research though literature on presumptive blood tests
I post here a bibliography research I made for PMF, so that is available to all JREF readers.
A few conclusion can be said in advance about specificity.
TMB is not more specific. It is just less sensitive to roughly the same array of substances reacting to luminol, in principle reacts to the all susbstances reacting with luminol (depending on the kind of false positive substances and the kind of support, in a variable degree). Definitely TMB is not "more specific" for blood and never considered as such.
In no case – according to literature - TMB is considered a mean suitable for cross testing luminol positive results.
More specific tests do exist, but are all less sensitive. Phenophtalein ("KM test") is actually a chemichal presumptive test said to be more specific than Luminol/TMB by some literature – by contrast, some literature considers it as highly sensitive and unspecific.
There is a highly specific haematic test (an "enzymatic" test for haemoglobin) but lacks sensitiveness in comparison to all chemical presumptive tests so that it can’t get close to their scale of operability.
Moreover: luminol is a direct test, meanis it is made on the surface to be examined. TMB is in most cases indirect: a sample must be collected from a stain and put in contact with a filter and a solution. This additional step diminishes drastically its sensitiveness, and can be determinant in the actual dilution of the sample. If the stain was made visible by means of chemioluminescent reagent as luminol, which employs a layer of liquid solution, the original blood/substance concentration in fact can be reduced and the solvent can literally “dilute out blood” (some of the material report this expression).
Now, the actual available literature about luminol and presumptive tests says much more. Conclusions and implications are many and interesting.
I don't want to encourage here people to post self-made researches, that was defined "
ab extracta" (from abstracts) by somebody. I tried to link here material readable for free. Many important studies on the topic seem to be related to two names, Creamer and Quickenden. Articles Webb and Gross seem available only in abstracts. Tobe, Watson, et al., Kent et al., seem to contradict some conclusions of Creamer.
1.
This is a comparative study on blood tests.
Luminol interference on Analysis
Luminol has “far the greatest sensitiveness” :
“Webb et al. [22] conducted a study where the luminol test was compared to four other forensic blood tests. These tests where phenolphthalein, LMG, Hemastix (Bayer) and a forensic light source. The luminol test used was found to have by far the greatest
sensitivity. Under laboratorial conditions CL was detected from luminol treated stains of the used hemoglobin solution (corresponding to blood) diluted up to 5•106 times. A comparably high sensitivity of the luminol test has been reported in other studies [22].
However the sensitivity is probably not as great under the conditions found at a crime scene and here, depending on several factors, perhaps one may “only” see blood diluted to about 1:10000 [14]”.
And it can produce false positives as well as false negatives:
Often these tests are based on the ability of hemoglobin to catalyse the oxidation of a chromogenic compound which produces a colour change [14]. Today the forensic use of about twenty such presumptive blood tests has been described [14]. They have all in common that they produce false positive and negative results to some degree, and therefore they are only presumptive [14].
Common substances that react to luminol are actually in a small number
In 2003 Creamer et al. [5] published a comprehensive study where the blood mimicking behaviour of 250 different substrates and compounds, common at crime scenes, had been measured on. In these test only 9 kinds of substrates or compounds were reported to give strong enough CL to be easy mistaken for blood.
These were preparations of turnip, parsnip, horseradish, bleaches (hypochlorite), copper metal, enamel paint, certain spray paints, furniture polish and interior fabrics in motor vehicles. In a separate study Quickenden et al.
[21] examined the interference with the luminol test for blood in motor vehicles. Also in motor vehicles only a few materials gave strong CL, without the presence of blood or hemoglobin solution.
[reading trhough articles you will see how the only kind of substances that can remain positive for days are metal salts, especially copper based composts; all other substances loose their capability to give positive result editor's note]
Substances are indeed recognizable by the color shade in luminol tests
(…) there are informative tables displaying intensities and wavelength shifts in
the CL [chemiluminescence, editor’s note] produced by the diverse materials.
This particular study relies on previous findings, cited, by which luminol doesn’t affect other presumptive tests (I note that other sources available on the internet apparently disagree with the latter finding).
Gross et al. [11] showedthat luminol treatment of bloodstains do not have a remarkable adverse effect on the use of neither the phenolphtalin (Kastle-Meyer) nor the tetramethylbenzidine tests. Other publications describes in a like manner that
the luminol test do not interfere with the subsequent use of other particular presumptive
blood tests [14].
