OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic 

DNA Casework Using Electrophoresis Platforms

1 

OSAC 2021-S-0003 

Standards for Determining 

Analytical and Stochastic 

Thresholds for Application to 

Forensic DNA Casework Using 

Electrophoresis Platforms

Human Forensic Biology Subcommittee 

Biology Scientific Area Committee 

Organization of Scientific Area Committees (OSAC) for Forensic Science 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

2 

OSAC Proposed Standard 

OSAC 2021-S-0002 

Standard for Determining Analytical 

and Stochastic Thresholds for 

Application to Forensic DNA 

Casework Using Electrophoresis 

Platforms 

Prepared by 

Human Forensic Biology Subcommittee 

Version: 2.0 

November 2023 

Disclaimer: 

This OSAC Proposed Standard was written by the Human Forensic Biology 

Subcommittee/Biology Scientific Area Committee of the Organization of Scientific Area 

Committees (OSAC) for Forensic Science following a process that includes an open comment 

period. This Proposed Standard will be submitted to a standards developing organization and is 

subject to change. 

There may be references in an OSAC Proposed Standard to other publications under development 

by OSAC. The information in the Proposed Standard, and underlying concepts and methodologies, 

may be used by the forensic-science community before the completion of such companion 

publications. 

Any identification of commercial equipment, instruments, or materials in the Proposed Standard 

is not a recommendation or endorsement by the U.S. Government and does not imply that the 

equipment, instruments, or materials are necessarily the best available for the purpose. 

To be placed on the OSAC Registry, certain types of standards first must be reviewed by a 

Scientific and Technical Review Panel (STRP). The STRP process is vital to OSAC’s mission of 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

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generating and recognizing scientifically sound standards for producing and interpreting forensic 

science results. The STRP shall provide critical and knowledgeable reviews of draft standards or 

of proposed revisions of standards previously published by standards developing organizations 

(SDOs) to ensure that the published methods that practitioners employ are scientifically valid, and 

the resulting claims are trustworthy. 

The STRP panel will consist of an independent and diverse panel, including subject matter experts, 

human factors scientists, quality assurance personnel, and legal experts, which will be tasked with 

evaluating the proposed standard based on a comprehensive list of science-based criteria. 

For more information about this important process, please visit our website at: 

https://www.nist.gov/topics/organization-scientific-area-committees-forensic-science/scientifictechnical-review-panels 

 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

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Forward: 

The individual minimum requirements for the determination and validation of analytical and 

stochastic thresholds are defined in this document. Such thresholds help to ensure credence in the 

reliability of the data obtained, while clearly conveying assumptions under which data will be 

evaluated during downstream interpretation. The goal is for the laboratory to consistently produce 

reliable and reproducible designations of allelic data and identify when allelic drop-out may have 

occurred supported by internal validation data and laboratory protocols. 

If a laboratory, as part of its data analysis methods, makes binary determinations regarding the 

detection or non-detection of peaks for casework, analytical thresholds are necessary. Similarly, if 

a laboratory, as part of its data analysis methods, makes binary determinations regarding the 

potential for allele drop-out in casework, stochastic thresholds are also necessary. 

Whenever a threshold is applied, it is possible that a classification error may occur. Intrinsic to 

any analytical threshold is the expectation that non-reproducible noise will produce some peaks 

that are incorrectly classified as alleles because they exceed the threshold, and that some true 

alleles will be undetected because they produce peaks below the threshold. Intrinsic to any 

stochastic threshold is the expectation that some errors will occur in determining whether allelic 

drop-out may have occurred. Some heterozygous genotypes will be erroneously classified as 

homozygous when the sister allelic peaks drops out and the second peak is above the stochastic 

threshold. Conversely, some homozygous genotypes will be erroneously classified as potentially 

heterozygous because the single peak is below the stochastic threshold. The advantage of 

determining thresholds based on statistical analysis of relevant empirical data, is that estimates can 

be made of the relative risk of these possible errors for a given threshold level. In setting 

thresholds, a statistically based approach must be employed by the laboratory to determine what 

proportion of these events are acceptable for the analysis of forensic casework. 

The draft of this standard was developed by the Human Forensic Biology Subcommittee of the 

Organization of Scientific Area Committees for Forensic Science. 

