Optothermal Measurement of Haemoglobin and Glucose in Blood

Prototype instruments are developed for determing haemoglobin and glucose content of blood based on optothermal spectroscopy. The optothermal instrument is described. The results of clinical haemoglobin and glucose tests show good agreement with standard methodes. For haemoglobin a method reproducibility of better than +2%.


INTRODUCTION
Optothermal spectroscopy has been shown to be very useful for determinig the light absorption properties of opaque or powdered substances which are difficult or impossible to analyse using transmission spectroscopy. One such substance is whole blood, where the high absorption and light scattering make transmission spectroscopy inappropriate.   (1) McQueen -8 3 (2) .

THE OPTOTHERMAL INSTRUMENT
A schematic representation of the optothermal instrument f o r haemoglobin analysis is shown in figure 1. The optical system consists of a light source, lens system, optical filter and light chopper. The open cell provides an electrical signal to the electronic system which consists of preamplifiers, lock-in amplifiers, electronic logic and a digital display. light through the collimating and focusing lenses. The color filter transmits light in the visible region. The mirror reflects the light upward toward the cell. Between the mirror and the cell the light is chopped at a frequency of 3 2 H z using a mechanical chopper.
The electrical signals from the cell are amplified in lock-in amplifiers, using the chopper frequency and phase as reference.
Following that the amplified signals are linearized in electronic logic circuits and the final output is transmitted to a digital display calibrated in haemoglobin or glucose concentration units. The instrument can be calibrated absolutely using a black sample, and a sample adjustment of the final amplifiers can be made routinely. The instrument requires about ten seconds to produce the measured value of the haemoglobin content on the digital display. Glucose is measured in forty seconds. Showing the components of the optical and mechanical systems and the optothermal cell.

Hemoglobin
For optothermal determination of haemoqlobin in blood there is no need for reagents or dilution of the blood. The instrument can clearly identify the haemoglobin peak in the absorption spectrum. One drop of blood is all required for measurement of clinically interesting concentration. (3) A comparison test of the optothermal instrument against the standard method (Van Kampen -Zijlstra) was carried out. Fresh samples o f whole blood with EDTA, citrate or heparin as anticoagulant were analysed for hemoglobin content after one to six hours storage.
Sampels were first sent for haemoglobin test using routine methodes with photometric instruments calibrated against haemoglobin standards. Each sample was numbered and had a separate protocol1 with the measurement results.
The haemoglobin content of the samples was then determined using the optothermal instrument in such a way that the previous results from the standard photometric analysis were not known to the laboratory personnel performing the analysis (blind test). In order to study the reproducibility of the optothermal instument results twenty determinations of the haemoglobin content of each sample were made. 616 sample were studied in this way between May and August 1983. The optother-ma1 instument was adjusted using a black sample daily. The results of this study are shown in figure 2.
Using the twenty determinations of each sample the reproducibility of the optothermal instrument results was determined to be better than '2%. Since no dilutions or reagents are required, this is the same as the method reproducibility.
Analysis of the 616 data points shown in figure 2 shows a 100% correlation between the standard cyanmethaemoglobin method and the optothermal method. No statistical difference could be detected between the two methods. There is no detec-  Besides haemoglobin other analyses can be carried out using optothermal spectroscopy. We have tested blood glucose. Comparisons were made to study the accuracy in a clinical study useing hexokinase as reference method. 4 8 0 samples were measured.
One drop of blood was added to reagent on the optothermal cell.
In 40 to 50 seconds the result could be read on the optothermal prototype instrument.

Results:
We found a good correlation between results of the routine method and the optothermal one.