The use of the luminol test has been found to have an strong adverse effect on subsequent forensic typing of serum protein markers [14].
2.
This is an abstract, probably of the cited article by Webb
“The luminol test was determined to be the most sensitive of the techniques, while Hemastix is a suitable alternative when the luminol test is not appropriate”: (only the abstract is readable)
http://www.ncbi.nlm.nih.gov/pubmed/16645959
3.
This study discusses effects of luminol on different materials, how this may influence the results in other tests and whether stains cleaned with soap or bleach are still visible though luminol.
http://projects.nfstc.org/workshops...ffect of Luminol on Presumptive Tests and.pdf
Among the conclusions, two points are clear: blood stains can still be visible even after a cleanup, and TMB can give false negatives (in accord with article cited in 1. ).
“ Prior to luminol testing, both PT and TMB performed well in detecting the blood on all surfaces which had not been washed.
Both tests gave positive results 7/7 times. Similar results were obtained on the surfaces which had been washed with either water or soap and water. (…) “
“ For those same surfaces tested with TMB, 3/7 gave strong positive results; one weak positive (tile) and three negative results (carpet, sheetrock-V, and sheetrock-H). The results for the PT test after the luminol treatment, both while the surface was still wet from the luminol treatment and after being allowed to dry, were similar to the results obtained before the luminol treatment. The only surface which gave different results after luminol treatment was the tile which was washed with bleach. Prior to luminol testing, the PT test on tile gave a weak positive result, while after luminol treatment the PT results were negative. Four of the surfaces which gave positive results with the TMB test before luminol treatment gave negative results after treatment (both wet and dry), and one of the surfaces which gave positive results before luminol treatment and after
luminol treatment (wet) gave negative results once the surface had dried.”
In this article there is also a table with lists of materials showing some false negatives ( - ) yielded by TMB on stains of blood at full concentration subsequently cleaned, under different conditions :
…
Tile PT TMB
Cleaned w/ soap and H2O PT: + + + TMB: + + +
Cleaned w/ 10% bleach PT: + - - TMB: w - -
….
Sheetrock
Cleaned w/ soap and H2O PT: + + + TMB: + - -
Cleaned w/ 10% bleach PT: - - - TMB: - - -
...
4.
This article is about interpretation of luminescence and the potential of specificity of luminol test by observing the light spectrum. It is straightforward to distinguish blood from bleach if you have a spectrometer (about naked eye I don't know what literature says). This is a study apparently on just two substances. Blood and bleach give different colour spectrums (it is in accord with study 1. ):
http://www.forensictv.net/Downloads...specificty_of_the_forensic_test_for_blood.pdf
"The spectra in Fig. 2 show that the spectral maxima for sodium hypochlorite and for haemoglobin are clearly different and would readily be distinguishable using relatively straightforward spectroscopic equipment. (…)
The spectral peak for the concentrated (150 g/L) haemoglobin is at 455 + - 2 nm which is red-shifted by ca. 25 nm from the sodium hypochlorite peak at 430 + - 3 nm."
5.
An abstract of the cited study (Gross): an article on degradation of DNA after TMB/luminol tests: “Effect of presumptive tests reagents on human blood confirmatory tests and DNA analysis using real time polymerase chain reaction” – only the abstract is readable:
http://www.ncbi.nlm.nih.gov/pubmed/20643520
6.
Evaluation of six presumptive tests for blood (pdf) Another comparative study. This study talks about “false positives” and other substances reacting with blood tests, a comparison (specificity, sensitiveness) of the common different presumptive tests.
http://projects.nfstc.org/workshops...ation of Six Presumptive Tests for Blood,.pdf
this study – in accord with the others - determines that luminol is the most sensitive:
" The luminol reagent reacted instantly, with both the 1:10,000 and 1:100,000 dilution factors producing a blue luminescence.
The luminescence lasted for close to a minute. [p. 104]"
"The Hemastixs reagent strips reacted with the 1:100,000 dilution by first causing a color reaction with the filter paper. At 1 min, one of the samples showed a color change on the filter paper of a green color. The rest of the samples showed this same reaction at between 1 min and 45 sec and 2 min. At 3 min and 45 sec, 17 of the reagent strips were a very light shade of green, (..) The remaining four strips registered a negative result at 4 min."