Keywords: analytical threshold, stochastic threshold, DNA, validation, signal, artifact, noise 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

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Table of Contents 

1 Scope ....................................................................................................................................... 6

2 Normative References ............................................................................................................. 6

3 Terms and Definitions ............................................................................................................. 6

4 Requirements ......................................................................................................................... 10

Annex A ........................................................................................................................................ 16

 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

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Standard for Determining Analytical and Stochastic Thresholds for Application to 

Forensic DNA Casework Using Electrophoresis Platforms 

1 Scope 

These standards set the requirements for forensic laboratories, which as part of their casework data 

analysis processes, are making determinations of: a) whether a peak in an electropherogram 

represents true signal or might be noise; and b) whether drop-out of a heterozygous sister allele to 

an observed peak either did not occur or might have occurred. 

This does not apply to probabilistic genotyping systems that calculate a probability-based 

analytical threshold as a part of its analysis process. 

This standard is applicable to forensic short tandem repeat (STR) DNA typing performed on 

electrophoresis platforms. 

2 Normative References 

The following reference is indispensable for the application of the standard. For dated references, 

only the edition cited applies. For undated references, the latest edition of the referenced document 

(including any amendments) applies. 

ANSI/ASB Standard 038, Standard for Internal Validation of Forensic DNA Analysis Methods. 

2020. 1st. Ed. 

3 Terms and Definitions 

For the purposes of this document, the following definitions apply. 
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and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

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3.1 

allele

Any of the forms of the same gene that occur at the same locus on a homologous chromosome but 

differ in base sequence. - Oorschot, R.A.H. and Ballantyne, K.N. “Capillary Electrophoresis in 

Forensic Biology.” Forensic Biology, 49-57. Elsevier Ltd., Macleod, VIC, Australia, 2013. 

3.2 

analytical threshold

The minimum height requirement at and above which detected peaks on a STR DNA profile 

electropherogram can be reliably distinguished from instrument background noise; peaks above 

this threshold are generally not considered noise and are either artifacts or true alleles. - J.M. 

Butler. Fundamentals of Forensic DNA Typing. Elsevier Academic Press, San Diego, CA, 2010. 

3.3 

artifact

A non-allelic product of the amplification process (e.g., stutter, non-templated nucleotide addition, 

or other non-specific product), an anomaly of the detection process (e.g., single or multichannel 

voltage spikes or “pull-up”), or a by-product of primer synthesis (e.g., “dye blob”) that may be 

observed on an electropherogram. Some artifacts may complicate the interpretation of DNA 

profiles when they cannot be distinguished from the actual allele(s) from a particular sample. - 

Scientific Working Group on DNA Analysis Methods, SWGDAM Interpretation Guidelines for 

Autosomal STR Typing. 2010. 

3.4 

control

Material of established origin that is used to evaluate the performance of a test or comparison. - 

ASTM E1732-12 Standard Terminology Relating to Forensic Science. ASTM International, West 

Conshohocken, PA, 2012. 
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and Stochastic Thresholds for Application to Forensic DNA 

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3.5 

coverage factor (k) 

Coverage factor (k factor) (Guide to Uncertainty of Measurement, GUM): numerical factor used 

as a multiplier of the combined standard uncertainty in order to obtain an expanded uncertainty. - 

NISTIR 6919, Recommended Guide for Determining and Reporting Uncertainties for Balances 

and Scales, Val Miller, State Laboratory Program, Weights and Measures Division National 

Institute of Standards and Technology, Technology Administration, U.S. Department of 

Commerce. 

3.6 

drop-out 

(1) Failure of an otherwise amplifiable allele to produce a signal above analytical threshold because 

the allele was not present or was not present in sufficient quantity in the aliquot that underwent 

polymerase chain reaction (PCR) amplification. (2) A hypothesis/postulate for the failure to 

observe one or more allelic peaks in an electropherogram that are expected for the assumed 

contributor(s) to a sample. – Scientific Working Group on DNA Analysis Methods, SWGDAM 

Guidelines for STR Enhanced Detection Methods. Scientific Working Group on DNA Analysis 

Methods, 2014. 

3.7 

empirical data 

Factual data that are based on actual measurement, observation, or direct sensory experience rather 

than on theory. - NFPA 921 Guide for Fire and Explosion Investigations. NFPA, 1 Batterymarch 

Park, Quincy, MA 02169-7471, 2014. 

