Standardization of reticulocyte counts in the athlete biological passport: A practical update.

INTRODUCTION: The athlete biological passport monitors blood variables over time to uncover blood doping. With the phasing in of a new series of blood analyzers, the Sysmex XN series, it was necessary to examine the comparability of results with the previously employed XT/XE series. A previous comparison between XN and XT/XE series suggested a small but significant bias between the two instruments in the measurements of RET%. Here, we examined the comparability of RET% on the XN and XT/XE platform using data collected over the first year since the transition. METHODS: The comparability of results obtained from XN and XT/XE instruments was assessed using three datasets: (i) 767 blood samples measured on both instrument series in 22 WADA-accredited laboratories, (ii) 27 323 samples measured on either instrument across 31 laboratories, and (iii) 119 clinical samples and 110 anti-doping samples measured on both instruments in a single laboratory. RESULTS: Analysis of the three datasets confirms the previous observation of a bias toward higher RET% values for samples measured on Sysmex XN instruments compared with the XT/XE series. Using data across a larger number of XN instruments and a larger athlete population, the current work suggests that the bias is proportional and slightly higher than previously observed across most of the range RET% values. CONCLUSION: A model is proposed for the comparison of data across XN and XT/XE technologies whereby the instrument bias increases proportionally with RET% measured on Sysmex XN Series, but where the rate of increase is negatively related to IRF%.

Use of Sysmex XN-10 red blood cell parameters for screening of hereditary red blood cell diseases and iron deficiency anaemia.

INTRODUCTION: In daily practice in haematology laboratories, red blood cell (RBC) abnormalities are frequent and their management is a real challenge. The aim of this study is to establish a "decision tree" using RBC and reticulocyte parameters from the SYSMEX XN-10 analyser to distinguish between patients with a hereditary RBC disease from iron deficiency anaemia and other patients. METHODS: We analysed results of complete RBC counts in a cohort composed of 8217 adults divided into 5 different groups: iron deficiency anaemia (n = 120), heterozygous haemoglobinopathy (n = 92), sickle cell disease syndrome (n = 56), hereditary spherocytosis (n = 18) and other patients (n = 7931). A Classification And Regression Tree (CART) analysis was used to obtain a two-step decision tree in order to predict these previous groups. RESULTS: Five parameters and the calculated RBC score were selected by the CART method: mean corpuscular haemoglobin concentration, percentage of microcytes, distribution width of the RBC histogram, percentage of nucleated red blood cells, immature reticulocytes fraction and finally RBC Score. When applying the tree and recommended flowchart, 158/166 of the RBC hereditary disease patients and 114/120 iron deficiency anaemia patients are detected. Overall, the correct classification rate reached 99.4%. Sensitivity and specificity for RBC disease detection were 95.2% and 99.9%, respectively. These results were confirmed in an independent validation cohort. CONCLUSION: Based on the XN-10 RBC and reticulocyte parameters, we propose a two-step decision tree delivering a good prediction and classification of hereditary RBC diseases. These results can be used to optimize additional reticulocyte analysis and microscopy review.

Evaluation of the Reticulocyte Production Index in the Pediatric Population.

OBJECTIVES: Since hematologic values vary with age in children, we evaluated the agreement between the "traditional" reticulocyte production index (RPI) and an RPI by age (RPI/A)-adjusted normal values. METHODS: A retrospective, observational, and analytical study was performed on CBCs of children with anemia younger than 18 years. The agreement and clinical repercussions of the RPI values were analyzed with an RPI/A developed with theoretical values for different ages. RESULTS: A total of 5,503 tests were analyzed and no systematic error between the two indices was found; however, there were significant proportional differences at higher values that resulted in lower RPI/A in children younger than 15 days and higher RPI/A in children aged 15 days and older. No agreement was observed at any age. The proportion of arregenerative anemia diagnosed using RPI/A was higher in children younger than 15 days and lower in those 15 days and older. CONCLUSIONS: RPI is not an adequate tool for evaluating the erythropoietic capacity of bone marrow in the pediatric population. The disagreement between the results can be explained by the difference in normal hematologic values between children and adults.

