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ABO, phosphoglucomutase and erythrocyte acid phosphatase typing of blood samples containing added fluoride
SCIENTIFIC & TECHNICAL
ABO, phosphoglucomutase and erythrocyte acid phosphatase typing of blood samples containing added fluoride R V WINCHESTER Institute of Environmental Health and Forensic Sciences, * Private Bag 92021, Auckland, New Zealand Journal of the Forensic Science Society 1993; 33: 159-164 Accepted 24 February 1992 Blood samples containing approximately 2% and 4% sodium fluoride have been stored at 4°C for up to 5 months and 4 weeks respectively. ABO typing by absorption-elution was successful on stains made from almost all of the samples. PGM subtyping by isoelectric focusing was successful on most samples, with clearer results from whole blood than from stains. Failure to type appears to be slightly more common at the higher preservative level and yields no result rather than an erroneous one. Apparent phenotypic changes in the 1+ and 1- bands have been seen after 5 months storage. E A P typing by isoelectric focusing was successful on most samples, again with clearer results from whole bloods than from stains. Possible errors can occur in samples containing C bands, but it is not clear whether they are caused by the presence of preservative. The results obtained are illustrated by two casework examples.
Des Cchantillons de sang qui contenaient approximativement 2% et 4% de fluorure de sodium on CtC stockCs h 4°C jusqu'h 5 mois et 4 semaines respectivement. Le typage ABO par absorptionClution rCussissait sur des taches faites avec presque tous les Cchantillons. Le typage PGM par focalisation isoClectrique rkussissait Cgalement sur la plupart des Cchantillons avec des rCsultats plus clairs h partir du sang qu'h partir de taches. Les Cchecs de typage paraissent ICghrement plus communs lorsqu'il y a un agent de preservation aux concentrations les plus ClevCes et ne produisent pas de rCsultat, plut8t qu'un resultat faux. Des changements phCnotypiques apparentes dans les bandes 1+ et 1- sont apparues a p r b 5 mois de stockage. Le typage E A P par focalisation isoClectrique Ctait Cgalement couronnC de succ&s sur la plupart des Cchantillons, de nouveau avec des rCsultats plus clairs avec le sang plut8t qu'avec les taches. Des erreurs possibles peuvent apparaitre dans des Cchantillons qui contiennent une bande C mais ce n'est pas clair si ce rCsultat est cause par la prCsence ou non d'agents conservateurs. Les rCsultats obtenus sont illustrCs par deux cas.
Blutproben mit 2% bzw. 4% Natriumfluorid-Anteil sind auf Typisierung nach 5 monatiger bzw. 4 wochiger Aufbewahrung bei 4°C untersucht worden. ABO-Typisierung mittels Adsorption-Elution war bei den meisten Proben erfolgreich; ebenfalls PGMtypisierung mittels Isoelektrischer Fokussierung, wobei Flussigproben im Gegensatz zu Proben von eingetrocknetem Blut klarere Aussagen ermoglichten. Schwierigkeiten der Typisierung sind wahrscheinlicher bei Proben mit hohem Konservierungsmittel-Anteil, wo keine bzw. falsche Ergebnisse auftraten. Bei Lagerzeiten uber 5 Monate waren erkennbare phanotypische Veranderungen in den 1 + und 1Banden zu sehen. EAP-Typisierung mittels Isoelektrischer Fokussierung war bei den meisten Proben ebenfalls moglich, wobei auch hier die Proben von Flussigblut im Gegensatz zu Proben von eingetrocknetem Blut besser abschnitten. Fehlergebnisse treten auf in Proben enthaltend C-Banden, wobei nicht genau feststeht, wieweit dafur der Einfluss der Konservierungsmittel verantwortlich ist. Die Ergebnisse der Untersuchung sind anhand von 2 KasuistikBeispielen illustriert. Se han almacenado a 4"C, muestras de sangre conteniendo fluoruro s6dico a1 2% y al 4% aproximadamente, durante 5 meses y 4 semanas respectivamente. FuC posible el tipaje ABO por absorcion-eluci6n de casi todas las manchas hechas con esas sangres. El subtipaje PGM por isoelectroenfoque fuC posible en la mayoria de las muestras con resultados mas claros en sangre entera que en manchas. La imposibilidad de tipaje parece ser ligeramente mas frecuente para el nivel de conservacion mayor, y mas que producir un resultado erroneo, imposibilita la obtencion de un resultado. Despues de 5 meses de almacenamiento se han visto cambios aparentes en el fenotipo en las bandas 1+ y I-. El tipaje E A P por isoelectroenfoque pudo realizarse con exito en la mayoria de las muestras, tambien con resultados mas claros en sangre entera que en manchas. Pueden ocurir errores en las muestras que poseen bandas C pero no esta claro si est5n causados por la presencia del conservante. Los resultados obtenidos se ilustran con dos ejemplos de casos reales.
Key Words: Blood; Grouping; Fluoride; Preservative; ABO; PGM; EAP. * Formerly DSIR Chemistry.
