Flow Cytometric Reticulocyte Analysis Research Articles

Paroxysmal nocturnal hemoglobinuria (PNH): higher sensitivity and validity in diagnosis and serial monitoring by flow cytometric analysis of reticulocytes

Flow cytometric analysis of GPI-anchored proteins (GPI-AP) is the gold standard for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Due to therapy options and the relevance of GPI-deficient clones for prognosis in aplastic anaemia detection of PNH is gaining importance. However, no generally accepted standard has been established. This study analysed the usefulness of a flow cytometric panel with CD58, CD59 on reticulocytes and erythrocytes, CD24/CD66b and CD16, FLAER on granulocytes and CD14, and CD48 on monocytes. Actual cut-off (mean + 2 SD) for GPI-deficient cells was established in healthy blood donors. We studied 1,296 flow cytometric results of 803 patients. Serial monitoring was analysed during a median follow-up of 1,039 days in 155 patients. Of all, 22% and 48% of 155 follow-up patients. showed significant GPI-AP-deficiency at time of initial analyses. During follow-up in 9%, a new PNH diagnosis, and in 28%, a significant change of size or lineage involvement was demonstrated. Highly significant correlations for GPI-AP deficiency were found within one cell lineage (r 2 = 0.61–0.95, p < 0.0001) and between the different cell lineages (r 2 = 0.49–0.88, p < 0.0001). Especially for detection of small GPI-deficient populations, reticulocytes and monocytes proved to be sensitive diagnostic tools. Our data showed superiority of reticulocyte analyses compared with erythrocyte analyses due to transfusion and hemolysis independency especially in cases with small GPI-deficient populations. In conclusion, a screening panel of at least two different GPI-AP markers on granulocytes, erythrocytes, and reticulocytes provides a simple and rapid method to detect even small GPI-deficient populations. Among the markers in our panel, CD58 and CD59 on reticulocytes, CD24/66b, and eventually FLAER on granulocytes as well as CD14 on monocytes were most effective for flow cytometric diagnosis of GPI deficiency.Electronic supplementary materialThe online version of this article (doi:10.1007/s00277-011-1177-4) contains supplementary material, which is available to authorized users.

Flow cytometric reticulocyte analysis using thiazole orange; clinical experience and technical limitations

Flow cytometric (FCM) reticulocyte analysis using thiazole orange (TO) is becoming an increasingly popular method for routine quantification of reticulocytes. The methodology is accurate, cost-effective and shows a high correlation with manual techniques. We describe our experience with the clinical application of FCM reticulocyte analysis in a general hospital setting over a 20-month period with special emphasis on technical limitations.

Reticulated platelets interfere with flow cytometric reticulocyte counts

As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis.

Reticulocyte parameters in hemoglobinopathies and iron deficiency anemia

Flow cytometric reticulocyte analysis allows the evaluation of reticulocyte maturity. New reticulocyte parameters have been used in the diagnosis and management of anemias, in the bone marrow transplant setting and in the monitoring of iron replacement or erythropoiet in therapy. Reticulocyte numbers and maturation levels have been studied in different hemoglobinopathies and the results have been correlated with the degree of ineffective erythropoiesis. In order to verify differences in reticulocyte parameters in various types of anemias and to test the absolute number of immature reticulocytes as a possible discriminating factor among various types of anemias, reticulocyte counts were performed on 219 samples from patients with sickle cell anemia (SS) (n= 62), hemoglobin S trait (n=9), Sbeta thalassemia (n=7), hemoglobin SC disease (n=11), beta thalassemia trait (n=33) and iron deficiency anemia (n= 47), and non-anemic individuals (n= 50). Mean fluorescence index (MFI) was defined as representative of the degree of reticulocyte immaturity and it was evaluated as a percentage and in absolute values. Reticulocyte counts and MFI values were significantly higher in SS, Sbeta thalassemic and SC groups when compared to controls, but not different among the three anemia groups. Patients with hemoglobin S trait, iron deficiency anemia and beta thalassemia trait showed reticulocyte parameters similar to the non-anemic group. There was no difference between the b thalassemic trait and iron deficiency anemia in relation to any parameters. MFI in absolute numbers were significantly higher in anemias that develop with the hemolytic process, although this was not evident in MFI percentage values. Our results showed that the erythoid expansion in sickle cell diseases (SS, SC and Sb thalassemia) leads to an enhanced immature reticulocyte release from bone marrow and that the phenomena is more evident by the MFI counting in absolute figures than in percentages. We concluded that the assessment of reticulocyte maturity provides interesting data about the pathophysiology and erythopoietic response in different types of anemias.

