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PubMed2001

Automated reticulocyte counting using the Sysmex RAM-1.

Reticulocyte counting using the Sysmex RAM-1 was evaluated. The results of 113 samples analysed on the Sysmex RAM-1 were compared with those from both flow cytometry (FCM) and a manual technique. Reticulocyte counting with the Sysmex RAM-1 showed excellent precision, with an overall coefficient of variance (CV) less than 5%, and almost no carry-over. Reticulocyte counts were stable after blood storage for 48 h at 4 degrees C and room temperature (RT). Linearity was demonstrated very well for Sysmex RAM-1 and FCM (rRAM=0.9998, rFCM=0.9974, P < 0.001) when reticulocyte counts were 5 x 109/l-480 x 109/l. Comparisons of methods showed a good relation for reticulocyte counts between Sysmex RAM-1 and manual counting (r=0.9760), FCM and manual counting (r=0.9623), and Sysmex RAM-1 and FCM (r=0.9527). Analysis of receiver operating characteristic curves showed that the true-positive rate (TPR) was 0.95 for Sysmex RAM-1, and 0.83 for FCM; the area under curve was 0.999 and 0.972 for Sysmex RAM-1, 0.990 and 0.900 for FCM. These findings confirm that Sysmex RAM-1 can make counting reticulocytes easier, more accurate and more reproducible.

Humans↗
PubMed1985

Analytic inaccuracy and imprecision in reticulocyte counting: a preliminary report from the College of American Pathologists Reticulocyte Project.

Precision and accuracy of reticulocyte counting appears to require significant improvement. Several poorly controlled analytic variables affect reticulocyte count performance including sample size, use of an ocular insert, interobserver variation in reticulocyte definition, and possibly the type of blood smear evaluated. These sources of imprecision and inaccuracy are discussed, and new data from the initial phases of the College of American Pathologists assessment of reticulocyte counting are presented. Recommendations for analytic goals for both manual and automated reticulocyte counting are provided.

Automation↗
PubMed1994

Yield of reticulocyte counts and blood smears in term infants.

Reticulocyte counts and blood smears are commonly recommended to evaluate jaundice in newborns. To investigate the results and diagnostic yield of these tests, we reviewed a computerized database and medical records of term newborns who had reticulocyte counts (n = 799) or blood smears (n = 781) within the first week after birth at two hospitals. Nearly a threefold difference was noted in reticulocyte counts between the two hospitals (median 8.0% vs 2.8%; P < .0001), apparently due to differences in laboratory methods. Among the patients with "abnormal" reticulocyte counts or blood smears (n = 192), isoimmunization was diagnosed in 54, presumed hemolysis of unknown etiology in two, G6PD deficiency in one, and pyropoikilocytosis in one. We conclude that better standardization of reticulocyte counts is needed. When ordered as screening tests for hemolysis in jaundiced infants, reticulocyte counts and blood smears seldom lead to diagnoses of hemolysis other than isoimmunization.

Bilirubin↗
PubMed2001

[Validity of reticulocyte count testing in reimbursement practice: a cost-effectiveness analysis].

Recently reticulocyte count testing has markedly decreased in Japan. We studied the current status of the reduction of reimbursement for reticulocyte count testing and the cost-effectiveness of the test in an academic hospital. The orders of complete blood counts(CBC) accompanied by reticulocyte count testing in Tokai University Hospital declined from 58.9 to 13.3% during the last decade. Among a total of 15,538 test orders for reticulocyte count in outpatients, full reimbursement was refused in 123(0.79%) during a one year period from April 1999 to March 2000. The cost-effectiveness of reticulocyte count testing was evaluated in 719 new outpatients who visited Tokai University Hospital during this period. A useful result(UR) of testing was defined as that which contributed to a change in a physician's diagnosis- or decision-making, relating to a "tentative initial diagnosis" obtained from patient history and physical examination alone. Cost-effectiveness for either CBC alone or in combination with reticulocyte count testing was evaluated by calculating clinical effectiveness(the number of URs yielded by a test per patient), economic efficiency(cost required for a test per UR generated), and the cost-effectiveness values(cost required for additional test(s) per additional UR generated). There was a wide disparity in the clinical effectiveness of reticulocyte count twisting among the hospital departments, ranging from 0.53 UR/patient(Gastroenterology-2) to 0(Oral Surgery, Neurology, Urology). Poor clinical effectiveness shown in particular hospital departments endorsed the increasing percentages in reimbursement rejection by the payer of health insurance. The test should be ordered more carefully in consideration of reimbursement and in regard to its cost-effectiveness among different patients groups.

