SEARCH · PubMed Health

Results for “Flow Cytometry”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 793 records · Page 44Linked to original sources

PubMed2001

Assessment of the human cellular immune response to T27K, a coccidioidal antigen preparation, by flow cytometry of whole blood.

Whole blood flow cytometry was performed among donors with various clinical forms of coccidioidomycosis using T27K, a coccidioidal antigen preparation protective in mice but not previously studied in humans. The median percent of CD3+ lymphocytes (CD3+) producing intracellular interferon-gamma (IFN-gamma) among healthy immune donors was 0.43%, significantly above that for non-immune donors (0.01%) and greater than that for subjects with other forms of coccidioidomycosis, including chronic pulmonary (0.11%), disseminated (0.09%) and concomitant human immunodeficiency virus (HIV) infection (0.07%) (P < or =0.002 for all). No increase in intracellular interleukin (IL)-10 production or apoptosis was noted in samples incubated with T27K. Among 14 HIV-infected patients with concomitant coccidioidomycosis, seven of eight patients whose peripheral blood CD4 concentration was > 200 cells microl(-1) had > 0.06% of CD3+ produce intracellular IFN-gamma, compared to none of six whose peripheral blood CD4+ lymphocyte concentration was < or =200 cells microl(-1) (P = 0.005). These data indicate that there is a specific human cellular immune response to T27K as a coccidioidal antigen and that this response can be categorized based on the clinical status of the coccidioidally infected patient.

AIDS-Related Opportunistic Infections↗

PubMed1985

Coexpression of T4 and T8 on peripheral blood T cells demonstrated by two-color fluorescence flow cytometry.

Using two-color fluorescence flow cytometry, we were able to detect the presence of small numbers of T4+T8+ cells (about 3%) in freshly isolated peripheral T cell populations derived from normal healthy donors. Coexpression of T4 and T8 was predominantly found on large blastlike cells and appeared to be related to activation. Stimulation of peripheral T cells with concanavalin A (Con A) for 5 days resulted in the generation of up to 60% of T4+T8+ cells. Coexpression was accompanied by a twofold increase in the number of T8 antigenic sites per cell. The T4+T8+ cells in lectin-stimulated cultures expressed high levels of the activation antigens T9, T10, and the IL-2 receptor but lacked T6, an antigen found on a majority of stage II thymocytes. Coexpression of T4 and T8 appeared to be a transitory process, because prolonged culture of T cells in the absence of lectin resulted in the loss of the T4+T8+ phenotype. Our data suggest that T cell activation in peripheral blood results in the generation of a T4+T8+ cell population which is distinct from previously described thymic and peripheral blood cells. Because T4 and T8 molecules may interact directly with MHC antigens, coexpression of these molecules may have an important role in immune function.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2006

A false expression of CD8 antigens on CD4+ T cells in a routine flow cytometry analysis.

The two-colour flow cytometry method applied in a routine enumeration of peripheral blood T lymphocyte subsets reveals that in some patients the entire population of CD4+ lymphocytes seems to express CD8 determinants as well. However, expression of the CD8 antigens on the cell surface is much lower in comparison with typical CD8+ cells. Moreover, in one-colour staining with an anti-CD8 antibody, cells with weak CD8 expression are not observed and only one typical population of CD8+ lymphocytes is seen. Investigating this phenomenon, we showed that after washing patient cells in RPMI before CD4/CD8 staining, the CD4+ T cell population did not show CD8 "co-expression". These results suggest that CD4+ lymphocytes, which seem to co-express CD8 antigen, in fact do not have this antigen on their surface. Moreover, after the addition of patient plasma to healthy donor cells prior to CD4/CD8 staining, a weak CD8 expression on normal CD4+ cells was noticed. Therefore we can assume that the agent(s) causing this phenomenon is/are present in the plasma of some patients. Altogether, these observations suggest that this phenomenon is nonspecific and probably results from cross-linking of anti-CD8 mAbs with anti-CD4 mAbs caused by factor(s) present in plasma of some patient. However, identification of that/these factor(s) requires further research.