Serum Transferrin Receptor Concentrations during Normal Pregnancy

Among the physiologic changes during pregnancy, the disproportionate increases in plasma volume and red cell mass produce a decrease in hemoglobin concentration. Pathologic anemia of pregnancy is mostly attributable to iron deficiency associated with increased requirements and inadequate intake (1), but physiologic changes also occur in iron markers. Iron plays an essential role in a spectrum of metabolic processes. Cellular iron uptake is facilitated by transferrin receptor (TfR)-mediated endocytosis (2). Serum TfR (sTfR) is a sensitive indicator of iron deficiency in inflammatory states and in the anemia of chronic diseases because its concentration is not influenced by the acute phase response (3). The sTfR concentration is closely related to erythroid TfR turnover; therefore, sTfR may be a useful marker to monitor erythropoiesis in various clinical situations (4). Erythropoiesis is a highly dynamic process and can be monitored by quantitative reticulocyte counting. Flow cytometric analysis of reticulocytes provides a quantitative reticulocyte measurement with high sensitivity and precision (5). The fluorescence intensity of reticulocytes is directly proportional to erythrocyte RNA content, and reticulocytes can be divided into three subpopulations by fluorescence intensity: low fluorescence reticulocytes, middle fluorescence reticulocytes, and high fluorescence reticulocytes (5)(6). The reticulocyte maturity index (RMI) is calculated from the proportion of reticulocyte subpopulations and can be used as the earliest and most sensitive predictor of bone marrow erythropoiesis (6). The sTfR concentration and the RMI during normal pregnancy have not been studied extensively, and the reported values for sTfR show a wide range of discrepancies. Therefore, in the present study, we investigated the sequential changes in sTfR concentrations according to gestational age in healthy pregnant women without anemia and iron deficiency. We …

Reticulocyte counts in laboratory animals using a manual and a flow cytometric procedure (sysmex R-3000™)

An evaluation of the flow cytometric procedure for reticulocyte counting was performed, together with a comparison to a reference manual counting method. An automated reticulocyte analyser, the Sysmex R-3000™ (Flow cytometric auramine O methods) was used to enumerate the absolute reticulocyte counts and the percentage of reticulocytes in blood obtained from rats, dogs and monkeys. Simultaneously, a maturation distribution was obtained by subdividing the reticulocytes into three maturation classes according to their fluorescence intensity. The reticulocyte counts and the maturation distributions can be determined rapidly and easily. The coefficients of variation (CV) for the within-day precision study in the reticulocyte counts of the three species were found to be 9.7%–20.5% for the micro-scopic method and 4.4%–9.4% for the flow cytometric method. A very good correlation was observed between the microscopic count and flow cytometric methods (r=0.958). In conclusion, because of high reproducibility and good agreement with the manual counting method for these species, flow cytometric analysis of reticulocytes is an attractive alternative procedure to micro-scopic enumeration in toxicological studies.

Flow Cytometric Reticulocyte Analysis:Multiinstitutional Interlaboratory Correlation Study

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.

Recombinant human erythropoietin in autologous blood donors: a dose‐finding study

A dose-finding study of recombinant human erythropoietin (rhEPO) was performed in 60 autologous donors who donated 2 units of blood prior to orthopaedic or vascular surgery. The correction of phlebotomy-induced anaemia was studied in four groups of 15 patients who received 500 U/kg, 250 U/kg, 125 U/kg or no (controls) rhEPO subcutaneously twice per week during a 3-week period. Haemoglobin concentration in the 500 U/kg, 250 U/kg and 125 U/kg group and in the controls reached respectively 99.2, 98.8, 91.9 and 87.1% of pre-phlebotomy value. Flow cytometric analysis of reticulocytes showed a steady increase of reticulocyte count as the dose increased. Maximal levels of high fluorescence reticulocytes which represent early changes of erythropoiesis were reached after 7 d and decreased thereafter in each group. Serum ferritin decreased significantly to approximately 50% of baseline values in all groups; no differences in the decrease of serum ferritin were observed between the patients who received rhEPO and the controls. No severe adverse events were observed. This study demonstrates a dose-related effect of rhEPO on erythropoiesis in autologous donors during the first 2 weeks. No further increase of reticulocytes was observed despite continued rhEPO therapy, which may be due to the inability of the mononuclear phagocytic system to release additional iron. To restore pre-phlebotomy haemoglobin concentration, a dose of 250 U/kg rhEPO was sufficient.

Predicting Bone Marrow Transplant Engraftment by Automated Flow Cytometric Reticulocyte Analysis

We measured reticulocyte counts and the middle fluorescence ratio plus high fluorescence ratio (MFR + HFR), a measurement of reticulocyte maturity, using a Sysmex R-1000 Automated Reticulocyte Analyzer on 32 allogeneic and 8 autologous marrow transplant patients for 44 days posttransplant. We compared instrumentgenerated data with visual reticulocyte counts and white blood cell (WBC) counts. The MFR + HFR yielded a measurable increase from baseline at 13.31 days, compared to 14.82 days for WBC count, 16.21 days for instrument-generated reticulocyte count, and 16.59 days for visual reticulocyte count. Instrument reticulocyte count precision improved upon visual count precision by five to tenfold. The MFR + HFR may be a sensitive measurement of regeneration.

