validation study of kpics spermfinder™ by nichevision forensics, llc for the identification of...

TECHNICAL NOTE

CRIMINALISTICS

Angela Mitchell,1 M.S.

Validation Study of KPICS SpermFinderTM byNicheVision Forensics, LLC for theIdentification of Human Spermatozoa*

ABSTRACT: Microscopic analysis for the identification of spermatozoa is commonly performed during the forensic examination of sexualassault evidence. Two widely utilized methods for the confirmation of the presence of spermatozoa are visualization of the cells via phase-contrastmicroscopy with wet mounted samples and bright field microscopy with histologically stained samples. The KPICS SpermFinder� by NicheVisionForensics, LLC accelerates this time-consuming process via an automated microscope with an algorithm designed to locate spermatozoa on a Christ-mas tree histologically stained microscope slide. Upon a qualified scientist’s review of the generated data, the KPICS SpermFinder� was able tolocate spermatozoa, typically finding on average 106.28% € 115.37% more spermatozoa than with manual examinations. The KPICS SpermFinder�provided the location of identified cells with reproducible results.

KEYWORDS: forensic science, forensic biology, sexual assault, spermatozoa, automation, KPICS SpermFinder�

In the forensic investigation of sexual assault cases, the scientistcommonly examines physical evidence for the presence of seminalmaterial. Microscopy is a powerful tool with which spermatozoacan be identified, thus confirming the presence of seminal material.Microscopic examinations are often time-consuming. In manyinstances, this type of examination must be conducted on numerouspieces of evidence within a case. An expedited process to performmicroscopic examinations and electronically document findingswould be advantageous to reducing sexual assault casework back-log and in creating an efficient form of documentation.

Semen, the fluid that is expelled during the male sex act, is com-prised of glandular secretions and cellular components. Spermato-zoa, the cellular component of semen, originate in the testis andcontain the male’s genetic information. A typical ejaculate contains1–6 mL of seminal material, averaging 3.5 mL, and contains c.50–100 million spermatozoa per milliliter. Human spermatozoa arecomprised of three major structures: the head, midpiece, and tail.An intact spermatozoan measures about 50–60 lm in total length.The head and midpiece are relatively of equal length, measuringroughly 4.6 lm long. The head is c. 2.6 lm wide and 1.5 lmthick. Morphological characteristics of a spermatozoan head includea flattened ovoid shaped cell body with an acrosome at the apicalportion. The side profile has a distinctive dolphin head shape (1–5).

Currently, the presence of spermatozoa is confirmed in theForensic Biology Section of the Allegheny County Office of theMedical Examiner Forensic Laboratory (ACOME FL) throughviewing three spermatozoa heads or one intact spermatozoan cellvia phase-contrast microscopic examination of wet mounted micro-scope slides prepared from questioned samples. Factors that canmake microscopic examination difficult can include the presence ofexcessive levels of epithelial cells, bacterial and cellular debris, orextremely low levels of spermatozoa. During cases of sexualassault, spermatozoa might not be present due to the use of prophy-lactics, biological degradation over time, lack of ejaculation, incom-plete ejaculation, vasectomy, a vas deferens obstruction, or othercases of sexual dysfunction (2–4).

This validation study investigates the utility of the KPICSSpermFinder� detection instrument by NicheVision Forensics,LLC (NicheVision, LLC, Akron, OH) in reducing examinationtime, creating electronic documentation of each sample’s micro-scopic examination, and increased spermatozoa detectability. TheKPICS SpermFinder� detection method involves histologicallystaining of an extract of a portion of the questioned sample on amicroscope slide then covering the sample with mounting mediumand a cover slip. The resulting slide is then placed onto the KPICSSpermFinder� detection instrument. The instrument scans the slideand utilizes an algorithm to identify potential spermatozoa basedon color, acrosome to nucleus color contrast density (the differencein color concentration between the clear acrosome and the rednucleus), and size. The KPICS SpermFinder� detection instrumentcreates an electronic image of the slide, electronic images of sper-matozoa candidates, and their x, y location on the sample micro-scope slide. The scientist then reviews this data to confirm thepresence of spermatozoa, if they are present in the sample, and sub-sequently generates a report for these findings. These reports areretained electronically as case documentation, creating a permanentrecord of the sample examination (6–8).

