A low-cost, accurate method for detecting reticulocytes at different maturation stages based on changes in the mitochondrial membrane potential.

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2-1 (MTGhigh/TMREhigh/FSbig), R2-2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.

Genotoxicity assessment of melamine in the in vivo Pig-a mutation assay and in a standard battery of assays.

The genotoxicity of melamine was evaluated with the combined Pig-a mutation/micronucleus assay, the bacterial reverse mutation assay, and the in vitro cytokinesis-block micronucleus assay (CBMN). Five groups of six- to eight-week-old male Sprague-Dawley (SD) rats were given three daily doses of vehicle control (100% pure sesame oil), melamine (500, 1000, and 2000 mg/kg) or positive control (N-ethyl-N-nitrosourea, ENU, 20 mg/kg) by oral gavage. Peripheral blood was sampled pre-dose (day -1) and at time points up to day 60. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies, on days -1, 15, 29 and 60, and micronucleus frequencies were measured in RETs on day 4. No significant increases in RBC(CD59-) or RET(CD59-) frequencies were observed for the melamine-treated group at any of the time points studied, but the positive control, ENU, induced statistically significant increases compared with the vehicle control. Similar results were obtained in the micronucleus assay. Melamine did not induce statistically significant increases in %MN-RET. In the bacterial reverse mutation assay, melamine was tested from 62.5 to 1000 µg/plate in tester strains TA97a, TA98, TA100, TA102, and TA1535, with and without metabolic activation, and no evidence of toxicity or mutagenicity was observed at any dose tested. In the in vitro CBMN assay, in Chinese hamster ovary (CHO) cells, melamine was tested (75, 150, and 300 µg/mL) in the presence and absence of S9 mix, and no positive increases in the number of cells containing micronuclei were seen. These results suggest that melamine does not exhibit significant genotoxic potential. These data could be valuable for risk assessment purposes and also for further characterizing the new in vivoPig-a gene mutation assay.

From stem cell to red blood cells in vitro: "the 12 labors of Hercules".

This article describes the research in progress that will permit the large-scale production of human red blood cells from hematopoietic stem cells. It also discusses the current state of this research, suggests the obstacles to be overcome to pass from the laboratory model to clinical practice, and analyzes the possible indications in the medium and long term. The potential interest of pluripotent stem cells as an unlimited source of red blood cells is considered. If it succeeds, this new approach could mark a considerable advance in the field of transfusion.

Development of a technique for quantification of reticulocytes and assessment of erythrocyte regenerative capacity in birds.

OBJECTIVE: To develop a reticulocyte classification scheme, optimize an avian reticulocyte staining protocol, and compare the percentages of reticulocyte types with polychromatophil percentage in blood samples from birds. SAMPLE POPULATION: Blood samples from a red-tailed hawk and 31 ill birds. PROCEDURES: A single blood sample obtained from a red-tailed hawk (Buteo jamaicensis) was used to optimize the staining protocol. For optimization of the staining protocol, 4 dilutions of whole blood with new methylene blue stain and 4 incubation times were evaluated. From samples submitted for avian CBCs, EDTA-anticoagulated whole blood samples from 31 ill birds were randomly selected and examined to compare polychromatophil and reticulocyte percentages. Reticulocyte staining was performed in all samples by use of a 1:3 (whole blood to new methylene blue) dilution with incubation for 10 minutes at room temperature (approx 22 degrees C); reticulocytes were assessed as a percentage of 1,000 RBCs by 2 independent observers. In Wright-Giemsa-stained blood smears, a polychromatophil percentage was similarly determined. RESULTS: 4 avian reticulocyte types were defined: ring-form reticulocytes, aggregate reticulocytes, and 2 subcategories of punctate reticulocytes. A reticulocyte-staining protocol was optimized. Interobserver and intraobserver variations in assessment of reticulocyte and polychromatophil percentages were not significant. A strong positive correlation (Spearman coefficient of rank correlation [rho] = 0.978) was identified between the percentage of polychromatophils and the percentage of ring-form reticulocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that quantification of ring-form reticulocytes provides an accurate assessment of erythrocyte regenerative capacity in birds.

Flow cytometric assessment of homeostatic aging of reticulocytes in rats.