JFSS 1993; 33(3): 159-1 64
Introduction Reference blood samples submitted to our forensic biology laboratory for blood grouping normally contain only an anticoagulant (usually EDTA, occasionally oxalate or citrate), and no preservative. Sometimes the only samples available are those containing added fluoride as a preservative. These samples are normally taken for blood alcohol measurement or toxicological studies. It was generally believed in our laboratories that preserved samples could not be used for blood grouping, but evidence on which this belief was based appeared to be very sparse. The aim of this study was to review the available information on ABO grouping, phosphoglucomutase (PGM) isoenzyme typing, and erythrocyte acid phosphatase (EAP) isoenzyme typing in samples containing preservative, and to carry out trials under the normal conditions used in our laboratory. The work was not initially intended as a trial of the ability to group preserved bloods which had been stored for long periods, but since much of the reviewed literature dealt with this aspect of the topic, some trials were subsequently carried out over extended storage periods. A BO typing Rees et al. [I] studied the persistence of ABH antigens in preserved blood samples stored for up to 416 days (approximately 14 months) at room temperature. The samples contained added sodium fluoride at a nominal concentration of 1 % and potassium oxalate (anticoagulant) at a nominal concentration of 0.25%. The true concentrations may have been up to 50% higher, as the pre-treated containers were sometimes not filled to the intended volume. ABO blood group status was determined by absorption-inhibition on the supernatant from diluted and centrifuged whole blood samples and by absorption-elution on stains made from the preserved bloods. The tests were carried out at 15 different intervals between 10 and 416 days from the taking of the original samples. The absorption-inhibition tests gave correct results for 148 out of 150 samples but failed in two cases to detect the A antigen in A,B samples. The absorption-elution method grouped all 150 samples correctly.
PGM isoenzyme typing Culliford [2] stated that "Fluoride i s . . . an inhibitor of PGM but the degree of inhibition produced is dependent on the concentration of substrate and other ions present. Sodium fluoride is often used as a preservative in blood samples but it has never been found to have adverse effects on the PGM
determination. This may well be because of the thorough washing of the red cells prior to lysis and use." Later he stated that the presence of fluoride had no effect on the ability to determine PGM type by gel electrophoresis provided that the cells could be washed. It should be remembered that subtyping was not at that time being routinely carried out in forensic laboratories. If the blood sample was lysed, and therefore could not be washed, the increased ionic strength of the sample distorted the patterns on the gel, but the author claimed that with experience the types could still be determined. Brinkmann [3], quoted in Gaensslen [4], claimed that samples of whole blood containing sodium fluoride, at an unspecified concentration, standing for up to a year at 4°C could undergo apparent phenotypic changes. In particular, some samples showed PGM "2-like'' patterns although they were either PGM 1 or 2-1 when fresh. Rees et al. [I], using the same series of samples as described above for ABO determinations, found that typing by gel electrophoresis was correct in all samples containing preservative for up to 32 days of storage at room temperature. After this time either changed results or no results were obtained depending on the original grouping. In particular, PGM 2-1 samples could be read as PGM 2 and caution was advised in interpreting results on any samples stored for greater than 5 weeks. Because samples were quite haemolysed even after only 10 days storage, it was not possible to remove the fluoride by washing. No reports have been located on the effects of preservatives on PGM subtyping using isoelectric focusing.
EA P isoenzyme typing Reimann and Willner [5], quoted by Gaensslen [4], reported successful typing of EAP "for a number of months in samples collected for blood alcohol determination", while Smerling [6] found the time limit to be at least 6 months. The preservative used in these studies was not stated. Brinkmann et al. [7] investigated EAP typing by gel electrophoresis in blood samples stored at 4°C containing 0.6% w/v sodium fluoride. All phenotypes except C, which was not included, were able to be determined in samples stored for up to 15 months. Older samples generally required larger amounts of material, and polyacrylamide was found to have advantages over starch as the gel medium. Some investigations were also carried out on stains but these were not made from preserved blood. Again, no JFSS 1993; 33(3): 159-164
reports have been found on the effects of preservatives on the determination of EAP types by isoelectric focusing. Materials and methods
Study using "blood alcohol" samples Blood collection bottles. Samples used in this part of the study were unwanted blood alcohol samples submitted in cases where blood alcohol determination was not required. Blood alcohol bottles supplied to doctors in our Medical Examination Kits contain 100 mg of sodium fluoride and 15 mg of potassium oxalate (dispensed into the bottles as a solution and subsequently dried). The final concentration of the two salts depends on the volume of blood put into the bottle. Estimates of the volume of blood in 12 of 18 samples used in this part of the study gave a mean of 4.9 ml (sd 1.0 ml, range 3-6 ml). Using a nominal volume of 5 ml of blood, the concentrations of sodium fluoride and of potassium oxalate will be 2% and 0.3% respectively. Sample details. Eighteen blood samples were selected from casework. These samples met the criteria that they were available as both unpreserved and preserved samples and that ABO, PGM and E A P typing had been carried out on the unpreserved samples. On arrival in the laboratory the preserved samples were stored at 4OC. For each sample, a single stain was made on clean cotton cloth. The interval between collection of the sample and making the stain varied from 12 to 147 days. Stains were air dried at room temperature and then stored at -20°C. At the same time as stains were made, samples of the blood, all of which were haemolysed, were also stored at -20°C. Stability of the various blood factors at -20°C has been assumed. Table 1 gives details of the samples. When the stains were made it was noted that most of the preserved samples were darker in colour than unpreserved samples. Many preserved samples had a thick, tarry consistency, making it necessary to spread the sample on the cloth. Neither the colour nor the consistency appeared to be related to the length of time the samples had been stored. These factors also did not appear to be related to the concentration of either preservative or anticoagulant, which would vary according to the volume of blood originally put into the bottle. Attempts were made to obtain washed red cells from some samples, but this was unsuccessful because of haemolysis. Hence whole blood grouping could not be attempted. JFSS 1993; 33(3): 159-1 fi4
TABLE 1. Details of preserved "blood alcohol" samples Sample no.
Days stored*
ABO groupt
PGM type?
EAP typet
* At 4°C until stain made and blood frozen.
t Of unpreserved sample. $Frozen samples of these bloods were not stored until 73 and 86 days respectively after the samples were taken.