1Allegheny County Office of the Medical Examiner, Forensic Laboratory,Forensic Biology, 1520 Penn Avenue, Pittsburgh, PA 15222.

*Instrumentation purchased and validation study supported throughNational Institute of Justice FY 2008 Forensic DNA Unit EfficiencyImprovement Program: 2008-DN-BX-K191. No conflicting interests or affil-iations between the author or agency and NicheVision Forensics, LLC toreport. The opinions, findings, and conclusions or recommendationsexpressed in this publication ⁄ program ⁄ exhibition are those of the authorand do not necessarily reflect those of the Department of Justice.

Received 13 Jan. 2011; and in revised form 13 May 2011; accepted 4June 2011.

J Forensic Sci, 2012doi: 10.1111/j.1556-4029.2012.02081.x

Available online at: onlinelibrary.wiley.com

2012 American Academy of Forensic SciencesPublished 2012. This article is a U.S. Government work and is in the public domain in the U.S.A. 1

TABLE 1—Sensitivity study samples comparison between KPICS SpermFinder� scientist reviewed results and manually examined bright field microscopyresults.

SampleDesignation

Preparation

KPICSSpermFinder�Results Run 1


Scientist 1Results

Scientist 2Results

Scientist 1to KPICS

SpermFinder�Percent

Difference*

Scientist 2 toKPICS


Difference*

AverageScientist to

KPICSSpermFinder�

PercentDifference*

SensitivityStudySamples

Number ofConfirmedPositives


V1 Couple 1_0 h oral swab 516 449 412 17.11 17.11V2 Couple 1_45 min oral swab 14 19 8 12 106.25 37.50 71.88V3 Couple 1_12 h vaginal swab 1212 1323 649 95.30 95.30V4 Couple 1_24 h vaginal swab 1322 1684 967 55.43 55.43V5 Couple 1_12 h menstrual

vaginal swab618 621 321 92.99 92.99

V7 Couple 1_24 h vaginal smear 369 364 230 321 59.35 14.17 36.76V8 Couple 1_12 h menstrual

vaginal smear973 1026 194 415.21 415.21

V10 Couple 2_6 h vaginal swab 2425 2445 1289 88.91 88.91V12 Couple 3_24 h vaginal swab 862 889 234 274.15 274.15V13 Couple 3_24 h vaginal swab 270 297 140 102.50 102.50V15 Couple 3_36 h vaginal swab 495 611 328 68.60 68.60V17 Couple 3_72 h vaginal swab 260 267 154 144 71.10 82.99 77.05V18 Couple 3_80 h vaginal swab 9 7 2 3 300.00 166.67 233.33V19 Couple 4_32 h vaginal swab 24 25 18 19 36.11 28.95 32.53V21 Vaginal swab + 1:10 semen

dilution880 814 495 71.11 71.11

V22 Vaginal swab + 1:100 semendilution

22 27 12 12 104.17 104.17 104.17

V23 Vaginal swab + 1:1000semen dilution

2 2 0 200.00

V24 Vaginal swab + 1:10,000semen dilution

4 4 1 1 300.00 300.00 300.00


5 7 1 500.00 500.00

V28 Rectal swab 0 0 0 0.00 0.00V29 Rectal swab + 1:1000

semen dilution6 5 4 37.50 37.50

V30 Oral swab 0 0 0 0.00 0.00V31 Oral swab + 1:1000 semen

dilution8 10 10 5 )10.00 80.00 35.00

V32 Menstruation vaginal swab 0 0 0 0.00 0.00V33 Menstruation vaginal swab

+ 1:1000 semen dilution2 4 3 0.00 0.00

V48 Vaginal swab + 1:10 semendilution

141 132 137 )0.36 )0.36


44 41 43 39 )1.16 8.97 3.91


3 2 1 150.00 150.00


2 3 1 150.00 150.00


2 2 0 200.00


540 532 464 15.52 15.52


41 53 30 56.67 56.67


1 0 0 100.00


2 2 1 100.00 100.00


0 0 0 0.00

V58 Couple 1_3.5 h oral swab 54 45 18 175.00 175.00V59 Couple 1_36 h menstrual

vaginal swab155 169 57 184.21 184.21

V60 Couple 3_1 h oral swab 38 45 29 43.10 43.10V62 Couple 3_90+ h vaginal swab 202 169 191 )2.88 )2.88V63 Couple 1_48 h vaginal swab 65 68 33 101.52 101.52V64 Couple 3_24 h oral swab 0 1 0 100.00Average 106.28Standard Deviation 115.37

*When the instrument found more spermatozoa than the scientist(s), the percent difference is a positive number. When the instrument found fewer spermato-zoa than the scientist(s), the percent difference is a negative number.