Adult↗

PubMed1987

Effects of the fungicide methyl-benzimidazol-2-yl carbamate (MBC) on mouse germ cells as determined by flow cytometry.

Dual-parameter (DNA, RNA) flow cytometry (FCM) measurements were made on testicular and epididymal sperm cells isolated from mice exposed by oral gavage to 0, 250, 500, or 1000 mg/kg X 5 d of the fungicide methylbenzimidazol-2-yl carbamate (MBC), which is known to bind with tubulin subunits and inhibit polymerization and microtubule formation. Effects of exposure to MBC were measured at 7, 24, and 39 d posttreatment. MBC had no effect on body weights, but testis weights and sperm parameters were altered, with few exceptions, only at the highest exposure level. Testis weights were reduced by about 25% at 7 and 24 d after exposure; recovery was observed by 39 d after treatment. FCM measurements of testicular cells showed relative percentages of certain testicular populations (round, elongating, and elongated spermatids) were different from the control pattern 7 and 24 d after treatment. The mean percent of cauda epididymal sperm head morphology abnormalities and the susceptibility of the nuclear DNA to denaturation were both elevated at 7, 24, and 39 d after exposure to 1000 mg/kg. The level of denaturation was determined by FCM measurements of the metachromatic shift in acridine orange (AO) stained sperm nuclei from green (native DNA) to red (single-stranded DNA) fluorescence and quantitated by the expression alpha t[red/(red + green] fluorescence. These data demonstrate that spermatogenesis is sensitive to high-dose MBC exposure resulting in an altered ratio of testicular cell types present, abnormal sperm head morphology, and an altered sperm chromatin structure.

Administration, Oral↗

PubMed1993

Detection of c-erbB-2 oncoprotein expression in breast tissue by multiparameter flow cytometry.

A novel technique in multiparameter flow cytometry (FCM) using dual laser excitation of three fluorescent dyes has been developed to differentiate breast epithelial cells from stromal components. This technique has been applied to determine the expression of the oncoprotein c-erbB-2 in breast epithelial tumors. SK-BR-3, a breast cancer cell line with c-erbB-2 overexpression, can be identified by FCM from a mixed cell suspension using a monoclonal anti-human cytokeratin antibody conjugated with fluorescein isothiocyanate. Using the mouse monoclonal anti-c-erbB-2 antibody, TA-1 (4.8 +/- 1.0 x isotype control), the c-erbB-2 oncoprotein overexpression in breast epithelial cells can be detected as an increase in indirect blue fluorescence by aminomethyl coumarin. MCF-7, a breast cancer cell line with normal c-erbB-2 expression, has baseline blue fluorescence (1.0 +/- 0.5 x isotope control). Twenty-one fresh breast specimens have been examined by FCM. Overexpression of c-erbB-2, defined by blue fluorescence ratio of TA-1/isotype control > or = 1.5 (> 3 SD from baseline), is detected in 0 of 3 patients with Stage I cancer, 5 of 14 patients with Stage II and III cancer, and 3 of 4 patients with proliferative disease. Patients with elevation of oncoprotein detected by FCM have corresponding RNA overexpression detected by Northern blot hybridization and increased gene amplification detected by Southern blot hybridization. FCM allows for the simultaneous identification of breast epithelial cells and the selective examination of these cells for the expression of c-erbB-2 oncoprotein, thus minimizing stromal contamination. This represents a novel application of FCM with potential for wide clinical applicability.

Biomarkers, Tumor↗

PubMedDate not supplied

[Breast cancer: prognostic value of flow cytometry and hormone receptors. Apropos of a Lebanese series of patients].

