OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

1

OSAC 2022-S-0041

Best Practice 

Recommendations for 

Internal Validation of 

DNA Extraction Methods

Human Forensic Biology Subcommittee

Biology SAC

Organization of Scientific Area Committees (OSAC) for Forensic Science
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

2

Draft OSAC Proposed Standard

OSAC 2022-S-0041 Best Practice Recommendations 

for Internal Validation of DNA Extraction Methods

Prepared by 

Human Forensic Biology Subcommittee

Version: 1.0

December 2022

Disclaimer:

This OSAC Proposed Standard was written by 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 (STR). The STR process is vital to OSAC’s mission of generating and recognizing 

scientifically sound standards for producing and interpreting forensic science results. The STR 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 STR 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. 

Keywords: internal validation, DNA extraction
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

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Table of Contents

1 4

2 4

3 4

4 6

5 7

5.1 7

5.2 7

5.3 7

5.4 10

5.5 11

5.6 12

Annex 14

Appendix A 14

References 14
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

1 Scope

This document provides best practice recommendations when performing an internal validation of 

DNA extraction methods, and is intended to be a companion document to ASB 038 Standard for 

Internal Validation of Forensic DNA Analysis Methods and ASB XXX Standard for Internal Validation 

of DNA Extraction Methods. This level of detail is intended to provide process experts a roadmap to 

bring DNA extraction methods online. 

2 Normative References

ASB 038 Standard for Internal Validation of Forensic DNA Analysis Methods

ASB XXX Standard for Internal Validation of DNA Extraction Methods

3 Terms and Definitions

3.1

Concordance

Agreement between DNA typing results.

3.2

Controls

Samples of known types, run in parallel with experimental, reference, or evidence samples that are 

used to demonstrate that a procedure is working correctly.

3.3

Contamination

Exogenous DNA or other biological material in a DNA sample, PCR reaction, or item of evidence; the 

exogenous DNA or biological material could be present before the sample is collected, or introduced 

during collection or testing of the sample.

3.4

Degradation

Fragmentation or damage of DNA by chemical, physical, or biological means.

3.5

Developmental Validation 

Accumulation of test data and determination of conditions and limitations of a new methodology; 

this generally occurs while the conditions and parameters are being worked out prior to the 

establishment of a defined assay, procedure or product.
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

3.6

DNA extraction

A technique used to release DNA from cells in a biological sample. 

3.7

Inhibition

Prevention of DNA synthesis during polymerase chain reaction by any substance that either directly 

interacts with DNA or interferes with the DNA polymerase.

3.8

Internal validation

Accumulation of test data within the laboratory for developing the laboratory standard operating 

procedures and demonstrating that the established protocols for the technical steps of the test and 

for data interpretation perform as expected in the laboratory.

3.9

DNA purification 

A process by which DNA is isolated from other cellular and non-cellular components using a 

combination of physical and/or chemical methods.

3.10

Polymerase chain reaction (PCR)

An enzymatic process by which a specific region of DNA is replicated during repetitive cycles that 

consist of the following: denaturation of the template; annealing of primers to complementary 

sequences at an empirically determined temperature; and extension of the bound primers by a DNA 

polymerase. 

3.11

Repeatability 

Precision and accuracy of results (e.g. quantitative and/or qualitative) of the same operator and/or 

detection instrument. 

3.12

Reproducibility 

Precision and accuracy of results (e.g. quantitative and/or qualitative) among different operators 

and/or detection instruments.

3.13

Sensitivity studies

Studies performed during validation of DNA or other test methods designed to define the limits of 

the method.
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

4 General Considerations

4.1 The laboratory shall evaluate the DNA extraction method using validated laboratory testing 

procedures or procedures intended for use in casework.

4.2 Reagent blank controls should be run with all sample sets throughout the validation 

process.

4.3 Applicable DNA quantitation and amplification controls should be run with all sample sets. 

4.4 Calibration and/or maintenance status of the laboratory instrumentation used should be 

verified prior to sample analysis to ensure the systems are operating optimally and within 

manufacturer’s parameters.

4.5 The laboratory should monitor results for the possibility of sample switch error or similar 

sample mishandling errors.