3.8 

internal validation 

The accumulation of test data within the laboratory for developing the laboratory standard 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

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operating procedures and determining the limits of the method(s). Internal validation 

demonstrates that the established protocols for the technical steps of the test and for data 

interpretation perform as expected in the laboratory. - ANSI/ASB Standard 038, 1st. Ed. 2020. 

3.9 

locus (loci)

A unique physical location of a gene (or specific sequence of DNA) on a chromosome; the plural 

of locus is loci (which is pronounced low-sigh). - J.M. Butler. Fundamentals of Forensic DNA 

Typing. Elsevier Academic Press, San Diego, CA, 2010. 

3.10 

noise

Meaningless output occurring in electronic equipment; it is random electronic variation that is 

generated by and intrinsic to the electronic circuitry. It ultimately establishes the smallest 

analytical signal that can be quantitatively measured with confidence. A part of a signal that is not 

the target signal. - ASB Technical Report 025 Crime Scene/Death Investigation – Dogs and 

Sensors Terms and Definitions. AAFS Academy Standards Board (ASB), 2017. 

3.11 

peak 

The visual image of an allele or other fluorescent signal on an electropherogram; for an allele, the 

peak height (Y axis) represents the amount of amplified DNA in the PCR and the peak position 

(X axis) represents the fragment length. - J.M. Butler. Fundamentals of Forensic DNA Typing. 

Elsevier Academic Press, San Diego, CA, 2010. 

3.12 

stochastic threshold 

The peak height value in a DNA electrophoretic profile above which it is reasonable to assume 

that, at a given locus, allelic dropout of a sister allele in a heterozygous pair has not occurred in a 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

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single source DNA sample; due to the possibility of shared alleles in mixed samples, the presence 

of allele peaks above the stochastic threshold is no guarantee that allele dropout did not occur in 

mixed DNA sample profiles. - Scientific Working Group on DNA Analysis Methods, SWGDAM 

Interpretation Guidelines for Autosomal STR Typing. 2010. 

3.13 

stutter 

An artifact of PCR amplification typically observed one or more repeat units smaller or larger than 

an STR allele in a DNA electrophoretic profile, may result from strand slippage during PCR 

amplification. A stutter peak is generally of lower relative fluorescent units (RFU) than the allele 

peak. - Scientific Working Group on DNA Analysis Methods, SWGDAM Interpretation 

Guidelines for Autosomal STR Typing. 2010. 

4 Requirements 

4.1 The laboratory shall have an analytical threshold1

 for each electrophoresis platform (e.g.,

distinct capillary electrophoresis (CE) models) used in casework that is established and tested 

using data generated during internal validation. 

NOTE: When multiple instruments of the same kind/model/platform are used for casework, data 

generated from each instrument should be considered due to potential variations in noise inherent 

to each instrument. 

4.1.1 The laboratory shall determine and document the acceptable proportion of noise peaks that 

will exceed the analytical threshold (e.g., as reflected by the number of standard deviations above 

1

 This does not apply to the dye channel used for the internal size standard. 
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the noise mean [RFU]). This establishes the laboratory’s predetermined expectation for acceptable 

performance of the analytical threshold. 

NOTE: As the number of standard deviations increases, the potential for allele non-detection also 

increases. Recognizing there is a tradeoff between the risk of allele non-detection and the risk of 

mistakenly labeling noise peaks as alleles, the analytical threshold should be set such that the 

probability that noise exceeds the analytical threshold is between 10-2 and 10-6 (e.g., k=2 to k=5). 

See Table 9 (Mönich et al., page 115) relating k value to probability that a randomly generated 

noise peak exceeds the analytical threshold. 2 

4.1.2 The laboratory shall establish and verify an analytical threshold based on internally 

generated empirical data acquired from the same electrophoresis platform, analysis software and 

DNA profiling chemistry utilized in casework. 

4.1.2.1 Validation studies used to establish an analytical threshold shall include samples of known 

composition (e.g., known genotype and negative controls3). Casework samples shall not be used 

to determine an analytical threshold. 

4.1.2.2 Acceptable positions on an electropherogram to interrogate when establishing analytical 

thresholds shall be those that exclude possible allele or artifact peaks of known origin (e.g., alleles 

and associated stutter products such as n-1, n-2, and n+1 positions, spectral pull-up peaks including 

those due to internal size standard, voltage spikes, unincorporated dye peaks). 

