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N C Bigelow

Publications and source records attributed to N C Bigelow.

10 recordsLinked to original sources
PubMed1998

Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry.

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 (r2 = 0.86) and superior precision with a CV < 15 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.

Adult↗
PubMed1996

Flow cytometric HLA-B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation.

The determination of HLA-B27 (B27) status is important in the diagnosis of ankylosing spondylitis, Reiter's disease, and other arthropathies. Flow cytometric (FCM) typing of B27 is a relatively new method which allows for rapid turnaround time and low cost. However, different leukocyte populations may manifest significant variation in staining intensity with B27 antibodies. Therefore, gating utilizing only light scatter properties of cells may lead to high readings in some B27-negative samples. Fluorescence gating on CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore, quantitative FCM measurements, as afforded through use of molecules of equivalent soluble fluorochrome (MESF) units, can minimize day-to-day and instrument-to-instrument variabilities in fluorescence measurements. We compared CD3 gating to light scatter gating and MESF analysis on 123 specimens in a 4-month period and found: 1) CD3 gating gave the lowest nonspecific B27 antibody binding among B27-negative subjects; 2) there was no significant difference in MESF values between CD3 gating and light scatter gating of B27-positive samples; 3) there was a wide range of B27 antibody binding fluorescence intensities among B27-positive subjects; 4) identification of patients with low B27 expression may require the use of CD3 gating in order to avoid costly confirmation testing; 5) fluorescent standard beads are stable for routine use in a clinical flow cytometry laboratory.

Antibodies, Monoclonal↗
PubMed1995

Flow cytometric reticulocyte maturity index: a useful laboratory parameter of erythropoietic activity in anemia.

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 413 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 (r2 = 0.041), hematocrit (r2 = 0.038), reticulocyte percentage (r2 = 0.078), 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.

Anemia↗
PubMed1994

Flow cytometric reticulocyte analysis. Multiinstitutional interlaboratory correlation study.

Reticulocyte analysis by flow cytometry offers precision and sensitivity greater than those of conventional morphologic methods and permits derivation of a reticulocyte maturity index. However, interlaboratory variability has not yet been reported. The authors analyzed 310 samples at eight sites using 11 instruments over a 4-month period to examine intermethod and interlaboratory variabilities. Stains included thiazole orange, ethidium bromide, and auramine O. Instruments included models by Coulter, Becton Dickinson, TOA Medical Electronics, and Ortho Diagnostics. The coefficient of variation (CV) among all sites and methods on these samples varied as a function of the reticulocyte percentage, ranging from a mean CV of 69% for samples with < .5% reticulocytes to 24.1% for those with > 2.5% reticulocytes. The best performance was observed with the TOA R-1000 dedicated reticulocyte analyzers, with a mean CV of 18.4% for samples with < .5% reticulocytes and 4.6% for samples with > 2.5% reticulocytes. The reticulocyte maturity index showed comparable intersite precision, with a mean CV of 16% for samples with > 2.5% reticulocytes with multipurpose flow cytometers and a mean CV of 7.3% with the TOA R-1000 instruments. Interclass correlations among all sites ranged from .79 to .99 for the reticulocyte counts and .41 to .88 for the reticulocyte maturity index. The authors conclude that flow cytometric reticulocyte analysis is more precise than manual reticulocyte analysis. With greater automation of this methodology, further interlaboratory standardization of reticulocyte counts and the reticulocyte maturity index can be achieved.

Adolescent↗
PubMed1994

Automated reticulocyte analysis. Clinical practice and associated new parameters.

Automated reticulocyte analysis, through continued instrument, software, and reagent developments, provides improved precision for the reticulocyte count. This rapid and simple testing assists in providing a cost effective and sensitive approach to the diagnosis and therapeutic monitoring of the anemic patient. As laboratory and clinical hematologist become familiar with the benefits and expanding clinical applications of this technology, the use of reticulocyte analysis will increase.

Autoanalysis↗
PubMed1993

Flow cytometric reticulocyte analysis and the reticulocyte maturity index.

