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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 3  |  Page : 949-955

Clinical significance of nucleated red blood cell count in pediatric patients with transfusion-dependent beta thalassemia


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission17-Jan-2020
Date of Decision05-Feb-2020
Date of Acceptance07-Feb-2020
Date of Web Publication30-Sep-2020

Correspondence Address:
Amira M.F. Shehata
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_14_20

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  Abstract 


Objectives
The aim of this study was to assess the count of nucleated red blood cells (NRBCs) as a potential marker of erythropoietic stress and transfusion therapy adequacy. To achieve this goal, we evaluated the performance of the Sysmex XN-1000 hematology analyzer and flow cytometry (FC) in counting NRBCs in comparison with manual microscopic counting (MC).
Background
NRBCs recognition by manual and automatic technologies has been widely evaluated. As transfusion of packed red blood cells is a principal supportive intervention for patients with thalassemia major, monitoring markers of an adequate transfusion therapy is essential in their management.
Patients and methods
NRBC percentages from 61 thalassemia major patients were analyzed by automated technologies, Sysmex XN-1000 analyzer and FC, and the results were compared with traditional MC as a reference method. Markers of ineffective erythropoiesis were estimated for all patients.
Results
Pretransfusion hemoglobin levels showed significant negative correlation with NRBC% obtained by Sysmex XN-1000 (r=−0.302, P = 0.018). Furthermore, NRBC% was positively correlated with reticulocyte% (r = 0.852, P < 0.001) and serum soluble transferrin receptor (r = 0.303, P = 0.017). The comparison of NRBCs% given by both automated methods versus MC showed strong and significant correlation (r2 = 0.999, P < 0.001). The degree of the agreement between methods was analyzed by the Bland–Altman plot; the mean bias between MC versus XN-1000 analyzer and MC versus FC were1.1 and − 0.4, respectively.
Conclusion
Monitoring of NRBC% by Sysmex XN-1000 analyzer has many advantages over manual counting and can help to optimize transfusion therapy for patients with beta thalassemia major.

Keywords: beta thalassemia major, flow cytometry, nucleated red blood cells, Sysmex XN.1000 hematology analyzer, transfusion


How to cite this article:
Shehata AM, Wanas SA, El-Hawy MA, Khalifa KA. Clinical significance of nucleated red blood cell count in pediatric patients with transfusion-dependent beta thalassemia. Menoufia Med J 2020;33:949-55

How to cite this URL:
Shehata AM, Wanas SA, El-Hawy MA, Khalifa KA. Clinical significance of nucleated red blood cell count in pediatric patients with transfusion-dependent beta thalassemia. Menoufia Med J [serial online] 2020 [cited 2020 Oct 20];33:949-55. Available from: http://www.mmj.eg.net/text.asp?2020/33/3/949/296649




  Introduction Top


Beta thalassemia represents a group of blood disorders characterized by reduced hemoglobin (Hb) levels and ineffective erythropoiesis. For patients with beta thalassemia major, chronic lifelong transfusion therapy is mandatory. For transfusion-dependent patients, the ideal pretransfusion Hb concentration should be maintained in the 9–10 g/dl range [1].

Nucleated red blood cells (NRBCs) are defined as immature forms of erythrocytes which normally present in the bone marrow but not in the peripheral blood of adults. The presence of circulating NRBCs in adults occurs in situations of hematopoietic stress such as severe infection, massive hemorrhage, marrow infiltration, or extramedullary hematopoiesis [2].

A correlation between the number of normoblasts in peripheral blood and the prognosis of various disorders was reported in many studies [3],[4],[5],[6]. In thalassemia syndromes, the count of NRBC in the peripheral blood reflected the degree of ineffective erythropoiesis [7]. It was suggested that NRBC count of less than 5% was an indicator of adequate transfusion therapy in transfusion-dependent thalassemia patients [8].

Therefore, precise detection and counting of NRBCs in such conditions is important. Manual microscopy is a commonly utilized tool of NRBCs enumeration in clinical laboratories [9]. This method is relatively simple but laborious, time-consuming, and imprecise especially with elevated normoblasts count [10].

