An Electronic Blood-Count Meter P. L. F R O M M E R

S T U D E N T M E M B E R A I E E

A direct-reading blood-count meter is de­scribed which yields results independent of particle size and coloration, rate of sampling, and quantity sampled. Errors from zeroing, changing sensitivity, and changing illumination are eliminated by a grid-current ratio amplifier.

TH E R E is a real need in the clinical laboratory for an automatic, accurate, and inexpensive instrument capable of determining the number of red and white

cells per unit volume of blood. 1 Before undertaking the discussion of the instrument that has been developed for this purpose, a clear understanding of certain physical properties of blood is necessary.

In human blood, the red cells, which are approximately disk shaped, average 0.0003 inch in diameter and 0.0001 inch in maximum thickness and have a normal count (number of particles per cubic millimeter) of 5 million; the white cells are on the order of twice as large but occur in far lower concentrations, around 7,000 per cubic millimeter. In clinical problems, the dimensions of the cells vary con­siderably—in a given sample there may be red cells with diameters ranging between 0.0001 and 0.0005 inch; 2 the coloration, specific gravity, and refractive index are also variables. 3 Red-cell counts may vary between 1 and 9 million, while white-cell counts may range from 1,000 to 200,000.

Because of the high initial concentrations, diluting the blood before making counts is standard practice. Red-cell counts neglect the error introduced by the presence of the comparatively few white cells; for white-cell counts, a diluent is used that destroys the red cells without affecting the white ones.

F i g . 1. B l o c k d i a g r a m o f t h e e l e c t r o n i c b l o o d - c o u n t m e t e r

P R I O R A R T

THE classical manual technique of counting blood cells consists of placing a dilute sample on a microscope slide

and counting the number of cells visible on certain ruled areas on the slide. I t is slow, fatiguing, and liable to human error. 4

T h e earliest automatic methods, depending upon the tur­bidity of the s a m p l e , 5 - 7 have been improved so that com­pensation for cell size is partially achieved over the small range in which the majority of normal red cells occur. 2

Counting particles individually as they appear in a limited microscope field has been tried frequently. 8 , 9 The elec­tronic scanning of a microscope field and counting the num­ber of pulses is the most recent development, 1 0 but the ex­pense of the necessary closed-loop television system is great. One embodiment of this scanning technique is the only auto­matic means that does not require uniform particle size for accurate results. In order to count large particles only once, this unit scans two adjacent lines of the raster simul­taneously and registers a count only when there is a signal from the lower beam and none from the upper one. 1 1

T H E N E W T E C H N I Q U E

ALL the previous ar t has dealt with efforts to determine the total number of particles in a given volume of

sample by inspecting a known quanti ty of solution and either counting individually the number of particles in it or by obtaining an over-all count as by measuring the light transmission. The principle of this instrument is entirely different—it is to determine the average volume of solution per single particle, which is the reciprocal of concentra­tion. 1 2

This is determined in the following manner : A very small inspection zone is observed—one so small that, as the sample passes through it, most of the time there is no particle in the zone. However, the instrument determines the fraction of time during which there is a particle in the zone. This fraction is a direct measure of the average volume per par­ticle according to the expression :

a v e r a g e v o l u m e v o l u m e o f i n s p e c t i o n z o n e X t o t a l t i m e o f i n s p e c t i o n

p a r t i c l e t o t a l t i m e p a r t i c l e s a r e p r e s e n t

Notice that the result is independent of the quanti ty sampled, the length of time of sampling, and the rate at which the sample is passed through the inspection zone. Revised text of the Best Student Prize Paper, which won the award for papers presented during the period August 1, 1953, to July 31, 1954.

P. L. Frommer, formerly at the University of Cincinnati, is now a student at Harvard Medical School, Boston, Mass.

This article was written on the basis of work done for a thesis in partial fulfillment of the requirements for the degree of electrical engineer at the University of Cincinnati, June 1954. The writer wishes to thank R. H. Engelmann and J. C. Frommer for their valuable ad­vice in the design of the blood-count meter and the Electric Eye Equipment Company, Danville, 111., for furnishing the parts for this project.

388 Frommer—Blood-Count Meter ELECTRICAL ENGINEERING

T H E I N S T R U M E N T

As shown in Fig. 1, the instrument that was constructed consists of a microscope, a photomultiplier tube view­

ing the microscope field, an amplifier, a clipper, and a d'Arsonval-type meter on which the concentration is read.