Cost-Benefit Analysis↗
PubMed1994

Do the reticulocyte maturation fractions and bone marrow reticulocyte count further help the classification of anemias?

BACKGROUND: Reticulocyte count plays a major role in anemic evaluation. The conventional method done by the manual supravital staining cannot subclassify the group of less than 2% of corrected reticulocyte count. The newly-developed flow cytometer provides different maturation fractions by measuring its fluorescent intensity. The reticulocytes are believed to shift to the circulation from the bone marrow earlier in more severe anemia. Therefore, the purpose of this study is to evaluate the role of reticulocyte maturation fractions and bone marrow reticulocyte in anemia classification. METHODS: By using a fully automated counter, the roles of the reticulocyte with maturation and their shifting from bone marrow were evaluated in anemias. Different groups of subjects (243 in total) including aplastic, nutritional, and infiltrative anemias and anemia due to excess destruction and blood loss were studied. Each subject had bone marrow examination for morphologic diagnosis and reticulocyte evaluation. RESULTS: Both the absolute count and the maturation fractions of reticulocytes showed significant difference among marrow infiltration, aplastic anemia, and hemolytic anemia. Both the absolute reticulocyte count and less mature fractions were lowest in aplastic group. The marrow reticulocyte counts and shift ratio to circulating blood added little benefit in the classification of anemias. CONCLUSIONS: The automated reticulocyte count with maturation fractions helps classify anemias, particularly for those with low reticulocyte count by the manual method.

Adolescent↗
PubMed1997

Comparison of a semi-automated new Coulter methylene blue method with fluorescence flow cytometry in reticulocyte counting.

A semi-automated new methylene blue method for reticulocyte counting was compared to the manual method and fluorescence flow cytometry. Over a wide range of reticulocyte counts (0.3-12.7%) the new methylene blue method was found to be comparable to that of fluorescence flow cytometry, with high precision. Between-run precision studies showed coefficients of variation (CVs) of 4.8, 6.9 and 14.5% for reticulocyte counts of 9.5, 2.4 and 0.7%, respectively. Within-run precision studies showed CVs of 8.6, 8.1 and 6.1% for reticulocyte counts of 0.9, 5.3, and 18.4%, respectively. The correlation coefficient between the Coulter method and the manual method was 0.83, and 0.85 between the Coulter method and flow cytometry. The possibility of extending the incubation time to at least 4 h and the insignificant changes observed with samples stored for up to at least 72 h at 4-8 degrees C make this new method most convenient and allow any laboratory having Coulter STKS or MAXM cell counters to perform reticulocyte counts in an efficient, reliable manner.

Cell Separation↗
PubMed1991

Comparison of a modified thiazole orange technique with a fully automated analyser for reticulocyte counting.

Two independent methods for quantitating reticulocyte counts were compared. One used a modified thiazole orange technique and a flow cytometer (Becton Dickinson FACS); the other was a fully automated whole blood analyser (Sysmex R1000). Both methods gave comparable results with a coefficient of variation of less than 5%. Samples measured using the R1000 showed a negligible decrease in the reticulocyte count over five days at room temperature, although there was evidence of continuing intracellular maturation: with thiazole orange there was an apparent increase. A practical reference range of 20-70 x 10(9)/l was established from 89 normal subjects. The close correlation between the two independent estimates indicates the validity of the quantitation of the reticulocyte count and shows that automation allows significant changes within and below the normal range to be detected with a degree of reliability which was not previously possible.

Benzothiazoles↗
PubMed1989

[Automated measurement of reticulocyte count by flow cytometry. II: Analysis of the blood containing abnormal erythrocytes or giant platelets].

