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1.
Clin Lab Haematol ; 23(3): 149-54, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11553054

RESUMO

Level of foetal haemoglobin (HbF) containing red cells (F cells) is a parameter for monitoring sickle cell anaemia (SS) patients undergoing treatment with HbF modulating drugs (including hydroxyurea (HU)). One convenient technique for F cell assay is flow cytometry. A flow cytometric method for the simultaneous assay of F cells, reticulocytes and HbF-containing reticulocytes (F reticulocytes) is described in this paper. These three parameters can be obtained within 2 h using double colour staining flow cytometry. Glutaraldehyde fixation, Triton X-100 permeabilization, monoclonal antibody to HbF Tri-colour conjugate (MoAb-HbF-TC; deep-red fluorescence) immuno-staining and thiazole orange (TO; green fluorescence) are employed. The red cell gate was set on forward scatter (FSC) and logarithmic side scatter (logSSC) for 50 000 cells on the flow cytometer. Fluorescent signals were acquired from fluorescent channel 1 (FL1; green) and (FL4; deep-red). Coefficient of variation percent (%CVs) of intra- and inter-assay were less than 9% and 15%, respectively. EDTA, citrate, heparin and CTAD anticoagulants are all suitable; the samples can be stored at 4 degrees C for up to 6 days. The method is a simple, rapid, convenient, reproducible and useful way of determining F cell, reticulocyte and F reticulocyte levels in sickle cell and thalassaemic patients.


Assuntos
Eritrócitos/química , Hemoglobina Fetal/análise , Citometria de Fluxo/métodos , Reticulócitos/química , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Anticoagulantes/farmacologia , Índices de Eritrócitos , Humanos , Hidroxiureia/farmacologia , Hidroxiureia/uso terapêutico , Reprodutibilidade dos Testes , Reticulócitos/citologia , Talassemia/sangue
2.
Hematology ; 6(1): 91-100, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-27419609

RESUMO

Red blood cells (RBCs) from sickle cell patients (SS) express thrombospondin receptor (CD36), contain ribonucleic acid (RNA, recognised as reticulocytes) and fetal haemoglobin (HbF, defined as F cells) in a higher proportion than RBCs from healthy individuals. The co-distribution of CD36, RNA and HbF on the same RBCs has not been demonstrated due to a lack of detection methods. A triple-colour staining flow cytometry for the co-distribution of CD36, RNA and HbF was developed. The method can simultaneously determine CD36-expressing RBCs (CD36 cells), RNA-bearing RBCs (reticulocytes), HbF-bearing RBCs (F cells), CD36-expressing reticulocytes (CD36 reticulocytes), CD36-expressing-F cells (CD36-F cells), HbF-bearing reticulocytes (F reticulocytes) and CD36-expressing-F reticulocjrtes (CD36-F reticulocytes). Mouse monoclonal antibody against CD36 (MoAb-CD36), antibodagainst mouse-immunoglobulin conjugated to biotin (Ab-Molg-Bi), streptavidin conjugated to rhodamine phycoerythrin (StA-RFE), MoAb against HbF conjugated to Tri-Colour® (MoAb-HbF-TC), Thiazole orange (TO), Glutaraldehyde and Triton X-100 were used. The procedure takes approximately 7 hours. The numbers of CD36 cells, reticulocytes and F cells obtaining from single and triple staining were well correlated and not significantly different. Intra- and inter-assay coefficient of variation percents (%CVs) of the triple-colour staining were less than 10 and 15% respectively. EDTA blood samples stored at 4°C for less than 3 days are suitable. The method trial was then employed on blood samples from SS and healthy individuals. The method is reproducible, objective and applicable for determination of co-distribution of other membrane and intracellular markers in RBCs.

3.
Cytometry ; 42(6): 389-93, 2000 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11135294

RESUMO

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.


Assuntos
Eritrócitos/química , Hemoglobina Fetal/análise , Citometria de Fluxo/métodos , Adulto , Anemia Falciforme/sangue , Anticoagulantes/química , Humanos , Reprodutibilidade dos Testes , Talassemia/sangue
4.
Am Clin Lab ; 18(10): 8-9, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10848432

RESUMO

The summarized data and other studies to date indicate that performance of the Pentra 60 is comparable to other hematology analyzers for all CBC and 5-Diff parameters. Comparative studies indicate good correlation for all the reportable CBC parameters, lymphocyte counts, and neutrophil counts with 24 hr stability on blood samples. Good intermethod correlations on monocyte and eosinophil counts were observed. Only basophil counts showed poor intermethod correlations, but this is expected on a statistical basis, and the counts are similar to those reported for other hematology analyzer performance studies. The combination of the MDSS and stepper motor fluidic system allows for low-volume blood sampling, compact size, and low operational noise level. The Pentra 60 is well suited for physician's office laboratories, medical clinics, small- or medium-size hospitals with less than 100 CBCs per day, and larger hospitals or reference laboratories that need back-up for a high-end automated hematology analyzer.


