The utility of `substrate controls' in relation to `contamination'

Forensic Science International85 (1997) 105–111

The utility of ‘substrate controls’ in relation to‘contamination’

Peter GillService Development, The Forensic Science Service, Priory House, Gooch Street North, Birmingham

B5 6QQ, UK

Received 20 April 1996; revised 30 September 1996; accepted 30 October 1996

Abstract

The use of ‘substrate’ controls in DNA analysis (specifically related to STRs) is criticallyexamined. It is concluded that the rationale for use is of limited value. Substrate controlsdo not test the possibility of contamination. 1997 Elsevier Science Ireland Ltd.

Keywords: Substrate control; Contamination; STRs; Inference; Relevance

1. Introduction

Historically, blood-grouping tests were compromised by possible spuriousresults from bacteria or non-specific reactions with the substrate e.g. cross-reaction with vegetable material. A control taken from a region close to the crimestain was routinely used to test such possibilities. If the substrate control gave apositive reaction which was the same result as the crime stain then the result fromthe crime stain was not reported. The original concept of the substrate controlwas to test for spurious reactions with environmental contaminants such asvegetable extracts, it was not intended to be used to test for a body fluid from ahuman source unassociated with a particular event or crime.

STRs differ from blood grouping tests in important respects:

1. STRs used in forensic analysis are human (or at least primate) specific.2. Adventitious results from bacteria or yeast have not been observed.0379-0738/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reservedPII S0379-0738( 96 )02085-3

106 P. Gill / Forensic Science International 85 (1997) 105 –111

3. Non-specific artefacts originating from substrate reactions are not observedwith the possible exception of reaction with some dyes [1].

2. Considerations

2.1. Definition of the ‘substrate’ control for STRs

Analysis of substrate controls adjacent to a crime stain has been suggested forSTRs [2] to test for the possibility of ‘contamination’. A substrate control couldbe defined as an area of garment which is adjacent to the crime stain, and isoutside the area indicated by a presumptive test for a given body fluid — apositive result from a substrate control would not be expected.

However, a negative presumptive test does not disprove the presence of a bodyfluid. This is because STRs are sensitive to levels of body fluid lower than thethreshold of the presumptive test and it is not uncommon for an STR result tooccur. It follows, therefore, that the area adjacent to a crime stain is neither‘substrate’ nor a control, it is simply an area of cloth adjacent to a crime stainwhere the presumptive test has failed to give a result. DNA may still be present insufficient quantity to amplify albeit in trace amounts. A possible use that a truesubstrate control may have, would be to test for the effect of a textile-dye — andthis type of control should be taken as far away from the crime stain as possible.For convenience the term ‘substrate’ control is retained in this paper. It is definedas previously stated.

To consider the merits of analysing material adjacent to a crime stain, themeaning of the word ‘contamination’ must first be made clear in the context ofPCR analysis.

2.2. Definition of ‘contamination’

There are 2 distinct causes:(1) Laboratory related cross-contamination occurs as a result of transfer of

DNA (either amplified or non-amplified) to a different test which then gives areaction seen as a spurious result or an unexplained mixture. The possibility ofcross-contamination is minimised by suitable laboratory set-up and by physicalseparation of the various processes. Reliance is placed upon the use of negativecontrols to test contamination of reagents and tubes — a blank sample is takenthrough the entire process in parallel with the case stain.

The conclusion from this is that the substrate control does not test contamina-tion arising from laboratory practise.

(2) If laboratory based contamination is discounted, the remaining alternativeto consider is that DNA has been transferred by means which are unassociatedwith a crime. Typically, this will occur before or after the crime has beencommitted. In this paper I use the term ‘passive transfer’ to describe such acondition since the act of contamination strongly suggests accidental transfer of

P. Gill / Forensic Science International 85 (1997) 105 –111 107

DNA unrelated to the environment of the wearer (by definition this would berestricted to the period of time after the crime i.e. after collection of the garmentby investigating officers and subsequent laboratory analysis). Transfer of DNArelevant to the crime which I define as ‘case-relevant transfer’ need not always berestricted to the period of time during which the offence was actually committed,although it seems logical to contend that in the majority of cases, contact with thesuspect would normally occur during a tightly-defined timeframe centred on theoffence itself.