4.6 Biological samples with known genotypes should be used where possible.

4.7 Suitability of a DNA extraction method for its intended use may not be solely defined by 

sensitivity, yield, or quality.

4.8 Additional studies should be performed on any modifications made to the DNA extraction 

method which could affect results (e.g. addition of chemicals beyond what is included in vendor 

kits, addition of BSA, change in incubation times, change to number of wash steps, etc.).

4.9 Additional studies should be performed on any modifications made to consumables used 

during the DNA extraction method which could affect the results (e.g. introduction of lyse and spin 

tubes).

4.10 Differential extraction methods should be evaluated to determine the effectiveness of the 

method for minimizing carry over, and ensuring DNA yields are maintained as determined during 

sensitivity evaluation.

4.11 Consistent preparation of sample replicates is needed to minimize variation.
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

5 Best Practice Recommendations for Internal Validation of DNA Extraction 

Methods

5.1 Concordance

5.1.1 Standard: The laboratory shall demonstrate concordance of the new DNA 

extraction method to previously validated methods or published results. 

5.1.2 Objective: The purpose of concordance testing is to demonstrate agreement 

between the results obtained from the DNA extraction method compared to previous 

results.

5.1.3 Considerations:

5.1.3.1 Concordance may be confirmed through a stand-alone experiment or using 

data generated from another experiment within the internal validation.

5.1.3.2 Concurrent comparison of the new extraction method to previously 

validated methods can inform the laboratory if differences in results are due to the 

extraction procedures or downstream variables.

5.1.4 Experimental Method:

5.1.4.1 Quantify the amount of DNA and perform DNA typing on all samples. 

5.1.4.2 Concordance should be evaluated by comparing the DNA typing results of 

the DNA extraction method to the results from a previously validated method or 

published results (e.g. proficiency test consensus profile, NIST Standard Reference 

Material (SRM) or purchased sample with published results).

5.1.5 Data Analysis and Results:

5.1.5.1 A comparison of observed typing results to the known values should be 

performed.

5.1.5.2 Observed discordance should be documented and if possible, a reason 

provided. 

5.1.6 Implementation:

5.1.6.1 Observed discordance may not invalidate the concordance study. Common 

reasons for discordance may include analysis of samples in the stochastic range, a 
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

null allele resulting from a primer binding site mutation, a difference in allele call 

due to different PCR primer sets, or a different method of fragment separation 

affecting resolution or migration.

5.2 References and Casework-like Samples

5.2.1 Standard: The laboratory shall evaluate the DNA extraction method using known 

references or casework-like samples and substrates representative of those typically 

analyzed by the testing laboratory. 

5.2.2 Objective: The purpose of testing representative samples is to assess the 

performance of the extraction method using different sample types, collection methods, and 

substrates to reflect those commonly submitted for laboratory analysis, including inhibited 

or otherwise challenging samples.

5.2.3 Considerations:

5.2.3.1 This study may be used to assess relative DNA extraction performance 

through comparison with another validated DNA extraction method or 

developmental validation.

5.2.3.2 The DNA extraction method being validated should generate reproducible 

results for pristine samples and consistent results for challenged samples.

5.2.3.3 Preparation of inhibited and adulterated sample sets should include control 

samples prepared using the same source of biological material in order to ensure 

comparable data sets. 

5.2.3.4 Mixture samples may be utilized to evaluate the ability of the method to 

recover DNA from minor contributors and to inform the laboratory on major/minor 

determination.

5.2.3.5 This study can assist in identifying optimal extraction 

volumes/weights/inputs, and/or appropriate sampling strategies for some 

extraction substrates.

5.2.4 Experimental Method:

5.2.4.1 Quantify the amount of DNA and perform DNA typing on all samples. 

5.2.4.2 The testing laboratory should analyze known and case-type samples 

representing those expected to be encountered by the laboratory for casework 

using the DNA extraction method. Samples may include:
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

5.2.4.2.1 Single source samples and samples containing more than one 

contributor in varying template amounts.

5.2.4.2.2 Diverse sample types (e.g. blood, saliva, semen, etc.).

5.2.4.2.3 Inhibited samples.

5.2.4.2.4 Adulterated samples (e.g. latent print processing reagents, gun oil, 

etc.).

