WO1992001934A1

WO1992001934A1 - Blood testing apparatus

Info

Publication number: WO1992001934A1
Application number: PCT/US1991/005026
Authority: WO
Inventors: Mark J. Pasula
Original assignee: Pasula Mark J
Priority date: 1990-07-17
Filing date: 1991-07-16
Publication date: 1992-02-06
Also published as: AU8419991A; EP0549599A4; JPH06502486A; EP0549599A1

Abstract

The blood testing apparatus includes a fluid flow control system (38) for moving a fluid containing blood cells through an electromagnetic field, and detecting the change in the electromagnetic field caused by the passing of the blood cell. The fluid flow control system includes a pump (40) and a series of valves (52, 70, 82), all controlled by a controller (43). The pump (40) draws the blood cells through the electromagnetic field. The size of the blood cell can be determined by the measurement of the value of the voltage change resulting from the passage of the blood cell through the electromagnetic field.

Description

BLOOD TESΗNG APPARATUS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of my earlier filed, and still pending, application Ser. No. 07/363,854, filed June 9, 1989, which is a continuation of Ser. No. 06/902,313, filed August 28, 1986 (now abandoned). Application Ser. No. 902,313 was itself a continuation-in-part of Ser. No. 06/547,767, filed November 1, 1983, now issued as U.S Pat. No. 4,614,722 on September 30, 1986.

This application is also related to my earlier filed application Ser. No. 06/913,940, filed October 1, 1986, now issued as U.S. Pat. No. 4,788,155 on November 29, 1988, which was also a continuation-in-part of the above-referenced applications Ser. Nos. 06/902,313 and 06/547,767.

Additionally, this application is a continuation-in-part of my co-pending application Ser. No. 07/453,869, filed December 20, 1989, which was also a continuation-in-part of my earlier filed application Ser. No. 07/363,854, referenced above.

BACKGROUND OF THE INVENTION

The present invention relates to medical testing apparatus and, more particularly, to apparatus for testing a subject's blood.

In my earlier patents (Pat. Nos. 4,614,722 and 4,788,155, the disclosures of which are hereby incorporated by reference) and patent application (Ser. No. 363,854, the disclosure of which is also incorporated by reference) I disclosed methods for testing a subject's blood for various maladies, including allergies. The described testing of the blood permits the diagnosis of the maladies by preparing two samples of the subject's blood.

The first sample is a control sample, and the second is a test sample. A test substance having a predetermined relationship with the malady under consideration is placed into the test sample, and the blood and the substance are given the opportunity to react. The blood cells in the two samples are counted and compared, both as to number and size-distribution. If a significant difference is observed between the blood cells present in the two samples, then it may be concluded that the subject has the malady for which he was tested.

I have also invented a kit for use in performing the referenced test, described in my co-pending application 07/453,869, filed Dec 20, 1989. The kit includes the various consumable products used in the performance of the test. To date, however, the test has been performed using pre-existing technology. No dedicated apparatus which is designed expressly for performing this test is known.

In addition, it would be useful to provide an apparatus which is capable of performing not only my patented test, but also of performing other blood tests, such as a hemoglobin or blood chemistry analysis, known as a CBC ("complete blood count"). Such a general purpose blood testing apparatus would have widespread utility in the field of diagnosis of a wide array of maladies.

OBJECTS AND SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a blood testing apparatus dedicated to the performance of the described test. It is a further object of the invention to provide a blood testing apparatus which is capable of performing the described test in combination with other blood tests.

It is a still further object of the invention to provide a blood testing apparatus capable of performing the described test, and still have general utility in the performance of other blood tests. Briefly stated, there is provided a blood testing apparatus which includes a fluid flow control system for moving a fluid containing blooα cells through an electromagnetic field, and detecting the change in the electromagnetic field caused by the passing of the blood cell therethrough. The fluid flow control system includes a pump and a series of valves, all controlled by a controller. The pump removes air from one side of a valve to cause a partial vacuum condition on one side of a valve, and pumps air into a line on the other side of the valve to cause a pressure differential across the valve. That pressure differential is then maintained while the opposing ends thereof are sealed, and the valve opened, so that a fluid flow will be initiated through the valve to equalize the pressure differential. The fluid flow draws blood cells through the electromagnetic field. The size of the blood cell can be determined by the measurement of the value of the voltage change resulting from the passage of the blood cell through the electromagnetic field. To avoid false readings, the measurement is taken at two discrete time intervals. The two measurements are compared and, if they differ by no more than a predetermined error factor, the reading is accepted as a true measurement of the blood cell. Other errors may be avoided by use of a separate continuous measurement of the voltage, so that, if the voltage remains perturbed for a period of time longer than expected for the blood cell to traverse the electromagnetic field, the readings may be discarded as erroneous. In accordance with these and other objects of the invention, there is provided an apparatus for testing a subject's blood, the apparatus comprising: means for retaining a first sample of the subject's blood, the first sample being a control sample; first means for counting blood cells in the first sample, thereby producing a first result; means for retaining a second sample of the subject's blood, the second sample being a test sample, and containing both the blood and a substance having a predetermined relationship with a malady for which the subject is being tested; second means for counting blood cells in the second sample, thereby producing a second result; and means for comparing the first and second results, thereby producing a comparison result; whereby the subject may be diagnosed as having the malady when the comparison result is greater than a predetermined amount.

