Electronic Method for Quantitation of Bleeding Time

Electronic Method for Quantitation of Bleeding Time E. J. WALTER BOWIE, B.M., ROBERT

B.CH., CHARLES A. OWEN, JR.,

M.D.,

J. HANSEN, B.S.E.E., AND JAMES ISAACSON, B.S.E.E.

Mayo Clinic and Mayo Foundation,

Rochester, Minnesota 55901

ABSTRACT Bowie, E. J. Walter, Owen, Charles A., Jr., Hansen, Robert J., and Isaacson, James: Electronic method for quantitation of bleeding time. Am. J. Clin. Pathol. 58: 255-259, 1972. A method for determining blood loss from standardized skin punctures is described. T h e volume of blood lost is measured by electrical conductance. The device allows the pattern of bleeding to be recorded continuously and the volume of blood lost to be directly recorded. The method is reproducible and has been standardized on a group of normal subjects. has long been considered an important test of the hemostatic mechanism. Reproducible results are possible with standardized skin punctures, 1 but, until recently, only the duration of bleeding could be measured. We recently described 2 a method for the continuous recording of the volume of exuding blood. The original method used a spectrophotometer to quantitate the hemoglobin in the blood lost. We have now simplified the technic and measure blood loss by electrical conductance.

BLEEDING TIME

Methods and Calculations The patient reclines on a couch so that his forearm is at approximately the level of his heart. A drop of blood, obtained from puncturing the pinna, is diluted 1:100 with distilled water in a hemocytometer and used to calibrate the machine. The Received August 4, 1971; received revised manuscript September 22, 1971; accepted for publication September 27, 1971. Supported in part by Research Grants HE-12512 and HE-13145 from the National Institutes of Health, U. S. Public Health Service.

calibration of the machine is discussed fully at the end of this section. The skin of the forearm is cleansed with 70% ethanol and permitted to dry. A blood pressure cuff on the upper arm is inflated to 40 mm. Hg and held at this pressure throughout the test. A puncture site midway between the elbow and wrist is chosen and a transverse incision, 1 to 2 mm. deep, is made with the Mayo lancet. 1 A transparent Lucite cube is immediately placed over the incision (Fig. 1) and taped lightly to the skin. Sterile distilled water at a temperature of 25 C. is sucked through the flow cube by a proportioning pump. Bubbles of air are introduced into the hemolyzed diluted blood in order to prevent "tailing." After the solution passes through a mixing coil, the bubbles of air are removed and the diluted blood passes through a conductance transducer. T h e conductivity is recorded continuously on a strip chart recorder. T h e area under the curve is integrated electronically and the volume of blood is indicated on a meter. Two Millipore filters (0.45 /*) are used in the system to sterilize the entering water

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FIG. 1. Lucite cube in position on forearm before tape is applied to hold it in position.

and air, respectively. After each use, the tubing and flow cube beyond the Millipore filters are sterilized as a single unit. The diagram of the flow system is shown in Figure 2. A block diagram of the electronic portion of the device is given in Figure 3. The 1,000-Hz sine-wave oscillator provides a constant voltage source for the flow cell. (An alternating source is used to avoid polarization effects on the electrodes of the flow cell.) The 1,000-Hz signal is applied to a low-leakage current (field effect transistor) operational amplifier through a sample of the patient's diluted blood in the flow cell. The output of the amplifier is rectified and then applied to a strip chart recorder and an integrator. The output voltage of the amplifier is inversely proportional to the effective resistance of the mixture of blood and distilled water between the two electrodes of the flow cell. Since the inverse of resistance is conductance, the amplifier output voltage is proportional to the number of ions in solution or the concentration of blood in the mixture of blood and distilled water. If the flow rate of the distilled water is kept constant, the amplifier output voltage will be directly proportional to the concentration of whole blood in the mixture. T o obtain the volume of blood, this voltage is integrated over the bleeding time interval.

The device is calibrated with a sample of the patient's own blood at a specified dilution by making use of the equation: t

V = R / C dt 0

in which V = volume of whole blood passing through flow cell; R = rate of flow of distilled water (must be high enough to provide a dilute solution of blood); C = concentration of blood in the solution; and t = time in minutes. If the concentration is held constant, the above equation reduces to: V = RCt This condition is met if the known dilution of the patient's blood sample is injected into the flow cell and allowed to remain there for the calibration time. Using the value of flow rate that would occur in an actual run, such as R = 0.1 ml./ min., and a calibration time of 5 min., the gain of the amplifier is adjusted so that the pen of the chart recorder is at half scale. Then, the integrator is allowed to integrate this value for 5 min. to check against the calculated value which, using the parameters given, would be: for R = 0.1. ml./min., C = 1/100 (100-fold dilution of the blood), and t = 5 min., V = (0.1 ml./ min.) (1/100) (5 min.) = 0.005 ml. or 5 /J.

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September 1972

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QUANTITATION OF BLEEDING TIME

Mixing coil

"BflflflflffD"

Proportioning pump Water orange-green 0.015 O.iml/min Air orange-white 0.025"

0.23ml/min orange-white 0.025" 0.23ml/min

orange-blue 0.01' 0.05ml/min

e e e

Flow cube T-__-, Millipore \ A 6 ! filters

|A9

-C3bypass

[Inlet for standard >

|

Debubbler C5 Electrode,

Conductance transducer

e Waste

FIG. 2. Diagram of complete apparatus.

Mixture of blood ond distilled water

Strip chart recorder (flow contour) Reset

Calibrate Electrodes 1000 Hz sine wave oscillator

Ideal rectifier

Integrator (operational amplifierL

Volume indicator (voltmeter)

Conductance transducer Calibrate FIG. 3. Block diagram of electronic portion of apparatus.

The integrator is adjusted by a cut-andtry process to give the value of 5 /*1. on its chosen calibration scale. This adjustment need be made only once, since any change in blood conductivity is accounted for when the calibration control is adjusted for half-scale output on the patient's own blood sample diluted 100-fold.

Results with New Technic Bleeding times and volumes of blood lost were measured on 26 normal adults (Fig. 4). Mean values were: bleeding time, 6.0 min.; blood loss, 5.3 j j . The reproducibility of the test was assessed by performing two or three consecu-

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BLOOD LOSS

10 8

T^ s