Comparison of automated method and photometric cyanmethemoglobin method for haemoglobin estimation

Background: Haemoglobin is one of the commonest tests required in pathology labs. There are many methods of haemoglobin estimation. Common methods used are sahli’s acid hematin method, photometric cyanmethemoglobin method with hemoglobin estimation by colorimeter, automated method by cell counter. Objective: To evaluate accuracy of hemoglobin estimation by manual photometric method versus automated method by hematology cell counters using the same sample at the same time. Materials and Methods: Blood samples of 460 adult patients and 72 children (<15 Yrs), including outdoor and indoor, between May 2019 to Aug 2019, attending H.I.M.S.were collected in EDTA tubes. Samples were properly mixed on blood shaker. Hemoglobin estimation done by photometric cyanmethemoglobin method by AIMIL digital colorimeter and by Mindray (BC5150) automated cell counter. Results: Results by photometric method showed higher mean value compared to automated method by 2.52%. Commercial control results showed 2.2% coefficient of variation by Mindray cell counter and 2.9% by photometer. Conclusion: Both methods are accurate with 2.52% more mean value in photometric method. When Haemoglobin is the only test required, photometric method is cost effective and feasible. If sample size is large as in tertiary care hospitals and multiple parameters like complete blood count (CBC) are needed, automated method is time effective and feasible. © 2020 Published by Innovative Publication. This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/)


Introduction
Hemoglobin comprises of four globin protein subunits, each having one polypeptide chain and one heme group. Oxygen binds reversibly to the ferrous iron atom in each heme group. 1,2 Main function of haemoglobin is to transport oxygen from lungs to the tissues, where oxygen is utilized for metabolism, 3 mainly to facilitate oxidative phosphorylation in mitochondria. 4 Anaemia is very common in our country, worldwide it affects over 800 million women and children, 5 also there are fair number of cases in advanced countries like US, 6 so haemoglobin is advised frequently to the patients especially in antenatal patients because of increased risk of complications. 7 Hb measurement is also essential for screening the eligibility of donors in blood banks. 8 The reference range for normal hemoglobin (according to WHO) is 13-18 gm/dl for men, 12-16 gm/dl for women. 9,10 Hemoglobin being one of the commonest investigations in laboratory, we require economical, feasible and accurate method. 11 Many methods are commonly used, sahli's acid hematin method, cyanmethemoglobin method by photometer, automated method by cell counters. 3 Different lab uses different methods depending on laboratory location, number of patients, availability of technical staff, problem of electricity, affordability. There is variation in hemoglobin measurement done by different instruments due to many reasons including type of sample. 10 Photometric cyanmethemoglobin method is based on principle that Drabkin reagent reacts with hemoglobin https://doi.org/10.18231/j.jdpo.2020.053 2581-3714/© 2020 Innovative Publication, All rights reserved. 273 in blood to form cyanmethemoglobin and developed colour is measured by photometry at 550 nm. Automated method on Mindray cell counter is based on principle of electrical impedance for cell counting and colorimetry for HB estimation. 12 Automated method uses a non cyanide hemoglobin method. 13

Aims and objective
The study were to evaluate accuracy, cost effectiveness, suitability and feasibility of photometric versus automated method of haemoglobin estimation.

Materials and Methods
We took 532 patient's (460 adults and 72 children) Hb estimation into consideration between May 2019 to Aug 2019 including indoor & outdoor patients attending Hind Institute of Medical Sciences, Safedabad, Barabanki, Uttar Pradesh. Adults were between 15 to 85yrs of age. Children were less than 15yrs upto 03 hrs old, newly born baby.
2 ml blood samples were collected in tubes containing K3EDTA anticoagulant. After proper mixing on blood shaker Hb estimation was done simultaneously both by Mindray 5 part hematology cell counter (BC-5150) and digital colorimeter by Aimil. For manual method 5 ml Drabkin's reagent was taken in test tube and 20 ul of blood was mixed and then waited for 5minutes at room temperature before taking absorbance on colorimeter at 550 nm filter against Drabkin , s solution. A standard curve is used to know the Hb concentration in the sample by measuring absorbance compared to standard control. Drabkin's solution contains cyanide which is hazardous during handling and disposal. 5 Drabkin's reagent was used from Arkray (formerly Span Diagnostics). Standards were also used from same company. For automated cell counter, control blood was used from Diagon. Standards (control) were run regularly. We did not include hemolyzed sample in our study.

Results
A total of 532 samples were processed for hemoglobin estimation. It takes 5 minutes by automated method while 15-20 minutes by photometric method. Photometer requires almost negligible maintenance while automated analyser regular maintenance.

Discussion
For a developing country like India, economically suitable methodology is the need of the hour. At our institute cost of the test by automated cell counter is Rs 100 whereas cost of manual photometric method is Rs 10 per test. Cost of Mindray Cell Counter is about Rs 4.0 lakh while AIMIL photometer cost only for Rs 9,500. When haemoglobin is the only test required, photometric method is cost effective and feasible. Automated method should be used where complete blood count (CBC), or multiple parameters are required. 14 International committee for standardization in haematology (ICSH) has recommended the'Drabkin' as the method of choice and have suggested all the other method should be adjusted to be comparable to this method 15 because of availability of internationally accepted reference  standard calibrator. 16,17 Periodic standards (controls) must be run to maintain accuracy both in manual & automated method. Hemoglobin estimation by automated cell counters is the next best method. 18 There is a significant and positive correlation between the manual and the automated method. 19 For accuracy proper sample collection and proper technique is very important. 20

Conclusion
Both methods of Hb estimation are accurate and for single parameter manual method is very cost effective.

Abbreviations
Hb: Hemoglobin HIMS: Hind Institute of Medical Sciences

Source of Funding
None.

Conflict of Interest
None.