Study using laboratory-prepared samples Samples are occasionally submitted in bottles intended for toxicological investigations. These bottles contain approximately twice the amount of sodium fluoride and nearly 4 times the amount of potassium oxalate as is present in the blood alcohol bottles. This part of the study was designed to investigate bloods preserved to these levels. Potassium oxalate and sodium fluoride were added, at two different concentrations, to relatively fresh blood (1-2 days) obtained from a blood transfusion centre. These samples already contained EDTA. The amounts of potassium oxalate added were calculated to give a total anticoagulant concentration (EDTA + potassium oxalate) approximately equal to that provided by potassium oxalate alone in the prepared blood alcohol and toxicology bottle. Sample preparation. Solutions of sodium fluoride (20%) and potassium oxalate (4%) were made up in distilled water. To one series of Eppendorf tubes (A) was added 0.1 ml of the sodium fluoride solution, and to another series (B), 0.2ml of sodium fluoride solution and 0.1 ml of potassium oxalate solution. The solutions were then dried at 37OC and 1ml of blood was added to each tube. The tubes were then gently agitated until all the salts were dissolved. The A series
contained 2% sodium fluoride and the B series 4% sodium fluoride and 0.4% potassium oxalate (together in both cases with the EDTA originally present). Samples were stored at 4°C and subsamples removed to make stains and to freeze at weekly intervals. Typing was carried out within 4 weeks of freezing the samples. A total of 19 samples was included in this part of the study, as detailed in Table 2. Typing in all 3 systems had previously been carried out on the unpreserved fresh samples.
Methods The methods used for ABO, PGM and EAP determinations were those normally used in the laboratory for forensic casework. ABO grouping was carried out only by absorption-elution on threads taken from stains, essentially using the method of Howard and Martin [8]. No attempt was made to distinguish A , and A, groups. PGM subtyping was carried out by an isoelectric focusing method based on that of Sutton and Burgess [9] but including the use of separators in the gel. E A P typing was by the isoelectric focusing method of Randall et al. [lo]. Blood solutions were made up from 20 microlitres of thawed blood, 2 microlitres of Triton X (15% in ethanol), and 60 microlitres of dithiothreitol ( D m ) solution (0.7%), while 5 x 3 mm portions of stains were extracted with 30 microlitres of D m solution. The same solutions and extracts were used for both PGM and EAP determinations. TABLE 2. Details of laboratory-preserved samples Sample no.
ABO group *
PGM fype *
EA P fype *
Results and discussion A BO grouping In the first study, all 18 samples were grouped correctly from stains using absorption-elution. It appears therefore that stains made from preserved blood containing up to about 2% added sodium fluoride can be successfully grouped after at least 5 months storage of the blood at 4°C. This result agrees with the work of Rees et al. [I].
In the second study, stains from samples 19, 20, 26, 30 and 34 were grouped one week after addition of preservative and storage at P C , and all grouped correctly. All 19 samples at both preservative levels were grouped 4 weeks after addition of preservative and storage at 4°C. All but 1 sample grouped correctly. Sample 27 at the higher fluoride level was grouped by one reader as AB and the other as B. Overall the results suggest that grouping of stains made from blood containing up to 4% sodium fluoride is generally reliable at least up to 4 weeks after addition of preservative.
PGM subtyping In the first study, all 18 samples of preserved blood which had been stored for up to 147 days (approximately 5 months) at 4OC before freezing, gave correct results in the PGM system. Sixteen of the 18 bloodstain samples were correctly grouped from stains made at varying intervals after storage at 4OC. Bands resulting from the stains were generally more distorted and streaked than those resulting from the bloods. The correct results from stains included sample 8 which was weak and not able to be confirmed 2 days later on a fresh extract. The remaining two samples (13 and 15) could not be determined with certainty because of excessive streaking. In these cases no result would have been reported in casework, and there was no likelihood of a wrong result being reported. The two samples unable to be typed from stains had been stored for 106 and 114 days, where other samples stored for up to 147 days had typed correctly. It appeared therefore that the presence of fluoride up to at least 2% in blood samples did not normally affect the ability to determine the PGM subtype, although occasional stains would not give a result.
* Of unpreserved sample. 162
Subsequent casework (see Case 2 below) suggested that apparent changes in the PGM 1 phecotype could occur on further extended storage. Some of the samples containing added preservative were therefore retyped at longer storage periods. Results are shown JFSS 1993; .?3(3): 159-164
in Table 3. As the earlier results showed that PGM typing was correct up to about 5 months after addition of preservative, it is apparent that at some time between about 5 and 8 months (150-230 days) changes occur which may make PGM typing unreliable. This is currently being investigated. In the second study, the 5 blood samples taken one week after addition of preservative all typed correctly at the lower fluoride level. All 19 samples at both preservative levels typed correctly after 4 weeks storage at 4OC. All 5 sample stains from 1 week at both preservative levels typed correctly. Samples from 4 weeks produced 3 instances of weak unreadable bands and 1 instance of no bands. At the lower fluoride level, sample 35 gave weak bands. At the higher fluoride level, samples 27 and 35 gave weak bands and sample 36 gave no bands. There is some evidence here that the effect of preservative is greater at the higher concentration. All of the weak results would have been reported as "no result" and again there would have been no likelihood of error.
EA P typing In the first study, 17 of the 18 blood samples were typed correctly. One blood sample (No 7. CB) was read by one reader as a probable CB and the other as B or CB. Sixteen of the 18 stains were typed correctly. The stain from sample 7 was read as a B by both readers. Sample 8 (CA), which was typed correctly from the blood, gave an inconclusive result from the stain. The quality of the bands obtained from the stains was in general inferior to that obtained from the blood samples. On the basis of these results it appeared possible that samples of either blood or stains containing C bands could yield erroneous results rather than no results. In TABLE 3. PGM determinations on preserved blood samples stored for long periods Sample no.
