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

Abstract

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...

Methodology-Dependent Variations in Reticulocyte Counts Using a Manual and Two Different Flow Cytometric Procedures

“…For instance, in flow cytometric techniques using thiazole orange for reticulocyte analysis a light scatter gate around the red blood cell population is utilized to minimize platelet and leukocyte contamination of the collected fluorescence signal (5). Drawbacks of flow cytometric reticulocyte analysis using thiazole orange are the reported v ejrors due to contamination with Howell-Jolly bodies or nucleated red blood cells and variances in staining conditions (9,14,18). However, in this study the higher reticulocyte counts obtained by FACScan compared to Sysmex R-3000 (tab.…”

Influence of the Clinical Status on Stress Reticulocytes, CD 36 and CD 49d of SSFA2Homozygous Sickle Cell Patients Followed in Abidjan

“…Stress reticulocytes correspond to the immature reticulocytes fraction (IRF) constituted by the sum of reticulocytes MFR and HFR [13]. The mean fluorescence intensity (MFI) was also measured.…”

Durchflußzytometrie in der Klinischen Diagnostik. Positionspapier der Arbeitsgruppe Durchflußzytometrie und Quantitative Mikroskopie der Deutschen Gesellschaft für Klinische Chemie und Deutschen Gesellschaft für Laboratoriumsmedizin

“…Bei im Vergleich zur mikroskopischen Technik deutlich höheren Präzision hat die Färbung mit einem RNA-Farbstoff, zumeist Thiazol O, ebenfalls die Möglichkeit eröffnet, eine Klassifizierung des Reifungsgrades des Retikulozyten anhand der Fluoreszenzintensität als,Maß des RNA-Gehaltes vorzunehmen [108][109][110][111]. Eine Subtypisierung von Retikulozyten ist auch anhand der Expression des Transferrinrezeptors (CD71) möglich [112].…”