• Users Online: 303
  • Print this page
  • Email this page

Table of Contents
Year : 2022  |  Volume : 15  |  Issue : 2  |  Page : 83-88

Haematology reference range evaluation for novel research parameters on the complete blood count analyzer sysmex XN-1000

Department of Pathology, Atal Bihari Vajpayee Institute of Medical Sciences, Dr. Ram Manohar Lohia Hospital, New Delhi, India

Date of Submission14-Dec-2021
Date of Decision30-Jan-2022
Date of Acceptance31-Jan-2022
Date of Web Publication04-Jul-2022

Correspondence Address:
Vijay Kumar
Department of Pathology, Atal Bihari Vajpayee Institute of Medical Sciences, Dr. Ram Manohar Lohia Hospital, New Delhi
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/hmj.hmj_81_21

Rights and Permissions

Background: In the field of haematology, there has been a consistent advancement in technology of the complete blood count (CBC) analyzers and hence, there has been an increase in the number of novel parameters that can be measured on these analysers. To put these newer parameters into routine clinical practice, it is essential to evaluate the reference ranges. Aims and Objective: To evaluate the reference intervals (RI) of the parameters which are calculated by Sysmex XN-1000, especially the newer research parameters. Materials and Methods: Blood samples from 150 clinically asymptomatic and apparently healthy individuals were assessed in the study and reference ranges for the same were evaluated on Sysmex XN-1000 CBC analyzer. Appropriate statistical tests were applied for the same. Results: The RIs of CBC parameters were evaluated in 123 adults (65 males and 58 females). Significant gender-dependent differences were found in red blood cells count, packed cell volume, haemoglobin (Hb), platelets and also in reticulocyte Hb and platelet-crit among the newer parameters. The other CBC reference ranges were also assessed from the remaining data. Conclusion: The present study, highlights the significance of different CBC parameters with special emphasis on newer research parameters. Although, the results of the current study are comparable with previous studies; further studies with larger sample sizes are needed for validation purposes in addition to the regional variation pertaining to these parameters.

Keywords: Complete blood count, reference interval, Sysmex XN-1000

How to cite this article:
Rahar S, Kumar V, Rao S, Gupta D. Haematology reference range evaluation for novel research parameters on the complete blood count analyzer sysmex XN-1000. Hamdan Med J 2022;15:83-8

How to cite this URL:
Rahar S, Kumar V, Rao S, Gupta D. Haematology reference range evaluation for novel research parameters on the complete blood count analyzer sysmex XN-1000. Hamdan Med J [serial online] 2022 [cited 2022 Aug 10];15:83-8. Available from: http://www.hamdanjournal.org/text.asp?2022/15/2/83/349808

  Introduction Top

Advancements in automated complete blood count (CBC) analyzers have led to more accurate, rapid and precise results in the field of haematology. Among the different analysers, Sysmex XN-1000 is being widely used in different laboratories which can measure the CBC parameters including a few newer parameters which are yet to come into routine clinical practice.[1]

Determination of reference intervals (RI) is crucial to separate healthy individuals from diseased persons (a patient). It is essential for the laboratories to provide correct, reliable and accurate information to the clinicians so that they can interpret the results appropriately to provide the correct treatment and further monitor the patients. Before introducing the novel parameters into routine clinical practice, there is a need to determine the RIs and validate them.[2],[3]

The main aim of the present study was to define the reference range of the parameters which are calculated by Sysmex XN-1000 CBC analyzer and especially stressing on the novel research parameters such as immature reticulocyte fraction (IRF), low fluorescence reticulocytes (LFR), medium fluorescence reticulocytes (MFR), high fluorescence reticulocytes (HFR), reticulocyte haemoglobin (Ret-He) and also some platelet indices such as mean platelet volume (MPV), platelet large cell ratio (P-LCR), platelet distribution width (PDW) and platelet-crit (PCT).