OBJECTIVE: The purpose of this work was to develop a flow cytometry method of obtaining RNA-thiazole orange signal distribution of reticulocytes (RET) from bone marrow aspirates and determine the distribution of age for RET in the blood and bone marrow. METHODS: The thiazole orange (an RNA dye) and LDS-751 (a DNA dye) composition was used to separate RET from other cell populations. LDS 751 was used to separate red blood cells (RBC) from nucleated cells. The differentiation between mature RBC and RET was done by means of the presence of residual RNA. It also served as a marker of age of RET. In vitro changes of the fluorescence signal of the RET population allowed us to obtain the unique relationship between the signal and the age for an individual cell and was used to transform the in vivo signal distribution into the age distribution. RESULTS: The determination of age was not possible due to the lack of knowledge of the RET signal distribution at birth. Instead, a relative time (RT) that a RET needs to become a mature RBC was used. The RT distribution of RET in blood and bone marrow was determined. The RT that an average RET spends in the blood is 0.43 days and ranges from 0 to 1.1 days. The time an average RET in bone marrow needs to become a mature RBC, is 1.36 days. The RET born with the highest signal needs about 1.8 days to become a mature RBC. About 37% reticulocytes die in the circulation. CONCLUSIONS: In summary, the thiazole orange staining can be used for determination of the RET age distribution. However, only the RT distribution can be determined. The proposed method was successfully applied to characterize in vivo aging of blood and bone marrow RET.

Automated human blood micronucleated reticulocyte measurements for rapid assessment of chromosomal damage.

This study evaluated the utility of human blood micronucleated reticulocyte (MNCD71+) frequency measurement as a cytogenetic damage biomarker. The analytical methodology was flow cytometry in conjunction with a previously described three color fluorescence labeling technique that includes anti-CD71 to focus analyses on the most immature fraction of reticulocytes [S.D. Dertinger, K. Camphausen, J.T. MacGregor, M.E. Bishop, D.K. Torous, S. Avlasevich, et al., Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood, Environ. Mol. Mutagen. 44 (2004) 427-435]. Blood specimens from 50 self-reported healthy adult volunteers were studied. In addition to MNCD71+ measurements, blood plasma folate and B12 levels were assessed, since these variables tend to influence other indices of cytogenetic damage. Time-course data are also provided for 10 cancer patients undergoing treatment. For these subjects, frequency of MNCD71+ was measured immediately before therapy, and daily during the first week of chemotherapy and/or fractionated radiotherapy. For the group of healthy volunteers, the variables of age, and folate and B12 levels demonstrated no significant effect on MNCD71+ frequency. In addition, no difference was observed between pre-treatment MNCD71+ values for cancer patients compared with healthy volunteers. Regarding chemotherapy and/or partial body radiotherapy, elevated frequencies were observed upon initiation of treatment for 9 of the 10 patients studied. Maximal effects were observed 3-5 days following initiation of therapy. The largest increases in frequency of MNCD71+ (up to 25.9-fold) were observed in those patients exposed to anti-neoplastic drugs, presumably due to the systemic red marrow exposure provided by these agents. Taken together, these data support the hypothesis that the MNCD71+ endpoint represents a valuable biomarker of cytogenetic damage that does not require cell culture or microscopy-based scoring.

Development of a large-scale chemogenomics database to improve drug candidate selection and to understand mechanisms of chemical toxicity and action.

Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.

Clinical utility of the IRF: assessment of erythroid regeneration following parvo B19 infection.

Parvo B19 (Fifth disease) is an erythrotropic virus which attaches through the 'P' globoside receptor on the surface of human red blood cells and precursors. This typically benign viral infection can cause a transient aplastic anemia in patients with underlying red cell disorders. In this case, a two-year-old child presents with severe aplastic anemia without evidence of underlying disease. Erythroid regeneration is monitored through the use of the immature reticulocyte fraction (IRF) and is demonstrated by the presence of high and medium fluorescence reticulocytes in the peripheral blood three to five days prior to the peak in absolute reticulocytes.

