Forensic Bloodstains and Physiological Fluid Analysis - ACS

15 Forensic Bloodstains and Physiological Fluid Analysis W. C. STUVER, ROBERT C. SHALER, and PETER M. MARONE P i t t s b u r g h a n d A l l e g h e n y C o u n t y C r i m e L a b o r a t o r y , Pittsburgh, P e n n .

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Downloaded by UNIV OF SOUTHERN CALIFORNIA on June 22, 2013 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/bk-1975-0013.ch015

RALPH PLANKENHORN Pennsylvania

State P o l i c e C r i m e L a b o r a t o r y , Greensburg, P e n n .

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The goal of f o r e n s i c serology is to i n d i v i d u a l i z e blood s t a i n s by i d e n t i f y i n g genetic markers whose population frequen- c i e s have been e s t a b l i s h e d . T h i s goal may soon be w i t h i n our reach. I t appears, how-ever, that t h i s will not be a one step a n a l y t i c a l procedure but a s e r i e s of analyses utilizing s e v e r a l components of the blood from which a profile of genetic markers can be e s t a b l i s h e d . Since these markers i n blood are i n h e r i t e d independent of one another and t h e i r frequencies w i t h i n a given population are known, the p r o f i l e obtained will permit a mathematical p r o b a b i l i t y or uniqueness to be c a l c u l a t e d . Thus, blood evidence will always remain i n the realm of p r o b a b i l i t y ; however, as with f i n g e r - p r i n t s , the p r o b a b i l i t y of two people having e x a c t l y the same profile may be so remote that a c o n c l u s i o n can be made as to its origin. Blood i s a multi-component system with formed elements of red and white blood c e l l s as w e l l as p l a t e l e t s , and a l i q u i d f r a c t i o n (plasma), each c o n t a i n i n g a vast array of biochemical c o n s t i t u e n t s . The f o r e n s i c s e r o l o g i s t has chosen three c l a s s e s of the blood c o n s t i t u e n t s f o r t h e i r genetic information and use i n i n d i v i d u a l i z a t i o n endeavors. These c o n s t i t u e n t c l a s s e s are 1) the blood grouping and typing antigens, 2) the polymorphic enzymes and 3) the polymorphic p r o t e i n s . The f a c t that blood grouping and/or typing antigens e x i s t has been known s i n c e Landsteiner discovered the ABO system around 1900 (1). Since then over 246 published antigens have been found; however, only three of these a n t i g e n i c systems, the ABO, MN and Rh, have r e c e i v e d crime l a b o r a t o r y acceptance (2). Until s e v e r a l years ago most crime l a b o r a t o r i e s d i d only ABO groupings; however, with the improvements of s p e c i f i c a n t i s e r a and the increased s e n s i t i v i t y of d e t e c t i o n techniques, the MN and Rh systems have a l s o been adopted as r e l i a b l e systems. The ABO, MN and Rh systems, u n l i k e l y many of the other a n t i -genic systems, have u s e f u l population f r e q u e n c i e s . For instance, the four groups belonging to the ABO system occur i n

