Simple method for differentiating between HLA and platelet-specific antibodies by flow cytometry.

Since platelets express both platelet-specific and class I HLA antigens, serum antiplatelet reactivity assessed by most platelet antibody techniques could be due to antibodies with either or both specificities. Flow cytometric analysis of sera for detection of antiplatelet antibody commonly employs a purified platelet preparation as target cells. A method is described for investigating sera for platelet antibodies by flow cytometry using a mixture of platelets and lymphocytes. The mixture of lymphocytes and platelets as target cells has the advantage of confirming the presence of the HLA antibodies in reactive sera. The concomitant use of platelets and lymphocytes treated with citric acid, pH3, or with chloroquine (to remove or alter surface HLA antigens without affecting platelet specific antigens) may further assist in identifying antiplatelet antibodies in alloimmunized patients. These techniques may also be useful in platelet crossmatching procedures.     

Calculating rabies virus neutralizing antibodies titres by flow cytometry

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton Dickinson FACSCalibur flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity.     

运用流式细胞术对细胞内雄激素受体检测的研究

目的建立一种灵敏度高、特异性强的细胞内雄激素受体检测方法。探讨原发性高血压患者雄激素受体变化与原发性高血压的关系。方法运用流式细胞术检测112例男性高血压患者(高血压组)及120例健康男性体检者(对照组)雄激素受体的状态。结果流式细胞术不仅能定性而且能定量检测外周血白细胞的雄激素受体状态,且所需外周血量少。同时结合雄激素受体含量,来判断与原发性高血压的关系。结论流式细胞术检测雄激素受体更敏感、更具特异性,能更确切地指导临床医生的判断和治疗。     

Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit insulin action.

Antibodies to the insulin receptor were prepared in BALB/c mice by immunization with IM-9 human lymphocytes, a cell type that has a large number of plasma membrane insulin receptors. The spleens of these mice were then removed, and their lymphocytes were fused to a mouse myeloma cell line, FO cells. After screening over 1,200 resulting hybrids, one stable hybrid was obtained that produced IgG1 antibodies directed towards the insulin receptor. This antibody blocked 125I-labeled insulin binding to its receptor by more than 90% in three human tissues: IM-9 cultured lymphocytes, freshly isolated adipocytes, and placenta membranes. In contrast, the antibody did not inhibit insulin binding to rat adipocytes and rat liver plasma membranes, suggesting that the antibody was species specific. In IM-9 cells, which had their proteins prelabeled with [35S]methionine, the antibody precipitated two polypeptides with molecular weights of 135,000 and 95,000; these molecular weights are identical to those previously identified as the alpha and beta subunits of the insulin receptor. The monoclonal antibody inhibited the actions of insulin on both human adipocytes and fibroblasts, suggesting that the antibody was an antagonist of insulin action. The present studies suggest, therefore, that monoclonal antibodies to the insulin receptor may provide new insights into the structure of the insulin receptor and its interaction with insulin.     

Detection of platelet-associated antibodies by flow cytometry in hematological autoimmune disorders

We describe our experience in the evaluation of platelet-associated immunoglobulins (PAIg) by flow cytometry in comparison to solid-phase assay in patients affected by idiopathic thrombocytopenic purpura and by Evans syndrome. Results show that the analysis of PAIg by flow cytometry is easy and reliable and correlates well with data obtained by the solid-phase technique. In addition, flow cytometry allows the evaluation of samples containing small numbers of platelets (<20000/mm³); the analysis is objective, not influenced by personal experience. Moreover, flow cytometry appears simple enough to be performed in a routine laboratory, and data might be retrieved to perform batch analysis. Our results appear to indicate that PAIg flow cytometry might be a sensitive tool for the evaluation of patients with autoimmune thrombocytopenia.     

