Some Results of the Development and Study of Instruments for Investigating the Process of Coagulation and Blood Analysis

I. N. Kraev

(Presented by the scientific seminar of the Faculty of Physics and Technology)

Biological analysis of blood and the study of the coagulating properties of blood have made it possible to clarify questions of the physiology and pathology of blood—questions which had seemed insoluble—blood consisting of plasma and formed elements of several types. Both biological analysis of blood, which consists in determining the number of blood cells of a given type in a sample, and knowledge of the mechanism of blood coagulation make it possible not only to elucidate the nature of a number of pathological states of the human organism, but also to develop rational and controllable methods of treatment and prophylaxis for a number of diseases.

Numerous questions connected with the treatment of various diseases and with the preservation of blood require a further expansion of research into the process of blood coagulation and blood analysis. Below are given the description and the results of the investigation of an instrument for determining the process of blood coagulation and of an electronic blood analyzer.

Electronic instrument for determining the process of blood coagulation

Although the process of blood coagulation consists of several phases closely interconnected with one another, as a number of investigators have shown, the principal phases of coagulation are three successive phases: 1) formation of active thromboplastin; 2) formation of thrombin; 3) formation of fibrin.

The first preparatory phase of coagulation, during which active blood and tissue thromboplastins are formed, lasts from 3 to 8 min. Then, under the influence of active thrombin (phase II), the process of formation of a fibrin clot begins (phase III), which is loose at the stages and subsequently gradually becomes denser.

With the completion of clot formation its contraction begins; this completes the process of blood coagulation. After the formation of the clot is completed, the latter may, to a greater or lesser degree, undergo lysis (liquefaction).

In various pathological states the time of clot formation, its density, and the time of liquefaction (dissolution) will differ, which assists in establishing a diagnosis; moreover, in establishing a diagnosis the results of the investigation of blood coagulation play a decisive role.

There exist test-tube methods for studying the coagulating properties of blood [1, 2] and mechanical methods using thromboelastographs [3, 4] (the designs of Hartert, Rogunov, and others). Mechanical thromboelastographs are based on recording the blood and the blood clot by rotating a cylinder (Hartert) or by the reciprocating motion of a plane in blood (Rogunov and coworkers). Blood coagulability is recorded on photographic film by a light beam.

All instruments currently in existence are quite complex in their design, inconvenient in everyday practice because of their dimensions, and cannot be transported to the patient’s bedside.

The author has carried out studies of a prototype instrument based on the principle of photometry. In the instrument, whose block diagram is shown in Fig. 1, indication of changes in the coagulating properties of blood is based on using the dependence of the degree of absorption of the light flux on the phase of blood coagulation.

The light flux, passing through a blood sample (volume \(V = 0.25\) ml) placed in a transparent cuvette, falls on a photoresistor connected into one arm of a measuring bridge.

From the magnitude of the bridge imbalance over time, one can judge the process of blood coagulation.

The use in experiments of cylindrical cuvettes with a flat bottom did not provide stable reproducibility of the readings, because the formation of the clot (its orientation) relative to the walls of the cuvette was not the same from one experiment to another.

The use of conical-type cuvettes, when clot formation occurs in the center of the cuvette, made it possible to obtain stable results.

The results of the experiment are shown in Fig. 2. The first phase recorded by the instrument (Fig. 2) includes the initial stage of the blood coagulation process—the formation of active tissue and blood thromboplastin—and is characterized by darkening of venous blood (curves 1, 2, 3, 4). Before the beginning of formation of the fibrin clot, the darkening stops for a short time (5–20 sec). During this period, thrombin is formed from prothrombin.

Fig. 1. Block diagram of an instrument for determining the process of blood coagulation.

The second and third phases are characterized by a sharp darkening of the blood, which lasts 15–45 sec and stops for 5–20 sec. During this period fibrin is formed from fibrinogen.

With the formation of the fibrin clot, the instrument records changes in the blood that cause its darkening. This process lasts 25–45 min until an equilibrium state is reached (5–10 min).

The fourth phase, recorded by the instrument, is characterized by lightening of the blood when retraction (contraction) of the fibrin clot begins. Separation of the clot from the walls of the cuvette occurs 30–55 min after the start of the experiment. Over 24 hours the clot contracts by up to 40%. Contraction of the clot is slowed or absent in

thromboembolism, hemophilic conditions, fibrinogenopenias, polycythemias, and others.

Numerous experiments with samples of the same blood, but in different quantities, showed that the character of the dependence of the instrument readings on time does not change. The more blood is used for the study, the longer the normal time. With a decrease in temperature, the time of formation of the coagulation phases is prolonged. Capillary blood (curve 5) coagulates more rapidly owing to the admixture of tissue thrombokinase.

Fig. 2. Experimental dependence of the instrument readings on time. 1, 2, 3, 4, 6—curves for venous blood; 5—curve for capillary blood.

An increase in blood coagulability is observed in painful irritations, blood loss, and in the postoperative and postpartum periods; a slowing is observed with increased anticoagulant activity of the blood and a decrease in the concentration of procoagulants, especially those that participate in the formation of thromboplastin.