2 JOURNAL OF FORENSIC SCIENCES

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Materials and Methods

Physiological fluid samples, including neat semen, oral swabs,vaginal swabs, rectal swabs, and postcoital samples were gener-ously supplied by forensic laboratory personnel. Canine semen wasfurnished by Dr. R. V. Hutchison of the Animal Clinic Northview,Inc. Equine semen was generously provided by Dr. Nicholas G.Loutsion of the Canon Hill Veterinary Clinic Inc.

Contrived samples were prepared by depositing c. 30 lL ofknown seminal material dilutions onto cotton swabs from the indi-cated orifice then allowing the swabs to air-dry. Postcoital samplesidentified by time interval and orifice swabbed were collected ontocotton swabs and allowed to air-dry. Smears were also preparedfrom a selection of the postcoital samples by rolling the swab headon a microscope slide directly after collection then allowing thesample to air-dry.

Microscope slide preparation was performed through extractionof the sample on the glass microscope slide using a small amountof water and teasing the substrate with forceps to increase extrac-tion efficiency. The extracted material was air-dried on the glassslide for c. 1 h at room temperature. In instances where smearswere examined, the smears were air-dried at room temperature forat least 1 h prior to histological staining. Once the samples werecompletely dried on the glass microscope slide, the sample areawas covered with Solution A: Kernechtrot Solution (SerologicalResearch Institute, Richmond, CA) and were then allowed to incu-bate at room temperature for 15 min. The slide was then gentlyrinsed with deionized water. The sample area was then coveredwith Solution B: Picroindigocarmine Solution (Serological ResearchInstitute) and allowed to incubate at room temperature for 15 sec.A final rinse was performed with absolute ethanol. One to threedrops of Cytoseal� 60 mounting medium (Richard-Allan Scien-tific, Kalamazoo, MI) was placed onto the sample area and coveredwith an appropriately sized cover slip. The resulting cellular mate-rial consisted of red nuclear material (spermatozoa head) with greenor blue cytoplasm material (spermatozoa tail and midpiece)(2,7–9).

Each prepared slide was examined by one or two scientists andat least twice by the KPICS SpermFinder� detection instrumentusing bright field microscopy at 400· magnification. Significantvariations in staining color density and spermatozoa detectabilitybetween orifice samples and donors were not observed when evalu-ated by the scientist and the KPICS SpermFinder� detectioninstrument. A false-negative rate (a negative percent differencevalue) was recorded when the detection instrument did not identifythe same number of spermatozoa as the scientist when comparingthe automated examination to the manual examination. When com-paring an automated examination to another automated examina-tion, a false-negative rate was recorded when the detectioninstrument did not identify the same number of spermatozoa as theprior run(s). Previously examined sexual assault slides were com-piled from three scientists of varied experience levels to provide anestimated average time spent to manually examine one slide. Statis-tical analysis was generated in EXCEL (Microsoft Corporation,Redmond, WA).

Results

Sensitivity Study

Due to the KPICS SpermFinder� detection instrument’s capabil-ities, spermatozoa were consistently observed in higher numberswhen examined with the instrument when compared to the scien-tist’s observations. Of the 55 slides examined, 92.72% had

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spermatozoa identified in numbers equal to or greater than scien-tist’s results when the KPICS SpermFinder� detection instrumentwas employed. In 7.27% of the slides examined, the KPICSSpermFinder� detection instrument found less spermatozoa thanthe scientist, but spermatozoa were positively identified on eachslide (Table 1; V31, V48, V49, and V62 exhibited false-negativerates). The KPICS SpermFinder� detection instrument finds sper-matozoa within a range of 500% to )10% of the scientist’s result-ing numbers by comparison. There was no instance where a false-negative slide was observed during this study; the required thresh-old to report a positive slide was reached in each slide containingspermatozoa.

