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1 Original Article Evaluation of nucleated red blood cell count by Sysmex XE-2100 in patients with thalassaemia or sickle cell anaemia and in neonates Sabrina Buoro 1, Mauro Vavassori 1, Silvia Pipitone 2, Anna Benegiamo 2, Eleonora Lochis 1, Sabina Fumagalli 1, Anna Falanga 3, Marina Marchetti 3, Alberto Crippa 1, Cosimo Ottomano 2, Giuseppe Lippi 2 1 Clinical Chemistry Laboratory, "Papa Giovanni XXIII" Hospital, Bergamo; 2 Diagnostic Blood Chemistry Unit, University Hospital of Parma, Parma; 3 Immunohaematology and Transfusion Medicine Unit, "Papa Giovanni XXIII" Hospital, Bergamo, Italy Background. Current haematology analysers have variable sensitivity and accuracy for counting nucleated red blood cells in samples with low values and in all those conditions characterised by altered sensitivity of red blood cells to the lysing process, such as in beta-thalassaemia or sickle-cell diseases and in neonates. The aim of our study was to evaluate the performance of the automated analyser XE-2100 at counting nucleated red blood cells in the above-mentioned three categories of subjects with potentially altered red blood cell lysis sensitivity and yet a need for accurate nucleated red blood cell counts. Materials and methods. We measured nucleated red blood cell count by XE-2100 in peripheral blood samples of 187 subjects comprising 55 patients with beta-thalassaemia (40 major and 15 traits), 26 sickle-cell patients, 56 neonates and 50 normal subject. Results were compared with those obtained by optical microscopy. Agreement between average values of the two methods was estimated by means of Pearson's correlation and bias analysis, whereas diagnostic accuracy was estimated by analysis of receiver operating characteristic curves. Results. The comparison between the two methods showed a Pearson's correlation of 0.99 (95% CI; ; p<0.001) and bias of (95% CI, ). The area under the curve of the nucleated red blood cell count in all samples was 0.98 (95% CI, ; p<0.001). Sub-analysis revealed an area under curve of 0.99 (95% CI, ; p<0.001) for patients with thalassaemia, 0.94 (95% CI, ; p<0.001) for patients with sickle cell anaemia, and 1.00 (95% CI, ) for neonates. Discussion. XE-2100 has excellent performance for nucleated red blood cell counting, especially in critical populations such as patients with haemoglobinopathies and neonates. Keywords: nucleated red blood cells, XE-2100, thalassaemia, sickle cell disease, neonates. Introduction Nucleated red blood cells (NRBC) are the precursors of red blood cells (RBC) and are physiologically confined to the bone marrow. Their presence in peripheral blood is a marker of abnormal erythropoiesis and/or hypoxic stress 1-9. NRBC are present in the peripheral blood in a number of conditions, including thalassaemia syndromes, myeloproliferative disorders (especially myelofibrosis) and bone marrow metastases of solid cancers. The presence of NRBC in peripheral blood can also be a marker of extramedullary haematopoiesis or generalised haematopoietic stress as occurs in septicaemia or massive haemorrhages generally followed by acute hypoxia 1-9. Several lines of evidence now attest that the presence of NRBC in peripheral blood also has a significant prognostic value. In patients with thalassaemia the number of NRBC is an indicator of inefficient erythropoiesis and may be useful for monitoring disease and for optimising transfusion therapy 2. In preterm and non-preterm neonates, the number and persistence of NRBC are directly correlated with increased mortality rate and clinical complications 4-6. Finally, in patients in an Intensive Care Unit, an increased number of these cells is an independent risk factor for mortality 7,8. In modern laboratory practice, determining NRBC count by optical microscopy (OM) of a smear of peripheral blood is time-consuming, requires specialised resources and is also plagued by a high degree of both intra- and inter-observer imprecision The latest generation of haematology analysers are based on different methods for NRBC enumeration, and the accuracy of the NRBC count largely depends upon technology and method Despite remarkable technological advances, current instruments have limited sensitivity and accuracy for 1