Previously, it was practically difficult to differentiate NRBCs from small lymphocytes by automated hematology analyzers; but with the recent technical development, it was possible to enumerate NRBC automatically with high reliability and short turnaround time [11]. In the XN-1000 hematology analyzer supplied by Sysmex Corporation both NRBCs and leukocytes are counted in the white cell nucleated (WNR) channel based on the flow cytometry (FC) technique and the final complete blood count (CBC) report shows a corrected number of leukocytes when NRBCs are recognized [12].

In the WNR channel, a nonionic detergent in the Lysercell WNR causes lysis of reticulocytes and mature erythrocytes; and disruption of the cell membrane of NRBCs and leukocytes. Then, a fluorescent dye in the Fluorocell WNR enters the cells and stains nucleic acids and organelles. The XN-1000 measures the fluorescence intensities as forward-scattered light (FSC) which reflects cell size and as the side fluorescent light (SFL), which reflects the internal complexity. These data are subsequently expressed as a 2D scattergram. On this scattergram, basophils have the highest FSC and SFL intensities and NRBCs the lowest SFL intensity compared with those of other leukocytes [13].

The use of FC for the detection and counting of normoblasts in the peripheral blood was initiated several years ago; it was more accurate and reproducible in comparison with the reference microscopy method [14].

In this study, we aimed to determine the importance of NRBC counts as a surrogate marker of ineffective erythropoiesis and adequate transfusion therapy in beta thalassemia major patients. We aimed also to evaluate the performance of the Sysmex XN-1000 hematology analyzer and modified FC method in counting NRBCs in comparison with manual microscopic counting (MC).


  Patients and Methods Top


The study included 61 pediatric patients with beta thalassemia major who were selected from the patients admitted to the Pediatric Department, Menoufia University for transfusion therapy in the duration between September 2018 and December 2018. All patients were on regular blood transfusion every 2–4 weeks. Most of the patients were on iron chelation therapy, mainly oral deferasirox, according to their serum ferritin levels. The guardians of all patients signed an informed consent. The research had been approved by our local medical ethics committee and was in accordance with the Helsinki Declaration.

A total of 61 patients were recruited for the study with a median age of 8.5 years (range, 1.5–18 years). Twenty-eight (45.9%) patients were women and 33 (54.1%) were men; 27.9% of patients were splenectomized. The median Hb level was 80 g/l (range, 40–112 g/l), other patient characteristics are illustrated in [Table 1].
Table 1: Clinical and laboratory characteristics of patients with thalassemia major

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Sampling

All blood samples had been collected from patients prior to transfusion and were analyzed in our Hematology Unit, Clinical Pathology Department, Menoufia University. A venous blood sample (5 ml) was withdrawn from each individual under aseptic condition by a clean venipuncture and then dispensed into two tubes: a measure of 3 ml of blood was delivered into dipotassium EDTA (K2-EDTA) vacutainer tubes for CBC, blood film, reticulocyte count, and FC analysis. A measure of 2 ml of blood was delivered into a plain tube, allowed to clot at room temperature before centrifugation for 10 min, and then the sera were separated and stored at –20°C for soluble transferrin receptor (sTFR) analysis.

Automated Sysmex XN-1000 hematology analyzer

Peripheral blood specimens of thalassemia patients were collected and processed within 2 h of collection from the patient. Automated CBC were analyzed by Sysmex XN-1000 hematology analyzer (Sysmex Corporation, Kobe, Japan). Sysmex XN-1000 gives both NRBCs % (NRBCs per 100 white blood cells) and absolute NRBC count (×109/l). Analyzer was regularly calibrated and maintained by the manufacturer and daily quality control was carried out by the laboratory staff.

Manual microscopic counting

After automated analysis, peripheral blood smears were made and stained with Leishman's stain. A 200-cell differential count using manual microscopy was performed by two independent observers. Then the average of both observers' NRBC counts was reported as the NRBC number per 100 white blood cells (WBCs) in accordance with the Clinical and Laboratory Standards Institute guideline [15]. MC was used as the reference method.