Casework Sample Study

Ten slides, which had been examined with phase-contrastmicroscopy prior to utilization in this validation study, were pre-pared by removing the existing cover slip once the sample had air-dried then staining according to the above protocol. The KPICSSpermFinder� detection system found spermatozoa at numbersequal to or higher than the manual examination of the sample.

When compared to examinations performed with phase-contrastmicroscopy, the KPICS SpermFinder� detection system found onaverage 1770.68% more spermatozoa. When compared to the man-ual examination with bright field microscopy, the KPICS Sperm-Finder� detection system found on average 214.10% morespermatozoa.

Contaminant Study

Contrived samples containing contaminants such as lubricants,cleansers, and yeast cells combined with known seminal materialdilutions were prepared according to protocols outlined in theMaterials and Methods section. The KPICS SpermFinder� detec-tion system found spermatozoa at numbers equal to or higher thanthe manual examination of the sample averaging 52.68% morespermatozoa observed.

Specificity Study

Contrived samples of diluted canine and equine seminal materialwere prepared with the preparation protocols outlined in the

TABLE 5—Contaminant study samples KPICS SpermFinder� scientist reviewed false-positive results comparison between automated examination runs.

SampleDesignation

Preparation



KPICSSpermFinder�False-Positive

Percent



KPICSSpermFinder�False-Positive

Percent

KPICSSpermFinder�

AverageFalse-Positive

PercentContaminationStudy Samples

Number ofCalled

Positives


Number ofCalled

Positives


V34 Vaginal swab+ 1:1000+ yeast cells

100,145 0 100.00 121,680 0 100.00 100.00

V35 Vaginal swab+ 1:1000+ douche

7167 7 99.90 7045 7 99.90 99.90

V36 Vaginal swab+ 1:1000+ Vaseline�*

3362 1 99.97 3692 2 99.95 99.96

V37 Vaginal swab+ 1:1000+ KY Jelly��

8088 14 99.83 7664 12 99.84 99.84

Average 99.92Standard Deviation 0.07

*Vaseline� (Unilever, Greenwich, CT).�KY Jelly� (Personal Products Company, Skillman, NJ).

TABLE 6—Specificity study samples KPICS SpermFinder� scientist reviewed false-positive results comparison between automated examination runs.

SampleDesignation

Preparation



KPICSSpermFinder�False-PositivePercent Run 1



KPICSSpermFinder�False-PositivePercent Run 2

KPICSSpermFinder�Average False-Positive Percent

SpecificityStudy Samples

Number ofCalled

Positives

Number ofConfirmed

AnimalSpermatozoa

Positives

Number ofCalled

Positives

Number ofConfirmed

AnimalSpermatozoa

Positives

V26 Vaginal swab+ 1:100 horsesemen dilution

6082 22 99.64 5364 21 99.61 99.62

V27 Vaginal swab+ 1:100 dogsemen dilution

9222 57 99.38 9012 48 99.47 99.42



Materials and Methods section. Animal spermatozoa were typicallynot detected because of their unique morphological characteristicsthat are inconsistent with the morphological characteristics ofhuman spermatozoa. Examination by the KPICS SpermFinder�detection instrument lead to human-specific identification of sper-matozoa upon data review by a qualified scientist.

Precision Study

Six samples (V2, V18, V23, V29, V33, and V36) were selectedfrom the prepared validation samples and run on the detectioninstrument six times each. It was determined that the KPICSSpermFinder� detection instrument found spermatozoa in higher

numbers than with manual examinations (Table 1; 106.28% morespermatozoa were found on average when using the detectioninstrument compared with manual examinations). On average31.86% of the total number of spermatozoa on each slide, as identi-fied through multiple runs, were not identified in each repeatedrun. Despite this discrepancy, higher numbers of spermatozoa aretypically observed in each run when compared to manual examina-tions (Tables 1 and 2).

Discussion

The KPICS SpermFinder� detection instrument employs an algo-rithm specific for the identification of the size, color contrast density

TABLE 8—Contaminant study samples comparison between KPICS SpermFinder� scientist reviewed results and manually examined bright field microscopyresults.

