IL34387A - Blood diagnostic methods employing radioactive tracer compounds and ion-exchange resin membrane - Google Patents

Description

BLOOD DIAGNOSTIC METHODS EMPLOYING RADIOACTIVE TRACER COMPOUNDS AND ION-EXCHANGE RESIN MEMBRANE m»3»opKvn ma-iaina s o'tf ηιη mn nna'«7 IUB'W The invention relates to the field of radioactive tracer compounds and more particularly to the use of such compounds in conjunction with ion-exchange resins for in vitro blood analyses.

It has been known to employ radioactive tracer compounds for in vitro determination of various diagnos-tically significant characteristics of blood serum. For example, in a manner more fully described hereinafter, thyroid hormone substances containing radioactive iodine have been used to measure' the total thyroxine content of blood serum and/or the thyroxine-binding-capacity of the serum proteins. (Similarly, compounds containing radio-; active iron have been used to measure the iron-binding-capacity of blood serum. These tests are based on measurr ing the amount of the radioactive tracer compound that is bound to the serum protein using a suitable radiation counter after removing the "free", that is unbound, tracer compound.

Thyroxine (hereinafter referred to as "T-4") containing a radioactive isotope of iodine is used as a tracer compound in determining the total thyroxine content of blood serum. For example, the serum proteins are first denatured and the stable T-4 is extracted with alcohol. A measured amount of radioactive T-4 bound to serum protein is then added to the extract. Exchange takes place between the stable T-4 and the radioactive T-4 so that, other factors being equal, the amount of radioactive T-4 that is bound to protein will be propor-tionaJ, to the amount of stable T-4 present in the original sample of blood serum. The actual amount of stable T-4 in an unknown sample can be then estimated by comparison with control samples to which known amounts of stable T-4 have been added.

To determine the thyroxine binding-index (TBI) of serum proteins, L-triiodothyronine containing radioactive iodine (hereinafter referred to as "T-3") is used as the tracer. T-3 is bound by globulin in the same manner as T-4 but less tightly, and therefor T^-4 is not significantly displaced by T-3. Thus when an excess of radioactive T-3 is added to a serum sample the amount that is bound to the serum protein is directly proportional to the TBI and the latter is determined by reference to a control sample containing a standard serum whose TBI is known.

The serum protein, thyroxine-binding globulin ("TBG") has a relatively high and specific affinity for binding the thyroid hormone substance thyroxine "T-4". It is also known that if radioactive T-4, that is T-4 containing radioactive iodine such as iodine-125, is added to a solution containing barbital buffered TBG, essentially all of the T-4 will be bound to the TBG.

If stable T-4 is then added to the TBG solution, the radioactive T-4 will be displaced from the TBG in proportion to the amount of stable T-4 that is added.

If another T-4 binding agent, such as an ion-exchange resin,, is now added to this system, it will bind the radioactive T-4 that has been displaced from the TBG.

The degree of radioactive T-4 displacement and hence the amount of stable thyroxine added to the TBG can be determined by removing the. ion-exchange resin from the system and comparing the radioactivity now emitted by the TBG solution with its original radioactivity. When increasing amounts of stable T-4 are added to solutions containing the same amounts of radioactive T-4 and TBG and treated as above, the radioactivity of the TBG solution decreases with each successive increase in added stable T-4. By plotting the radioactivity of the treated sample against the amount of stable T-4, a graph is ob-. tained like that shown in Figure 1. If unknown amounts of T-4 are added to the TBG solution and treated in an Identical manner, the amount of T-4 thus added can be read from the graph.

For this determination, diluted blood serum is customarily used as the source of TBG. It has not . been the practice to subject it to any further treatment.

In order to determine T-4 in blood serum, the serum T-4 must first be separated from the binding proteins. This is, accomplished, by denaturing the proteins with alcohol. The denatured proteins release most (approximately 80 ) of their bound T-4 which is removed in the alcoholic supernatant after centrifugation.

In methods known heretofore, this alcoholic extract is evaporated to avoid possible errors Introduced by alcohol in the test mixture. Generally, 30 to 120 minutes are required to complete this step, depending upon the number of samples tested , The lron-bindlng-capacity of serum is determined in much the same way as the thyroxine -bindlng-capa-city using a tracer compound containing a radioisotope of iron, such as a radioactive ferric ammonium citrate.

