• Users Online: 478
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
Year : 2022  |  Volume : 12  |  Issue : 3  |  Page : 312-315

Comparison of haemoglobin estimation of blood donors by specific gravity method, HemoCue method and automated haematology cell analyzer

Department of Pathology, Sri Devaraj Urs Medical College, Kolar, Karnataka, India

Date of Submission27-May-2022
Date of Acceptance05-Aug-2022
Date of Web Publication15-Sep-2022

Correspondence Address:
Dr. Subhashish Das
Department of Pathology, Sri Devaraj Urs Medical College, Kolar, Karnataka
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/aihb.aihb_100_22

Rights and Permissions

Introduction: Pre-donation haemoglobin (Hb) screening is among the foremost test done on blood donors to determine whether an individual is fit to donate with the intention of preventing inadvertent donation from an anaemic donor. The aim of the study was to compare the efficacy of the three common Hb estimation methods, namely, copper sulphate (CuSO4) method, HemoCue photometer and automated cell counter in reporting the Hb levels of blood donors. Materials and Methods: CuSo4 specific gravity method, HemoCue and automated cell analyzer (Sysmex XN-550) were used to determine the Hb levels in blood samples of 500 donors. Descriptive statistics were used to analyse the demographic details of the donor. Kappa statistics were used to determine the level of agreement between the three methods of Hb estimation. Results: HemoCue was found to be more sensitive (86.21%), whereas CuSo4 (97.88%) was found to be more specific. Kappa agreement was good between CuSo4 and Sysmex XN-550 (0.703), whereas it was moderate between HemoCue and Sysmex XN-550 (0.458). Conclusions: The CuSO4 method is still viable for Hb estimation among blood donors. Thus, it can be utilised as the primary screening method; however, follow-up testing with HemoCue or automated cell analyzer can be done to minimise unnecessary deferrals and false acceptance.

Keywords: Blood donation, copper sulphate, haemoglobin estimation, HemoCue

How to cite this article:
Choudhari N, Das S, Kumar R. Comparison of haemoglobin estimation of blood donors by specific gravity method, HemoCue method and automated haematology cell analyzer. Adv Hum Biol 2022;12:312-5

How to cite this URL:
Choudhari N, Das S, Kumar R. Comparison of haemoglobin estimation of blood donors by specific gravity method, HemoCue method and automated haematology cell analyzer. Adv Hum Biol [serial online] 2022 [cited 2023 Feb 7];12:312-5. Available from: https://www.aihbonline.com/text.asp?2022/12/3/312/356099

  Introduction Top

One of the fundamental purposes of a blood transfusion service is to offer safe and high-quality blood products for patients and to do so, we must avoid collecting blood from an anaemic donor. As a result, pre-donation haemoglobin (Hb) testing for blood donors is critical to protect the health of transfusion recipients.[1]

According to the Drugs and Cosmetic Act, 1940 and the criteria mentioned in the Directorate General of Health Services Technical Manual, 2003, only blood donors with Hb levels of ≥12.5gm/dl are eligible for whole blood donation.[2],[3]

Despite various methods for Hb estimation, no single technique has emerged as the most appropriate and ideal for a blood donation setup. A highly accurate method in a blood donor setting is more likely to be expensive. In a developing country like India, it is not possible to use such a method for screening so many blood donors' samples. On the other hand, a less accurate and cheaper method may give false results which may lead to either donation of blood by an anaemic subject or loss of eligible donors. Therefore, there is a requirement to adopt a cost-effective and time-saving Hb estimation method that delivers accurate laboratory results.[1]

The copper sulphate (CuSo4) specific gravity method has been traditionally used and is still being used for donor screening at many blood centres in India due to its easy availability and cost-effectiveness. However, it does not provide an acceptable degree of accuracy with many studies showing deferral of subjects who failed the test but were not anaemic. It is, therefore, important to determine anaemia amongst them using the standard diagnostic method so that there is no loss of any potential donors.[4],[5]

Another method uses a new generation of Hb photometers, the HemoCue. HemoCue photometer calculates the Hb concentration in g/dl and displays the results as a digital reading in 15–45 s.[6] Similarly, the Sysmex XN-550 is an automated, compact and haematology analyzer designed to generate a full blood count with a standard five-part white blood cell differential and an immature granulocyte count, as well as an optional reticulocyte and optical platelet counts.[6]

The present study aims to compare the efficacy of three Hb estimation methods, namely, the CuSO4 method, HemoCue photometer and automated cell counter (Sysmex XN-550) in reporting the Hb levels of blood donors.