  Materials and Methods Top

This study was a prospective cross-sectional observational study which was conducted at a Tertiary Care Hospital in Delhi after obtaining the Institutional Ethical Clearance (File No. 360 [09/2020]/IEC/ABVIMS/RMLH 478). In this study, 150 clinically symptomless and apparently healthy individuals were included after taking consent. These individuals were those who came for their routine health check-ups or for pre-employment examination and were ≥18 years of age. A detailed clinical history regarding any past medical/surgical illness or use of any prescribed medications or history of blood transfusion in the previous 1 month was taken. Vital signs were recorded such as blood pressure, heart rate, respiratory rate, temperature and body mass index which were all within the normal limits. The paediatric age group (below 18 years) was excluded since the hematological parameters have a different reference range from adults.

A volume of 5 ml blood sample was collected in EDTA and plain vacutainers and was processed within 2 h of collection for the haematological analysis. CBC, erythrocyte sedimentation rate, serum iron, Vitamin B12 and folic acid levels were assessed from the samples collected. Only those individuals were included in the study who revealed a totally normal report of all the tests mentioned above with no flagging in the CBC report.

Out of 150 individuals evaluated, finally a total of 123 samples (65-male, 58-female) were considered to take part in the study after excluding the individuals whose values were out of the normal range for the above tests.

Calibration of Sysmex XN-1000 CBC analyzer was done according to the manufacturer's guidelines and daily controls were run according to the College of American Pathology guidelines.

Statistical analysis

The data normality was checked using Shapiro- Wilk test. The categorical variables were expressed in the form of number and percentage (%) while the quantitative data with normal distribution were expressed as the means ± 2 standard deviation (SD) and the data with non-normal distribution as median with 2.5th and 97.5th percentiles (interquartile range). The variables which were quantitative and not normally distributed; were analysed using Mann- Whitney test and independent t-test were used for comparison between males and females.

The data collected were entered in Microsoft EXCEL and were analysed using the Statistical Package for the Social Sciences (SPSS software, IBM manufacturer, Chicago, USA, version 21.0.), and a P < 0.05 was considered statistically significant.

  Results Top

A total of 123 healthy individuals (65-men, 58-women) were assessed and the RIs for varied CBC parameters were calculated. The age range of these individuals was 18–73 years with a mean age of 41.06 ± 14.15.

Shapiro–Wilk test was used to test the normality of data for all the CBC parameters and P < 0.05 was applied for significance. Parameters such as TLC, MCV, MCH, MCHC, RET, IRF and MFR along with platelet and platelet parameters showed a normal distribution so the range was depicted using mean ± SD, while the parameters such as Hb, packed cell volume (PCV), red blood cells (RBC), RDW, HFR and Ret-He were distributed non-normally or showed skewed data, therefore, the range was represented using median, interquartile range and range as 2.5–97.5 percentile [Table 1].
Table 1: Reference ranges for parameters reported on Sysmex XN-1000

Click here to view

[Table 2] depicts the separate reference ranges for men and women for these hematological parameters. For this Mann- Whitney U-test was used and if P > 0.05 then the null hypothesis was retained indicating that the reference ranges are similar for both men and women which was seen in TLC, MCV, RDW, RET, IRF, LFR, MFR, HFR, PDW, MPV, P-LCR and if the P < 0.05 then the null hypothesis was rejected which indicates that the reference ranges are different for men and women which was seen in Hb, PCV, RBC, MCH, MCHC, platelet count, Ret-He, PCT parameters.
Table 2: Differences in reference ranges between men and women subpopulations

Click here to view

Among the newer parameters studied in this study, reference ranges of Ret-He and PCT showed considerable differences between men and women while other parameters like PDW, P-LCR, LFR, MFR, HFR and IRF had the same RIs for both the genders.

  Discussion Top

Many studies are conducted on reference ranges of CBC analysers but due to varying population dynamics, it is important to conduct a study which determines the local RIs reflecting the parameters in that population.