Assuntos
Contagem de Células Sanguíneas/instrumentação , Hematologia/instrumentação , Estudos de Avaliação como Assunto , Humanos , Contagem de Leucócitos/instrumentação
5.
Transfusion ; 38(8): 749-56, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9709783

RESUMO

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.


Assuntos
Anticorpos Monoclonais , Contagem de Eritrócitos , Sangue Fetal/citologia , Hemoglobina Fetal/análise , Transfusão Feto-Materna/sangue , Citometria de Fluxo , Adulto , Animais , Feminino , Hemoglobina Fetal/imunologia , Transfusão Feto-Materna/diagnóstico , Humanos , Recém-Nascido , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Sensibilidade e Especificidade
6.
Cytometry ; 26(4): 286-92, 1996 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-8979028

RESUMO

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.


Assuntos
Complexo CD3/imunologia , Citometria de Fluxo/métodos , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Antígeno HLA-B27/classificação , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Citometria de Fluxo/instrumentação , Antígeno HLA-B27/imunologia , Humanos , Modelos Lineares , Controle de Qualidade
7.
Cytometry ; 22(1): 35-9, 1995 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-7587731

RESUMO

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.


Assuntos
Anemia/sangue , Técnicas de Laboratório Clínico , Eritropoese/fisiologia , Citometria de Fluxo , Reticulócitos/citologia , Anemia/patologia , Senescência Celular/fisiologia , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
8.
Am J Clin Pathol ; 102(4): 468-77, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7942604

RESUMO

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.


Assuntos
Citometria de Fluxo/métodos , Garantia da Qualidade dos Cuidados de Saúde , Reticulócitos/patologia , Reticulócitos/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Benzotiazóis , Senescência Celular , Criança , Pré-Escolar , Feminino , Citometria de Fluxo/instrumentação , Corantes Fluorescentes , Humanos , Lactente , Laboratórios , Masculino , Pessoa de Meia-Idade , Quinolinas , Análise de Regressão , Reprodutibilidade dos Testes , Contagem de Reticulócitos , Tiazóis
9.
Hematol Oncol Clin North Am ; 8(4): 617-30, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7961284

RESUMO

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.


Assuntos
Autoanálise , Contagem de Reticulócitos , Doenças Hematológicas/diagnóstico , Humanos , Contagem de Reticulócitos/instrumentação
11.
Ann N Y Acad Sci ; 677: 281-92, 1993 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-8494215

RESUMO

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.


Assuntos
Anemia/diagnóstico , Citometria de Fluxo/métodos , Reticulócitos/fisiologia , Anemia/sangue , Anemia/terapia , Envelhecimento Eritrocítico , Citometria de Fluxo/normas , Humanos , Laboratórios/normas , Monitorização Fisiológica/métodos , Controle de Qualidade , Reticulócitos/citologia
12.
Cytometry ; 14(3): 318-26, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8472608

RESUMO

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.


Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes , Reticulócitos , Tiazóis , Anemia/sangue , Benzotiazóis , Contagem de Células/métodos , Diferenciação Celular , Índices de Eritrócitos , Citometria de Fluxo/normas , Hematologia/métodos , Humanos , Laboratórios/normas , Quinolinas , Estatística como Assunto
13.
Pathobiology ; 58(2): 99-106, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-1694437

RESUMO

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.


Assuntos
Reticulócitos , Laranja de Acridina , Benzotiazóis , Contagem de Células Sanguíneas , Diferenciação Celular , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Quinolinas , RNA/metabolismo , Reticulócitos/citologia , Tiazóis
14.
Arch Pathol Lab Med ; 113(6): 684-9, 1989 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2471489

RESUMO

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.


Assuntos
Citometria de Fluxo/métodos , Reticulócitos/citologia , Tiazóis , Benzotiazóis , Transplante de Medula Óssea , Contagem de Células , Sobrevivência Celular , Análise Custo-Benefício , Eritropoese , Citometria de Fluxo/economia , Citometria de Fluxo/normas , Corantes Fluorescentes , Humanos , Controle de Qualidade , Quinolinas , RNA/análise , Reticulócitos/análise , Reticulócitos/fisiologia , Transplante Autólogo
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