True PGM type*
Apparent PGM type from blood stored at 4°C -20°C
Total days stored t
* Of unpreserved sample. ?The frozen samples had been stored at -20°C for the last 133 days of the storage period. Earlier storage was at 4OC. JFSS 1993; 33(3): 159- 164
particular CB could be read as B and CA possibly as BA. The two samples in this series where mistypings could have occurred had been stored for 52 and 66 days respectively before stains were made, but as no other samples containing C bands were available at the time it was not possible to determine whether errors might arise from storage effects or from difficulties in reading any samples containing C bands. This latter problem can sometimes occur even with fresh samples not containing any preservative. Results from the remaining samples containing 2% fluoride, that is, the B, BA and A phenotypes, were satisfactory up to 147 days for both blood and stains. In the second study, of 4 bloods sampled one week after the addition of 2% fluoride, 3 (samples 19, 26 and 34) typed correctly. Sample 20 (C) was read by one reader as B and the other as CB. At 4 weeks after addition of preservative at both 2% and 4% levels, all 19 samples were typed correctly, including the C type. After one week at the 2% fluoride level, stain samples 19, 26 and 34 typed correctly. At the 4% fluoride level after 1 week these three samples together with sample 30 (of which there was insufficient at the 2% level), typed correctly. Sample 20 typed as either B or CB at both fluoride levels. All 19 samples typed correctly at both preservative levels after 4 weeks. Applications to casework
Case 1 In a recent homicide case, blood from the suspect was submitted in a blood alcohol bottle, and that from the victim in a toxicology bottle, with a higher level of preservative. The samples were received in the laboratory 3 days (suspect) and 2 days (victim) after collection and were stored at 4OC. Whole blood grouping on the partially haemolysed samples was attempted 2 days after receipt and unexpectedly yielded a result (ABO type B) from the suspect. The victim's blood was too haemolysed to group. Stains were made at this time and both samples grouped by absorption-elution 5 days later. The victim was ABO type A and the type B from the suspect was confirmed. PGM subtyping of the preserved whole blood samples 2 days after receipt gave a probable 2 + 1+ for the suspect and a 2 + 1 - with possible additional bands at the anode for the victim. Neither sample gave a result from the stain 5 days later. The suspect gave a clear 2 + 1 from the blood but no result from the stain after a further 2 days, while the victim gave an inconclusive result from the stain with possible
+
additional bands in the 2 region. No clear results were obtained from casework items from the victim. EAP typing of the blood 2 days after receipt gave clear results of B (suspect) and BA (victim) which were confirmed on tests of the stains after a further 5 days. The BA result from the victim was also confirmed by stains from casework items. The PGM results on blood from the suspect are largely as expected from the trials but the inability to group the stains after only 5 days was unexpected. There seemed to be a possibility that the higher fluoride concentration in the sample from the victim could affect the PGM typing by causing the appearance of additional bands. EAP results appear to be unaffected by preservative at either concentration. Case 2 This case required the typing of an old blood alcohol sample which the defendant in a drunken driving case alleged might not have been his own. Quality control procedures make it extremely unlikely that there was any exchange of samples. The defendant provided for comparison a fresh unpreserved blood sample which was typed as ABO type A, PGM type 1 and EAP type B. The questioned blood alcohol sample taken approximately 8 months earlier and stored mainly at 4°C was typed as ABO type A and EAP type B. PGM typing showed a probable 1- from the blood and a from a stain made approximately 2 probable 1 weeks earlier and stored at -20°C. Repeat determinations approximately 2 weeks later gave no result for the stain and a clear 1- for the blood. These results are consistent with the changes seen under control conditions (Table 3) and indicate that caution is needed in interpreting PGM results from old
+
+
preserved samples. A common origin between the two samples in the case could not be excluded.
References 1. Rees B, Howard H D and Strong SK. The persistence of blood group factors in stored blood samples. Journal of the Forensic Science Society 1975; 15: 43-49. 2. Culliford BJ. The examination and typing of bloodstains in the crime laboratory. Washington: US Department of Justice, 1971. 3. Brinkmann B. Interkonversion des PGM-Phanotypes unter besonderen Bedingungen. Beitraege zur Gerichtlichen Medizin 1974; 32: 141-144. 4. Gaensslen R. Sourcebook in Forensic Serology, Immunology and Biochemistry. Washington: US Department of Justice, 1983. 5. Reimann W and Willner G. Identifizierung gelagerter Alkoholblutproben durch Bestimmung der Typen der Sauren Erythrozytenphosphatase (SEPh). Deutsche Zeitschrift fuer die Gesamte Gerichtliche Medizin 1968; 64: 33-38. 6. Smerling M. Bestimmung der Sauren Erythrozytenphosphatase an Alten Blutalkoholproben und in Blutspuren. Archiv fuer Kriminologie 1969; 144: 161-166. 7. Brinkmann B, Gunnemann M and Koops E. Investigations on the decay of acid phosphatase types in stored blood stains and blood samples. Zeitschrift fuer Rechtsmedizin 1972; 70: 68-71. 8. Howard H D and Martin PD. An improved method for ABO and MN grouping of dried bloodstains using cellulose acetate sheets. Journal of the Forensic Science Society 1969; 9: 28-30. 9. Sutton JG and Burgess R. Genetic evidence for four common alleles at the phosphoglucomutase-1 locus (PGM,) detectable by isoelectric focusing. Vox Sanguinis 1978; 34: 97-103. 10. Randall T, Harland WA and Thorpe JW. A method of phenotyping erythrocyte acid phosphatase by isoelectric focusing. Medicine, Science and the Law 1980; 20: 43-47.
