Reticulocytes and Reticulated Platelets: Simultaneous Measurement in Whole Blood by Flow Cytometry

Rapi, Stefano; Ermini, Angela; Bartolini, Luca; Caldini, A; Genovese, A; Miele, Alba; Buggiani, A.; Fanelli, Alessandra

doi:10.1515/cclm.1998.036

Cited by 10 publications

(4 citation statements)

References 16 publications

(9 reference statements)

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“…Our study did not specifically address the interferences by leucocytes, erythrocyte inclusions (nuclei in immature erythrocytes, Howell-Jolly bodies, plasmodia) and major thrombocytes or thrombocyte aggregates which are also stained by the dyes we employed (7,11,(20)(21)(22). Although these interferences in principle exist, they are very unlikely, because in all three methods effective gating procedures are used which allow the discrimination between erythrocytes and other blood cells.…”

Section: Discussionmentioning

confidence: 99%

Determination of Reticulocytes: Three Methods Compared

Siekmeier

Bierlich²,

Jaross³

2000

CCLM

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Determination of reticulocytes in peripheral blood is a valuable tool for getting information about erythropoiesis of an individual. For many years, reticulocyte numbers were quantified manually by means of a microscope after staining with supravital dyes. However, this method is tedious and shows low reproducibility. Therefore, several methods for the automated determination of reticulocytes have been established in laboratory routine within the last years. The aim of this study was to compare three of these automated methods for reticulocyte analysis. Reticulocytes were determined in 130 subsequent routine samples by means of an ABX Pentra 120 Retic haematological analyser, a Coulter EPICS XL MCL flow cytometer and a Coulter STKS haematology system, using the fluorescent dye thiazole orange or the supravital dye new methylene blue for reticulocyte staining, respectively. The reticulocyte concentrations were slightly lower for the Coulter STKS haematology system (mean +/- SD 1.89+/-1.32%) when compared with the Coulter EPICS XL MCL flow cytometer or the ABX Pentra 120 Retic haematological analyser (2.11+/-1.25% and 2.12+/-1.15%, respectively). The correlations between all methods were significant (r(s) > or = 0.843, p < 0.001). Small intercepts were, however, observed in the correlation plots between the values obtained by means of the Coulter STKS haematology system and those obtained by the other two methods. Within-batch coefficients of variation were 6.0%, 6.9% and 7.8% for the ABX Pentra 120 Retic haematological analyser, the Coulter STKS haematology system and the Coulter EPICS XL MCL flow cytometer, respectively. The corresponding between-batch coefficient of variation values were 6.8%, 4.9% and 5.3% as well as 14.1%, 7.6% and 6.1% for the low, medium and high control levels determined by means of the ABX Pentra 120 Retic haematological analyser and the Coulter STKS haematology system, respectively. These data suggest that all three methods allow the efficient and reliable determination of reticulocyte counts under clinical routine conditions. However, although the obtained data are very similar, differences exist which should be taken into account for the normal values of the different methods.

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Section: Discussionmentioning

confidence: 99%

Determination of Reticulocytes: Three Methods Compared

Siekmeier

Bierlich²,

Jaross³

2000

CCLM

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“…This wide range can be explained by lack of standardization of the methods. Many factors have been identified that contribute to this analytical issue: the type and concentration of fluorescent dye, incubation time and temperature, fixation, RNAse treatment and the flow cytometric data analysis, including gating and threshold settings [17,18,[39][40][41]. One of the major problems is that platelets show non-RNAspecific binding of fluorescent dye, resulting in background staining, which is size-dependent [39,42,43].…”

Section: Reticulated Platelet Methods -Flow Cytometrymentioning

confidence: 99%

Reticulated platelets: analytical aspects and clinical utility

Hoffmann

2014

Clinical Chemistry and Laboratory Medicine (CCLM)