In the context of the substrate control, passive or case-relevant transfer ofDNA from an unknown source may be observed in addition to the major stainbeing investigated; the STR profile may or may not be the same as the suspect. Ifwe consider a case where a victim has been assaulted by a single suspect andgarments from both individuals have been DNA profiled, both passive andcase-relevant transfer of DNA from several different sources can occur over aperiod of time i.e. before, during and after the offence. Accordingly, it would notbe surprising to detect small amounts of DNA originating from the victim orperpetrator in areas outside an identifiable crime stain. Interpretation of such aresult is normally straightforward, and does not compromise the result from thecrime stain itself.

We next need to consider the implications of the presence of a trace amount ofan unknown DNA type which matches neither victim nor suspect. There are twopossibilities.

1. The DNA has been transferred during the course of the crime from aperpetrator who is different from the suspect.

2. The DNA was transferred by passive means before or after the crime.

Examples which involve passive transfer of DNA from a source other than thevictim could involve transfer via saliva spray — someone sneezing onto thegarment is a possibility.

Experiments carried out by Lygo et al. [2], and Comey and Budowle [3] to testfor passive transfer of DNA onto a garment or other substrate as a result ofcoughing, perspiration or handling demonstrated that this type of cross-transfer isunusual (but can occur). Furthermore, the amounts of DNA transferred are likelyto be low.

The difficulty the forensic scientist faces is that he has no information aboutwhen transfer occurred. He can only state whether or not an observed DNAprofile is consistent with a given scenario. It does not follow that simply becausethere is a DNA profile which cannot be explained that the DNA is a result of‘contamination’. The forensic scientist may not be in a position to ascertain therelevance of an unknown profile in the context of what is known about the case.However, it may be possible that further information could later become availablewhich then explains its presence.

The conclusion that can be drawn is that the presence of trace amounts of DNAon areas of the garment which match the victim or suspect is to be expected.


These traces may be found in the ‘substrate’ control but it is unlikely that anydifficulty in interpretation will occur as a result. On the other hand, if the DNAprofile in the control area does not match a known individual then the relevanceto the case is difficult to assess since it is not known when or how the transferoccurred.

3. Interpretation of positive ‘substrate control’ results

To establish if a result from a ‘substrate control’ affects interpretation, it isworth listing all possible scenarios in a typical case where a garment worn by thevictim is analyzed for the presence of a body fluid. In all cases it is assumed thatthe STR profiles of victim and suspect have been established and are differentfrom each other otherwise the interpretation would be different.

Table 1 illustrates some possible inferences which can be drawn from a rangeof scenarios involving results from testing an area adjacent to the crime stain. Theresults from none of the scenarios affect the interpretation of the main bloodstain. Under certain circumstances the interpretation may be clarified; forexample, if a mixture is present, we have found that proportions vary within thecrime stain such that sampling at the edge or outside the edge of a crime stainmay reveal just one component.

4. Relevance of evidence

To generalise is always difficult because the circumstances of every case aredifferent and cannot be considered in isolation from other evidence. In addition,evidence is only useful if it is relevant to the case; the possibility of transfer byinnocent or passive means must also be considered in an investigation [4].Unexpected results may be explained if further information becomes availableduring the investigation of a case. In practise, the scientist may wish to explorethe following 2 different possibilities.

Given that the DNA profile matches the suspect what is the chance of seeingthe evidence if:

1. the profile originated from an unknown perpetrator of the crime;2. the profile originated as a result of passive transfer from an unknown

individual (ignoring the possibility of laboratory contamination as this has beendealt with earlier).