JFSS 1993; 33(3): 159- 164
ABO, phosphoglucomutase and erythrocyte acid phosphatase typing of blood samples containing added fluoride R V WINCHESTER Institute of Environmental Health and Forensic Sciences, * Private Bag 92021, Auckland, New Zealand Journal of the Forensic Science Society 1993; 33: 159-164 Accepted 24 February 1992 Blood samples containing approximately 2% and 4% sodium fluoride have been stored at 4°C for up to 5 months and 4 weeks respectively. ABO typing by absorption-elution was successful on stains made from almost all of the samples. PGM subtyping by isoelectric focusing was successful on most samples, with clearer results from whole blood than from stains. Failure to type appears to be slightly more common at the higher preservative level and yields no result rather than an erroneous one. Apparent phenotypic changes in the 1+ and 1- bands have been seen after 5 months storage. E A P typing by isoelectric focusing was successful on most samples, again with clearer results from whole bloods than from stains. Possible errors can occur in samples containing C bands, but it is not clear whether they are caused by the presence of preservative. The results obtained are illustrated by two casework examples.
Des Cchantillons de sang qui contenaient approximativement 2% et 4% de fluorure de sodium on CtC stockCs h 4°C jusqu'h 5 mois et 4 semaines respectivement. Le typage ABO par absorptionClution rCussissait sur des taches faites avec presque tous les Cchantillons. Le typage PGM par focalisation isoClectrique rkussissait Cgalement sur la plupart des Cchantillons avec des rCsultats plus clairs h partir du sang qu'h partir de taches. Les Cchecs de typage paraissent ICghrement plus communs lorsqu'il y a un agent de preservation aux concentrations les plus ClevCes et ne produisent pas de rCsultat, plut8t qu'un resultat faux. Des changements phCnotypiques apparentes dans les bandes 1+ et 1- sont apparues a p r b 5 mois de stockage. Le typage E A P par focalisation isoClectrique Ctait Cgalement couronnC de succ&s sur la plupart des Cchantillons, de nouveau avec des rCsultats plus clairs avec le sang plut8t qu'avec les taches. Des erreurs possibles peuvent apparaitre dans des Cchantillons qui contiennent une bande C mais ce n'est pas clair si ce rCsultat est cause par la prCsence ou non d'agents conservateurs. Les rCsultats obtenus sont illustrCs par deux cas.
Blutproben mit 2% bzw. 4% Natriumfluorid-Anteil sind auf Typisierung nach 5 monatiger bzw. 4 wochiger Aufbewahrung bei 4°C untersucht worden. ABO-Typisierung mittels Adsorption-Elution war bei den meisten Proben erfolgreich; ebenfalls PGMtypisierung mittels Isoelektrischer Fokussierung, wobei Flussigproben im Gegensatz zu Proben von eingetrocknetem Blut klarere Aussagen ermoglichten. Schwierigkeiten der Typisierung sind wahrscheinlicher bei Proben mit hohem Konservierungsmittel-Anteil, wo keine bzw. falsche Ergebnisse auftraten. Bei Lagerzeiten uber 5 Monate waren erkennbare phanotypische Veranderungen in den 1 + und 1Banden zu sehen. EAP-Typisierung mittels Isoelektrischer Fokussierung war bei den meisten Proben ebenfalls moglich, wobei auch hier die Proben von Flussigblut im Gegensatz zu Proben von eingetrocknetem Blut besser abschnitten. Fehlergebnisse treten auf in Proben enthaltend C-Banden, wobei nicht genau feststeht, wieweit dafur der Einfluss der Konservierungsmittel verantwortlich ist. Die Ergebnisse der Untersuchung sind anhand von 2 KasuistikBeispielen illustriert. Se han almacenado a 4"C, muestras de sangre conteniendo fluoruro s6dico a1 2% y al 4% aproximadamente, durante 5 meses y 4 semanas respectivamente. FuC posible el tipaje ABO por absorcion-eluci6n de casi todas las manchas hechas con esas sangres. El subtipaje PGM por isoelectroenfoque fuC posible en la mayoria de las muestras con resultados mas claros en sangre entera que en manchas. La imposibilidad de tipaje parece ser ligeramente mas frecuente para el nivel de conservacion mayor, y mas que producir un resultado erroneo, imposibilita la obtencion de un resultado. Despues de 5 meses de almacenamiento se han visto cambios aparentes en el fenotipo en las bandas 1+ y I-. El tipaje E A P por isoelectroenfoque pudo realizarse con exito en la mayoria de las muestras, tambien con resultados mas claros en sangre entera que en manchas. Pueden ocurir errores en las muestras que poseen bandas C pero no esta claro si est5n causados por la presencia del conservante. Los resultados obtenidos se ilustran con dos ejemplos de casos reales.
Key Words: Blood; Grouping; Fluoride; Preservative; ABO; PGM; EAP. * Formerly DSIR Chemistry.
