Quantitative Flow Cytometry Method Research Articles

DEVELOPMENT OF A SPIKE-IN FLOW-CYTOMETRY METHOD FOR THE QUANTITATION OF FETAL HEMOGLOBIN (HBF) IN INDIVIDUAL RED BLOOD CELLS OF SICKLE CELL DISEASE (SCD) PATIENTS

HbF is the most relevant modulator of the severity of SCD, inhibiting the polymerization of the mutant sickle hemoglobin (HbS). Despite its clinical relevance, a straightforward method to quantify and identify protective levels of HbF in individual red blood cells (RBCs) is still lacking. Here, we sought to develop a fluorescent-labeled RBC “Spike-In”(Spk) control (Ctr), to be mixed with patient samples, to serve as an internal reference in a novel quantitative flow-cytometry method. The Spk-Ctr was generated by mixing equal numbers of CFSE-labeled and fixed HbF Neg adult and HbF Pos cord blood (CB) RBCs, whose mean corpuscular HbF (MCHbF) mass was pre-calculated, based on the %HbF (HPLC-derived) and the MCH (hematology analyzer), using the formula: MCHbF=(%HbFxMCH)/100. Spk-Ctr was mixed with a similarly-fixed Test Sample, composed of 10% HbF Pos (prepared with the same RBCs as the Spk-Ctr), permeabilized, stained with an anti-HbF PE conjugate, and acquired by a flow cytometer. The anti-HbF PE Median Intensity Fluorescence (MIF) was obtained for gated HbF Pos RBCs in both the Ctr CFSE Pos and Test CFSE Neg gated populations. A conversion factor (CF) was calculated by dividing the pre-calculated MCHbF by the MIF obtained for HbF Pos RBCs from the CFSE Pos Spk-Ctr. By multiplying the anti-HbF PE fluorescence intensity of each RBC in the test sample by the CF, an absolute HbF/RBC values (in pg of HbF per RBC) can be obtained, which can be used to generate a distribution histogram to calculate the percentage of RBCs above a protective HbF/RBC threshold. The Ctr CB used had 73.2% HbF and a MCH of 36.4pg, resulting in a MCHbF of 26.64pg. The anti-HbF PE MIF of the Spk-Ctr HbF Pos RBCs was 25,166a.u. (arbitrary units), resulting in a CF of 0.001058. The anti-HbF PE MIF of the HbF Pos RBCs of the Test Sample was 24,146a.u., leading to an estimated MCHbF of 25.56pg; a deviation of only 4% from the expected (known) MCHbF. In contrast, when samples were permeabilized and stained in separate tubes, this variation was 27%. Recently, a work described a laborious method requiring a standard curve, generated with rare samples from subjects with hereditary persistence of HbF, and calibration beads. This method correct only for experimental variations arising from flow-cytometry acquisition and bath-to-bath antibody affinity/fluorescence differences, but not from experimental variability associated with sample handling of the test patient samples and those used in the standard curve. Our method eliminates the complexity associated with the use of a cumbersome standard curve, as the Spike-In Ctr and patient cells are subjected to the exact same procedures (in the same tube). Thus, experimental variability (derived from differences in, pipetting, incubation time, temperature, affinity/fluorescence of antibody batches, instrument or acquisition settings, etc.) is not expected to interfere with the quantitation. Once fully developed and validated, our method may serve as an auxiliary tool for the clinical management of SCD patients, contributing to the prediction of complications (allowing preventive measures to be adopted), or allowing the monitoring of the response to therapeutic approaches based on the induction of HbF (including hydroxyurea).

Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS‐CoV‐2 spike protein

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID‐19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer‐friendly non‐adherent Jurkat T‐cell line that stably expresses the full‐length native spike “S” protein of SARS‐CoV‐2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self‐cleaving sequence, allowing to accurately quantify the presence of anti‐S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double‐staining with the test sera and anti‐EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti‐S antibodies with protective neutralizing activity are long‐lasting and can be detected in sera 8 months after infection.