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approximately the f o l l o w i n g percentage frequencies: 0 - 44%, A - 44%, Β - 8% and AB - 3%. The MN system has three groups having the f o l l o w i n g frequencies: M - 30%, MN - 50% and Ν - 20%, and the Rh system b a s i c a l l y has a f i v e component antigen system g i v i n g r i s e to eight gene complexes or agglutinogens (3). Phenotyping using Rh a n t i s e r a can be q u i t e u s e f u l i n obtaining i n d i v i d u a l i z i n g information. Recent advances have a l s o been made i n shortening the pro cedure f o r o b t a i n i n g blood group antigen information. A quick, r e l i a b l e procedure f o r the ABO grouping used i n the P i t t s b u r g h and Allegheny County Crime Laboratory i n v o l v e s a maximum of 45 minutes (4). This includes a 10 minute preparation and c o l l e c t i o n of threads, a 10 minute antibody incubation, a 3 minute wait, a 10 minute e l u t i o n and a 10 minute r o t a t i o n and examination p e r i o d . Thus by shortening an otherwise lengthy technique, a s e r o l o g i s t can e f f i c i e n t l y process more samples i n a given period of time with l e s s m a t e r i a l waste, thus p e r m i t t i n g f u r t h e r analy s i s on the same sample. In a d d i t i o n , another advantage of t h i s technique i s that i t uses only three bloodstained threads from the questioned source m a t e r i a l to accomplish what normally would take s i g n i f i c a n t q u a n t i t i e s of blood. The second main c l a s s of blood c o n s t i t u e n t s used as genetic markers are the polymorphic enzymes. The enzymes of i n t e r e s t to the f o r e n s i c s e r o l o g i s t are p r i m a r i l y l o c a t e d w i t h i n the red blood c e l l and are commonly r e f e r r e d to as isoenzymes. These can b r i e f l y be described as those enzymatically a c t i v e p r o t e i n s which c a t a l y z e the same biochemical r e a c t i o n s and occur i n the same species but d i f f e r i n c e r t a i n of t h e i r physicochemical p r o p e r t i e s . (This d e s c r i p t i o n does not exclude the t i s s u e isoenzymes that occur w i t h i n the same organism; however, our c o n s i d e r a t i o n deals only with those of the red blood c e l l i n p a r t i c u l a r . ) The occurrence of multi-molecular forms of the same enzyme ( i s o enzymes) has been known f o r s e v e r a l decades ; however, i t was not u n t i l the Metropolitan P o l i c e Laboratory of Scotland Yard adapted e l e c t r o p h o r e t i c techniques to d r i e d blood a n a l y s i s that these systems were catapulted to the prominence they p r e s e n t l y r e c e i v e (2). For many of the f o r e n s i c s e r o l o g i s t s i n the United States, the use of e l e c t r o p h o r e s i s and isoenzyme determination i s a r e c e n t l y - i n h e r i t e d c a p a b i l i t y shared by only a few l a b o r a t o r i e s . Many isoenzymes have been i d e n t i f i e d from v a r i o u s human t i s s u e sources ; however, our c o n s i d e r a t i o n w i l l d e a l with s i x e r y t h r o c y t i c systems that have received r o u t i n e crime l a b o r a t o r y s t a t u s . These are phosphoglucomutase (PGM), adenylate kinase (AK), adenosine deaminase (ADA), glucose-6-phosphate dehydro genase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD) and e r y t h r o c y t i c a c i d phosphatase (ΕΑΡ). The PGM system has r e c e i v e d the greatest amount of a t t e n t i o n f o r three reasons. F i r s t , i t i s a very s t a b l e enzyme and pro duces an e a s i l y i n t e r p r e t e d zymogram; second, i t s population frequencies are very u s e f u l s i n c e the three phenotypes (commonly


