| Abstract|| |
Background: The hemoglobincyanide method (HiCN) method for measuring hemoglobin is used extensively worldwide; its advantages are the ready availability of a stable and internationally accepted reference standard calibrator. However, its use may create a problem, as the waste disposal of large volumes of reagent containing cyanide constitutes a potential toxic hazard. Aims and Objective: As an alternative to drabkin`s method of Hb estimation, we attempted to estimate hemoglobin by other non-cyanide methods: alkaline hematin detergent (AHD-575) using Triton X-100 as lyser and alkaline- borax method using quarternary ammonium detergents as lyser. Materials and Methods: The hemoglobin (Hb) results on 200 samples of varying Hb concentrations obtained by these two cyanide free methods were compared with a cyanmethemoglobin method on a colorimeter which is light emitting diode (LED) based. Hemoglobin was also estimated in one hundred blood donors and 25 blood samples of infants and compared by these methods. Statistical analysis used was Pearson`s correlation coefficient. Results: The response of the non cyanide method is linear for serially diluted blood samples over the Hb concentration range from 3gm/dl -20 gm/dl. The non cyanide methods has a precision of + 0.25g/dl (coefficient of variation= (2.34%) and is suitable for use with fixed wavelength or with colorimeters at wavelength- 530 nm and 580 nm. Correlation of these two methods was excellent (r=0.98). The evaluation has shown it to be as reliable and reproducible as HiCN for measuring hemoglobin at all concentrations. The reagents used in non cyanide methods are non-biohazardous and did not affect the reliability of data determination and also the cost was less than HiCN method. Conclusions: Thus, non cyanide methods of Hb estimation offer possibility of safe and quality Hb estimation and should prove useful for routine laboratory use. Non cyanide methods is easily incorporated in hemobloginometers by using very minute quantities of reagents and test sample; hence, these methods can be used to provide point of care testing in blood banks for hemoglobin (Hb) estimation in blood donors.
Keywords: Alkaline borate, alkaline hematin detergent- 575, digital colorimeter (LED based), quarternary ammonium compounds
|How to cite this article:|
Shah VB, Shah BS, Puranik G V. Evaluation of non cyanide methods for hemoglobin estimation. Indian J Pathol Microbiol 2011;54:764-8
| Introduction|| |
Hemoglobin (Hb) is an essential test carried out in all the laboratories. Hemiglobincyanide (HiCN) method performed satisfactorily for many years. With the advent of laboratory automation, a new diversity of methods arose. The manual HiCN method was phased out as a routine method and gradually became a reference method.  Other factors like legislation affecting the transportation of reagents and control of substances such as cyanides, which are hazardous to health have also led to the development of methods which do not employ cyanide for the determination of hemoglobin. 
Several investigators using Sodium Lauryl Sulfate (SLS) have reported satisfactory performance of hemoglobin estimation when compared with the widely used hemiglobincyanide (HiCN) method as a reference.  These methods are being widely used in automated cell counters. ,,
The alkaline hematin method has been evaluated extensively by authors in other countries and East Africa. ,
In our country, approximately 70% of laboratories still use manual HiCN method for Hb estimation especially in rural areas. Hence, the need to have non cyanide methods for Hb estimation in our country to avoid the environmental pollution by cyanide reagents.
There are no Indian studies on comparison of alkaline hematin methods with HiCN.
We formulated a simple, novel cyanide free cheap reagent from locally available materials using quarternary ammonium compounds as lyser in an alkaline borate solution.
The hemoglobin estimation by the AHD-triton and alkaline borate method were compared with hemoglobin estimation by HiCN method.
We further evaluated the scope of non cyanide methods of Hb estimation for testing Hb of blood donors in outdoor blood camps on light emitting diode (LED) based portable battery operated colorimeter.
| Materials and Methods|| |
The study was carried out in the department of pathology of a tertiary public care hospital in a Mumbai city. Fresh blood anticoagulated with ethylene diaminetetraacetic acid -dipotassium (EDTA-K2) was used. Capillary blood by finger prick method was used for hemoglobin estimation in blood donor camps.