Reticulocyte analysis has evolved to one of the accepted and routinely practiced clinical applications of flow cytometry technology. Similar to CD4 measurements, FCM technology has contributed to documented improvements in reticulocyte counting precision over previous microscope-based techniques. The ability to FCM instruments to quantitate fluorescence intensity has been utilized to derive a parameter of reticulocyte maturity, which we have termed the reticulocyte maturity index. The FCM-derived RMI parameter offers an additional perspective to assess the erythropoietic response in anemic patients over reticulocyte counting alone and provides further insight into the differential diagnosis of anemia. Clinical utility of the RMI has been reported in monitoring expensive, new-technology therapies, such as bone marrow transplantation and erythropoietin therapy. FCM reticulocyte analysis is still not fully mature, but is in a state of continued evolution. Needs exist for (i) improved software for data analysis, (ii) newer automation techniques to improve interlaboratory correlations, and (iii) further validation and refinement of the reticulocyte maturity index parameter.

Anemia↗
PubMed1993

Proposal for standardization of flow cytometric reticulocyte maturity index (RMI) measurements.

Flow cytometric (FCM) reticulocyte analysis is more accurate, sensitive, and reproducible relative to previously employed manual microscopic methods in clinical laboratory hematology. FCM reticulocyte analysis using RNA binding fluorochromes additionally allows for the quantification of fluorescence intensity or population distribution of the reticulocyte RNA content. Viewed from the perspective of erythroid maturation, quantification of the fluorescence intensity distribution provides a reticulocyte maturation index (RMI). We performed a systematic study of 18 different methods to express thiazole orange stained reticulocyte fluorescence intensity, compared to standard mean fluorescence intensity quantification, using 185 anemic and non-anemic human blood samples. The method best correlating with the mean fluorescence intensity RMI on 2 different FCM instruments (R2 = 0.93 and 0.86) was a ratio of the highly fluorescent reticulocytes, defined using a normal adult population, and the total number of reticulocytes (HFR%). In contrast to mean fluorescence intensity measurements, a HFR% RMI parameter can provide similar units of expression (0.01-1.00) with good correlation between different FCM instruments (R2 = 0.76). We conclude the HFR% method of RMI expression provides a superior means of interlaboratory standardization and clinical comprehension of this useful diagnostic parameter in clinical laboratory hematology.

Anemia↗
PubMed1990

Clinical flow cytometric reticulocyte analysis.

The flow cytometric (FCM) analysis of reticulocytes in a clinical laboratory can be accomplished using acridine orange (AO), thiazole orange (TO), auramine O, thioflavin T, pyronin Y, dimethyl-oxacarbocyanine or transferrin receptor assays. AO and TO are vital stains, show good correlation with microscopic reticulocyte determinations and, when compared to each other, give an excellent correlation. The coefficient of variation is below 5% and batch analysis on blood samples stored for 96 h further reduces manpower needs. Finally we have used the mean fluorescent intensity of TO as a reticulocyte maturity index (RMI). In bone marrow transplant patients, the RMI has been the earliest indicator of marrow engraphment. Using the RMI we have been able to define three patterns of engraphment: early, delayed and failed. Although a variety of standards will be required, the clinical FCM reticulocyte analysis promises to be the preferred and accepted method in the clinical laboratory.

Acridine Orange↗
PubMed1989

Flow cytometric reticulocyte quantification using thiazole orange provides clinically useful reticulocyte maturity index.

Flow cytometric reticulocyte quantification with thiazole orange has been reported to be of potential utility in a clinical hematology laboratory. We have instituted this technique into routine clinical testing for 18 months and we describe this experience. Flow cytometric analysis provided not only reproducible, cost-effective reticulocyte quantification, but a quantitative reticulocyte maturity index proportional to the amount of RNA in the reticulocytes. The reticulocyte maturity index measurement represents an independent parameter of erythropoiesis, which provided clinically valuable information regarding bone marrow engraftment in patients following autologous bone marrow transplantation. The findings of this study demonstrate the clinical utility of thiazole orange reticulocyte analysis and indicate the diagnostic importance of the reticulocyte maturity index measurement in the evaluation of erythropoietic activity.

Benzothiazoles↗