JFSS 1993; 33(3): 159-1 64
Introduction Reference blood samples submitted to our forensic biology laboratory for blood grouping normally contain only an anticoagulant (usually EDTA, occasionally oxalate or citrate), and no preservative. Sometimes the only samples available are those containing added fluoride as a preservative. These samples are normally taken for blood alcohol measurement or toxicological studies. It was generally believed in our laboratories that preserved samples could not be used for blood grouping, but evidence on which this belief was based appeared to be very sparse. The aim of this study was to review the available information on ABO grouping, phosphoglucomutase (PGM) isoenzyme typing, and erythrocyte acid phosphatase (EAP) isoenzyme typing in samples containing preservative, and to carry out trials under the normal conditions used in our laboratory. The work was not initially intended as a trial of the ability to group preserved bloods which had been stored for long periods, but since much of the reviewed literature dealt with this aspect of the topic, some trials were subsequently carried out over extended storage periods. A BO typing Rees et al. [I] studied the persistence of ABH antigens in preserved blood samples stored for up to 416 days (approximately 14 months) at room temperature. The samples contained added sodium fluoride at a nominal concentration of 1 % and potassium oxalate (anticoagulant) at a nominal concentration of 0.25%. The true concentrations may have been up to 50% higher, as the pre-treated containers were sometimes not filled to the intended volume. ABO blood group status was determined by absorption-inhibition on the supernatant from diluted and centrifuged whole blood samples and by absorption-elution on stains made from the preserved bloods. The tests were carried out at 15 different intervals between 10 and 416 days from the taking of the original samples. The absorption-inhibition tests gave correct results for 148 out of 150 samples but failed in two cases to detect the A antigen in A,B samples. The absorption-elution method grouped all 150 samples correctly.
PGM isoenzyme typing Culliford [2] stated that "Fluoride i s . . . an inhibitor of PGM but the degree of inhibition produced is dependent on the concentration of substrate and other ions present. Sodium fluoride is often used as a preservative in blood samples but it has never been found to have adverse effects on the PGM
determination. This may well be because of the thorough washing of the red cells prior to lysis and use." Later he stated that the presence of fluoride had no effect on the ability to determine PGM type by gel electrophoresis provided that the cells could be washed. It should be remembered that subtyping was not at that time being routinely carried out in forensic laboratories. If the blood sample was lysed, and therefore could not be washed, the increased ionic strength of the sample distorted the patterns on the gel, but the author claimed that with experience the types could still be determined. Brinkmann [3], quoted in Gaensslen [4], claimed that samples of whole blood containing sodium fluoride, at an unspecified concentration, standing for up to a year at 4°C could undergo apparent phenotypic changes. In particular, some samples showed PGM "2-like'' patterns although they were either PGM 1 or 2-1 when fresh. Rees et al. [I], using the same series of samples as described above for ABO determinations, found that typing by gel electrophoresis was correct in all samples containing preservative for up to 32 days of storage at room temperature. After this time either changed results or no results were obtained depending on the original grouping. In particular, PGM 2-1 samples could be read as PGM 2 and caution was advised in interpreting results on any samples stored for greater than 5 weeks. Because samples were quite haemolysed even after only 10 days storage, it was not possible to remove the fluoride by washing. No reports have been located on the effects of preservatives on PGM subtyping using isoelectric focusing.
EA P isoenzyme typing Reimann and Willner [5], quoted by Gaensslen [4], reported successful typing of EAP "for a number of months in samples collected for blood alcohol determination", while Smerling [6] found the time limit to be at least 6 months. The preservative used in these studies was not stated. Brinkmann et al. [7] investigated EAP typing by gel electrophoresis in blood samples stored at 4°C containing 0.6% w/v sodium fluoride. All phenotypes except C, which was not included, were able to be determined in samples stored for up to 15 months. Older samples generally required larger amounts of material, and polyacrylamide was found to have advantages over starch as the gel medium. Some investigations were also carried out on stains but these were not made from preserved blood. Again, no JFSS 1993; 33(3): 159-164
reports have been found on the effects of preservatives on the determination of EAP types by isoelectric focusing. Materials and methods
Study using "blood alcohol" samples Blood collection bottles. Samples used in this part of the study were unwanted blood alcohol samples submitted in cases where blood alcohol determination was not required. Blood alcohol bottles supplied to doctors in our Medical Examination Kits contain 100 mg of sodium fluoride and 15 mg of potassium oxalate (dispensed into the bottles as a solution and subsequently dried). The final concentration of the two salts depends on the volume of blood put into the bottle. Estimates of the volume of blood in 12 of 18 samples used in this part of the study gave a mean of 4.9 ml (sd 1.0 ml, range 3-6 ml). Using a nominal volume of 5 ml of blood, the concentrations of sodium fluoride and of potassium oxalate will be 2% and 0.3% respectively. Sample details. Eighteen blood samples were selected from casework. These samples met the criteria that they were available as both unpreserved and preserved samples and that ABO, PGM and E A P typing had been carried out on the unpreserved samples. On arrival in the laboratory the preserved samples were stored at 4OC. For each sample, a single stain was made on clean cotton cloth. The interval between collection of the sample and making the stain varied from 12 to 147 days. Stains were air dried at room temperature and then stored at -20°C. At the same time as stains were made, samples of the blood, all of which were haemolysed, were also stored at -20°C. Stability of the various blood factors at -20°C has been assumed. Table 1 gives details of the samples. When the stains were made it was noted that most of the preserved samples were darker in colour than unpreserved samples. Many preserved samples had a thick, tarry consistency, making it necessary to spread the sample on the cloth. Neither the colour nor the consistency appeared to be related to the length of time the samples had been stored. These factors also did not appear to be related to the concentration of either preservative or anticoagulant, which would vary according to the volume of blood originally put into the bottle. Attempts were made to obtain washed red cells from some samples, but this was unsuccessful because of haemolysis. Hence whole blood grouping could not be attempted. JFSS 1993; 33(3): 159-1 fi4
TABLE 1. Details of preserved "blood alcohol" samples Sample no.
Days stored*
ABO groupt
PGM type?
EAP typet
* At 4°C until stain made and blood frozen.
t Of unpreserved sample. $Frozen samples of these bloods were not stored until 73 and 86 days respectively after the samples were taken.
