Effects of storage conditions on forensic examination of blood samples and bloodstains stored for 20 years

Legal Medicine, 2016

Authors

Journal

Legal Medicine

DOI

10.1016/j.legalmed.2016.01.003

Study Design

Addressed Question

Stability of DNA and mRNA markers from blood after storage for 20 years

Activity Context

None

Depositor & Contact

Depositor Characteristics

N/A

Criteria for Shedder Status

N/A

Previous Activities

N/A

Contact Scenario

sampling blood in heparin - absorption of blood on cotton gauze - storage

Primary Substrate

Primary Substrate Type

cotton gauze, plastic tube

Primary Substrate Material

CottonPlastic

Deposit

absorbing blood from heparin blood sample

Delay

N/A

Secondary Substrate

Secondary Substrate Type

N/A

Secondary Substrate Material

N/A

Secondary Substrate Contact

N/A

Further Transfer

N/A

Sampling

Background DNA on Sampled Surface

Negative (Assumed)

Sampling Time

delayed

Persistence

time: 20 years storage at temperature: RT, 4°C, -20°C, -80°C

Sampling Method

direct extraction

Sampling Area

1x1 cm sample cotton gauze or 30 µl blood

Laboratory Analysis

Extraction

RNA: Rneasy Mini kit, High capacity RNA-to-cDNA kit; DNA: EZ1 DNA Investigator kit

DNA Quantification

DNA (quantification and assessment of degradation): KAPA Human Genomic DNA Quantification and QC kit, StepOne Plus Real-Time PCR system

Input for Profiling

RNA: 2 µl cDNA, DNA: maximum quantity that yielded appropriate electropherogram

Profiling

RNA: RealTime PCR amplification of HBB, ACTB, GAPDH and 18S-rRNA; DNA: AmpFlSTR Identifiler Plus PCR amplification kit, ABI 310 Genetic Analyzer, GeneMapper ID software, threshold: 150 rfu

Reference Samples

N/A

Profile Interpretation and Mixture Analysis

determination whether information was obtained from loci, no comparison to reference profiles

RNA Data Interpretation

detection of HBB, 18S, ACTB, GAPDH mRNAs based on RNA-specific Ct thresholds

Results

DNA Quantity

N/A

Profile Quality

mostly full profiles

Parameter Used for Comparison

Leucomalachite-green testing and anti-human Hb testing results; expression of HBB, GAPDH, ACTB mRNA and 18s-rRNA (Ct values); Degradation ratios (129:41 bp fragment, 305:41 bp fragment); number of STR loci detected

Summary of Results

Leucomalachite and anti-human Hb testing positive for all samples; blood mRNAs not detected from stored blood samples, but detected in all stored bloodstain samples, higher detection levels from samples stored below -20°C; bloodstains stored at RT and 4°C significantly more degraded than fresh blood, no degradation in samples stored below -20°C; STR profiling possible from all samples, few STR loci missing from samples stored at RT; Conclusion: to prevent nucleic acid degradation during long-term storage, samples should be stored below -20°C and as bloodstains

Raised Questions

N/A

Cautionary Remarks

causes for higher DNA/RNA stability from bloodstains than from blood not evaluated; data on qPCR experiment incomplete (cf. MIQE guidelines)

Field Reference

Publication

Title

The full title of the publication as it appears in the journal or conference proceedings. Together with Authors and Year, this allows unambiguous identification and retrieval of the source document.

Year

The calendar year in which the study was published. Relevant for assessing the currency of the findings, as methods and analytical sensitivity in forensic genetics have evolved considerably over time. Older studies may reflect lower detection thresholds or outdated profiling kits.

Journal

The name of the peer-reviewed journal or conference in which the study was published. The publishing venue is relevant for assessing editorial standards and scientific credibility, which is particularly important given that publication quality in forensic genetics varies widely.

DOI

The Digital Object Identifier (DOI) of the publication. A DOI provides a persistent link to the original source document and allows unambiguous retrieval regardless of changes in journal URLs or publisher. Format: 10.XXXX/...

Authors

The surname(s) and initial(s) of all authors who contributed to the publication, as listed in the original study. Useful for locating the full source and assessing author expertise or institutional affiliation.

Study Design

Addressed question

A concise summary of the central scientific question the study aimed to answer. This helps users quickly assess whether the study is relevant to their specific case question — for example, whether it investigates direct skin-to-object transfer, secondary transfer via an intermediary, persistence over time, or the influence of a particular variable on DNA yield.