Assessment of hematologic progenitor engraftment by complete reticulocyte maturation parameters after autologous and allogeneic hematopoietic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Hematopoietic restoration after marrow ablation is initiated by the erythroid compartment. However, the absolute microscope counts or corrected percentage of reticulocytes have proven to be poor markers of hematopoietic engraftment. Some reports have highlighted the usefulness of automatic flow cytometry methods to determine highly fluorescent reticulocytes, or mean fluorescence index. In this series of 60 hematopoietic stem cell transplants, we sought the normal kinetics throughout the post-transplant period of the following reticulocyte maturing parameters: highly fluorescent reticulocytes (RETH), immature reticulocyte fraction (IRF), mean fluorescence index (MFI) and also mean reticulocyte volume (MRV). DESIGN AND METHODS: Sixty consecutive patients undergoing allogeneic bone marrow (30 cases) and autologous mobilized stem cell transplantation (30 cases) were studied. Parameters of reticulocyte maturation were measured every other day from the beginning of the conditioning regimen until myeloid engraftment. RESULTS: Nadir values for the analyzed reticulocyte parameters were found between days +4 and +7 and thereafter, increases in these reticulocyte parameters appeared earlier than the rise in neutrophils. We considered erythroid engraftment to have occurred on the day when RETH reached 3%, IRF 10%, MFI 10 and MRV 110 fL. These cut-offs were assigned considering the 25% quartile for each parameter on the day that the myeloid engraftment occurred. The median engraftment days for RETH were +9 and +16, for IRF +9 and +13, for MFI +9 and +13 and for MRV +11 and +13 in autologous and allogeneic procedures, respectively. When compared to standard neutrophil engraftment, IRF and MFI engraftment occurred significantly earlier in all patients. Remarkably, we found a statistical correlation between the day a reticulocyte parameter reached its cut-off and the subsequent day of absolute neutrophil count (ANC) recovery for MFI after allogeneic transplants and for MRV after autologous procedures (p < 0.001 and p= 0.02, respectively). Of all the clinical parameters tested, only the number of infused CD34 cells showed a statistical influence on erythroid engraftment in autologous transplant. INTERPRETATION AND CONCLUSIONS: Early reticulocytes appear sooner than neutrophils after both autologous and allogeneic transplants, and any determined reticulocyte parameter can reliably measure this fraction. Nevertheless, our results show that MRV and MFI cut-offs are useful for determining subsequent myeloid engraftment. These findings could be relevant to decision-making in those patients with primary graft failure heralded by an absence of increasing values of MFI and MRV, indicating very low production of reticulocytes from the graft, who could, therefore, benefit from earlier rescue therapy.

Automated reticulocyte counting and measurement of reticulocyte cellular indices. Evaluation of the Miles H*3 blood analyzer.

This study evaluated reticulocyte counting and measurement of reticulocyte cellular indices with the Miles H*3 blood analyzer, a new instrument that combines the Technicon/Miles technology for blood cells counting with a staining technique allowing counting of reticulocytes, quantification of staining intensity and measurement of reticulocyte cellular indices. Reticulocyte counts obtained with the Miles H*3 analyzer were compared with those obtained by manual counting, flow cytometry (thiazole orange method) and by the Sysmex R-3000 (Baxter Diagnostics) reticulocyte analyzer. Reticulocyte counting with the Miles H*3 showed excellent precision, and linearity in the range tested (1.1-49% and 1-72% reticulocytes, respectively, with two different protocols) with no significant carryover. Reticulocyte counts were stable after storing blood samples for 72 hours at 4 degrees C. Comparison of the four different methods, showed an acceptable intraclass correlation between Miles H*3 and Sysmex R-3000 (intraclass correlation coefficient, [ri] = .952), Miles H*3 and flow cytometry (ri = .922), and Sysmex R-3000 and flow cytometry (ri = .938). There was no satisfactory correlation between any of the three automated methods and the values obtained with manual counting of reticulocytes (ri = .538-.755), consistent with the well known imprecision of the manual technique. For a group of normal pediatric subjects, age 1-10, we obtained the following values (+/- SD) of reticulocyte indices: mean corpuscular volume 97.6 +/- 4.7 fL; cell hemoglobin concentration mean 28.2 +/- 1.4 g/dL; cell hemoglobin content 26.7 +/- 1.6 pg. We determined the direct cost, including depreciation, of the manual and instrumental methods. Cost/test varied from $1.61 for manual method to $6.03 for the Sysmex R-3000. Cost/test for flow cytometry and Miles H*3 were $3.34 and $3.49, respectively.

Alternative method of gestational age assessment by the measurement of human erythrocyte differentiation antigen expression.