SampleDesignation

Preparation



Scientist 1Results

Scientist 2Results

Scientist 1to KPICS


Difference*

Scientist 2to KPICS


Difference*

Average Analystto KPICS


Difference*ContaminationStudy Samples



V34 Vaginal swab + 1:1000semen dilution+ yeast cells

0 0 0 0.00 0.00

V35 Vaginal swab + 1:1000+ douche

7 7 4 6 75.00 16.67 45.83

V36 Vaginal swab + 1:1000+ Vaseline��

1 2 1 1 50.00 50.00 50.00

V37 Vaginal swab + 1:1000+ KY Jelly��

14 12 7 4 85.71 225.00 155.36


*When the instrument found more spermatozoa than the scientist(s), the percent difference is a positive number. When the instrument found fewer sperma-tozoa than the scientist(s), the percent difference is a negative number.

�Vaseline� (Unilever, Greenwich, CT).�KY Jelly� (Personal Products Company, Skillman, NJ).

TABLE 7—Casework study samples comparison between KPICS SpermFinder� scientist reviewed results and manually examined bright field microscopyresults.

SampleDesignation

Preparation



Scientist 1Results

Scientist 1to KPICS

SpermFinder�Percent Difference*

Phase-ContrastPositives

Phase-Contrastto KPICS


Difference*CaseworkStudy Samples



V38 Case 1-rectal swab 1 1 1 0.00 1 0.00V39 Case 2-vaginal swab 56 37 30 55.00 16 190.63V40 Case 3-vaginal swab 582 1163 338 158.14 18 4747.22V41 Case 4-rectal swab 512 537 108 385.65 18 2813.89V42 Case 5-rectal swab 159 197 68 161.76 18 888.89V43 Case 6-vaginal swab 50 60 11 400.00 3 1733.33V44 Case 7-vaginal swab 9 9 3 200.00 3 200.00V45 Case 8-rectal swab 238 193 120 79.58 11 1859.09V46 Case 9-mattress cutting 66 70 14 385.71 9 655.56V47 Case 10-mattress cutting 1050 1026 250 315.20 22 4618.18

Phase-contrast to KPICSSpermFinder�

Average 1770.68Standarddeviation

1769.67

Scientist to KPICSSpermFinder�

Average 214.10Standarddeviation

148.69



between the acrosome and nucleus of the spermatozoa, and colorproduced by Christmas tree staining of spermatozoa. The algorithm’sparameters took into account differences in staining lot products(color density variations because of different lot numbers), samplecharacteristics (location collected from and presence of contami-nants), viewed morphological characteristics (spermatozoa degrada-tion and orientation), and variations between scientist’s stainingtechnique. These less stringent parameters led to fewer false-negativerates, and high false-positive percentages. High false-positive per-centages were not detrimental to the analysis of the sample(Tables 3–6). Through verification of the focus confidence and theslide scan area, the resulting candidate images were representative ofthe sample area examined. Discrepancies observed in comparisonsbetween manual and automated examinations (Table 1) and the pre-cision study (Table 2), where all spermatozoa were not identified ineach run, could have been due to environmental factors such asvibrations during the sample’s data collection. When the instrumentwas capturing images of the sample for analysis, any movement thatwould cause a blurred image was detrimental, thus variations infalse-positive numbers were observed between automated examina-tion runs. These vibrations could not be eliminated but were reducedwith the installation of an antivibration platform to the microscope.

It was observed that the KPICS SpermFinder� detection instru-ment provided significantly clearer contrast when compared to thescientist’s bench top microscope (Leica DMLS; Leica Microsys-tems, Wetzlar, Germany), leading to the identification of more sper-matozoa. Samples with high levels of debris and dense cellularmaterial were examined with the detection instrument and spermato-zoa were observed between these layered cells. Due to the improvedoptics, the detection instrument found spermatozoa in sampleswhere the scientist originally reported the sample to be negative orinconclusive with manual examination (Tables 1 and 7). In the case-work sample study, it was concluded that when comparing phase-contrast manual examinations to the detection instrument, the detec-tion instrument on average found 1770.68% more spermatozoa.When comparing bright field manual examinations to the detectioninstrument, it was determined that on average 214.10% more sper-matozoa were observed (Table 7). With scientist review of the mor-phological characteristics of spermatozoa in generated candidateimages, it was concluded that the KPICS SpermFinder� detectioninstrument equipped with the Leica DM5500B microscope wassuperior to the current manual examinations. The nature of the slide

