GB1600146A - Chemical reagent compositions for serum iron test - Google Patents

Description

(54) CHEMICAL REAGENT COMPOSITIONS FOR SERUM IRON TEST (71) We, ENVIRONMENTAL SCIENCES ASSOCIATES, INC. a corporation organized under the laws of the Commonwealth of Massachusets, U.S.A., and having a principal place of business at 45 Wiggins Avenue, Bedford, Massachusetts, U.S.A., do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to chemical reagent compositions for serum iron tests.

Background of the invention One clinical or biomedical test of significance is the test for iron in blood or in blood serum. This test can be performed both to determine the total iron binding capacity of serum and to determine the amount of iron actually present. Both of these tests are of importance in connection with various medical conditions characterized by either too much or too little iron in the blood or serum. In ordinary conditions when the blood is low in red blood cells there is an automatic response in the generation of an increased quantity of transferrin, an iron binding component of serum, thus increasing the capacity of serum to transport iron within the body. In many cases, however, the increase of transferrin is not effective either through lack of sufficient iron in useable form in the diet, or as a consequence of one of several diseases or medical conditions.In such instances, from one cause or another, the body is deficient in iron or is characterized by excessive iron.

It is known that iron deficiency ordinarily evidenced in the form of anemia, can be a serious medical problem. It is also known, but less widely recognized, that certain long-range consequences may result from iron deficiencies or excesses at critical times such as, for example, the first few weeks of a baby's lifetime or perhaps, even more important, during the pre-natal period. It is generally thought that about 10% or more of the population in the United States is iron deficient and perhaps seriously so, and that the population elsewhere in the world is at least as generally iron deficient. Reliable information about such generalized iron deficiency is not readily available, and one of the reasons for this lack of information is the ponderous nature of serum iron analysis.As now practiced, serum iron testing is generally performed in a medical laboratory using a relatively large sample such as, for example, about 1/2ml. of serum for which 2mls. of blood is necessary. Normal testing takes about 1/2 hour per sample and the ordinary procedure is to collect samples and to run a number of them once a quantity of samples has been collected.

The test is at best cumbersome as a medical test, requires too large a sample of blood, and usually requires skilled operators and an expensively equipped laboratory. In the final analysis, testing for iron is a clinical test ordinarily performed on adults with recognizable problems and is performed in most cases as confirmation of a physician's tentative diagnosis. It is not ordinarily performed as a routine generalized test and is poorly adapted to large scale or widespread screening of large numbers of people. It is too cumbersome and too time consuming. In particular, present serum iron testing which requires too large a blood sample is not well adapted to widespread screening of children and particularly of infants.

General nature of the invention The present invention enables a quick and easy way of testing for iron in blood or serum using microsamples of serum and producing reliable results in a matter of seconds.

According to the present invention a chemical reagent composition for releasing iron from serum for electrochemical testing comprises a substantially iron-free mixture of a lower aliphatic alcohol, HCI between about 5J/2 Formal and about 81/2 Formal, and about 200 parts per million silver ion.

In use of the composition according to the invention a small sample of blood serum such as typically 5 or so microliters and up to about 100 microliters is added to the composition which releases iron from its serum bonding and separates the transfer potentials of iron and its most usual interfering element, copper.

If total iron-binding capacity is being measured, the serum is first fully saturated with iron, as by mixing it with an iron-containing ion exchange resin.

The hydrochloric acid used in the composition is preferably about 7 Formal. The lower aliphatic alcohol is preferably isopropanol. Methanol and ethanol have been found nearly as effective as isopropanol but have the disadvantage that they are more expensive, and they are more volatile and therefore more difficult to handle. Higher alcohols such as butanol and the like are operable, but are less compatible with the relatively strong hydrochloric acid employed. Other materials such as acetonitrile and acetone also are operable but are less satisfactory partly because of less satisfactory performance and partly because of cost, volatility. toxicity and the like.

