BACKGROUND:The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (P = 0.86) and superior precision with a CV c15 percent for blood samples with >0.1 percent fetal cells.Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies. he detection of circulating fetal cells in maternal blood represents an important area of laboratory support to the obstetrical management of ABBREVIATIONS: ELISA = enzyme-linked immunosorbent assay; FMH = fetomaternal hemorrhage; HAT = hypoxanthine aminopterin thymidine; Hb = hemoglobin; HbA = hemoglobin A; HbF = hemoglobin F; PBS = phosphate-buffered saline; PBS-BSA = PBS with bovine serum albumin; RBC(s) = red cell(s); RhIG = Rh immune globulin; WBC(s) =white cell(s). From the Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan.

The primary purpose of the study was to examine circulating neutrophils and monocytes and their plasma membrane expression of CD64, CD11b, and CD18 after two bouts (B1 and B2) of eccentric exercise. Subjects (n = 10) performed 25 forced-lengthened contractions of the forearm flexors on two occasions separated by 3 wk. Blood samples were obtained before exercise and at 1.5, 6, 12, 24, 48, 72, and 96 h of recovery. CD64, CD11b, and CD18 expression was determined via direct immunofluorescence and used as an indicator of neutrophil and monocyte activation. Creatine kinase activity (B1 = 1,390, B2 = 108 U/l), myoglobin (B1 = 163, B2 = 41, ng/dl), and muscle soreness and tenderness were higher (P < 0.01) after B1 compared with B2. Neutrophils at 6, 12, and 96 h were higher (P < 0.05) for B1 vs. B2. CD11b expression on neutrophils was 2.7-fold higher at 72 h for B1 vs. B2. CD64 expression on neutrophils at 72 and 96 h was 1.4- and 1.9-fold higher, respectively, for B1 vs. B2. At 72 and 96 h, CD18 and CD64 expression on monocytes was 1.3-fold higher for B1 vs. B2. The observed changes were not significantly correlated with changes in creatine kinase activity or myoglobin. In conclusion, the adaptation to eccentric arm exercise was associated with a reduction in circulating neutrophils and a lower state of neutrophil and monocyte activation.

Flow cytometric reticulocyte quantification with thiazole orange (TO) was used to study erythropoiesis in 20 patients following autologous bone marrow transplantation for the treatment of acute myelogenous leukemia. Flow cytometric reticulocyte analysis provided not only the reticulocyte percentage and absolute reticulocyte count but a quantitative reticulocyte maturity index (RMI) proportional to the amount of RNA in the reticulocytes. The RMI values, but not the reticulocyte percentage or absolute counts, correlated temporally with the rise in the absolute neutrophil counts in the posttransplantation period. In the majority of patients (12/20), the RMI value was the earliest indicator of bone marrow engraftment. The findings of this study demonstrate an important clinical utility of TO reticulocyte analysis by flow cytometry and indicate the diagnostic importance of the RMI measurement in the evaluation of erythropoietic activity in bone marrow transplant patients.

Flow cytometric reticulocyte analysis is superior to manual reticulocyte counting with respect to precision and sensitivity. Furthermore, because the fluorescence intensity of reticulocytes is directly proportional to the erythrocyte RNA content, flow cytometric analysis using thiazole orange gives a quantitative reticulocyte maturity index (RMI). Previous studies have demonstrated that the RMI parameter is the earliest indicator of bone marrow engraftment following transplantation. In the present study, we analyzed the correlation of the RMI to standard red cell parameters, reticulocyte percentage, and absolute reticulocyte count in 41 3 anemic patients. The correlation of RMI to serum erythropoietin (Epo) and serum transferrin receptor (TfR) was analyzed in a subset of anemic blood samples. We found weak correlations between the RMI and hemoglobin (rZ = 0.0411, hematocrit (r2 = 0.0381, reticulocyte percentage (r2 = 0.0781, and absolute reticulocyte count (r2 = 0.087). Stronger correlations were observed between the RMI and Epo (r2 = 0.181) and the TfR (r2 = 0.191). The results indicate that the RMI represents a cost-effective measurement of erythropoietic activity and provides an additional parameter to classify anemic patients into categories of high and low erythropoietic activity, especially in hypoproductive anemias. o 1995 Wiley-Liss, Inc.Key terms: Erythropoiesis, erythrocyte, red cell, erythropoietin, transferrin receptor, thiazole orange It has recently been shown that flow cytometric reticulocyte analysis is more precise, more sensitive, and less costly than manual reticulocyte counting ( 5,6,8,10,15, 24,28). It is likely that this method will find widespread clinical use in the future for these reasons. In addition to accurate reticulocyte enumeration, because the measured fluorescence intensity is directly proportional to the amount of RNA in the immature erythrocytes, this method has the ability to quantitate reticulocyte maturity. Although reticulocyte maturation has been studied for over a century, only the flow cytometrically derived RMI appears to allow clinical utility (18). This quantitation of RNA in the reticulocytes using fluorescence intensity of thiazole orange-stained whole blood samples, termed the reticulocyte maturity index (RMI), has been shown to be an early predictor of bone marrow engraftment in transplant patients (2,1523). The parameter has also been reported to distinguish iron deficiency anemia from anemia of chronic disease (34). A similar RMI parameter can be derived from the Sysmex R series' dedicated reticulocyte analyzers based on the fluorescence intensity of auramine 0 reticulocyte staining, which has also been reported to be an early predictor of bone marrow engraftment (3,9,19,22) and parallels the increase in circulating CD34+ cells following granulocyte colony-stim-0 1 9 9 5 Wiley-Liss, Inc.ulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) priming prior to peripheral stem cell harvesting (27,29). However, no information on the ...