It has also been known to employ ion^-exchange resins to adsorb excess tracer compound that is not bound to the serum protein in the sample. Such ion-rexchange resins have been used in the form of granules or In the form of a synthetic sponge which has been impregnated with the resin. In either case, difficulties in separating the resita from the liquid sample are the source of significant errors. For example, when using granular resins, errors may result from particles of resin which inadvertently remain in the liquid. In the case of resin-impregnated sponges, the sponge must be carefully squeezed and rinsed in order to remove all of the liquid sample before accurate radio-activity counts can be made. This separation is time consuming and subject to error. For routine testing of large numbers of blood samples, a simpler, quicker and more accurate method of adsorbing and measuring the excess tracer compound that is not bound to the serum protein is needed.

The present invention is broadly directed to methods of In vitro blood analysis which^ cpmprise the steps of preparing a liquid sample containing blood serum protein and a predetermined amount of a radioactive tracer compound capable of being bound by the serum protein and by an ion-exchange resin, adding a relatively thin strip of a membrane consisting essentially of an ion-rexchange resin to the sample, maintaining the membrane in contact with the liquid sample for a predetermined length of time, separating the membrane from the sample and determining the relative amounts of the tracer compound bound to the serum and to the membrane.

The present invention is also directed to the method of determining the thyroxine content of blood serum using radioactive thyroxine as a tracer compound which comprises the steps of adding to a known quantity of a reagent consisting essentially of a solution containing thyroxine -binding globulin and radioactive thyroxine, an alcoholic extract of a sample of blood serum whose thyroxine content is to be determined and an ion-exchange resin, maintaining the globulin solution in contact with the resin for a predetermined length of time, separating the anion exchange resin from the globulin solution, and comparing the radioactivity of the globulin solution with that of globulin solutions to which have been added known amounts of stable thyroxine dissolved in the same volume of alcohol from the .same source as that present in said alcoholic serum extract. The invention also includes a special reagent for use in carrying out the above-described method and a kit far facilitating the practice of the method.

The serum proteins include . globulin, albumin and prealbumin which have the capacity to bind and transport thyroxine, a hormone produced by the thyroid gland. The tracer material is a substance such as thyroxine which is bound by the serum substance and which contains a radioactive element such as a radioisotope of iodine. The tracer compound may be either a naturally occurring blood constituent or a synthetic substance which has similar properties and behaves in a similar manner, such as for example L-trllodothyronlne which resembles the natural hormone thyroxine. Alternatively, the tracer compound may be one that yields an element such as Iron that Is also normally present in blood serum, as a protein-bound constituent.

The sole function of the ion-exchange resin is to adsorb free, tracer compound so that It can be removed from the sample. Since the membrane is quite thin, CO herent, and essentially monoabsorbent, it is easily and quickly removable from the sample, using for example, a pair of forceps. The amount of tracer compound bound to the protein in the liquid sample or the free tracer compound in the resin membrane can then be determined by counting the radioactivity in one or the other using a conventional radiation counter. Since the amount of radlpactlve tracer compound originally added to the sample is known, it is sufficient to measure the radioactivity in either the resin membrane or the liquid sample, from which radioactivity in the other can be calculated. Ordinarily it is most convenient to count the radioactivity in the liquid sample, i.e., the tracer compound bound to the serum protein.

The ion-exchange resin membranes employed in the present invention are relatively thin strips, sheets or films of a solid hydrous gel consisting of an insoluble polymeric matrix to which are attached dissociable cationic or anionic groups, the gel being preferably reinforced with some suitable fibrous material. Ely "dissociable cationic group" is meant a group containing a dissociable anion capable of exchanging with the anion to be adsorbed, e.g., a radioactive, iodide ion, Similarly a "dissociable anionic group" contains an exchangeable cation. Many useful resin membranes of this kind are known, as for example those described in United States patents Nos . 2 , 730, 768, 2 , 780, 604 , 2, 800, 445 and 2 , 860, 097. In one membrane of this type, for example, the matrix is a copolymer of vinyl, compounds, the dissociable cationic groups are quaternary ammonium groups and the reinforcement consists of loosely woven fibers of a copolymer of vinyl chloride and acrylonitrile sold under the trade designation "Dynel". pesirably, thin strips of membrane are used in the practice of the invention and the thickness thereof may range, for example, from about 5 mils to about 50 mils, .