We have examined the influence of erythrocytes containing inclusion bodies, nucleated red cells or giant platelets on the measurement of reticulocyte count by automated machine, R-1000. Correlation of the reticulocyte count between automated and conventional method was extremely good in the blood containing red cells with Jolly bodies, Pappenheimer bodies or basophilic stippling . However, correlation was poor when the sample contained the nucleated red cells. Reticulocyte count was decreased in the blood with significant amounts of nucleated red cells. Since nucleated red cells themselves are not counted as reticulocytes in the machine, this was considered to be due to increased young reticulocytes which frequently appeared with nucleated red cells. Both cold agglutinated red cells and giant platelets apparently influenced the reticulocyte count by the R-1000. These results suggest that red cells with Jolly bodies, Pappenheimer bodies or basophilic stippling do not influence the automatic counting of reticulocytes. Although nucleated red cells, cold agglutinated red cells and giant platelets affected the reticulocyte count, the machine shows abnormal flags in most of above cases (except highly agglutinated red cells), so that one can recount reticulocytes by conventional method. We conclude the machine can safely count the reticulocytes even in the blood containing abnormal red cells or platelets.

Blood Platelets↗
PubMed1990

Evaluation of reticulocyte counts by flow cytometry in a routine laboratory.

Reticulocyte counting using flow cytometry and a membrane-permeable fluorescent dye, thiazole orange, was evaluated as an alternative to the conventional manual method. The flow cytometric method was more precise (mean c.v. of 4.3%) than the imprecise manual technique (mean c.v. of 22.4%). Linearity was highly acceptable (r = 0.99) over the reticulocyte count range of 1.8-30.1%. Flow cytometry allows processing of large numbers of samples with reduction in technologist time, and the improved precision and tenfold increase in the number of cells counted should considerably improve the clinical utility of the reticulocyte count.

Adult↗
PubMedDate not supplied

Performance of reticulocyte counts in stored blood specimens in a pediatric population utilizing new methylene blue.

Although reticulocyte counts can be reliably performed for up to 48 h after storage in EDTA, it is unclear whether this is applicable to the pediatric age group. In order to evaluate this, manual reticulocyte counts were performed on 20 specimens from pediatric patients stored at 4 degrees C for up to 24 h post collection. Samples were evaluated at 1-3, 6, 12, 18, and 24 h after storage in EDTA vacutainer tubes at 4 degrees C. The age of the subjects ranged from 1 day to 9 years with a median age of 3 years. Patients' reticulocyte counts ranged from 0 to 27% (5.89 +/- 7.21). No clinically significant changes were evident in the reticulocyte count over 24 h after specimen collection. The mean of the 20 specimens at 1-3 h was 5.50 and at 24 h was 5.40 (P > .05). The standard deviation of the mean values ranged from 7.03 to 7.26 (P > .05). The results indicate that reticulocyte counts may be performed on previously drawn blood held at 4 degrees C for up to 24 h post collection in a pediatric population without significant difference from baseline values.

Blood Preservation↗
PubMed1995

[Evaluation of the Coulter method for reticulocyte count].

Although the automation of reticulocyte counts was developed in the beginning of the 80's, the reticulocyte modules integrated in the hematology analysers have been only recently made available. The purpose of our study is the evaluation of the reticulocyte module included in the Coulter STKS, performed in the Necker-Enfants-Malades hospital laboratory. Regarding the technical conditions of use, this simple and user-friendly method can be easily adapted to routine use. Results, as compared to those obtained with two reference methods, microscopy and cytofluorometry, are accurate if the operator's attention is drawn by some causes of interferences, generally flagged by the instrument.

Evaluation Studies as Topic↗
PubMed1993

Errors in automated reticulocyte counts due to Heinz bodies.

Serial reticulocyte counts on a patient with an unstable haemoglobin haemolytic anaemia whose red cells contained Heinz bodies were obtained by two automated methods. One false low and several false high counts were obtained with standard threshold settings; each was accompanied by activation of the appropriate alarm. With the FACScan a bimodal red cell distribution was found, corresponding to the proportions of Heinz body positive and negative cells. This case illustrates the fact that Heinz bodies can interfere with automated reticulocyte counting methods, and to a degree that could be clinically important.

Anemia, Hemolytic↗
PubMed1992

Evaluation of erythropoiesis after bone marrow transplantation: quantitative reticulocyte counting.

