METHOD FOR SCREENING BLOOD TO CONTROL AIDS TRANSMISSION

Abstract: Blood collected for storage and subsequent transfusion is tested for AIDS transmission potential by determining whether purine salvage enzyme activities are abnormal in the red blood cells. If the adenosine deaminase (ADA) and/or the purine nucleoside phosphorylase (PNP) activities are elevated, the blood is suspect and should not be used unless other tests establish its safety.

Description: METHOD FOR SCREENING BLOOD TO CONTROL AIDS TRANSMISSION GRANT REFERENCE - (This invention was developed in part under NIH/DHHS Grant No. 1 R43 Al 21209-01.) FIELD OF INVENTION, BACKGROUND AND PRIOR ART The field of the invention relates to tests for 10 screening blood to control transmission of the acquired im¬ munodeficiency syndrome (AIDS) . The serious new disease called acquired immuno- l ~ deficiency syndrome (AIDS) continues to spread at an alarm¬ ing rate. It has been found to be caused by a transmissibl viral agent. The probable cause is a human retrovirus as¬ sociated with adult leukemia/lymphoma called HTLV-III or 20 LAV. Popovic, et al. (1984) Science, 224:497-500; and Laur- ance, et al. (1984) N. Engl. J. Med. , 311 (2) .1269-1273. I has been shown that AIDS can be transmitted through blood o plasma. Marx (1983) Science 219:42-43; and "Facts About 25 AIDS," (Tauber, 1984), U.S. Public Health Service Bulletin. There is an urgent need for rapid, accurate, and relatively inexpensive tests for screening blood to control the transmission of AIDS. Three potentially useful diag¬ 30 nostic tests have been reported in the literature. The first test relates to the fact that patients with this syn¬ drome have essentially non-functioning cellular immune sys- 35 terns due to a quantitative defect in T-cells, particularly in the helper/inducer subset. This leads to a reversal of the normal helper-to-suppressor T-cell ratio among periph¬ eral blood lymphocytes. In this regard, researchers have noted the depressed ratio of 0KT-4+ (Leu-3+) (helper/ . - ¬ cytes. Gottlieb (1981) N. Engl. J. Med. , 305:1425-1431. However, reduced measurements of 0KT-4/0KT-8 ratios have been reported in a variety of other disease processes, which diminishes the specificity of this diagnostic tech¬ nique in screening for AIDS. Recently, Hersh, et al. (1983) N. Engl. J. Med. , 308: 45-86, reported a consistent elevation in the levels of serum thymosin 1 in eight patients. Further follow-up with a larger patient population needs to be performed be¬ fore this marker can be used to accurately assess AIDS. In addition, this test takes over one day and is difficult to perform. More recently, a radio-immunoprecipitation test was developed for the detection of antibodies to the major core protein of lymphadenopathy virus (LAV) . Kalyanaraman et al. (1984) Science, 225: 321-323. The presence of anti¬ body against core protein was, found in only 42% of serum samples from AIDS patients. Rubin, et al. (1981), I muno- harmacol, 3: 307-312. However, Sarngadharan, et al. (1984) Science, 224:506-508, has reported that 88% of AIDS patients examined have antibodies against p41, the presumed envelope antigen of HTLV III. 79% of pre-AIDS patients examined demonstrate antibodies to the same antigen. Tests were per¬ formed using an ELISA format. Such ELISA based tests, how¬ ever, will take approximately four hours to perform, and may exhibit a high false positive rate which will necessitate confirmation. This invention describes a blood screening method based on determination of purine salvage enzyme activity in red blood cells, which levels have been found to be abnormal in AIDS patients. More specifically, the enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) are elevated in a high percentage of AIDS-diagnosed It is known that children afflicted with the syn¬ drome of severe combined immunodeficiency disease (SCIDS) have an inherited deficiency of the enzyme adenosine de¬ aminase (ADA) in red blood cells. See Snyder et al. (1983) N. Engl. J. Med. , 308:45. Although the virol etiology of AIDS is quite different than " the genetic abnormality causin SCIDS, it was conceived that abnormal ADA or PNP levels might be an indication of AIDS. It has now been demon¬ strated that this abnormality does exist in AIDS patients, both ADA and PNP in red cells being elevated. This eleva¬ tion is clearly distinguishable from the SCIDS abnormality where these enzymes are depressed in red blood cells. ADA enzyme activity is normally present in a • variety of tissues including erythrocytes, leucocytes, liver, kidney, and skin. The highest activity is present in lymphoid tissue, specifically the thymus . The role of ADA in the thymus and other lymphoid tissues is not clear. Peripheral blood lymphocytes after stimulation by PHA (phytohemagglutinin) have an increased activity of ADA per cell as well as increases in other enzymes of the purine salvage pathway. An increase in serum as opposed to red blood cell ADA activity has been reported in patients with bronchi.