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Use of the XE-2100 in a Patient with Cold Auto-Immune Hemolytic Anemia

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Use of the XE-2100 in a Patient with Cold Auto-Immune Hemolytic Anemia Sysmex Journal International Vol.10 No.1 (2000) Use of the XE-2100 in a Patient with Cold Auto-immune Hemolytic Anemia Gudrun STAMMINGER and Lothar BEIER Klinikum Chemnitz gGmbH, Institut für Laboratoriumsmedizin, Flemmingstraße 2, 09009 Chemnitz, Germany. The analysis of specimens, drawn from patients with cold auto-immune hemolytic anemia (caused by elevated levels of anti-I on the red blood cell (RBC) membrane) is, in general, a difficult problem for automated hematology analyzers. In such patients, because of aggregation of the RBCs, which occurs mainly at temperatures lower than 30°C, it is not possible to measure the RBC count and hematocrit accurately. During an evaluation of the XE-2100 in our laboratory we studied a patient with this disorder. During the first weeks after admission to the Haematology Clinic very marked aggregation was observed in all specimens from the patient despite pre-warming of the EDTA blood to 37°C. The patient received 13 transfusions of pre-warmed, leukocyte-poor red cells and cyclosporine A. Two weeks later the activity of anti-I decreased. The result was recovery from the hemolytic disease, detected very early by the system and accom- panied by an improvement of the cell counts. Erythropoietic activity was easily followed by the nucleated red blood cell (NRBC) counts and reticulocyte (RET) parameters generated by the XE-2100. During the first two weeks the NRBC counts were increased but the RETs remained low. During recovery the picture was completely reversed. RETs and especially their immature fractions increased very markedly and NRBCs disappeared. EDTA associated platelet (PLT) clumping was detected by use of the IMI Channel. On using lithium heparin as anticoagulant the PLT count became normal. In the XE-2100 newly developed principles for counting and characterization of blood cells are introduced as well as already well established methods. From our point of view, at this time, the XE-2100 is the most appropriate system for diagnosis of diseases associat- ed with an elevated number of NRBC in the peripheral blood, e.g. the immune hemolytic anemias. (Sysmex J Int 10 : 3 - 12, 2000) Key Words Automated Hematology Analyzer, XE-2100, Nucleated Red Blood Cell (NRBC), Reticulocyte Count, Auto-Immune Hemolytic Anemia INTRODUCTION Laboratory diagnosis of cold 3-6) A cold auto-immune mechanism produces a special type auto-immune hemolytic anemia of hemolytic anemia. IgM auto-antibodies are involved; 1) Complate blood cell (CBC) including the reticulocyte they are complete and complement binding. They have count, the RBC count and hematocrit are decreased, mainly the specificity “I”, rarely “i”. Normally these the MCV falsely elevated and the MCH and MCHC antibodies are only active in the cold (4°C), but in severe are falsely decreased. The reticulocyte number may cases with a high antibody titer their thermal amplitude is be increased depending on the status of the disease wider and results in agglutination of (RBC) and haemoly- and its severity. Depending on the severity of the dis- sis in vivo1, 2). ease, many erythrocyte aggregates are found. The It is possible to differentiate primary and secondary types monospecific direct antiglobulin test (DAT) is positive of the cold agglutinin disease. In vivo the antibody causes (complement C3D-specifity). macrsocopically visible clumping of the RBCs at a tem- 2) The titer of anti-I is increased. perature below 37°C. This phenomenon can lead to 3) More nonspecifically increased analytes are: bilirubin, some severe problems in the laboratory diagnosis: LDH, AST, serum- hemoglobin. 1) Problems in determining blood groups and in perform- 4) Haptoglobin is decreased. ing cross matching. 5) Bone marrow examination is necessary for the evalua- 2) Measurements from photometry based assays may be tion of the erythropoiesis and to search for possible influenced by the hemolysis. causes of the disease (e.g., lymphoma). In most cases 3) Erythrocyte aggregation adversely affects the mea- erythropoiesis is increased to compensate for vigorous surement of the RBC count, the hematocrit, the RBC hemolysis. indices and the reticulocyte count2, 3). 