Standardization of reticulocyte counts in the athlete biological passport.

INTRODUCTION: The percentage of circulating reticulocytes (RET%) is a useful marker of blood doping in the context of the Athlete Biological Passport (ABP). The viability of the ABP depends on the comparability of sample data obtained across multiple laboratories for a given athlete. With the recent introduction of a different technology for the measurement of reticulocytes, the goal of this study was to compare currently employed Sysmex XT/XE analyzers to the recently introduced Sysmex XN analyzer. METHODS: RET% differences were searched in two independent data sets, the first consisting of 95 369 RET% values coming from 29 laboratories located in five continents as part of routine testing for the ABP, the second from a targeted study involving 510 samples analyzed on both a Sysmex XT and XN analyzers by two different laboratories. RESULTS: A relatively small but significant bias of 0.27 ([0.22-0.35] 95% CI) for the first data set and 0.19% ([0.16-0.22] 95% CI) for the second data set was observed with Sysmex XN analyzers returning higher values than Sysmex XT/XE analyzers. This bias appears constant over most of the range of RET% measured in elite athletes. CONCLUSION: When RET% values are obtained for the same athlete with different technologies (XT/XE vs XN), an adjustment of RET% emanating from the XT/XE instruments through a decrease of 0.22% within the ABP calculated ranges appears to be sufficient to integrate the results from the two technologies.

Reticulocyte count and extended reticulocyte parameters by Mindray BC-6800: Reference intervals and comparison with Sysmex XE-5000.

INTRODUCTION: In this study, analytic performance (imprecision, carryover, time stability) and diagnostic efficiency of Mindray BC-6800 analyzer to quantify reticulocytes and extended reticulocyte parameters was evaluated. Moreover, reference intervals on adult population were determined. Results were compared with those obtained by Sysmex XE-5000 analyzer. METHODS: One hundred and eighty-four healthy adults of both sexes, and 368 subjects affected by various pathologic conditions (nutritional anemias before and after treatment, hemolytic and posthemorragic anemias, acute and chronic inflammations, malignancy under therapy, and beta thalassemia trait) were selected. RESULTS: Reference intervals were as follows: reticulocytes (×109 /L): 23.2-93.2; immature reticulocyte fraction: 0.015-0.14; mean reticulocyte hemoglobin equivalent (RHE) (pg): 30.9-35.7; mean reticulocyte volume (fL): 97.8-118. Imprecision on reticulocyte count at all concentrations was close to analytic goal based on within-subject biological variation. Carryover (2.3%) was negligible, and time-stability was excellent up to 8 hours. CONCLUSION: When compared with XE-5000, BC-6800 shows a good overall correlation on counting despite evidence of difference in the upper limit of reference intervals (93.2 vs 101.3). Comparison of diagnostic efficiency of extended parameters shows a good total agreement of RHE (91.6%).

Reference values for reticulocyte parameters and hypochromic RBC in healthy children.

INTRODUCTION: New erythrocyte and reticulocyte parameters available on modern hematology analyzers have shown to be useful markers in children. However, pediatric method-specific reference ranges for these indices are sparse. We aim to establish pediatric reference values for reticulocyte parameters and hypochromic red blood cells in healthy children with the ADVIA 2120 hematology analyzer. METHODS: Prospective study of 311 healthy children aged from 6 months to 18 years old with normal hematological parameters. ADVIA 2120 hematology analyzer (Siemens Healthcare Diagnostics, Amadora, Portugal) was used for whole-blood hematological measures. The sample population was grouped according to age into three cohorts, and gender distribution was used for participants aged ≥12 years. RESULTS: Age- and gender-specific reference limits of reticulocyte hemoglobin content (CHr or Ret-He), difference between the reticulocyte and erythrocyte hemoglobin content (Delta-He), immature reticulocyte fraction (IRF) and percentage of hypochromic red blood cells (%Hypo) are provided. Delta-He, IRF and %Hypo showed statistically significant gender differences in the 12-17 years group. CHr presented no significant gender variation within all age groups (median 30.8 ± 1.7 pg). CONCLUSION: Establishing reliable pediatric reference intervals for these novel hematological parameters may offer valuable prospects for clinical practice and research in the pediatrics field.