In U.S. Patent Specification No. 4090926, it is disclosed that certain trace impurities can be released from a biomedical sample. In that U.S. Patent Specification it is disclosed that trace amounts of lead can be detected and measured electrochemically by releasing lead from its complexing in the sample by treatment including the use of calcium and chromium ions. According to the method disclosed in that U.S Patent Specification, the reagent is added to a sample such as a sample of blood and the sample can then be promptly analyzed by electro-chemical methods without difficult and time consuming sample preparation. The results of the analysis are available almost immediately after the sample is taken. The reagents disclosed in that U.S.Patent Specification are not operable to release iron from serum, and prior to the present invention there were no releasing agents for serum iron compatible with electrochemical analysis.

The use of the invention in apparatus for and methods of blood serum iron tests will now be described by way of example with reference to the accompanying drawings in which: Figure 1 is a front perspective view of the apparatus; Figure 2 is a front view, partially in section, of a cell of the apparatus of Figure 1; Figure 3 is an end cross section of the stirring member of Figure 2; Figure 4 is a front cross section of the electrode of Figure 2; Figure 5 is a diagrammatic view of a proof of flow detector used in the apparatus of Figure 1; Figure 6 is a block diagram of controls and functions of the apparatus of Figure 1.

The apparatus and methods to be described form the subject of our Patent Application No. 7900370 (Serial No. 2012435).

In Figure 1 is illustrated apparatus generally designated 10 for the measurement of iron in serum or blood. The apparatus includes a base 11. Mounted on base 11 by means of upright support 12 is a cabinet 13 whose front face acts as a control panel 14. Mounted on the panel are various control mechanisms including a display panel 16, function buttons including a standby button 17, an "autoblank" control button 18, an "autoblank set" button 19, and a calibration knob 20. Also positioned on the control panel 14 is an off-on button 22, a flow indicator 23 suitably labelled to show that a prior sample is flushed out and a new test may be started. Also on the control panel are a start button 24, and a "running" indicator 25.

For convenience it is preferred that the controls be combinations of push-buttons and indicating lights, and in the actual apparatus such combination buttons and lights are used.

Depending from the bottom of cabinet 13 is a cell assembly 27 indicated in outline and shown in Further detail in Figure 2. Positioned on base 11 are two containers 28 and 29, suitably connected by plastic tubing or the like to the cell block. Container 28 receives flushed cell contents at the end of each run and container 29 holds a supply of fresh cell liquid or electrolyte.

In use and operation the off-on button 22 is first activated. Ordinarily, the apparatus will be left running in a standby condition overnight and will be turned off if it is to be left idle for a period of a week or more. At the start of each week, or for purposes of abundant caution at the start of each day, the apparatus may be calibrated. It is first operated with the calibration button in operating position to standardize the electronics as will be hereinafter described. A blank sample of reagent is run first. Then the "auto-blank" button 19 is set, holding the calibration. Next a standard sample of known iron concentration is introduced into the cell 27 and the apparatus runs through a cycle.When it has been properly standardized, the calibration knob 20 is adjusted so that the reading in the display panel 16 corresponds with the known quantity in the standard calibration sample.

In repetitive runs the cell 27 is constantly filled with an electrolyte and the cell stirring apparatus is constantly in operation to keep the cell continuously uniform and mixed. A known quantity of a test sample is then pipetted into a cell 27. The running indicator 25 lights to show that the test is in operation. In the present apparatus the display panel is a digital display which counts to zero and then up to the number of micrograms per 100 mililiters of serum (g%). When the digital display stops counting the test is complete.

After a timed waiting period the cell electrolyte containing the sample is flushed into container 28 and a new supply of electrolyte is introduced into the cell from container 29.

When the start test indicator 24 lights up again, the apparatus is ready for a next sample.

The entire test consumes less than one minute, the largest portion of which is the preliminary mixing time.

Figure 2 shows the cell assembly of the apparatus of Figure 1 comprising generally a cell block 33 and an electrode 34 mounted therein. The cell block comprises a suitable mounting piece such as. for example, a plastic block having screw threads 35 or other mounting means at the upper end. A vertical channel or cylindrical hollow 36 runs through the cell block and communicates with the interior of electrode 34. Two passage ways, the first an inlet passage 38 to receive a sample to be tested which may, for example, be by means of a pipette (not shown) inserted into channel 38. and the second an outlet passage way 39 for cell liquid.