Activity context

Describes the specific activity or scenario in which DNA transfer was studied — particularly whether the transfer occurred in the context of a criminal act (e.g. shooting, stabbing, sexual contact) or a routine social or professional interaction (e.g. handshake, speaking, shared laundry). This is a key filter for activity-level evaluation: it allows users to identify studies relevant to the specific scenario alleged in a case, enabling a comparison of alternative hypotheses about how a trace came to be deposited.

Valid values

noneshootingmanual strangulationassaultsexual contactkissingstabbingburglarytheftscratchingwashing machinecaseworkbitingprofessionalspeakingmailsocial contactwildlife crimeexplosive devicescar accidents

Category

The overarching category describing what aspect of DNA trace formation is examined in the study. "Primary deposit" covers direct (skin-to-surface) contact; "Transfer scenario" covers further movement of DNA after the initial deposit (secondary, tertiary transfer, etc.); "Background DNA" addresses pre-existing DNA levels on surfaces unrelated to the case event; "Persistence" covers how DNA degrades or survives over time under varying conditions; "Recovery" covers methods used to collect DNA from surfaces for subsequent analysis. Most studies fall into one or two categories.

Valid values

Primary depositTransfer scenarioBackground DNAPersistenceRecovery

Specifications

A more granular classification of the specific variable or sub-topic examined within the study's category. For example, within "Primary deposit" a study might focus on "individual characteristics" (e.g. shedder status), "contact" (type or duration), or "surface" (material properties). Within "Persistence", it specifies the exact degrading factor studied, such as time, temperature, humidity, or water immersion. Note: "BG on..." entries describe what surface type background DNA was examined on — they do not indicate whether background DNA was actually present on sampled surfaces (see the "Background DNA on sampled surface" column for that).

Valid values

individual characteristicscontactsurfacebodily origintransfer sequenceprevious activitiestransfer via vectortransfer via handshakeBG on skin/other body locationsBG on personal itemsBG on public itemsBG on clothingBG in professional environmentPersistence with timePersistence with temperaturePersistence with firing/explosionPersistence with irradiationPersistence with environmental conditionsPersistence with packaging and transportPersistence with regular activitiesPersistence with further contactPersistence with water immersionPersistence with decontaminationPersistence with latent print enhancementSamplingExtractiondirect PCRDNA profilingRNA profilingvisualisationPersistence with microbial growthPersistence with humidity

Variables of interest

The specific parameters that were deliberately varied between experimental conditions in order to assess their effect on DNA transfer, persistence, or recovery. For example, a study might vary contact duration, applied pressure, surface material, or number of wash cycles. Understanding which variables were tested helps determine how directly the study's findings are applicable to a specific case scenario.

Stringency of Control

Indicates how closely the study's conditions were controlled or how realistically they reflected actual case scenarios. "Controlled" = a laboratory experiment with standardised, reproducible conditions. "Reality" = samples derived from real casework or uncontrolled environments (e.g. public surfaces). "Close to realistic" = a semi-controlled experiment that attempts to mimic real-world conditions. This distinction is critical for evaluating how transferable the findings are to real cases: highly controlled studies offer internal validity but may not reflect true-to-life variation, while casework-based studies have ecological validity but lack experimental rigour.

Valid values

controlledrealityclose to realistic

Number of Individuals

The total number of human participants (donors or depositors) involved in the study. A larger sample size generally increases the statistical reliability of findings and accounts for known inter-individual variation in DNA shedding behaviour. Studies with very few participants (e.g. n < 5) should be interpreted with caution. "n.s." indicates the number was not specified in the publication.

Replicates per Individual and condition

The number of times each experimental condition was repeated per participant. A higher number of replicates reduces measurement noise and increases confidence in the reported results. Where replicates are low or absent, findings may not be robust. "n.s." indicates this was not specified in the publication.

Nucleic Acid

The type of nucleic acid analysed in the study. "DNA" refers to standard autosomal nuclear DNA (the most common). "mtDNA" (mitochondrial DNA) is inherited maternally and is present in higher copy numbers per cell, making it useful for degraded samples. "Y-Chromosome" refers to studies specifically analysing multiple Y-chromosomal markers, relevant in mixed-source traces involving male contributors. "RNA" can be used for body fluid identification. "Methylome" refers to methylation patterns, relevant for tissue-type identification or age estimation. The nucleic acid type affects how findings should be interpreted and whether they are applicable to the analytical method used in a given case.