OBJECTIVES: This study has three objectives. 1) First to establish the influence of flow cytometry and hormone receptors on overall survival and disease free survival of stage I to IIIA breast cancer patients. 2) Second to search for associations between the classical prognostic factors: age, menopausal status, histological and nuclear grading, tumor diameter, node metastases, stage, estrogen and progesterone receptors (ER, PR), as well as ploidy and S phase fraction. 3) And third to evaluate the effect of flow cytometry and hormone receptors on the response to neoadjuvant chemotherapy in stage IIIB and IV patients. MATERIAL AND METHODS: The series is composed of 150 patients with primary invasive breast cancer (149 females and one male), diagnosed at the St George Hospital Pathology Department and in whom an evaluation of the hormone receptors and a measuring, by flow cytometry, of ploidy and S phase fraction (SPF) were performed. Among them, 68 were followed for a mean period of 45 months. The disease free survival and the overall survival were recorded as well as the response to neoadjuvant chemotherapy. RESULTS: The authors did not find any statistically significant difference in survival between diploid and aneuploid tumors, tumors with SPF above and below 8%, those who are ER+ and ER- and those who are PR+ and PR-. But it seemed that diploid, low SPF and positive hormone receptors tumors had a tendency toward a better disease free survival. The multivariate analysis using the Cox proportional hazards identified the tumor diameter as being the only independent prognostic factor (p = 0.046). The nuclear grade reached a limit of significance (p = 0.08). Diploidy, low SPF and positivity of ER and PR were associated one to the other and to low histologic and nuclear grades. Finally, neither flow cytometry parameters nor hormone receptors were predictive of a susceptibility to neoadjuvant chemotherapy.

Adult↗

PubMed1995

Detection of human growth hormone receptors on IM-9 cells and peripheral blood mononuclear cell subsets by flow cytometry: correlation with growth hormone-binding protein levels.

We have developed a method using flow cytometry to identify fluorescein-conjugated GH receptors (GHR) on IM-9 lymphocytes and circulating peripheral blood mononuclear cell subsets. Binding to IM-9 cells and peripheral blood mononuclear cells was concentration dependent and could be competitively blocked by the addition of unlabeled human GH, but not by the addition of rat or bovine GH or human insulin or PRL. Using two-color flow cytometric analysis, fluorescein-conjugated human GHR were readily detected on more than 90% of B lymphocytes and monocytes, but only variably on T lymphocytes. B Lymphocytes and monocytes had approximately 6000 GHR/cell. Using two-color flow cytometry, we identified GHR on circulating B lymphocytes in subjects with GH deficiency (n = 9), precocious puberty (n = 6), and Turner syndrome (n = 5) and in seven subjects with miscellaneous disorders, including familial short stature, bone dysplasia, Crohn disease, congenital adrenal hyperplasia, and acromegaly. The percentage of B lymphocytes expressing GHR in subjects with GH deficiency (mean +/- SD, 95 +/- 9%), precocious puberty (91 +/- 15%), and Turner syndrome (84 +/- 15%) was not different from that in normal volunteers (90 +/- 12%; n = 14). In 10 subjects, serum GH-binding protein levels were assayed simultaneously with B lymphocyte GHR. GH-binding protein was normal in all (mean, 1255 pmol/L; range, 773-1809). There was a good correlation between GHR expression on B lymphocytes and GH-binding protein levels (r = 0.75; P = 0.01). We postulate that GHR found on circulating B lymphocytes may contribute to the pool of receptors identified in serum as GH-binding proteins. Two-color flow cytometry appears to be an effective method for the detection of GHR on circulating peripheral blood mononuclear cell subsets. The evaluation of GHR on circulating B lymphocytes may prove to be a useful means of evaluating GH-GHR interactions in subjects with growth disorders.

Adult↗

PubMed1998

New method for quantitative determination of fetal hemoglobin-containing red blood cells by flow cytometry: application to sickle-cell disease.