2 Buoro S et al the NRBC count in samples with low values and in all those conditions in which red blood cells have altered sensitivity to the lysing process utilised by the different automated haemocytometers 12,13. This altered sensitivity, which is physiological in neonates, can also characterise patients with haemoglobinopathies, liver and kidney diseases or those undergoing chemotherapy. In such cases, the analysers may misclassify NRBC as white blood cells, thus leading to potential overestimation of the leucocyte count, underestimation of NRBC, or both. In the worst scenario, the analysers might even be unable to detect the presence of NRBC accurately 14. The XE-2100 haemocytometer (Sysmex Co., Kobe, Japan) has a specific channel for NRBC counting based on an optical fluorescence system associated with a specific lysing agent which is responsible for selective lysis of red blood cells. As a result of this lysing process, the nuclei of NRBC and white blood cells remain intact and their nucleic acids (i.e., DNA and RNA) are finally stained with the polymethine fluorescent dye. In the dedicated channel, the instrument differentiates NRBC from white blood cells and stroma by cellular dimension and fluorescence intensity. This study was aimed to evaluate the performance of the automated analyser XE-2100 at counting NRBC in patients with beta-thalassaemia (trait and major), in patients with sickle cell anaemia and in neonates. These subjects share a potential altered sensitivity to lysis of red blood cells and a need for accurate NRBC count 2-6. Materials and methods Blood samples This study included a total of 187 peripheral blood samples anticoagulated in K3EDTA-coated tubes (3 ml, 5.4 mg K3EDTA, Becton Dickinson Italy, Milan, Italy), analysed in the clinical chemistry laboratory of the general hospital of Bergamo and the diagnostics unit of the University Hospital of Parma. In both centres, the specific CLSI H03-A6 blood collection procedure was used, as recommended by Lima-Oliveira et al. 15, in order to prevent venous stasis. In detail, the blood samples were from 55 patients with beta-thalassaemia (median age 30 years; range, 4-49), 26 patients with sickle cell anaemia (median age 20 years; range, 1-30) and 56 neonates (aged less than 1 week). The control group consisted of 50 healthy adults (median age 47 years; range, 20-60) selected for the absence of manifest illnesses and with complete blood count (with differential white blood cell analysis), creatinine, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glucose, C-reactive protein and ferritin within the limits of the conventional reference ranges. The study was performed in compliance with the Helsinki Declaration and in accordance with the terms of all local legislation. Nucleated red blood cell count The peripheral blood samples were analysed by the XE-2100 haemocytometer (Sysmex Co.) within 2 hours of blood sampling, as specified by the manufacturer. The XE-2100 has a specific channel for NRBC counting based on an optical fluorescence system associated with a specific lysing agent which is responsible for selective lysis of red blood cells. NRBC counts are expressed both as percent and absolute count ( 10 9 /L). The XE-2100 instruments are identical, so that the commutability of results is explicitly guaranteed. The quality control materials are also the same. The coefficient of variation, assessed from internal quality control of performance, was below 9.5% in the two laboratories in the hospitals of Bergamo and Parma. A smear of peripheral blood stained with May Grunwald and Giemsa dye (Carlo Erba Reagents S.p.A. Milan, Italy), was produced for each sample: the NRBC were counted by two experts, in accordance with the CSLI standards 16, using the reference standard for manual counting by OM. NRBC counts are expressed as percent number of 100 white blood cell count. Samples are considered positive by OM when NRBC 1/200 white blood cells (or 0.5%) in accordance with the criteria described in the Standard International Consensus Group for Haematology (ISLH) 17. As for the comparative evaluation between the two methods, we used the mean value of the two operators' OM count. Whenever there was not complete agreement between the two operators on classification