Quantitative detection of target cells using unghosted cells (UGCs) of DxH 800 (Beckman Coulter).

BACKGROUND: In the Retic channel of DxH 800 (Beckman Coulter), the red blood cells (RBCs) resistant to hemoglobin clearing are counted as unghosted cells (UGCs). The aim of this study was to evaluate that the UGC is a surrogate marker for both the detection and counting of target cells. METHODS: In total, 1181 samples including 22 from iron deficiency anemia (IDA) patients, 95 from jaundice, 2 from sickle cell anemia, 3 from thalassemia, 1 cord blood, and 269 from normal controls were analyzed. Slides were prepared from all samples except normal controls and target cells were counted for correlation analysis of target cell counts to UGCs. RESULTS: The normal control samples showed 0.01% (0%-0.01%) UGCs, and the reference range was set at ≤0.02%. The IDA samples showed 0.015% (0.01%-0.03%) UGC count and 0.05% (0%-0.2%) target cell count. The jaundice samples showed 0.98% (0.1%-5.36%) UGC count, and 1.4% (0.1%-7.0%) target cell count. The two sickle cell anemia samples showed 0.41% and 3.74% UGC counts and 0.4% and 11.5% target cell counts. A cord blood sample showed 0.01% UGCs and 0% target cells. The three thalassemia samples showed 0.01%, 1.99%, and 7.82% UGC counts and 0%, 1.4%, and 15.5% target cell counts. The samples showing poikilocytosis other than target cells showed normal UGC count (≤0.02%). The positive predictive value of UGCs was 58.2% (124/213) and the negative predictive value was 96.8% (674/696). The UGC counts were well correlated to the manual target cell counts (r=0.944, p=0.000). CONCLUSIONS: This study demonstrates for the first time in the literature that a hematological parameter obtained automatically every time a reticulocyte counting is performed can be used to both screen for the presence of target cells and reliably quantify them.

Reference intervals for red cell variables and platelet counts in infants at 2, 5 and 13 months of age: a cohort study.

AIMS: To derive reference values for red cell variables and platelet counts from a cohort of infants sampled at precise ages during the first 13 months of life. METHODS: Blood counts, reticulocyte counts and zinc protoporphyrin concentrations were obtained from healthy term infants of North European ancestry at 2, 5 and 13 months of age. RESULTS: Mean cell volume (MCV) and mean cell haemoglobin (MCH) values did not differ significantly between 5 and 13 months and MCH concentration was unaffected by age. Values of all other variables at any one age differed significantly from those at the other two. Haemoglobin, mean cell haemoglobin, zinc protoporphyrin and platelet values (95% ranges) at 2 (n=119), 5 (n=97) and 13 months (n=42) were, respectively, 91-125, 101-129 and 105-133 g/L; 28.6-33.1, 24.5-28.7 and 24.3-28.7 pg; 36-116, 25-91 and 27-57 micromol/mol haem; and 216-658, 241-591 and 209-455×10(9)/L. At 2 and 5 months, respectively, 26.9% and 10.8% of subjects had platelet counts >500×10(9)/L. Reticulocyte counts at 2 months and MCV and MCH values at 5 months were significantly higher in girls. In boys, red cell distribution width values were significantly higher at 5 months, and zinc protoporphyrin values at both 2 and 5 months. CONCLUSIONS: These findings indicate the value of obtaining reference data at precise ages during infancy and confirm and extend earlier reports indicating a gender difference in laboratory measures used to assess iron status in early infancy.

[Automated hematology analysers and spurious counts Part 3. Haemoglobin, red blood cells, cell count and indices, reticulocytes]. / Anomalies et erreurs de détermination de l'hémogramme avec les automates d'hématologie cellulaire Partie 3. Hémoglobine, hématies, indices érythrocytaires, réticulocytes.