In patient N, formation of the blood-coagulation phases was not observed (curve 6).

Since the blood-coagulation time characterizes the coagulability of the blood as a whole, it does not always reflect the true state of the blood-coagulating system. Test-tube methods for studying the blood-coagulation time do not reveal insignificant shifts in the functional state of the blood-coagulating system in various diseases. This is explained by the fact that these methods register only the time required for the formation of such an amount of thrombin as is capable of causing the conversion of fibrinogen into fibrin.

Comparison of the results of determining the blood-coagulation time by an instrument based on the principle of photometry and by the methods of Morawitz and Lee-White showed their complete agreement.

The method proposed by the author makes it possible, more sensitively than the others, to detect disturbances of blood coagulability in various states of the organism.

The instrument described is simple and has small dimensions and weight. Use of the instrument described makes it possible to carry out mass studies of the process of blood coagulation with high accuracy. The instrument is provided with a thermostat for maintaining specified temperature conditions.

Electronic Blood Analyzer

The analyzer, whose block diagram is shown in Fig. 3, is intended for the rapid determination of the number of red blood cells (erythrocytes) in the blood.

The method for determining the relative volume concentration of erythrocytes in the blood is based on measuring the conductivity of a blood sample in a Wien bridge.

As can be seen from the block diagram (Fig. 3), a 10 kHz generator, through an emitter follower, feeds the bridge circuit. Two arms of the bridge are fixed resistances. For automatic compensation of changes in ambient temperature, the third arm of the bridge is a thermistor.

Fig. 3. Block diagram of the analyzer.

The unknown resistance, consisting of the blood sample in a chamber of a definite volume, constitutes the fourth arm of the bridge. Balance of the bridge corresponds to a zero reading (absence of erythrocytes). The unbalance signal is fed to the input of the amplifier and, through a detector circuit, reaches the recording instrument. A microammeter M-24, graduated directly in units of erythrocyte concentration, was used as the recording instrument.

Studies carried out on the dependence of the readings of the recording instrument on the generator frequency showed that the optimum frequency is \(f = 10\) kHz; below this frequency polarization affects the readings, and above it capacitive conductivity does.

The data obtained agree with Wagner’s data [5], obtained for a dielectric of heterogeneous structure on the basis of Maxwell’s theory [6].

The sensor of the instrument is a pipette made of organic glass, with platinum electrodes mounted inside it.

A blood sample of volume \(V = 20\ \text{mm}^3\) is placed in the space between the electrodes. The use of organic glass makes it possible to monitor the filling of the space between the electrodes by the sample.

The instrument was calibrated according to the data of the centrifuge method. Blood samples for calibration were obtained by mixing donor blood plasma with erythrocytes separated by means of a centrifuge, in various proportions.

For each proportion, a blood sample was taken into the sensor and a reading was made. For each sample, up to 10 instrument readings were taken; in this case, as the experiments showed, the scatter of the instrument readings was less than 1%.

This method made it possible to monitor changes in the instrument readings as the erythrocyte concentration was increased from 0 to 80%.

The accuracy of this calibration was checked on a large number of whole-blood samples, the relative volume concentration of erythrocytes in which was determined by centrifugation for 30 min at a speed of 3000 revolutions per minute.

The calibration curve of the blood analyzer is presented in Fig. 4. As a result of the studies carried out, it was established that the electrical conductivity of blood is an accurate indicator of the relative volume concentration of erythrocytes.

Fig. 4. Dependence of the readings of the recording instrument on the erythrocyte concentration.

The instrument described (Fig. 5) satisfies the need for precise transfer of the relative erythrocyte-concentration meter, and makes it possible to carry out the necessary measurements quickly.

The laboratory tests of the electronic blood analyzer that were conducted made it possible to determine its principal technical characteristics and demonstrated the possibility of using it for purposes of analyzing the composition of blood.

Use of the indicated analyzer makes it possible:

1. To reduce the analysis time to 1 min.
2. To increase the accuracy of measurement by eliminating operator errors.

Fig. 5. Photograph of the analyzer. 1—sensor, 2—secondary instrument, 3—scarifier.

1. To carry out mass investigations of blood composition.

2. The instrument is simple, has small dimensions and weight, which increases the efficiency of working with it, reduces the cost of the design, and facilitates its adjustment.

REFERENCES

1. V. P. Baluda, V. N. Malyarovskii, I. A. Oivin. Laboratory Methods for Investigating the Blood Coagulation System. State Publishing House of Medical Literature. Moscow, 1962.

2. I. Todorov. Clinical Laboratory Investigations in Pediatrics. State Publishing House “Medicine and Physical Culture,” Sofia, 1960.

3. A. Z. Serikova. Experience in the use of thromboelastography in clinical practice. Terapev. arkhiv, issue 11, 1959.

4. G. A. Rogunov. The domestic thromboelastograph and its application. Klinicheskaya meditsina, vol. 39, No. 3, 77–81, 1961.

5. K. Wagner, Arch. Elektrotechnik, 2, 1914.

6. J. Maxwell, Treatise on Electrocity und Magnetism, Oxford Uneversity Press, Cambridge Eng., 1873.