  Materials and Methods Top

Study population

This is a prospective observational study conducted over 6 months from January 2021 to June 2021 in a tertiary health care centre. This study was ethically approved by the Institutional Ethics Committee (SDUMC/KLR/IEC/745/2020–21). This study followed ethical standards delineated in the Helsinki declaration 1975, with an update in 2013.

Study size

A convenient sample of 500 human blood samples was obtained from blood donors. The inclusion criteria included consenting donors aged 18 years and above who provided blood samples of a minimum 2 ml volume. The exclusion criteria included all the samples that were seropositive, insufficient or haemolysed.

The procedure for sample collection

Capillary blood samples were collected by deep finger prick on the index or middle finger of the left hand using a dry sterile lancet (Unilet Excelite II, England) after disinfecting with ethanol and massaging the finger to facilitate blood flow. The first drop was wiped away and the second drop was used for testing by CuSo4 method and HemoCue method (HemoCue AB, Ängelholm, Sweden). Two millilitres of venous blood samples were collected into EDTA Vacutainer tubes and were analysed on the automated cell counter as soon as possible.

Blood sampling and analysis of Hb were performed only by doctors and technicians who were trained for the instruments on a few pilot samples using the three methods before commencing the study.

Testing of all eligible donor samples was analysed immediately or within 30–60 min of collection. The blood sampling and analysis of Hb were first estimated by CuSo4, followed by HemoCue, and finally by the automated cell analyzer. Results of CuSO4 were interpreted as pass or fail at Hb cutoff of ≥12.5 g/dl while HemoCue readings were considered to pass when the readings were ≥12.0 g/dl and fail below 12.0 g/dl.

Every day, following the standard operating procedure, the working CuSO4 solution was created (specific gravity 1.053) and standardised. Every day, the HemoCue photometer's functionality was tested by measuring the control cuvette according to the manufacturer's recommendations. Quality control and calibration of the automated haematology analyzer were performed according to standard operating procedures using stabilised control reagents provided by the manufacturer.

Statistical analysis

Data were entered into a Microsoft Excel data sheet and were analysed using SPSS 22 version software (SPSS Statistics - IBM Data Science Community USA). Categorical data were represented in the form of frequencies and frequency percentages. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of each method were also calculated. Kappa statistics were used to check the level of agreement between the tests.

  Results Top

Most of the donors were males (95.4%) and between 21 and 30 years (52.6%). Around 80% were voluntary blood donors. Furthermore, 70% of the samples were collected from outdoor camps as shown in [Table 1]. [Table 2] shows the deferral data of the three methods against the cell counter method.
Table 1: Demographic details of the blood donors and the site of sample collection

Click here to view
Table 2: Deferral data of the three haemoglobin estimation methods in different levels of haemoglobin level according to cell counter method

Click here to view

According to this data, the highest deferrals were seen in the CuSo4 (7.6%) method followed by HemoCue (6.4%) and automated cell analyzer (6.2%). CuSO4 falsely accepted four of 500 (0.8%) donors and falsely deferred 11 of 500 (2.2%) donors. The false acceptance with HemoCue was 5 of 500 (1%), and false deferral was 6 of 500 (1.2%) donors.

The measure of agreement between CuSo4 and Sysmex XN-550 was found to be good with a κ = 0.703 (P < 0.001). Whereas the measure of agreement between HemoCue and Sysmex XN-550 was found to be moderate with a κ = 0.458 (P < 0.001).

The sensitivity of the HemoCue (86.21%) method was found to be higher than that of the CuSO4 (75.86%) method, whereas specificity was higher in the case of CuSO4 (97.88%) when compared to the HemoCue (91.30%) method.

The PPV was higher in the case of CuSO4 (68.75%) when compared to HemoCue (37.88%), whereas the NPV was higher in the HemoCue (99.08%) method when compared to CuSo4 (98.50%). Accuracy was found to be higher in CuSO4 (96.60%) than HemoCue (91%).

  Discussion Top

An acceptable Hb screening method should be available for blood collection to accept as many potential donors as feasible and avoid any unnecessary deferrals. Any new approach for Hb screening that is introduced must reduce time and money, and it must be validated against gold standard methods.