-ti carcinoma and in other malignancies. Increased serum enzyme activity has also been re¬ ported in patients with infectious mononucleosis , hepatitis and hepatocellular jaundice. ADA as normally found in the organs previously mentioned would be expected to increase as a result of an increase in cell turnover due to malignancy or inflammation. Additionally, release of intracellular en¬ zymes due to necrosis could also cause an increase in serum enzyme activity. No increase in erythrocyte ADA or PNP ac¬ tivity has been reported as a result of such pathologic con¬ ditions. Further, one would not expect an elevation of ADA or PNP in the erythroid cell line by a disease process at a distant site. There have been two reports of increased ADA ac- tivity in red cells from a few patients with hereditary hemolytic anemia. Valentine, et al.. (1977) Science, 195: 783-785. Additionally, increased erythrocyte ADA activity, has been reported in patients with congenital hypoplastic anemia (Diamond-Blackfan Syndrome). Glader and Diamond, Blood, (1981) 58:42A. Significantly, these researchers verified no increase in red cell ADA activity in young RBC's (i.e., in patients with hemolytic anemia), in fetal red cells, in red cells from patients receiving steroid therapy, in patients with stress erythropoiesis , or in the syndrome of transient erythroblastopenia of childhood. No such in- crease has been reported for acute inflammatory processes, neoplasia, or chronic inflammatory disorders as reflected by red cell ADA activity. AIDS patients examined in the study leading to the present invention demonstrated no reticulocytosis which could account for increased ADA ac- tivity. Reticulocytosis not involving hereditary hemolytic anemia is not associated with increased red cell ADA ac¬ tivity. DETAILED DESCRIPTION The method of this invention is designed for use in conjunction with the operation of blood banks wherein human blood is collected, tested, stores, and administered to human subjects as blood or plasma. For presently ap¬ proved practices reference may be made to authoritative descriptions, such as "ABB Standards," 11th Edition, Paul J. Schmidt, Editor (Published by American Association of Blood Banks, Arlington, VA) . As part of present standard pro¬ cedures, samples of the collected blood are obtained for testing. More specifically, at the time of collection of a unit of blood (i.e., 450 ml with 63 ml of anticoagulant) two test tubes are also filled with the donor's blood: (1) one tube with anticoagulant (hepariή or ' citrate) , which is used to type the blood for ABO and RH antigens, and (2) a tube of clotted blood to test for hepatitis and syphilis. Since only a small amount of blood is required for typing, the excess can be used from the uncoagulated tube to test for RBC enzyme levels. Al ernatively, if needed a third tube can be collected with anticoagulant to provide the red blood cells for enzyme testing. For example, the sample may provide from 10 to 20 ml of heparinized blood. The RBC's are recovered by centrifuging, washed, and resuspende in a suitable aqueous buffer. The resuspended RBC's are ruptured by a freeze-thaw procedure, and the clarified supernatant recovered by centrifuging, which is used for the enzyme assays. determine whether the purine salvage enzyme (PSE) content of the RBC's is elevated above a normal level. The PSE level may be determined specifically with respect to adenosine de¬ aminase (ADA) , and/or purine nucleoside phosphorylase (PNP) . If elevated levels are detected in the supernatant prepared as described, the blood unit should be discarded, unless it is determined to be free of AIDS virus. If the ADA and/or PNP levels are in the normal ranges, AIDS transmission po¬ tential is not indicated. The blood unit, unless excluded by other tests, can be added to the blood bank. The screen¬ ing test of this invention can be used in conjunction with other tests for AIDS transmission potential or AIDS diagno¬ sis. In practicing the screening method of the present invention an understanding of purine catabolism is helpful. For. this purpose the catabolism of adenosine, inosine and guanosine by ADA and PNP is represented by the following chart. CATABOLISM OF ADENOSINE, INOSINE AND GUANOSINE BY ADA & PNP (1) II ADENOSINE GUANOSINE Key to Abbreviations : ADA = adenosine deaminase PNP = purine nucleoside phosphorylase XO = xanthine