The thing about the TMB tests is this. I have posted cites sfrom forensic manuals where they test a Luminol positive with another presumptive test. If that is negative, the assumption is it is not blood and nothing further is done. The reason for this is because of Luminols high sensitivity to a lot of things and they want to make sure it is blood it is reacting to. In Stefi's case, instead of making the assumption it was not blood, she made the assumption it was. This makes no sense to me. The SOP is do a confirmatory test after two positive presumptive tests. I can't imagine an assumption of blood after the negative TMB test without even attempting a confirmatory test. My guess is that she did test them and that they were negative, yet again.
As I've written quite a few times in the recent past, there is a primary reason why the proper 2-step TMB test has much higher specificity for blood than Luminol (i.e. it returns far fewer false positives than Luminol).
The reason is this: TMB and Luminol both react to oxidants that may be present in the substance being tested. TMB reacts by changing colour, and Luminol reacts by luminescing for a short time.
However, blood is not an oxidant.
That's why both tests - Luminol and TMB - also require the presence of peroxide (usually hydrogen peroxide, H2O2). In the presence of the heme ions within red blood cells, the peroxide is subjected to a peroxidase reaction which releases an oxygen ion that then oxidises the heme ion, and produces water (H2O) as a byproduct.
This oxidised heme ion then can act as the oxidant in the reaction with either TMB or Luminol.
So the first important thing is this:
neither Luminol or TMB will react to blood without the additional presence of peroxide
Still with me?! (Hope so!)
Now, the reason why this above point is so important is that both Luminol and TMB will react to ANY oxidant - it's only if that oxidant is the oxidised heme ion that's the product of the peroxidase process on the peroxide that it will react to the presence of blood.
And this leads to important point number two:
Luminol testing applies the Luminol and the peroxide at the same time - it's therefore impossible to tell whether any luminescence reaction is the result of oxidised heme ions (via the interaction with the peroxide) or whether it's the result of direct oxidation by another oxidant.
In other words, if your Luminol glows, you're unable to distinguish whether you're looking at blood or an oxidant (examples of oxidants include many acids, bleach, and a variety of common household cleaning products).
But in the two-step TMB test, you're able to screen for oxidants because the application of the TMB alone forms part one of the test. Remember that blood does not oxidise TMB unless peroxide is also present. Therefore, if the application of TMB alone produces a colour change, this clearly shows that an oxidant (i.e. NOT BLOOD) is present.
Now, if the TMB does not change colour in the first step (i.e. the application of the TMB alone), this is good news if you're looking for blood. It's good news because what you do then is apply part two of the test: you apply peroxide on top of the TMB. If the TMB now changes colour, it tells you that you very likely have blood, since the colour change is almost certain to have occurred as a result of the peroxide oxidating the heme ion, which in turn oxidised the TMB.
So the final important point is this:
the two-step TMB test gives the ability to screen out all latent oxidants (in a way that the Luminol test cannot do), and if it shows negative to step one then positive to step two, then the substance under test almost certainly contains blood.
The scientific definition of the above point is that the two-stepTMB test has extremely high specificity for blood. The Luminol test has low specificity for blood - owing to its inability to distinguish between blood and any oxidants present. And this is EXACTLY why forensic scientists follow up a positive Luminol test with a 2-step TMB test: positive to Luminol tells them that blood might be present (but equally a whole variety of different oxidants might be present instead), and a 2-step TMB test then tells them with far higher confidence whether or not blood is present.
So if a sample tests positive for Luminol, it should then be subjected to the 2-step TMB test. If this 2-step test gives a positive in the first step, then it's likely that the substance causing the Luminol positive was not blood, but was instead an oxidant of some sort. If the first step gives a negative, but the second step gives a positive, then it's very likely that the substance being tested is blood. The tester should then move on to a confirmatory test for blood, and potential DNA analysis if possible.