The Sysmex XN-1000 is a CBC analyzer which uses impedance-based counts for measuring RBCs and platelets which includes direct current technology and hydrodynamic focusing. Hb is calculated by a calorimetric technique using the sodium lauryl sulphate Hb method. While the other parameters like white blood cell count and reticulocyte count use fluorescence flow cytometry technology using a semiconductor laser which classifies cells by irradiating them with a laser beam and analysing their forward scattered light, side scattered light and side fluorescent light.[1],[4],[5],[6],[7]

The Clinical and Laboratory Standards Institute (CLSI) has outlined a method for calculating the RIs in which reference individuals from a reference sample group are obtained from the reference population on which the reference limits are calculated. Many different methods can be used to establish the RIs. However, we have applied the basic approach wherein 120 persons from the reference sample group who are healthy are taken into account. The values from these individuals are then evaluated by non-parametric methods to determine the 2.5 and 97.5 percentiles which form the 95% RI. Although this technique has a disadvantage in that it does not exclude the extreme values from healthy individuals.[1],[2],[8]

Other methods for RI determination are; the truncation method which uses an 80% confidence interval as a reference range, another method is a scientific method known as the robust statistical method which estimates the center of distribution and gives lower points to extreme values. But the best approach for the determination of RIs is by multicenter RI studies which require taking samples from different centers with variable distribution of age, sex and race. This approach is difficult to achieve since it requires a huge amount of funds and time.[1],[2],[8]

The presence of reticulocytes in the peripheral smear helps to evaluate the erythropoietic activity of bone marrow therefore is useful in diagnosing cases of anaemia. CBC analyzers use the flow cytometric method to assess the reticulocyte maturity wherein the fluorescence intensity of the reticulocytes is directly proportional to the content of RNA present whereas conventional microscopy is unable to determine the immature reticulocytes in the peripheral smears which are prepared.[9]

In the analyser, reticulocytes are divided into three classes according to fluorescent intensity; HFR which denotes the young or immature reticulocytes while LFR and MFR correspond to maturing reticulocytes. IRF is defined as the sum total of the cells of MFR and HFR (%).[9],[10]

IRF is a newer parameter that requires more research so that it can be used in routine practice. IRF can act as an early marker of bone marrow engraftment of haematopoietic stem cell transplant and bone marrow regeneration after chemotherapy.[10],[11] IRF also rises before the rise of reticulocyte count to check the treatment response in cases of nephropathy, HIV infection and myelodysplastic syndromes when treated with erythropoiesis-stimulating agents.[10],[12] A study showed that IRF could also be helpful in the diagnosis of hereditary spherocytosis (HS) when combined with reticulocyte count, as, in HS a high reticulocyte count is noted without an elevated IRF to the same degree.[10],[13] This parameter is also useful to monitor the efficaciousness of treatment in megaloblastic or iron deficiency anemia (IDA) since it rises before the reticulocyte counts.[10]

Ret-He measures the Hb content of the reticulocyte. It directly assesses the iron incorporation into erythrocyte Hb and hence estimates the recent functional availability of iron into erythron when other biochemical parameters are unable to provide any information in this regard.[14]

Its major uses are in:

  • It can detect IDA earlier especially when it coexists with other chronic disorders like rheumatoid arthritis or during any inflammatory process when the biochemical markers used for evaluating iron status like serum iron, transferrin or ferritin can be extremely unbalanced due to chronic diseased state[10],[14]
  • In functional iron deficiency (FID) iron stored in the body is not able to comply with the demands of increasing erythropoiesis, like during the treatment with erythropoiesis-stimulating agents. A high/normal value of ferritin along with a lower Ret-He value can be suggestive of FID[10],[14]
  • It can also be used in monitoring the treatment efficacy in IDA and iron deficiency detected while recovering from megaloblastic anaemia.[10]

Some limitations of using Ret-He are also noted. It is reduced in thalassaemia syndromes while it is raised in IDA patients with confounding megaloblastic anaemia or use of drugs which cause macrocytosis like hydroxyurea used in treating patients of sickle cell disease due to an increase in mean reticulocyte volume.[10]

Platelets or thrombocytes have a major role in haemostasis. On light microscopy, a blood smear is stained to examine the platelets for size, shape, number and clumping. Microscopic examination together with platelet parameters on automated analysers can help to improve the knowledge about platelet disorders. The different platelet parameters which can be measured using automated analysers are MPV, PDW, P-LCR and PCT.[15]

MPV measures the average size of platelets and indicates platelet activity and function.[16],[17] An association has been found between elevated MPV levels in diabetes mellitus, hypertension, atherosclerosis, acute coronary syndrome and stroke.[16],[17],[18],[19] MPV usually increases when there is platelet destruction as is seen in immune thrombocytopenic purpura (ITP) or in cases where immature platelets are released in the circulation while MPV is decreased when there is ineffective platelet formation in the bone marrow.[3],[15]