The probabilities of chance association for both scenarios are identical. If theSTR result has occurred as a result of passive transfer then the evidence stillsupports the hypothesis that the DNA actually originated from the suspecthimself, but under circumstances unassociated with the crime. The inference maybe qualified if the stain has been demonstrated to be sperm, blood or saliva by


Tab

le1

Alis

tof

poss

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crim

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test

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cent

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ime

stai

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ossi

ble

infe

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es

Mat

ches

susp

ect

Neg

ativ

eN

ode

tect

able

DN

Aou

tsid

est

ain

area

.

Mat

ches

susp

ect

Mat

ches

susp

ect

The

stai

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ffus

edin

toth

e‘c

ontr

ol’

regi

onan

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ata

leve

lun

dete

ctab

leby

apr

esum

ptiv

ete

stor

has

orig

inat

edfr

oma

body

fluid

diff

eren

tto

the

crim

est

ain.

Mat

ches

susp

ect

Mat

ches

vict

imT

here

isso

me

back

grou

ndD

NA

pres

ent

whi

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have

orig

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other tests. If the result has been obtained from an area outside the main stainarea, then it may not be possible to qualify the type of body fluid which gave riseto the result and this may affect the inference. The evidence still supports thehypothesis that the suspect has deposited cells on the garment but in the absenceof information defining the body fluid type, the relevance of the information maybe different to that obtained from the crime stain proper.

5. Conclusions

The rationale for using substrate controls appears to have never been clear, buttheir use is widespread. It is the intention of this paper to critically examine thereasons to carry out a substrate control test in the context of DNA STR analysis.If the control is taken from an area adjacent to a crime stain and a presumptivebody fluid is negative, then this does not properly test the substrate.

Contamination implies accidental transfer of DNA. By definition this can onlyhappen during the period of time after the garment has been collected by officersinvestigating the crime and subsequent laboratory analysis (i.e. unrelated to boththe environment of the wearer and to the crime itself). Transfer of DNA whilethe garment is still worn (by victim or suspect) before and after the crime which isunassociated with the event is best described as innocent or passive transfer (i.e.relevant to the environment of the wearer, but unassociated with the crime itself).

If taken from an area adjacent to a crime stain which is apparently free of bodyfluid, the substrate control is neither substrate nor a control region becausetransfer of trace amounts of DNA can be detected in regions where a presumptivetest for body fluids gives a negative result. The substrate control cannot giveinformation about contamination, and it may not be known whether the traceamount of DNA present was transferred as a result of case-relevant or passivetransfer. The substrate control might help with certain types of interpretation —for example where the components of a mixture are unevenly spread through thefabric.

The results of DNA tests may not tell us when the transfer of DNA occurred,particularly if trace amounts are found and the body fluid type is not determined.Simply because a DNA profile matches a suspect, does not mean that transferoccurred during the course of the crime. The possibility of passive or innocenttransfer must always be considered within the context of what is known about agiven case.

Acknowledgments

I am grateful to I.W. Evett, R. Fourney, C. Kimpton, R. Sparkes, P. Lincolnand D.J. Werrett for helpful discussions and also to the referees who made veryuseful comments on the original manuscript.


References

[1] A. Urquhart, C.T. Chiu, T. Clayton, T. Downes, R. Frazier, S. Jones, C. Kimpton, M.V. Lareu,E. Millican, N. Oldroyd, C. Thompson, S. Watson, J. Whitaker and P. Gill, Multiplex STRsystems with fluorescent detection as human identification markers. Proceedings of the FifthInternational Symposium on Human Identification 1994, Promega Corp., 1995, pp. 73–83.

[2] J.E. Lygo, P.E. Johnson, D.J. Holdaway, S. Woodroffe, J.P. Whitaker, T.M. Clayton, C.P.Kimpton and P. Gill, The validation of short tandem repeat (STR) loci for use in forensiccasework. Int. J. Legal Med., 107 (1994) 77–89.

[3] C.T. Comey and B. Budowle, Validation studies on the analysis of the HLA DQ alpha locususing the polymerase chain reaction. J. Forensic Sci., 36 (1991) 1633–1648

[4] I.W. Evett, Establishing the evidential value of a small quantity of material found at a crimescene. JFSS, 33 (1993) 83–86.

The utility of `substrate controls' in relation to `contamination'

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