Drabkins reagent was obtained from Perfect Biologicals.
The AHD reagent was prepared by dissolving 25 grams of Triton X-100 in one liter of 0.1 mole of NaOH (Reagent Grade) with PH of 11.8.
The Alkaline- Borate reagent was prepared by adding 10 gm of MyTAC (Myristyl trimethyl ammonium chloride) which is a quarternary ammonium compounds in 5 mmoles/L sodium tertraboronate (Borax) and 5 mmoles/L NaOH with a pH of 11.3.
The study design was as follows:
- The absorption curves of the hemochromogens formed in non cyanide methods were recorded with a scanning spectrophotometer (Genesis UV10) between 400-700 nm. Fresh blood (20 μl) was added to the reagent (4 ml) and mixed thoroughly. The absorption curves were recorded within 20 seconds.
- Calibration of hemoglobin: Primary whole blood traceable calibrator (Biorad) was used to calibrate the colorimeter for Hb estimation with HiCN method. Secondary blood standard was prepared by HiCN method. These secondary standards were used to calibrate AHD method and alkaline borate method for Hb estimation. The factor obtained was as follows; 34.3 in HiCN method, 43.3 in AHD method, and 26.7 in alkaline borate method.
- HiCN Method: Add 20 μl blood in 4 ml Drabkins reagent (1:200 dilution). Wait for 10 minutes and the readings are taken at 530 nm by colorimeter.
- AHD Method: Add 20 μl blood in 3 ml AHD reagent (1:151 dilution). Wait for three minutes and the readings are taken at 580 nm by colorimeter.
- Alkaline borate Method: Add 20 μl blood in 4 ml of alkaline borate reagent(1:200 dilution). Wait for three minutes and take the reading at 530 nm by colorimeter.
- Reaction time and kinetics:
The timed kinetics of three methods of Hb estimation was done by running the freshly prepared Hb samples on the semiautoanalyser and the readings were recorded at the interval of three minutes for 60 minutes.
- Linearity: The linearity was checked on Hb concentrations from 3 gm% to 20 gm% by three methods. The whole blood was concentrated by removing the supernatant plasma and the value was adjusted to 20 gm by HiCN method and then serial dilutions were prepared up to 3gm%.
- Stability: The stability was tested by recording the readings of Hb concentrations of five samples after 4 hours, 24 hours, and 48 hours up to four days.
- Precision: The mean, standard deviation (SD) and coefficient of variation (CV) was calculated for all three methods. The Hb sample of known concentration (11 gm%) was prepared by adding 20 μl of blood in four ml of reagent. The sample is mixed and absorbance readings were taken 20 times consecutively of single sample and Hb concentrations were calculated. The mean, SD, and CV are calculated on these 20 readings. This gave the instrument measurement uncertainty.
The same sample was pipetted 20 times and 20 readings were taken and Hb concentrations were calculated. The mean, SD, and CV were calculated on these 20 readings which gave total system measurement uncertainty (pipetting + instrument).
The cost of reagents used in cyanide Drabkin`s method and cyanide free methods, AHD, and alkaline borate method were evaluated.
- Two hundred test blood samples were studied by these three methods which had varying Hb concentrations and also included 50 samples of high leukocyte counts greater than 20,000 per μl. Twenty five (25) blood samples of infants were also tested and compared by these methods.
The statistical analysis used in the study is Pearson`s Correlation Coefficient. Correlations were ranked as excellent (r=0.93-0.99), good (r=0.80-0.92), fair(r=0.59-0.79) and poor (r<0.59).
Semiautoanalyser, Kanad Vidyut (Model Shreerang 01) was used for timed kinetic studies. Genesis UV Spectrophotometer was used for measuring absorption spectra of reagents. The Digital Colorimeter instrument LED based (light emiting diode) of Model HANS 356 (Kanad Vidyut) was used in our study in blood camps which is portable and can be battery operated. The capillary blood drawn by bold finger prick is used i.e., 5 μl of blood added to 1 ml of alkaline borate reagent predispensed in cuvettes.