The bottom of channel 36 is recessed to receive electrode 34. This electrode 34 is in the form of a hollow cylinder. and the inner surface of the electrode and the inner surface of channel 36 are flush and as smooth as possible so as to minimize the material caught therebetween. In actual practice the electrode is permanently mounted in the cell block by suitable means such as. for example, by an epoxy resin or the like, and the inner surface of the joint between the two is machined smooth.

At the bottom of electrode 34 is a seal and connector device 46 which may, for example, be in the form of a plastic plug molded to the electrode 34 having a screw thread connection 35a for connecting a pipe or hose thereto and having a channel 49 extending therethrough.

A continuous passage is thus formed. and electrolyte or other contents of the cell can be flushed out by passing fresh electrolyte or other liquid in through channel 49 and out through outlet 39 in the cell block above the electrode.

The electrode is shown in further detail in Figure 4 and comprises a generally cylindrical body or block having mounted therein the first electrode segment 42, a second electrode segment 43, a third electrode segment 44. and a reference electrode segment 45. The first three electrode segments 42, 43. and 44 are suitable electrode material such as graphite and the reference electrode segment is silver and each comprises a cylindrical ring embedded into the electrode body 41. As presently constructed, the electrode body is a molded resin such as. for example, epoxy resin with the electrode segments molded and properlv spaced therein. The electrode segments are spaced apart by a narrow gap so as to be insulated one from the other and the inner surface of the entire electrode 34 is a smooth as possible.

Electrical connections (not shown in Figure 4) are provided to each of the electrode segments and are suitably connected to the apparatus by means of a four wire lead terminating in a four-pronged plug as shown in Figure 2.

Positioned within electrode 34 is a stirring means 50 mounted on and rotatable by means of rod 51 which ordinarily will be graphite so as to be electrochemically neutral. At least the lower end of stirring means 54 is slightly wedge-shaped or cone-shaped, and is generally close fitting within electrode 54. A diagonal groove 52 which is better seen in Figure 3 runs along the surface of the stirrer 50. When rotated in the direction shown by arrow 54 groove 52 creates this high degree of mixing or turbulence closely adjacent to the electrode surface.

A plastic tube, pipe or the like (not shown in Figure 2) connects the cell assembly 27 to the apparatus. At a selected point along the tube and preferably within cabinet 13 a portion of this tube 40 has a flow detector illustrated diagrammatically in Figure 5. At one point along tube 40 an emitter 55 or other light source is positioned near a window in the tube.

The window may be a transparent insert or the tube itself may be transparent. Opposite the emitter a detector 56 is positioned adjacent a similar window in tube 40. When the tube 40 is empty or filled with a gas the beam of light 57 from the emitter is quite diffuse. When the tube is filled with a liquid such as the cell electrolyte flowing through the tube, the liquid acts as a lens and increases the sharpness of focus of light beam 57. Detector 56 is adjusted for a threshold such that it can determine the presence of liquid in the tube 40 and the length of time such liquid is present. The signal from the detector is employed to indicate that there has been flow of liquid through tube 40 for a sufficient time to accomplish flushing out of cell electrolyte after a single run so as to remove the sample therefrom.

In Figure 6 is shown a block diagram of electrical controls for the apparatus and of the fluid flow thereof. A cell 27 such as cell of Figure 1 is connected to have a reagent or electrolyte conveyed therethrough in individual analysis quantities. A pump 60 pumps air through a line 61 from a reagent container 62. A reagent valve 63 controls flow of the reagent to cell 27. Referring to Figure 2, the reagent flows into lower channel 49 and thus into and through the cell 27. Another fluid line 65 is positioned to carry the reagent or other liquid from the cell 27 past an optical sensor 68 such as, for example, the sensor shown in Figure 5. Line 65 then conveys the liquid to a drain container 60. A vacuum line 70 returns to pump 60. It is observed that the flow of the liquid through cell 27 flows into the bottom of the cell and out through outlet 39 positioned above the cell.It is also observed that inlet channel 38 in cell 27 is located somewhat above outlet channel 39 so that liquid normally flows out channel 39 rather than channel 38.

