technology transfer workshop forensic lmd research studies at rosalind franklin university of...

Technology Transfer Workshop

Forensic LMD Research Studies at Rosalind Franklin University of

Medicine and ScienceNorth Chicago, IL

Christine T. Sanders

[email protected]


How did this start?

• 2003 - Sanders investigates the applicability of LMD for forensic use as a thesis topic

• Jan 2004 - Sanders and Peterson at RFUMS write NIJ grant proposal to investigate LMD technology for separation of sperm from mixtures.

• Feb 2004 - AAFS presentation of preliminary data• June 2004 - Awarded two year grant #2004-DN-BX-K215• Several presentations made throughout the grant period• Published paper in July 2006, JFS• July/Aug. 2007 - NFSTC Technology Transfer Workshop

• Second manuscript in progress


Separation Methods

• Preferential Lysis (differential extraction)

• Flow Cytometry

• Microchip separation

• Membrane Filtration

• Magnetic Antibodies

• Y- chromosome (non-physical separation)

Laser Microdissection


Studies for LMD Development• Histological staining study

• DNA isolation study

• Yield Evaluation qPCR

• Mixture separation study

• Low Copy Number study (LCN)

• Comparative study

• Case Study


Technical Obstacles• Optimizing LMD cutting parameters

• LMD microscope slides

• Environmental conditions of instrument

• “hanging chads”

• Static

• Collection buffer

• Etc…


Histological Staining for LMD

Not StainedE-CellsSperm

63x 40xHistological staining study considerations:

–Visually discriminate sperm and epithelial cells–Effect on downstream analysis


Histological Staining Part 1

Stains tested:

• Hematoxylin / Eosin (H&E)

• Christmas Tree stain (nuclear fast red / picroindigocarmine)

• Acridine Orange

• Wright Stain (azure blue / eosin)

• Methyl Green

Evaluation:

•Stains were evaluated for ability to ID sperm

•LMD collected cells were isolated with Qiagen QIAamp

extraction

•STR/Profiler Plus analysis performed


AcridineOrange

Not Stained

Christmas Tree

Hematoxylin/Eosin

Technology Transfer WorkshopMicroscopic ID scores of sperm & epithelial cells

Histological Stain Sample UNSTN H&E CTS MG WRT AO

Spermatozoa 1 + / - + + - - - + 2 + / - + + + - - - + 3 + / - + + + - - - +

Buccal Cells 1 + / - + + + - - - + / - 2 - + + + - - - - 3 + / - + + + - - - - +

UNSTN = not stained. H&E = hematoxylin/eosin. CTS = nuclear fast red/picroindigocarmine. MG = methyl green. WRT = Wright's stain. AO = acridine orange.

- - : cannot ID or highly challenging- : poor+ / - : satisfactory+ : good+ + : excellent


RFU Levels of Stained Cells

0%

20%

40%

60%

80%

100%

1Pe

rce

nt

RF

Us

No

rma

lize

d

to U

ns

tain

ed

Sa

mp

les

H&ECTS

62% 43%

Stained specimens exhibited RFU values significantly lower than unstained specimens (P < 0.01)


• Methyl green, Wright’s stain not suitable for LMD

• Cells stained with Acridine orange resulted in no amplified product

• Christmas tree stain & Hematoxylin/Eosin: – Good stains for sperm ID. – Significantly lower RFU values than unstained control– However, genotyping could still be obtained with 300

sperm cells or 150 E-cells– H&E best choice

Part I: Histology Study Summary

Results prompted second phase of histology study (Part II)


Part II: Histology StudyStains Tested:• H&E Modified - shorter exposure times• Nuclear Fast Red - nuclear dye• SYBR 14/Propidium Iodine - fluorescent duel stain• PSA/PI - fluorescein-conjugated Pisum sativum agglutinin

(FITC-PSA) & propidium iodide

Evaluation:

• Stains were evaluated for ability to ID sperm

• LMD collected cells were isolated with Lyse-N-Go

extraction

• STR/Profiler Plus analysis performed


RFU Differences: Stained vs. Unstained LMD Cells


Part II: Histology Study Summary

• SYBR 14/Propidium Iodine

– No STR results could be obtained - not suitable for DNA analysis. PI tests indicate SYBR 14 as the problem agent.

