Carsten Hohoff, Ph.D. 21 min

How Efficient is Your DNA Extraction Method? A Multi-year, Multi-lab Review of GEDNAP Proficiency Test Results


The German DNA Profiling Group (GEDNAP) proficiency tests are one of the largest, independent, blind trial, stain-DNA profiling quality control exercises in Europe and possibly the world. Each year GEDNAP hosts two proficiency tests and opens them to any laboratory - private institute, university institute, or governmental - from any country worldwide. During this presentation, we will analyze the extraction efficiency data obtained from the GEDNAP proficiency tests. This presentation aims to provide valuable insights into the efficiency of different DNA extraction methods and encourage laboratories to critically evaluate their current practices. By learning from the experiences of a diverse range of laboratories, we can collectively work towards enhancing the accuracy and reliability of DNA profiling in forensic investigations.



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[MUSIC PLAYING]

0:03

Hello, and welcome to hits.

0:13

My name is Stefan Koeneman.

0:16

I'm a field application scientist for Thermophicia

0:19

Scientific.

0:21

And today, I'm with Dr. Kasnowoff,

0:24

who is the lead manager of the Institute of Forensic Genetics

0:28

in Amsterdam, and he is the host of the Getna Proficiency

0:33

Tests.

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Kasn, today, will have the talk.

0:39

How efficient is your DNA extraction method?

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A multi-year multi-lab review of Getna Proficiency Test

0:47

Results.

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And I'm very glad that Kasnowoff will present this talk today.

0:54

Stefan, thank you very much for this kind introduction.

0:56

Welcome to hits 2024.

0:59

I will give a brief overview of the Getna Proficiency Test.

1:03

The scheme of my presentation is shown here.

1:05

I will give a brief introduction.

1:07

I will review the history and the background

1:09

of the Getna Proficiency Test.

1:11

And then I will focus on the extraction module.

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And then at the end, I will give some ideas on perspectives.

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The focus of my presentation will be regarding Module 9.

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This is extraction efficiency of the Getna Proficiency Test.

1:28

I will briefly introduce the aim and the scheme of the module.

1:32

I will present recent results.

1:33

And I will do a review of the data of the last years.

1:37

Here, we have plotted the evaluation

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of the extraction yield regarding a five-year period,

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well, because we have two Getna Proficiency Test per year.

1:50

So we are showing Getna 54 till Getna 64.

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And we see that always the thermophistic scientific

2:00

prephylar chemistry has performed best

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and always provided the highest yield of human nuclear DNA

2:08

throughout all proficiency tests using various substrates

2:14

from swaps to FDA cards to cellulose cards.

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And even without any carrier.

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To start with, Getna is an acronym.

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It stands for German DNA Profiling.

2:28

This name was invented in energy to the Atna European DNA

2:32

Profiling in the 1990s.

2:35

Already proficiency tests in the 1980s,

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they were organized by the German Society of Legal Medicine

2:42

for serological markers, ABO blood groups, et cetera,

2:46

that were used in forensic casework.

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And then DNA markers were incorporated later.

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And Professor Brinman then founded the German Stain

2:56

Commission and founded these proficiency tests

3:01

that we now call a jet nap.

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From the beginning, also laboratories

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from the neighboring countries were invited to participate,

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mainly from Denmark, the Netherlands, Austria,

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and Switzerland.

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I joined the team of Professor Brinman in 1999

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and since the year 2000, I'm involved in the evaluation,

3:21

presentation, and preparation of the jet nap proficiency test.

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Already in 2001, the scheme was reviewed by Bruce Padolia

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at that time, FBI, and his final evaluation statement

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was it's probably one of the best proficiency test

3:37

teams in the world.

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So we are closely connected to the European and mainly German

3:44

forensic community.

3:46

A brief overview on the Institute, the IFMG,

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is now the host of the jet nap proficiency test.

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We are a fully accredited forensic casework

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Institute, according to ISO 17025.

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The ISO 17043 is in preparation for the proficiency test.

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The accreditation body suggested to make one year

4:09

before we apply.

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So what are the aims of this proficiency test

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and of any proficiency test?

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So there's a manual that lays down

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that the aim is that correct results are reported

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and the correct nomenclature is used.

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That was an issue mainly in the first years

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when there were various nomenclature out there.