5.2.4.2.5 Degraded samples, including differential degradation in mixed 

samples.

5.2.4.2.6 Different substrates.

5.2.4.2.7 Different DNA collection methods (e.g. swabbing, tape-lift, cuttings, 

etc.).

5.2.5 Data Analysis and Results:

5.2.5.1 Evaluate the data and determine if the extraction method is compatible with 

samples tested. 

5.2.5.2 Compare the amount of DNA recovered to expected quantities or results 

from previous methods, if applicable.

5.2.5.3 DNA typing results from samples should be compared to any previous 

results, where applicable.

5.2.5.3.1 If comparison with a previous method and using the same samples 

is not possible, then comparison with validation data from the previous 

method using similar samples should be performed. 

5.2.5.4 The method’s ability to remove or mitigate PCR inhibition should be 

determined. 

5.2.6 Implementation:

5.2.6.1 The results from this study should be used to inform laboratory procedures 

and workflow. 

5.3 Inhibition and Degradation

5.3.1 Standard: The laboratory shall determine the susceptibility of the DNA extraction 

method to the introduction of inhibition or degradation.

5.3.2 Objective: The purpose of this study is to determine if the DNA extraction method 

introduces inhibition or causes degradation. 
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

5.3.3 Considerations:

5.3.3.1 Quantitation methods that provide information related to sample 

degradation or inhibition may assist in sample evaluation.

5.3.4 Experimental Method:

5.3.4.1 Quantify and perform DNA typing on all samples. 

5.3.4.2 Samples should be single-source DNA samples of good quality and not 

limited in concentration. 

5.3.5 Data Analysis and Results:

5.3.5.1 DNA typing results should be evaluated to determine if any adverse effect is 

observed that may be attributable to the extraction chemistry (e.g. signal reduction, 

partial or complete inhibition, allelic or locus dropout, etc.). 

5.3.6 Implementation:

5.3.6.1 The data from this study shall inform the laboratory as to the applicability of 

this extraction method to case samples.

5.4 Sensitivity:

5.4.1 Standard: The laboratory shall determine the sensitivity of the DNA extraction 

method.

5.4.2 Objective: Sensitivity studies are used to assess the ability to obtain reliable results 

from a range of DNA quantities.

5.4.3 Experimental Method:

5.4.3.1 Quantify and perform DNA typing on all samples. 

5.4.3.2 Dilutions of single source biological material should be extracted in 

triplicate.

5.4.4 Data Analysis and Results: 

5.4.4.1 Analyze the resulting data using the laboratory’s analysis parameters. 
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

5.4.4.2 Evaluate the DNA typing results from replicate extractions to determine at 

what sample dilution a full DNA profile is no longer obtained and when the DNA 

profile obtained is no longer interpretable.

5.4.4.3 Compare the DNA typing results from replicate extractions to determine 

consistency of the DNA extraction method.

 

5.4.4.4 Compare recovered quantities with previous methods, if possible, to 

determine if the method has comparable performance with the previously validated 

method.

5.4.4.4.1 If comparison with a previous method and using the same samples 

is not possible, then comparison with validation data from the previous 

method using similar samples should be performed. 

 

5.4.5 Implementation:

 

5.4.5.1 The laboratory should evaluate the DNA yields to determine the suitability 

for use with casework. 

5.5 Reproducibility and Repeatability:

5.5.1 Standard: The laboratory shall demonstrate the precision and accuracy of the DNA 

extraction method through repeatability and reproducibility studies.

5.5.2 Objective: The method should consistently yield comparable quantities of DNA 

with minimal operator-to-operator variability, when used for the same type and amount of 

sample, and when performed using the same equipment. 

5.5.3 Considerations:

5.5.3.1 Samples should be homogeneous to the extent possible.

 

5.5.4 Experimental Method:

5.5.4.1 Samples should be single-source DNA of good quality and not limited in 

concentration.

5.5.4.2 Quantify all samples.

5.5.4.3 The laboratory should determine the reproducibility of the method through 

repeated tests by different individuals, when practicable. 

 
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

5.5.4.4 The laboratory should determine the repeatability of the method through 

repeated tests by the same individual.