[editor’s note: positive reaction is a blue color; a 1/ 100.000 dilution equates to 45/50 erytrocytes per mm3 of liquid ; negative controls with TMB also give a green reaction after several minutes, so late or faint green is considered negative]
Another finding was that bleach doesn’t react with luminol after 18h
“ Contrary to the literature findings, this study found that luminol only reacted with blood and the metal salts. Bleach gave no reaction, but this could be because the bleach solution was only 5% concentration, and that it was not tested right away but first allowed to dry for at least 18 h “ (..)
bleach diminishes with time also its interference, aka inhibition of blood own luminescence:
“ Kent et al. (20) noted that when bleach-treated blood is left for several days, the interference by bleach is diminished. “ (..)
Also, several of the substances – like potato – do not react with luminol any more when dry, contrarily to blood:
“This could once again be due to the substances’ drying time before testing” (..) .
Interesting: according to this study, luminol is actually MORE specific than TMB (Hemastix)
“ (..) Based on this, the best overall presumptive blood test in this study was luminol. It had the greatest sensitivity and specificity. It did not destroy the DNA, and it could be reapplied. Its only drawback is that it must be used in near or complete darkness. Leuchomalachite green was found to be as specific to blood as luminol, but its sensitivity was 10 times less, and it destroyed the DNA. Phenolphthalein had equal sensitivity to most of the other tests, but was extremely unspecific, and the amount of recoverable DNA is reduced when this test is used. HemastixTM [TMB] were easy to transport and use, were sensitive, but not very specific although specificity could be increased if the strips were looked at rather than the reaction on the stain.”
[I note that some other studies instead seem to consider phenolphtalin (“Kastle-Meyer test”) as more specific than TMB, editor’s note]
7.
This .ppt (from centralia.edu/academics ) is a classroom text
CENTRALIA
8.
This is another classroom .ppt text: note that these lessons maintain that luminol affects subsequent tests, this one specifies the warning that luminol can "dilute out blood” and therfore affect other subsequent tests
here:
MARSHALL
or here:
http://www.science.marshall.edu/murraye/2008 Forensics Lectures/Crime Scene Forensic DNA.ppt
9.
The abstract of another article highlights the superior sensitiveness of luminol test :
“A sensitive method for determination of serum hemoglobin based on iso-luminol chemiluminescence”
T. Olsson , a, K. Bergströma and A. Thorea
aDepartment of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, S-141 86 HuddingeSweden
Received 16 November 1981. Available online 20 January 2003. The article is old and searchable on http://www.sciencedirect.com/
“ Abstract:
A simple and rapid method for determination of serum hemoglobin is described. Hemoglobin may be determined in serum within the range of 0.02–400 mg/1 by the sensitive chemiluminescent iso-luminol reaction. The iso-luminol assay was considerably more sensitive than the conventional colorimetric procedure based on tetramethylbenzidine. Precision and accuracy were higher with the iso-luminol assay especially at low levels of hemoglobin. The correlation between the luminescent and colorimetric method was linear but the colorimetric determinations resulted in higher concentrations of hemoglobin. This discrepancy was probably caused by non-heme serum iron which interfered more strongly with the colorimetric method. “
10.
More articles emphasize the greater sensitiveness of luminol. In this research luminol was found to be about 5 times more sensitive than TMB (Hemastix). But the sensitiveness of TMB decreases considerably if it is used with the “indirect” method, though paper/cotton sample:
http://lem.ch.unito.it/didattica/infochimica/2006_Luminolo/determinazionesangue.html
“ Based on the results presented here, the luminol test is clearly the most sensitive blood detection technique commonly used by forensic investigators. The Hemastix® test was the next most sensitive, followed by the KM and LMG techniques. The Polilight® was by far the least sensitive technique, being 50 000 times less sensitive than the luminol test and 10 times less sensitive that the next least sensitive technique investigated here (LMG). Another interesting finding was that the sensitivity of the KM, LMG and Hemastix® tests decreased considerably when applied to filter paper or cotton swabs of bloodstains. While the amount of blood transferred from a stain to a swab may vary considerably, depending on the investigator, this result clearly shows that it is favourable to test a bloodstain directly. The sensitivity of the luminol test was found to be 1:5 000 000 for both the bloodstained cloth and haemoglobin solutions. This was consistent with previously reported literature values [14, 22]. No literature results were found regarding the sensitivity of either the Hemastix® or Polilight® tests for blood; however, previous literature on the active reagent of the Hemastix® test, TMB, determined its sensitivity to be 1:1 000 000 for diluted haemoglobin solutions “