The capillary method of Hb measurement in field situations for CuSO4/HemoCue is more practical than the venous sampling approach,[7] but because our reference method was based on venous samples, only venous samples were employed in this work to maintain homogeneity and have near true values.[8] In addition, venous samples outperform capillary samples in terms of sensitivity, specificity, PPV, NPV and other performance metrics.[9],[10]

Furthermore, because donor acceptance procedures are based on venous Hb standards rather than capillary Hb values, all these devices favoured venous sampling over capillary for Hb measurement. CuSO4 has been a traditional method of donor Hb screening notwithstanding its disadvantages.[11] To ensure accurate results, a CuSO4 solution with an appropriate specific gravity should be used, along with other technical procedures. Because each drop of blood added to the solution affects the specific gravity, the solution should be changed daily, or at least every 25 tests.[12] CuSO4 falsely accepted four of 500 (0.8%) donors for blood donation in the present study, which is less compared with the 5% reported by Malukani et al.[13]

CuSO4 falsely deferred 11 of 500 (2.2%) donors, which was in contrast to 29% reported by Sawant et al.[14] This discrepancy could be attributable to a variety of factors, including preparation, quality control and storage, as well as technical ability and diligence, which can differ from person to person. These factors were a source of concern when the semi-quantitative methodology was used.

The false acceptance with HemoCue was five of 500 (1%), which is lower than that (6%) reported by Patel et al.[15] HemoCue falsely deferred six donors (1.2%), whereas Patel et al. reported 3.3%.[15]

In the present study, HemoCue (86.21%) was found to be more sensitive than CuSO4 (75.86%). This is in accordance with the study by Tondon et al.[16] whereas it contrasts with the study by Rout et al.[17] who found both the methods equally sensitive. The specificity of CuSo4 (97.88%) was higher than that of HemoCue (91.30%). This is in contrast with the findings of Rout et al. and Tondon et al. who found the specificity of HemoCue higher than that of CuSo4.[17]

When choosing a method for Hb estimation in blood donors, the PPV of the screening test is critical since the goal is to ensure the safety of blood donors while also avoiding unnecessary deferral of potential blood donors.[18] In the present study, PPV was found to be high in CuSo4 (68.75%) than that of HemoCue (37.88%).[Table 3] This is in contrast with the findings of Rout et al. and Tondon et al.[16],[17] NPV is important for donor safety. In the present study, NPV was similar for CuSO4 (98.50%) and HemoCue (99.08%). This is in accordance with the study by Tondon et al. and Rout et al.[16],[17]
Table 3: Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of copper sulphate method and HemoCue in comparison with Sysmex XN-550

Click here to view

In the case of measures of agreement, HemoCue when compared with Sysmex XN-500 showed moderate agreement (κ = 0.458). This is in accordance with the findings of Yadav et al. who also found moderate agreement between the two testing methods (κ = 0.697).[19] In contrast, the measure of agreement was good between the CuSO4 method and Sysmex XN-550 with a κ = 0.703 in the present study.

It is also worth mentioning that the HemoCue apparatus costs around 35000 Indian Rupees (INR), whereas each disposable microcuvette costs around 30 INR. CuSo4 powder (500 g) costs only 175 INR and can be used to test 2000–2500 samples (assuming 159.63 g is utilised for about 750–800 tests), costing around 0.06–0.08 INR for each test. Our findings show that HemoCue is roughly 500 times more expensive than the CuSO4 technique.

Minimal inter-observer and inter-instrument errors were among the study's strengths. The equipment was subjected to adequate quality control and was regularly inspected. All three instruments were subjected to daily internal quality checks.

Limitations of the study

The study's shortcomings include the limited sample size and the fact that it was done in a single location. A larger sample size and a multicentre investigation could yield more conclusive results.

