EvidenceProf Blog

Editor: Colin Miller
Univ. of South Carolina School of Law

Friday, January 8, 2016

An Introductory Post on the EDTA Testing Done in the Steven Avery Case

Several readers have asked me questions about the ethylenediaminetetraacetic acid (EDTA) evidence used in the Steven Avery/"Making a Murderer" case. This is an introductory post, which will focus upon the use of this evidence at Avery's trial.

This all comes from the Brief of Plaintiff-Respondent (the State of Wisconsin) in State v. Avery, 2010 WL 4736754 (Wis.App. II Dist. 2010). In 2005, Teresa Halbach was murdered. Apparently, blood that matched Steven Avery's DNA profile was found in the cargo and ignition areas of Halbach's vehicle. "Avery's defense at trial was 'that police officers who had access to a vial containing Steven Avery's blood, which was located in the clerk of court's office, planted Steven's blood in Ms. Halbach's car...."

According to the State,

The coup de grâce to the blood-planting theory was delivered by Dr. Marc LeBeau, who is the unit chief of the Chemistry Unit at the Federal  Bureau of Investigation Laboratory in Quantico, Virginia (321:73). Dr. LeBeau testified that blood collection tubes typically contain a preservative or anticoagulant agent (321:90-91). The type of agent is indicated by the color of the tube's stopper (321:91). Purple-stoppered blood collection tubes use ethylenediaminetetraacetic acid (EDTA) as the anticoagulant agent (id.). Avery's blood was in a purple-stoppered tube (321:94).

Dr. LeBeau testified that the FBI laboratory tested a number of items of evidence for the presence of EDTA, including swabs taken from the bloodstains in Ms. Halbach's RAV4, control swabs taken from areas near the bloodstains, and Avery's blood from the purple-stoppered tube (321:94-99, 103, 114). The testing protocol used by the FBI was able to detect the presence of EDTA in a sample as small as one microliter of EDTA preserved blood, a minute amount equivalent to about one-fiftieth of a drop (321:129).

According to Dr. LeBeau, if the bloodstain swabs from Ms. Halbach's vehicle tested positive for EDTA and the control swabs tested negative, that would be an indication that the blood came from a purple-stoppered tube and had been planted (321:127). On the other hand, if EDTA were not found on the bloodstain swabs, that would suggest that the blood came from active bleeding and not from an EDTA-preserved tube (id.).

Dr. LeBeau testified that the FBI Laboratory was unable to identify any presence of EDTA in the bloodstain swabs or the control swabs from Ms. Halbach's vehicle (321:133-34). The blood from the tube containing Avery's blood, in contrast, contained “significant amounts of  EDTA in it” (321:134). Dr. LeBeau testified, to a reasonable degree of scientific certainty, “that the bloodstains that were collected from the RAV4 could not have come from the EDTA tube” (321:135).

So, that's the State's position. (1) Purple-stoppered blood collection tubes use EDTA to preserved blood; (2) Avery's blood sample was in a purple-stopped blood collection tube; (3) Avery's blood sample had "significant amounts of EDTA in it;" and (4) the FBI Laboratory was unable to identify any presence of EDTA in the bloodstain swabs or the control swabs from Ms. Halbach's vehicle. Therefore, to a reasonable degree of scientific certainty, “the bloodstains that were collected from the RAV4 could not have come from the EDTA tube."

In response,

the defense called Janine Arvizu, an independent contractor who works as a laboratory quality auditor (324:5-6). Ms. Arvizu agreed that when the FBI's testing protocol produces a positive result, that is a valid indication that there is EDTA in the sample (324:23). She also testified that if the result were negative, she could not tell whether that meant that there was no EDTA or that the level of EDTA was below the testing method's detection limit (324:23-24). In Ms. Arvizu's opinion, it was “quite plausible” that the bloodstains swabbed from the RAV4 contained EDTA, “but the lab simply was not able to detect it” (324:59). However, Ms. Arvizu did not testify that EDTA was present in the swabs. Nor did she explain why, if Avery's blood vial was the source of the bloodstains in the vehicle, the EDTA levels in those bloodstains would have been below the FBI's detection limit given the FBI's finding that the blood in the vial contained significant amounts of EDTA (324:5-104).