Hemoglobin was estimated in one hundred blood donors; in 50 blood donors, Hb was done by AHD-triton method and in 50 blood donors by alkaline borate method. These values were later compared with HiCN method.
| Results|| |
- Absorption Spectra: The [Figure 1] shows absorption curve formed by the cyanide reagent with peak absorption at 540 nm. In comparison, AHD reagent had absorption peak at 580 nm whereas alkaline borate had two absorption peaks at 530 nm and 575 nm.
|Figure 1: The absorption curve formed by the cyanide reagent shows peak absorption at 540 nm, whereas AHD reagent shows absorption peak at 580 nm and alkaline borate has absorption peak at 530 nm|
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- Kinetics: The sharp fall of optical density was seen after third reading corresponding to 10 minutes in HiCN method indicating the conversion of Hb to HiCN takes at least 10 minutes. In AHD and alkaline borate method, reaction takes place immediately in less than three minutes as seen from the graph in [Figure 2].
|Figure 2: Kinetics Graph: Three different Hb concentrations of 8 gm%, 9 gm%, and 12 gm% measured by AHD method, HiCN method, and alkaline borate method, respectively. The X axis shows 20 consecutive readings taken with interval of 3 minutes for 60 minutes. The sharp fall of optical density seen after third reading corresponds to 10 minutes in HiCN method. In AHD and alkaline borate, reaction takes place in less than three minutes|
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- Precision: The results of analysis done 20 times on one sample of Hb concentration of 11gm% is given below [Table 1],[Table 2]
The standard deviation and coefficient of variation obtained by these three methods is within normal range of acceptability.
- Linearity: The [Figure 3] shows a linear graph of varying Hb concentrations from 3-20 gm%.
|Figure 3: The absorbances of varying Hb concentrations from 3-20 gm% showed a linear graph for all the three methods|
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- Stability: The stability of hemochromogens formed by the three methods was observed by recording absorption readings at 530 nm and 580 nm for four days consecutively. The cyanide free reagent methods produced stable absorbance readings remaining unchanged till four days which is depicted in [Figure 4].
|Figure 4: The cyanide free reagent methods produced stable absorbance readings remaining unchanged till four days|
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The running cost of reagents for one test in AHD-triton, alkaline borate method and HiCN method is 10 paise, 30 paise, and 100 paise, respectively.
- The correlation between HiCN method and AHD-Triton, HiCN method, and Alkaline borate was done using Pearson`s correlation coefficient. Excellent correlation was observed between the non cyanide methods and HiCN method which is depicted in [Figure 5].
The correlation coefficient of 0.96 and 0.97 is statistically significant at P<0.000 which indicates that the calculated value of r is unlikely to have resulted from random chance.
|Figure 5: Correlation plot for hemoglobin levels (g/dl) in 200 blood samples, determined using HiCN method with AHD-triton and alkaline borate method|
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The possible effect of leukocyte counts on Hb measurement was examined by comparing the correlation and from a series of blood samples with high leukocyte counts (>20,000 WBC/μl). The correlation coefficient was r=0.97 indicating no effect of leukocytes on Hb estimation. Hb results of infants by HiCN method was compared by non cyanide methods and the correlation was excellent, though r value was slightly on lower side (r=0.94). This is explained as the fetal Hb is resistant to alkaline reagents used in non cyanide methods.
| Scope|| |
The cyanide free methods were utilized for screening blood donors at the camp site. The LED digital colorimeter was used and calibrated with the whole blood standard and a factor was derived which was fed into the colorimeter (factor for AHD-575 was 43.3 at wavelength 580nm, factor for alkaline borate method was 26.7 at wavelength 530nm). The Hb concentrations were obtained immediately by the direct readings. These results were later compared with standard HiCN methods in the laboratory. There was excellent correlation (r=0.97).