There is a clear need for improved indicators of infection or sepsis to increase the sensitivity and specificity of both diagnosis and therapeutic monitoring. One of the effects of inflammatory cytokines on the innate immune response is the rapid up-regulation of CD64 expression on the neutrophil membrane. We and others have hypothesized that the measurement of neutrophil CD64 expression might represent an improved diagnostic indicator of infection and sepsis. In this study we assessed the relative ability of flow cytometric neutrophil CD64 measurements, neutrophil counts, myeloid immaturity differential counts, and flagging on an automated hematology analyzer to correlate with the presence of infection, as determined by a retrospective clinical scoring system of infection or sepsis. A total of 160 blood samples were randomly selected to derive equal proportions of the 3 categories of flags on a Coulter STKS blood counter that indicate the presence of a myeloid left shift. The patients for these samples were scored by retrospective chart review and placed into 4 groups on the basis of likelihood of infection, sepsis, or severe tissue injury. Neutrophil CD64 expression demonstrated a superior sensitivity (94.1%), specificity (84.9%), and positive predictive likelihood ratio (6.24), compared with neutrophil counts (sensitivity, 79.4%; specificity, 46.8%; positive predictive likelihood ratio, 1.49), band counts (sensitivity, 87.5%; specificity, 43.5%; positive predictive likelihood ratio, 1.55), myeloid immaturity fraction (sensitivity, 94.6%; specificity, 84.5%; positive predictive likelihood ratio, 2.12), and flagging on an automated hematology analyzer (sensitivity, 94.1%; specificity, 40.5%; positive predictive likelihood ratio, 1.58). Relative to the other laboratory parameters, the neutrophil CD64 parameter also provided the best separation of the 4 clinical groups. The findings indicate that neutrophil CD64 expression as determined by quantitative flow cytometry is an improved diagnostic indicator of infection/sepsis relative to current laboratory indicators of relative or absolute myeloid cell counts or hematology analyzer flagging algorithms.

Assay of fetal hemoglobin (HbF) and/or HbF containing red blood cells (F+ cells) is essential for monitoring sickle cell and thalassemic patients, especially during treatment with HbF stimulators. Some previous flow cytometric methods contain several washing steps. This simplified method contains no washing step and takes less than an hour to perform. The %F+ cells in five mixtures of fetal red blood cells with adult red blood cells were nonsignificantly different in the original and simplified procedure. The %F+ cells of 12 patients compared in these two procedures were also not significantly different. The intra- and interassay %CVs do not exceed 3% and 7% respectively. EDTA, citrate, or heparin is suitable as anticoagulant and the samples can be stored at 4 degrees C for up to 2 weeks. The %F+ cells and %HbF [by high-performance liquid chromatography (HPLC)] of 83 samples were highly significantly correlated regardless of diagnosis. In conclusion, this new simplified flow cytometric method for F+ cells is simple, convenient, rapid, reproducible, and could be applied for monitoring sickle cell and thalassemic patients as an alternative to HPLC, where this is unavailable. It can also be applied as a fetal cell assay in fetomaternal hemorrhage.