According to feature of the invention, there is further provided a method of counting blood cells contained in a fluid, the method comprising the steps of: establishing an electromagnetic field in the fluid; measuring a baseline level of the electromagnetic field in the fluid; drawing the fluid through the electromagnetic field at a controlled rate of flow, thereby causing the blood cells to traverse the magnetic field; measuring the level of the electromagnetic field at a first time interval after the electromagnetic field varies from the baseline level; measuring the level of the electromagnetic field at a second time interval after the electromagnetic field varies from the baseline level; comparing the level of the electromagnetic field at the first and second time intervals; and accumulating a level of the electromagnetic field corresponding to each change in the level of the electromagnetic field from the baseline; whereby, when the comparison results in finding a difference between the level of the electromagnetic field at the first and second time intervals which is greater than a predetermined error factor, the accumulation of that individual level is discarded as erroneous.

The above, and other objects, features and advantages of the present invention will become apparent from the following description read in conjunction with the accompanying drawings, in which like reference numerals designate the same elements.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a front elevation of a blood testing apparatus in accordance with the invention, shown partly in cross-section.

Fig. 2 is a schematic diagram of a fluid pressure control apparatus which comprises a portion of the blood testing apparatus of Fig. 1.

Fig. 3 is a detail cross-section of a lower portion of an aperture tube used in the inventive blood testing apparatus.

Fig. 4 is a further detail of the lower portion of the aperture tube shown in Fig. 3. Figs. 5a and b are waveforms of voltage readings taken the blood testing apparatus of Fig. 1.

Fig. 6 is a circuit diagram of a counting circuit of the inventive blood testing apparatus.

DETAILED DESCRD7TION OF THE PREFERRED EMBODIMENT

Referring now to Fig. 1, there is shown, generally at 10, a blood testing apparatus, rα accordance with the invention. Blood testing apparatus 10 may be used to test a subject's blood in a variety of ways, including my patented antigen-leukocyte cellular antibody test (ALCAT), a hemoglobin analysis, and a blood chemistry test and a standard complete blood count (CBC).

In practice, however, CBC and ALCAT are not usually performed at the same time, although they may be performed by the same machine at different times, depending on the needs of the facility in which the apparatus is deployed.

Since the techniques required for standard hemoglobin, CBC and blood chemistry testing are commonly known to those of ordinary skill in the art, I will direct my explanation to the use of blood testing apparatus 10 for the ALCAT test. Inventive blood testing apparatus 10 includes a housing 12, and a platform 14 disposed in an opening 16 therein. A testing subassembly 18 depends from an upper surface 20 of opening 16.

Testing subassembly 18 includes a red blood cell aperture tube 22, a hemoglobin nozzle 24, a plurality of chemistry nozzles 26, and a white blood cell aperture tube 28. To utilize blood testing apparatus 10, blood is drawn from a subject. The amount of blood to be drawn depends on the use to be made of the subject apparatus, since it is not necessary that the technician make full use of its capabilities. If, for example, the technician intends to perform 50 food allergy tests (a common amount) by means of the ALCAT test, approximately 10 ml of blood is needed. The desired amount of blood is drawn from the subject into a vacutainer containing an anticoagulant, preferably a citrate, such as those used in performing standard PT or PTT blood tests. Sodium citrate is a suitable anticoagulant.

The preferred method of drawing blood is by venipuncture into a 3.8% solution, sterile, non-buffered sodium citrate blood collection tube having a 4.5 ml draw. Such tubes are commercially available from Curtis Matheson Scientific, Inc., among others. To draw 10 ml, two such tubes would be used.

Once the blood is drawn, the vacutainer is inverted several times, without violent shaking, to ensure the mixture of the blood and anticoagulant without foaming. The blood is then placed into a container containing a diluent, such as normal saline, and mixed. My preferred method of mixing the blood and diluent is by means of magnetic stirring for one minute at a moderate speed. This ensures that the blood cells in the diluted solution are evenly distributed. The blood should be diluted to a desired concentration, depending on the precise methodology to be followed. This is a matter of design choice, and may be made by the user depending on the precise tests to be performed. For example, the ALCAT test may be used to measure changes in white blood cells, red blood cells or platelets, all of which have different concentrations in whole blood. White blood cells are ordinarily found in concentrations of from 4500 to 11,000 per micro-liter, while red blood cells and platelets may each have concentrations of between 3,600,000 and 5,000,000 per micro liter. Thus, if it is desired to measure a projected total of approximately 5000 white blood cells, for example, whole blood should be diluted by a factor of 500, while to reach the same concentrations, for red blood cells or platelets, the whole blood would have to be diluted by a factor of 50,000.

Dilution is a common procedure, and may be accomplished in any desired fashion. In the preferred method, an external diluter, such as commercially available from Sequoia Turner is preferred. Once the desired dilution is achieved, the technician may ready the blood for testing.

