DNA recovery after sequential processing of latent fingerprints on copy paper

Abstract Forensic examiners must determine whether both latent fingerprint development and DNA profiling can be performed on the same area of an evidence item and, if only one is possible, which examination offers the best chance for identification. Latent fingerprints can be enhanced by targeting different components of fingerprint residues with sequential chemical treatments. This study investigated the effects of single‐reagent and sequential latent fingerprint development processes on downstream DNA analysis to determine the point at which latent fingerprint development should be stopped to allow for DNA recovery. Latent fingerprints deposited on copy paper by one donor were developed using three sequential processes: 1,8‐diazafluoren‐9‐one (DFO) → ninhydrin → physical developer (PD); 1,2‐indanedione‐zinc (IND‐Zn) → ninhydrin → PD; and IND‐Zn → ninhydrin → Oil Red O (ORO) → PD. Samples were examined after the addition of each chemical treatment. DNA was collected with cotton swabs, extracted, quantified, and amplified. DNA yields, peak heights, number of alleles obtained, and percentage of DNA profiles eligible for CODIS upload were examined. DNA profiles were obtained with varying degrees of success, depending on the number and type of treatments used for latent fingerprint development. The treatments that were found to be the least harmful to downstream DNA analysis were IND‐Zn and IND‐Zn/laser, and the most detrimental treatments were DFO, DFO/laser, and PD. In general, as the number of treatments increase, the opportunities for DNA loss or damage also increase, and it is preferable to use fewer treatments when developing latent fingerprints prior to downstream DNA processing.


| INTRODUC TI ON
Disciplines within the forensic field must often work together to ensure the most comprehensive examination of evidence is accomplished. In today's crime laboratories, it is not uncommon for fingerprint examiners and DNA analysts to visually examine evidence to determine the best course of action prior to processing and to develop a joint plan of action for downstream forensic testing of the item of interest.
Because latent fingerprint and forensic DNA testing can be considered destructive processes, the questions often asked are whether or not both examinations can be performed on the same area and, if only one is possible, which is likely to offer the best chance for identification. Previous studies have shown that partial to full DNA profiles can still be obtained from a single fingerprint on paper substrates after latent print processing [1]. Various groups have even demonstrated that while the quality of DNA profiles may be negatively affected, quantifiable DNA can be obtained from fingerprints treated with single development reagents such as ninhydrin, 1,8-diazafluoren-9-one (DFO), and 1,2-indanedione-zinc (IND-Zn) [1][2][3][4][5].
Latent fingerprints are primarily comprised of eccrine sweat released from pores between the friction ridges, sebaceous secretions from other areas of the body, and contaminants from the environment thus producing an impression of the ridges when deposited [6].
Eccrine sweat is comprised of a complex variety of compounds [7], including water (20%-70%) [8,9] and quantifiable amounts of amino acids that react with reagents, such as DFO [10], ninhydrin [11], and IND-Zn [12], through distinct mechanisms. Additionally, fingerprint residues contain sebaceous oils that are transferred to the fingers when an individual touches sebaceous gland-rich areas such as the face and scalp [6]. When deposited on porous surfaces, the lipids found in these sebaceous oils can be detected by chemicals such as physical developer (PD) or Oil Red O (ORO). Because each of these development processes reacts with different components found in fingerprint residue, it has been determined that the development of latent fingerprints can be enhanced by a targeted sequential processing scheme whereby different attributes of fingerprint residues are developed in a stepwise manner [13,14]. While various studies have been conducted to determine the downstream effects of single-process latent fingerprint development on DNA analysis [1][2][3][4][5]15,16], the extent to which routine sequential latent fingerprint development processes affect the recovery of DNA is unknown.
The aim of this study was to evaluate the effects of single-reagent and sequential latent fingerprint development processes on downstream DNA analysis. If an effect was determined, the secondary goal was to determine at which step in the process the DNA was affected. Three sequential processes for developing latent fingerprints on paper were evaluated: (1) DFO (visualized with a 532 nm laser), followed by ninhydrin, followed by PD [14,[17][18][19]; (2) IND-Zn (visualized with a 532 nm laser), followed by ninhydrin, followed by PD [14,20]; and (3) IND-Zn, followed by ninhydrin, followed by ORO, followed by PD [13,17]. DFO was popularized as a component of single-reagent and sequential processes in the 1990's [21] and became the second most widely used reagent for developing latent fingerprints on porous surfaces by 2004 [22]. IND-Zn has been identified as the successor to DFO [19,20,[23][24][25]; however, DFO is still used by some practitioners. DFO [26] and IND-Zn [20] may both be followed with ninhydrin to develop additional ridge details. Further treatment with reagents like PD and ORO can sometimes develop additional latent prints not previously visualized [13].

| Solution preparation
The materials and methods for preparing the chemical solutions used for latent fingerprint development can be found in Table 1.

| Fingerprint preparation
A total of 144 fingerprints were prepared on standard-weight copy paper (Staples) by one donor over the course of 9 days. Fingerprints were obtained with informed consent. A single donor was used to mitigate DNA yield variability associated with different individuals' propensities to shed epithelial cells [27]. Paper was cut into 1.5″ × 2.0″ sections and was decontaminated by short-wave UV irradiation in a Spectronics Spectrolinker™ XL-1500 with a 254 nm bulb. Substrates were placed on a desktop scale, and the index, middle, and ring fingers from both hands were used to deposit fingerprints by pressing down until 2 kg was reached after a total substrate contact time of approximately 1 s. Next, the finger was removed from the substrate. To mimic real-world evidentiary-handled documents, fingerprints were not charged with additional sebaceous oils, handwashing was kept to a minimum, and at least 2 h elapsed after handwashing and between fingerprint depositions.

