Enumeration of Reticulocytes Using Fluorescence-Activated Flow Cytometry

Corash, Laurence; Rheinschmidt, Margaret; Lieu, Sandi; Meers, Patricia; Brew, Elizabeth

doi:10.1159/000157131

Cited by 24 publications

(13 citation statements)

References 10 publications

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“…Clin ical laboratories have differing FCM instrumentation availability, different volumes of reticulocyte tests, dif ferent patient populations, and different regional clinical practice patterns. Thioflavin T has the disadvantage of requiring great attention to the staining times, but its emission and excitation spectral properties are well suited for those laboratories equipped with mercury arcbased flow cytometers [2,14]. Tanke et al [21][22][23] have thoroughly documented the feasibility of pyronin Y for FCM reticulocyte enumeration.…”

Section: Fluorescent Dyesmentioning

confidence: 99%

Clinical Flow Cytometric Reticulocyte Analysis

Davis

Bigelow²

1990

Pathobiology

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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.

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Section: Fluorescent Dyesmentioning

confidence: 99%

Clinical Flow Cytometric Reticulocyte Analysis

Davis

Bigelow²

1990

Pathobiology

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“…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.…”

mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

1995

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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 ...

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“…FCM retic can be performed with a number of methods [10,25], utilizing fluorescent dyes such as thiazole orange [4,8,9,12,17], thioflavin T [5,19,21], ethidium bromide [51, pyronin Y [24,271, and acridine orange [10,19,22,23,. In addition, TOA Medical Electronics (Kobe, Japan) markets a dedicated, semiautomated reticulocyte counter using flow cytometric tech- Laboratory methods of quality control of clinical FCM retic analysis have been proposed using refrigerated rabbit blood [5,61, normal human samples [9,10,3 11, and commercial reticulocyte control reagents (Streck Laboratories, Omaha, NB).…”

mentioning

confidence: 99%

“…In addition, TOA Medical Electronics (Kobe, Japan) markets a dedicated, semiautomated reticulocyte counter using flow cytometric tech- Laboratory methods of quality control of clinical FCM retic analysis have been proposed using refrigerated rabbit blood [5,61, normal human samples [9,10,3 11, and commercial reticulocyte control reagents (Streck Laboratories, Omaha, NB). Hence, the ability to standardize and control reticulocyte enumeration by flow cytometric methods is a reality and is generally practiced by clinical laboratories performing these assays.…”

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

et al. 1993

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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. 0 1993 Wiley-Liss, Inc.Keyterms: Red blood cell, anemia, erythrocyte, fluorescent dyes, blood, eryth-ropoiesis Clinical flow cytometric reticulocyte (FCM retic) analysis is now widely practiced in clinical laboratories in the United States, Europe, and Japan. In terms of numbers of patients, FCM retic analysis likely represents the most common clinical application of fluorescence-based flow cytometry. Interlaboratory standardization of this assay is currently of great concern in laboratory hematology. A subcommittee of the National Committee for Clinical Laboratory Standards is developing guidelines for this form of clinical testing. The American Medical Association CPT coding system now recognizes reticulocyte analysis by flow cytometry as a diagnostic medical test, distinct from manual microscopic counting methodology.FCM retic can be performed with a number of methods [10,25], utilizing fluorescent dyes such as thiazole orange [4,8,9,12,17], thioflavin T [5,19,21], ethidium bromide [51, pyronin Y [24,271, and acridine orange [10,19,22,23,33-351. In addition, TOA Medical Electronics (Kobe, Japan) markets a dedicated, semiautomated reticulocyte counter using flow cytometric technology and the fluorescent dye auramine 0 on the Sysmex R-1000 instrument [16,28,291. All of these methods of reticulocyte enumeration give excellent correlation (R2 range: 0.80-0.95) with one another [lo].Laboratory methods of quality control of clinical FCM retic analysis have been proposed using refrigerated rabbit blood [5,61, normal human samples [9,10,3 11, and commercial reticulocyte control reagents (Streck Labo...

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Enumeration of Reticulocytes Using Fluorescence-Activated Flow Cytometry

Cited by 24 publications

References 10 publications

Clinical Flow Cytometric Reticulocyte Analysis

Clinical Flow Cytometric Reticulocyte Analysis

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

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

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