Reticulocyte quantification by flow cytometry, image analysis, and manual counting

Schimenti, Kerry J.; Lacerna, Kathleen; Wamble, Alison; Maston, Lisa; Iaffaldano, Carol; Straight, Margaret; Rabinovitch, Albert; Lazarus, Hillard M.; Jacobberger, James W.

doi:10.1002/cyto.990130808

Cited by 37 publications

(23 citation statements)

References 31 publications

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“…Reticulocytes were stained with thiazole orange, 45 purified on a FACScan flow cytometer (BD-Biosciences) and plated after limit dilution at one cell per 3 wells in a 96-well plate. Plates were stored at Ϫ80°C for 1 hour and cells were thawed into 2.5 L lysis buffer (1.6% IGEPAL/NP-40; Sigma, St Louis, MO).…”

Section: Single-cell Expression Level Analysismentioning

confidence: 99%

Correction of a mouse model of sickle cell disease: lentiviral/antisickling β-globin gene transduction of unmobilized, purified hematopoietic stem cells

Levasseur

Ryan

Pawlik

et al. 2003

Blood

185

169

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Although sickle cell anemia was the first hereditary disease to be understood at the molecular level, there is still no adequate long-term treatment. Allogeneic bone marrow transplantation is the only available cure, but this procedure is limited to a minority of patients with an available, histocompatible donor. Autologous transplantation of bone marrow stem cells that are transduced with a stably expressed, antisickling globin gene would benefit a majority of patients with sickle cell disease. Therefore, the development of a gene therapy protocol that corrects the disease in an animal model and is directly translatable to human patients is critical. A method is described in which unmobilized, highly purified bone marrow stem cells are transduced with a minimum amount of self-inactivating (SIN) lentiviral vector containing a potent antisickling ␤-globin gene. These cells, which were transduced in the absence of cytokine stimulation, fully reconstitute irradiated recipients and correct the hemolytic anemia and organ pathology that characterize the disease in humans. The mean increase of hemoglobin concentration was 46 g/L (4.6 g/dL) and the average lentiviral copy number was 2.2; therefore, a 21-g/L /vector copy increase (2.1-g/dL) was achieved. This transduction protocol may be directly translatable to patients with sickle cell disease who cannot tolerate current bone marrow mobilization procedures and may not safely be exposed to large viral loads. IntroductionSickle cell disease (SCD) is an autosomal recessive disorder that affects more than 300 000 individuals worldwide and more than 70 000 in the United States. The molecular basis of the disease is an AϾT transversion in the sixth codon of the ␤-globin gene. 1,2 This mutation results in the substitution of a valine residue for glutamic acid on the surface of hemoglobin S (HbS; ␣ 2 ␤ 2 S ) tetramers. The valine creates a hydrophobic projection that fits into a natural hydrophobic pocket formed on hemoglobin tetramers after deoxygenation. 3,4 The interaction of tetramers results in the formation of HbS polymers/fibers that cause red blood cells (RBCs) to become rigid and nondeformable and to occlude small capillaries. 5-10 These vaso-occlusive events cause severe tissue damage that can result in strokes, splenic infarction, kidney failure, liver and lung disorders, painful crises, and other complications. 11,12 Cycles of erythrocyte sickling also cause the cells to become fragile, and lysis produces chronic anemia. Although sickle cell anemia was the first hereditary disease to be understood at the molecular level, 1,2 there is still no adequate long-term treatment or cure. Current measures focus on induction of fetal hemoglobin to inhibit polymer formation, treatment and prevention of infections, palliative measures to control pain, and surgical treatment of complications. 12 Allogeneic bone marrow transplantation is the only available cure, but this procedure is limited to a minority of patients with an available, histocompatible donor. 13 Autologous trans...