Valid values

DNARNAmtDNAY-ChromosomeMethylome

Bodily origin

Specifies the biological source of the DNA in the study — i.e. the cell type and/or body location from which the deposited material originated (e.g. buccal cells, skin cells, spermatozoa, saliva, blood, vaginal epithelial cells, nasal secretions). This is relevant because different cell types vary considerably in the amount of DNA they shed, how they transfer, and how well they persist on surfaces. "Trace" is used when the bodily origin is unknown or uncharacterised, as is common with touch DNA.

Depositor & Contact

Depositor characteristics

Any individual-level characteristics of the DNA-depositing person(s) that were recorded or considered in the study, such as age, sex, skin condition, hygiene habits, or shedder status classification. Inter-individual variation is one of the most significant sources of variability in DNA transfer research. Where depositor characteristics are unspecified ("n.s."), the generalisability of findings may be limited. "n.a." = not assessed (i.e. characteristics were not considered in the study design).

Criteria for shedder status

When study participants were classified as "good", "normal", or "poor" DNA shedders, this column records the criterion or method used to make that classification (e.g. DNA yield from a standardised handshake on a defined surface). Shedder status is a well-documented source of inter-individual variation and has significant implications for evaluating the likelihood that a given person would have deposited a detectable trace. "n.s." = criteria not specified; "n.a." = shedder status was not assessed in this study.

Previous activities

Any activities performed by the depositor prior to the experimental contact that may have influenced DNA transfer — for example, hand washing, physical exercise, eating, or prior handling of objects. Previous activities can either enhance DNA availability on the skin (e.g. friction) or reduce it (e.g. recent handwashing). Where not controlled for, this represents a potential confound. "n.s." = not specified; "n.a." = not assessed.

Contact scenario

A step-by-step description of the sequence of events in the contact scenario as performed in the study. This includes the nature, duration, and order of contacts between persons and/or objects. For transfer studies, this is essential context: the number of transfer steps and the nature of each contact directly affect DNA yield and profile quality. Understanding the full scenario helps assess whether the study is analogous to the circumstances of a specific case. "n.s." = not specified; "n.a." = not assessed.

Primary Substrate

Primary substrate type

The type of object or surface that received the initial (primary) DNA deposit through direct contact. Where a precise item cannot be identified, an umbrella term is used: e.g. "firearms" (including pistols, rifles, revolvers), "knife", "cartridges", "fabric", "tools", "points of entry" (door handles, window frames), "body parts", "body fluids", or "personal items" (mobile phones, glasses, bags). This column describes what was touched, while "Primary substrate material" describes what it is made of. "n.s." = not specified.

Primary substrate material

The physical material composition of the primary substrate. Surface material is a key variable in DNA transfer and retention: porous materials (e.g. cotton, denim) tend to retain DNA differently from smooth non-porous surfaces (e.g. glass, metal, plastic). This affects both the amount of DNA deposited and the efficiency of subsequent sampling. Relevant when comparing study findings to the material of an item in a specific case.

Valid values

plasticwoodpaperglasscottonskinskin and nailvariousvinylmucosametaln.s.adhesiveairleatherfabricaluminiumsquirrel hairfibreglassbrassnickel-plated brasspolyesterduststonesteelnickelnylondenimwoollatexnitrilemelamineacetaterubberfoodanimal bodiesplexiglassceramic

Deposit

Describes the time and manner in which the primary DNA deposit was made — for example, whether it involved a single brief touch, prolonged grip, rubbing, or forceful contact, and whether the contact was performed once or multiple times. The nature of the primary deposit fundamentally influences how much DNA is transferred and how reliably a profile can be obtained. "n.s." = not specified; "n.a." = not applicable (e.g. in background DNA studies where no intentional deposit was made).

Delay (conditions)

The time elapsed between the primary deposit and a subsequent contact (secondary transfer), along with any storage or environmental conditions during that interval. This column specifically concerns delays between contacts in a transfer chain, not the delay before sampling (which is covered under "Persistence (conditions)"). Delay duration and conditions (e.g. temperature, humidity, exposure) can affect the amount of DNA available for onward transfer. "n.s." = not specified; "n.a." = not applicable.

Secondary Substrate

Secondary substrate type

The type of object or surface that received DNA via secondary (indirect) transfer — i.e. after DNA had already been deposited onto a primary surface and was then transferred onward. The same umbrella terms apply as for the primary substrate (firearms, knife, fabric, body parts, etc.). Secondary transfer is a key mechanism by which DNA can reach a scene or object without direct contact by the person of interest. "n.s." = not specified.