Returning to Fig. 1, a tray 30 is positioned atop platform 14 in use. Tray 30 includes a plurality of blood cell testing receptacles 32 and chemistry testing receptacles 34. Blood cell testing receptacles 32 and chemistry testing receptacles 34 may be identical, except that blood cell testing receptacles include a disk 36 which may contain a substance used in the performance of my patented test, as will be described below. However, it is preferred that blood chemistry testing receptacles 34 be slightly smaller than blood cell testing receptacles 32, since the tests performed therein require the use of less serum or blood than those performed in blood cell testing receptacles 32. Once the blood is diluted to the desired concentration, it is dispensed into blood cell testing receptacles 32 and diluted serum or blood is deposited into chemistry testing receptacles 34. Once placed in a blood cell testing receptacle 32, the blood cells will be given the opportunity to react with the respective disk 36 therein. If the particular blood cell testing receptacle 32 is intended for use as a control, then disk 36 contains no substance for reaction, and is neutral to the blood. It is preferred that about 0.5 ml of the blood/suspension mixture be dispensed into each blood cell testing receptacle 32. That amount of liquid should contain the desired concentration of blood cells to be counted.

Once the blood has been placed in tray 16, it must be given the opportunity to react. This is accomplished by incubating the blood, after being placed in contact with disks 36, at approximately body temperature (36.6° Celsius) for 45 minutes. After that period, the blood specimens should be removed from the incubator, and permitted to stand at room temperature for an additional 30-45 minutes, for a total period of 75-90 minutes of incubation. If the test is to be performed on either red blood cells or platelets, then the blood is ready to be examined. However, if white blood cells are to be analyzed, then the red blood cells in the suspension must be lysed. The rationale for this procedure has been explained in depth in my earlier patents.

Lysing is accomplished by adding a lysing agent, preferably a mixture of 6 ml of ALCALYSE, sold by Amtl Corp., and 1000 ml of Isoton II sold by Coulter Electronics, Inc. This mixture, 1006 ml, is sufficient for testing for 50 different food allergies. More or less may be prepared depending on the intended application. It will be appreciated by those of ordinary skill in the art that the amount of lysing agent added must be considered when calculating the concentration of blood cells to be measured, since the addition of the liquid lysing agent dilutes the blood solution. As stated above, the precise concentration of blood cells per unit volume is a matter of design choice.

After the lysing agent has been prepared, it must be dispensed into blood cell testing receptacles 32. It is important that the lysing agent be dispensed into the different blood cell testing receptacles 32 at carefully monitored intervals. The lysing agent described will act on the red blood cells in approximately 30 seconds. It will also act on the white blood cells in approximately 50-55 seconds. Since it is desired that the white blood cells not be affected by the lysing agent, measurement must be performed during the window of opportunity which exists after the red blood cells are lysed, and before the white blood cells are lysed.

Furthermore, the inventive apparatus will perform two or three tests per minute, so the lysing agent must be dispensed into each blood cell testing receptacle 32 approximately 30 seconds before it is to be inserted into blood cell testing apparatus 10, so that the blood therein will be ready for testing at the appropriate time.

Thus, the technician should dispense the lysing agent into blood cell testing receptacles 32 at approximately 30 second intervals, to allow sufficient time for the lysing agent to react with the red blood cells, but insufficient time for it to react with the white blood cells, thereby ensuring an accurate count of the white blood cells.

Once the lysing has taken place, the actual tests may be performed.

In order for the desired tests to be performed, the contents of receptacles 32 and 34 must be brought into contact with the lower ends of the various components of testing subassembly 18. This may be accomplished either by lowering testing subassembly 18 or raising platform 14. Either may be performed in any well-known manner, and it is not believed that this simple construction need be described in any further detail here.

Once the blood cell solution has been prepared, the blood cells are ready for counting. The actual counting process, detailed below, requires the movement of blood cells at a controlled rate, and so it is necessary to provide means for the controlled movement of the fluid blood suspension. This fluid control apparatus is shown in Fig.

2, generally at 38.

Fluid control apparatus 38 includes a pump 40 driven by a motor 42, controlled by a motor controller 43. Pump 40 has a positive pressure line 44 which splits at a junction 46 into two separate lines, a vent 48 to atmosphere, and a line 50. Vent 48 and line 50 are both controlled by a valve 52, so that when vent 48 is open to the atmosphere, line 50 is closed, and when line 50 is open, vent 48 is closed. A pressure sensor 54 senses the pressure on line 50. Line 50 terminates in an air bottle 55, which is coupled to an electrolyte reservoir 56, by a line 57.

A line 58 runs from the bottom of electrolyte reservoir 56 through one hole of a two-hole stopper 60 to the bottom of an aperture tube 62 filled with an electrolyte solution 64, such as Isoton II manufactured by Coulter Electronics, Inc. Aperture tube 62 is conventionally made of a non-conductive material, such as quartz glass, or a polyethylene, and are widely available commercially. It is here noted that two-hole stopper 60 may have further holes than are here discussed, for example for additional measuring conduits or fluid supply conduits. For our purposes, here, however, only the two holes are needed for an understanding of the invention.