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found i n the B r i t i s h population) have the f o l l o w i n g percentages: PGM - I - 58%, PGM 2-1 - 36% and PGM - 2 - 6%; and t h i r d , phosphoglucomutase can be found i n other f o r e n s i c a l l y important p h y s i o l o g i c a l f l u i d s , namely, semen and v a g i n a l s e c r e t i o n s (5). This l a s t dimension has been extremely u s e f u l i n f u r t h e r i n d i v i d u a l i z i n g seminal s t a i n s found on garments and/or bed c l o t h i n g a s s o c i a t e d with sexual a s s a u l t cases. Adenylate kinase has a l s o received q u i t e a b i t of a t t e n t i o n because i t can be i d e n t i f i e d on the same e l e c t r o p h o r e t i c zymogram as PGM, thereby a f f o r d i n g a d d i t i o n a l isoenzyme information from the same blood sample, (Figure 1). This a b i l i t y to o b t a i n m u l t i p l e isoenzyme information from a s i n g l e e l e c t r o p h o r e t i c zymogram i s not new. Publications dealing with human genetic studies have l i s t e d PGM, AK, ADA and 6-PGD being determined on the same e l e c t r o p h o r e t i c zymogram (6, 7). Another isoenzyme with s u b s t a n t i a l i n t e r e s t i s e r y t h r o c y t i c a c i d phosphatase (ΕΑΡ) (8, 9, 10). T h i s system has three autosomal a l l e l i c genes termed A, Β and C. These can be homo zygous or heterozygous g i v i n g r i s e to BA, CA and CB phenotypes. Each of these phenotypes i s e a s i l y d i s t i n g u i s h e d using s t a r c h g e l e l e c t r o p h o r e s i s with very u s e f u l population frequencies of approximately A - 13%, Β - 35%, C - 0.2%, BA - 43%, CA - 3%, CB - 6%. E r y t h r o c y t i c a c i d phosphatase has been found to remain v i a b l e f o r many months a f t e r drying and s u c c e s s f u l typing can be performed on a minimum of s e v e r a l threads (9), (Figure 2). A major disadvantage of many of the other isoenzymes, not mentioned, and which could be i d e n t i f i e d i n blood, i s the f r e quency of v a r i a n t s (11). When u t i l i z i n g these other systems i n screening blood samples to f i n d d i f f e r e n c e s between the v i c t i m and suspect blood types, the odds are against the examiner. Many have at l e a s t 98% of the population tested belonging to one of the isoenzyme v a r i a n t s ( i . e . , phosphohexose isomerase). By c o n t r a s t , i f the s e r o l o g i s t should f i n d that the v i c t i m s blood does have a r a r e v a r i a n t then the p r o b a b i l i t i e s of the questioned blood s t a i n being from the v i c t i m are very high and of extreme value as a form of a s s o c i a t i v e evidence. The t h i r d main c l a s s of c o n s t i t u e n t s used as genetic markers i n the blood are polymorphic p r o t e i n s (2, 11). Hemoglobin and the haptoglobins c o n s t i t u t e the most important members of t h i s c l a s s i f i c a t i o n . The haptoglobins are Alpha^ g l o b u l i n s which are r e s p o n s i b l e f o r binding f r e e homoglobin released i n t o the plasma a f t e r d e s t r u c t i o n of red blood c e l l s . G e n e t i c a l l y , they e x i s t i n three forms, H I, H 2 and H 2-1, with the f o l l o w i n g population d i s t r i b u t i o n ? H Ï - 14%, H 2-1 - 53%, and H 2 - 32%. Once again these f r e q u e n c i e l are usefu? i n screening blBod f o r d i f f e r e n c e s , (Figure 3). Hemoglobins can be u s e f u l to the f o r e n s i c s e r o l o g i s t , f o r example, i n d i f f e r e n t i a t i n g f e t a l blood i n cases of abortions, s i n c e f e t a l hemoglobin i s d i f f e r e n t from adult hemoglobin on a molecular l e v e l , and a l s o as an a n t h r o p o l o g i c a l marker f o r 1

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Figure 1. Separation of PGM and AK electrophoresis was done for 22 hr at 6.5 V/cm at 4°C in a 1 mm, 14% starch gel prepared in J M Tris, EOT A, maleic acid, MgCL = 7.4 tank buffer diluted 1:10. The PGM side of the gel was stained at 1-2 hours before the AK using an agar overlay technique at 37°C. The visualized bands are precipitated with formazan.

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Figure 2. Erythrocytic acid phosphatase sche matic. A schematic drawing illustrating typical results of an ΕΑΡ determination in a 13%, 1mm starch gel prepared in 0.24M NaILPO 0.15M trisodium citrate tannic acid buffer di luted 1:100. The electrophoresis is carried out for 416 hr at approximately 410 V . The gels are stained by the fluorescence produced after enzymatic hydrolysis by methylumbelliferyl phosphate at 37 °C for 116 hr. h