• H&E Modified and Nuclear Fast Red

– Good stains for sperm ID. – No significant difference in RFUs obtained from that of unstained

controls

• PSA-FITC/PI– Good duel stain but staining consistency difficult to control

between samples. STR genotypes can be obtained from PSA-FITC stained cells. (more optimization required)


PSA-FITC/PI stain on a PEN slide


• Lyse-N-Go : commercial lysis buffer (low manipulation)

Series of heating and cooling incubations 8C-97C

• Microlysis: commercial lysis buffer (low manipulation)

Series of 65C and 96C incubations• Qiagen QIAamp: commercial kit

DNA binding membrane columns• Chelex

DTT (dithiothreitol) added to all three protocols

DNA isolation study considerations:–Low manipulation, Small volume, Purity

DNA Isolation Study

Technology Transfer WorkshopDetection of Loci Using

Three Isolation Methods


Total PCR Product Detected

*


DNA Isolation Study Summary

• MicroLYSIS method was not suitable for LMD with forensic STR analysis

• QIAamp performed best for collection of stained epithelial cells

• Both Lyse-N-Go and QIAamp performed well for isolating DNA from LMD collected sperm cells – Lyse-N-Go provides a low manipulation method and

inexpensive– QIAamp provides a cleaner DNA extract but higher

manipulation required and higher cost.


DNA Yield Study• LMD process may provide an estimate of DNA

quantity.

• Efficiency of DNA extraction process must be considered

• Two methods of DNA quantification performed from LMD collected cells extracted with the Qiagen QIAamp® protocol– Real-Time qPCR– Relative amounts of STR PCR product to a

standard curve


Sample % Yield

by real-time qPCR

% Yield

by STR RFUs

300 sperm (n=5) 22.9 + 4.8 % Off scale

150 sperm (n=5) 13.3 + 2.3 % Off std curve

80 sperm (n=5) 16.3 + 5.4 % 25.1 + 6.6%

40 sperm (n=5) 18.5 + 3.8 % 22.0 + 5.6%

20 sperm (n=5) 12.2 + 4.7 % 20.1 + 7.8%

10 sperm (n=5) 14.8 + 0.8 % 23.6 + 6.6%

5 sperm (n=4) 17.8 + 2.9 % Off std curve

AB Pos control

0.0313 -1ng

13.0 + 2.0 %

(n=6)

27.5 + 4.7%

(n=5)

Yield of DNA Extraction (sperm)


Sample % Yield by real-time qPCR

150 epithelial (n=5) 37.5 + 2.8 %

80 epithelial (n=5) 45.4 + 6.8 %

40 epithelial (n=5) 37.5 + 4.2 %

20 epithelial (n=5) 40.5 + 7.0 %

10 epithelial (n=5) 32.9 + 5.3 %

5 epithelial (n=5) 41.4 + 8.6 %

2 epithelial (n=5) 40.7 + 11.0 %

Yield of DNA Extraction (e-cells)


DNA Yield Study: Summary

• Extraction efficiency surprisingly low but consistent

• Cells can easily be counted during LMD collection, starting DNA material calculated, and then final DNA quantity can be estimated factoring in extraction efficiency prior to PCR

• Laborious and sample consuming DNA quantification step can be eliminated when using LMD.


Mixture Study

• Mixtures were prepared with the equivalent of half a female oral cotton swab + 1µl of semen.

• Collection amounts of 75, 150 and 300 sperm cells were recovered from the mixtures.