Secondary substrate material

The physical material of the secondary substrate — i.e. the surface onto which DNA was indirectly transferred. As with primary substrates, the material influences how much DNA is retained and how retrievable it is. This is especially relevant for evaluating scenarios where indirect deposition is offered as an alternative hypothesis to direct contact. "n.s." = not specified.

Valid values

plasticwoodglassskinn.sadhesivesquirrel hairfibreglassacetateoilcottongelatinesiliconewooldenimleathernylonpolyestersilknitrilelatexpapercardboardbonemetalfabric

Type of secondary contact

A description of how the secondary transfer event occurred — including the nature, duration, and force of the contact between the primary and secondary substrate. For example, whether it was a brief touch, a prolonged rub, a forceful grip, or incidental contact. The mechanics of the secondary contact directly affect transfer efficiency, which is central to evaluating whether indirect DNA deposition is a plausible alternative explanation. "n.s." = not specified; "n.a." = not applicable.

Further transfer

Describes any additional transfer steps beyond the secondary contact — i.e. tertiary or higher-order transfer events, including the substrates involved and the nature of each contact. Longer transfer chains are generally associated with decreasing DNA quantities at each step, but even tertiary transfer has been shown to produce detectable and sometimes interpretable DNA profiles under certain conditions. "n.s." = not specified.

Sampling

Background DNA on sampled surface

Records whether background DNA (pre-existing DNA from unrelated individuals or prior contact events) was detected on the sampled surface, and how this was established. "Present" = background DNA was found. "Negative (Confirmed)" = absence was confirmed through dedicated control sampling. "Negative (Assumed)" = no controls were run, but background DNA was assumed to be absent. "Sampled" = the study specifically sampled surfaces to characterise background DNA levels (i.e. the study IS about background DNA). Background DNA is an important confounding factor: it can complicate mixture interpretation and challenge attributions of a trace to a specific person or event.

Valid values

PresentNegative (Confirmed)Negative (Assumed)Sampled

Sampling time

Indicates whether the surface was sampled immediately after the transfer scenario or after a defined delay. Sampling immediately after deposition provides a baseline for DNA quantity and quality, while delayed sampling introduces persistence as an additional variable. Understanding when sampling occurred is essential for correctly interpreting reported DNA quantities and profile qualities. "n.s." = not specified; "n.a." = not applicable.

Persistence (conditions)

When sampling was delayed, this column specifies the duration of the delay and the conditions under which the item was stored or exposed during that period — for example, indoor ambient conditions, outdoor weather exposure, specific temperatures, humidity levels, or UV irradiation. Persistence data is critical for cases involving a delay between the alleged contact event and the recovery of the trace (e.g. a weapon recovered days after the incident). "n.s." = not specified; "n.a." = no delay before sampling.

Sampling method

The technique used to collect DNA from the surface, such as wet/dry swabbing, tape lifting, cutting out a fabric section, or scraping. The sampling method directly affects DNA recovery efficiency and can interact with substrate material. Some methods are better suited to porous surfaces, others to hard non-porous ones. Differences in sampling method between the study and the case being evaluated may limit the comparability of findings. "n.s." = not specified; "n.a." = not assessed.

Sampling area

Describes the location on the item from which the sample was taken and, where reported, the size of the sampling area (e.g. 4 cm²). Sampling area affects the total amount of DNA collected and is relevant for interpreting DNA quantities: a larger area may yield more DNA irrespective of transfer efficiency. Where multiple areas were sampled, each may be listed separately. "n.s." = not specified; "n.a." = not assessed.

Laboratory Analysis

Extraction

The method used to release and purify DNA from the sample, including any concentration or clean-up steps (e.g. organic extraction, Chelex, differential lysis for mixed intimate samples, solid-phase extraction). Extraction efficiency varies by method and by sample type, and can strongly influence downstream DNA quantities and profile quality. Differences in extraction approach between studies should be considered when comparing DNA yield results. "n.s." = not specified; "n.a." = not assessed.

DNA Quantification

The method used to measure the amount of DNA in the extracted sample before profiling, such as quantitative PCR (qPCR/real-time PCR), digital droplet PCR (ddPCR), or fluorometry. Quantification results inform decisions about how much DNA to input into the PCR amplification step and can indicate whether a sample is likely to yield a full or partial profile. "n.s." = not specified; "n.a." = not assessed (some studies skip quantification and proceed directly to profiling).