6) Microbiological and virological diagnostics (possible − 3 − Sysmex Journal International Vol.10 No.1 (2000) Mycoplasma or EBV infection) Mycoplasma pneumo- at different stages of the illness. During the first period niae infection is sometimes accompanied by a marked of the disease the RBC count and the HCT could not be increase in anti-I titer. used for diagnosis. Once treatment became effective the measurements became reliable. Treatment Evaluation of the XE-2100 Avoidance of cold is the most important treatment to pre- th rd vent activation of the antibody mechanism. Transfusion –results between July 26 and September 3 of pre-warmed, washed and filtered leukocyte- poor red Hemoglobin (Fig. 6) cells may be necessary. Successful treatment is heralded The hemoglobin concentration decreased to 38 g/L dur- by reversal of the indicators of hemolysis and a rise in ing the first days after admission because of active hemoglobin concentration and hematocrit3-6). hemolysis. Hemolysis stopped following transfusion of At the time of our evaluation of the XE-2100 a patient pre-warmed, washed and filtered RBC-concentrates and (G.U.) was admitted to hospital. While all routine hema- treatment with Cyclosporine A. The patient received 13 tological measurements were made on the Sysmex SE- units of leukocyte-poor red cells with the blood group O 9500 analyzer, the patient’s specimens were also ana- RhD-positive. Two weeks after admission the hemoglo- lyzed on the XE-2100. The results from the latter are pre- bin concentration started to show a sustained rise. sented and discussed. RBC (Fig. 7), HCT (Fig. 8) (Pictures 1-3) Because of extreme RBC aggregation it was not possible MATERIALS AND METHODS to measure an accurate count. As recovery commenced two weeks after admission, the RBC count and hemat- ocrit started to increase. Similar results were obtained Specimens from the SE-9500/RAM-1 and the XE-2100. Blood anti-coagulated in K2EDTA and pre-warmed to 37°C was used for routine blood count measurements. MCH and MCHC (Fig. 9) During the admission, EDTA associated platelet clump- The Wintrobe-Indices clearly reflect the progress of the ing was noted and subsequently lithium heparin anticoag- disease. At the start the MCH and MCHC were extremely ulant was used for the CBC and reticulocyte measure- high, however, with successful treatment they returned to ments. the normal ranges. The high reproducibility of these K2EDTA was used for the bone marrow specimen. results is noted. Reticulocytes (Fig. 10) Methods About 8-10 days after admission the number of reticulo- For the investigation of the CBC, NRBC and reticulo- cytes increased both in relative and absolute counts (see cytes the XE-2100 (Sysmex Corporation, Kobe, Japan) also Picture 4). Later and during regeneration both was used. counts were very high as expected. The instrument was Microscopic examination of the blood smear and the able to measure these counts continuously. bone marrow was performed after staining by the The different reticulocyte maturity fractions varied espe- Pappenheim method (May-Grünwald/Giemsa-stain). cially during the first days. It is not possible to give an explanation for this fact. Effects of the matrix (RBC- aggregates) may be partly responsible. Erythropoiesis RESULTS may also have been variable during this time. The high fluorescence ratio (HFR) fraction was high during the All parameters, values necessary for diagnosis were first period but, as expected, it fell as recovery proceeded. increased (Table 1). The monospecific DAT was posi- Throughout the course of the disease, the immature retic- tive (anti-C3D) and the anti-I titer was 1:2056. ulocyte fraction (IRF) maintained an inverse relationship to the low fluorescence ratio (LFR) of the reticulocyte Figs. 1-5 are examples of measurements by the XE-2100 population (Fig. 11). Table 1 Important results for diagnosis of cold auto-immune hemolytic anemia in G.U. on admission. Analyte Patient result Reference range in health Bilirubin (Total) 135.4 µµ mol/L 3.4 - 22.2 mol/L LDH 61.2 µ kat/L 5.2 - 10.3 µ kat/L AST (GPT) 1.14 µ kat/L < 0.58 µ kat/L Serum-hemoglobin 40 µ mol/L 0 - 9 µ mol/L − 4 − Sysmex Journal International Vol.10 No.1 (2000) Fig. 1 Sample 9 (27/07/99) Fig. 2 Sample 2 (02/08/99) − 5 − Sysmex Journal International Vol.10 No.1 (2000) Fig. 3 Sample 9921900755 (09/08/99) Fig. 4 Sample 2 (20/08/99) – EDTA-blood with PLT clumps − 6 − HGB (g/L) 100 120 20 40 60 80 0 Jul. 27 29 Aug. 2 Fig. 6 5 HGB onXE-2100 9 11 HGB Date 16 18 Sysmex JournalInternationalVol.10No.1(2000) 20 24 Fig. 5 27 Sample 9924501604(03/09/99) Sept. 1 3 − 7 − RBC (×1012/L) 0.5 1.5 2.5 3.5 0 1 2 3 4 Jul. 27 29 Aug. 2 Fig. 7 5 RBC onXE-2100 9 11 RBC Date 16 18 20 24 27 Sept. 1 3 Reticulocytes (‰) MCH (pg) 100 150 200 250 100 120 140 160 180 200 50 20 40 60 80 0 0 Jul. 27 Jul. 27 29 29 Aug. 2 Aug. 2 5 5 9 9 RET‰ 11 11 MCH Date Date 16 16 HCT 0.05 0.15 0.25 0.35 0.1 0.2 0.3 0.4 18 18 0 Sysmex JournalInternationalVol.10No.1(2000) 20 20 Jul. 27 29 24 Fig. 9 24 Fig. 10 27 27 Aug. 2 Fig. 8 MCH andMCHConXE-2100 Sept. 1 Sept. 1 5 Reticulocytes onXE-2100 3 3 HCT onXE-2100 9 − 8 11 HCT Date − Reticulocytes (×1012/L) MCHC (g/L) 16 1000 1500 2000 2500 0.05 0.15 0.25 0.35 0.45 500 0.1 0.2 0.3 0.4 0 0 18 Jul.
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