The results of the NIST Quantitation

Study 2004 and how it led to the

production of SRM 2372 Human

Quantitation Standard.

Margaret C. Kline

NIST

Applied Genetics Group

Disclaimers

Funding: Interagency Agreements 2003-DN-R-121 and

2008-DN-R-121 between the National Institute of Justice

and NIST Office of Law Enforcement Standards.

Points of view are mine and do not necessarily represent

the official position or policies of the US Department of

Justice or the National Institute of Standards and Technology.

Certain commercial equipment, instruments and materials are

identified in order to specify experimental procedures as completely as

possible. In no case does such identification imply a recommendation or

endorsement by the National Institute of Standards and Technology nor

does it imply that any of the materials, instruments or equipment identified

are necessarily the best available for the purpose.

Background: Pre-QS04 Interlaboratory

Quantitation Performance

Design

Log(Design)

n

Nf

Q3

Q2

Q1

Min

Max

Rng

Xs

Low

High

Rng

Xmax

8

4

2

1

1/2

1/4

1/8

1/16

1/32

N ~ 108N ~ 82N ~ 432004

QN PMO RSTU VW X A B E C F D G H

1999 2001

Rep

ort

ed

[D

NA

], n

g /

L

Diamond symbols are the

NIST [DNA] values.

Boxes enclose the central

50 % of the reported

values.

The horizontal line within

the box is the median value

The y-axis is scaled as

factors of 2.

Most of the central 50 %

values fell within a factor of

2 except the lowest [DNA]

tested in the 1999 study.

Results of the 2001 study

indicated an improvement

of the [DNA] comparability.

Why QS04: Quantitation Study 2004?

• The STR genotyping kits were requiring a tighter

controlled range of input DNA.

• Quantitative PCR (qPCR) methods were starting

to be used the forensics labs along with the

traditional Slot Blot methods.

• Lower [DNA] needed to be tested for

comparability between labs.

• What happens with different tube materials?

NIST Quantitation Study 04

Consisted of:

- 8 DNA extracts labeled A – H

- (1.5 ng/µL, 0.5 ng/µL, 0.16 ng/µL, 0.05 ng/µL)

- A – D Dilutions of a multi-source lyophilized DNA

- E – H Dilutions of a single-source male DNA

- Shipped Dec 2003 –Jan 2004 to 84 laboratories

- Labs were requested to use multiple methods

with multiple analysts

We received:

- Data from 80 Labs (95 % participation)

- Total of 287 sets of data

- Participants used 19 different quantification methods

(primarily variations of Quantiblot and qPCR)

Blot PCR-RT

A B E C F D G H

Picogreen

1

0.1

0.01

HGDFCEBA

Total

0.01

0.1

1

HGDFCEBA

AluQuant

Distributions

of among

participant

results

N = 287 N = 187 N = 60

H

N = 13 N = 12

H – Supplied in PFA tube

BodeQuant N=1

and Yield gels N=15 not shown

A

B E

C F

G D

qPCR

Quantiblot

TMB, ECL

ACES

10

different

methods

7

aa

a a

a

a aa

ppp

p

p

ppa p

-3 -2 -1 0 1 2 3

Direct,

Non-blot

TE

E

T

T TTT TT T

T T

TT T

TTT

T T T TTT

T

TTF

F

F

DD

DE E

E

E

E

E

EEE

EE

E

EEE EE

EE

A

AA

AA

TFDEA

-3 -2 -1 0 1 2 3

Direct,

Blot

2

98 76

655

43 1

1

1

0

00

0

0 0000 00

00

00

0

000

6 510

-3 -2 -1 0 1 2 3

PCR

RT

Concordance

Ap

pare

nt

Pre

cis

ion

QS04: Among-Participant Results

“Bold” characters represent the median performance

of all results submitted for a particular method

The 3 reference semi-circles:

- inner-most delimits a total comparability of 1 sd from perfect

agreement with the consensus medians for all samples

- middle 2 sd

- outer 3 sd

a=agarose, p=Picogreen, A=ACES, T=QuantiblotTMB, E=QuantiblotECL

Established Technologies “New” Technologies

qPCR

aa

a a

a

a aa

ppp

p

p

ppa p

-3 -2 -1 0 1 2 3

Direct,

Non-blot

TE

E

T

T TTT TT T

T T

TT T

TTT

T T T TTT

T

TTF

F

F

DD

DE E

E

E

E

E

EEE

EE

E

EEE EE

EE

A

AA

AA

TFDEA

-3 -2 -1 0 1 2 3

Direct,

Blot

2

98 76

655

43 1

1

1

0

00

0

0 0000 00

00

00

0

000

6 510

-3 -2 -1 0 1 2 3

PCR

RT

Concordance

Ap

pa

ren

t P

recis

ion

Concordance

Ap

pare

nt

Pre

cis

ion

0 = Quantifiler 1 = Alu RT-PCR 5 = BRCA1 6 = CFS-HUMRT

Kline, et al. (2005) J. Forensic Sci. 50(3):571-578

Interlaboratory Comparisons

60 data sets Laboratory Performances with Real-Time PCR Methods

Comparing results from

8 different samples using

10 different methods

qPCR Facts

• qPCR is RELATIVE to the standards used to

generate a calibration curve.