It will also be appreciated by those of ordinary skill in the art, that aperture tube 62 could be either red blood cell aperture tube 22 (Fig. 1) or white blood cell aperture tube 28 (also Fig. 1). The procedures for operation of each aperture tube is the same, and so they will be described generically. It is believed that a single fluid control apparatus 38 could control two aperture tubes 62, operating in parallel, but only one will be described, for clarity.

A lower end of aperture tube 62 is placed in blood cell testing receptacle 32. Blood cell testing receptacle 32 contains a blood cell suspension 66. A line 68 runs from a top of aperture tube 62 through a second hole in two-hole stopper 60 past a valve 70 to the bottom of a waste container 72. A waste line 74 runs from a top of waste container 72 to a balance container 76. A further line 78 runs from balance container 76 past a vacuum sensor 80 through a valve 82 to a junction 84. Vacuum sensor 80 senses the negative pressure in line 78. Junction 84 splits line 78 between a line 86 which serves as an input to pump 40, and a vent 88 to atmosphere.

Valve 82 controls the opening of line 78 and vent 88, so that when line 78 is open, vent 88 is closed, and when vent 88 is open, line 78 is closed.

Valves 52, 70 and 82 are controlled by controller 43. The outputs of pressure sensor 54 and vacuum sensor 80 are input to controller 43.

To operate fluid control apparatus 38, the technician actuates its operation by means of input from controller 43. In the preferred embodiment, controller 43 would be an XT- or higher class computer containing a suitable program. The program will vary depending on the precise computer used and the application for which it is intended, and so the program is not reproduced here. Those of ordinary skill in the art, having this disclosure available, and without undue additional experimentation, would be able to formulate a satisfactory program for any desired application.

Before pump 42 is actuated, and before the initial operation of blood testing apparatus 10 and fluid control apparatus 38, all lines are at ambient pressure, aperture tube 62 is completely filled with electrolyte 64 and electrolyte reservoir 56 contains a volume of electrolyte as well. Air bottle 55, electrolyte reservoir 56, waste container 72 and balance container 76 are all sealed to the atmosphere.

Once pump 40 is actuated, controller 43 causes valve 52 to close vent 48, thereby opening line 50. Controller 43 also closes valve 70, si itting off line 68 and causes valve 82 to close line 78, thereby opening vent 88. This means that pump 40 draws ambient air from vent 88, past junction 84, along line 86, through pump 40, along lines 44 and 50 to air bottle 55. Thin flow of air increases the pressure in air bottle 55, which then increases the pressure in the top of electrolyte reservoir 56 (along line 57). The downward pressure on the surface of electrolytes in electrolyte reservoir 56 causes electrolytes to flow along line 58 into aperture tube 62, and then out of aperture tube 62 along line 68 to valve 70. Since valve 70 is closed, the pressure from line 50 to valve 70 increases above atmospheric pressure. This pressure is reflected in the pressure along line 50, which is measured by pressure sensor 52 and input to controller 43. Once the desired level of pressure is reached, preferably atmosphere plus one-third (appx. 220 mm of Hg), controller 43 actuates valve 52 to shut lines 50 and 58, and open vent 48. This causes the positive pressure to be sealed between valves 52 and 70. After the positive pressure is sealed to the left (in Fig. 2) of valve 70, controller

43 actuates valve 82 to close vent 88 and open line 78. Pump 40 then draws air from line 74, through balance container 76, along lines 78, 86 and 44 to vent 48. Thus, a negative (vacuum) pressure is induced in the top of waste container 72, suctioning the material from the right of valve 70 along line 68. Thus, a pressure differential is formed on opposite sides of valve 70. When the vacuum pressure on line 78 reaches the desired level, preferably atmosphere minus one-third, as measured by vacuum sensor 80 and input to controller 43, controller 43 actuates valve 82 to close line 78 and open vent 88. Pump 40 may then be shut off.

At this point, there is a completely sealed positive pressure line between valves 52 and 70, and a completely sealed vacuum pressure line between valves 70 and 82.

There is an additional factor, not heretofore addressed, which impacts slightly on the pressure build-up. Reference is now made to Fig. 3.

Fig. 3 shows a detail of a lower portion of aperture tube 62, suspended in blood cell testing receptacle 32 (not separately shown in Fig. 3). Electrolyte 64 is shown within aperture tube 62, and blood suspension 66 is shown on the exterior of aperture tube 62.

Aperture tube 62 is not completely solid. It has a small aperture 90 therein which provides communication between the interior and exterior of aperture tube 62. A jewel 91 having an aperture 92 is disposed in the mouth of aperture 90 on the exterior side of aperture tube 62. Aperture 90 provides the sole means of communication between the interior and exterior of aperture tube 62.

