Sysmex XN-HPC: study of reference intervals and clinical decision limits in healthy allogeneic donors mobilised with G-CSF

Abstract

The Sysmex XN series haematopoietic progenitor cell (XN-HPC) is a novel tool for assessing stem cell yield before allogeneic haematopoietic stem cell transplantation. This study aimed to establish a reference interval (RI) for XN-HPC in peripheral blood allogeneic transplant donors following granulocyte colony-stimulating factor (G-CSF) stimulation and determine its clinical significance. All specimens were analysed using Sysmex XN-20. Samples were collected and analysed using non-parametric percentile methods to define the RIs. Quantile regression was used to explore the dependency of the RIs on sex and age. Samples were included in clinical decision limits for apheresis based on receiver operating characteristic curve analysis. The non-parametrically estimated RI for XN-HPC was 623.50 (90% confidence interval [CI90%] 510.00–657.00) to 4,144.28 (CI90% 3,761.00–4,547.00). The RIs for the XN-HPC were not age-dependent but were sex-dependent. The RI for males was 648.40 (CI90% 582.00–709.00)–4,502.60 (CI90% 4,046.00–5,219.00) and for females was 490.90 (CI90% 311.00–652.00)–3,096.90 (CI90% 2,749.00–3,782.00). Comparisons based on XN-HPC values between the poor and less-than-optimal groups, good and less-than-optimal groups, and good and non-good groups had areas under the curve of 0.794 (P < 0.001), 0.768 (P < 0.001), and 0.806 (P < 0.001), respectively, indicating a good predictive value for mobilisation effectiveness. XN-HPC data exceeding 3974 × 10⁶/L suggested that a sufficient number of stem cells could be collected clinically. Values > 5318 < 10⁶/L indicated 100% mobilisation effectiveness. We established an RI for XN-HPC in peripheral blood allogeneic transplant donors following G-CSF stimulation and determined clinical decision thresholds for mobilisation efficiency.

Introduction

Peripheral blood haematopoietic stem cell transplantation (PBSCT) is an established treatment for several haematological malignancies [1, 2]. A sufficient mobilisation of stem cells should result in a significant increase in the number of haematopoietic stem cell yields in peripheral blood to ensure adequate collection during the harvesting process [3, 4]. The abundance of stem cells is closely associated with post-transplant haematopoietic reconstitution, immune reconstitution, transplant-related complications, relapse rate, disease-free survival, and overall survival at 5 years [5–9]. Typically, the adequacy of mobilisation is assessed by monitoring the CD34 cell dose in the transplant [10, 11]. Based on different thresholds of CD34 cell dose, mobilisation effectiveness is determined as follows: Less than 2 × 10⁶ cells/kg of recipient body weight indicates poor or suboptimal mobilisation, while greater than 5 × 10⁶ cells/kg of recipient body weight indicates successful mobilisation. Further subdivision into 2–5 × 10⁶ cells/kg of recipient body weight indicates less-than-optimal mobilisation, and > 5 × 10⁶ cells/kg of recipient body weight indicates good mobilisation [12].

In recent years, the Sysmex XN series haematopoietic progenitor cell (XN-HPC) has emerged as a crucial ex vivo diagnostic parameter for quantifying haematopoietic progenitor cells before allogeneic haematopoietic stem cell transplantation, owing to their strong correlation with CD34 + cell counts [13–16]. XN-HPC is a haematologic parameter evaluated using the Sysmex XN-20 fully automated cell counter to assess haematopoietic stem cells and the haematopoietic progenitor cell yield. Its detection principle is based on flow cytometry, in which a lytic reagent dissolves lipid components on the cell membrane and labels nucleic acids. The lipid content on the surface of primitive progenitor cells is relatively low, resulting in less nucleic acid fluorescence labelling and weaker side-scattering fluorescence detection, forming the specific detection of cell clusters [17, 18]. XN-HPC offers short detection cycles, low cost, minimal operational bottlenecks, and optimal timing for peripheral blood stem cell collection. These inherent attributes enrich the prospects for pre-transplantation, transplantation, and post-transplantation applications [19, 20]. In particular, with the interpretation of the role and function of the CD34 + cell dose, XN-HPC may contribute to a more comprehensive understanding of their significance [15, 21–23].

As the use of this biomarker has become increasingly widespread, there is a growing demand for the clinical interpretation of haematopoietic stem cell transplantation donors as a representative healthy population. Therefore, the purpose of this study was to establish a reference interval (RI) for XN-HPC in peripheral blood cells after mobilisation in allogeneic transplantation donors and to determine its threshold in clinical decision limits (CDLs) to enhance the efficacy and safety of allogeneic transplantation mobilisation and collection.