The diluted sample is placed upon a special microscope slide. A channel down the length of the slide permits the cover glass to enclose a constant known depth of sample. Then the inspected volume is the depth of this sample times the area viewed by the photomultiplier. Movement of the sample through the inspection zone is accomplished by manually moving the entire slide with respect to the micro­scope objective. Movement need not be along any par­ticular path, nor does it have to occur at constant speed.

The microscope is dark-field i l luminated; the photo­multiplier tube viewing it generates current pulses during the presence of particles in the inspection zone. A grid-current ratio amplifier is used to discriminate these pulses above the "dark current" due to stray illumination. T h e output of this amplifier is fed to a clipper, which generates constant current during signal from the photomultiplier, and zero current otherwise. A d-c microammeter deter­mines the average value of this pulsating current.

Note that this average current is equal to the amplitude of the pulses multiplied by the fraction of time that they are present, which is the same as the fraction of time that par­ticles are in the inspection zone. Since it has been shown that this is a measure of volume per particle, it is possible to calibrate the microammeter in units of concentration.

Compensation must be made in the meter calibration for the random distribution of particles, which occasionally re­sults in the simultaneous presence of more than one par­ticle within the inspection zone. This is a simple problem in the mathematics of probability. Also, to assure ac­curacy, the width of the inspection zone is made great in comparison to particle width so that the signal rise time, which is dependent upon particle size, is small relative to the total time of the signal.

O P T I C S

THE optics of the instrument were built around a standard microscope, as shown in Fig. 2. In order to obtain

sharp contrast between the signal from a particle and the background, dark-field illumination is employed. This means that the field seen through the microscope appears black and any particle in the field shows up as a point of light. This is done very simply on any microscope by plac­ing a mask in front of the center of the condenser lens, thus permitting only those beams of light to illuminate the slide that will not pass directly into the objective lens. How­ever, any particle on the slide will cause dispersion of the light falling upon it and some of this dispersed light will fall upon the objective, which directs it to the photomultiplier.

The field inspected by the photomultiplier is limited by a mask in the plane to which the objective normally focuses the slide. This permits the microscope to be brought to focus by the regular eyepiece, which is then removed and replaced by the phototube chamber and its mask.

To avoid 120-cycle noise, the slide is illuminated by a lamp fed by half-wave rectified resistance-capacitance fil-

F I E L D FOCUSED ON M A S K

DARK FIELD S T O P

LIGHT F R O M L A M P

F i g . 2 . O p t i c a l s y s t e m f o r t h e m e t e r

tered direct current. Provision must be made to shield the microscope from intense stray illumination such as direct sunlight.

T H E A M P L I F I E R

THE very small amount of light dispersed by a single blood cell requires a highly sensitive detector, so a

photomultiplier is a natural choice. But the sensitivity of a photomultiplier varies greatly both with time and with applied voltage. For instance, 1-per-cent change in ap­plied voltage may cause a 20-per-cent change in output current. There are also such variations in the optical sys­tem as lamp brightness and microscope adjustments. How­ever, experience has shown that a signal to dark-current ratio of 2:1 and a signal to noise ratio of 5:1 is approxi­mately maintained. Thus the best method of determining a signal is by comparing it to the dark current, not to an abso­lute value. The grid-current ratio amplifier accomplishes this and thereby completely eliminates the sensitivity ad­justments and dark-current nulling commonly encountered in sensitive phototube circuits. 1 3 This is accomplished ac­cording to the following theory :

Because of the Maxwell distribution of velocity of elec­trons emitted by the cathode, if grid current flows in a tube the grid will assume a voltage 1 4

Vc=a+b logIc

where a and b are constants. Then if Icl is the grid current due to stray illumination

MAY 1955 Frommer—Blood-Count Meter 389

5V Ve> V|Q> 6 .3V v^> V|?>

F i g . 3. S c h e m a t i c d i a g r a m o f t h e p h o t o m u l t i p l i e r , a m p l i f i e r ,

c l i p p e r , a n d m e t e r f o r t h e e l e c t r o n i c b l o o d - c o u n t m e t e r

and Ic2 is the grid current when a signal is present, the peak-to-peak alternating voltage on the grid will be

V c 2 - V c l T ( a + b l o g Ic2)-(a+b l o g Icl)

= b(log 7 c 2 - l o g Icl)

= b l o g y 1

ICl

Then, assuming linear operation, the peak-to-peak al­ternating voltage on the plate will be

E b i - E b l = c l o g y ^ -

where c is a constant. Note that the output of this grid-current ratio amplifier

is proportional to the ratio of signal to dark current, not to the absolute value of the signal amplitude. This has been verified experimentally by varying the voltage to the photo-multiplier from —670 to —1,200 volts, which varies the sensitivity by a factor of 100, with the output of the grid-current ratio amplifier remaining constant within 3 per cent. In other applications, this grid-current principle has been found useful for grid currents in the range from 10~~3 to 10~~9 ampere.