In the preferred embodiment, aperture tube 62 has walls which are approximately 1 mm thick, and, at its lowest end, has an exterior diameter of approximately 10 mm. Thus, aperture 90 has a length of approximately 1 mm. Aperture 90 also has a diameter of approximately 1 mm. Jewel 91 has an exterior diameter at least as great as the interior diameter of aperture 90, so that it completely fills aperture 90, and a thickness of approximately 300 microns. Aperture 92 of jewel 91 is shaped generally as a funnel, with a generally cylindrical portion having a diameter of between 120 and 200 microns, and most preferably approximately 140 microns. The cylindrical portion is positioned centrally in jewel 91 on the side thereof which is on the exterior of aperture tube 62, and extends approximately half-way through jewel 91. The remainder of aperture 92 widens at an angle of approximately 45°. As will be described in detail below, blood cells will travel the length of aperture

90 for counting, but fluid may also flow therethrough. Accordingly, a buildup of pressure on the interior of aperture tube 62 will force electrolyte 64 to exit aperture 90 and flow into blood cell testing receptacle 32. However, aperture 90 is not shown to scale in Fig. 3. In fact, the length of aperture 90 is on the order of 120-140 microns, and its diameter is 100 microns. The diameter thereof is much larger than the diameter of a blood cell (white blood cells may be as large as 10 microns), but many times smaller than the diameter of line 58, which is approximately 3 mm. Thus, while a small amount of electrolyte 64 will be forced outward through aperture 90, it is a very small amount when compared to the volume of fluid being forced into aperture tube 62 along line 58, so that the outward flow of electrolyte 64 through aperture 90 does not lessen greatly the pressure which builds up in aperture tube 62.

The outward flow through aperture 90 has a salutary effect, however. While the pressure causes a small volume of flow through aperture 90, that volume is under (relatively) high pressure, so that it clears aperture 90 of any blockage which may exist, thereby ensuring that any measurement which is later taken will be accurate.

Returning now to Fig. 2, it will be remembered that pump 40 causes a pressure differential on opposite sides of valve 70, which is closed prior to measurement.

Once the desired pressure differential across valve 70 is reached (after approximately one minute), the actual process of measurement may take place. Here, reference is made again to Figs. 1 and 3. A pair of electrodes 93 and 93' are used to take the measurements used by blood testing apparatus 10. A first electrode 93 comprises a single strand of insulated wire, preferably platinum wire, which winds about the bottom of aperture tube. An exposed end thereof is located on the side of aperture tube 62 removed from aperture 90. It should also be disposed at a height lower than aperture 90, to ensure that, if aperture 90 is covered by liquid, then so is electrode 93. This establishes the means for providing an electrical connection with the interior of aperture tube 62, as will be described presently.

The other electrode 93' is best seen in Fig. 1. Electrode 93' is suspended within aperture tube 62, at a position removed from aperture 90, preferably in the range of from eight to ten centimeters. This distance renders it less likely that electrode 93' will be disturbed by the turbulence near aperture 90. One of electrodes 93 and 93' is attached to a voltage generating means (not shown in Fig. 3) and the other to a voltage detecting means (also not shown in Fig. 3). Which of pair of electrodes 93 and 93' generates the voltage, and which measures it, is not relevant. It is noted that, in the preferred embodiment, the two electrodes will alternate as anode and cathode, so that the electrons which flow therebetween will flow in alternating directions between measurements. This prevents the build-up of contaminants which would tend to accrete if only one electrode served as the cathode. The voltage across aperture 90 is identified as V. It will be appreciated that, while the discussion of the preferred embodiment is directed to the measuring of a voltage across aperture 90, it would be possible to perform the equivalent measurement by taking the reading of the current which passes through aperture 90. These would be equivalent readings.

When a voltage is produced by one of pair of electrodes 93, current will flow evenly through electrolyte 64 or blood suspension 66, since they are both conductive. However, since aperture tube 62 is non-conductive, the only available conductive path between electrodes 93 and 93' is along the electrolytes found in aperture 90. When aperture 90 contains only electrolytes, there is a constant voltage, of known magnitude, which follows aperture 90. This establishes a constant baseline voltage (see V_B in Fig. 5a) which may be used as a comparative reference voltage. When a blood cell 94 (not shown to scale) enters aperture 90, it perturbs the voltage, and dampens V. Voltage V is monitored by controller 43. Controller 43, therefore, may ascertain the level of V prior to the commencement of any measurements, so that controller 43 may first measure and then remember that level, i.e. V_B.

Thus, controller 43 may correct V_β for different measurements of different specimens, and, essentially, re-calibrate blood cell testing apparatus 10 at each measurement, since the baseline voltage V_B for each specimen will be newly determined, and established uniquely for that specimen.

At the time when controller 43 closes valves 52 and 82, sealing off lines 50, 58, 68, 74 and 78 from the atmosphere, no appreciable pressure exists on electrolytes 66 in blood cell receptacle 32, so that it remains essentially at rest.

When measuring is to start, after closure, controller 43 opens valve 70, allowing the negative pressure in waste container 72 and balance container 76 to draw electrolyte 64 from aperture tube 62. Since line 58 leading to electrolyte container 56 is closed by valve 52, the fluid movement along line 68 causes suction in aperture 90 (see Fig. 3), thereby drawing blood suspension 66 through aperture 90, carrying with it some blood cells to be counted. Since the pressure in line 78, the diameter of aperture 90 and viscosity of blood suspension 66 are all known, the rate of flow of blood suspension 66 through aperture 90 is also known. Thus, the rate of flow of any solid particle, such as a blood cell, along aperture 90 is known. The flow rate is quite fast, relative to the size of the blood cells, and, with the parameters of the preferred embodiment, is in the range of 5 meters per second (m/s).