A new method to quantify red blood cells (RBCs) containing fetal hemoglobin (HbF), or F cells, by flow cytometry was developed. The use of formaldehyde as fixative agent and sodium dodecyl sulfate (SDS) to permeabilize fixed RBCs resulted in a low staining background, a negligible Hb leakage, and minimal cell clumping. HbF was detected by immunofluorescence with a murine monoclonal antibody directed to the gamma chain of human Hb. The accuracy of the F-cell count was evaluated on mixed-field populations of adult and cord RBCs. The results of flow cytometry were highly correlated with HbF dosage in the hemolysate by high-pressure liquid chromatography (HPLC) (R > 0.99). The sensitivity of the method allowed the study of HbF distribution at the single-cell level in normal controls and patients with sickle-cell disease (SCD). All patients exhibited a heterocellular distribution of HbF, with highly variable percentages of F cells and non-F cells. As an additional and valuable biological parameter in SCD, the mean HbF content per F cell could be deduced from the measurement of HbF level by HPLC and F-cell count by flow cytometry. Results were unchanged after storing the cells for several weeks in an optimized resuspending solution. Since it does not need uncommon reagents or devices and allows the simultaneous detection of membrane antigens by two-color flow cytometry, this method is convenient for routine laboratories as well as for research purposes.

Adult↗

PubMed1996

Immunocytochemistry and flow cytometry evaluation of human megakaryocytes in fresh samples and cultures of CD34+ cells.

Adhering platelets on the cell surface can give misleading results when doing flow cytometry analysis of platelet/megakaryocyte-specific glycoprotein (GP) antigens to enumerate megakaryocytes (MK) in mobilized peripheral blood (PB), apheresis products, or normal bone marrow (BM). For adequate quantification and characterization of human MK, we examined samples with parallel flow cytometry and immunocytochemistry. MK expression of GP IIb/IIIa (CD41a), GP Ib (CD42b), GP IIIa (CD61), CD45, CD33, and CD11b, and their light scatter properties were evaluated. Fresh samples of low density mononuclear cells (MNC) or purified CD34+ cells contained 10-45% of platelet-coated cells. Platelet-coated cells decreased dramatically after several days of incubation in a serum-free medium supplemented with stem cell factor, IL-3, IL-6, and/or GM-CSF. Between d 9-12, flow cytometry detected a distinct CD41a+ MK population, 8.3 +/- 1.3% in BM CD34 cell cultures (n = 7) and 13.1 +/- 2.1% in PB CD34 cell cultures (n = 14), comparable to immunocytochemistry data (7.8 +/- 1.9% and 16.4 +/- 2.6%, respectively). CD41a stained a higher proportion of MK than CD42b or CD61, while CD42b+ or CD61+ cells contained more morphologically mature MK than CD41a+ cells in cultures containing aplastic serum. When fluorescence emission of CD41a was plotted against forward-light scatter (FSC), subpopulations of small and large MK were observed. Such subpopulations overlapped in CD41a intensity and side-light scatter (SSC) property. Most MK co-expressed CD45 (98.8% positive) but not CD33 (80.7% negative) or CD11b (88.9% negative). Our data indicate that flow cytometry can be used effectively to identify MK. However, caution should be taken with samples containing adherent platelets.

Adult↗

PubMed1996

Clinical evaluation of cell deoxyribonucleic acid content measured by flow cytometry in bladder cancer.

PURPOSE: We evaluated the clinical value of flow cytometry in bladder cancer. MATERIALS AND METHODS: Deoxyribonucleic acid (DNA) content was measured by flow cytometry in 275 untreated patients with bladder tumor followed for 1 to 8 years. Four pathological parameters (stage, grade, observed vascular invasion and associated carcinoma in situ) and 3 flow cytometric parameters (ploidy, number of aneuploid cell lines and DNA index) were defined. RESULTS: Univariate survival analysis showed that every parameter, when considered separately, was a significant prognostic factor (p < 0.0001 in call cases). Multivariate analysis showed that stage (p < 0.0001), DNA index (p < 0.01) and associated carcinoma in situ (p < 0.05) were independent, significant prognostic factors. However, ploidy and DNA index enhanced prognostic information above the traditional stage and grade only in patients with a stage pT1, grade 3 tumor (p < 0.05). Retrospectively, different therapeutic decisions could have been made using DNA content only in 4% of cases. CONCLUSIONS: In patients with bladder cancer DNA content is an independent predictor of survival but its clinical usefulness is limited.