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And the jet nap proficiency had to harmonize the nomenclature

4:35

because in Europe, there are so many countries

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and there are national DNA databases

4:39

that talk to each other during the night.

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So the nomenclature is an issue.

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And on the other hand, the aim of the participants

4:46

might be a bit different.

4:48

They have to take part in any kind of proficiency test

4:52

according to the ISO 17025.

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And they need a certificate, especially in Germany,

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there is a market, there are tenders for the German police

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and only laboratories that have successfully participated

5:06

in a proficiency test can apply for those tenders.

5:10

Now let's move on.

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What do we send out?

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We send out, for example, blood samples on cellulose pads

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or on forensic swaps.

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As you can see here, we have to produce

5:20

two to 300 samples at the same time.

5:23

Now let's move on to the evaluation.

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There are at least three important groups.

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If a participant has a group one result,

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he has reported the given values.

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And if this laboratory has reported an error,

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this is categorized as group four.

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So this will be shown in the presentation

5:44

or in the certification.

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Let's move on to the participants.

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Here's an example of JETNAP 62.

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We had a total of 181 different laboratories

5:55

from 44 different countries.

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The majority of participants has been from Germany with 61.

6:02

The majority of laboratories is from Europe.

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The proficiency test is open to any laboratory

6:08

that wants to participate.

6:11

Up to now there are 12 modules.

6:13

The focus is on STRs, autosomal ones,

6:15

genosomal ones and the melogianin.

6:18

We have a module that is called stain characterization

6:21

regarding presumptive testing and confirmatory testing.

6:25

The focus of this presentation is the extraction efficiency,

6:29

but we also offer MTDNA sequence analysis

6:32

by statistics and forensic DNA phenotyping.

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Now let's move on to the extraction efficiency module.

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The aim is a benchmarking.

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Of course, the question is,

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how does my DNA extraction method perform

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in comparison to the methods of other participants?

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The focus is on automated DNA extraction methods,

6:56

but since JETNAP is very open to any laboratory,

7:00

also smaller laboratories that use

7:02

a manual extraction method can participate.

7:07

So there was one particular state,

7:09

it's called stain five that is sent out in triplicate.

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To the laboratories, it is expected that they do the extraction

7:17

and then send the three extracts to the IFMG

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for DNA quantitation.

7:23

The sample in this year was a female healthy volunteer.

7:28

The white blood cell concentration was measured

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and then also the DNA concentration was measured

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and we sent out this sample in two different concentrations

7:39

for JETNAP 66 and for JETNAP 67.

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As you can see here,

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at the first proficiency test in 2023.25 nanogram per swap

7:52

we have sent out and for the JETNAP 67,

7:58

approximately one nanogram per swap we have sent out.

8:03

The blood was diluted with a dilution buffer PBS

8:07

with rutide block that is a very famous blocking reagent used

8:12

for thasan blotting and so on.

8:14

So this reduces binding and interaction of DNA

8:19

with polystyrene surface.

8:21

We have changed the substrate from year to year.

8:26

So we were under auspices of the German stain commission

8:31

and they decided from year to year

8:36

which substrate to use.

8:38

I will talk on this item a bit later.

8:42

So in 2023 swaps again were chosen

8:46

as the carrier of the blood stain.

8:47

As you can see here,

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that is how we prepared the dilution on swaps

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and this is the outcome.

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So this is sent out to the participants.

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We have the mean yield, we have a standard deviation

9:01

and we have on the left hand side in the left column

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the acronymized participant like L101595

9:10

has used the manual extraction.

9:12

The mean yield is shown and the standard deviation.

9:16

And here we have a summary and a comparison.

9:19

So 11 laboratories used the manual extraction method

9:22

while 31 laboratories used an automated approach.

9:27

The mean yield for the automated laboratories

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was 0.23 nanogram and the expected value was 0.25.

9:34

So that was quite good.

9:36

Although we had a couple of laboratories

9:38

that did not extract any DNA at all.

9:43

And then the other proficiency test,

9:44

we had four times the amount of DNA.

9:48

So roughly one nanogram per swap that was sent out.

9:52

And here you can see the results again

9:56

that was sent out to the participants.

9:58

So you can do a benchmarking to find out

10:00

where they are and how much DNA they

10:04

extracted.