oxidase GDA = guanine deaminase (1) Based on Cowan and Amnann, (1981) Clinics in Haem oLo y 10(1):139. In the catabolism of adenosine by Pathway I, the enzyme ADA utilizes adenosine to produce inosine and ' ammonia (NH,). Inosine in turn is catabolized by PNP to hypoxan- thine, which is a substrate for xanthine oxidase (XO) . The product xanthine is further converted in the presence of oxygen (0 2 ) to uric acid. According to Pathway II, purine guanosine is catabolized by PNP to guanine, which is acted on by guanine deaminase (GDA) to form xanthine and ammonia. As with Pathway I, xanthine is converted to uric acid by xanthine oxidase (XO) . Pursuant to Pathway I, ADA activity can be deter¬ mined using adenosine as the substrate, by measuring the absorption of any of the degradation products: NH- , inosine, hypoxan hine, xanthine, and uric acid. Similarly, PNP ac¬ tivity can be determined by using inosine as a substrate ac¬ cording- to Pathway I and quantitating the amount of hypo¬ xanthine, xanthine or uric acid formed. PNP can also be determined using guanosine as a substrate following Pathway II, and detecting the production of guanine, NH-, , xanthine, or uric acid. Detailed descriptions for determining ADA levels include chro atographic and spectrophotometric methods. The chromatographic assay employs radio-labeled adenosine and determines the quantity of radioactive product inosine. See, for example, Coleman and Hutton (1975), Bioche . Med. 13:46-55; and Coleman, et al. (1978), J. Biol. Chem. 253 (5) :1619-1626. The spectrophotometric methods quantitate the formation of the products inosine, guanine, or uric acid by monitoring the enzyme catalyzed reaction at specific wavelengths of absorption for these compounds. The spectro- p n....„ . See, for example, Kalkar (1947) J. Biochem. , 167:429-443; Hopkinson, et al. (1969) Human Genet. 32:361-367; and Cowan, et al. (1982) Cellular Immunol. 67:121-128. For example, inosine may be tected at 265 nm and uric acid at 293 nm. For a description of an automated colorimetric determination of uric acid, see Gochman and Schmitz (1971) , Clinical Chem. 17:1154-1159. In this procedure uricase-generated hydrogen peroxide is measured by oxidative coupling of 3-methyl-2- benzothiazolinone hydrazone and N, N-dimethylaniline in the presence of peroxidase to produce a stable, sensitive inda- mine dye. In another procedure, uric acid can be oxidized to allantoin and carbon dioxide by phosphotungstic acid, which is reduced in the reaction to tungsten blue and de- tected spectrophotometrically. This invention and the results obtainable thereby are further illustrated by the following examples. EXAMPLE I Materials and Methods Sub ects Blood samples were obtained from 19 patients with clinically documented AIDS: 9 adults with Kaposi's sarcoma, 7 patients with opportunistic infections (pneumocystis carinii) , and four patients with both Kaposi's sarcoma and opportunistic infection. All had evidence of a T-cell as- sociated immunosuppression documented by reduced T4/T8 and depressed in vitro T cell responses to pokeweed mitogen and PHA. Additionally, four patients with lymphadenopathy syn¬ drome (an earlier clinical form of AIDS) were investigated as well as eleven "healthy" homosexuals at risk for AIDS. Sixteeri randomly selected normal heterosexual controls were also studied. Measurement of Enzyme Activities Heparinized peripheral blood (10-20 ml) was ob¬ tained from human adults and centrifuged over a Hypaque- Ficoll cushion. Peripheral blood mononuclear cells (PBMC) were removed and RBC's were washed three times with PBS, and the RBC pellet was resuspended in 50 mM KPO, pH 7.4 in 12 x 75 ml glass tubes. The suspension was freeze-thawed four times in a dry ice-acetone bath and centrifuged in a microfuge (Beckman) for 1 min. The supernatant was stored at -70° (Revco) until the assays were performed. Pre¬ liminary studies showed enzyme. activity stable for up to six months when prepared in this manner. ADA activity (Vmax) was determined using the spectrophotometric method of uric acid production as described by Hopkinson, et al. (1969), Hum. Genet. 