So, that's the defense position. A "positive" FBI test is a valid indication that there is EDTA in a sample, but a "negative" FBI test does not necessarily mean that there is no EDTA in the sample because the level of EDTA could simply be "below the testing method's detection limit."

Those are the basic facts, at least as presented by the State of Wisconsin. This leaves the question of the reliability and EDTA testing. In future posts, I will address how EDTA testing has been treated by the courts, the FBI, etc.



| Permalink


From what I gather that is why they stopped using this testing method all together due to its unreliability.

Posted by: NavyMom | Jan 8, 2016 7:54:12 AM

And the lab confirmed that they don't puncture the caps of sample vials. The vial has a separate top under the purple cap that is permeable by needles, as with IV bags and port-o-caths. Whoever handled the sample didn't know anything about lab viles.

Posted by: Megan Pawlak | Jan 8, 2016 9:53:01 AM

Very happy to see Colin sinking his teeth into this

Posted by: LG | Jan 8, 2016 10:06:33 AM

I have to take back my assertion. After some looking around, the tube from 1985 had a conventional stopper and I can't find information. Yet on how blood was extracted from such a vial. It did not have the permeable top as modern vials do!

Posted by: Megan Pawlak | Jan 8, 2016 10:45:09 AM

I'm no expert but I did work in hospital and research labs in England in the 1980s and 90s and did some blood taking. Thoughts: 1. You don't make a hole in a vacutainer stopper when you take blood - you only put the stopper (conventional or hemoguard) onto the tube after you've filled it; 2. Agree with Megan P, that's definitely a conventional stopper not a hemoguard; 4. No lab tech in their right mind would try shoving a metal needle throught a conventional stopper, that's three kinds of risky - you'd (put your gloves on and) remove the stopper and withdraw your sample for testing with a long plastic pipette dropper.

Posted by: WLJ | Jan 8, 2016 12:36:52 PM

3. (!) in any case, putting a hypodermic needle through a hemoguard doesn't leave a big hole that leaks blood, they bit you puncture is rubber, not plastic

Posted by: WLJ | Jan 8, 2016 12:59:05 PM

Error - apologies all for multiple commens - meant to say you don't make hole in vacutainer *cap* when taking blood. You obvs do make hole in rubber stopper, but as per 3. it's self-sealing

Posted by: WLJ | Jan 8, 2016 1:50:57 PM

Here's my big question. While the blood in the vial had "significant amounts of EDTA in it" it was in a sealed tube, inside a styrofoam container, inside a box. The blood found in the car was presumably exposed to air, sunlight and increases/decreases in temperature. Is it possible the samples taken from the car may not show detectable amounts of EDTA because of evaporation or burn off of the EDTA?

Posted by: stellarangel | Jan 8, 2016 2:48:38 PM

If my recollection of the show is correct, the FBI had stopped doing the EDTA test about 10 years prior to the Avery trial. However, when the prosecution asked, they were able to pull together procedures/protocols/equipment/methods etc in a matter of weeks in order to run this test. There was a motion regarding the admissibility of this seemingly off-the-cuff test, and the judge ruled it admissible. Is this how scientific testing in a court of law normally works? Because it seems wack to me. (As does the constant refreshing of this site. I've typed this comment 3 times and I hope it makes it through this time!)

Posted by: Beth | Jan 8, 2016 8:07:32 PM

Can someone please answer stellarangel's question? Will EDTA oxidize or degrade being exposed to heat, air, humidity etc????

Posted by: Jason | Jan 8, 2016 11:14:32 PM

Thanks for all of the responses. As I noted, this is just a preliminary post, and I will get into the specifics in later posts. I am still very much getting up to speed on the subject because, as some of you have noted, this isn't something that the FBI has done very often.