The path of travel of a blood cell measured by the inventive apparatus is shown in Fig. 4, and the voltages measured over time are shown in Fig. 5a., to which concurrent reference are now made.

As shown in Fig. 4, as blood cell 94 is drawn through aperture 90, it follows a path from the exterior of aperture tube 62 to the interior thereof, along the path shown by arrows 96, 98 and 100. Various positions of blood cell 94 as it is drawn into, through and beyond aperture 90 are designated in Fig. 4 by the time at which they occur, with a capital "T", followed by a subscript. The voltage readings across pair of electrodes 93 at those same times are shown in Fig. 5a.

When blood cell 94 is not within aperture 90 (for example when it is to the left of position T₀ in Fig. 4), it does not affect the voltage read by pair of electrodes 93 across aperture 90. This is reflected by the steady line V_B, the baseline voltage shown in Fig. 5a. In the preferred embodiment, the voltage between electrodes 93 and 93' is approximately 180 volts.

As the pressure change across aperture 90 begins to pull blood suspension 66 through aperture 90, blood cells begin to be pulled therethrough as well until, at a time T_j, blood cell 94 is completely within aperture 90.

From time T₀ to time T_l5 as blood cell 94 gradually enters aperture 90, voltage V dips (reflected by an increase in the negative voltage ordinate in Fig. 5a). Once completely within aperture 90, the resistance of blood cell 94 continues to depress the voltage until blood cell 94 begins to leave aperture 90 at a time T₂. After blood cell 94 leaves aperture 90, it continues to perturb V until blood cell 94 moves a sufficient distance away at a time T₃ that it no longer affects V.

The resistivity of blood cells is known, and it is proportional to the volume of the cell. Thus, the difference between V_B and the measured maximum voltage (the plateau between times T_j and T₂) determines the size of the blood cell which has passed through aperture 90.

This renders the act of counting the blood cells in varying size ranges quite simple, since each blood cell will act as a unit pulse with a maximum voltage indicative of its size. Mere counting of pulses of differing sizes will yield the desired results.

Some possible errors should be anticipated, however, and corrections should be implemented.

First, it will be appreciated that the above method is designed to count all particles that enter aperture 90, not simply blood cells, and that it will be assumed that all particles are blood cells. This assumption may lead to errors. When measuring white blood cells, for example, there may be a problem.

The measurement of white blood cells involves the lysing of red blood cells, which produces some cell fragments. These fragments will be smaller than white blood cells, and could skew the results by adding false readings of small "blood cells". Other particles which may be present in the blood suspension, e.g. minor contaminants which do not otherwise affect the readings taken, may also skew the results in that fashion.

These types of errors may be corrected by setting a minimum threshold voltage (shown as voltage V_t in Fig. 5a) for readings. Unless the measured voltage differs from

V_B by an amount greater than V_t, no measurement is taken. In the preferred embodiment, V_t is approximately 5 volts. Once controller 43 has measured V_B, as described above, it may then determine the appropriate V_t for the specimen, since V_t will preferably be a fixed difference from baseline voltage V_β. Another source of error may result from natural turbulence within aperture tube 62. Even with a smooth and strong suction pulling electrolyte 64 through line 58, the path taken by some blood cells may bring blood cell 94 sufficiently close to the exit of aperture 90 that it may affect the voltage reading. Such a path is shown in Fig. 4 by arrows 102, 104 and 106. Such a path may be followed by a blood cell which strikes the opposing wall of aperture tube 62 (not seen in Fig. 4), and then passes near the interior end of aperture 90. Given the rate of travel of blood cell 94 through aperture 90, this is to be expected.

If, at a time T₄, blood cell 94 passes near the interior end of aperture 90, the value of V may change. If the change is small, it will not exceed V_t, and no reading is made.

However, if blood cell 94 passes sufficiently close to aperture 90 to reduce V to a level greater than V_t, the returning blood cell may be erroneously counted as an additional blood cell, of a smaller size. It is possible to avoid an error of this type by means of a two-stage counting process. Once V is detected as having changed to a degree greater than V_t, at a time tø, a timer is initiated, to measure V at a time t_j after the initial entry of a particle into the area near aperture 90. The duration of t_t is selected so that it is greater than the period of time for blood cell 94 to move from a position at which it perturbs V to a degree greater than V_t to a position wholly within aperture 90 so that the perturbance is at or near its maximum, i.e. after T_j. A suitable delay for the preferred embodiment is 8 micro-seconds.

This measurement gives a single reading of the maximum level of V due to the presence of blood cell 94 in aperture 90. However, as seen in the waveform on the right portion of Fig. 5a, that could lead to an erroneous counting of a "shadow" of blood cell 94 on the exterior of aperture 90.