PDW refers to the variability in the size of platelets. It is gaining attention due to its utility to distinguish between secondary thrombocytosis and primary thrombocytosis cases like myeloproliferative disorders. It is also useful in hypoproductive thrombocytopenia (aplastic anemia) and hyperdestructive thrombocytopenia (ITP).[3]

P-LCR is the % of platelets that is more than the normal value of platelet volume of 12fL in the total platelet count. It reflects the platelet activity. It is increased in thrombocytopenia while decreased in thrombocytosis. It also has been related to antiplatelet antibodies.[15]

PCT detects platelet quantitative abnormalities and can act as an efficacious screening tool. It is the volume occupied by platelets in blood as %. PCT = platelet count × MPV divided by 10,000. PCT is decreased in thrombocytopenia while increased in thrombocytosis.[15],[20]

Some spurious results can be obtained if the analyser counts the fragmented RBCs, microcytes, leukaemic cell fragments and cryoglobulins as platelets therefore, microscopic evaluation is essential along with the analyser readings.[3]

In our study, few deviations or differences in the reference ranges were found in some of the parameters when compared to other studies [Table 3].[1],[15],[21] Statistically significant difference in reference ranges was found between men and women for Hb, RBC, PCV, MCH, MCHC and platelets along with PCT and Ret-He similar to other studies.[1],[22] In this study, we failed to notice any significant statistical difference in values of MPV and P-LCR between males and females as compared to the study by Ali et al.[15]
Table 3: Comparing the present study with other studies in the context of newer parameter ranges

Click here to view

There are differences between RIs in different populations and such differences reflect the significance of establishing the reference ranges in each laboratory according to the local or native population. Environment and nutritional factors might be playing a major part in these differences.[22]

  Conclusion Top

We reported the RIs for IRF, LFR, MFR, HFR, Ret-He, PDW, MPV, PCT and P-LCR to explore and promote more research and guide patient care and treatment. Since these parameters are generated by the analyzer Sysmex XN-1000 but are not routinely described on the CBC reports nor are they asked by the clinician, so it does not put any additional burden of cost. It is important for the laboratories to render accurate reports to the treating physician which is essential to provide a conclusive diagnosis and implement the correct intervention. It is important to determine the reference range of a native population as it reflects or considers the population to which the tests are to be applied.


The limitation of this study was the small sample size that was seeable of prices concerned in doing iron, Vitamin B12 and folic acid studies. However, the method for calculating the RIs was robust enough to calculate the RIs according to the CLSI guidelines. Another limitation was that the age-related differences were not assessed for RIs.

Ethical clearance

This study was a prospective cross-sectional observational study which was conducted at a Tertiary Care Hospital in Delhi after obtaining the Institutional Ethical Clearance (File No. 360 [09/2020]/IEC/ABVIMS/RMLH 478).