Methods

Participants

This study was conducted at the State Key Laboratory of Experimental Hematology, the National Clinical Research Center for Blood Diseases, and the Haihe Laboratory of Cell Ecosystem of the Institute of Hematology & Blood Diseases Hospital. It involved the prospective collection of data and specimens from PBSCT donors admitted from December 2022 to December 2023. The inclusion criteria comprised having at least one report with numerical XN-HPC test results and mobilisation with granulocyte colony-stimulating factor (G-CSF). The exclusion criteria, established according to reference standards, included mobilisation with non-G-CSF alone and missing XN-HPC data. Clinical decision-making exclusion criteria, in addition to the RI exclusion criteria, comprised selecting the non-first-day collection specimen type and excluding the CD34 + count. The study was approved by our hospital’s ethics committee following the guidelines of the Helsinki Declaration, and all participants provided written consent after being informed of the study’s purpose.

XN-HPC determination

XN-HPC measurements were conducted using a Sysmex XN-20 fully automated haematology analyser with the white precursor cell flow cytometry channel. This channel represents a population of haematopoietic progenitor cells (HPCs) with weak corresponding fluorescence signals, strong forward scattered light signals, and weak side scattered light signals. The detection method was referenced as “HPC measurement and Sysmex workflow” [13].

Statistical analysis

Data were analysed using the MedCalc application 22.021 (Mariakerke, Belgium), SPSS 26.0 software (IBM Inc., USA), and GraphPad Prism 8.0 (GraphPad, USA). Descriptive statistical analysis was initially performed on the variables, with continuous variables reported as mean ± standard deviation and median (P25–P75) according to their probability distribution, whereas categorical variables were expressed as frequencies. Non-parametric tests, including the Mann–Whitney U and Kruskal–Wallis tests, were used for analysis. The Kolmogorov–Smirnov test was used to determine whether the variables were normally distributed. The 2.5th and 97.5th percentiles, determined using the non-parametric percentile method described in Clinical Laboratory Standards Institute (CLSI) guideline C28-A3, were reported as the lower and upper limits of the RI, respectively, providing a 90% confidence interval as the reference limit. Percentile regression (2.5th and 97.5th percentiles) was used to determine sex and age dependence. Receiver operating characteristic curve analysis was conducted to establish different CDLs for XN-HPC based on Youden’s index using the CD34 × 10⁶/kg recipient weight as the gold standard for mobilisation effectiveness.

Results

Basic characteristics of the study population and samples

A total of 346 samples meeting the criteria were included, of which 298 had XN-HPC data on the first apheresis, with corresponding sample numbers of 189 and 298 for the non-first-apheresis and first-apheresis groups, respectively. Among the included donors, 73% were male. The mobilisation regimen was almost exclusively G-CSF alone, with only 4% of the patients additionally administered plerixafor. Analysis between the first-apheresis and non-first-apheresis groups revealed significant differences in mononuclear cell count, CD34 + cell count, and XN-HPC count (all P < 0.001), but not in white blood cell count. The median (quartiles) XN-HPC in the first-apheresis group, 1,998.50 (1,360.00–2,851) × 10⁶/L, was significantly higher than that in the non-first-apheresis group, 1,342.00 (888.50–1,875.50) × 10⁶/L. After applying the inclusion and exclusion criteria, 78 XN-HPC results were excluded, leaving 409 results for the study and analysis of RIs. Based on the strong correlation between XN-HPC and CD34 + cell dose in the first-apheresis group and the completeness of CD34 + cell data, 294 cases were analysed for CDL exploration and establishment (Table 1).

Table 1.

Characteristics of investigated donors and apheresis

	First apheresis	Non-first-apheresis	P-value
No. of donors	298	346
Age, median (IQR)	35 (23–45)	35 (23–46)
Male, n (%)	215 (73%)	243 (70%)
BMI, Mean ± SD	24.16 ± 19.30	24.09 ± 4.40	-
Mobilizers, n (%)
G-CSF only	298(100%)	333 (96%)
Others	–	13 (4%)
No. of specimens	298	189
WBC × 109/L, Median (IQR)	245.49 (204.44–312.94)	254.26 (214.42–323.46)	0.17
MNC × 109/L, Median (IQR)	10.19 (8.02–13.30)	8.06 (6.14–10.92)	< 0.0001
CD34 +  × 106 cells/kg, Median (IQR)	4.27 (2.83–6.50)	2.15 (1.45–3.12)	< 0.0001
XN-HPC × 106/L, Median (IQR)	1998.50 (1360.00–2851.00)	1342.00 (888.50–1875.50)	< 0.0001