Erythroid regeneration is an important and separate element in the engraftment process in allogeneic and autologous bone marrow transplantation (alloBMT, autoBMT). Qualitative visual reticulocyte counting has proved inadequate in the evaluation of erythropoiesis after BMT but automated flow cytometry now allows the reliable quantitation of reticulocytes even to very low levels. Reticulocyte counts and highly fluorescent reticulocyte (HFR) counts (very early reticulocytes) were estimated daily in recipients of 22 autoBMT and 14 alloBMT using a Sysmex R-1000 automated reticulocyte counter. Marrow ablation caused an immediate and rapid fall in both the reticulocyte count and the HFR. Measurable numbers of reticulocytes persisted throughout the hypoplastic period, but HFR fell to zero in the majority of both the autoBMT and alloBMT. HFR rose significantly after a median time of 14 d post-autoBMT, and 12 d post-alloBMT. Attainment of 15 x 10(9)/l reticulocytes and 0.5 x 10(9)/l HFR at day 21 post-transplant was associated with ultimate engraftment in 100% cases. Inadequate engraftment was seen in the majority of patients whose responses fell below these levels. Graft-versus-host disease was associated with a transient slight reduction in reticulocyte count. Neither episodes of infection nor blood transfusions had any significant impact on trends of reticulocytes or HFR. Automated flow cytometric reticulocyte counting has been shown to provide an accessible measure of erythroid activity which may be of predictive value in the management of patients following bone marrow transplantation.

Blood Cell Count↗
PubMed1991

Reticulocyte counting in thalassemic and other conditions with the R-1000 Sysmex analyzer.

Precise reticulocyte counts are difficult to obtain by the manual method when their percentage in the blood is low or normal. In these instances, rapid reticulocyte counting by flow cytometry appears to offer more accuracy and precision. The purpose of this study was to establish reticulocyte counts in heterozygous beta-thalassemia for reference purposes and to evaluate the performance of the recently introduced apparatus R-1000 (Sysmex) in the very heterogeneous thalassemic and sickle-cell syndromes. We studied a total of 364 samples; 102 heterozygous beta-thalassemia carriers, 180 normal matched controls, 36 patients with thalassemia major or intermedia, and 46 patients with various sickle-cell syndromes. Reticulocyte counts (both as percentage and as total number) were higher in heterozygous beta-thalassemia than in normal controls (p less than 0.001) and showed an inverse correlation with the respective hemoglobin values (p less than 0.001). These results confirm the proposed slightly increased erythropoietic activity in heterozygous beta-thalassemia carriers. A drawback of the technique is that the reticulocyte-platelet discrimination error is signaled frequently in all conditions displaying a marked red cell heterogeneity, especially when these are associated with high reticulocyte numbers. This calls probably for readjustment of the corresponding algorithm. In addition, all these conditions show a significantly increased auramine-O mature red-cell nonspecific fluorescence.

Anemia, Sickle Cell↗
PubMed1993

Reticulocyte count with maturation fractions in pancytopenic evaluation by a fully automated counter.

Using a fully automated reticulocyte counter, the roles of the reticulocyte count with maturation in pancytopenia were evaluated. Different groups of pancytopenia including aplastic anemia, infiltrative marrow disorder, hypersplenism, and megaloblastic anemia were recruited. All patients had bone marrow examinations for morphological diagnosis and reticulocyte evaluation using an automated counter. The roles of these parameters were then analyzed statistically in the differential evaluation among these conditions. The following subjects were studied: 292 normal subjects, 67 cases of aplastic anemia, 69 cases of marrow infiltration by different malignancies, 35 cases of hypersplenism, and 13 cases of megaloblastic anemia. The results showed that the absolute reticulocyte counts were lowest in the groups of aplastic anemia and megaloblastic anemia and highest in hypersplenism. Both showed significant differences from the infiltrative groups. The maturation fractions were most immature in the group of marrow infiltration and are significantly different from the other groups. It was concluded that the highest absolute reticulocyte count (> 0.09 10(12)/L) obtained in pancytopenic patients suggests it to be a case of hypersplenism. The lowest counts (< 0.03 10(12)/L) with lowest immature fractions (< 10%) suggest the groups of aplastic or megaloblastic anemias. The highest immature fractions (> 30%) with a nearly normal reticulocyte count favor the group of marrow infiltration.

Adolescent↗
PubMed1996

Follow-up of leucocyte and reticulocyte counts for the prediction of early graft failure after non-HLA-identical BMT in children.