10:06

Here you can see the summarized evaluation.

10:09

Only six laboratories took part

10:11

in the manual extraction method

10:14

and 26 used automated extraction solutions.

10:18

And the mean yield was 0.76 nanogram.

10:21

The expected value was 0.99.

10:23

So that was quite close.

10:25

The aim is the benchmarking.

10:29

So we tried to find out the influence

10:32

of the hardware and the chemistry

10:35

on the extraction yield.

10:36

And with those numbers that we have,

10:41

so 26 different laboratories,

10:43

we often found unique combinations

10:46

of a hardware and the chemistry.

10:48

So the diversity of combinations was quite high.

10:52

And that means on the other hand,

10:53

we have a very low statistical power

10:55

to find out which combination per se is very efficient.

11:02

But what we could see is that the prepfiler

11:05

did outstanding the well

11:08

in this proficiency test.

11:11

You can see here we have evaluated the extraction yields

11:15

regarding the JETNA proficiency test 54.

11:19

As you can see here,

11:21

the prepfiler chemistry performed very well

11:25

and exceeding the extraction yields

11:27

of other solutions significantly.

11:32

Moving to the next proficiency test, JETNA 55,

11:37

still the prepfiler chemistry performed best.

11:42

But there were other successors

11:44

that were nearly as good.

11:48

Moving to the recent proficiency test, JETNA 66.

11:52

Again, we found out that the prepfiler chemistry

11:55

performed best has the highest yield

11:58

compared to other companies.

12:00

And if you remember, JETNA 67,

12:05

we had more DNA four times the amount

12:10

than with JETNA 66.

12:12

And again, the combination of laboratories

12:16

that use the prepfiler chemistry had by far

12:20

the highest yield in human nuclear DNA.

12:25

So we did an evaluation of 10 years time

12:29

between JETNA 54 and 64.

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So every year we have two proficiency tests.

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And as you can see here,

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the extraction yield is shown at the rise

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from year to year and from proficiency test

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to proficiency test because in previous years,

12:48

the stain commission had decided

12:50

that between the proficiency test,

12:55

we should have a significantly difference

12:58

in the expected DNA concentration.

13:03

And also there was an influence of the carrier.

13:07

From year to year, we changed.

13:08

So we started with FDA cards,

13:12

then we moved on to swaps.

13:16

And in 2022, the stain commission decided

13:20

that no carrier at all should be used.

13:22

So we pipetted the blood into the polypropylene tube

13:27

in the epind off tube and let it dry there to send out

13:30

to find out if the carrier has an influence

13:33

with the extraction chemistry

13:34

because it was clear to the stain commission

13:39

that the extraction yield with other solutions

13:44

except from prepfiler was significantly lower.

13:48

And the stain commission, of course,

13:50

was interested in maximizing the yield,

13:53

not to lose evidential value

13:55

because if an extraction method performs

14:00

not so good in the proficiency test,

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one can also expect that it does not perform well

14:08

in casework scenarios.

14:10

And here we see the median of all the years

14:15

from JETNAP 54 to 61.

14:18

Again, there is a high success rate,

14:22

a very good extraction yield of the prepfiler chemistry

14:27

compared to other chemistry solutions as well.

14:32

So this was a rather preliminary data evaluation.

14:35

Stefan and I, we know each other since 2006

14:40

and we aim to do a more thorough data evaluation soon

14:44

and will present a dot on one occasion or another.

14:51

And there are additional investigations.

14:54

There are questions about sample purity, of course,

14:59

and the downstream genotyping results.

15:03

And the principle of JETNAP is that we are in close contact

15:07

with the participants.

15:08

So we are just preparing a questionnaire,

15:11

how to proceed to ask what substrates, what carriers

15:16

should be used in the next proficiency tests,

15:20

what about the reasonable concentrations that are used.

15:24

And so we will take this into account

15:26

and then set up the next extraction efficiency samples

15:31

in 2024.

15:34

And if you would like to participate, please contact me.

15:38

You can register at jetnap.org,

15:41

or you can write an email to me and my team

15:47

So thank you very much for your attention

15:50

and thank you, of course, to the anonymized donors

15:54

of the samples.

15:55

So this is very important that we get new samples

15:59

from year to year.

16:00

Again, thank you very much.