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As shown in the schematic diagram of Fig. 3, the photo-cathode of the photomultiplier is given a high negative voltage, such as —900 volts. Nine dividing resistors to ground provide proper voltages for the first eight dynodes, the eighth dynode in this case assuming a potential of —100 volts. The plate is brought to + 1 5 0 volts ; the ninth dynode is connected directly to the grid of the ratio amplifier. Such a connection is necessary because the ratio amplifier re­quires current flowing into its grid. The floating ninth dynode with respect to the rest of the photomultiplier is done so that the rest of the photomultiplier and the high voltage may be definitely referenced to ground. With the circuit used, the current to the grid of the ratio amplifier is the difference between the plate current of the photo­multiplier and the current between the last two dynodes.

In addition to the stable operating point thus described, there is a stable condition in which the ninth dynode assumes a potential slightly more negative than the eighth dynode. Under these conditions, grid current to the ratio amplifier is completely cut off due to its highly negative grid and the plate current of the photomultiplier is cut off for lack of secondary emission. Between these two points of stable equilibrium, there is a point of unstable equilibrium at which the second­ary emission of the ninth dynode exactly equals the current reaching it from the eighth dynode. T h e undesirable stable condition is prevented by connecting the ninth dynode to the cathode of a diode, the plate of which is maintained around — 10 volts.

The operation of the grid-current ratio amplifier has al­ready been discussed. T h e signal from its load resistor is capacitor coupled to the first triode of the clipper stage where a diode provides d-c restoration. This grid is given a positive potential with respect to ground so that cathode-follower action in this triode develops sufficient voltage across the common cathode resistor so that the grounded grid triode is kept cut off in the absence of signal. The signals to the first triode, which are all negative pulses, are of suffi­cient amplitude to drive that tube to cutoff, permitting cur­rent pulses of constant amplitude in the second triode. T h e capacitor from B+ to the plate of this triode causes the load resistor and the series microammeter to be affected only by the average plate current, that is, the average value of the current pulses. Thus the microammeter reads the fraction of time that a signal is present according to the expression

^ave ^ Λη t o t a l t i m e s i g n a l s a r e p r e s e n t

c t o t a l t i m e

J i g . 4 . P o w e r s u p p l y f o r t h e e l e c t r o n i c b l o o d - c o u n t m e t e r

7 M A X is made, say, five times the current required for full-scale deflection on the microammeter, so that if particles are present one fifth of the time, full-scale deflection will result. If 7 M A X were made many times the current required for full-scale deflection, scale changes could be accom­plished by switching to appropriate meter shunts. In any case, going back to the optical system, a different objective lens or a different mask could be used to change the in­spected volume and thereby change the scale.

I t should be noted that since the load resistor of the last triode acts only upon the average current, the plate voltage will be that corresponding to the average plate current. Since the amplitude of the current pulses is slightly depend-

390 Frommer—Blood-Count Meter ELECTRICAL ENGINEERING

ent upon the plate voltage, compensation must be made in the meter scale for the slight decrease in current pulse amplitude when current pulses occur for a greater fraction of time.

In practice, the first triode of the clipper must be given sufficient positive bias that the amplified Johnson noise from the photomultiplier will not permit conduction in the second triode. Also, the amplitude of the signal coupled from the ratio amplifier must be adjusted so that the proper portion of the signal pulse is clipped out by the clipper stage.

T H E P O W E R S U P P L Y

THE schematic diagram of Fig. 4 shows a conventional full-wave condenser-input filtered-5+ supply capable

of delivering 400 volts with a 100-milliampere current drain. This is far more power than is necessary, for only 20 milli-amperes is used for the voltage-regulator tube, regulated 150 volts, and 25 milliamperes is used for the regulated — 650- to — 1,350-volt 1.35-milliampere supply. This high voltage is the triple resistor-capacitor filtered half-wave rectified output of a tickler coil r-f high-voltage oscillator. Regulation is achieved by controlling the B+ to the oscil­lator through a series tube, the grid of which is controlled by the amplified voltage appearing between ground and the reference tap on the voltage divider going between the regu­lated + 150 volts and the negative output. T h e output may be varied by adjusting the variable resistor in the upper arm of this divider. Regulation is maintained within 0.2 per cent from zero to full load (1-megohm load) at any out­put voltage.