preparations could offer an additional explanation for the observeddiscrepancies besides the previously discussed contrast differencesbetween microscopes. In phase-contrast microscopy a wet mountedslide is prepared from the sample, thus allowing cellular material tomove within the water under the cover slip during the manualexamination. Spermatozoa present in the wet mounted slide couldhave been moving in the area outside of the scientist’s field of viewor moving under other cellular material or debris. This movementmay explain why a sample with low concentrations of spermatozoawould be identified as being negative for spermatozoa. In histologi-cally stained slides, the sample is fixed to the slide by air drying,preventing the movement of cellular material. Due to the stationarycellular material a higher number of spermatozoa were observedwhen compared to wet mount samples.

Chemical contaminants present in some samples make identifica-tion of spermatozoa challenging for the scientist. Samples contain-ing chemical contaminants, such as lubricants and cleansers, wereexamined by the KPICS SpermFinder� detection instrument todetermine the chemical’s impact on results (Table 8). No significanteffects were observed. Inconsistent staining results may be pro-duced in the presence of petroleum-based contaminants because ofhydrophobic interactions preventing contact between the cellularmaterial and the water-based staining solutions, but do notadversely affect results. In samples containing petroleum-basedlubricants, sampling was conducted to limit the amount of lubricantpresent to minimize this issue. Significantly, higher false-positiveresults were observed when evaluating samples with high levels ofyeast cells on the detection instrument. This was because of themanner in which yeast cells stained red similar to a spermatozoannucleus. Yeast cells could be differentiated from spermatozoa whenreviewed by a qualified scientist.

The detection system’s algorithm considered the contrast betweenthe acrosome and the nucleus of the human spermatozoan head.This contrast can occur in objects other than spermatozoa resultingin a false-positive finding. In some instances, the contrast betweenthe animal spermatozoa and the surrounding materials produced afalse-positive result (Table 9). Animal spermatozoa have uniquemorphological characteristics and thus do not consistently cause afalse-positive result to be reported. Although the detection systemis capable of detecting human spermatozoa, the generated candidateimages must be reviewed by a qualified scientist to determine thespecies origin of the spermatozoa.

TABLE 9—Specificity study samples comparison between KPICS SpermFinder� scientist reviewed results and manually examined bright field microscopyresults.

SampleDesignation

Preparation



Scientist 1Results

Scientist 2Results

Scientist 1to KPICS


Difference*

Scientist 2to KPICS


Difference*

Average Analystto KPICS


Difference*

SpecificityStudySamples

Number ofConfirmed Animal

SpermatozoaPositives

Number ofConfirmed Animal

SpermatozoaPositives

V26 Vaginal swab+ 1:100 horsesemen dilution

22 21 23 21 )6.52 2.38 )2.07

V27 Vaginal swab+ 1:100 dogsemen dilution

57 48 1397 )96.24 )96.24

Average )51.38Standard Deviation 63.44



A precision study was conducted on the detection system byscanning the same sample area of six slides six times each. It wasdetermined that the KPICS SpermFinder� detection instrumentperformed to a reproducibility rate of 68.14%. While the instrumentdid not find each spermatozoan in each run, it did find spermatozoain each run typically in higher numbers than the scientist did manu-ally (Tables 1 and 2). It was found that the instrument generatedon average 98.95% false-positive results during the precision study.This false-positive average varied from sample to sample and con-tinually changed within multiple runs of the same sample. Thesediscrepancies in reproducibility and false-positive results could bedue to previously discussed vibration issues during the image col-lection process.