At the end of the incubation period the resin membrane, to which a portion of the radioactive tracer compound is now bound, can be removed from the liquid sample very quickly and easily, as for example by grasping it with a pair pf tweezers. As the membrane is withdrawn the tip may be touched to the side of the test vial in order to remove any adherent droplets of liquid sample.

It is not necessary to rinse the membrane since the film of liquid sample on the surface of the membrane is too small to have any significant effect on the test results, and unlike resin-impregnated sponges, the membranes have essentially no absorbent capacity. Moreover, because of the physical integrity of the resin membrane, there is no danger of erroneous results caused by resin particles remaining in the liquid sample.

The diagnostic tests of the invention are based upon comparing samples of blood serum with control samples ' < whose values are known. Therefore, It is only necessary that both unknown and control samples be incubated under identical conditions, the most important of which are duration and temperature. The use of a constant temperature both or chamber is not necessary. The simplicity of the present method facilitates Incubating a number of unknown samples together with a control sample under essentially identical conditions with respect to time and temperature. Precise control of these variables is there -fore less critical than with the methods formerly known and used .

The vessel for containing the liquid sample may be of any suitable inert material, such as glass or plastic, and should be . of a form and with dimensions such that it fits into the well of a scintillation counter.

The following examples further illustrate the invention.

Example 1 Determining Total Thyroxine Content of Blood Using Thyroxine-1-125 Bound to Globulin In the following example the matrix of the ion-exchange membrane is a copolymer of vinyl compounds, the dissociable cationic groups being quaternary ammonium groups and he reinforcement consisting of loosely woven fibers of "Dynel" (a copolymer of vinyl chloride and acrylonltrile) . A commercially available membrane of this kind is type AR-111 manufactured by Ionics Incorporated, Watertown, Massachusetts. The membrane has a thickness of 24 mils, contains 34$ by weight of the resin and has a capacity of 1.8 milli-equivalents per gram of A- dry resin. A strip of this membrane approximately 1 x 4 .5 centimeters has many times the capacity required to adsorb the free T-4 in the following tests.

To 1 cc. of blood serum contained In a centrifuge tube Is added 2 cc. of 95$ ethanol, and the contents are well mixed to denature the serum proteins. The tube , Is then centrlfuged at 2500 rpm for 4 -5 minutes. Next, 0.3 ml. of the supernatent liquid Is removed and transferred to a 4 ml. test vial and to this is added 3 ml. of a pH 8.6 barbital buffer solution containing 0.64$ sodium chlcrlde, 0.23$ barbital sodium, and 0.6$ barbital.

A series of reference samples containing 0, , 10, 15 and 20 mlllimicrograms of stable T-4 in 0.2 ml. of ethanol is also prepared. To each is added 3 ml. of the barbital buffer solution.

To each of the test vials prepared as abpve is then added 1 ml. of a barbital buffer solution containing 2$ human serum to which is bound T-4 containing a known amount of iodine-125, e.g., 0. 1 microcurie. A strip of the ion-exchange resin membrane is also added to each vial. The dimensions of the membrane should be such that it fits easily in the vial without binding.

The vials are then rotated for 1 hour at room temperature, opened, and the membrane is immediately removed from the vial with a fprceps. It is not necessary to rinse the membrane as the amount of liquid adhering to it is of no practical significance.

The radioactivity of each vial is then measured by means pf a scintillation counter. The radioactivity of each standard reference sample is graphed against the ' - . ' ' ·' A- number of mlllimlcrograms of stable thyroxine which It contains.

The number of mlllimlcrograms of stable thyroxine present In the unknown serum sample or samples can then be read from the standard graph. This value Is divided by 0,80, since ethanpl extracts approximately 80$ of the thyroxine present in the serum. The corrected value then represents the amount of the thyroxine present In each 0.1 ml. of the serum, or the number of micrograms of thyroxine per 100 ml. of serum.