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i d e n t i f y i n g negroid blood. Approximately 10% of the negroid p o p u l a t i o n r e t a i n a form of hemoglobin r e f e r r e d to as s i c k l e c e l l (11). S i c k l e c e l l hemoglobin (Hb-S) i s caused by a replacement of glutamic a c i d with v a l i n e at the s i x t h p o s i t i o n on the beta chain. Several e l e c t r o p h o r e t i c techniques have proven u s e f u l f o r d i f f e r e n t i a t i n g the v a r i o u s hemoglobins A, F, S, D and Ε (12). Present research i s being c a r r i e d out to f u r t h e r the metho dology of the a n t h r o p o l o g i c a l c l a s s i f i c a t i o n of b l o o d s t a i n s . Two isoenzymes, peptidase A and g l u t a t h i o n e reductase, have been reported to have polymorphic forms i n negro populations and l i t t l e or no v a r i a n t s i n Caucasians (13, 14, 15). Thus, should the r a r e v a r i a n t be demonstrated i n a s t a i n , there would be a high p r o b a b i l i t y concerning the e t h n i c o r i g i n of the blood. In a d d i t i o n , research has been i n i t i a t e d i n t o the use of Gm and Inv typing to a s s i s t i n the a n t h r o p o l o g i c a l c l a s s i f i c a t i o n of blood s t a i n s (2, 17). For example, the combination of Gm f a c t o r s 1, 2, 17 and 4, 22 i s found i n Caucasians, whereas Gm f a c t o r s 1, 6 and 11 are found i n negroes and Gm 1, 4 and 17 are found s t r i c t l y i n mongoloids. Gm and Inv are amino a c i d sequences o c c u r r i n g i n the l i g h t and heavy chains of immunoglobulins (16, 18). A n t i b o d i e s s p e c i f i c to the Gm and Inv groups are found i n some p a t i e n t s s u f f e r i n g from rheumatoid a r t h r i t i s and i n some healthy people. So f a r , 23 Gm types and 3 Inv types have been found. The success of Gm and Inv typing w i l l depend on the q u a n t i t y of s t a i n , and the s p e c i f i c i t y , q u a l i t y and a v a i l a b i l i t y of the a n t i s e r a , (Figure 4). Gm and Inv typing w i l l not only be an asset i n anthropo l o g i c a l t e s t i n g , but w i l l a l s o be v a l u a b l e i n i n d i v i d u a l i z i n g blood s i n c e only c e r t a i n combinations of Gm and Inv types are found i n any one person's blood. For i n s t a n c e , the i n d i v i d u a l ' s blood may type p o s i t i v e f o r Gm 1, 4, 17, 22, whereas another person may type as 1, 5, 12 and 21. Current research i n v o l v e s the use of radioimmunoassay to q u a n t i t a t e t e s t o s t e r o n e and estrogen i n d r i e d blood samples (22, 24). The u l t i m a t e goal of t h i s research w i l l be to d e t e r mine the sexual o r i g i n of the s t a i n s . In the past, researchers have attempted t h i s by i d e n t i f y i n g Barr bodies and Y chromosomes using d i f f e r e n t i a l f l u o r e s c e n c e s t a i n i n g with quinacine; however, these t e s t s r e q u i r e d a s u b s t a n t i a l amount of blood deposited as a t h i n f i l m on a non-porous surface and are t h e r e f o r e l i m i t e d i n t h e i r a p p l i c a t i o n (19, 20, 21). The s e n s i t i v i t y and b a s i c technique of radioimmunoassay w i l l permit the a n a l y s i s of blood s t a i n s on v i r t u a l l y any surface and should a l s o be a p p l i c a b l e to very small ones. Forensic s e r o l o g i s t s have had l i t t l e success i n i d e n t i f y i n g menstrual blood. With the increase i n the number of sexual a s s a u l t s (rape i n p a r t i c u l a r ) taking place each year, the a n a l y s t confronts the problem of menstrual blood i d e n t i f i c a t i o n more o f t e n . A recent p u b l i c a t i o n reported the i d e n t i f i c a t i o n of