Allogeneic BMT using a non-genotypically HLA-identical donor may be curative for children suffering from lethal haematological diseases, who lack a genotypically HLA-identical donor. Unfortunately, graft failures are often seen, especially after T cell depletion of the graft. We studied whether untimely decreased counts of leucocytes and reticulocytes in peripheral blood might predict graft failure at an early stage. Fifty-five recipients of a non-genotypically HLA-identical BM graft were included in the study; data from these children were compared with those of 77 recipients of a genotypically HLA-identical BM graft. Time-related reference values of peripheral blood leucocyte and reticulocyte counts were established in graft recipients with proven donor-origin recovery after BMT. Graft failure after nonHLA-identical BMT was observed in 16 out of 55 children (29%) and after HLA-identical BMT in one out of 77 (1.3%). With respect to early graft failure, the predictive value of granulocyte numbers falling below the lower limit of the reference values and a rapid decline of reticulocyte numbers after their appearance in peripheral blood was 100% (95% confidence intervals of 83-100% and of 80-100%, respectively). Early immunosuppressive intervention was applied in six patients and was successful in three of them.

Adolescent↗
PubMed1997

Evaluation of the reticulocyte count portion of technicon H*3 blood analyzer: lowering the test expense by reducing the reticulocyte reagent.

Automated reticulocyte counting has become an essential instrument of the hematology laboratory. This automatic technique has lead to diminishing labour tasks and to significant improvements in accuracy and precision compared with the manual microscopic methods. In any event, it adds a considerable expense to the laboratory budget. Here, we report the modified method of applying the new mixture of 1 microL of whole blood with 1 mL of reticulocyte reagent, which we evaluated for its accuracy and precision, instead of using the mixture of 3 microL of whole blood with 3 mL of reticulocyte reagent recommended by the company. We demonstrated the accepted accurate and precise results of percentage and absolute number of retculocyte count, low-stained reticulocyte count and its corpuscular indices; the mean reticulocyte corpuscular volume (MCVr), mean reticulocyte corpuscular hemoglobin concentration (CHCMr), and mean reticulocyte hemoglobin content (CHr). These suggested that, for every red cell assessed, the number, the cell volume, hemoglobin content and concentration are accurately and precisely measured by the modified method while the sub-populations of reticulocyte count and distribution width of reticulocyte indices are variable. In conclusion, our results provided the information that 1) the modified method can be used as a routine test and it provides accurate and precise results; 2) with the modified method, two-thirds of the expense spent for reticulocyte reagent can be saved; 3) it should not be used for research purposes.

Indicators and Reagents↗
PubMed2000

Automated immature reticulocyte counts are early markers of engraftment following autologous PBSC transplantation in patients with lymphoma.

The traditional indicators of engraftment following PBSC transplantation (PBSCT) are the rising total WBC count and ANC. Reticulocytes may be an earlier indicator, since as reticulocytes mature, there is a gradual loss of cellular RNA, which can be measured using methylene blue and light scatter with an Abbott CD 3500 automated counter (Abbott Laboratories, Maidenhead, U.K.). Reticulocytes can be divided into three fluorescence ratios depending on the amount of light scatter generated, high, medium, and low. The most immature are the high fluorescence reticulocytes (HFR). Standard engraftment parameters together with HFR were measured in a homogeneous group of 25 patients with lymphoma after PBSCT using a standard conditioning protocol. An ANC of 0.5 x 10(9)/L was achieved after a median of 10 days (mean 11.2 days, range 9-22). The recovery of the HFR to 2% of the total reticulocytes was significantly shorter, with a median of 8 days (mean 7.5 days, range 6-10) (p < 0.0001). The values of HFR to 2% preceded the ANC of 0.5 x 10(9)/L in 24 of the 25 patients by a median of 3 days (mean 3.8 days, range 2-12 days). On this basis, it can be determined that in 96% of cases, engraftment was indicated earlier by HFR measurement. The HFR to 2% even preceded the ANC of 0.1 x 10(9)/L in 23 of the 25 patients, showing that engraftment was indicated earlier in 92% of patients. Immature reticulocytes appearing in peripheral blood can be reliably measured by automated cytometers, and HFR can, therefore, be used as an earlier indicator of engraftment following PBSCT. This information provides the opportunity for earlier cessation of antibiotics and growth factors and could lead to earlier discharge from hospital, with cost savings.

Adult↗