Example 2 Determining Thyroxine Binding Capacity A membrane of the same kind described In Example 1 Is used in the following test. The following additional reagents are also employed: (¾) Incubation Vials - each containing 3.5 ml.

Buffer Solution and approximately 0.1 microcuries of T-3 containing iodine-131.

The buffer solution had the following compo-r sition: 2.42$ trie (hydroxy methyl) amlno- methane in aqueous solution whose pH was adjusted to 7.2 by adding approximately 15 ml. of concentrated hydrochloric acid per liter of the solution, (b) Standard Control Serum - (a serum whose thyroxine-binding-capacity is "normal") .

The serum samples to be tested are obtained by withdrawing about 5 ml. of venous tylood, allowing it to clot in a test tube and removing the serum. The radlo-activity of the Incubation vial containing 3.5 ml. buf- fer and iodine-I3I T-3 is first counted and this value is recorded as the PreTest Count. Exactly 0.5 ml. patient serum is then added to the incubation vial. For each series of tests, exactly 0.5 ml. of control serum is added to an incubation vial, in the same manner as for the unknown serum sample. A strip of the resin membrane is added to each vial which is capped and incubated for 2 hours at room temperature, the vial being rotated gently to insure that all of the liquid sample comes in contact with the resin membrane. After the incubation period, the resin membrane is removed from each vial wi1;h forceps, allowing the strip to touch the inside lip of the vial as It is withdrawn to remove any drops of liquid clinging to the strip. Each unknown serum and the control serum are counted In a well counter for 1 minute and the value is recorded as the "postcount " .

The T-3 index, a convenient measure of the thyroxine-binding-capacity, is calculated as follows: I¾estU"ount x 100 - ≠ uptake of serum uptake for the unknown serum T _ , d Jo uptake for the control serum ^ naex The status of an individual's thyroid function Is proportional to the degree of saturation of available T-3 binding sites in the serum proteins. For example, In a patient with a hyperactive thyroid the available sites are filled to a greater extent than in the person with a functionally normal thyroid. Similarly, a hypothyroid individual exhibits more unfilled binding sites than does the euthyroid patient.

Example 3 For the following test the ion-exchange resin membrane is the same as that described in Examples 1 and 2.

To a vial containing 3 ml. of a buffer solution (see Example 1) was added approximately 7 ugm of stable iron, a radioactive tracer compound containing approximately 0.1 uc of iron-59, and 1 ml. of blood serum. The iron was added in the form of ferric ammonium citrate.

The serum-buffer solution is mixed and allowed to stand at least 10 minutes at room temperature.

A strip of ion-exchange membrane is added to the vial which is capped and incubated for 30 minutes at room temperature while rotating it gently to insure thorough contact with the resin membrane.

After the Incubation period, the resin strip is removed and the solution assayed for radioactivity. The activity in this vial is compared to that in a standard which contains 1 ml. of water in place of the blood serum.

The LIBC of the serum sample may be calculated using the following equation: ^ugm Fe/ml. serum (LIBC) = Serum Counts per Min. x μ Fe in Standard Fe standard cpm.

Figure 1 is a graph depicting an illustrative standard curve obtained by plotting the radioactivity of treated thyroxine-binding globulin samples against amounts of stable thyroxine, Using the method of the present invention exemlified below the time re uired to determine th roxine in blood serum is substantially shortened since evaporation of the alcoholic extract is unnecessary. Moreover, using the special TBG reagent disclosed herein, the accuracy of the method is substantially improved because the slope of the line shown in Figure 1 is steeper than that obtained with untreated dilute TBG reagent under the same conditions.

Example 4 About 10 ml. of blood whose thyroxine is to be determined is withdrawn and allowed to coagulate. The serum is removed and 1 ml. is added dropwise to 2 ml. of 95$ ethanol in a centrifuge tube and the contents are well mixed to denature the serum proteins. This alcoholic mixture is then centrifuged at 2500 rpm for 4-5 minutes and 0.3 ml. of the supernatant liquid is withdrawn and transferred to a suitable test vial which contains 4 ml. of the special TBG solution whose preparation is described hereinafter.

In the same manner, several control samples are prepared. In place of the ethanolic serum extract, 0.3 ml. of an ethanolic solution containing a known amount (e.g.,. 0, and 12 nanograms) of stable thyroxine are added.