32:361-367. Briefly, ADA activity was deter- mined in a reaction mixture consisting of 0.795 ml 50 mM KPO, buffer, 0.005 ml xanthine oxidase, 0.1 ml of varying concentrations of adenosine (lOmM) , 0.005 ml PNP (purine nucleoside phosphorylase) and 0.1 ml of the extract. The PNP was diluted 1:20 in buffer prior to addition to the re¬ action mixture. This mixture was incubated at 37° C for 45 sec. in a water bath prior to being placed in a recording spectrophotometer. Contaminating ADA activity in the PNP was determined and subtracted from the measured ADA ac¬ tivity in the cell extracts. The change in optical density was read at 293 nm and was recorded over a 15 minute period. This activity was linear as functions of time and hemoglobin concentration of the extract. Hemoglobin concentrations were measui.:- y convert ng emog o n to cyanomet emog o n. Mean corpuscular hemoglobin concentration is assumed to be constant. The statistical analyses used Student's t test. The results are expressed as nanomoles of uric acid pro¬ duced per mg of hemoglobin per hour. The average normal ADA level was taken as less than 30 nmoles/mg/hr. Results The data is summarized in Table A. Of the 19 patients with AIDS, 16 (84%) had a significant increase in RBC ADA enzyme activity. As a subgroup, patients with both Kaposi's sarcoma and opportunistic infections had the high¬ est increase in enzyme activity, with average activities three times greater than control subjects. Patients with either Kaposi's sarcoma or opportunistic infections alone had enzyme levels approximately two-fold higher than ' con¬ trols. Significantly, 4 of 4 patients with lymphadenopathy syndrome (100%) and 5 of 11 "normal" healthy homosexuals (45%) demonstrated increased ADA activity. A two tailed Student's t test was used to assess the statistical validity of the data. Overall, adenosine deaminase activity was significantly increased in AIDS patients p<.004. None of the AIDS patients had hemolytic anemias or increased reticulocyte counts. Discussion The results demonstrate a direct relationship be¬ tween the enzyme adenosine deaminase and the disease AIDS. Levels of ADA are elevated from two to three-fold in 84% (16/19) of the patients investigated. This increase was seen in AIDS patients regardless of the symptoms, i.e., Kaposi t. sarcoma, opportunistic infections, or a combination of the two. This increase does not appear related to an in¬ crease in reticulocytes in these patients and hemolytic anemia and reticulocytosis is not observed as a part of the constellation of symptoms associated with AIDS. Signifi¬ cantly, a subpopulation of "healthy" homosexuals had a sig¬ nificant increase in ADA activity. These subjects were at high risk to get AIDS and as such it appears this test al¬ lows for early detection of the disease. Because of its two to three year prodrome, it is important to detect early AIDS (i.e., lymphadenopathy patients as well as patients at risk) so that blood products containing HTLV III are not transmitted to healthy recipients. t_> in o LΠ o uι TABLE A ADA ACTIVITY IN RED BLOOD CELLS l-KOM l'ATIENTS WITH AIDS AND CONTROL Nι>. ui " No. of Patients Mean STD Error rouj> 1'al i i-nl : With Elevated ADA (1) ADA Activity of Mean (nmoles uric acid/ingllb/hr) AI S Kaposi's sarcoma (KS) 9 7 36.3 Opportuni tic Infection (01) 6 5 34.0 KS and 01 . . 53.3 AIDS (All) ιy 16 39.0 Lymphadenopαihy Syndrome (I.ΛS) . 42.7 Control Healthy heterosexual v lu i ers 16 2 22.9 2. . Heal hy homosexuals at risk 11 5 32.6 3.9 (1) I! I L-V.II ι-d ADA .I". I t v i i y i ι I )iι mi. ■> 1.. • -./ui)',/ In" This example describes an alternate procedure for determining ADA in red blood cells (RBC's). The RBC's are suspended in 50 mM potassium phosphate buffer, pH 7.0, 2.5"% Triton X-100 and 1 mM mercaptoethanol at a concentration of o 1 x 10 cells /ml. The cell suspension is homogenized with 50 strokes with a motor driven teflon pestle in a glass Dounce homogenizer. The homogenate is centrifuged at 7000 x G for 10 minutes and the supernatant fractions assayed for adenosine deaminase activity (ADA) . The ADA.activity can be measured by the chromato¬ graphic method previously described. Coleman, et al. (1978) J. Biol. Chem. 253:1619-1626. This consists of measuring the conversion of + C adenosine to inosine. The standard reaction mixture is composed of 50 mM potassium phosphate buffer, pH 7.0, and O.lmM C adenosine (Amersham Corp., Arlington Heights, IL.) Five to 25. ul'* of supernatant frac¬ tion is added to the appropriate amount of reaction mixture to yield a final volume of 100 ul. After incubation at 35°C, a 20 ul aliquot is spotted onto a strip of Whatman DE-81 chromatography paper. Unlabeled carrier inosine is spotted at the origin. Strips are 23 cm x 10 cm and each strip has 12 channels for product application. Strips are developed in 1 mM ammonium formate by descending chromatography. The chromatography step is complete in 30 to 60 minutes. The DE-81 strips are dried at 105°C, the inosine spot is cut out and counted in a toluene -0.47.BBOT cocktail in a scin¬ tillation counter. ne un t o enzyme act v ty s equa to one nmo e C inosine produced per minute. Separation of the sub¬ strate from product on DE-81 paper provides a rapid, sensi- tive procedure for assaying this