Standard estimates of gestational age are dependent on such subjective data as maternal recollection of last menstrual period, ultrasound examination of the fetus, and the postnatal physical and neurological examination (Ballard score). We hypothesized that a quantitative, objective laboratory test using flow cytometric analysis of erythroid differentiation antigens could be useful to predict gestational age. For this study erythrocyte samples were obtained within 24 hours of birth from 25 infants (gestational ages 20 to 41 weeks) who met the criteria that traditional estimates of gestational age, such as the Ballard score, fetal ultrasound, and maternal estimate of last menstrual period all agreed within 1 week for the assessed infant's gestational age. Study measurements included reticulocyte count and determination of the percentage of erythrocytes that expressed the 5F1 and 20.3 erythropoietic differentiation antigens. Linear regression analysis indicated that the best correlations with gestational age were reticulocyte count (R2 = .354) and the reciprocal of the percentage of erythrocytes expressing the 5F1 antigen (R2 = .470). When both variables were incorporated into a linear regression model, the predictability of gestational age achieved an R2 = .608. Through this study we have established the feasibility and methodology of using fetal and newborn erythrocytes to provide an objective assessment of gestational age by flow cytometric analysis of erythroid differentiation antigen expression. This methodology will allow for an independent assessment of gestational age when fetal blood sampling is performed for other prenatal diagnostic studies. Further investigation is needed to identify other erythroid differentiation markers that would improve the accuracy of our model to predict gestational age.

Evaluation of the Sysmex R-1000. An automated reticulocyte analyzer.

The new fully automated reticulocyte analyzer, Sysmex R-1000 (TOA Medical Electronics, Kobe, Japan), was evaluated for its routine use in the Hematological Laboratory at the University Hospital Basel, Switzerland. The operating characteristics, such as within-run precision, linearity, and carryover, fulfilled the manufacturer's specifications and are excellent. Correlation with the standard method, manual reticulocyte counting, is linear for normal and high values. For low reticulocyte counts the regression points show a deviation from their linearity. An absolute zero value is not obtained by the R-1000. The R-1000 measures total RNA content of each cell and expresses the value as low fluorescence ratio (LFR), medium fluorescence ratio (MFR), and high fluorescence ratio (HFR). The analysis of this ratio resolves the problem of zero reticulocytes: A fraction of less than 0.002 (0.2%) with an LFR of 100% represents aplasia; a shift of the intensity of fluorescence to HFR heralds regeneration. Results of samples stored at room temperature remain stable and within the range of the within-run precision for up to 12 hours, when stored at 5 degrees C for more than 48 hours. The authors conclude that the R-1000 is easy to operate, fulfills the criteria for accuracy and precision, and is highly suitable for daily routine use in a large central hematologic laboratory.

Flow cytometric reticulocyte quantification using thiazole orange provides clinically useful reticulocyte maturity index.

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.

Flow cytometric reticulocyte counting with thioflavin T in a clinical hematology laboratory.

Flow cytometric measurement of reticulocyte counts, using the cationic dye thioflavin T, was implemented in a clinical hematology laboratory. Results obtained correlated well with manual reticulocyte counts using new methylene blue, but the reproducibility of counts done with the flow cytometer was much greater than that with manual counts. The new procedure permitted a reduction in laboratory staff, but required an intensive period of training for technologists. Utilization of the cytometer required about four hours per day of machine time for 60 counts, permitting implementation of other flow cytometric assays in the clinical laboratory.

Assessment of toxicity of o-nitrochlorobenzene in rats following a 4-week inhalation exposure.

o-Nitrochlorobenzene (ONCB) is a chemical intermediate used for the synthesis of various industrial chemicals. To evaluate the subchronic toxicity of this compound, three groups of 15 male and 15 female Sprague-Dawley rats were exposed to ONCB vapor 6 hr/day, 5 days/week for 4 weeks at target concentrations of 10, 30, or 60 mg/m3. A control group of 15 animals/sex was exposed to room air in a separate inhalation chamber. Concentrations of ONCB in the chambers were determined at least three times a day using a uv spectrophotometer. Parameters monitored in this study included observation for signs of toxicity, body weights, ophthalmoscopic exam, hematology, and clinical chemistry. At necropsy, selected organ weights were recorded and over 35 tissues/animal were examined microscopically for all control and high-exposure level animals. No mortality was observed in this study. Mean body weights of all groups were comparable to controls. Animals exposed to the mid and high concentrations of ONCB showed a significant increase in blood methemoglobin and a significant decrease in hemoglobin, hematocrit, and red blood cell counts. Spleen and liver weights (absolute and relative to body weight) were significantly increased for these two groups. Microscopic changes, observed only in the spleen, included increased degree of extramedullary hematopoiesis and hemosiderosis. These data suggest that the toxicity of ONCB is comparable to that of its structural analog, p-nitrochlorobenzene. Thus these two compounds should have similar work-place exposure limits.