A strip of ion-exchange resin membrane is then added to each test vial. Since thyroxine is amphoteric, the ion-exchange resin may be either anion-selective or cation-selective. Many such resins are available in the form of membranes. For example, a commercially available anion-selective resin suitable for the purposes of this invention is designated "AR-lll" (manufactured by Ionics Incorporated, Watertown, Massachusetts).

After the resin strips are added, the vials are capped and rotated for exactly 1 hour at room temperature . It Is convenient to use one of the commercial rotators es pecially designed for this purpose . Since the resin uptake of radioactive thyroxine is a function of rotation time, it is essential that the rotation time be the same for both the unknown and control samples . At the end of the 1 hour rotation time, the resin strip is removed with a forceps and discarded . The radioactivity of each vial is then counted and recorded as the "post-count " . A minimum of 10, 000 counts per minute will insure an error of less than 2$. The ' "pre -count " is also advantageously determined by counting an unused vial containing the TBG-radlpactive T-4 solution. The pre-count/post -count ratio is calculated for each of the samples . The values for the standard samples are then plotted against the amount of added T-4, a straight line is drawn through the points , and the thyroxine content of the unknown serum samples is read from the result ing graph (see Figure 1 ) . Since the alcohol extracts approximately 80$ of the T-4 from the serum, the value read from the graph should be divided by the extraction efficiency (approximately 0, 8) to give the actual thyroxine content of the serum. This corrected value then represents the amount of T-4 (nanograms ) in each 0.1 ml . of serum whi ch is numerically equivalent to the number of /Jgm. of T-4 per 100 ml . (/em . ) of patient serum.

The special TBG solution Is conveniently prepared by pass ing the blood serum through a column containing a suitable anion<-exchange res in which is capable of adsorbing the thyroxine but not the ser um protein.

A suitable procedure for preparing the special TBG solution Is as follows. To 137 ml. of serum Is added 328Ο ml. of a pH 8.6 barbital buffer solution containing 0.64$ sodium chloride,. 0.23 barbital sodium, and 0.6$ barbital. A small amount of radioactive thyroxine (approximately .24 milllcuries) is also added to serve as a tracer for determining the efficiency of the extraction.

An extraction column for this purpose is prepared by adding a sufficient amount of resin to a suit-able glass column to provide a bed volume of 1200 ml. and a flow rate of approximately 25 ml. per minute. For this purpose, a strongly basic quaternary ammonium type resin is suitable. For example, a commercially available resin of this kind designated "IRA-400" (manufactured by the Rohm and Haas Company) is satisfactory, but other anion-selective or cation-selective resins may be used instead.

The resin is added to the column as a water slurry and a layer of water is always maintained above the resin. The column is connected to a reservoir containing the serum solution to be extracted, and flow of the serum through the column is begun. Periodically a 1 ml. sample of the eluate is removed and its radioactivity counted to measure the efficiency of the extraction.

The radioactivity of the eluate should be 10$ or less that of the stock solution. The extracted serum solution is then preferably passed through a 0.22 micron filter. More (approximately .30 milllcuries) radioactive thyroxine is then added to the eluate and the latter is diluted with six times its volume of the aforementioned buffer solution. The resulting solution should have a pH of 8.6.

The usefulness and accuracy of the method is Increased if the analyst Js provided with a kit containing all the special reagents and other supplies required. For example, a useful kit consists of a number of ml. test vials made of glass and provided with screw caps. The vials should include 4 cc. of the above TBG solution and should be matched for radioactivity. The standard samples should contain known amounts of stable thyroxine (e.g., 0 and 12 nanograms) in 0.3 mlt of alcohol. A container of alcohol from the same source used to prepare the control samples should also be supplied. The kit should also include the required ion-exchange resin, the latter preferably being in the form of membrane strips. The other necessary equipment such as syringes, radiation counter and rotator are standard laboratory equipment.

Claims (25)

1. The method of in vitro analysis of blood which comprises: preparing a liquid sample containing blood serum protein and a predetermined amount of a radio active tracer compound capable of being bound by the serum protein and by an ion-exchange resin; adding a relatively thin strip of a membrane consisting essentially of an ion-exchange resin to the sample; maintaining said membrane in contact with the liquid sample for a predetermined length of time; separating the membrane from the sample; and determining the relative amounts of the tracer compound bound to the serum protein and to the membrane .