Abstract 4474: A quantitative flow cytometry method for the measurement of HLA-DR expression level on monocyte subsets

Abstract There is growing evidence that solid tumor cancer is often associated with a systemic immunosuppressive environment which correlates with poor responses to immunotherapy. The frequency of CD14+HLA-DRlo/neg monocytes, also called monocytic myeloid derived suppressor cells (M-MDSCs), is often used as a flow cytometry based biomarker for the measurement of the level of immunosuppression in cancer immunotherapy research. While this approach has been demonstrated to work in clinical investigation studies with well-controlled cytometer settings and analysis methods, it is difficult to produce consistent results and compare measurements from different laboratories due to lack of standardization. The BD Horizon™ Dri Monoset panel is designed to measure the absolute counts and relative frequency of classical, intermediate and nonclassical monocyte subsets for research use. The assay uses whole blood samples analyzed by flow cytometry using a consensus reagent panel and gating strategy published by an international organization. When used in combination with BD Quantibrite™ beads, the level of HLA-DR expression on each subset of monocytes can be quantified in antibody bound per cell (ABC) units independent of the cytometer used to perform the assay. Evaluating 10 normal blood donor samples, our data demonstrates using the BD Horizon™ Dri Monoset panel in combination with BD Quantibrite beads, quantification of HLA-DR expression level on monocyte subsets provides consistent flow cytometry results. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD, the BD Logo, Horizon, and Quantibrite are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved. Citation Format: Wei Huang. A quantitative flow cytometry method for the measurement of HLA-DR expression level on monocyte subsets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4474.

Monitoring CD49d Receptor Occupancy: A Method to Optimize and Personalize Natalizumab Therapy in Multiple Sclerosis Patients.

In natalizumab-treated relapsing-remitting MS (RRMS) patients, various extended interval dosing strategies are under evaluation to minimize severe treatment-associated side effects, mainly progressive multifocal leukoencephalopathy development. Up to now, it has not been presented any approach, even in form of assay design, to determine the optimal percentage of CD49d receptor occupancy (RO) associated with a favorable clinical, radiological, and immunological response. A multiparametric quantitative flow cytometry method was settled to measure CD49d RO on peripheral blood lymphocytes. The analytical protocol was tested in a 6-month follow-up from 19 RRMS patients treated with the natalizumab standard dosing of every 4 weeks or an extended-interval dosing of every 6 weeks. Extended natalizumab dose schedule promoted an increase of CD49d molecules per cell surface and a reduction of CD49d RO levels. The reduction observed on CD49d RO was not only depending on dose schedule but also on individual parameters such as body mass. Interestingly, individual clinical outcome was apparently the same between the different dose schedules or even better with the extended interval dosing. Following up CD49d RO levels with a well-regulated monitoring work scheme is crucial to further identify over-/under-treated patients and to define a safe, personalized natalizumab regimen. © 2017 International Clinical Cytometry Society.

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity.

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.

Abstract 2908: A first-in-human phase I study of SAR650984, a humanized anti-CD38 antibody in patients with CD38+ hematological malignancies: Preliminary PK and PD results of escalation phase