Enumeration of platelets by multiparameter flow cytometry using platelet-specific antibodies and fluorescent reference particles

Summary The correct enumeration of platelets is still an elusive matter. This is mainly due to the fact that commercial instruments which are used for platelet counting cannot discriminate platelets from other cellular particles and precipitates that cause similar signals. Visual (chamber counting) methods are still frequently used in routine laboratories to verify low automated platelet counts (< 50 × 10/l) despite obvious technical and statistical drawbacks. The following report shows how platelet counts can be measured by multiparameter flow cytometry with the help of reference particles (fluorescent latex beads) and platelet-specific antibodies i.e. anti-GPIIb/IIIa(CD41a), anti-GP Ib-α (CD42b) and anti-GP IIIa (CD61). The linearity of this method was highly satisfactory and the observed imprecision was within acceptable limits. At a platelet concentration of 10 × 10⁹/l the coefficient of variation (CV, n = 10) ranged from 5.3% (PCV = 0.456) to 5.6% (PCV = 0.148). Accuracy was evaluated by comparing results to the ICSH-selected method for platelet counting. The correlation of both methods was significant (P < 0.005) and Passing-Bablok's linear regression analysis showed no systematic differences between the two methods. Comparisons of this new platelet counting technique were also performed with routine visual methods, automated blood analysers (Technicon H-1, Sysmex E-5000) and a different flow cytometric method using only forward and side light scatter properties of platelets for their discrimination. The linear correlation of all methods was significant (P < 0.01) at platelet concentrations above 50 × 10⁹/l. At lower platelet concentrations, our new platelet counting technique correlated significantly only with the visual and the forward/side scatter methods. These findings stress the necessity to confirm low platelet counts by automated blood analysers and suggest that using multiparameter flow cytometry with platelet-specific antibodies may be a proficient way to do so. The possibility of using this technique as a reference method is discussed.     

Detection of minimal residual disease in acute lymphoblastic leukemia by flow cytometry with monoclonal antibodies.

To detect more precisely the minimal residual disease in acute lymphoblastic leukemia (ALL), two-color flow cytometric analysis for the detection of cell-surface antigen (CD10; CALLA) and nuclear terminal deoxynucleotidyl transferase (TdT) was performed in the six patients with CALLA-positive ALL coexpressing TdT. In all patients, the leukemic blasts coexpressed Ia (HLA-DR), CD9, CD19, CD20, CD24, and CD10. Five of six patients achieved complete remission, but one has so far relapsed. No leukemic blasts (CD10+, TdT+) were detected at the time of complete remission. During maintenance chemotherapy, leukemic blasts coexpressed C10 and TdT were found 2.32% in the patient's peripheral blood by two-color analysis, whereas no obvious leukemic cells were recognized morphologically. The patient relapsed leukemia with the same phenotype 4 weeks after the examination. On the basis of our findings, we suggest that two-color flow cytometric analysis with the use of these antibodies is quite valuable to detect the minimal residual leukemic cells in a patient with ALL. The reduction of leukemic cells below the threshold of detection of methods currently available appears to be necessary to achieve a cure in ALL. Hence accurate diagnosis of ALLs with monoclonal antibodies (MAbs) should contribute substantially to the development of an effective form of therapy for their cure.     

Liver cell-membrane specific antibodies in autoimmune hepatitis patients detected by flow cytometry

In this study, we identified autoantibody in patients with autoimmune hepatitis against liver cell membrane using flow cytometry. After incubation of one of the hepatoblastoma cell lines, Hep G2, with serum from a patient, and the addition of FITC-labeled anti-human Ig antibody, anti-membrane antibodies were analyzed by flow cytometry. By our method, the antibody in serum can react only with autoantigens on the cell surface. Furthermore, propidium iodide staining enabled us to exclude the possibility of crossreaction of antibodies against dead cells. The relative fluorescence intensity in 12 patients with autoimmune chronic active hepatitis was significantly higher than that in 20 normal subjects, six primary biliary cirrhosis (PBC), 18 chronic viral hepatitis, and 11 systemic lupus erythematosus (SLE). The normal level of fluorescence intensity provided by sera from the SLE patients indicated that antibody binding to the liver cell membrane was not derived from Fc-mediated immune complex capture. These findings demonstrated that this flow cytometric technique provides a simple and accurate method for the detection of autoantibodies against liver cell membrane.     