 

5.5.5 Data Analysis and Results:

5.5.5.1 A comparison of DNA quantitation values should be performed. 

5.5.5.1.1 The range, mean and standard deviation values for the 

concentration of DNA in the extracted product should be calculated.

5.5.5.1.2 The laboratory should evaluate inconsistencies in DNA 

concentrations.

5.5.6 Implementation:

5.5.6.1 The laboratory should evaluate the data and determine if the method 

produces consistent results and suitability for use with casework. 

5.6 Contamination:

5.6.1 Standard: The laboratory shall determine the susceptibility of the DNA extraction 

method to the introduction of exogenous DNA. 

5.6.2 Objective: The purpose of the contamination study is to inform the laboratory as to 

the susceptibility of the DNA extraction method to the introduction of exogenous DNA 

originating from reagents, consumables, laboratory personnel, instrumentation, workflow, 

and environment.

5.6.3 Considerations: 

5.6.3.1 The following should be considered for the origin of contamination:

5.6.3.1.1 Consumables (e.g. tubes, reagents, pipette tips, etc.)

5.6.3.1.2 Laboratory personnel (e.g. appropriate personal protective 

equipment (PPE), training, sample handling, etc.)

5.6.3.1.3 Laboratory environment (e.g. possible new cleaning 

schedule/procedure, HVAC systems, workflow, dedicated lab space, etc.)

5.6.3.1.4 Laboratory workflow (e.g. automation equipment, hoods, 

centrifuges, etc.)

5.6.3.1.5 DNA sampling method (e.g. M-vac, swabs, tape lift, etc.)

5.6.3.2 Additional controls may be processed to test the system based on the 

variables described in the considerations above.
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

5.6.4 Experimental Method: Not applicable

5.6.5 Data Analysis and Results:

5.6.5.1 All validation samples, including the extraction (reagent) blank controls, 

should be evaluated for the presence of exogenous DNA.

5.6.5.2 If observed, the source of the contamination should be characterized, 

attribution attempted and the rate of occurrence estimated.

5.6.6 Implementation:

5.6.6.1 The results of this study will identify the point(s) in the laboratory process 

where contamination event(s) occur and should be used to establish laboratory 

policies concerning the following:

5.6.6.1.1 Laboratory procedures to prevent and detect contamination.

5.6.6.1.2 Control measures (e.g. PPE).

5.6.6.1.3 Procedural requirements concerning controls (e.g. placement and 

number).

5.6.6.1.4 Corrective measures required when contamination is detected. 

5.6.6.1.5 Systemic problems that may require further improvements.

5.6.6.1.6 Contamination tolerance interpretation guidelines.

5.6.6.1.7 Laboratory environmental requirements (e.g. design, workflow, 

cleaning, instrument maintenance, etc.).
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

Annex 

Appendix A

Cross function study - This table is intended to assist laboratories with efficiently utilizing sample 

data generated across the multiple studies outlined in this document. The x-axis is a category list of 

the samples in process and the y-axis is the listed outline of each study described in this document.

References

Lee GM, Thornthwaite JT, Rasch EM. Picogram per Cell Determination of DNA by Flow 

Cytofluorometry. Analytical Biochemistry 1984;19137:22 l-6.

Guidelines and Considerations for Evaluating or Validating Extraction Chemistry. Applied 

Biosystems. Forensic News. July, 2010.

ENSFI. Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling 

Process. http://www.enfsi.eu/sites/default/files/documents/minimum_validation_guidelines_in_d 

na_profiling_-_v2010_0.pdf

Lee SB, Shewale JG (2017). DNA Extraction Methods in Forensic Analysis. In Encyclopedia of 

Analytical Chemistry, editors John Wiley & Sons, Ltd. DOI: 10.1002/9780470027318.a1104m.pub2.
OSAC 2022-S-0041 Best Practice Recommendations for

Internal Validation of DNA Extraction Methods

SWGDAM Validation Guidelines for Forensic DNA Analysis Methods. 

https://www.swgdam.org/_files/ugd/4344b0_813b241e8944497e99b9c45b163b76bd.pdf. 

Accessed July 27, 2022.