of samples (positive or negative for presence of NRBC in respect to ISLH criteria), a conjoint OM revision with NRBC count was performed, until full inter-operator accordance was reached on the result. Statistical analysis Agreement between the average count by OM (performed by two independent operators) and those obtained with the XE-2100 were evaluated using Pearson's correlation, and estimation of bias. Diagnostic accuracy was evaluated by analysis of the receiver operating characteristic (ROC) curve and values for the sensitivity, specificity and correct classification (CC) were determined. The statistical analysis was performed with Analyse-it (Analyse-it Software Ltd., Leeds, UK). Results Out of the total 187 samples from patients or neonates, 93 (49.7%) were positive by OM for the presence of NRBC and 101 (54%) by XE-2100; 34/93 (36.6%) by OM and 37/101 (36.6%) by XE-2100 were from patients with thalassaemia, 21/93 (22.6%) by OM and 21/101 (20.8%) by XE-2100 were from patients with sickle cell anaemia, and 38/93 (40.9%) 2

3 Automated NRBC counts in haemoglobinopathies and neonates by OM and 41/101 (40.6%) by XE-2100 were from neonates (Table I). Of the 101 samples from healthy adult controls, all were negative by OM and 2/101 (~2%) were positive by XE-2100 (Table I). For a better evaluation of the performance of the instruments with respect to OM, the samples were divided into two classes: the first class (C1) 161/187 (86%) had low NRBC counts (from 0.0 to 10.0%) while the second class of samples (C2) 26/187 (14%) had high counts (from 11% to 335%). Inter-operator conjoint OM revision was performed for only 16 of the total 187 samples (8.5%), due to very low NRBC counts (comprised between 0.5 and 1.0%), until complete inter-operator agreement was reached. Correlation between instrumental and manual counts The correlation between OM and XE-2100 counts was tested on the whole sample cohort, exhibiting NRBC counts between /L or 0% and /L or 355%. The Pearson's correlation coefficient was 0.99 (95% CI, ; p<0.001), and the mean bias was 0.61 (95% CI, from 1.5 to 0.3) (Figure 1A and 1B). In the class of samples with a low count, the Pearson's correlation coefficient was 0.90 (95% CI, ; p<0.001), the mean bias was 0.02 (with 95% CI from 0.15 to 0.10) (Figure 1C and 1D). In the class of samples with a high count, the Pearson's correlation coefficient was 0.98 (95% CI, 0.88 to 0.97; p<0.001) and the mean bias was (95% CI from to 4.53) (Figure 1E and 1F). Diagnostic accuracy Analysis of the ROC curve for all samples showed an area under curve (AUC) of 0.98 (95% CI, from 0.97 Table I - NRBC count by XE-2100 and by optical microscopy (OM) in peripheral blood samples from 187 subjects, by categories. Groups of subjects % NRBC >0 by OM to 1.00; p<0.001). The sub-analysis of the ROC curve in the three groups of patients revealed an AUC of 0.99 (95% CI, from 0.98 to 1.00; p<0.001) for patients with thalassaemia, 0.94 (95% CI, from 0.85 to 1.00; p<0.001) for patients with sickle cell anaemia and 1.00 (95% CI, from 1.0 to 1.0) for the neonates (Table II and Figure 2A-2D). In the whole population, a NRBC of 0.2% for XE displayed a CC with OM of 94.7% (9 false positive cases and 1 false negative case) with a sensitivity of 98.9% and a specificity of 90.4%. In the group of patients with thalassaemia, the CC with OM was 94.5% (3 false positive cases), the sensitivity was 100% and the specificity was 85.7%. In the group of patients with sickle cell anaemia the CC with OM was 92.3% (1 false positive case and 1 false negative case), the sensitivity was 95.2% and the specificity was 80.0%. In the group of neonates, the CC with OM was 94.6% (3 false positive cases), the sensitivity was 100% and the specificity was 83.3%. In the group of controls there were two false positive samples. By using a NRBC cut-off of 0.5%, the XE-2100i displayed a CC with OM of 96.4% in the group of thalassaemic patients (2 false positive cases), a sensitivity of 100% and a specificity of 90.5%. In the group of neonates, the CC with OM was 100% and both the sensitivity and the specificity were 100%. Discussion This study is the first to have evaluated the performance of XE-2100 in enumerating NRBC in patients with a potential altered sensitivity to lysis of RBC and