The insulin receptor: characterization and regulation using insulin-antiinsulin antibody complexes as a probe for flow cytometry

The primary approach for the characterization of the insulin receptor has been through the study of its interaction with 125I-labeled insulin. Recently, we demonstrated that insulin receptors can also be identified by flow cytometry using antibodies to the receptor. In the present study, we characterized the insulin receptor on human lymphoblastoid cells (IM-9) and studied its regulation using insulin and antiinsulin antibodies as a probe for flow cytometry. The mean peak fluorescence of the cells treated with insulin followed by antiinsulin serum was 30-50 U above the control value. There was a close correlation between [125I]insulin binding and peak fluorescence. Fish insulin, which has about 50% the affinity of porcine insulin for the insulin receptor but does not bind to antiinsulin antibodies, did not enhance antiinsulin antibody binding, but competed for the pork insulin-antiinsulin antibody complexes in a dose-dependent manner. Exposure of IM-9 cells to insulin or antireceptor antibodies resulted in reduction in the number of insulin receptors. Cells down-regulated with 10(-6) M insulin or a monoclonal antibody to the insulin receptor had 40% of the [125I]insulin binding of the control cells and 40-50% of the peak fluorescence when insulin-antiinsulin was the probe for the immunofluorescence studies. Cells down-regulated with human autoantibodies to the receptor had 4% [125I]insulin binding and 10% peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical fluorescent peak. These date indicate that flow cytometry with insulin-antiinsulin antibody complexes provides a new approach to the measurement of insulin receptors, since it provides direct measurement of the occupied receptor.     

Sensitive detection technique of myeloperoxidase precursor protein by flow cytometry with monoclonal antibodies

This report describes the analysis of culture cells and blast cells separated from the heparinized bone marrow and whole blood of patients with acute leukemias by means of a density-gradient technique (Ficoll-sodium metrizoate d = 1.077 g/cm³). Cell-surface antigens were analyzed by a fluorescence-activated cell sorter using a panel of monoclonal antibodies (MAbs). The blast cells and culture cells were fixed by 3% paraformaldehyde in phosphate-buffered saline. A low level of expression of MPO precursor protein was found in THP−1. K-562 and HEL, MEG-01, erythro-megakaryocytic leukemia cell lines, Jurkat, MOLT-3, MOLT-4, RPMI8402, ATL-5, T-cell leukemia cell lines, Raji, Daudi. BALL-1, B-cell leukemia cell lines, and AGNK1 showed negative reaction. The de novo MPO-negative acute leukemias, middle level of expression of MPO precursor protein, was found in the blasts of MPO-negative AML (AML, M0), which coexpressed CD13, CD33, CD34, and CD38. A high level of expression of MPO protein was found in all cases of AML, M1, and M2. The MPO expression was not found in all cases of acute lymphoblastic leukemia. The highest level of MPO expression was found in cases of AML, M3, and AML, M3v, suggesting the diagnostic value for this type of leukemia. The detection of MPO precursor protein by flow cytometric analysis with monoclonal antibodies is essential for the determination of lineage and precise diagnosis of acute unclassifiable leukemia, and should contribute substantially to the development of an effective form of therapy and cure. Am. J. Hematol. 58:241–243, 1998. © 1998 Wiley-Liss, Inc.     

Monoclonal antibodies to the human insulin receptor that activate glucose transport but not insulin receptor kinase activity.

Three mouse monoclonal antibodies were produced that reacted with the alpha subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.     

Evidence that human and porcine insulin differently affect the human insulin receptor: studies with monoclonal anti-insulin receptor antibodies.

Binding studies have been carried out with radioiodinated monoclonal antibodies directed to various epitopes of the insulin receptor in order to detect differences between human and porcine insulin in the interaction with the human insulin receptor. Human insulin was more effective that porcine insulin at inhibiting the binding of 125I-MA-5 to IM-9 cells, Hep-2 human larynx cells and human placenta membranes. On the contrary, human and porcine insulin showed similar inhibitory effect on the binding of two other labeled anti-insulin receptor monoclonal antibodies, thus ruling out the possibility that results were due to experimental artifacts. Although several interpretations are possible, data reported suggest that human insulin and porcine insulin might differently affect the insulin receptor, even if, the biological significance of these findings remains unknown.     

Decrease in the insulin receptor protein level by anti-insulin receptor antibodies: roles of tyrosine kinase activity and receptor internalization