Flow cytometric analysis

The FC method was modified from the previously reported study by de Vries et al. [16], using a triple-labeling method with monoclonal antibody to CD45, labeled with Peridinin Chlorophyll Protein Complex (CD45-PerCP, Clone 2D1; BD Biosciences, San Jose, California, USA), antibody to CD71, labeled with fluorescein isothiocyanate (CD71-FITC, Clone L01.1; BD Biosciences) and antibody to glycophorin A, labeled with phycoerythrin (glycophorin A-PE, clone GA-R2; BD Biosciences).

Each blood sample (100 μl) was pipetted into a round-bottom tube and incubated in the dark at room temperature for 15 min in the presence of 20 μl CD45 (PerCP), CD71-FITC, and glycophorin A (PE). The samples were then treated by 1 ml of lysing solution and left for 10 min in the dark at room temperature. After that, centrifugation of samples was done at 2000 rpm for 5 min, the supernatant was discarded, and the samples were washed twice using 2 ml of phosphate buffered saline. Lastly, samples were ready for analysis after being suspended in 0.2 ml of phosphate buffered saline.

The NRBC percentage was determined by FC method analysis on an FACS Calibur (Becton Dickinson Immunocytometry Systems, San Jose, California, USA); gating was done on the basis of side scatter (SSC)/ forward scatter (FSC) and SSC/CD45 (R1: included white blood cells, normoblasts, unlysed red blood cells and debris, R2: included CD45-normoblasts, unlysed red blood cells, and debris while R3: included CD45 + while blood cells) and 10 000 events were acquired.

NRBCs were recognized as (CD45−, double + CD71 and glycophorin A) and WBC identified as (CD45 + cells). The percentage of NRBCs related to the WBC number was calculated by the formula: NRBCs%=(NRBC events/WBC events)×100 [Figure 1].
Figure 1: Triple staining method for the identification of NRBCs by flow cytometry. NRBC, nucleated red blood cells.

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Number of NRBCs per 100 WBC (NRBC%) results obtained from Sysmex XN-1000 and from FC method were compared with manual NRBCs% as the reference method.

Evaluation of markers of erythropoietic activity

Serum-sTFR was estimated by Cobas Integra 400 plus analyzer (Roche Diagnostics, Mannheim, Germany). Reticulocyte production index (RPI) was calculated using the equation: RPI = corrected reticulocyte percentage/maturation correction, where corrected reticulocyte percentage = reticulocyte percentage × (patient's hematocrit/normal hematocrit) and the maturation correction was 1 for hematocrit of 36–45%, 1.5 for hematocrit of 26–35%, 2 for hematocrit of 16–25%, and 2.5 for hematocrit less than 15%.

Statistical analysis

Bland and Altman plot, Passing–Bablok regression analysis, correlation, and intraclass correlation coefficients (ICC) were used for statistical comparisons between NRBCs% obtained by Sysmex XN-1000 and modified FC method with NRBCs% obtained by reference MC method. Spearman's correlation test was used to evaluate the association between NRBCs% and other parameters. Mann–Whitney test was used for comparison between two groups regarding quantitative variables. SPSS software package (version 20;IBM Corp., Armonk, New York, USA) and XLSTAT (Addinsoft, New York, New York, USA) was used for data analysis. P value less than 0.05 was reported statistically significant.


  Results Top


Comparison of nucleated red blood cells% determined by Sysmex XN-1000 analyzer with nucleated red blood cells% determined by microscopic counting method

The comparison of NRBC% given by both methods showed a high and significant correlation (r2, coefficient of determination = 0.999, P < 0.001). Excellent reliability found between both methods with ICC was calculated as 1.000 [95% confidence interval (CI), 1.000–1.000].

Passing–Bablok regression analysis yielded the equation y = 0.264 + 0.984×, in which the slope was 0.984 (95% CI, 0.978–0.989) and the intercept was 0.264 (95% CI, 0.100–0.312). Passing–Bablok regression analysis did not show significant proportional (slope) error, but constant (intercept) error was found between both methods [Figure 2].
Figure 2: Passing–Bablok and Bland–Altman analysis of NRBCs % obtained by the Sysmex XN-1000 analyzer versus NRBC% obtained from the reference MC method. In the Passing–Bablok regression analysis, the dotted line represents the zero-bias line; the black line represents mean bias. In the Bland–Altman analysis, the bold black line and the dotted lines represent the mean bias and 95% limits of agreement, respectively. NRBC, nucleated red blood cells.