Abstract SAR650984 is a novel humanized monoclonal antibody specifically targeting the CD38. , A phase 1 dose escalation study is being conducted in patients with confirmed CD38+ hematological malignancies to determine safety of SAR650984, to establish the MTD and the biologically active dose, and to characterize the PK and PD after repeated IV administration. Methods: The study design was composed of 2 parts. The first one was an accelerated phase with 1 to 3 patients per dose cohort from 0.0001 mg/kg to 0.1 mg/kg and the second phase includes 3 to 7 patients per dose cohort between 0.3 and 20 mg/kg every 2 weeks. An additional 6 patient cohort was added at 10 mg/kg every week. Results: 34 out of the 39 treated patients were Relapsed Refractory Multiple Myeloma (MM) patients. SAR650984 was well tolerated. Overall Response Rate (CR + PR) for dosing cohorts of ≥1mg/kg is 32% (2 MR, 5 PR, 2 CR of 28). Pharmacodynamics were evaluated by measuring CD38 density and its occupancy by the drug in bone marrow on cancer cells, using a validated quantitative flow cytometry method. This parameter has been correlated with the dose and exposure levels. Receptor Occupancy (RO) could be detected starting from the 1 mg/kg dose. From 5 mg/kg dose level, preliminary results demonstrated receptor occupancy greater than 70% (maximum observed: 97.7%). From 0.0001 to 0.01 mg/kg doses, no or limited PK parameters could be calculated. The PK profile was not linear with a decrease in clearance when the dose was increased suggesting target mediated clearance. Based on individual modeling, a moderate to high variability (CV% = 49 to 130%) was observed on estimated systemic total clearance, regardless of the dose group. At the10 mg/kg Q2W, the shape of the PK profile suggests saturation of the target. All MM patients (N=6) at cycle 1 had SAR650984 plasma trough levels consistently above the 10 µg/ml level which is associated with anti-tumor activity in mouse xenograft studies (Cssmin of the lowest pharmacological active dose). At 20 mg/kg Q2W, in 2/3 MM patients, SAR650984 plasma trough levels from cycle 2 were above the concentration needed to eradicate the tumor (129 µg/mL) in the same animal model, based on the Simeoni et al modelling approach. These preliminary phase 1 safety and efficacy results are associated with a favorable PK/PD profile and justify the progression of the program to phase 2. NCT : NCT01084252 Citation Format: Marie-Laure L. Ozoux, Hélène Guillemin, Marie-Hélène H. Pascual, Sylvaine Cartot-Cotton, Christine Veyrat-Follet, Delphine Valente, Maxime Moulard, Christine Mauriac, Antoine Deslandes, Dorothee Semiond. A first-in-human phase I study of SAR650984, a humanized anti-CD38 antibody in patients with CD38+ hematological malignancies: Preliminary PK and PD results of escalation phase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2908. doi:10.1158/1538-7445.AM2014-2908

In vitro induction of apoptosis, necrosis and genotoxicity by cosmetic preservatives: application of flow cytometry as a complementary analysis by NRU

Preservatives are used in cosmetics to prevent microbial contamination; however, some preservatives are not free of allergenic and cytotoxic potential. Allergenicity and cytotoxicity potential values are major aspects of preservative safety, which determine limitations and maximum concentration dose in a cosmetic product. The purpose of this study was to investigate and compare the in vitro apoptosis, necrosis and genotoxicity-inducing potential of five different types of preservatives: Phenoxyethanol (PE), Propylparaben (PP), Methylparaben (MP), Benzyl Alcohol (BA) and Ethylhexyl Glycerine (EG). In vitro experiments were carried out on human dermal fibroblasts by a quantitative flow cytometry method, using specific cell markers (Annexin V, Propidium Iodide and H2AX). We compared the resulting cell viability by means of neutral red uptake (NRU) and established the IC(50) . Our results showed that PE, PP, MP and BA have similar cytotoxic mechanisms (high apoptosis and necrosis levels only at the test concentration of 1%), whereas EG showed only an apoptosis pathway. For genotoxicity, both parabens yielded the highest values. Results obtained by flow cytometry for necrosis were comparable to those produced by NRU; however, NRU does not distinguish apoptosis from necrosis. We propose that flow cytometry is a more sophisticated methodology for understanding the cytotoxic mechanisms of cosmetic preservatives and can be used to complement the NRU.

Early Expression of FcγRI (CD64) on Monocytes of Cardiac Surgical Patients and Higher Density of Monocyte Anti‐Inflammatory Scavenger CD163 Receptor in “On‐Pump” Patients

Objective. Activation of innate immunity cells is inseparably linked to cardiac surgical operation. The aim of this study was to assess the kinetics in the expression of receptor for Fc part of IgG, FcRI (CD64), and scavenger receptor CD163 on peripheral blood cells of cardiac surgical patients and to examine the effect of cardiac bypass as a separable influence on the systemic acute inflammatory response. Methods. Forty patients, twenty in each group, were randomly assigned to CABG surgery performed either with “on-pump” or without “off-pump” cardiopulmonary bypass. Standardized quantitative flow cytometry method was used to determine the expression of surface markers. Results. The density of CD64 molecule on monocytes reached maximum on the 1st postoperative day () whereas the peak for CD64 molecule expression on granulocytes was postponed to the 3rd postoperative day (). The expression of CD163 scavenger molecule on monocytes reached maximum on the 1st postoperative day (). The density of CD163 molecule on monocytes on the 1st postoperative day is significantly higher in “on-pump” patients in comparison with “off-pump” patients (). Conclusion. In cardiac surgical patients the expression of activation marker FcR1 (CD64) on monocytes is increased earlier in comparison with granulocytes in both “on-pump” and “off-pump” patients. The expression of scavenger molecule CD163 on monocytes is significantly higher in “on-pump” patients.