16:01

- Awesome, thank you so much, Kasten.

16:06

So you already talked a little bit about other substrates

16:11

that you used.

16:13

Why do you finally have chosen swaps

16:17

as the substrate for the extraction efficiency test?

16:21

- So we followed the advice

16:23

of the German Stain Commission to take swaps in 2023.

16:27

And we are open, we will ask the community

16:31

what they would expect in 2024, what substrate?

16:35

Either we stick on with swaps

16:36

or we move to another substrate,

16:39

which is forensically relevant.

16:44

Well, I've worked a lot with the prep file chemistry, of course.

16:49

And of course, I'm happy, the results of the prep file

16:54

are very good.

16:55

I also think that the extracts are very clean.

16:59

You said, yeah, there's a discussion sometimes

17:03

about the yield that an extraction method is providing

17:09

and the quality of the DNA extracts they're providing.

17:13

And I think prep file also has a very good quality.

17:17

Do you think that one of the two would be enough

17:21

to be successful in the get-up trials?

17:24

- Well, until recently, we only focus on the quantitation

17:32

and we provide a certificate of participant or participation.

17:37

We have thought about moving

17:41

to a more sophisticated module

17:43

and take also the quality into account

17:46

made me to offer a general typing part as well

17:50

to find out how the extracted DNA performs.

17:54

It is a bit more complicated than to evaluate, of course,

17:57

than just to compare the quant results.

18:02

In the beginning, the stain commission

18:04

was not in favor of that

18:05

because they thought quantification is the only thing

18:09

that is relevant, but of course,

18:11

it's both quantity and quality.

18:14

Quantity is essential because we are working with casework,

18:18

with stains that sometimes are present only in tiny amounts

18:22

and it must be the aim of any laboratory

18:24

to optimize the extraction process to yield,

18:28

to have a yield as high as possible.

18:31

And then the next step,

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the quality should be also good

18:35

that a general typing approach is easily done

18:38

in the downstream process.

18:40

Yeah, makes sense.

18:42

And yeah, finally, last question from me.

18:46

So, getnap is out for years.

18:51

But finally, there are other proficiency tests.

18:55

Why should people choose the getnap?

18:59

- We have a long-standing experience with that.

19:03

So we work more than 25 years with this getnap scheme

19:07

in close interaction with the forensic community

19:10

of Germany and the neighboring countries.

19:12

So if you're not from these countries,

19:14

it would also make sense to have a look

19:17

if that is a format that can improve the quality

19:20

of your laboratory.

19:21

Traditionally, we have a difference

19:23

to other proficiency tests.

19:24

So we send out three reference samples and four stains

19:28

that are not related in a case scenario so far.

19:32

So we will do a questionnaire to the community

19:34

and ask if they want to change the procedure.

19:37

So we are open to any cooperation with the community,

19:41

of course, and find out what is the best way.

19:43

There's a bias.

19:45

As a provider of the proficiency test,

19:48

you must take care that it is not too easy

19:50

while the participants might aim

19:54

to have an easy proficiency test.

19:56

And so our history is that we do not offer

20:00

a very easy proficiency test.

20:02

We send out samples that are sometimes difficult to analyze.

20:06

Sometimes they contain variants as in real cases.

20:10

- Makes sense.

20:11

Kasten, thank you very much.

20:13

Was great working with you.

20:15

I'm happy to have an next project,

20:18

possibly in the future.

20:19

Thank you very much.

20:22

- Thank you, Chawan, for the opportunity to present

20:24

in the hits.

20:25

Thank you very much.

20:26

(upbeat music)

20:28

- The Applied Biosystems,

20:35

HID Nimbus Presto Q&A System Software

20:38

makes deck setup foolproof.

20:40

It's used for sample purification

20:41

and for automated quant and amp setup.

20:44

And it can normalize your DNA samples

20:46

and calculate dilution schemes.

20:48

It's easy to use and offers a wizard

20:50

that takes you through the process

20:51

with guidance mode and short videos.

20:54

It can calculate reagent needs

20:55

based on your selected input and output,

20:57

chemistries and dilution volumes.

21:00

The software eliminates the need for manual calculations

21:02

so you can just set up your run,

21:04

walk away and have confidence in your results.

21:06

(upbeat music)

21:09

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