Aged↗

PubMed1999

Utility of flow cytometry in subtyping composite and sequential lymphoma.

Composite lymphoma (CL) is defined as more than one distinct lymphoma variant occurring in the same anatomic site, and sequential lymphoma (SL) is defined as different lymphoma variants occurring at different sites or at different times in the same patient. The utility of flow cytometry immunophenotyping in evaluating CL and SL has only been investigated in a few single-case studies. To further define the utility of flow cytometry in evaluating these tumors, records were searched at two institutions. Cases representing high-grade progression of low-grade lymphoma were excluded. For each CL/SL, clinical data was obtained and morphology was evaluated in routinely processed H&E-stained tissue sections. Tumor components were subtyped using revised European-American classification (REAL) criteria. Follicle center components were graded using modified Rappaport criteria. Immunophenotype was determined using two-color flow cytometry and paraffin-section immunostains. Four cases were identified. Case 1, nodal follicle center, follicular, grade III plus marginal zone CL, showed two discrete populations of monoclonal B-cells that differed in their expression of CD10. Case 2, cutaneous lymphoplasmacytoid lymphoma followed by mesenteric non-Hodgkin's lymphoma (lymphoplasmacytoid plus follicle center, follicular, grade III) plus Hodgkin's disease CL, showed CD5-/CD10-/CD19+/kappa+ cells by flow cytometry in both tissue samples. The Hodgkin's disease component showed CD3-/CD15-/CD20-/CD30+ Reed-Sternberg cell variants in paraffin-section immunostains. Case 3 represented nodal follicle center lymphoma, follicular, grade I (CD3-/CD5-/CD10-/CD19+/kappa+) followed by cutaneous anaplastic large T-cell lymphoma (CD2+/CD4+/CD5+/CD19- cells with partial expression of CD3 and CD7). Case 4 represented cutaneous follicle center lymphoma, follicular, grade I (CD5-/CD10+/CD19+/CD23+/lambda+) followed by bone marrow B-cell small lymphocytic lymphoma (CD5+/CD10-/CD19+/CD23+/kappa+). Results show that flow cytometry is a potentially useful adjunct in characterizing CL and SL.

Aged↗

PubMed2005

Validation of large particle flow cytometry for the analysis and sorting of intact pancreatic islets.

BACKGROUND: Accurate quantification of total islet yield is an essential step prior to transplantation and for research. The standard method of manually determining an islet equivalent (IEQ) count is subjective and prone to error. We evaluated Complex Object Parametric Analyzer and Sorter (COPAS) large particle flow cytometry for the determination of islet equivalent counts and purities of islet preparations. METHODS: Initial validation of the sensitivity and accuracy of the COPAS flow cytometer was performed by analysis and sorting of uniform polystyrene microspheres with sizes similar to islets. Human and Rhesus monkey islets were stained with the zinc-specific fluorescent dye Newport Green to discriminate islet from nonislet tissue. Islet sizes were extrapolated from standard curves obtained using microspheres from which individual islet volumes were calculated. IEQ counts on six islet preparations were performed by the standard manual method and compared with results obtained by automated COPAS flow cytometry. RESULTS: The COPAS flow cytometer was highly accurate in the detection and measurement of both polystyrene microspheres and islets. IEQ counts determined by COPAS flow cytometry were consistent with manual counts although subject to error when assessing preparations with significant numbers of islets embedded within acinar tissue. Size-specific islet sorting with retention of morphology and dithizone staining was also shown using the COPAS flow cytometer. CONCLUSIONS: COPAS large particle flow cytometry provides a novel automated approach for quantification of intact islets and determination of islet equivalent yield. In addition, the ability to analyze and sort islets based upon user defined criterion opens unique avenues for experimentation.