| Discussion|| |
Hemoglobin estimation is one of the most frequently used procedure in clinical laboratories. In blood banks, Hb estimation is done in all the blood donors to assess their suitability for donating blood. In 1966, International Committee for Standardization in Hematology (ICSH) started as an Expert Panel on Hemoglobinometry. , The HiCN method recommended by ICSH has been used for years because it provides accurate and precise results. ,, However, the hemiglobincyanide method has the following disadvantages: it employs Drabkin`s reagent which contains potassium cyanide and is photosensitive. With the advent of laboratory automation, a new diversity of methods arose. The manual HiCN method was phased out as a routine method and gradually became the reference method with which the current methods should agree. 
In our country, majority of the laboratories, especially in rural areas, still use manual HiCN method for Hb estimation. The main cause for concern is the presence of cyanide in the reagent having toxic effects which are well known. The HiCN reagent has potential risk to laboratory personnel who handle the reagents. The laboratory effluents in the drainage containing cyanide is harmful to aquatic life in low concentrations and is dangerous if allowed to enter and contaminate domestic water tanks, irrigation water supplies, and lakes.
A novel lauryl sulfate hemoglobin colorimetric method has been described and evaluated for use in automated analysers.  The pigment formed is stable for only four hours at room temperature, and hence, the method is not ideal for manual use in busy health facilities.
Newer hemoglobinometers which measure the hemoglobin concentration of lysed blood directly without dilution are now available; however, they are expensive and use disposable cuvettes, both of which make them unsuitable for use in developing countries.
The AHD- triton method like hemiglobincyanide method involves diluting small volumes of blood (which must be accurately pipetted) in a lysing solution (AHD reagent) prior to a colorimetric measurement. The AHD-triton reagent is not toxic or photosensitive and also the AHD reagent is stable for eight months at 4-8* celsius which agrees with findings of Zander et al.
The basic reagent for AHD method is Triton X- 100, which is a non-ionic detergent, causing the lysis of red blood cells.
The alkaline borate method uses quarternary ammonium compounds and is cheaper than AHD. The main advantage of this method is that the reading can be taken at wavelength of 530 nm (which is also the same in Drabkins method). Hence, same hemoglobinometer (colorimeter) designed for the hemiglobincyanide method is also suitable for use with alkaline borate method.
The reaction is very fast in non-cyanide methods, takes less than three minutes whereas conversion time in HiCN method takes 9-10 minutes.
The cost of reagents used in Hb estimation by these three methods was evaluated and cost was 10 paise, 30 paise, and 100 paise for AHD method, alkaline borate method, and HiCN method, respectively.
Some newer colorimeters are fitted with light emitting diode (LED) that emits light at fixed nm- 530 nm, 580 nm, and 630 nm.  The LED based instruments are inherently stable because there is very little drift and the factor derived is stable for a long time and do not require frequent calibration. Our instrument was calibrated and factor was fed in the instrument. We used our LED colorimeter in blood camps and tested the Hb by non cyanide methods for the donors. The Hb readings were then compared with standard hemiglobincyanide method. The correlation was excellent (r=0.97).
However, some hemoglobinometers designed for the hemiglobincyanide method are also suitable for use with non cyanide methods offering flexibility in their utility. The hemoglobinometers equipped with LED`s and health centers that already have such instruments can therefore easily use non cyanide methods for Hb estimation.
| Conclusion|| |
The cyanide free reagents are acceptable and desirable for routine laboratory analysis both from its safety standpoint and its stability in determining hemoglobin. The flexibility and low cost of non cyanide reagents offers practical advantages which clearly warrant its introduction into health facilities and also in providing point of care testing in blood banks.
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Vinaya B Shah
Flat no 38, Building No 2, K K Marg, Govt Colony, Next to Race Course, Haji Ali, Mumbai - 400 034
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]