| Fingerprint processing
Latent fingerprints were treated with single-reagent or sequential latent fingerprint development processes (Table 2). Eight replicates were processed with either DFO, ninhydrin, ORO, • Sequential processing may be considered depending on the number and type of treatments used.
• Results demonstrated that fewer treatments are preferable.
• DFO and PD are not recommended when performing downstream DNA analysis.
or PD, and 10 replicates were processed with either DFO/laser, IND-Zn, or IND-Zn/laser. Three sequential processes were exam- 1,8-Diazafluoren-9-One DFO was prepared following the Federal Bureau of Investigation's (FBI) guidelines for developing latent fingerprints [18]. Samples were briefly soaked in the reagent, air dried, and placed in an Isotemp ® model 106G oven (Thermo Fisher Scientific) for 20 min at 100°C for development.

1,2-Indanedione-Zinc
IND-Zn was prepared following Ramotowski [28]. Samples were briefly soaked in the reagent, air dried, and placed in an environmental

Ninhydrin
Ninhydrin was prepared following Ramotowski [28]. Samples were briefly soaked in the reagent, air dried, and placed in an environmental chamber (Caron) for 15 min at 80°C and 65% relative humidity for development.

Oil Red O
Oil Red O was prepared by combining ORO and sodium hydroxide solutions following guidelines from Beaudoin [30]. Samples were im-

| Sample collection
DNA was collected directly from the paper with 100% cotton Bode SecurSwabs (Bode Technology) by wetting the tip of the swab with 1-2 drops sterile DNA grade water from a 3 ml AddiPak brand water vial, swabbing the area where the fingerprint was deposited and/ or visualized, turning the swab to the dry portion of the swab head, and swabbing the area again. To determine where in the sequential process the DNA may be affected, DNA was collected after each treatment was added.

| Data analysis
The effects of the single-reagent and sequential latent fingerprint with a match rarity of at least one in ten million [32]  This variation in processing did not lead to statistically significant differences in DNA yield, number of alleles obtained, or peak height for either chemical treatment.

| Sequentially treated latent fingerprints, DFO/ Laser → Ninhydrin → PD
Latent fingerprints treated sequentially with DFO/laser, ninhydrin, and PD were compared with the untreated latent fingerprints using the aforementioned metrics ( Figure 2   This study did not determine whether decreases in DNA yield and STR profile quality were due to DNA loss resulting from immersion of the samples during latent fingerprint development; DNA degradation resulting from exposure to high heat, high humidity, incompatible pH, or deleterious chemicals; or a combination of these factors. Heat induces DNA degradation through a variety of mechanisms that can lead to strand breakage [34], including hydrolysis [35,36], deamination [37,38], depurination [39], depyrimidination [40], and oxidation [41][42][43]. Degradation rates resulting from these mechanisms increase as temperature and incubation times increase and pH decreases [44].  [45], and DNA concentration [45].  [42,43]. When planning to perform downstream DNA processing of latent fingerprints, these treatments should be avoided both in both single-reagent and sequential processes.

| Sequentially treated latent fingerprints, IND-Zn → Ninhydrin → ORO → PD
To determine the effect of the laser treatment on DNA recovery,  however, other methods, such as cutting samples from the paper, may prove more effective. Cotton swabs can retain up to 50% of the recoverable DNA [61]. Furthermore, DNA extraction can result in the loss of ≥72% of the initial template amount [62,63], and alternative processing methods may improve the overall quality of the STR profiles. In particular, direct amplification, a method in which a cutting or swab is added directly to an amplification reaction without prior extraction or quantification, has been identified as an effective method for improving DNA profiles from low-yield samples [64].

| CON CLUS IONS
Single-reagent and sequential fingerprint development treatments can be used to visualize latent fingerprints on paper items for examination by fingerprint examiners and for targeted DNA collection by DNA analysts. While latent fingerprint enhancement can help provide more information during an investigation, the number and types of fingerprint development treatments that are used can negatively impact the ability to obtain DNA from the fingerprints. In particular, the use of single-reagent and sequential latent fingerprint development treatments containing DFO and PD are not recommended when performing downstream DNA analysis as these treatments have been found to be detrimental to DNA processing. Prior F I G U R E 5 (A) An untreated fingerprint generated a partial profile with 34 alleles. The DNA yield was 0.065 ng, and the average peak height was 228 ± 204 RFU. (B) A fingerprint treated with IND-Zn generated a partial profile with 33 alleles. The DNA yield was 0.055 ng, and the average peak height was 182 ± 144 RFU. (C) A fingerprint treated with IND-Zn + ninhydrin generated a partial profile with 29 alleles. The DNA yield was 0.069 ng, and the average peak height was 185 ± 171 RFU [Color figure can be viewed at wileyonlinelibrary.com] to examination, fingerprint examiners and DNA analysts should determine which forensic analyses will be performed to facilitate the selection of a single-reagent or sequential fingerprint development treatment that maximizes fingerprint visualization and minimizes interference with the development of CODIS eligible DNA profiles.
Although selection of appropriate development treatments can minimize the opportunities for DNA loss and damage, the development of CODIS-eligible DNA profiles is not guaranteed due to the variable amounts of DNA contained within fingerprints.

ACK N OWLED G M ENTS
The authors thank Irvin Rivera and Alison Moran for their work processing the latent fingerprint and DNA samples, respectively.
Additionally, they would like to thank Kelli Lewis, the laboratory di-