A cell potential control 70 is set or adjusted to apply two potentials, one to the first electrode segment 42 and another to the second electrode segment 43. A reference potential is applied to the silver electrode segment 45, and a ground or other potential applied to third electrode segment 44 provides a source of current to the cell. The cell potential control thus applies a first potential 72 to the first electrode segment 42 of Figure 2 and applies a second potential 73 to the second electrode segment 43 of Figure 4. The first potential is such as to measure iron plus copper, and the second is such as to measure copper alone, all as described in greater detail elsewhere herein. The current or signal from the electrodes in the cell 27 is then fed to a current convertor subtractor 75 with three variable gains for adjustment.The signal then goes to a signal accumulator 76. to a calibration linking circuit 77 which also has a variable gain or cahbrator 78. The signal from the calibration blanking circuit 77 then is fed to a readout 80 and. in turn, to an autoblank control 81. The signal from the autoblank control is returned to the calibration blanking circuit 77. When the calibration is correct, an autoblank set 82 is operable to fix the circuits.

A control synchronizer 85 activates the pump and valve timing control 86 and also an analog timing control 87. The analog timing control as activated by control synchronizer 85 is in the ready position and is activated for analysis by a start analysis control 88 which appears on the apparatus as start test button 24.

Optical sensor 68 whose operation is illustrated in Figure 5 directs a signal to flow sense circuit 19 which in turn sends a signal to pump and valve timing control 86 and analog timing control 87. As described in connection with Figure 5, if the flow through line 65 is inadequate for complete flushing of cell 27, the signal from flow sense circuit 90 operates to turn off pump 60 or close valve 63 or both, and to inactivate analog timing control 87 so that an analysis cannot be started without resetting the apparatus.

A power supply 91 operated from an A/C power source 92 supplies a positive voltage through line 93, a negative voltage through line 94, and a ground potential through line 95 which are supplied to the cell potential control 70. The cell potential control 70 can be controlled by potential set 96.

The analyzer consists of two sections: analog circuiting for converting, conditioning and displaying the electrochemical signal; and reagent handling circuitry for automatic sample changing at the end of an analysis.

The analysis cycle is controlled by two sequential timers 87. The first timing interval (30 seconds) is initiated after the start analysis switch 88 is depressed. This sequence is used to bring the cell to equilibrium. The second interval (20 seconds) is the concentration measurement. During this time the electrochemical signal is converted and displayed. The now preferred operation displays the "count down" or "count up" digitally during the measurement. Cell reference potential is controlled by potentiastat circuit 70 and is set by control 96. This potential is applied between the reterence electrode 45 and test electrode 42 (see Figure 4). A difference potential is seen between test electrode 43 and the reference. This potential is set by Offset 2 control operating on current converter subtractor 75. The equivalent potential becomes [Eset l - EoffsetJ.

During the measurement interval the cell currents are fed into current to voltage converter circuit 75 and gain controlled by potentiometers "Gain 1" and "Gain 2" The difference of the resulting voltages is taken and fed into the accumulator circuit 76 and integrated during the measurement interval. The integrated voltage then has the autoblank value subtracted from it and a gain applied to it by calibrate circuitry 77.

The resultant value is then displayed on the readout 80 in direct units of micrograms of iron per 100ml (g%) of serum.

Cell reagent is changed automatically in two ways. The first is when the unit is switched from the standby to run position, and the second occurs at the end of each analysis cycle.

Pump and valve timers are set on by the control sync circuit 85 from a trigger signal received by the standby control switch or the analysis cycle timer. The solenoid valve 63 is used to control reagent flow into the cell. The pump supplies pressure (4 psi nominal) to reagent supply 62 and vacuum (7" Hg) to drain reservoir 69. The pressure forces clean reagent through the valve into cell 27. This increase in cell volume is taken off through the drain line to the drain reservoir 69. The reagent inlet valve is timed on for 8 seconds. The pump is on for an additional 2 seconds to drain any excess reagent above a set level from the cell.