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Section: Single-cell Expression Level Analysismentioning

confidence: 99%

Correction of a mouse model of sickle cell disease: lentiviral/antisickling β-globin gene transduction of unmobilized, purified hematopoietic stem cells

Levasseur

Ryan

Pawlik

et al. 2003

Blood

185

169

View full text Add to dashboard Cite

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“…Tlie availability of flowcytometers in routine hospital laboratories has provided an attractive tool for automated determination of the reticulocyte count (8)(9)(10)(11)(12)(13)(14). By using flow cytometry one has the obvious advantage of being able to classify a larger number of cells in a shorter period of time s compared to manual methods, resulting in a rather low statistical inter-operator Variation.…”

Section: Introductionmentioning

confidence: 99%

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

Houte¹,

Bartels²,

Schoorl³

et al. 1994

CCLM

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This paper describes a comparison between the microscopic brilliant cresyl blue and the flow cytometric thiazol orange (FACScan) and auramine O (Sysmex) methods for enumeration of reticulocytes.The mean intra-assay coefficients of Variation for the microscopic, FACScan and Sysmex methods were established to be 33, 9 and 5% respectively. A rather poor correlation was observed between the microscopic count and both flow cytometric methods (FACScan r = 0.61; Sysmex r = 0.57). However, the correlation between the flow cytometric methods was satisfactory (r = 0.79).Reference ranges for the reticulocyte count, corresponding with the 2.5th and 97.5th percentile, were determined for the microscopic (8-30%o), FACScan (ll-27%o) and Sysmex (8-18%o) methods. Sysmex R-3000 methodology definitely revealed the lowest and narrowest reference ränge.In conclusion, because of higher reproducibility, flow cytometric analysis of reticulocytes is an attractive alternative procedure for the microscopic enumeration method.

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“…Heute verfügen alle großen Blutbildautomaten über die Möglichkeit der durchflusszytometrischen Retikulozytenzählung, so dass präzise Messungen in allen größeren Laboratorien mit vertretbarem Zeitaufwand und zu akzeptablen Preisen 2010;105:538-43 (Nr. 8), © Urban & Vogel, München 538 ÜBERSICHT möglich sind [2][3][4]. Damit ist die Voraussetzung geschaffen, die Retikulozytenzahl als einen wesentlichen Parameter zur pathophysiologisch orientierten Differentialdiagnose der Anämien zu verwenden.…”

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“…Insbesondere wird häufig übersehen, dass zur Beurteilung der normalen oder nicht normalen Funktion der Erythropoese die Retikulozytenzahl nur unter Berücksichti-gung der Hämoglobinkonzentration oder des Hämatokrits möglich ist. Methoden der Bestimmung der Retikulozytenzahl mittels Durchflusszytometrie sind geräteabhängig unterschiedlich, liefern aufgrund der um ein Vielfaches höheren Zahl der beurteilten Zellen aber mit hoher Präzision vergleichbare Ergebnisse, die sich sowohl im Normalbereich als auch bei verminderten und hohen Retikulozytenzahlen nicht wesentlich von der manuellen Methode unterscheiden [4,8]. Die Vergleiche mit besonders sorgfältiger manueller Zählung entsprechen allerdings nicht der allgemeinen Praxis, da bei dieser insbesondere reife Retikulozyten mit nur gering ausgeprägter SGF der Zählung entgehen können und systematische Unterschiede zwischen verschiedenen Untersuchern beobachtet werden.…”

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Die Bestimmung der Retikulozytenzahl: Eine alte Methode gewinnt neue Bedeutung

Heimpel

Diem²,

Nebe

2010

Med Klin

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In clinical practice, the reticulocyte count is the most useful method to estimate red cell life span and red cell production. However, experience of hematologists as well as surveys have shown that counting of reticulocytes is often neglected in the diagnostic work-up of unclassified anemias, and many physicians have difficulties in interpreting the results. Formerly, this was partly due to the low precision of manual counts. Today, this method is largely replaced by flow cytometry, available in almost all larger systems for automated blood count analysis, at low costs and sufficient analytic precision. Interpretation is supported by calculated parameters such as the Reticulocyte Production Index. Here, the authors discuss the limits of the analytic methods and the problems of interpretation in the context of the laboratory profile and the clinical setting.

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Reticulocyte quantification by flow cytometry, image analysis, and manual counting

Cited by 37 publications

References 31 publications

Correction of a mouse model of sickle cell disease: lentiviral/antisickling β-globin gene transduction of unmobilized, purified hematopoietic stem cells

Correction of a mouse model of sickle cell disease: lentiviral/antisickling β-globin gene transduction of unmobilized, purified hematopoietic stem cells

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

Die Bestimmung der Retikulozytenzahl: Eine alte Methode gewinnt neue Bedeutung

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