• qPCR instruments use a selected Cycle

Theshold (CT) for calculations.

• The premise is that at 100% PCR efficiency

you have a doubling of the PCR product.

• Therefore

1 CT = [DNA]/2 to [DNA]

2.

QS 04 Indicators

• Ten different qPCR methods were used to

evaluate DNA samples distributed in the NIST

Interlaboratory DNA Quantitation Study 2004

(QS04).

• These methods appeared to have some bias

relative to each other.

• Is the bias method- or standard-based?

Interlaboratory Performance

Design

Log(Design)

n

Nf

Q3

Q2

Q1

Min

Max

Rng

Xs

Low

High

Rng

Xmax

8

4

2

1

1/2

1/4

1/8

1/16

1/32

N ~ 108N ~ 82N ~ 432004

QN PMO RSTU VW X A B E C F D G H

1999 2001

Rep

ort

ed

[D

NA

], n

g /

L

PFA tube

All quantitation methods results

12

0.01

0.1

1

QS04: Lesson Learned

Results for the sample

in PFA (ie, Teflon) tubes

were consistently close

to the nominal DNA

concentration of 50

pg/µL .

SRM 2372

Human DNA Quantitation Standard

Components

A: Male/single donor/RNased/NIST (52.5 ng/µL)

B: Female/multiple donors/NIST (53.6 ng/µL)

C: Mixture/male & female/commercial (54.3 ng/µL)

Quantities supplied: 110 μL of Human Genomic DNA

Certification Decadic Attenuance (Absorbance) by a US National Reference

Spectrophotometer

Homogeneity by a Cary 100 Bio Spectrophotometer

Validation of conventional [DNA] by Interlaboratory Study and NIST

qPCR studies.

Interlaboratory Study

• 32 laboratories participated

• This limited study was advertised at the NIJ

Grantees meeting, June of 2006

• All laboratories provided data

(Thank You!)

• Net result of the study: the SRM materials are

appropriate for use with different qPCR methods

Interlaboratory Data

Each symbol represents the

average qPCR [DNA] results

reported for a given method by

one participating laboratory.

The cross at each {A/C,B/C} pair

represents approximate 95 %

confidence intervals for the two

ratios for the method as

implemented at that laboratory.

The diagonal line represents the

expected behavior when

measurements deviations from

the consensus value are

consistently biased.

Method dependent bias is

present.

2.5

2.25

2

1.75

1.5

1.25

1

4/5

4/6

4/7

4/8

4/9

4/10

4/10 4/9 4/8 4/7 4/6 4/5 2.52.2521.751.51.251

A / C

B / C

Qfiler

CFS

Sybr_ALU

Probe_ALU

Picogreen

CADOJmonoTH01

CADOJ3TH01

CADOJ3CSF

95% Predicition

NIST Quantitation Interlaboratory Studies

1999 Duewer DL, Kline MC, Redman JW, Newall PJ, Reeder DJ.

NIST Mixed Stain Studies #1 and #2: Interlaboratory Comparison of DNA

Quantification Practice and Short Tandem Repeat Multiplex Performance with

Multiple-Source Samples. J Forensic Sci 2001;46(5):1199-1210.

2001 Kline MC, Duewer DL, Redman JW, Butler JM.

NIST Mixed Stain Study #3: DNA Quantification Practice and its Influence on Short

Tandem Repeat Multiplex Performance.

Anal Chem 2003;75(10):2463-2469.

2004 Kline MC, Duewer DL, Redman JW, Butler JM.

Results from the NIST 2004 DNA Quantitation Study.

J Forensic Sci, 50(3): 571-578.

2007 Kline, M.C., Duewer, D.L., Travis, J.C., Smith, M.V., Redman, J.W., Vallone, P.M.,

Decker, A.E., Butler, J.M. (2009) Production and certification of NIST Standard

Reference Material 2372 Human DNA Quantitation Standard.

Anal. Bioanal. Chem. 394: 1183-1192.

http://www.cstl.nist.gov./biotech/strbase/interlab.htm

Thanks for your attention

Funding:

Interagency Agreement between

National Institute of Justice and

NIST Office of Law Enforcement

Standards

margaret.kline@nist.gov