Quantitative Measurement of CD52 Expression and Alemtuzumab Binding in Adult Acute Lymphoblastic Leukemia (ALL): Correlation with Immunophenotype and Cytogenetics in Patients (Pts) Enrolled on a Phase I/II Trial from the Cancer and Leukemia Group B (CALGB 10102).

The feasibility of incorporating Alemtuzumab (A) into frontline therapy of adult ALL pts was evaluated in a recently completed Phase I/II clinical trial, CALGB 10102. Pts were considered CD52+ and eligible to receive A during post-remission therapy if at least 10% of lymphoblasts had detectable expression of CD52 as measured by qualitative flow cytometry analysis in a CALGB reference laboratory. Using this qualitative assay, we have determined that 212 of 302 (70%) pts were CD52+ and eligible to receive A. Interestingly, 40/40(100%) pts with t(9;22) were CD52+. To better characterize CD52 expression level on lymphoblasts at the time of diagnosis and to obtain insights into subset specific therapies in adults with ALL, we developed a quantitative flow cytometry method to measure CD52 antigen expression and A binding using custom conjugated clinical grade A antibody. Results of this quantitative assay are expressed in arbitrary units of Antibody Bound per Cell (ABC). To determine whether ABC correlated with immunophenotype and specific cytogenetic subsets of adult ALL, we assessed ABC in a randomly selected subset of 88 untreated pts entered onto CALGB 10102. Seventy-two (81%) pts had precursor-B cell (pre-B) ALL and 16 (19%) had precursor T-cell (pre-T) ALL. The median ABC for pre-B cases was 37,178 (range: 1,545–228,247) and was significantly higher than the median ABC of 15,585 (range: 5,231–53,409) for pre-T cases (p=0.01). Interestingly, ABC on both pre-B and pre-T lymphoblasts was significantly lower than the median ABC on residual normal B-lymphocytes of 135,047 (range: 30,277–1,214,141), and on residual normal T -lymphocytes with a median of 70,139 (range: 20,621–557,859) that were present in the diagnostic bone marrow specimen (p<0.001). ABC by cytogenetic subset was also evaluated with results tabulated below for some of the commonly recurring abnormalities:ABC on lymphoblasts differed significantly across the cytogenetic subsets examined (p<0.001). The highest ABC levels were present in pts with del 9p and the lowest ABC levels were detected in pts with t(4;11). In conclusion, CD52 expression and A binding are significantly higher in pts with pre-B ALL than those with pre-T ALL. However, overall CD52 expression and A binding is significantly lower on lymphoblasts in comparison with normal residual lymphocytes present in the diagnostic bone marrows. We also demonstrate for the first time that differential CD52 expression and A binding occurs in specific cytogenetic subsets of adults with ALL. Quantitative PCR analysis of minimal residual disease following A treatment is being performed to determine whether pretreatment ABC levels identify pts who benefit from A therapy.CD52 ABC in Common Cytogenetic SubsetsCytogenetic SubsetsCD52 ABC on BlastsTotal number of cases studies (n=39N(%)MedianRangeLoss of 9p*61467,323.5(13,702–206,952)t(9;22)(q34;q11.2)143237,558.5(11,771–135,085)Normal karyotype122736,127.5(5,829–111,443)t(4;11)(q21;q23)**7163,826(1,685–4,950)* 5 patients had del(9p), 1 patient had i(9)(q10) ** including 1 patient with t(11;19)(q23;p13.3)

PRAME Protein Is Aberrantly Expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma.