• PCR amplification was performed using standard 28 cycles

• Extended PCR was performed by amplifying half of the PCR product an additional 6 cycles

Technology Transfer Workshop** * *

* * * **

* *

**** *AmpFlSTR

Profiler Plus

DNA mixture:sperm cells + female epithelial cells (*)


300 Sperm cells

separated by LMD


“150 sperm cells” from a mixtureStandard PCR Extended PCR


“75 sperm cells” from a mixture

Standard PCR Extended PCR


Detection of Profiler Plus Alleles

• Under standard PCR conditions (28 cycles)– “75 sperm” 71+12% alleles

– “150 sperm” 96+3% alleles

– “300 sperm” 100% alleles

• Under “extended cycles” PCR (34 cycles)– “75 sperm” 100% alleles

– “150 sperm” 100% alleles


Allelic Balance

• Heterozygote peak height ratio: Height of the lower peak divided by the height of the higher peak, expressed as a percentage

• Under standard PCR conditions (28 cycles)– “75 sperm” 79.3+2.9%– “150 sperm” 81.8+4.3%– “300 sperm” 82.0+1.4%

• Under “extended cycles” PCR (34 cycles)– “75 sperm” 67.0+13.2%– “150 sperm” 85.2+6.7%


Summary: Mixture Study

• LMD separation of sperm cells from a epithelial cell mixture results in a single semen donor genotype

• The lower limit of detection using ABI user guide’s PCR protocol (standard conditions) is ~75-150 sperm cells.

• Extended cycle analysis can extend the lower limit of detection


LCN Study of Mixtures

• Prepared mixtures of human female oral swabs (epithelial cells) and male semen (sperm cells). Equivalent to 1 swab + 1µl of semen

• Collected 80, 40, 20, 10 and 5 sperm cells by laser microdissection

• Profiler Plus PCR amplification was performed using 34 & 38 cycles. (+6 & +10 cycles)

Technology Transfer Workshop“80 sperm cells” at 34 PCR cycles

Technology Transfer Workshop“40 sperm cells” at 34 PCR cycles


“20 sperm cells” at 34 cycles


“10 Sperm Cells” at 34 PCR Cycles


80

40

20

10

5

LMD Collected Cells from a Mixture (34 cycles)


Percent of Profiler Plus Profile Detected from LMD Collected Cells (34 cycles)

N=5 N=5

***

N=5N=5 N=5

Technology Transfer WorkshopPercent of Profiler Plus Profile Detected from LMD Collected Cells (34 & 38 Cycles)


Epithelial Cell Carryover : Outlier?

STR plots from LMD collected sperm cells with epithelial cell DNA carryover. Female donor alleles (indicated by asterisks) were detected in the 40 and 80 sperm cell collections from one of the slide smears in this study.


LCN Mixture Study Summary

• Minute numbers of sperm cells can be separated and recovered by LMD from epithelial cell mixtures.

• STR genotyping can be obtained with as little as 5 sperm cells captured by LMD using increased PCR cycles.


PCR mastermix added directlyto collection/extraction tube

Collected 20-30 sperm cells by LMDwith Lyse-N-Go Extraction

9µl smeared on PEN slide for LMD

Maximum volume of extractinput into PCR

qPCR DNA Quanification

Differential Extractionwith Organic Purification

25µl used for Preferential Lysis

Mixtures of sperm cells and female epithelial cells1:5, 1:10, 1:20, 1:40, 1:80 & 1:160 (SP:EC)

Comparative Study

AmpFlSTR Profiler Plus for 34 cycles


LMD PL

Plots of Sperm Fractions LMD vs. PL 1:5 Cell Mixture Ratio


LMD PL

Plots of Sperm Fractions LMD vs. PL 1:160 Cell Mixture Ratio

Technology Transfer WorkshopLMD vs. PL : Detection of Male Donor Genotype


LMD vs. PL : Female Carryover

Technology Transfer WorkshopComparison of PCR Product Quantity : LMD vs. PL of Sperm Fraction

* ** *


PCRCapillary Electrophoresis

Data Analysis

DNA Quantification 4 hrs.

DNA IsolationChelex: 70 min. or

Organic: 2 hrs.

Preferential Lysis Separation3- 4 hrs.

Microscopic ID

PCRCapillary Electrophoresis

Data Analysis (single source genotype)

DNA IsolationLyse-N-Go 20 min.

Microscopic IDLMD ~5 cells/min.

Sexual Assault Kit Swab:Elute Cells & Prepare Smear

Preferential Lysis Method

LMD Method

Work Flow Chart: Comparing Methods


Comparative Study Summary

• LMD provides improved separation and detection of sperm from epithelial cell DNA over the preferential lysis method at higher e-cell to sperm-cell ratios.