A flow sense circuit 68 consisting of optical sensor 56 and sense circuit 90 monitors the cell drain line 65 during the reagent flushing cycle. If there is no reagent flow or if a low amount of reagent passes through the cell, the sense circuit will reset the pump and valve timers and inhibit the operator from starting an analysis A audio alarm and indicator light (light 23, Figure 1) are activated at this time. A new cycle cannot be started until the operator places the instrument in the standby condition which resets the sense circuit.

As described in Figure 5, the flow sense circuit 90 consists of an optical sensor (LED 55 and phototransistor assembly 56 placed at the cell drain line. Its output changes from a low voltage level (line empty) to a higher voltage level (reagent flowing). The level change is sensed and integrated during the first 4 seconds of the reagent cycle. If the integrator voltage is below a preset level at the end of the 4 second interval, instrument lockout is activated.

In the autoblanking operation when a blank concentration reading is taken and is to be nulled out of future readings, the unit is switched from "run" to "autoblank". The autoblank set switch is depressed, starting a 4 second timer. The binary coded decimal output from the display is latched in the circuit. This BCD number is then converted from a digital to an analog signal.

An analog voltage of correct polarity and magnitude is fed to the calibration circuitry and subtracted from the concentration analog voltage resulting in a zero output to the display.

The electrochemical reactions which take place and are measured by the two electrodes are the oxidation or reduction of ferric or ferrous ion and the reduction of cupric to cuprous ion. Since deposition of material on anode and cathode do not occur, these are termed "charge transfer" rather than electrolytic reactions. Associated with electrode 42 in Figure 4 is the reduction of ferric ion (Fe+3) to ferrous ion (Fe+2) together with the reduction of cupric ion (cm+2) to cuprous ion (Cu+'). At electrode 43 there occurs the reduction of cupric ion to cuprous ion and the oxidation of ferrous ion to ferric ion. As a matter of choice. electrode 42 is selected for the higher potential.The signal at the one electrode is subtracted from the other with the following result: (A) Fe+3 + e- < Fe+2; Cu' + e- Cu+ and (B) Fe+2 < Foe+3 e e; Cu± < Cu+' - e.

by subtraction A - B = Fe+3 and Fe+2; Cu ) 0.

As can be seen. the reduction of cupric to cuprous ion is cancelled out in the logic with the result that the total of iron content is the signal which is fed to the digital or other readout.

The present apparatus employs potential means for electrode 42 variable from 0 to 1 volt and set for about 460 millivolts. and potential means variable for about 0 to 300 millivolts for offset for electrode 42 and set for 250 millivolts.

At the counter electrode, or electrode 44, there may be a reaction, but the principal purpose is to provide current to the cell. This reaction is primarily the reduction of hydrazine to produce nitrogen.

Included in the reagent or matrix in accordance with the invention is an extremely minute quantity of silver ion in the range of about 200 parts per million which assists in the operation of the silver reference electrode 45. The reference potential is the silver ion potential, maintained by reference electrode 45. Accordingly, the reagent which in its presently preferred composition includes 7 Formal HCl in isopropanol together with 200 parts per million silver ion provides an electrochemical composition which releases iron from serum or its iron binding components to make the iron available to electrochemlcal techniques, which separates the charge transfer potentials of iron and copper, and which given reproducible results in the analysis of serum iron by electrochemical or electrolytic techniques in microliter sample quantities.

Claims (4)

WHAT WE CLAIM IS:

1. A chemical reagent composition for releasing iron from serum for electrochemical testing comprising a substantially iron-free mixture of a lower aliphatic alcohol, HCI between about 51/2 Formal and about 8l/2 Formal, and about 200 parts per million silver ion.

2. A composition according to claim 1 wherein the alcohol is isopropanol.

3. A composition according to claim 1 or 2 wherein the HC1 concentration is about 7 Formal.

4. A chemical reagent composition for releasing iron from serum substantially as hereinbefore described.