2. . The method of claim 1 wherein the determination is made by counting the radioactivity in the serum protein and/or counting the radioactivity in the membrane

3. . The method of claim 1 or 2 wherein the blood serum protein is selected from the group consisting of globulin, albumin and prealbumin.

4. The method of claim 1, 2 or 3 , wherein the radioactive tracer compound is a thyroid substance.

5. The method of claim 4 wherein the thyroid substance contains radioactive iodine selected from iodine-125 or iodine-131.

6. The method of claim 4 wherein the thyroid substance is selected from the group consisting of thyroxine containing iodine τ125 and L-triiodothyronine containing iodine -131.

7. The method of claim 1, 2 or 3 wherein the radioactive tracer compound is ferric ammonium citrate containing iron-59.

8. The method of any of the preceding claims wherein the membrane has a thickness of from about 5 mils to about 50 mils „

9. The method of any of the preceding claims wherein the membrane is a solid hydrous gel comprising an insoluble polymeric matrix to which are attached dissociable ionic groups .

10. The method of claim 8 wherein the gel is reinforced with a fibrous material.

11. The method of claim 8 wherein the dissociable Ionic groups are quaternary ammonium groups.

12. The method of any of the preceding claims wherein the membrane is maintained in contact with the liquid sample at about room temperature.

13. The method for determining the thyroxine content. of blood serum using radioactive thyroxine as a tracer compound which comprises the steps of adding to a known quantity of a reagent consisting essentially of a solution containing thyroxine -binding globulin and radioactive thyroxine, (a) an alcoholic extract of a sample of blood serum whose thyroxine content is to be determined, and (b) an ion-exchange resin, maintaining the said globulin solution in contact with the said resin for a predetermined length of time; separating the anion-exchange resin from the globulin solution; and comparing the radioactivity of the globulin solution with that of globulin solutions to which have been added known amounts of stable thyroxine dissolved in the same volume of alcohol from the same source as that present in said alcoholic serum extract .

14. . 1 „ The method according to claim 13 wherein said reagent comprises a buffered solution of radioactive thyroxine and blood serum from which most of the naturally occurring thyroxine has been extracted.

15. . The method according to claim 13 or 14 wherein the radioactive thyroxine is iodine-125 .

16. The method according to claim 13 , l^ or 15 wherein the globulin solution is maintained in contact with the resin by rotating a. sealed vessel containing said solution and resin.

17. The method for preparing a reagent for use according to claim 13 in determining the thyroxine content of blood serum/which comprises passing diluted blood serum through a column containing an ion-exchange resin to extract most of the naturally occurring thyroxine from the serum, and adding radioactive thyroxine to the resulting eluate.

18. A reagent for use in determining the thyroxine content of blood serum which comprises a buffered solution of radioactive thyroxine and blood serum from which most of the naturally occurring thyroxine has been extracted.

19. A reagent according to claim 18 wherein the radioactive thyroxine contains iodine-125 .

20. A kit for determining the thyroxine con- ' serum according to claim 13 tent of blood -pAas-ffla-/comprising a buffered solution of blood serum containing not more than about 20$ of the normal amount of thyroxine and a sufficient amount of thyroxine-Γ1"2^ to act as a tracer, two control samples containing a known amount of thyroxine dissolved in a predetermined volume of alcohol, a quantity of alcohol from the same source used to prepare the control sample, and a plurality of resi membrane strips whose dimensions permit them to fit loosely in the text containers.

21. The method of in vitro analysis of blood as claimed in claim 1, substantially as herein described.

22. The method for determi ng the thyroxine content of blood serum using radioactive thyroxine as a tracer compound as claimed in claim 13, substantially as herein described.

23. The method for preparing a reagent for use in determining the thyroxine content of blood serum as claimed in claim 17, substantially as herein described.

24. A reagent for use in determining the thyroxine content of blood serum as claimed in claim 18, substantially as herein described.

25. A kit for determining the thyroxine content of blood serum as claimed in claim 20, substantially as herein described. S. HOROWITZ & CO. r AGENTS FOR APPLICANTS