  Conclusions Top

We have shown that the CuSO4 approach is still valid. Therefore, it can be used as the primary screening method; however, to avoid unnecessary deferrals, follow-up testing can be done with HemoCue or an automated cell analyzer. This study may be useful to blood centres with limited resources, particularly for camp donations where mass Hb screening is performed.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Whitehead RD Jr., Mei Z, Mapango C, Jefferds ME. Methods and analyzers for hemoglobin measurement in clinical laboratories and field settings. Ann N Y Acad Sci 2019;1450:147-71.  Back to cited text no. 1
Myers DJ, Collins RA. Blood donation. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2022. Available from: http://www.ncbi.nlm.nih.gov/books/NBK525967/. [Last accessed on 2022 Aug 05].  Back to cited text no. 2
Pehlic V, Volken T, Holbro A, Jirout Z, Drexler B, Buser A, et al. Long-term course of haemoglobin and ferritin values in high-frequency donors of whole blood and double erythrocyte apheresis. Transfus Med Hemother 2021;48:71-8.  Back to cited text no. 3
Freeman AM, Rai M, Morando DW. Anemia screening. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2022. Available from: http://www.ncbi.nlm.nih.gov/books/NBK499905/. [Last accessed on 2022 Aug 05].  Back to cited text no. 4
Chaparro CM, Suchdev PS. Anemia epidemiology, pathophysiology, and etiology in low- and middle-income countries. Ann N Y Acad Sci 2019;1450:15-31.  Back to cited text no. 5
Małecka M, Ciepiela O. A comparison of Sysmex-XN 2000 and Yumizen H2500 automated hematology analyzers. Pract Lab Med 2020;22:e00186.  Back to cited text no. 6
Jain A, Chowdhury N. Comparison of the accuracy of capillary hemoglobin estimation and venous hemoglobin estimation by two models of HemoCue against automated cell counter hemoglobin measurement. Asian J Transfus Sci 2020;14:49-53.  Back to cited text no. 7
  [Full text]  
Lim MD. Dried blood spots for global health diagnostics and surveillance: Opportunities and challenges. Am J Trop Med Hyg 2018;99:256-65.  Back to cited text no. 8
Shreffler J, Huecker MR. Diagnostic testing accuracy: Sensitivity, specificity, predictive values and likelihood ratios. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2022. Available from: http://www.ncbi.nlm.nih.gov/books/NBK557491/. [Last accessed on 2022 Aug 05].  Back to cited text no. 9
Vaghani H, Mehta R, Desai K, Duseja S, Mehta T. Effect of non-surgical periodontal therapy on glycosylated haemoglobin levels in diabetics and non-diabetic healthy controls with periodontitis. Adv Hum Biol 2016;6:95.  Back to cited text no. 10
  [Full text]  
Bell S, Sweeting M, Ramond A, Chung R, Kaptoge S, Walker M, et al. Comparison of four methods to measure haemoglobin concentrations in whole blood donors (COMPARE): A diagnostic accuracy study. Transfus Med Oxf Engl 2021;31:94-103.  Back to cited text no. 11
Sharma R, Sharma S. Physiology, blood volume. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2022. Available from: http://www.ncbi.nlm.nih.gov/books/NBK526077/. [Last accessed on 2022 Aug 05].  Back to cited text no. 12
Malukani P, Gajjar M, Gonsai R, Bhatnagar N, Goswami R. Study of newer invasive and non-invasive methods of haemoglobin estimation in blood donor screening – A study on 200 donors. Int J Cur Res Rev 2014;6:2-30.  Back to cited text no. 13
Sawant RB, Bharucha ZS, Rajadhyaksha SB. Evaluation of hemoglobin of blood donors deferred by the copper sulphate method for hemoglobin estimation. Transfus Apher Sci 2007;36:143-8.  Back to cited text no. 14
Patel AJ, Wesley R, Leitman SF, Bryant BJ. Capillary versus venous haemoglobin determination in the assessment of healthy blood donors. Vox Sang 2013;104:317-23.  Back to cited text no. 15
Tondon R, Verma A, Pandey P, Chaudhary R. Quality evaluation of four hemoglobin screening methods in a blood donor setting along with their comparative cost analysis in an Indian scenario. Asian J Transfus Sci 2009;3:66-9.  Back to cited text no. 16
[PUBMED]  [Full text]  
Rout D, Sachdev S, Marwaha N. Evaluation of new non-invasive & conventional invasive methods of haemoglobin estimation in blood donors. Indian J Med Res 2019;149:755-62.  Back to cited text no. 17
[PUBMED]  [Full text]  
Das S, Harendra Kumar M. Comparative evaluation of quality parameters of platelet stored in additive solution versus plasma. Adv Hum Biol 2022;12:163.  Back to cited text no. 18
  [Full text]  
Yadav K, Kant S, Ramaswamy G, Ahamed F, Jacob OM, Vyas H, et al. Validation of point of care hemoglobin estimation among pregnant women using digital hemoglobinometers (HemoCue 301 and HemoCue 201+) as compared with auto-analyzer. Indian J Hematol Blood Transfus 2020;36:342-8.  Back to cited text no. 19


  [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 Downloaded92    
    Comments [Add]    

Recommend this journal