PRAME (Preferentially Expressed Antigen in Melanoma) gene is located on chromosome 22 (22q11.22) and is expressed at low levels by normal adrenal, ovarian and endometrial cells. In contrast, its expression has been demonstrated at high levels in several types of cancers, such as in acute myeloid and lymphoid leukemias and multiple myeloma . We recently have reported that PRAME is also expressed in lymphoproliferative diseases and its transcripts were detected in 26 out 58 patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Nevertheless, the expression of PRAME protein in normal and neoplastic tissues is unknown. In order to address this question, we produced a monoclonal antibody against PRAME protein. A 562-nt fragment of the PRAME cDNA that includes the region that codes for the immunogenic 9-peptide was clone in pCR2.1-TOPO vector in DH5 alpha cells and subcloned into a pET24a expression vector, and the 196-amino acid peptide was expressed in BL21(DE3) cells as His-taged protein. The monoclonal antibody (MoAb) against PRAME was generated from splenocytes from mice immunized with the purified peptide. We then analyzed PRAME expression by flow cytometry in samples of peripheral blood (PB, n=15), bone marrow (BM, n=3) from healthy donors; in tonsils (n=3) from patients submitted to tonsilectomy for non malignant diseases and in PB samples from 26 CLL and 7 MCL patients. PRAME positive cells represented less than 15% of cells from normal PB, BM and tonsil cells. In contrast, 25 out of 26 CLL and 6 out of 7 MCL cases presented more than 20% of PRAME + cells [mean 59% (range:20–95%) and 75% (20–94%) for CLL and MCL, respectively]. The lymphoid cells from normal BM and tonsils that expressed PRAME were CD19+CD10+ CD27+ CD38± TdT− cIgM− CD5− suggesting that PRAME is expressed early during lymphoid ontogenesis and that its expression in CLL and MCL cells is aberrant. Furthermore, PRAME expression in normal lymphocytes was dimmer than in CLL and MCL cells. To quantify PRAME expression we evaluated PRAME Specific Antibody Binding Capacity (SABC) by a quantitative flow cytometry (QFC) method. The mean values of SABC were of 10,339 sites per cell (range 3,075 to 24,665) and of 14,191 sites per cell (range 5,059 to 20,679) for CLL and MCL, respectively. In contrast, in normal PB lymphocytes SABC values ranged from 1,628 to 1,781 sites per cell. Even in the two cases of CLL and MCL that had less than 20% PRAME+ cells, PRAME SABC was 3,075 and 12,347 sites per cell respectively, therefore significantly higher than the observed in normal PB lymphocytes. To evaluate the sensitivity of the QFC method, we established a cut off value for PRAME SABC based on the highest value detected in normal PB cells and then serially diluted tumor MCL cells marked with PRAME in normal PB lymphocytes. Based exclusively on PRAME expression, we were able to detected neoplastic MCL cells up to a 1:1000 dilution. In conclusion, PRAME protein is strongly expressed in the neoplastic clone in most patients with CLL and MCL. This finding supports the suggestion that this antigen may be further explored as a target for diagnostic, to detect minimal residual disease detection and for therapeutic approaches.

Detection of antilineage specific leucocyte antibodies by a quantitative immunocytometry method in sera from candidates for renal allografts

A sensitive and quantitative flow cytometry method for detecting antileucocyte antibodies was developed to study retrospectively samples from candidates using frozen donor cells and frozen recipient sera. This immunofluorescence method was used to compare levels of reactivities of serum antibodies before and after donor specific blood transfusion treatment and after renal transplantation. The results demonstrate that the flow cytometry crossmatch is a sensitive and accurate method which should be used prospectively before precluding transplantation in the presence of a positive B cell standard crossmatch. Antibodies detected by flow cytometry before transplantation could be responsible for an early acute rejection episode. Finally, combination of flow cytometry crossmatch and standard crossmatch assays might thus be useful before precluding transplantation in living related donors.

Impaired Fcα receptor expression is linked to increased immunoglobulin A levels and disease progression in HIV-1-infected patients