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menstrual blood s t a i n s based on the e l e c t r o p h o r e t i c separation and q u a n t i t a t i o n of l a c t a t e dehydrogenase (LDH) isoenzymes (25). They n o t i c e d a s i g n i f i c a n t e l e v a t i o n i n the LDH-4 and LDH-5 f r a c t i o n s or stained bands. The a c t i v i t y of these 4 and 5 bands i s reported to remain f o r two weeks a f t e r the blood d r i e s . Recent work i n t h i s l a b o r a t o r y has r e s u l t e d i n the development of LDH s and PGM s on the same p l a t e . Through t h i s technique, i n formation can be obtained concerning the menstrual o r i g i n of the blood and a l s o , p o s s i b l y , information regarding the PGM type of the c o n t r i b u t o r . This can be a l s o very meaningful when the menstrual blood i s mixed with semen and the r e s u l t a n t mixture dep o s i t e d on the suspect's c l o t h i n g . In a recent case, such a mixture was t e s t e d . The woman was a PGM-I and the perpetrator a PGM 2-1. The PGM study of the s t a i n revealed not only the "a" and "c bands of the PGM-I type but a l s o the b and "d" bands from a PGM-2 or PGM 2-1 semen source. Another i n t e r e s t i n g dimension to the LDH isoenzyme system r e s i d e s i n the f a c t that during the process of spermatoagenesis an LDH isoenzyme i s formed that when separated by s t a r c h e l e c t r o p h o r e s i s i s found midway between the LDH^ and LDH^ bands. It i s r e f e r r e d to as the LDH band (26). E f f o r t s are being made to i d e n t i f y t h i s band i n semen. This could p o s s i b l y be a s o l u t i o n to the dilemma of i d e n t i f y i n g seminal s t a i n s i n cases where the perpetrator i s azospermatic, aspermatic or has had a vasectomy ( i . e . , he i s n a t u r a l l y or a r t i f i c i a l l y incapable of producing spermatozoa), (Figure 5). In the event that the LDH system does not solve the problem of seminal s t a i n i d e n t i f i c a t i o n , research has been i n i t i a t e d to produce s p e c i f i c a n t i s e r a to c e r t a i n antigens only found i n seminal f l u i d . P r e l i m i n a r y work i n d i c a t e s that at l e a s t f i v e c o n s t i t u e n t s of seminal f l u i d can be e l e c t r o p h o r e t i c a l l y separated and a n t i g e n i c a l l y introduced i n t o r a b b i t s (28, 29). (Commercial a n t i s e r a now on the market have proven u n s a t i s f a c tory (27).) E l e c t r o p h o r e s i s has become a most v i t a l technique f o r the separation and i d e n t i f i c a t i o n of the genetic markers i n blood. In a d d i t i o n to blood, Adams and Wraxall (30), of the M e t r o p o l i t a n P o l i c a Laboratory, have a p p l i e d acrylamide e l e c t r o p h o r e s i s to the i d e n t i f i c a t i o n of various sources of a c i d phosphatase (AP) a c t i v i t y found when v a g i n a l swabs or washings are being tested f o r the presence of seminal f l u i d . This technique i s capable of d i f f e r e n t i a t i n g between v a g i n a l and seminal ( p r o s t r a t i c ) a c i d phosphatase s i n c e p r o s t r a t e AP moves f a s t e r i n the g e l . D i f f e r e n t i a t i o n between v a g i n a l AP and p r o s t r a t i c AP has e x p l a i n ed cases where i t has been found that s i g n i f i c a n t AP l e v e l s are present while no spermatozoa can be l o c a t e d m i c r o s c o p i c a l l y . In one p a r t i c u l a r case, the high AP l e v e l was due to the v a g i n a l enzyme and no bands of p r o s t r a t i c AP were detected. Thus the presence of high l e v e l s of AP does not n e c e s s a r i l y confirm the presence of seminal f l u i d . This t e s t i s i n agreement with the 1


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I- Hp - Ite


COMPLEXES

0

Figure 3. Haptoglobin separation schematic. A schematic illustrating typical results of a Hp determination run in 8 mm, 10% starch gel prepared with tris citric acid buffer at pH = 8.6. The tank buffer is boric acid, pH = 7.9. The electrophoresis is run at 100 V for 17 hr at4°C. The Hp-Hb complexes are stained by virtue of the peroxidase reaction of hemoglobin which gives a color reaction with benzidine.