BMI, body mass index; SD, standard deviation; IQR, interquartile range; G-CSF, granulocyte colony-stimulating factor

Establishment of the XN-HPC reference range

Table 2 shows the RI for XN-HPC after G-CSF mobilisation in 409 cases, established as 623.50–4,144.28 × 10⁶/L, with males accounting for 70% of the sample. Non-parametric statistics were used to determine the median, lower limit, and upper limit of XN-HPC in the different subgroups. Percentile regression was employed to explore sex and age dependence in the XN-HPC RI, with parameter estimates at various percentiles all > 0.01, indicating no age dependence but sex dependence. However, distinct RIs for children (399–4,031 × 10⁶/L) and adults (653.50–4,161.25 × 10⁶/L) have been described. Sex dependence was evident at percentiles 0.25, 0.75, and 0.9 (P-values: 0.007, < 0.001, and 0.001, respectively), with the male RI (648.40–4,502.60 × 10⁶/L) exhibiting a rightward skew and broader spread compared to the female RI (490.90–3,096.90 × 10⁶/L).

Table 2.

Reference intervals of XN-HPC

Subgroup	No. of specimens	Median (IQR), × 106/L	Lower limit (90%CI), × 106/L	Upper limit (90%CI), × 106/L
All	409	1569.00 (1080.00–2538.50)	623.50 (510.00–657.00)	4144.28 (3761.00–4547.00)
Gender
Male	287	1691.00 (1188.75–2732.75)	648.40 (582.00–709.00)	4502.60 (4046.00–5219.00)
Female	122	1358.50 (914.00–1980.00)	490.90 (311.0–652.00)	3096.90 (2749.00–3782.00)
Age, years
Aged ≤ 18	75	1284.00 (907.50–2002.25)	399.00 (110.00–623.00)	4031.00 (2843.00–5219.00)
Aged > 18	334	1606.50 (1182.00–2664.00)	653.50 (582.00–733.00)	4161.25 (3782.00–4608.00)

IQR, interquartile range; CI, confidence interval

Establishment of mobilisation effect-related CDL for XN-HPC

Analysis based on the CD34 + cell count on the first day of collection, defining mobilisation effectiveness, revealed proportions of poor mobilisation, less-than-optimal mobilisation, and good mobilisation donors of 14% (41/294), 45% (133/294), and 41% (120/294), respectively. Sex, body mass index, and XN-HPC counts were significantly different among the three groups. Pairwise comparisons among the three groups revealed significant differences (all P < 0.0001). Comparisons based on XN-HPC values between the poor and less-than-optimal groups, good and less-than-optimal groups, and good and non-good groups had areas under the curve of 0.794 (P < 0.001), 0.768 (P < 0.001), and 0.806 (P < 0.001), respectively, indicating a good predictive value for mobilisation effectiveness. Correspondingly, when the XN-HPC data exceeded 3,974 × 10⁶/L, a sufficient number of haematopoietic stem cells could be collected clinically, whereas a value exceeding 5318 × 10⁶/L indicated 100% effectiveness of mobilisation in this study (Table 3, Fig. 1).

Table 3.

Clinical decision limits for XN-HPC

	Poor	Less-than-optimal	Good	P-value
CD34 +  × 106 cells/kg	< 2	2 to < 5	≥ 5
No. of donors	41	133	120
Age, Mean ± SD	43 ± 17	32 ± 14	30 ± 12	0.1350
Male, (%)	51%	70%	83%	< 0.0001
BMI, Mean ± SD	22.96 ± 4.25	23.58 ± 4.31	25.16 ± 4.14	0.0035
XN-HPC × 106/L, Median (IQR)	960.00 (695.25–1510.00)	1670.00 (1259.75–2435.00)	2715.50 (2002.50–3505.50)	< 0.0001
AUC		0.788	0.800
Optimal Cut-off (Sensitivity, Specificity)	–	1029 (89.47, 60.98)	2154 (76.44,72.50)
Diagnostic Cut-off (Sensitivity, Specificity)	–	3974 (3.73, 100.00)	5318 (3.31,100.00)

BMI, body mass index; AUC, area under the receiver operating characteristic curve

Fig. 1.