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Quantification of the degree of the agreement between NRBC% obtained by the two methods was analyzed by the Bland–Altman plot; the mean bias was 1.1 with 95% limits of agreement between − 7.9 and 10.1 [Figure 2].

Comparison of nucleated red blood cells% determined by flow cytometry method with counts determined by microscopic counting method

Passing–Bablok regression analysis yielded the equation y = 0.412 + 0.997×, in which the slope was 0.997 (95% CI, 0.990–1.012) and the intercept was 0.412 (95% CI, 0.347–0.620). Passing–Bablok regression analysis did not show significant proportional (slope) error but constant (intercept) error was demonstrated between both methods, especially with high NRBCs% [Figure 3].
Figure 3: Passing–Bablok and Bland–Altman analysis of NRBCs % obtained by flow cytometry versus NRBC% obtained from the reference MC method. In the Passing–Bablok regression analysis, the dotted line represents the zero-bias line; the black line represents the mean bias. In the Bland–Altman analysis, the bold black line and the dotted lines represent the mean bias and 95% limits of agreement, respectively. MC, microscopic counting; NRBC, nucleated red blood cells.

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High correlation (r2 = 0.999, P < 0.001) was found between NRBCs% obtained by FC versus MC and ICC was calculated as 1.000 (95% CI, 1.000–1.000).

Bland–Altman plot demonstrated a mean bias of only − 0.4 (95% limits of agreement between − 8.4 and 7.6) [Figure 3].

Nucleated red blood cells count as a surrogate marker of adequate transfusion therapy in beta thalassemia major patients

To assess the significance of NRBCs as a marker of ineffective erythropoiesis and transfusion adequacy, correlation analysis was performed between NRBCs% and both of Hb and markers of ineffective erythropoiesis.

In all patients, pretransfusion Hb levels showed significant negative correlation with NRBC% obtained by MC (r=−0.304, P = 0.017), Sysmex XN-1000 (r=−0.302, P = 0.018), and FC (r=−0.306, P = 0.016).

NRBCs% obtained by Sysmex XN-1000 was positively correlated with reticulocyte % (r = 0.852, P < 0.001), RPI (r = 0.780, P < 0.001), and sTFR (r = 0.303, P = 0.017).

Then patients were grouped according whether they were splenectomized or not Hb level negatively correlated with NRBCs% obtained by MC, XN-1000, and FC in both splenectomized (r=−0.662, −0.658, −0.660 respectively, P = 0.004) and nonsplenectomized patients (r=−0.626, −0.627, −0.632, respectively, P < 0.001).

Analysis revealed that NRBCs%, reticulocyte %, and RPI were significantly increased in the splenectomized group in comparison with the nonsplenectomized group (P < 0.001) while no significant differences were found between both groups regarding Hb level (P = 0.357) and sTFR (P = 0.435) [Table 2].
Table 2: Clinical and laboratory characteristics differences in nonsplenectomized and splenectomized thalassemia patients

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Further analysis was conducted by categorizing the patients into two groups based on NRBC%; group I included thalassemia patients with NRBCs of less than 5%, and group II included patients with NRBCs equal to or more than 5%. It was found that pretransfusion Hb was significantly increased (P = 0.013) while reticulocyte%, RPI, and sTFR were significantly decreased (P < 0.001, P < 0.001, P = 0.032, respectively) in group I in comparison with group II [Table 3].
Table 3: Relationship between nucleated red blood cells% and characteristics of thalassemia patients

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  Discussion Top


The presence of NRBCs in the peripheral blood of adult is abnormal finding reflecting bone marrow irritation or infiltration and is associated with worse prognosis [17]. The main goal of treatment in patients with beta thalassemia, especially transfusion-dependent patients, is to reduce morbidity and improve the quality of life. The major challenge for these patients is to provide adequate transfusion therapy with the avoidance of transfusion-related complications [18].