4.1.2.3 When modifications to the instrument are made that have the potential to impact the noise 

output of the instrumentation, the performance of the analytical threshold shall be verified and 

2

 Mönich, U.J., Duffy, K., Medard, M., Cadambe, V., Alfonse, L.E. and Grgicak C. "Probabilistic characterisation 

of baseline noise in STR profiles." Forensic Science International: Genetics 19 (2015): 107-122. 

3

 Negative amplification and Reagent blank controls are acceptable sample types providing that they contain no 

indication of amplified product. 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

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documented (e.g., performance check following change in laser and/or recalibration of the 

instrument). 

4.1.3 The laboratory shall assess statistically-based analytical thresholds for each dye channel. 

A number of statistical methods to establish analytical threshold(s) have been described in the 

scientific literature.4 Relevant references are provided in Annex A Bibliography. Laboratories 

employing a single global analytical threshold for all dye channels shall provide statistical support 

(e.g., based on 1-way analysis of variance [ANOVA] showing no statistically significant 

differences [p<0.05] in noise across dye channels). 

4.1.4 If the laboratory employs rounding (e.g., to the nearest unit of 5 or 10 RFU), the 

implications of this rounding regarding the chance of mistaking noise for signal and the chance of 

not labeling a true allele in low template samples shall be documented. 

4.2 The laboratory shall have a stochastic threshold5

 for each electrophoresis platform (e.g.,

distinct CE models and DNA profiling chemistry) used in casework that is established and verified 

using data generated during internal validation. 

NOTE: When multiple instruments of the same kind/model/platform are used for casework, data 

generated from each instrument shall be evaluated to identify potential variations inherent to each 

instrument. 

4.2.1 The laboratory shall determine and document the acceptable proportion of false 

homozygotes (drop-out) that will appear above the stochastic threshold (e.g., as reflected by the 

4

 Methods based on an extreme value calculation (e.g., 2X peak to trough difference) do not address the statistical 

confidence of a given analytical threshold. In addition, such methods can be easily skewed by outlier data and thus 

do not meet the requirements of this standard. 

5

 Determination of a stochastic threshold does not apply to the dye channel used for the internal size standard. 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

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number of standard deviations above the mean). This establishes the laboratory’s predetermined 

expectation for acceptable performance of the stochastic threshold. 

NOTE: As the number of standard deviations increases, the potential for true homozygote 

detection decreases. Recognizing there is a tradeoff between detecting true homozygotes and the 

risk of mistakenly labeling a heterozygote with drop-out as a homozygote, the stochastic threshold 

should be set such that the probability that drop-out exceeds the stochastic threshold is between 

10-2 and 10-6 (e.g., k=2 to k=5).

4.2.2 The laboratory shall establish a stochastic threshold based on internally generated empirical 

data acquired from the same electrophoresis platform, analysis software, and DNA profiling 

chemistry utilized in casework. 

NOTE: Though laboratories using probabilistic genotyping systems are not required to establish 

or apply stochastic thresholds, they are still required to conduct validation studies that inform the 

laboratory of stochastic issues (e.g., allele drop-out). 

4.2.3 Validation studies of allelic drop-out used to establish a stochastic threshold shall include 

dilution series of single source samples of known genotype with a high level of heterozygosity and 

a range of differences in sister allele separation within each locus. The dilution series shall include 

DNA quantities around which allelic drop-out is likely to occur. 

NOTE: The use of larger data sets improves the accuracy of the stochastic threshold. Stochastic 

events are, by definition, random. 

4.2.4 If processes are utilized to increase or decrease sensitivity (e.g., changes to amplification 

cycle number, injection time, and post-amplification purification or concentration of amplified 
OSAC 2021-S-0003, Standard for Determining Analytical 

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products), the laboratory shall perform additional studies to determine the appropriate stochastic 

threshold(s) for the method(s) employed. 

NOTE: For any profile generated using a reduced sensitivity method, where all interpreted peaks 

remain above the default stochastic threshold, the laboratory may evaluate whether or not the 

stochastic threshold implemented for routine data analysis is applicable to the decreased 

sensitivity method. 

4.2.5 A number of methods to calculate a stochastic threshold have been described in the 

scientific literature. Relevant references are provided in Annex A Bibliography. The method 

selected must be supported by both the scientific literature and empirical data generated during 

internal validation by the laboratory.6

 The laboratory shall document the desired level of statistical 

confidence (e.g., as reflected by the number of standard deviations above the mean) for 

establishing a stochastic threshold. 