H-CHAINS

Figure 4. Schematic illustrating the positions of the Gm and Inv sites on the IgG molecule

L-CHAIN

I I I

I I I I I I I I I 11 I

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Figure 5. Lactic acid dehydrogenase schematic. This schematic illustrates the value of LDH isozyme patterns for the identification of menstrual blood and seminal material.



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argument that many f o r e n s i c s e r o l o g i s t s have made, namely that the chemical a c i d phosphatase t e s t can only be regarded as a presumptive t e s t u n t i l the s p e c i f i c source of the AP can be identified. In c o n c l u s i o n , f o r e n s i c serology has made great advances during the past 2-3 years and the next few years promise to be even more worthwhile. Since blood i s so complex, i t presents so many avenues f o r i n v e s t i g a t i o n that i t w i l l continue to be a f e r t i l e area f o r meaningful f o r e n s i c research. Literature Cited 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

I s s e t t , P. D., "Applied Blood Group Serology", Spectra B i o l o g i c a l s , Oxnard, C a l i f o r n i a (1970). C u l l i f o r d , B. J . , "The Examination and Typing of Bloodstains i n the Crime Laboratory", N a t i o n a l I n s t i t u t e of Law Enforce -ment and C r i m i n a l J u s t i c e , Washington, D. C. (1971). I s s e t t , P. D., in r e f . 1, p. 58. Unpublished r e s u l t s from t h i s l a b o r a t o r y . C u l l i f o r d , B. J., i n r e f . 2, p. 106. Goedde, W. H. and Brinkmann, H. G., HUMANGENTIK (1972) 15(3), 277. Hopkinson, D. A., Spencer, Ν. and H a r r i s , Η., NATURE 199, 969 (1969). Wraxall, Β. G. D., IV I n t e r n a t i o n a l Meeting of F o r e n s i c Sciences, Edinburgh, Scotland (1972). Wraxall, B. G. D., personal communication. G i b l e t t , R., "Genetic Markers i n Human Blood", B l a c k w e l l Scientific P u b l i c a t i o n s , Oxford (1969). Wraxall, B. G. D., J . FOR. SCI. SOC. (1972) 12(3), 452. Lewis, W. H. P. and H a r r i s , Η., NATURE (1967) 215. Kaplan, J . D. and B e n t l e r , Ε., NATURE (1968) 217, 256. Lewis, W. H. P., NATURE (1971) 230, 215. Grubb, R. and L a u r e l l , C. Β., ACTA PATHOL. MICROBIOL., SCAND. (1956) 39, 390. Blanc, Μ., G o i t y , R. and Ducos, J . , J . FOR. SCI. (1971) 16(2), 176. Popartz, C., L e n i o r , J . and Rovart, L., NATURE (1961) 189, 586. Pearson, P. L. and Bobrow, Μ., NATURE (1970) 226, 78. Renard, S., J . FOR. SCI. SOC. (1971) 11, 15. P h i l i p s , A. P. and Gaten, Ε., LANCET (1971), Aug. 14, 371. Furuyama, S., Mayes, D. M. and Nugent, C. A., STEROIDS (1970) 16(4), 415. Hillier, S. G., Brounscy, B. G. and Cameron, E. H. D., STEROIDS (1973) 21(5), 735. Boon, D. A., Keenan, R. Ε. and Slaunwhite, J r . , W. R., STEROIDS (1972) 20(3), 269. Asaon, M., Oya, M. and Hayakawa, Μ., FORENSIC SCIENCES (1972) 1,

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25. 26. 27. 28.

SCIENCE

Khalap, S., unpublished communication. Baxter, S. J . , MED. SCI. LAW (1973) 13(3), 155. B e l i n g , C. and Li, T., BERT. STERIL, (1973) 24(2), 132. Hara, M., Kuyanag, Y., Inoue, I. and Fukuyama, T., NIPPON HORGUKU ZASSHI (1971) 25(4), 322. Wraxall, B. G. D., personal communication.


29.

FORENSIC


Forensic Bloodstains and Physiological Fluid Analysis - ACS

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