Several situations lead to abnormal haemoglobin measurement or to abnormal red blood cells (RBC) counts, including hyperlipemias, agglutinins and cryoglobulins, haemolysis, or elevated white blood cells (WBC) counts. Mean (red) cell volume may be also subject to spurious determination, because of agglutinins (mainly cold), high blood glucose level, natremia, anticoagulants in excess and at times technological considerations. Abnormality related to one measured parameter eventually leads to abnormal calculated RBC indices: mean cell haemoglobin content is certainly the most important RBC parameter to consider, maybe as important as flags generated by the haematology analysers (HA) themselves. In many circumstances, several of the measured parameters from cell blood counts (CBC) may be altered, and the discovery of a spurious change on one parameter frequently means that the validity of other parameters should be considered. Sensitive flags allow now the identification of several spurious counts, but only the most sophisticated HA have optimal flagging, and simpler ones, especially those without any WBC differential scattergram, do not share the same capacity to detect abnormal results. Reticulocytes are integrated into the CBC in many HA, and several situations may lead to abnormal counts, including abnormal gating, interference with intraerythrocytic particles, erythroblastosis or high WBC counts.

Manual and automated reticulocyte counts.

Manual reticulocyte counts were examined under light microscopy, using the property whereby supravital stain precipitates residual ribosomal RNA versus the automated flow methods, with the suggestion that in the latter there is greater precision and an ability to determine both mature and immature reticulocyte fractions. Three hundred and forty-one venous blood samples of patients were analyzed of whom 224 newborn and the rest adults; 51 males and 66 females, with ages between 0 and 89 years, as part of the laboratory routine for hematological examinations at the Clinical Laboratory of the Hospital Universitário do Oeste do Paraná. This work aimed to compare manual and automated methodologies for reticulocyte countings and evaluate random and systematic errors. The results obtained showed that the difference between the two methods was very small, with an estimated 0·4% systematic error and 3·9% random error. Thus, it has been confirmed that both methods, when well conducted, can reflect precisely the reticulocyte counts for adequate clinical use.

Under-filled blood collection tubes containing K2EDTA as anticoagulant are acceptable for automated complete blood counts, white blood cell differential, and reticulocyte count.

Current laboratory standards from Clinical Laboratory Standards Institute (CLSI) and manufacturer's (Becton Dickinson) data indicate that under-filling K(2)EDTA blood collection tubes can result in erroneous hematology values. To accommodate under-filled tubes and reduce collection volumes while optimizing our automation, we explored the acceptable limit of under-filled tubes for hematology values. We collected 8.0 ml of blood from 30 normal adult volunteers. Each donation was aliquoted in the following volumes: 4.0, 2.0, 1.0, 0.5 ml x 2. These samples were analyzed within 1 h of blood collection on Sysmex XE-2100 (Sysmex America Inc., Mundelein, IL, USA) for complete blood count, reticulocyte, and white blood cell differentials. Results of the under-filled tubes were compared to those of the standard volume. The Deming regression analysis show excellent correlation for all parameters between each under-filled blood collection volume compared to a standard 4 ml volume. The Bland and Altman analysis shows good agreement between both 1.0 and 2.0 ml compared to a 4.0 ml volume. The 0.5 ml compared to a 4.0 ml volume, however, shows increased variation on many parameters. In addition all three collection volumes show negative bias compared to the standard volume for platelet count, but the difference is considered insignificant with a percent difference of 5.5%, 3.2%, and 1.5% for 0.5, 1.0, and 2.0 ml collection volume respectively. Finally for 0.5 ml collection volume we noticed a low level of false positive flagging rate for white blood cell. Acceptable complete blood count values of under-filled powdered K(2)EDTA tubes can be obtained with as little as 1.0 ml of blood.

In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei.

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes. In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.

ISO/IEC 17025 Sysmex R-500 hematology reticulocyte analyzer validation.