Animals↗

PubMed1985

Evaluation of colonic neoplasia by flow cytometry of endoscopic biopsies.

Colonoscopy is a valuable and frequently used method in the evaluation of colonic neoplasia. Flow cytometry is a technique that can be used to diagnose malignancy. In this study, flow cytometry was used to evaluate colonoscopic biopsies taken from patients with suspected colonic neoplasia. Nineteen colonic biopsies were obtained and evaluated by this technique. Aneuploidy was demonstrated in six patients with carcinoma of the colon. In addition, abnormal DNA histograms were noted in two premalignant conditions (colonic adenoma and inflammatory bowel disease). The results show that flow cytometry can be applied to colonic biopsies and suggest that it may be of use in the diagnosis of malignant and premalignant conditions of the colon.

Adenoma↗

PubMedDate not supplied

Reagents for measuring intracellular enzyme activity with flow cytometry.

The line of enzyme substrates described, together with the flow cytometer, bring a simplified method of measuring intracellular enzyme activity to the research laboratory. They are optimized for use in flow cytometry but can also be adapted to other fluorescent detection methods (e.g., spectrofluorometry, fluorescent microscopy, and image analysis) (Figure 5). The large number of enzymes that can be measured with the reagents (currently 37), together with the automation and multiparametric capabilities of flow cytometry, offer many advantages over conventional methods of cell characterization. They also expand the potential of functional assays for studying many normal physiological and disease processes.

Animals↗

PubMed2005

Evaluation of apoptosis and necrosis induced by statins using fluorescence-enhanced flow cytometry.

The purpose of this study was to evaluate the apoptosis and necrosis induced by five kinds of statins in IM-9 human lymphoblasts with fluorescence-enhanced flow cytometry using avidin-biotin complex. IM-9 human lymphoblasts (2 x 10(4) cells/cm2) were seeded into tissue culture plates and incubated with five kinds of statins. Statin-treated cells were first incubated with biotin-annexin V, followed by addition of avidin-FITC and propidium iodide, and then subjected to flow cytometry. The fluorescence intensity was enhanced using an avidin-biotin complex system, resulting in successful separate determination of the statin-induced apoptosis and necrosis by flow cytometry, which enabled us to quantitatively evaluate the statin-induced cell damage. Flow cytometric analysis results in the intensity of statin-induced apoptosis in IM-9 cells as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. The intensity of statin-induced necrosis in IM-9 cells was expressed as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. The total damage of IM-9 cells induced by five kinds of statins were expressed as the sum of both percentages of apoptosis and necrosis as follows: atorvastatin cerivastatin>fluvastatin simvastatin>pravastatin. Our studies show that fluorescence enhancement with avidin-biotin complex is useful for the identification and quantitation of annexin-positive apoptosis cells and thus, the fluorescence-enhanced flow cytometry was shown to be applicable for screening of statins as new anti-leukemia agents.

Apoptosis↗

PubMed2000

[Detection of minimal residual disease in acute leukemias by flow cytometry].

Detection of minimal residual disease in acute leukemias by flow cytometry has become an interesting tool for the analysis of minimal residual disease in acute leukemias. Some complementary studies can be used for this purpose such as conventional cytogenetics, FISH and molecular biology (PCR and Southern Blot). Flow cytometry study can detect smoldering blast cells in patients with acute leukemias who appear to be in complete remission by conventional morphologic criteria (< 5% of blast cells in bone marrow aspirate sample). This method can recognize 1 blast in 10(-4) mononuclear bone marrow cells considering the characteristics of immunophenotype at diagnosis (aberrant, asynchronous and overexpressed phenotypes) or using the analysis cell maturation to identify normal and abnormal patterns. These differences can be distinguished when the cells occupy the 'empty spaces' where no normal cells are evident. The principal objective of detecting low levels of tumor cells is to obtain more information about the efficacy of the treatment in order to: 1) design adapted post-remission protocols for high risk patients, 2) predict relapses previous to the clinical manifestations, 3) study the quality of stem cells harvested for autologous bone marrow transplantation.