Expression of immunoglobulin (Ig) A Fc receptors (Fc alpha R) and their saturation by endogenous IgA were studied on blood monocytes and neutrophils to evaluate the role of Fc alpha R in the formation of increased serum levels of IgA and IgA-immune complexes (IgA-IC) observed during HIV-1 infection. Peripheral blood samples were obtained from 45 patients at different stages of HIV-1 infection and from 22 healthy volunteers. This study was performed using a quantitative flow cytometry method in which blood cells were stained with anti-Fc alpha R monoclonal antibodies (MAb) recognizing epitopes outside the IgA-binding site and with F(ab')2 fragments of anti-IgA antibodies. Immunoprecipitations of radiolabelled surface Fc alpha R molecules were analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis under glycosylated and deglycosylated conditions. This study reveals a diminished surface expression of Fc alpha R on blood monocytes of HIV-1-infected patients, which follows disease progression. Fc alpha R molecules on patients' neutrophils have a higher apparent molecular mass (60-90 kD) with normal protein core, suggesting expression of receptors with altered carbohydrate moieties. Increased levels of serum IgA significantly correlate with decreased levels of Fc alpha R in HIV-1-infected patients. Surface Fc alpha R molecules are saturated by endogenous IgA1 in both cell types. These findings suggest that defective expression and/or altered glycosylation of Fc alpha R may result in receptor saturation, impairment of IgA catabolism and diminished clearance of IgA-IC in HIV-1-infected patients. Fc alpha R expression represents a new marker for disease progression.

Impaired Fcα receptor expression is linked to increased immunoglobulin A levels and disease progression in HIV-1-infected patients

Objectives: Expression of immunoglobulin (Ig) A Fc receptors (FcαR) and their saturation by endogenous lgA were studied on blood monocytes and neutrophils to evaluate the role of FcαR in the formation of increased serum levels of lgA and IgA-immune complexes (IgA-IC) observed during HIV-1 infection. Methods: Peripheral blood samples were obtained from 45 patients at different stages of HIV-1 infection and from 22 healthy volunteers. This study was performed using a quantitative flow cytometry method in which blood cells were stained with anti-FcαR monoclonal antibodies (MAb) recognizing epitopes outside the IgA-binding site and with F(ab')2 fragments of anti-IgA antibodies. Immunoprecipitations of radiolabelled surface FcαR molecules were analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis under glycosylated and deglycosylated conditions. Results: This study reveals a diminished surface expression of FcαR on blood monocytes of HIV-1-infected patients, which follows disease progression. FcαR molecules on patients' neutrophils have a higher apparent molecular mass (60-90kD) with normal protein core, suggesting expression of receptors with altered carbohydrate moieties. Increased levels of serum IgA significantly correlate with decreased levels of FcαR in HIV-1-infected patients. Surface FcαR molecules are saturated by endogenous IgA1 in both cell types. Conclusion: These findings suggest that defective expression and/or altered glycosylation of FcαR may result in receptor saturation, impairment of IgA catabolism and diminished clearance of IgA-IC in HIV-1-infected patients. FcαR expression represents a new marker for disease progression.

Flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin, its subunits, and fragments on human cancer cells.

A quantitative flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin (hCG), its subunits, and fragments on human cancer cells was developed using a double-antibody reaction; a flow cytometry with a 2-W argon laser, standard settings, and filters for fluorescein isothiocyanate use; commercially available software; and the ectopic hCG producer CCL 2 HeLa cells from the American Type Culture Collection (ATCC) as a cell control to standardize the reagents and for overall quality control. Twenty-two monoclonal antibodies (MoAb) and immunoglobulin G fractions from three rabbit polyclonal antisera were tested for effects of antibody concentration (titration), reproducibility at different levels of epitope expression, and variability of epitope expression to select appropriate primary antibodies. Based on the results of the various tests, three polyclonal immunoglobulin G antibodies and a panel of nine MoAb directed to epitopes located in five different regions on the hCG molecule were selected as first antibodies. Their specificity was determined by using two unrelated MoAb of the same isotype at the same concentration to replace the primary MoAb and by a competition experiment. The unrelated MoAb also were used for the selection of the appropriate control fluorescence profile needed for the software. The unique characteristics of this method were: the use of living cells, standardized reagents, internal and external quality control, and the highest sensitivity, which could detect as few as 10(3) molecules of fluorochrome per cell. Serial analyses of the ATCC CCL 2 HeLa cells and two of its variants and of the eutopic hCG producer JEG-3 choriocarcinoma cells revealed the expression of membrane-associated epitopes of intact hCG, its subunits, and fragments by a high percentage of the cells, indicating that the expression of these sialoglycoproteins by these two different types of cancer cells is a common phenotypic characteristic.