Input for Profiling

The amount of nucleic acid (DNA or RNA) used as input for the PCR amplification step prior to profiling. This is typically reported in nanograms (ng) or picograms (pg), or described as "a.p.m.i." (as per manufacturer's instructions) when the exact amount is not stated. The input amount affects profile completeness and the likelihood of artefacts such as allelic drop-out or drop-in. "n.s." = not specified; "n.a." = not assessed.

Profiling

The method used to generate the DNA or RNA profile from the amplified material, including the commercial kit used (e.g. GlobalFiler, PowerPlex Fusion), the capillary electrophoresis platform, the analysis software (e.g. GeneMapper), and any specific settings or thresholds applied. Profiling methodology determines which genetic markers are typed, the sensitivity of detection, and the interpretation standards applied. Differences in profiling kits and thresholds between studies and cases can affect comparability. "n.s." = not specified; "n.a." = not assessed.

Reference samples

Describes how and from whom reference samples (known DNA profiles for comparison) were obtained in the study — for example, buccal swabs collected immediately before the experiment, or blood samples taken under controlled conditions. Reference samples are necessary to determine whether a recovered trace profile matches an individual and to assess mixture composition. The timing and method of reference collection can affect profile quality. "n.s." = not specified; "n.a." = not assessed.

Profile interpretation and mixture analysis

Describes the approach used to interpret the DNA profiles obtained, including how mixed profiles (containing DNA from more than one contributor) were analysed. This may include the software or probabilistic genotyping tool used (e.g. STRmix, TrueAllele, LRmix Studio), the number of contributors assumed, and any thresholds or statistical frameworks applied. The interpretation method has a significant impact on reported match statistics and on whether a contributor could be included or excluded. "n.s." = not specified; "n.a." = not assessed.

RNA data interpretation

Where RNA analysis was performed (e.g. for body fluid identification), this column describes the method used to interpret the RNA expression data — for example, which tissue-specific markers were targeted, what expression thresholds were applied, and whether the interpretation was qualitative or quantitative. RNA analysis can supplement DNA profiling by identifying the biological source of a trace. "n.s." = not specified; "n.a." = RNA analysis was not performed or not applicable.

Results

DNA Quantity

Reports the range or average DNA quantity obtained from samples in the study, typically expressed in nanograms per microlitre (ng/µl) or total nanograms recovered. These values provide an indication of how much DNA can realistically be expected under the experimental conditions studied, and can be used as a benchmark for comparing with case sample yields. Large variation between individuals and conditions is common. "n.s." = not specified; "n.a." = not assessed.

Profile Quality

A qualitative or semi-quantitative characterisation of the DNA profiles obtained — for example, whether full single-source profiles, partial profiles, or mixed profiles were produced; whether allelic drop-out or drop-in was observed; and whether profiles were suitable for database searching or individual comparison. Profile quality is directly relevant to assessing the evidential value of a trace and is often more informative than DNA quantity alone. "n.s." = not specified; "n.a." = not assessed.

Parameter used for comparison

Specifies which measurable outcome(s) were used to compare results across conditions or individuals in the study — for example, DNA quantity, number of alleles detected, percentage of full profiles obtained, or likelihood ratio values. Understanding what was compared is essential for interpreting the reported findings and for assessing whether the study's conclusions are supported by its data. "n.s." = not specified; "n.a." = not assessed.

Summary of results

A concise, curated summary of the most important findings and trends from the study, as interpreted in the context of activity-level evaluation. This goes beyond restating the abstract: it highlights which conditions produced detectable DNA, which variables had the greatest impact, and what the results mean for interpreting DNA traces in forensic practice. Where results were mixed or dependent on specific conditions, this is noted. "n.s." = not specified; "n.a." = not assessed.

Raised questions (by authors)

Future research directions, open questions, or unresolved issues explicitly identified by the study's authors. These may include gaps in the current experimental design, variables not addressed, or broader questions about DNA transfer or persistence that the study was unable to resolve. Awareness of these gaps is relevant when assessing whether current scientific knowledge is sufficient to support a definitive opinion on a case question.

Cautionary remarks

Critical observations about the study's limitations, methodological weaknesses, or constraints on the generalisability of its findings — as identified through expert review by the DNA-TrACK-Web curators. This may include concerns about sample size, unrealistic experimental conditions, uncontrolled confounders, or potential issues with analytical methods. These remarks are essential for responsible use of the database: even a well-designed study may have specific limitations that affect how its results should be weighted in a particular case. "n.s." = not specified; "n.a." = no specific cautionary remarks identified.