NEW APPROACH TO HAEMOGLOBINOMETRY BASED ON ...

Physiological Measurements NEW APPROACH TO HAEMOGLOBINOMETRY BASED ON REFLECTANCE PHOTOMETRY

V.Koul, N.Debnath, S.Anand and S.K.Guha

Centre for Biomedical Engineering, Indian Institute of Technology and All India Institute of Medical Sciences, New Delhi, INDIA ABSTRACT This paper describes a new approach to haemoglobinometry based on reflectance photometry using pretreated cell strip. A disposable cell strip is designed to absorb standardised volume of blood from a finger prick without any volumetric measuring apparatus. Pretreatment of the cell oxidises haemoglobin to methaemoglobin, thereby eliminating the errors of Tallqvist's method. The behavior of blood cells on chemically treated as well as untreated filter paper is studied by scanning electron microscopy. The principle behind the proposed method is used to fabricate a small, cheap and portable haemoglobinometer using LED's and suitable sensors. This technique provides a simple and reliable means of ascertaining haemoglobin content in blood for routine as well as field use. INTRODUCTION Haemoglobin percentage in blood is a diagnostic parameter for knowing different kind of anaemia. The conventional clinical methods [l,2] are cumbersome for field use. This is because these techniques involve diluting fluids, pipettes and use of standards. Further ; fairly long time is required for a chemical reaction to be completed. The optical wedge method [33 and Tallqvist's filter paper technique L43 are of little clinical use as the colour matching gives erratic results. In this paper, experimental investigations leading to the development of haemoglobinometer based on reflectance photometry using chemically pretreated filter paper are presented. The scanning electron microscopy is used to study the behavior of blood cells. The choice of chemical used is confirmed by spectroscopic studies. In addition, colorimetric estimation using 640 nm filter is also presented. EXPERIMENTAL INVESTIGATIONS Blood samples Blood samples for this study were routinely obtained from the clinical chemistry laboratory. Specimens were collected by venipuncture into EDTA, containing

evacuated blood collection tubes and kept at 4 degree cent. before and during transport to the lab. within 24 hrs. of collection. Further, capillary blood samples were obtained by finger prick with a spring loaded sterile lancet, Colorimetric estimation To 0.02 ml of blood was added 4 ml of N/10 HC1 and 0.1% NaN02 separately. Brownish red colour so obtained showed gradual increase in optical density with an increase in Hb content as shown in Table I. TABLE I Optical Density

Hb content g/dl 6.25 8,00 9-50 12,50 14.00

0.1$ NaN02 0.090 0,110 0.140 0.160 0.185

N/10 HC1 0.0?5 0.100 0.135 0.1?0 0.190

In practice HC1 cannot be used as it destroys the fibres of the filter paper. Spectroscopic studies Spectrophotometric studies are conduted on (a) thin film of blood, (b) oxygenated blood and (c) blood treated with NaN02, in the spectral range from 400-800 nm. The absorption peak of (a) and (b) is found at 548 nm which is characteristic of oxy-Hb. The absorption peak of sample (c) was found at 640 nm which is due to the conversion of haemoglobin to methaemoglobin. Scanning electron microscopy It has been previously reported[5H that the passage of blood through the pores of filter paper lead to RBC rupture with release of haemoglobin which subsequently gets oxidised to oxyhaemoglobin. In the present investigations such a phenomenon is not observed. Figure 1 shows the scanning electron micrograph which indicates that there is no rupture of the cells : only some RBC's are seen in disfigured form. When blood is treated with 2% NaN02, RBC's are ruptured and Hb is released which gets oxidised to met-Hb. The

IEEE ENGINEERING IN MEDICINE & BIOLOGY SOCIETY 10TH ANNUAL INTERNATIONAL CONFERENCE—1847

RBC rupture is clearly observed in the SEM shown in Fig.2.

of the blood. A block diagram of the haemoglobinometer is shown in Fig.3CONCLUSIONS Experimental investigations carried out have resulted in the development of a simple and reliable haemoglobinometer for routine as well as field use. This haemoglobinometer is based on reflectance photometry using chemically treated filter paper. Pretreatment converts blood to a stable form i,e. ' methaemoglobin, thereby eliminating the errors of Tallqvist's method. The accuracy and reproducibility are further improved by measureing the reflectance of methaemoglobin.

Fig.l Scanning electron micrograph of blood passed through untreated filter paper ; X 2500

ACKNOWLEDGEMENT Authors are thankful to Department of Electronics for financial assistance. REFERENCES

Fig.2 Scanning electron micrograph of blood treated with 2% NaN02 ; X 5000 Cell strip The cell of 6 mm diameter is made of Whatman filter paper No.4, which has been selected to obtain uniformity in the spread of blood. It is soaked into 2f0 NaN02 solution for 2 minutes and allowed to dry at room temperature. Development of haemoglobinometer On the basis of above investigations, a haemoglobinometer has been developed. It consists of four red coloured LED's which are arranged symmetrically and placed 90 degree apart. The LED are slightly inclined so as to concentrate the light beam at the centre of the hole where cell strip is placed. A single LDR detector of diameter 8 mm having resistance of 20 K ohms is placed at the centre of four LED's and is pointing towards the cell. The arrangement is made in such a way that no direct light falls on it from the LED's. As mentioned earlier, the optical density, hence colour of the cell changes with a change in the Hb content of the blood. The detector resistance which depends on the colour of the cell is therefore monitored and calibrated directly to indicate the Hb content

1.

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D.L.Drabkin and J.M.Austin, " Spectrophotometric studies :Spectrophotometric constants for common haemoglobin derivatives in human, dog and rabbit blood",J.Biol.Chem.,98,pp719-721, 1932.

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P.0nesti,N.S.Stenhouse and H. J.Woodlife," Comparison of three simple haemoglobinometers", Med.J.Aust.,l,pp683686,1969.

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P.C.Elwood and A.Jacobs," Haemoglobin estimation: A comparison of different techniques",Brit.Med.J.,1,pp20-2^,1966.

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S.K.Guha and S.Mahajan," Infrared reflectance from whole bllod soaked filter paper and implications in haemoglobinometry",Med. and Biol. Eng, and Comput.,2l,pp97-l00,1983.

RESISTANCE VOLTAGE CONVERTER ALEDS

Fig.3

Block diagram of the haemoglobinometer

1848—IEEE ENGINEERING IN MEDICINE & BIOLOGY SOCIETY 10TH ANNUAL INTERNATIONAL CONFERENCE