The Sysmex R-500 (R-500) Hematology Analyzer is a bench-top system appropriate for the analysis of limited batches of blood samples. The R-500 provides percentage proportional (RET%), absolute reticulocyte (RET#), and absolute red blood cell (RBC#) counts. The system was validated at the Doping Control Laboratory of Athens, according to the International Committee for Standardization in Hematology, International Standards Organization (ISO/IEC) 17025, and World Antidoping Agency (WADA) specifications. The instrument calibration was performed according to the manufacturer and validation parameters comprised linearity, precision, uncertainty (intermediate and long-term precision), comparability, effect of drift, carryover, stability, and accuracy. The linearity and the comparability studies for RET#, RET%, and RBC# were expressed in regression factors (R2) and coefficients of correlation [r(x, y)], respectively. For the precision studies, the coefficients of variation for RET#, RET%, and RBC# were 9.49%, 9.83%, and <1.5%, respectively. For the intermediate precision studies, the coefficients of variation for RET#, RET%, and RBC# were 3.1%, 3.6%, and 0.6%, respectively. Carryover was found to be negligible. Sample stability was demonstrated at both room temperature and at 4 degrees C over a 24-hour period. Comparability studies for the R-500 were performed using a Sysmex SE-9500. The total evaluation led to the conclusion that the R-500 is an accurate and precise analyzer and because of to its relatively limited size, it can be considered a portable instrument, capable to be used in sports competition and training sites, where doping control and health tests are conducted. The analytical methodology of RET% measurement by the R-500 has been incorporated into the Doping Control Laboratory of Athens' Scope of Accreditation according to the ISO/IEC 17025 and WADA specifications.

European multi-center evaluation of the Abbott Cell-Dyn sapphire hematology analyzer.

This study presents the results of performance evaluations of the Cell-Dyn Sapphire (CD-Sapphire) undertaken by 3 study sites in Europe. These studies focused on the routine blood count analyses with specific consideration of precision and imprecision, linearity, inter-instrument correlations, and white blood cell differential and flagging efficiencies. The CD-Sapphire was compared to the Cell-Dyn CD4000, Bayer Advia 120, Beckman Coulter GenS, and reference microscopy.

Standardization of reticulocyte values in an antidoping context.

The lack of standardization of reticulocyte results hinders the ability of sports authorities to recognize the telltale fluctuations over time that are typical for athletes using illegal blood doping to improve their performance. Therefore, the aim of the present study was to devise a tenable approach for antidoping authorities to quantify, instrument bias. We evaluated reticulocyte data derived during a 42-week period from 210 hospital patient blood samples measured in duplicate simultaneously on up to 11 hematology analyzers located in a single laboratory. We found that square root transformation of reticulocyte values enabled quantification of interinstrument bias by using the mean reticulocyte value of a cohort of approximately 54 subjects as a de facto calibration agent. We also demonstrated that measurement precision associated with low reticulocyte values was not inferior to that associated with higher values.

Effect of altitude on second-generation blood tests to detect erythropoietin abuse by athletes.

BACKGROUND AND OBJECTIVES: ON- and OFF-model scores derived from blood parameters sensitive to erythropoiesis have been shown to be a useful tool to identify athletes who are currently injecting erythropoietin to enhance performance or those who have recently stopped doing so. We investigated changes in blood parameters and model scores during and after exposure to terrestrial and simulated altitudes. DESIGN AND METHODS: We retrospectively evaluated changes in hematologic data collected from 19 elite cyclists who lived and trained 2690 m above sea level for 26-31 days, from six elite Kenyan runners who lived 2100 m above sea level but descended to compete at sea level competitions, and from 39 well-trained subjects who resided at sea level but slept at a simulated altitude of 2650-3000 m for 20-23 days of either consecutive or intermittent nightly exposure. RESULTS: Upon ascent to a terrestrial altitude, ON- and OFF-model scores increased immediately, mainly because of an increase in hemoglobin concentration. Scores had not returned fully to baseline three weeks after return to sea level, because of the persistence of the raised hemoglobin concentration for the ON and OFF scores and a fall in reticulocyte percentage for OFF scores. Effects were smaller or negligible for simulated altitude. For Kenyan runners, ON- and OFF-model scores decreased within seven days of descent to sea level. INTERPRETATION AND CONCLUSIONS: Our results reinforce the notion that caution should be exercised when interpreting blood results from athletes who have recently been exposed to either terrestrial or simulated altitude, and appropriate allowance should be made for the effect of altitude on blood model scores.