Flow Cytometry↗

PubMed1995

Flow cytometry: its use in pediatric renal transplantation utilizing polyclonal induction.

Induction protocols for pediatric renal transplant recipients commonly utilize polyclonal or monoclonal agents in sequence with additional immunosuppression. Polyclonal agents Minnesota antilymphoblast globulin (MALG), anti-thymocyte globulin (ATGAM) effect both T and B cells while monoclonal agents (OKT3) are T-cell specific. Flow cytometric analysis of T-cell subsets has become the marker of adequacy of immunosuppression during OKT3 therapy, yet to date no marker exists to measure the adequacy of immunosuppression with polyclonal agents. At the University of Michigan, flow cytometric analysis in pediatric renal transplant recipients undergoing polyclonal induction reveals that CD3, CD4, and CD8 suppression initially occurs. As opposed to OKT3, a rebound of CD3 cells occurs despite daily use of a polyclonal agent in sequence with additional immunosuppressives. During these analyses, a single kidney was lost which flow cytometry failed to predict. No significant difference in flow cytometric patterns occurred when comparing ATGAM to MALG induction. Flow cytometry utilized during polyclonal induction in pediatric renal transplant recipients revealed a variety of T-cell suppression patterns but failed to demonstrate persistence of suppression. Despite this lack of suppression as indicated by flow cytometry, a 97% 1 year allograft survival rate exists in the pediatric transplant program at the University of Michigan Medical Center. Therefore, the role of flow cytometry during polyclonal induction has yet to be well defined.

Antibody Formation↗

PubMed1996

Use of a directly conjugated monoclonal anti-D (BRAD-3) for quantification of fetomaternal hemorrhage by flow cytometry.

BACKGROUND: Determination of the volume of fetal D-positive cells in the circulation of D-negative women after delivery is carried out to determine whether additional prophylactic anti-D should be given to the mother. Although the Kleihauer-Betke test is still widely used to calculate the fetomaternal hemorrhage, increasing use is being made of flow cytometry. STUDY DESIGN AND METHODS: A conjugated monoclonal anti-D was prepared by labeling purified BRAD-3 (IgG3) with fluorescein isothiocyanate (FITC-BRAD-3). This reagent was used to label D-positive red cells by a one-step procedure: 5 microL of washed cells were incubated with 50 microL of FITC-BRAD-3 (50 micrograms/mL) at 37 degrees C for 30 minutes; then the cells were washed and 500,000 events were analyzed by flow cytometry. RESULTS: The FITC-BRAD-3 reagent effectively labeled D-positive cells. The percentage of D-positive cells in mixtures containing more than 0.04 percent D-positive cells in D-negative cells was accurately determined by using this reagent and flow cytometry. Although the Kleihauer-Betke test was more accurate than this one-step flow cytometric method at quantifying fetomaternal hemorrhage of < 1 mL, the flow cytometric method was more accurate in the 1- to 7-mL fetomaternal hemorrhage range of 1 to 7 mL (whole-blood equivalents). Analysis of 175 clinical samples for fetomaternal hemorrhage gave consistent quantification results with the three methods used: the Kleihauer-Betke test, flow cytometry with FITC-BRAD-3, and flow cytometry with polyclonal anti-D followed by FITC-anti-IgG. CONCLUSION: Labeling of samples with FITC-BRAD-3 was simple and rapid. By flow cytometric analysis, good separation of D-positive from D-negative cells was obtained, and fetomaternal hemorrhage of > 1 mL was quantified accurately.

Antibodies, Monoclonal↗