Chapter 16 Reticulocyte Analysis and Reticulocyte Maturity Index

The traditional laboratory methodology of reticulocyte analysis for the past century has been the manual microscopic counting of erythrocytes or red cells on supravitally stained blood samples. Although slide-based methods, such as new methylene blue staining, do provide clinically useful information, the lack of precision in these subjective assays is well documented. In particular, the poor precision of manual counting methods does not allow for clinically useful sequential monitoring of patients with low reticulocyte levels or for therapeutic monitoring of drug therapies for either erythropoietic inhibition or enhancement. Methods of flow cytometric reticulocyte analysis for both clinical and experimental applications have been reported over the past years utilizing a variety of fluorescent dyes. Thiazole orange (TO) has distinct advantages over other nucleic acid binding dyes for reticulocyte analysis using commercial multipurpose flow cytometry (FCM) instruments and is currently utilized by the vast majority of clinical laboratories. TO reticulocyte analysis offers the ability to use easily reticulocyte enumeration and a stable, reproducible fluorescence intensity measurement over a 60-min period. Furthermore, the fluorescence signal provides a high quantum yield or good separation of the reticulocyte (positive) from the mature red blood cell (negative) populations.

Flow cytometric reticulocyte analysis and the reticulocyte maturity index.

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.

Flow-Cytometric Analysis of Reticulocytes in Normal Cord Blood

We performed precise reticulocyte counts and a reticulocyte maturation study according to ribosomal RNA (rRNA) content, in normal cord blood. For this purpose we analyzed 35 cord blood specimens corresponding to a mean gestational age of 39.2 weeks (range 38-41). In all specimens complete blood counts were performed with the H*1 (Technicon, Bayer) analyzer. Reticulocyte maturation study and counts were conducted by the R-1000 (Sysmex) reticulocyte analyzer. We obtained the following measurements (mean +/- 1 SD): reticulocyte percentage 3.11 +/- 0.75% and reticulocyte absolute count 137.3 +/- 33.3 x 10(9)/l. There was no correlation between the reticulocyte counts and the duration of gestation or the type of labor (normal, forceps assisted or cesarean section). Reticulocyte subpopulations with different rRNA content and maturation were expressed as reticulocytes with high (HFR), median (MFR) and low (LFR) fluorescence of the fluorochrome auramine bound to rRNA. Normal cord values were: HFR% = 13.6 +/- 2.4, MFR% = 22.5 +/- 2.4 and LFR% = 63.9 +/- 4.3. Reference maturation subpopulations were assessed for comparison in 180 samples of healthy adults (90 males and 90 females) and were found HFR% = 1.0 +/- 0.8, MFR% = 9.7 +/- 3.3 and LFR% = 89.2 +/- 3.4. The HFR% and MFR% were significantly higher while LFR% was significantly lower (p < 0.001) in cord compared to adult blood, denoting a shift to more immature reticulocyte forms. The above values are provided as normal reference data of the reticulocyte maturation profile in full-term cord blood.

Clinical Flow Cytometric Reticulocyte Analysis

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.

Utility of flow cytometric reticulocyte quantification as a predictor of engraftment in autologous bone marrow transplantation

Flow cytometric reticulocyte quantification with thiazole orange (TO) was used to study erythropoiesis in 20 patients following autologous bone marrow transplantation for the treatment of acute myelogenous leukemia. Flow cytometric reticulocyte analysis provided not only the reticulocyte percentage and absolute reticulocyte count but a quantitative reticulocyte maturity index (RMI) proportional to the amount of RNA in the reticulocytes. The RMI values, but not the reticulocyte percentage or absolute counts, correlated temporally with the rise in the absolute neutrophil counts in the posttransplantation period. In the majority of patients (12/20), the RMI value was the earliest indicator of bone marrow engraftment. The findings of this study demonstrate an important clinical utility of TO reticulocyte analysis by flow cytometry and indicate the diagnostic importance of the RMI measurement in the evaluation of erythropoietic activity in bone marrow transplant patients.

Changes in erythropoiesis due to radiation or chemotherapy as studied by flow cytometric determination of peripheral blood reticulocytes.

Flow cytometric determination of time dependent changes of numbers of reticulocytes in peripheral blood were investigated as a parameter for changes in erythropoiesis induced by radiation- or chemotherapy. Rats irradiated or treated with drugs (such as e.g. cyclophosphamide 100 mg/kg, vincristin 0.2 mg/kg, or mitomycin C 1.0 mg/kg) showed clear changes in erythropoietic activity. Reticulocyte numbers decreased rapidly until day 3-4 after treatment; this period was followed by a gradual increase and normal control values were seen at day 8-11. Radiation effects of doses as low 0.5 Gy could be detected in such a way. Similar studies were performed with patients with ovarian tumors treated with cis-platinum, a drug that may cause non-immune haemolysis. During prolonged treatment some patients showed increasing numbers of reticulocytes, measured at the first day of each hospitalization period, whereas leucocyte and platelet counts stayed more or less constant. Increasing numbers of reticulocytes generally indicates stimulation of erythropoietic activity of the bone marrow (due to increased blood loss); in this study increasing numbers often preceeded a decrease in hemoglobin values later on. Flow cytometric analysis of reticulocytes is therefore a potentially useful tool to detect changes in erythropoiesis, and considered more sensitive for the early recognition of patients that develop anemia, than hemoglobin measurements only.