• LMD Does not require a DNA quantification step

• Single sample processing more rapid using LMD


Case Studies using Low Copy #• 4 case studies

– Non-probative or adjudicated cases– Originating crime lab performed organic differential

extraction and attempted typing of 13 core loci– Case “A” - public masturbation (tissue paper recovered

from the scene) – Cases “B”, “C” & “D” - sexual assaults (vaginal swabs

obtained)• Case “A” - ample sperm available

– 30 sperm collected by LMD followed by LCN analysis• Case “B”, “C” & “D” - Difficulty locating sperm and

limited sample available. – modified the LMD protocol to include a “mini-lysis”– 18-30 sperm collected by LMD followed by LCN analysis

Technology Transfer WorkshopLocus Case A sp

LMD Case A sp

original PL results

Case B sp LMD**

Case B sp original PL

results

Case B suspect

Case B victim

D3S1358 16, 17 16, 17 17 15, 16, 17 16, 17 15, 16 vWA 17 17 15, 18 15, 16, 18 15, 18 15, 16 FGA 21, 23 21, 23 19, 24 19, 22, 24 19, 24 22, 25

AMEL X, Y X, Y X, Y X, Y X, Y X D8S1179 12, 14 12, 14 10, 13 10, 13, 14 10, 13 14, 17 D21S11 28, 31 28, 31 30.2, 31 30, 30.2, 31 30.2, 31 30, 31.2 D18S51 18 18 15, 17 15, 16, 17 15, 17 16 D5S818 9, 12 9, 12 8, 12 8, 12, 13 8, 12 13 D13S317 11, 12 11, 12 12, 14 12, 14 12, 14 12 D7S820 8, 12 8, 12 9 8, 9, 10 9, 10 8, 12

Locus Case C sp

LMD** Case C sp

original PL results

Case C victim*

Case D sp LMD**

Case D sp original PL

results

Case D victim*

D3S1358 15 15 16 15 15, 16 15 vWA 16, 18 16, 18 16 18 16, 18 17, 18 FGA 24, 25 24 24

AMEL X, Y X, Y X X X, Y X D8S1179 13, 15 13, 15 10, 14 12 12, 13 12, 13 D21S11 28, 31.2 28 30, 31.2 32.2 28, 32.2 28, 30 D18S51 14, 16 13 D5S818 11, 12 11, 12 10, 11 10 10, 13 8, 11 D13S317 10, 13 9, 10 13 12, 13 8 D7S820 11, 12 10

* Partial profile of victim assumed from the epithelial cell fraction of vaginal swab ** LMD performed after “mini-PL” sp = sperm fraction

Technology Transfer WorkshopA Closer Look at Case B

Locus PL - Sperm Fraction

Male

Exemplar

10 Sperm LMD (LCN)

30 Sperm *LMD (LCN)

D3 15, 16, 17 16, 17 17 ***

VWA 15, 16, 18 15, 18 15 15, 18

FGA 19, 22, 24 19, 24 19, 24 19, 24

AMEL X , Y X, Y Y X, Y

D8 10, 13, 14 10, 13 13 10, 13

D21 30, 30.2, 31 30.2, 31 31 30.2, 31

D18 15, 16, 17 15, 17 17 15, 17

D5 8, 12, 13 8, 12 13** 8, 12

D13 12, 14 12, 14 12 12, 14

D7 8, 9, 10 9, 10 9 ***

Carryover from victim shown in red; Shared alleles underlined


Case Studies Summary• LMD’s performance

– Case “A” - similar result to PL– Case “B” & “C” - improved results – Case “D” - worse results

• This study may not be the best case study comparison test of LMD to the PL method due to other variations in the analysis (i.e. LCN vs. STD PCR, and “mini-lysis”)

• Larger case study population required in a forensic lab setting to make final evaluation of LMD


Research Team• RFUMS

– Christine T. Sanders– Daniel A. Peterson– Emily Reisenbigler

• LAPD– Nick Sanchez

• University of Central Florida– Jack Ballantyne

• Northern Illinois Regional Crime Lab– Kenneth Pfoser

technology transfer workshop forensic lmd research studies at rosalind franklin university of...

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