Study using laboratory-prepared samples Samples are occasionally submitted in bottles intended for toxicological investigations. These bottles contain approximately twice the amount of sodium fluoride and nearly 4 times the amount of potassium oxalate as is present in the blood alcohol bottles. This part of the study was designed to investigate bloods preserved to these levels. Potassium oxalate and sodium fluoride were added, at two different concentrations, to relatively fresh blood (1-2 days) obtained from a blood transfusion centre. These samples already contained EDTA. The amounts of potassium oxalate added were calculated to give a total anticoagulant concentration (EDTA + potassium oxalate) approximately equal to that provided by potassium oxalate alone in the prepared blood alcohol and toxicology bottle. Sample preparation. Solutions of sodium fluoride (20%) and potassium oxalate (4%) were made up in distilled water. To one series of Eppendorf tubes (A) was added 0.1 ml of the sodium fluoride solution, and to another series (B), 0.2ml of sodium fluoride solution and 0.1 ml of potassium oxalate solution. The solutions were then dried at 37OC and 1ml of blood was added to each tube. The tubes were then gently agitated until all the salts were dissolved. The A series
contained 2% sodium fluoride and the B series 4% sodium fluoride and 0.4% potassium oxalate (together in both cases with the EDTA originally present). Samples were stored at 4°C and subsamples removed to make stains and to freeze at weekly intervals. Typing was carried out within 4 weeks of freezing the samples. A total of 19 samples was included in this part of the study, as detailed in Table 2. Typing in all 3 systems had previously been carried out on the unpreserved fresh samples.
Methods The methods used for ABO, PGM and EAP determinations were those normally used in the laboratory for forensic casework. ABO grouping was carried out only by absorption-elution on threads taken from stains, essentially using the method of Howard and Martin [8]. No attempt was made to distinguish A , and A, groups. PGM subtyping was carried out by an isoelectric focusing method based on that of Sutton and Burgess [9] but including the use of separators in the gel. E A P typing was by the isoelectric focusing method of Randall et al. [lo]. Blood solutions were made up from 20 microlitres of thawed blood, 2 microlitres of Triton X (15% in ethanol), and 60 microlitres of dithiothreitol ( D m ) solution (0.7%), while 5 x 3 mm portions of stains were extracted with 30 microlitres of D m solution. The same solutions and extracts were used for both PGM and EAP determinations. TABLE 2. Details of laboratory-preserved samples Sample no.
ABO group *
PGM fype *
EA P fype *
Results and discussion A BO grouping In the first study, all 18 samples were grouped correctly from stains using absorption-elution. It appears therefore that stains made from preserved blood containing up to about 2% added sodium fluoride can be successfully grouped after at least 5 months storage of the blood at 4°C. This result agrees with the work of Rees et al. [I].
In the second study, stains from samples 19, 20, 26, 30 and 34 were grouped one week after addition of preservative and storage at P C , and all grouped correctly. All 19 samples at both preservative levels were grouped 4 weeks after addition of preservative and storage at 4°C. All but 1 sample grouped correctly. Sample 27 at the higher fluoride level was grouped by one reader as AB and the other as B. Overall the results suggest that grouping of stains made from blood containing up to 4% sodium fluoride is generally reliable at least up to 4 weeks after addition of preservative.
PGM subtyping In the first study, all 18 samples of preserved blood which had been stored for up to 147 days (approximately 5 months) at 4OC before freezing, gave correct results in the PGM system. Sixteen of the 18 bloodstain samples were correctly grouped from stains made at varying intervals after storage at 4OC. Bands resulting from the stains were generally more distorted and streaked than those resulting from the bloods. The correct results from stains included sample 8 which was weak and not able to be confirmed 2 days later on a fresh extract. The remaining two samples (13 and 15) could not be determined with certainty because of excessive streaking. In these cases no result would have been reported in casework, and there was no likelihood of a wrong result being reported. The two samples unable to be typed from stains had been stored for 106 and 114 days, where other samples stored for up to 147 days had typed correctly. It appeared therefore that the presence of fluoride up to at least 2% in blood samples did not normally affect the ability to determine the PGM subtype, although occasional stains would not give a result.
* Of unpreserved sample. 162
Subsequent casework (see Case 2 below) suggested that apparent changes in the PGM 1 phecotype could occur on further extended storage. Some of the samples containing added preservative were therefore retyped at longer storage periods. Results are shown JFSS 1993; .?3(3): 159-164
in Table 3. As the earlier results showed that PGM typing was correct up to about 5 months after addition of preservative, it is apparent that at some time between about 5 and 8 months (150-230 days) changes occur which may make PGM typing unreliable. This is currently being investigated. In the second study, the 5 blood samples taken one week after addition of preservative all typed correctly at the lower fluoride level. All 19 samples at both preservative levels typed correctly after 4 weeks storage at 4OC. All 5 sample stains from 1 week at both preservative levels typed correctly. Samples from 4 weeks produced 3 instances of weak unreadable bands and 1 instance of no bands. At the lower fluoride level, sample 35 gave weak bands. At the higher fluoride level, samples 27 and 35 gave weak bands and sample 36 gave no bands. There is some evidence here that the effect of preservative is greater at the higher concentration. All of the weak results would have been reported as "no result" and again there would have been no likelihood of error.
EA P typing In the first study, 17 of the 18 blood samples were typed correctly. One blood sample (No 7. CB) was read by one reader as a probable CB and the other as B or CB. Sixteen of the 18 stains were typed correctly. The stain from sample 7 was read as a B by both readers. Sample 8 (CA), which was typed correctly from the blood, gave an inconclusive result from the stain. The quality of the bands obtained from the stains was in general inferior to that obtained from the blood samples. On the basis of these results it appeared possible that samples of either blood or stains containing C bands could yield erroneous results rather than no results. In TABLE 3. PGM determinations on preserved blood samples stored for long periods Sample no.
