Evaluation of reticulocyte counts by flow cytometry in a routine laboratory

Abstract

Reticulocyte counting using flow cytometry and a membrane-permeable fluorescent dye, thiazole orange, was evaluated as an alternative to the conventional manual method. The flow cytometric method was more precise (mean c.v. of 4.3%) than the imprecise manual technique (mean c.v. of 22.4%). Linearity was highly acceptable (r = 0.99) over the reticulocyte count range of 1.8-30.1%. Flow cytometry allows processing of large numbers of samples with reduction in technologist time, and the improved precision and tenfold increase in the number of cells counted should considerably improve the clinical utility of the reticulocyte count.

Contagem de reticulócitos de cães saudáveis ou anêmicos pela citometria de fluxo

“…Vários fatores podem influenciar a reticulocitometria por microscopia de luz, dentre as quais citam-se as dificuldades técnicas na identificação dos reticulócitos, o tamanho da amostragem (total de células contadas: ideal 1.000 células, mínimo 500 células), tipo e qualidade do esfregaço sangüíneo, variação e qualidade da coloração, além, logicamente, do tempo gasto na análise das amostras e da baixa reprodutibilidade do método (Ferguson et al, 1990;Rowan et al, 1996;Nobes e Carter, 1990;Riley et al, 2001). A contagem de reticulócitos com o uso do citômetro de fluxo tem a vantagem de enumerar uma grande quantidade de células (10.000 a 50.000 células) em um curto espaço de tempo e com pouca variação de ordem técnica (Nobes e Carter, 1990;Brow e Wittwer, 2000;Riley et al, 2002).…”

“…A contagem de reticulócitos com o uso do citômetro de fluxo tem a vantagem de enumerar uma grande quantidade de células (10.000 a 50.000 células) em um curto espaço de tempo e com pouca variação de ordem técnica (Nobes e Carter, 1990;Brow e Wittwer, 2000;Riley et al, 2002). A maior desvantagem é a contagem superestimada de reticulócitos, causada por possível interferência de inclusões em eritrócitos, como os eritrócitos nucleados, os corpúsculos de Howell-Jolly, corpúsculo de Heinz e os parasitas intracelulares (Ferguson et al, 1990;Rowan et al, 1996;Brow e Wittwer, 2000;Riley et al, 2001). Uma grande variedade de fluorocromos pode ser usada na citometria de fluxo.…”

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

“…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.…”

“…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.…”

“…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).…”