enzyme. Using the descending chromatography and the conditions described adenosine travels with the solvent front and inosine moves a short distance from the origin in 30 to 60 minutes. Hypo- xanthine and xanthine move slightly behind inosine. EXAMPLE III In conjunction with the experiments reported in Example I, PNP activity was determined. The procedure used was as follows. Lysates from red blood cells were obtained as previously described above. Briefly, for measuring PNP activity the reaction mixture consists of 0.795 ml 50 mM KPΘ, buffer, 0.005 ml xanthine oxidase (Sigma X450) of vary¬ ing concentrations of inosine (0.1-10 mM) and 0.1 ml of ly- sate. This mixture is incubated at 37°C for 2 min. in a water bath prior to being placed in a recording spectro- photometer (Gilford) . The change in optical density is rea at 293 nm over a 15 mm period. Vmax is determined from Lineweaver-Burke plots at substrate saturation. Statistical analysis of data is as previously described. The data is summarized below in Table B. As show by the data of Table B, PNP levels were significantly ele¬ vated especially in AIDS patients with KS and 01. Impor¬ tantly, highest elevations of PNP occurred in patients with earlier manifestations of AIDS, i.e., lymphadenopathy syn¬ drome and in patients at risk. PNP determinations may therefore be used instead of or in addition to termination of the ADA levels. PNP may also be measured using guanosine as a sub strate and detecting uric acid production spectrσphot ' o- metrically at 293 nm. Lysates from RBC's were obtained as previously described. The same method as in Example III was used but guanosine was substituted for inosine. TABLE B PNP Activity in Red Blood Cells From Patients with AIDS and Control No. of Mean STD Error Group Patients PNP Ac ivicv of Mean (nmoles uric acid/mg/Rb/hr) AIDS Kaposi's sarcoma (KS) 1887 104 Opportunistic Infection (01) 6 1824 223 KS and 01 4 2148 143 Lymphadenopathy Syndrome 2179 158 Control Healthy heterosexual volunteers 1863 120 Healthy homosexuals at risk 11 2255 105

Claims: CLAIMS I CLAIM: 1. ■ In the operation of blood banks wherein human blood is collected, tested, stored, and transfused to human subjects as blood or plasma, the method of screening col¬ lected blood for potential transmission of the acquired i - munodeficiency syndrome (AIDS) , comprising obtaining a test sample of red blood cells (RBC's) from blood collected from an individual donor prior to transfusion use thereof, deter¬ mining whether the purine salvage enzyme (PSE) content of the sampled RBC's is elevated above a normal level, and dis¬ carding blood having RBC's with elevated PSE. 2. The method of claim 1 in which said PSE level is determined only with respect to adenosine deaminase (ADA) , 3. The method of claim 1 in which said PSE level is determined only with respect to purine nucleoside phos¬ phorylase (PNP) . 4. The method of claim 1 in which said PSE level is determined with respect to both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) . 5. The method of claim 1 in which said determina- tions are made by enzymatic conversion of substrates con¬ taining adenosine for ADA detection, and inosine or guanine for PNP detection, said substrates being formulated to pro¬ duce uric acid as the final product, and detecting the amount of uric acid produced. 6. The method of claim 2 in which said determina¬ tion is made by enzymatic converstion of a substrate con¬ taining adenosine, said substrate being formulated to pro¬ duce uric acid as the final product, and detecting the amount or pro uce . 7. The method of claim 3 in which said determina¬ tion is made by enzymatic conversion of a substrate contain- ing inosine or guanine, said substrate being formulated to produce uric acid as the final product, and detecting the amount of uric acid produced. 8. In the operation of blood banks wherein human blood is collected, tested, stored, and transfused to human subjects as blood or plasma, the method of screening col¬ lected blood for potential transmission of the acquired im¬ munodeficiency syndrome (AIDS) , comprising obtaining a test sample of red blood cells (RBC's) from blood collected from an individual donor prior to transfusion use thereof, deter¬ mining whether the purine salvage enzyme (PSE) content of the sampled RBC's is elevated above a normal level, said PS level being determined with respect to adenosine deaminase (ADA) and/or purine nucleoside phosphorylase (PNP) , and dis¬ carding blood having RBC's wit elevated PSE.

Inventors: WIDDER KENNETH J, WIDDER, KENNETH, J.
Applicants: MOLECULAR BIOSYSTEMS INC, MOLECULAR BIOSYSTEMS, INC.
Application year: 1986
Main classification: C12Q1
IPC codes: C12Q1, G01N33