BACKGROUND Quantitation of adult erythrocytes (RBC) containing fetal hemoglobin (F cells) is of potential clinical utility in evaluating erythropoietic disorders, such as myelodysplasia and hemoglobinopathies, and in monitoring F‐cell augmenting therapy. F‐cell counting methodologies include fluorescence microscopy and flow cytometry. Previous flow cytometric methods have employed an isotype antibody control to distinguish F cells from non‐F cells. We investigated the feasibility of using the orange autofluorescence signal (FL2) in glutaraldehyde‐fixed RBC to substitute for fluorescein isothiocyanate (FITC)‐labeled isotype control antibody use in F‐cell quantitation. METHODS Our previously published method for fetal red cell detection in fetomaternal hemorrhage was used, employing a FITC‐labeled anti‐hemoglobin F (HbF) monoclonal antibody reagent. Blood samples with varying F‐cell counts were quantitated for F cells using both immunofluorescence microscopy and flow cytometry comparing FITC‐labeled isotype to FL1 thresholding defined by FL2 autofluorescence. RESULTS F cell percentages obtained by using an FL2 defined threshold for FL1 gating correlated well with expected values in diluted blood samples (r2 = 0.994, slope = 1.019, intercept = 0.24), values obtained using an isotype control (r2 = 0.996, slope = 1.012, intercept = −0.17), and microscopic immunofluorescence counts (r2 = 0.989, slope = 0.999, intercept = −0.72). F‐cell quantitation by the isotype control and FL2 autofluorescence methods was also comparable in 40 blood samples (r2 = 0.994, slope = 1.014, intercept = 0.03). Intra‐assay, interobserver, and interinstrument precision with this autofluorescence gating method exhibited low imprecision (coefficient of variation <14%). CONCLUSION This novel method is a more objective and less laborious alternative for F‐cell quantitation by flow cytometry compared to using an isotype control or microscopy, thereby providing a more robust methodology for clinical studies and consideration as a laboratory reference method for F‐cell counting. Cytometry (Comm. Clin. Cytometry) 42:239–246, 2000. © 2000 Wiley‐Liss, Inc.

Background: Quantitation of adult erythrocytes (RBC) containing fetal hemoglobin (F cells) is of potential clinical utility in evaluating erythropoietic disorders, such as myelodysplasia and hemoglobinopathies, and in monitoring F-cell augmenting therapy. F-cell counting methodologies include fluorescence microscopy and flow cytometry. Previous flow cytometric methods have employed an isotype antibody control to distinguish F cells from non-F cells. We investigated the feasibility of using the orange autofluorescence signal (FL2) in glutaraldehyde-fixed RBC to substitute for fluorescein isothiocyanate (FITC)-labeled isotype control antibody use in F-cell quantitation.Methods: Our previously published method for fetal red cell detection in fetomaternal hemorrhage was used, employing a FITC-labeled anti-hemoglobin F (HbF) monoclonal antibody reagent. Blood samples with varying F-cell counts were quantitated for F cells using both immunofluorescence microscopy and flow cytometry comparing FITC-labeled isotype to FL1 thresholding defined by FL2 autofluorescence.Results: F cell percentages obtained by using an FL2 defined threshold for FL1 gating correlated well with expected values in diluted blood samples (r 2 ‫؍‬ 0.994, slope ‫؍‬ 1.019, intercept ‫؍‬ 0.24), values obtained using an isotype control (r 2 ‫؍‬ 0.996, slope ‫؍‬ 1.012, intercept ‫؍‬ ؊0.17), and microscopic immunofluorescence counts (r 2 ‫؍‬ 0.989, slope ‫؍‬ 0.999, intercept ‫؍‬ ؊0.72). F-cell quantitation by the isotype control and FL2 autofluorescence methods was also comparable in 40 blood samples (r 2 ‫؍‬ 0.994, slope ‫؍‬ 1.014, intercept ‫؍‬ 0.03). Intra-assay, interobserver, and interinstrument precision with this autofluorescence gating method exhibited low imprecision (coefficient of variation <14%).Conclusion: This novel method is a more objective and less laborious alternative for F-cell quantitation by flow cytometry compared to using an isotype control or microscopy, thereby providing a more robust methodology for clinical studies and consideration as a laboratory reference method for F-cell counting. Cytometry (Comm. Clin. Cytometry) 42:239 -246, 2000.