a need for accurate NRBC counting, such as subjects with beta-thalassaemia (trait and major) or sickle cell anaemia and neonates 2-6. In fact, the pool of circulating NRBC in peripheral blood is a reflection of the degree of ineffective erythropoiesis, which is present in sickle cell anaemia and neonates but especially in patients with thalassaemia syndromes. On the other hand, NRBC are absent in thalassemia traits, this disease being characterised by effective erythropoiesis. Thus, NRBC counts could be useful to optimise follow-up and transfusion therapy, especially in patients with severe thalassaemia 2. The accuracy of NRBC enumeration in the peripheral blood is emerging as an essential aspect of laboratory haematology and these parameters should now be regarded as a powerful tool for diagnosis, prognostication and therapeutic decision making, especially in neonates and in particular categories of conditions (i.e., emergency care, sickle cell disease and thalassemia). NRBC counting by OM is challenging, especially in patients with peripheral blood smears containing few or very few NRBC 10-12,18, because of the inherent limits of manual counting as clearly stated in the CSLI standard 16. % NRBC >0 by XE-2100 All subjects analysed 49.7% (93/187)* 54% (101/187) Patients 36.6% (34/93)* 36.6% (37/101) with thalassaemia** Patients with 22.6% (21/93)* 20.8% (21/101) sickle cell anaemia Neonates 67.8% (38/93)* 40.6% (41/101) Healthy adult controls 0% (0/93)* ~2% (2/101) *On total amount of NRBC-positive cases by OM counting (93 patients); on total amount of NRBC-positive cases by XE-2100 counting (101 patients); **the thalassaemia group includes subjects with thalassaemia major (40 subjects) and thalassaemia trait (15 subjects): the group with thalassaemia major had % NRBC >0 to 36.6% (34/93) by OM and 35.6% (36/101) by XE-2100, whereas the group with thalassaemia trait showed 0% NRBC by OM and 0.99% (1/101) by XE NRBC: nucleated red blood cells. 3

4 Buoro S et al Figure 1 - Correlation between the % NRBC determined by XE-2100 and OM for all samples. (A) the Pearson's correlation coefficient was 0.99 (95% CI, 0.98 to 0.99; p<0.001). (B) bias was 0.61 (95% CI, from 1.5 to 0.3). Correlation between the % NRBC determined by XE-2100 and OM for samples with a low count (<10%). (C) the Pearson's correlation coefficient was 0.90 (95% CI, 0.87 to 0.99; p<0.001). (D) bias was 0.02 (95% CI from 0.15 to 0.10). Correlation between the % NRBC determined by XE-2100 and OM for samples with a high count. (E) the Pearson's correlation coefficient was 0.98 (95% CI, 0.88 to 0.97; p<0.001). (F) bias was (95% CI from to 4.53). NRBC: nucleated red blood cells; OM: optical microscopy; CI: confidence interval. 4

5 Automated NRBC counts in haemoglobinopathies and neonates Table II - ROC analysis of the different groups of subjects. ROC analysis Groups of subjects AUC 95% CI, from-to p All subjects analysed <0.001 Patients with thalassaemia (major + trait) <0.001 Patients with sickle cell anaemia <0.001 Neonates ROC: receiver operating characteristic; AUC: area under curve; CI: confidence interval. Figure 2 - ROC curve analysis. (A) all samples analysed; (B) only subjects with thalassaemia; (C) only subjects with sickle cell disease; (D) only neonates. ROC: receiver operating characteristic. 5

6 Buoro S et al The capability of modern automated haemocytometers to enumerate NRBC throughout a broad range of values carries significant advantages for modern medical laboratories, since it overcomes the main drawbacks of manual counting, i.e., the long turnaround time, the relative inaccuracy and imprecision, along with the need to train the personnel appropriately. The limit of detection of NRBC by modern haemocytometers varies according to the specific features of the analysers and the technology used, and is typically between 1% and 2% 18. Analysis of the data obtained in this study shows that the XE-2100 provides good correlation vs OM for NRBC counting (r=0.99; p<0.001) also exhibiting a clinically negligible bias ( 0.61) and does not affect correct clinical classification of examined subjects, especially at low NRBC counts (0-10%) in whom the bias was 0.02 (95% CI from 0.15 to 0.10) (Figure 1D). The good correlation obtained in our population demonstrates