4.3 Following the completion of the laboratory’s internal validation study, all data, data 

analyses, and calculations used to determine the analytical and stochastic thresholds shall be 

documented in the final validation report. 

4.3.1 The validation report shall include the following information: 

a) a record of predetermined specifications and quality attributes (i.e., statistical confidence 

level/error rate7

) for accepting and implementing the thresholds(s) into operations. 

6

Methods based on the largest surviving allele do not directly address the probability of allele drop-out at the stochastic 

threshold. Therefore, these methods are not recommended for determining a stochastic threshold. Thresholds shall be 

established based on statistical analysis, and skewed (i.e., non-symmetrical) data must be appropriately transformed 

prior to further analysis. 

7

 Type 1 Error, the rejection of a true null hypothesis (e.g., a 99% confidence level has a 1% error rate). In the context 

of the analytical threshold, this represents the probability that an instrument noise peak will exceed the analytical 
OSAC 2021-S-0003, Standard for Determining Analytical 

and Stochastic Thresholds for Application to Forensic DNA 

Casework Using Electrophoresis Platforms

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b) a description of the samples, test methods, electropherograms and data used to calculate 

the threshold(s). 

c) any formulae or theory applied to compute the thresholds. 

d) reference literature as appropriate. 

4.3.2 The validation report, all data, data analyses, and calculations used to determine the 

analytical and stochastic thresholds shall be maintained by the laboratory. 

5 Conformance 

Documented conformance to these requirements needs to be: (1) approved by the laboratory’s 

DNA Technical Leader or other appropriate personnel (2) communicated to all analysts during 

training, and (3) made readily available for review (e.g., by auditors or inspectors, stakeholders 

who use reports generated by the laboratory, etc.). 

To demonstrate conformance with this standard, the laboratory shall meet the requirements in the 

ANSI/ASB Standard 038, Standard for Internal Validation of Forensic DNA Analysis Methods. 

First Edition. 2020. 

 

threshold. In the context of the stochastic threshold, this represents the probability that a true heterozygous peak will 

exceed the stochastic threshold while the sister allele has dropped out. 
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and Stochastic Thresholds for Application to Forensic DNA 

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Annex A 

(informative) 

Bibliography 

Gilder, J., Doom, T., Inman, K. and Krane, D. Run-specific limits of detection and quantitation 

for STR-based DNA testing. J For. Sciences, 2007, 52:(1):97-101. 

Bieber, F.R., Buckleton, J.S., Budowle, B., Butler, J.M. and Coble, M.D. "Evaluation of forensic 

DNA mixture evidence: protocol for evaluation, interpretation, and statistical calculations using 

the combined probability of inclusion." BMC genetics 17.1 (2016): 125. 

Butts, E.L., Kline, M., Duewer, D., Hill, C., Butler, J.M. and Vallone, P. "NIST validation 

studies on the 3500 Genetic Analyzer." Forensic Science International: Genetics Supplement 

Series 3.1 (2011): e184-e185. 

Gill, P., Puch-Solis, R. and Curran, J. "The low-template-DNA (stochastic) threshold—its 

determination relative to risk analysis for national DNA databases." Forensic Science 

International: Genetics 3.2 (2009): 104-111. 

Mönich, U.J., Duffy, K., Medard, M., Cadambe, V., Alfonse, L.E. and Grgicak C. 

"Probabilistic characterisation of baseline noise in STR profiles." Forensic Science 

International: Genetics 19 (2015): 107-122. 

Puch-Solis, R., Kirkham, A.J., Gill, P., Read, J., Watson, S. and Drew, D. "Practical 

determination of the low template DNA threshold." Forensic Science International: Genetics 5.5 

(2011): 422-427. 

Rowan, K.E., Wellner, G.A. and Grgicak, C.M. "Exploring the impacts of ordinary laboratory 

alterations during forensic DNA processing on peak height variation, thresholds, and probability 

of dropout." Journal of forensic sciences 61.1 (2016): 177-185. 

Federal Bureau of Investigations Quality Assurance Standards (QAS) for Forensic DNA Testing 

Laboratories https://www.fbi.gov/file-repository/quality-assurance-standards-for-forensic-dnatesting-laboratories.pdf

NISTIR 6919, Recommended Guide for Determining and Reporting Uncertainties for Balances 

and Scales, Val Miller, State Laboratory Program, Weights and Measures Division 

National Institute of Standards and Technology, Technology Administration, U.S. Department of 

Commerce.