An r-f oscillator high-voltage supply was chosen because of the low-ripple output possible with low-capacitance fil­tering and the resulting safety to life, as well as for its adapt­ability to regulation. However, with the amplifier that has been developed, regulation of the high voltage is proba­bly unnecessary.

C O N C L U S I O N S

THE instrument that has been constructed appears to meet the requirements for a useful particle-concentra­

tion meter.

1. Simplicity of operation requires the technician to move a slide under the microscope objective and to read the blood count directly from the deflection of a meter needle—there are no zeroes to set, nulls to balance, or size compensation factors to apply. White-cell counts may be taken by using a smaller dilution and reading the meter on a different scale.

2. Low cost is inherent in a system that requires only a 5-tube amplifier and a 6-tube power supply, all using only standard inexpensive components. There are no optical or mechanical parts not already found on all standard micro­scopes, except the slide, the phototube housing, and the light mask. The whole instrument may be considered a micro­scope accessory.

3. The accuracy of the instrument has not been tested, but there appear to be no inherently inaccurate parts in the electronics, while the optics in combination with the elec­tronics have the advantage of yielding results substantially

independent of particle size and coloration, and rate of sampling.

4. The versatility of the instrument makes it useful not only for both red- and white-cell counts, but for micro­scopic-particle counts in general—for dust and plankton counts, and for impurity and colloid counts in chemical solutions.

R E F E R E N C E S

1. The Present State of Photoelectric Erythrocyte Count, L. L. Bloom. American Journal of Clinical Pathology, vol. 23, 1953, Baltimore, Md., pp. 798-800.

2. Appraisal of Instrument for Counting Erythrocytes by Scatter Photometry, F. S. Brackett, C. F. T. Mattern, B. J . Olson. Ibid., pp. 731-45.

3. Photoelectric Determination of Erythrocyte Count, L. L. Bloom. Ibid., vol. 15, 1945, pp. 85-93.

4. Error of Estimate of the Blood Cell Count as Made in the Hemocytometer, J . Berkson, T. B. Magath, M. H u m . American Journal of Physiology, Washington, D. C , vol. 128, 1940, p. 309.

5. The Relation of Red Cell Diameter and Number to the Light Transmission of Sus­pensions, E. Ponder. American Journal of Clinical Pathology, vol. 3, 1935, pp. 99-106.

6. The Use of Photoelectric^Turbidometry in the Determination of Red Cell Counts, Hematocrit and Hemoglobin, J . H. Wittlock. Blood, New York, Ν. Y., vol. 2, 1947, p. 463.

7. A Method for Computing the Red Cell Count on the Photoelectric Colorimeter, G. M. Parker, H. E. Spicer, H. Porter. American Journal of Clinical Pathology, vol. 14, 1944, p. 37.

8. Photoelectric Counting of Individual Microscopic Plant and Animal Cells, C. Lagercrantz. Nature, London, England, vol. 161, 1948, p. 25.

9. An Apparatus for Counting Small Particles in Random Distribution with Special Reference to Red Blood Corpuscles, H. S. Wolf. Ibid., vol. 163, 1950, p. 967.

10. Human Blood Cells Counted Accurately by T V Camera Used as "Eye" of Com­puter. Electrical Engineerings vol. 73, March 1954, pp. 288-9.

11. Flying-Spot Microscope, F. Roberts, J . E. Young, D. Cawley. Electronics, New York, Ν. Y., vol. 36, 1953, pp. 137-9.

12. Method and Apparatus for the Counting of Particles per Unit Volume in a Fluid, Joseph C. Frommer. U. S. Patent Application 272,590, filed February 20, 1952.

13. Method of and Device for Determining Physical Quantities, Joseph C. Frommer. U. S. Patent 2,517,554.

14. Thermionic Emission (book), A. L. Reiman. John Wiley and Sons,Inc, New York, Ν. Y.. 1934, pp. 41-6 .

Miniature Transistor Preamplifier

D u M o n t t r a n s i s t o r p r e a m p l i f i e r c a n a m p l i f y 1 , 0 0 0 t i m e s

e l e c t r i c s i g n a l s o f a m i l l i o n t h o f a v o l t a n d p o w e r o f a

b i l l i o n t h o f a w a t t

MAY 1955 Frommer—Blood-Count Meter 391