the optimal performance of XE-2100 even in patients with altered sensitivity to lysis of red blood cells and NRBC. This evidence is in line with that previously reported by Gulati et al. 12 in non-specific types of samples. The AUC obtained from analysis of the ROC curve also appears excellent, under all circumstances (Figure 2A-2D). The XE-2100 shows a slightly better performance in sickle-cell anaemia subjects, with an AUC of 0.94, even if this value is probably influenced by the relatively small number of such patients investigated (26 subjects). For discrimination of positive samples with XE- 2100, analysis of the ROC curve defined as a NRBC cut-off of 0.2% (Figure 2A-2D). At this threshold, the CC of XE-2100 vs the OM was always good, being 94.1% in the whole population (with 9 false positive cases and 1 false negative case), 94.5% in samples taken from patients with thalassaemia (3 false positive cases), 94.6% in neonates (3 false positive cases) and 92.3% in samples drawn from patients with sickle cell anaemia (1 false positive and 1 false negative cases). The percent of false positive cases is lower than that reported in another study (i.e., 15.1% in the sample with NRBC percentages ranging between 0.15% and 1.9%) 12. It is also noteworthy that the 0.5% of false negative cases among the samples from patients with sickle cell anaemia with OM counting 1% is significantly lower than that reported in another study (i.e., 6.8%) 12. However, this sample would have been subjected to microscopic analysis in accordance with the ISLH standards 17. More specifically, reticulocytosis accompanied by an alarm of "RET Abnormal scattergram" was present in the patient with sickle cell anaemia. An adjustment of the thresholds for discriminating positive samples in XE-2100 may be effective to improve the analytical performance in patients with thalassaemia and in neonates. Accordingly, the NRBC cut-off of 0.5% increased the CC with OM to 96.4% and 100% in these two populations, respectively. However, since the presence of circulating NRBC in peripheral blood is always a marker of abnormal erythropoiesis and/or hypoxic stress, it is preferable to select a lower instrumental cut-off threshold for the count of these cells, in order to maximise sensitivity rather than specificity, for screening purposes. Conclusion On the basis of our data, we can conclude that the XE-2100 performs excellently with regards to NRBC counting, especially in critical populations such as patients with haemoglobinopathies and neonates. The discrimination cut-off that we identified (NRBC 0.2%) significantly enhances the diagnostic sensitivity. This appears to be the most reasonable choice, since screening of samples from unknown patients is commonplace in routine laboratory practice. Automated determination of NRBC counts in thalassaemic patients can also provide a useful indicator of the degree of ineffective erythropoiesis (major vs traits), allowing better control of the need for transfusion therapy. Authorship contributions We guarantee that each Author participated sufficiently in the work to take public responsibility for the content, including substantial contribution to the conception, design, and execution of the study, or analysis and interpretation of data. All Authors were involved in drafting the article or revising it critically, and read and approved the final version. Disclosure of conflicts of interests All Authors declare that they have no conflicts of interest, including any financial, personal or other relationships with other people or organisations that could inappropriately influence, or be perceived to influence, their work. References 1) Danise P, Manconi M, Barella F, et al. Evaluation of nucleated red blood cells in the peripheral blood of hematological diseases. Clin Chem Lab Med 2011; 50: ) Danise P, Amendola G, Di Concilio R, et al. Nucleated red blood cells and soluble transferrin receptor in thalassemia syndromes. Clin Chem Lab Med 2009; 47: ) Kwon MJ, Nam MH, Kim SH, et al. Evaluation of the nucleated red blood cell count in neonates using the Beckman Coulter UniCel DxH 800 analyzer. Int J Lab Hematol 2011; 33: ) Christensen RD, Henry E, Andres RL, Bennett ST. Reference 6

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