Recently, with enormous technological evolution in hematology analyzers, automated NRBCs counting has become available. The essential goal of this study was to assess the NRBC count as a reliable marker of erythropoietic stress and adequate transfusion in pediatric patients with beta thalassemia major. To achieve this goal, accurate and effective NRBC counting was important. So, we evaluated two automated technologies in comparison with traditional manual counting to assess their performance in NRBC counting.

Being widely available, routinely used, and simple technique, it was more reasonable to use manual counting, performed in accordance with the Clinical and Laboratory Standards Institute guideline, as a reference method to assess NRBCs% obtained by XN-1000 analyzer and modified FC methods.

The results showed excellent correlation and good agreement between the reference MC method and both XN-1000 analyzer and FC method in counting NRBC.

Comparison analysis between different methods indicated that the NRBCs% by XN-1000 analyzer was underestimated when compared with the reference MC method. This higher NRBCs% obtained by MC may be related to nonhomogeneous distribution of cells across the slide and the variation in the examined fields. However, NRBCs% by the FC method was mildly overestimated when compared with the reference MC method.

The use of manual MC method for NRBC% has many disadvantages including prolonged time for slide preparation, the possibility that blood cells are not homogeneously distributed across the slide, prolonged time for interpretation, observers' subjectivity, and delay in reporting results [19]. Therefore, the MC method is unsuitable for laboratories processing many specimens with normoblastemia, especially with high count and when accurate NRBC count is required for therapy monitoring.

The present study revealed that NRBC% results obtained by the XN-1000 analyzer had strong correlations and agreement with the reference method. This finding was consistent with the previous results reported by Tantanate and Klinbua [20]. Nevertheless, the use of the XN-1000 analyzer in NRBC% analysis has many advantages including short turnaround time and efficiency. Therefore, automated NRBC% analysis by the XN-1000 analyzer could replace the traditional MC method.

FC with the use of CD45 monoclonal antibody and propidium iodide were previously used for NRBCs counting [14]. In this study, we utilized the triple immunostaining technique for NRBCs using CD45, CD71, and GpA. We aimed to properly differentiate NRBC (CD45−/CD71+/GpA+) from unlysed erythrocytes (CD45−/CD71−/GpA+). NRBCs% by the FC method was mildly overestimated when compared with the reference MC method. These findings were inconsistent with Houyhongthong et al.[21], who reported that the NRBC count by MC was overestimated when compared with the FC method. These conflicting results may be explained by the different protocols used. Although this FC technique showed excellent agreement and highly significant correlations with the reference MC methods, it has some drawbacks including the long turnaround time and high cost.

Thalassemic patients included in our study had a median Hb level of 80 g/l, indicating that most patients had poor control of their chronic anemia. The analysis of data from this study demonstrated a negative correlation between NRBC% and Hb levels regarding all patients and when they were categorized according to the presence or surgical removal of spleen. Moreover, NRBC% was positively correlated with reticulocyte %, RPI, and sTFR. Similar to our study results, a study by Karakukcu et al. [22] reported negative correlation between NRBC% and Hb.

Analysis showed that NRBCs%, reticulocyte %, and RPI were significantly increased in the splenectomized group in comparison with the nonsplenectomized group while no significant differences were found between both groups regarding Hb level and sTFR. These findings were consistent with the Danise et al. [23] study that reported significant difference of NRBC count between the splenectomized and the nonsplenectomized group with absence of difference between both groups as regard sTFR levels.

Further analysis revealed that pretransfusion Hb was significantly increased while reticulocyte %, RPI, and sTFR were significantly decreased in thalassemia patients with an NRBC of less than 5% in comparison with patients with NRBC equal to or more than 5%. These findings demonstrated that an NRBC of less than 5% could be used as an indicator of suppressed ineffective erythropoiesis and so adequate transfusion therapy.


  Conclusion Top


NRBCs can be used as a perfect and simple marker of erythropoietic stress. Thus, NRBCs counting can be used as a tool for selecting the appropriate pretransfusion Hb concentration required to suppress ineffective erythropoiesis for each thalassemia patient. Measuring NRBC% in the peripheral blood of thalassemic patients by Sysmex XN-1000 analyzer offers several advantages, including a faster turnaround time, labor savings, and cost effectiveness.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
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