True PGM type*
Apparent PGM type from blood stored at 4°C -20°C
Total days stored t
* Of unpreserved sample. ?The frozen samples had been stored at -20°C for the last 133 days of the storage period. Earlier storage was at 4OC. JFSS 1993; 33(3): 159- 164
particular CB could be read as B and CA possibly as BA. The two samples in this series where mistypings could have occurred had been stored for 52 and 66 days respectively before stains were made, but as no other samples containing C bands were available at the time it was not possible to determine whether errors might arise from storage effects or from difficulties in reading any samples containing C bands. This latter problem can sometimes occur even with fresh samples not containing any preservative. Results from the remaining samples containing 2% fluoride, that is, the B, BA and A phenotypes, were satisfactory up to 147 days for both blood and stains. In the second study, of 4 bloods sampled one week after the addition of 2% fluoride, 3 (samples 19, 26 and 34) typed correctly. Sample 20 (C) was read by one reader as B and the other as CB. At 4 weeks after addition of preservative at both 2% and 4% levels, all 19 samples were typed correctly, including the C type. After one week at the 2% fluoride level, stain samples 19, 26 and 34 typed correctly. At the 4% fluoride level after 1 week these three samples together with sample 30 (of which there was insufficient at the 2% level), typed correctly. Sample 20 typed as either B or CB at both fluoride levels. All 19 samples typed correctly at both preservative levels after 4 weeks. Applications to casework
Case 1 In a recent homicide case, blood from the suspect was submitted in a blood alcohol bottle, and that from the victim in a toxicology bottle, with a higher level of preservative. The samples were received in the laboratory 3 days (suspect) and 2 days (victim) after collection and were stored at 4OC. Whole blood grouping on the partially haemolysed samples was attempted 2 days after receipt and unexpectedly yielded a result (ABO type B) from the suspect. The victim's blood was too haemolysed to group. Stains were made at this time and both samples grouped by absorption-elution 5 days later. The victim was ABO type A and the type B from the suspect was confirmed. PGM subtyping of the preserved whole blood samples 2 days after receipt gave a probable 2 + 1+ for the suspect and a 2 + 1 - with possible additional bands at the anode for the victim. Neither sample gave a result from the stain 5 days later. The suspect gave a clear 2 + 1 from the blood but no result from the stain after a further 2 days, while the victim gave an inconclusive result from the stain with possible
+
additional bands in the 2 region. No clear results were obtained from casework items from the victim. EAP typing of the blood 2 days after receipt gave clear results of B (suspect) and BA (victim) which were confirmed on tests of the stains after a further 5 days. The BA result from the victim was also confirmed by stains from casework items. The PGM results on blood from the suspect are largely as expected from the trials but the inability to group the stains after only 5 days was unexpected. There seemed to be a possibility that the higher fluoride concentration in the sample from the victim could affect the PGM typing by causing the appearance of additional bands. EAP results appear to be unaffected by preservative at either concentration. Case 2 This case required the typing of an old blood alcohol sample which the defendant in a drunken driving case alleged might not have been his own. Quality control procedures make it extremely unlikely that there was any exchange of samples. The defendant provided for comparison a fresh unpreserved blood sample which was typed as ABO type A, PGM type 1 and EAP type B. The questioned blood alcohol sample taken approximately 8 months earlier and stored mainly at 4°C was typed as ABO type A and EAP type B. PGM typing showed a probable 1- from the blood and a from a stain made approximately 2 probable 1 weeks earlier and stored at -20°C. Repeat determinations approximately 2 weeks later gave no result for the stain and a clear 1- for the blood. These results are consistent with the changes seen under control conditions (Table 3) and indicate that caution is needed in interpreting PGM results from old
+
+
preserved samples. A common origin between the two samples in the case could not be excluded.
References 1. Rees B, Howard H D and Strong SK. The persistence of blood group factors in stored blood samples. Journal of the Forensic Science Society 1975; 15: 43-49. 2. Culliford BJ. The examination and typing of bloodstains in the crime laboratory. Washington: US Department of Justice, 1971. 3. Brinkmann B. Interkonversion des PGM-Phanotypes unter besonderen Bedingungen. Beitraege zur Gerichtlichen Medizin 1974; 32: 141-144. 4. Gaensslen R. Sourcebook in Forensic Serology, Immunology and Biochemistry. Washington: US Department of Justice, 1983. 5. Reimann W and Willner G. Identifizierung gelagerter Alkoholblutproben durch Bestimmung der Typen der Sauren Erythrozytenphosphatase (SEPh). Deutsche Zeitschrift fuer die Gesamte Gerichtliche Medizin 1968; 64: 33-38. 6. Smerling M. Bestimmung der Sauren Erythrozytenphosphatase an Alten Blutalkoholproben und in Blutspuren. Archiv fuer Kriminologie 1969; 144: 161-166. 7. Brinkmann B, Gunnemann M and Koops E. Investigations on the decay of acid phosphatase types in stored blood stains and blood samples. Zeitschrift fuer Rechtsmedizin 1972; 70: 68-71. 8. Howard H D and Martin PD. An improved method for ABO and MN grouping of dried bloodstains using cellulose acetate sheets. Journal of the Forensic Science Society 1969; 9: 28-30. 9. Sutton JG and Burgess R. Genetic evidence for four common alleles at the phosphoglucomutase-1 locus (PGM,) detectable by isoelectric focusing. Vox Sanguinis 1978; 34: 97-103. 10. Randall T, Harland WA and Thorpe JW. A method of phenotyping erythrocyte acid phosphatase by isoelectric focusing. Medicine, Science and the Law 1980; 20: 43-47.
JFSS 1993; 33(3): 159- 164