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Sehgal KK, Tina D, Choksey U, Dalal RJ, Shanaz KJ. Reference range evaluation of complete blood count parameters with emphasis on newer research parameters on the complete blood count analyzer Sysmex XE-2100. Indian J Pathol Microbiol 2013;56:120-4.  Back to cited text no. 1
[PUBMED]  [Full text]  
Mave V, Kulkarni V, Bharadwaj R, Khandekar M, Gupta A, Gupte N. Determination of a reference interval in a population. Natl Med J India 2012;25:33-4.  Back to cited text no. 2
Farias MG, Schunck EG, Dal Bó S, de Castro SM. Definition of reference ranges for the platelet distribution width (PDW): A local need. Clin Chem Lab Med 2010;48:255-7.  Back to cited text no. 3
Briggs C. Quality counts: New parameters in blood cell counting. Int J Lab Hematol 2009;31:277-97.  Back to cited text no. 4
Briggs C, Harrison P, Machin SJ. Continuing developments with the automated platelet count. Int J Lab Hematol 2007;29:77-91.  Back to cited text no. 5
Pfaeffli J. Reference limits for the automated haematology analyser sysmex XE-2100. Sysmex J Int 2002;12:18-23.  Back to cited text no. 6
Pekelharing JM, Hauss O, de Jonge R, Lokhoff J, Sodikromo J, Spaans M, et al. Haematology reference intervals for established and novel parameters in healthy adults. Sysmex J Int 2010;1:1-9.  Back to cited text no. 7
Edward AS, Basil TD, Miller WG, D'Orazio P, Eckfeldt JH, Evans SA, et al. How to Define and Determine Reference Intervals in the Clinical Laboratory; Approved Guideline. 2nd ed., Vol. 20. Wayne, Pennsylvania: NCCLS; C28-A2; 2000. p. 1-59.  Back to cited text no. 8
Watanabe K, Kawai Y, Takeuchi K, Shimizu N, Iri H, Ikeda Y, et al. Reticulocyte maturity as an indicator for estimating qualitative abnormality of erythropoiesis. J Clin Pathol 1994;47:736-9.  Back to cited text no. 9
Piva E, Brugnara C, Spolaore F, Plebani M. Clinical utility of reticulocyte parameters. Clin Lab Med 2015;35:133-63.  Back to cited text no. 10
Davis BH. Immature reticulocyte fraction (IFR): By any name, a useful clinical parameter of erythropoietic activity. Lab Hematol 1996;2:2-8.  Back to cited text no. 11
Dunlop LC, Cohen J, Harvey M, Gallo J, Motum P, Rosenfeld D. The immature reticulocyte fraction: A negative predictor of the harvesting of CD34 cells for autologous peripheral blood stem cell transplantation. Clin Lab Haematol 2006;28:245-7.  Back to cited text no. 12
Mullier F, Lainey E, Fenneteau O, Da Costa L, Schillinger F, Bailly N, et al. Additional erythrocytic and reticulocytic parameters helpful for diagnosis of hereditary spherocytosis: Results of a multicentre study. Ann Hematol 2011;90:759-68.  Back to cited text no. 13
Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret He) and assessment of iron-deficient states. Clin Lab Haematol 2006;28:303-8.  Back to cited text no. 14
Ali U, Gibbs R, Knight G, Tsitsikas D. Sex-divided reference intervals for mean platelet volume, platelet large cell ratio and plateletcrit using the Sysmex XN-10 automated haematology analyzer in a UK population. Hematol Transfus Cell Ther 2019;41:153-7.  Back to cited text no. 15
Schneider DJ. Abnormalities of coagulation, platelet function, and fibrinolysis associated with syndromes of insulin resistance. Coron Artery Dis 2005;16:473-6.  Back to cited text no. 16
Coban E, Adanir H. Platelet activation in patients with Familial Mediterranean Fever. Platelets 2008;19:405-8.  Back to cited text no. 17
Bath P, Algert C, Chapman N, Neal B; PROGRESS Collaborative Group. Association of mean platelet volume with risk of stroke among 3134 individuals with history of cerebrovascular disease. Stroke 2004;35:622-6.  Back to cited text no. 18
Greisenegger S, Endler G, Hsieh K, Tentschert S, Mannhalter C, Lalouschek W. Is elevated mean platelet volume associated with a worse outcome in patients with acute ischemic cerebrovascular events? Stroke 2004;35:1688-91.  Back to cited text no. 19
Chandrashekar V. Plateletcrit as a screening tool for detection of platelet quantitative disorders. J Hematol 2013;2:22-6.  Back to cited text no. 20
Sachdev R, Tiwari AK, Goel S, Raina V, Sethi M. Establishing biological reference intervals for novel platelet parameters (immature platelet fraction, high immature platelet fraction, platelet distribution width, platelet large cell ratio, platelet-X, plateletcrit, and platelet distribution width) and their correlations among each other. Indian J Pathol Microbiol 2014;57:231-5.  Back to cited text no. 21
[PUBMED]  [Full text]  
Ambayya A, Su AT, Osman NH, Nik-Samsudin NR, Khalid K, Chang KM, et al. Haematological reference intervals in a multiethnic population. PLoS One 2014;9:e91968.  Back to cited text no. 22


  [Table 1], [Table 2], [Table 3]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  In this article
Materials and Me...
Article Tables

 Article Access Statistics
    PDF Downloaded59    
    Comments [Add]    

Recommend this journal