136

147

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Reticulated platelets are immature platelets circulating in blood; they reflect the activity of megakaryopoiesis in the bone marrow. Therefore, they can be used as a non-invasive test in patients with thrombocytopenia in various clinical conditions. The preferred method of analysis is by flow cytometry. However, there is an evident lack of analytical standardization, making it difficult to compare results obtained in different laboratories. Currently, two types of hematology analyzers are on the market offering fully automated measurement of reticulated or immature platelets: the high end analyzers manufactured by Sysmex (XE-and XN-series) and Abbott (CELL-DYN Sapphire). Although the methods are essentially different and cannot be used interchangeably, both have been proven to have clinical utility. Reticulated or immature platelet assays are useful for the differential diagnosis of thrombocytopenia and for monitoring bone marrow recovery after chemotherapy or stem cell transplantation. These assays may aid clinicians in platelet transfusion decisions when recovery from thrombocytopenia is imminent. In addition, preliminary findings indicate that there is a rationale for reticulated or immature platelets for risk stratification in acute coronary syndromes and for monitoring the effect of treatment with antiplatelet drugs in patients with coronary artery diseases. The aim of this paper is to present the present technology available for measuring reticulated platelets as well as an overview of the current status of clinical application. This overview also indicates that more research is needed before reticulated or immature platelet assays can be applied in other clinical conditions than thrombocytopenia and after transplantation.

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“…Some assays use whole blood and others use platelet‐rich plasma. Monoclonal antibodies specific to platelets used to delineate reticulated platelets are varying and normal counts of reticulated platelets are reported to vary from 1% to 20% (2–16).…”

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confidence: 99%

Aberrant increase in the immature platelet fraction in patients with myelodysplastic syndrome: a marker of karyotypic abnormalities associated with poor prognosis

Sugimori

Kondo

Shibayama

et al. 2008

European J of Haematology

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Reticulated platelets are RNA-rich immature platelets and are thought to reflect the ability of bone marrow to produce platelets (1). Reticulated platelets can be measured by flow cytometry using thiazole orange (2). However , the measurement of the reticulated platelets has not been included in routine examinations because of the complexity of the procedure. Furthermore, the standard method for measurement of reticulated platelets has not been established so far. Some assays use whole blood and others use platelet-rich plasma. Monoclonal antibod-ies specific to platelets used to delineate reticulated plate-lets are varying and normal counts of reticulated platelets are reported to vary from 1% to 20% (2-16). A recent software program using the Sysmex XE2100 fully automated complete blood count analyzer (Sysmex, Kobe, Japan) has facilitated the measurement of RNA-rich immature platelets automatically. Although this instant assay lacks specificity, the measurement of the immature platelet fraction percentage in total platelets (IPF%) may be useful in ascertaining whether thrombo-cytopenia is caused by reduced platelet production or increased metabolic turnover because of platelet destruction. Indeed, IPF% has been successfully utilized to differentiate ITP and aplastic anemia (AA) (17). The measurement of IPF% has also been shown to be useful in predicting platelet recovery following Abstract Objectives: Some patients with myelodysplastic syndrome (MDS) show a marked increase in the percentage of immature platelet fraction (IPF%) despite the absence of severe thrombocytopenia. To determine the significance of such an unbalanced increase in the IPF%, we investigated the IPF% and other laboratory findings of 51 patients recently diagnosed with MDS. Method: Subjects consisted of 80 healthy males, 90 healthy females, and 51 patients with MDS and 20 patients with idiopathic thrombocytopenic purpura (ITP). The IPF and IPF% were determined using a Sysmex XE-2100 system loaded with IPF Master software (XE IPF Master, Sysmex). Platelet counts were measured simultaneously. Results: IPF% and platelet counts of these patients ranged from 1.1% to 25.1% (median, 5.3%) and from 6 to 260 · 10 9 ⁄ L (median, 71 · 10 9 ⁄ L), respectively. Twelve patients showed platelet counts more than 50 · 10 9 ⁄ L with 10% or more IPF%. All of the 12 patients had chromosome abnormalities including monosomy 7 and complex abnormalities involving 7 or 5q. In the other 39 patients who did not show the aberrant IPF% increase, chromosomal abnormalities were seen only in seven patients and none of them had chromosome 7 abnormalities. The IPF% of two patients increased to more than 10% in association with the appearance of monosomy 7. Conclusions: These findings suggest that a high IPF% in MDS patient may be a marker for karyotypic abnormalities with a poor prognosis, including chromosome 7 abnormalities.

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Reticulocytes and Reticulated Platelets: Simultaneous Measurement in Whole Blood by Flow Cytometry

Cited by 10 publications

References 16 publications

Determination of Reticulocytes: Three Methods Compared

Determination of Reticulocytes: Three Methods Compared

Reticulated platelets: analytical aspects and clinical utility

Aberrant increase in the immature platelet fraction in patients with myelodysplastic syndrome: a marker of karyotypic abnormalities associated with poor prognosis

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