To investigate the mechanism of severe impairment of insulin action in type B insulin resistance, we extracted IgG from the serum of a patient with type B insulin resistance (B-IgG) and analyzed the inhibiting effect of B-IgG not only on insulin signaling but also on IGF-I signaling in Chinese hamster ovary (CHO) cells expressing human insulin receptor or human IGF-I receptor. Preincubation with 1 mg/ml B-IgG prevented insulin-induced phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) but did not alter the IGF-I-induced phosphorylation of the IGF-I receptor and IRS-1. ¹²⁵I-insulin binding was inhibited by 93% after preincubation with BigG at 37° C and was recovered up to 50% of the control value by acid washing. However, when cells were preincubated with B-IgG at 4° C, the insulin binding completely recovered the control value by acid washing. ¹²⁵I-IGF-I binding was not altered by B-IgG preincubation. Immunoblot study revealed that the protein level of the insulin receptor was strongly decreased by preincubation with 1 mg/ml B-IgG at 37° C, but never at 4° C. The IRS-1 protein level did not change by B-IgG preincubation. In order to know the role of the insulin receptor internalization in the inhibiting effect of B-IgG, we employed CHO cells expressing mutant insulin receptors which do not undergo internalization (CHO-K1018R). B-IgG incubation of CHOK1018R at 37° C failed to decrease the protein level of the insulin receptor. The present data indicate that IgG from the diabetic patient with type B insulin resistance decreased insulin receptor protein level, probably due to the enhanced degradation rate of the insulin receptor, in which insulin receptor tyrosine kinase activity and internalization are required for this process. This effect of B-IgG was specific for the insulin receptor with no effect on either IGF-I receptor or IRS-1, as reflected by the IGF-I effectiveness on glycemic control in this patient. Received: 26 September 2001 / Accepted in revised form: 12 July 2002 Correspondence to K. Eguchi     

Analysis of the cellular DNA and RNA content in acute leukemias by flow cytometry

Summary In the present study bone marrow samples from 573 patients with newly diagnosed acute myeloid (AML) and lymphoblastic or undifferentiated leukemias (ALL/AUL), were analysed for their cellular DNA und DNA/RNA content, respectively, by means of flow cytometry. From 237 patients with AML 35.4% revealed aneuploid DNA stemlines. While no relation of DNA aneuploidy with other pretherapeutic parameters, including FAB subtype, white blood cell count, lactate dehydrogenase, S-phase index and percentage of blasts in the bone marrow, was observed, cases with aneuploid DNA stemlines revealed a tendency towards longer remission duration. In ALL/AUL 21.8% of 280 patients expressed DNA aneuploidies, which were less frequently found in T-cell ALL (11.1%) as compared to common(C)-ALL (21.4%) or null-cell(null)-ALL (23.5%). DNA aneuploidy was not related with other clinically defined risk factors such as age, white blood cell count, and rapid achievement of remission. Patients with DNA indices <1.0, however, tended to have shorter remissions. A significant difference in RNA indices was observed between AML and ALL/AUL with median values of 14.4 and 10.1, respectively (P<0.05). These data indicate the usefulness of flow cytometric analyses of cellular DNA and RNA content for the characterization and classification of acute leukemias, complementing the identification of clinical risk factors, immuno-phenotyping and cytogenetics.Abbreviations AML acute myeloid leukemia - ALL acute lymphoblastic leukemia - AUL acute undifferentiated leukemia - T-ALL, C-ALL and Null-ALL T-cell, common and Null-cell ALL     

The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria

We have characterized the erythrocytes, granulocytes, and platelets of 54 patients with paroxysmal nocturnal hemoglobinuria (PNH) with antibodies to glycosylphosphatidylinositol-anchored proteins (anti- CD55, anti-CD59, and anti-CD16) and flow cytometry to establish the usefulness of this technique in the diagnosis of this disorder. All patients demonstrated either completely (PNH III) or partially (PNH II) deficient red cells and granulocytes. Anti-CD59 best demonstrated PNH II red cells, which were present in 50% of the patients. The proportion of abnormal granulocytes was usually greater than the proportion of abnormal red cells; 37% of the patients had >80% abnormal granulocytes. Anti-CD55 did not delineate the erythrocyte populations as well as did anti-CD59. Either anti-CD55 or anti-CD59 could be used equally well to analyze granulocytes; anti-CD16 did not demonstrate cells of partial deficiency. Platelets could not be used for detailed analysis as the normal and abnormal populations were not well distinguished. Flow cytometry of erythrocytes using anti-CD59 or of granulocytes using either anti-CD55 or anti-CD59 provides the most accurate technique for the diagnosis of paroxysmal nocturnal hemoglobinuria; it is clearly more specific, more quantitative, and more sensitive than the tests for PNH that depend upon hemolysis by complement (the acidified serum lysis [Ham] test, the sucrose lysis test, and the complement lysis sensitivity [CLS] test).