This erroneous count may be avoided by use of a second reading of V taken after a period of time t₂ after t₀. This reading should be taken at a point in time in which a blood cell which actually traverses aperture 90 would be expected to be still within aperture 90, but at which a blood cell which is merely passing by on the exterior of aperture 90 will have left. Under the preferred operating conditions, it is expected that a blood cell will traverse the length of aperture 90 in approximately 25 micro-seconds, so a suitable duration of t₂ would be 16 micro-seconds from t₀. The second measurement is compared with the first, and, if the second measurement is also greater than V_t, then the two measurements are averaged to produce a mean measurement of the size of blood cell 94. If the second measurement is less than V_t, then both readings are discarded. This eliminates, therefore, the false readings due to the passing of a blood cell near an electrode 93, and renders the count more accurate. It is noted that the concentration of blood cells per unit volume is sufficiently low in blood suspension 66 that it is not expected that a true reading of the size of a first blood cell within aperture 90 would be masked or altered by the presence of a second blood cell passing near electrode 93 on the exterior of aperture 90, nor by two blood cells passing through aperture 90 simultaneously.

The statistical likelihood of either event occurring is small, and, even if either event does occur, one or even two such occurrences would be insufficient to alter the true count in any statistically significant fashion. A further type of error might occur if a particle too large to fit through aperture

90, such as a hair, becomes lodged in the opening of aperture 90, clogging it. This would cause a sustained drop in V, since the large particle would impact severely on the voltage across aperture 90. In this case, simply measuring V at t₁ and t₂ would not guarantee accuracy. As shown in Fig. 5b, if a particle lodges in aperture 90, V could be perturbed for an indefinite period. Readings taken at t_j and t₂ will appear to show that an actual particle is in aperture 90, providing a false reading. To account for such a possible error, a separate monitoring may be performed.

An error condition such as described has the characteristic that the value of V will be perturbed at a generally constant level for an indefinite period of time. It is possible, however, that the change in V would be less than V_t, and so would not register with the primary measuring technique. Accordingly, a second, independent measurement of V is made each time V crosses a second, lower, threshold V_t>, which, in the preferred embodiment is approximately 10 millivolts. Second threshold voltage V_t» may be determined by controller 43 at the same time, and in the same manner as controller 43 determines V_t.

Once V is perturbed to a value greater than V_t», a timer is actuated. The timer is set to re-measure V at a time t₃ after a blood cell would be expected to have finished the journey through aperture 90. In the preferred embodiment, a suitable interval for t₃ would be 50 micro-seconds, twice the expected time it would take for a blood cell 94 to go completely through aperture 90.

If the measurement of V at time t₃ is greater than V_t», then it is presumed that the change in V measured the occurrence of an error condition, and remedial steps must be taken.

First, the error condition is relayed to controller 43 for alerting the operator of blood testing apparatus 10 of the error. Any conventional warning signal, such as flashing lights, sound, etc. may be used to alert the technician. Second, it is preferred that fluid control apparatus 38 correct the error condition.

Since such an error would be the result of a blockage of aperture 90, that blockage must be cleared. As noted earlier, when valves 52 and 70 close lines 58 and 68, respectively, the positive pressure retained in aperture tube 62 causes a rapid outflow of fluid through aperture 90, this will clear almost any type of blockage that may arise. In addition, to prevent recurrence of the error condition, the warning signal generated by controller 43 alerts the technician to intervene. Specifically, the technician should move the blood cell testing receptacle in which the error condition occurred, so that any debris which may have clogged aperture 90 will be removed from aperture 90, and then, after the blood cell testing receptacle is moved, that debris will be in a location removed from aperture 90.

Thus, the error condition may be detected, corrected and then avoided quickly and with only minimal intervention by the technician.

If aperture 90 becomes clogged a second time, or if the initial attempts to remedy the clog is ineffective for any reason, the sample would have to be discarded, as the sample will be unreliable.

A circuit useful in performing the various measurements is shown in Fig. 6, generally at 108. Circuit 108 includes a first comparator 110 having two inputs, V and V_t (from controller 43). When V exceeds V_t, a pulse is generated by comparator 110 to actuate a delay circuit 112. Delay circuit 112 is set to generate two different enabling signals, a first after a delay equal to t_l5 and a second after a delay equal to t₂. It will be appreciated that two separate delay circuits could also be utilized. That is strictly a matter of design choice. The first enable signal enables an amplifier 114 which receives V, and outputs a signal representative of the magnitude of V after time t_χ. That signal is delayed by a delay circuit 116 for a period of time equal to the difference between t_x and t₂. The delayed first signal is input to comparators 118 and 120. The second enable circuit enables an amplifier 122 which receives V, and outputs a signal representative of the magnitude of V after time t₂. That signal is also input to comparators 118 and 120, and to a divider 124 as well.

Comparator 118 takes the difference between V at times t_j and t₂, and generates a signal representative of that difference to a comparator 126. Divider 124 takes its input, the value of V at time t₂, and divides it by a predetermined amount. This predetermined amount is the error level acceptable to the user, and is preferred to be approximately ten per cent.

The output of divider 124 is input to comparator 126. Comparator 126 compares the difference between the measured values of V at times t^ and t₂, and the error level from divider 124. Jf the difference in the measured values of V is greater than the error level set in divider 124, then an error condition exists, and an error signal is generated. It will be appreciated that, in accordance with the above teachings, the measurement of V at the two different times serves to render the count more reliable. In the case of a blood cell bouncing back, as shown in Fig. 4, to generate the erroneous reading shown in the right of Fig. 5a, the difference between the two readings will be the entire value of the first measurement, so that will greatly exceed the acceptable error level as generated by divider 124. The purpose of the comparison made here is to ensure that minor statistical changes in V do not cause the circuit to discard true readings.

