody that recognizes a conserved epitope (eg. clone 3F8, CALTAG). CD16 is monomorphic on NK cells. CD24 and CD48 are better in that respect. CD59 versus side scatter allows to differentiate among the major leukocyte subsets. Non red cells in the erythrocyte scatter gate (platelets, debris etc.) and red and debris in the platelet gate are significant and require immunological gating. The testing of CD34 stem cells (for autologous transplantation) with CD59 (expressed on all maturation stages) requires some experience. Diagnosis should be made in the peripheral blood as bone marrow esp. shows the problems mentioned above. Monitoring every 6 month (?) is meaningful as the proportion of affected cells varies over time. Strict quality control (including always a normal control sample) and education are important because of the severity of the diagnosis. Each laboratory needs to set up its own threshold value of GPI negative cells. I want to stop here as the message might has got already too long. I think you will have got a feeling about the laboratory diagnosis of PNH. Therefore, everybody needs to decide on his own whether he is able to perform such a test esp. when the frequency is low. More details are published in: Nebe CT, Paroxysmal Nocturnal Hemoglobinuria: Clinical Aspects and Flow Cytometric Analysis, in: Aspects of the Flow-Cytometric Analysis of Red Blood Cells, K. Gutensohn, H.-H. Sonneborn and P. Kühnl (eds.), 81-94, Clin. Lab. Publications (1997) Regards Thomas Nebe Dr.med. C. Thomas Nebe Universitaetsklinikum Mannheim Zentrallabor Theodor-Kutzer-Ufer 1-3 D-68167 Mannheim * +49 621 383-3485 * +49 621 383-3819 e-mail: thomas.nebe@ikc.ma.uni-heidelberg.de http://www.ma.uni-heidelberg.de/inst/ikc/
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