The required amount of scientist time to examine sample slideswas evaluated through compiling an estimated average time spentper slide from three scientists of varied experience levels. Manualexamination of a sample slide takes on average 1–2 h of a scien-tist’s time to complete. The automated detection instrument typi-cally examines one slide in c. 2 h and can be run overnight thusallowing the scientist to perform other tasks during data collection.Review of the generated data by the scientist typically takes c.20 min but can take up to 90 min. Variations in data review timesare largely due to the number of generated positive results perslide. This number is expected to change between samples, basedon the analysis parameters of the instrument and variation in sam-ple size. As with manual examinations, review of data generatedby the instrument may also be lengthened by high levels of epithe-lial cells, bacteria, yeast cells and debris present in the sample.

Conclusions

The KPICS SpermFinder� detection instrument by NicheVisionForensics, LLC validation study determined that the detectioninstrument performs as well as, and in most cases better than, aqualified scientist utilizing phase-contrast microscopy or manualbright field microscopy for identification of spermatozoa. Auto-mated microscope slide examinations typically took less scientisttime, permitted the scientist to perform other tasks during data col-lection, could be run overnight, and detected significantly morespermatozoa. The detection instrument documented the x, y coordi-nates of spermatozoa with reproducible results. When necessary,the reexamination of samples was facilitated with the use of theelectronic record produced by the detection instrument and theavailability of the histologically stained slide. The detection instru-ment was capable of detecting spermatozoa in the presence ofchemical contaminants. With data review by a qualified scientist,spermatozoa species identification is confirmed. The utility of theKPICS SpermFinder� detection instrument has been proven toprovide superior analysis and documentation of microscopically

examined samples and is a valuable alternative to the current man-ual phase-contrast microscopy method.

Acknowledgments

The author extends her appreciation to Medical Director Dr.Karl Williams, Forensic Laboratory Director Robert Huston,and Forensic Biology Manager Janine Yelenovsky for validationstudy support and manuscript review. The author would alsolike to extend her gratitude to Ms. Sara Bitner, Ms. Anita Kozy,Mr. Walter Lorenz, Mr. Thomas Meyers, Mr. Aaron Schneider,and Ms. Janine Yelenovsky of Forensic Biology in the Alle-gheny County Office of the Medical Examiner Forensic Labora-tory for miscellaneous sample preparation and assistance overthe duration of the validation study. Additionally, staining pro-tocol assistance and training from Mr. Dale Laux of the OhioAttorney General Bureau of Criminal identification and Investi-gation in Richfield, Ohio was invaluable and deeply appreciatedduring this validation study. The author is particularly apprecia-tive to Ms. Sara Bitner, Ms. Anita Kozy, Mr. Thomas Meyers,and Ms. Janine Yelenovsky for assistance with manuscript prep-aration and review.

References

1. Chang TSK. Seminal cytology. In: Geer JH, editor. Proceedings of aForensic Science Symposium on the Analysis of Sexual Assault Evi-dence; 1983 July 6–8; Quantico, VA. Washington DC: U.S. Departmentof Justice, 1983;45–56.

2. Baechtel FS. The identification and individualization of semen stains. In:Saferstein R, editor. Forensic science handbook, Vol. 2. EnglewoodCliffs, NJ: Prentice Hall, 1988;347–92.

3. Gaensslen RE. Sourcebook in forensic serology, immunology and bio-chemistry. Washington, DC: U.S. Department of Justice, 1983;149–82.

4. Hafez ESE, editor. Human semen and fertility tegulation in men. St.Louis, MO: C.V. Mosby Company, 1976.

5. Allery JP, Telmon N, Mieusset R, Blanc A, Rouge D. Cytological detec-tion of spermatozoa: comparison of three staining methods. J ForensicSci 2001;46:349–51.

6. Armogida L, Meles V. SpermFinder training basic. Akron, OH: NicheVi-sion, LLC, 2009.

7. NicheVision, LLC. KPICS SpermFinder glossary V.1.14. Akron, OH:NicheVision Forensics, LLC, 2010.

8. NicheVision, LLC. KPICS SpermFinder owners manual. Akron, OH:NicheVision Forensics, LLC, 2008.

9. Serological Research Institute. Christmas tree stain R540. Richmond,VA: Serological Research Institute, 2009.

Additional information reprints and requests:Angela Mitchell, M.S.Allegheny County Office of the Medical ExaminerForensic Laboratory, Forensic Biology1520 Penn AvenuePittsburgh, PA 15222E-mail: [email protected]


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