Posted by: Colin Miller | Jan 9, 2016 3:54:15 AM

Hi Colin. While watching Making a Murderer I couldn't help but wonder why the defense didn't ask for a Frye hearing regarding the EDTA evidence? Hope to hear your take on this in an upcoming blog. Especially if the test had to be reformatted just for this case.

Posted by: Courtney | Jan 9, 2016 9:43:37 AM

This article states the The defense expert did not explain why the amount of EDTA would be below detectable levels in the blood sample tested and yet at higher levels in the vial it was compared against. That seems fairly obvious to me. The blood in the car had been exposed to oxygen and heat for a substantial period of time, whereas the blood it had been tested against, the blood in the vial, had been stored in a glass vial at specific temps to preserve it. It makes no sense that blood, once taken out of the tube and smeared on car upholstery, would have anywhere near the same amount of EDTA that it would when stored properly.

Posted by: Claudia Miles | Jan 9, 2016 12:19:03 PM

What caused the puncture mark in the top of the tube?

Posted by: Laura Hurst | Jan 9, 2016 3:16:06 PM

Colin, this is a very interesting blog post discussing the problems with the EDTA testing. It's definitely worth a read.


Posted by: Tracie Strunsee | Jan 9, 2016 7:28:40 PM

As a phlebotomist of 11 years, testing for EDTA was the first thing that came to mind. im wondering also how the blood smears were tested as well as the blood from the vial? Additionally, if a tube is overfilled during collection it can leak a bit as well as leaving a needle hole but I'd think after all those years, any blood on the top of the tube would fall off after it dries. Since EDTA binds to calcium and metals, would the amount that was left on the vehicle if it came from the vial be reduced when it's been out in the elements??

Posted by: Laura | Jan 10, 2016 2:03:58 PM

Tracie, thank you for adding the link. It confirmed what I was thinking.

Posted by: Laura | Jan 10, 2016 2:34:36 PM

Just wanted to give my $0.02 as a chemist on the ETDA evaporation theory. I just looked it up quickly and EDTA has a (predicted) boiling point of 614 C. This means it is highly unlikely any would evaporate even if exposed for a long period of time.


Posted by: Brandon | Jan 10, 2016 9:50:43 PM

How long does it take for the ETDA to mix with the blood? - Is it pretty quick/does it need shaking/would there be time to remove some blood from the vial before it was fully mixed?

Posted by: Cupcake | Jan 11, 2016 4:26:04 AM

Hi Collin. Thanks for writing about the EDTA. I am also curious as to the validity of the test and am curious to see what you discover in your further research.

Posted by: Emily | Jan 11, 2016 6:52:03 AM

I was curious what the source of the DNA was on the key? If blood, was this tested for EDTA? Would go a long way to supporting the framing claims.

Posted by: Mike B | Jan 11, 2016 10:14:38 AM

Hi Colin,

I do not necessarily think that EDTA would degrade much in the sun. It is really just a salt. However, in order to remove the dried blood from the swab they would need to dilute it into buffer. I am not sure how this testing was done, but there would be no way that the concentration of dissolved blood would be the same as in the vial after dilution into buffer. If the limit of detection is at one drop of concentrated blood, it is highly likely that this would be too weak to test in a diluted sample. I believe that this was a cleverly designed strategy to deceive the jury into thinking that they were able to develop an accurate test, when they did not. I would certainly think that this work should be reviewed by an independent lab.

Posted by: San | Jan 11, 2016 11:12:30 AM

Does anyone know what specific test the FBI used to test for EDTA?

Posted by: Jim | Jan 11, 2016 1:16:01 PM

What about testing the blood found on the outside of the vial for EDTA.

Posted by: todd | Jan 12, 2016 8:35:45 PM

Has anyone explained WHY the vial had the puncture mark in the top?
Why would someone do that? Why not just take the cap off then put it back on?
That does not make sense to me.

Posted by: nique | Jan 13, 2016 8:02:44 AM

Post a comment