It will be appreciated that either measured voltage could be used as the reference voltage for comparator 126, and that some level other than ten per cent could be used, within wide ranges. The precise choices made by those of ordinary skill in the art will be a matter of design choice.

Returning to comparator 120, comparator 120 takes the inputs of the two measured voltages, and averages them, generating a signal representative of the average of these two signals, and, hence, the measured size of the blood cell which passed through aperture 90 at the relevant time. It would be possible to take instead ether measured voltage as well, but it is believed that the average of these two signals will result in the more accurate measurement.

The average measurement is input to an amplifier 128, which has a disable input of the error signal generated by comparator 126. Thus, when the comparison indicates that the reading is erroneous, amplifier 128 is disabled, and does not pass that reading on for accumulation.

All actual measurements pass through amplifier 128 to an accumulator 130, which stores the value of each signal, and thus maintains information indicative of the number of blood cells actually counted, and of their respective sizes. This information is passed on to a memory 132, unless a further flow error is detected. Flow errors, such as a blockage, will be detected by the second part of circuit 108.

A comparator 134 monitors V constantly, and compares it to the second threshold voltage, V_t>. When V exceeds that value, a triggering signal is sent to a delay circuit 136, which sends an enable signal to a comparator 138 after a time t₃. Comparator 138 compares V and V_t>, and, if V is greater than that value, for the reasons specified above, an error condition exists, and an error signal is generated. This signal clears accumulator 130, and actuates controller 43 to generate a suitable warning, and to institute the reverse flow through aperture 90 to clear the blockage.

Controller 43 then resets the blood testing apparatus, the technician moves the blood cell testing receptacle, and a new measurement may begin. The actual counting that is to be performed is to measure the number of blood cells per unit volume of whole blood, usually one micro-liter. It will be appreciated that the actual raw count must be related to this number. For example, it is preferred that, in the preferred embodiment, for white blood cells, that the count be made for a diluted volume which is l/500th of the concentration of blood cells in whole blood. Thus, the actual count is performed to determine the number of white blood cells in 500 micro- liters of the 1:500 diluted suspension, so that the raw count corresponds directly to the number of blood cells per micro-liter in whole blood. For white blood cells or platelets, with the higher concentrations noted above, it is preferred that the count be made over the same volume, and then multiplied by 100, to allow for the dilution factor of 1:50,000. If the user prefers to dilute the whole blood to a greater or lesser degree, to take longer or shorter measurements, it is a simple matter to calculate the actual concentration of blood cells, and may be done without undue experimentation. This apparatus may enable the user to perform the ALCAT test with a hitherto unknown simplicity, and accuracy.

The remaining components of blood cell testing apparatus 10, used to perform hemoglobin, blood chemistry and CBC are well known and understood, and so will not be detailed here.

Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

What is claimed is:

1. Apparatus for testing a subject's blood, said apparatus comprising: means for retaining a first sample of said subject's blood, said first sample being a control sample; first means for counting blood cells in said first sample, thereby producing a first result; means for retaining a second sample of said subject's blood, said second sample being a test sample, and containing both said blood and a substance having a predetermined relationship with a malady for which said subject is being tested; second means for counting blood cells in said second sample, thereby producing a second result; and means for comparing said first and second results, thereby producing a comparison result; whereby said subject may be diagnosed as having said malady when said comparison result is greater than a predetermined amount.

2. The apparatus of Claim 1, further comprising: means for introducing said substance into said second sample.

3. The apparatus of Claim 1, further comprising: means for lysing a portion of said blood cells in said first sample.

4. The apparatus of Claim 1, further comprising: means for lysing a portion of said blood cells in said second sample.

5. The apparatus of Claim 4, further comprising: means for lysing a portion of said blood cells in said first sample.

The apparatus of Claim 1, wherein said means for retaining a second sample includes means for containing a plurality of different test samples, each containing a different substance having a predetermined relationship with a respective one of a plurality of different maladies; wherein said means for counting blood cells in said second sample includes means for counting blood cells in each of said plurality of different test samples, thereby generating a respective plurality of second results; and wherein said means for comparing includes means for comparing each of said second results with said first result; whereby said subject may be diagnosed with respect to each of said plurality of maladies.

7. A method of counting blood cells contained in a fluid, the method comprising the steps of: establishing an electromagnetic field in said fluid; measuring a baseline level of the electromagnetic field in said fluid; drawing said fluid through said electromagnetic field at a controlled rate of flow, thereby causing said blood cells to traverse said magnetic field; measuring the level of said electromagnetic field at a first time interval after said electromagnetic field varies from said baseline level; measuring the level of said electromagnetic field at a second time interval after said electromagnetic field varies from said baseline level; comparing the level of said electromagnetic field at said first and second time intervals; and accumulating a level of said electromagnetic field corresponding to each change in the level of said electromagnetic field from said baseline; whereby, when said comparison results in finding a difference between said level of said electromagnetic field at said first and second time intervals which is greater than a predetermined error factor, said accumulation of that individual level is discarded as erroneous.