Clinical decision limits of XN-HPC on different mobilisation effects. A Comparison of XN-HPC level among three groups of participants: poor, less-than-optimal, and good. **** indicates P-value < 0.0001. B, C, and D: The area under the curve (AUC) representing the clinical decision limits of XN-HPC under different mobilisation effects. Specifically, B depicts the comparison between less-than-optimal and poor groups, C illustrates the comparison between good and less-than-optimal groups, and D shows the comparison between Good and Less-than-optimal + Poor groups

Discussion

In this study, we successfully established an RI for XN-HPC in donors for allogeneic haematopoietic stem cell transplantation (HSCT) following G-CSF mobilisation, marking a significant milestone in China. We also investigated the CDLs associated with the different mobilisation effects of XN-HPC. The calculated RI for XN-HPC was found to be 623.50–4,144.28 × 10⁶/L, within the biological reference interval. However, the clinical interpretability of this indicator warrants further exploration both domestically and internationally. Notably, our study revealed sex dependence in the RI of XN-HPC, but no age dependence. To characterise mobilisation efficiency and processes, we defined various mobilisation effects. For instance, we determined that an XN-HPC result of 3,974.00 × 10⁶/L on first apheresis indicates a sufficient number of stem cells for collection, while a result of 5,318.00 × 10⁶/L signifies good mobilisation, necessitating no additional collection processes.

Our previous research corroborated the correlation between XN-HPC and CD34 + cell counts, establishing XN-HPC as a biomarker for quantitative haematopoietic progenitor cells before allogeneic HSCT, consistent with findings from international studies [13]. However, further exploration is required to ascertain its interpretational applicability in mobilisation effectiveness and treatment response monitoring. The integration of reporting RIs and CDLs into clinical laboratory reports, as emphasised by ISO 15189, highlights the importance of accurately interpreting clinical laboratory test results to enhance clinical decision-making capabilities. RIs and CDLs are valuable interpretation tools. When adopting the non-parametric method recommended by the CLSI to calculate the RI limits, the RI can be expressed as the lower limit value (2.5th percentile) to the upper limit value (97.5th percentile) [24]. CDL, also known as the medical decision level, is established based on clinical laboratory test indices to delineate various clinical decision thresholds. It is important to note that while a clinical laboratory test index establishes a unique RI in a specific population, there may be multiple CDLs because different clinical decision contexts necessitate the corresponding CDLs. Optimising mobilisation can reduce transplant costs and mortality rates [25]. The optimal dose of stem cells was found to be 5 × 10⁶ cells/kg, which facilitates the early transplantation of neutrophils and platelets and reduces the utilisation of medical resources [26]. Therefore, the development and implementation of clinical decision values for XN-HPC based on different mobilisation effects are imperative in clinical practice.

Defining mobilisation failure based on the CD34 + cell count on the first day of collection, this study identified a proportion of 14% (41/294), categorising them as experiencing poor mobilisation. This was an additional finding of this study. Notably, this rate exceeded the mobilisation failure rates reported in recent studies, which range from 2 to 11%. Several factors may have contributed to this disparity, including potential data loss, which led to a relative increase in the observed proportion. Additionally, it is plausible that some donors did not attain the optimal collection time, which further influenced outcomes [27–31]. This further underscores the necessity of delving into the significance of XN-HPC data in stem cell transplantation, with its clinical interpretability currently under investigation. Concerning the exploration of optimal cut-offs in the CDL, it is worth noting the potential practical limitations of its application. Consequently, our clinical participants exhibited the highest specificity. For instance, when XN-HPC exceeds 3,974 × 10⁶/L, it fulfils the needs of recipients with CD34 + cell counts > 2 × 10⁶ cells/kg. Similarly, when XN-HPC surpasses 5,318 × 10⁶/L, additional medical assistance for donors becomes unnecessary.

This study has two primary limitations. First, the number of children recruited was less than the minimum recommended number of 120 children per age group. Owing to this limitation, age-specific RI may be obscured despite hypothesis testing. Second, this was a single-centre, small-sample study. Extensive multicentre research is needed to gain recognition from more research teams.

In summary, we established, for the first time in China, an RI of XN-HPC in donors for allogeneic haematopoietic stem cell transplantation after G-CSF mobilisation and the related CDLs. This tool can predict and guide mobilisation effectiveness, haematopoietic reconstruction, and post-transplant complications. We believe that our results will serve as a reference for other laboratories.

Author contributions

All authors contributed to the study conception and design. The first draft of the manuscript was written by Lunhui Huang, and Binbin Lin. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, Grant/Award Numbers: 2022-I2M-C&T-B-092, 2021-I2M-1-023.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare that they do not have any conflicts of interest.

Ethical approval

The study was approved by our hospital’s ethics committee following the guidelines of the Helsinki Declaration.

Consent to participate

All participants provided written consent after being informed of the study’s purpose.