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Tests for the presence of blood in or on artworks: this article describes and evaluates alternative forensic procedures that were used to test for the presence of blood in a red inscription inside of the lid of an antique wooden memento box that is attributed to the property of and is inscribed and signed "Frida Kahlo".
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Application and Evaluation of Three Methods to Screen for the Presence of or Use of Blood on Works of Art
-- Daniel Friedman
The owners of this item and the extensive collection of which it is a part, Carlos Noyola (an antiquarian) and Leticia Fernandez (an art conservator), expressed an interest in evaluating the possibility that artist Frida Kahlo used actual blood, perhaps mixed with other paint pigments, to write the inscription or illustration found inside the lid of this box.
Interest in this object derives both from its presence in a large collection of material attributed to Frida Kahlo, in the contents of the box, that included a paper annotated and signed "Property of Diego Rivera", but principally, as it served as a well-preserved 1940's artifact whose inscription referred to human blood.
Our initial hypothesis was that although the Kahlo-inscription refers to her blood (sangre), and although she used the blood image frequently in her paintings, drawings, and in sketches included in many letters and on pages in her diaries, the inscription on this box was probably comprised of a red paint pigment or perhaps a red ink, in any case preserved by having been in a closed container for more than 50 years.
We speculated that had actual blood been used, and considering that the box is approximately 60 years old, the inscription should have been browner in color. However because some artists are believed to have on occasion mixed blood or still other body fluids into paint pigments, we wanted to perform a definitive test for evidence of blood on this object.
With advice from crime scene forensic experts Judy and Don Doje, educators and crime scene forensic suppliers in Ocoee, Florida, we selected several tests that could be performed in the laboratory established by Noyola/Fernandez in the Centro de Investigación Frida Kahlo (CIFK) in San Miguel de Allende, Guanajuato, Mexico.
Three different forensic tests for blood were considered for use in detecting the presence of blood on or in works of art:
The first two of these were applied, tested and documented in procedures described in this article. We adapted and tested three sample preparation methods using the Hemastix® Reagent Strip and one sample preparation method using the Phenolpthalein Forensic Presumptive Blood Test. Because of the negative results of the first two tests, the Luminol® test for blood on surfaces was held in reserve for future use.
Using the Bayer Hemastix® Reagent Strip to Test for the Presence of Blood in or on Works of Art
This test was designed for medical use to screen for minute amounts of blood in human urine and will respond to trace levels of hemoglobin as low as 0.015-0.062 mg/dL of free hemoglobin, with lesser concentrations being detected under some circumstances. The test is equally sensitive to myoglobin as to hemoglobin. Detecting blood at such dilute concentrations is approximately equivalent to detecting 5-20 intact red blood cells per microliter of fluid.
The test procedure for examining the Frida Kahlo wooden box used a sterile swab to collect pigment or other particles from the surface being examined. The sample-collecting swab is used dry (or if necessary dampened with sterile water), touched to the sample area, and then dampened (if a dry swab was used), and touched to the blood test sensing area of the Hemastix® reagent strip.
For the purpose of testing for the presence of blood in or on works of art, such as Frida Kahlo's inscription on the memento box in this study, the sample collection method for use with the Hemastix® reagent strip was tested using three different methods:
Areas of the Frida Kahlo memento box tested and test controls were selected as follows:
Table of Results of Tests for Blood in Frida Kahlo's Signature & Inscription Using the Hemastix® reagent strip
The table below summarizes the results of all three variations using the Bayer Using Hemastix® reagent strips to test for the presence of blood. The test results were consistent among all three sample preparation methods, but the "Dry Swab" sampling method was found to be the least invasive and to produce the least difficulty with an overloaded sample while the "Dilute Method" risks a false negative and is generally not recommended.
False Negative Results using Hemastix® Reagent Strips to Test Artworks
Hemastix® Method #1 (wet swab), #2 (dilute method), and even #3 (dry swab) can all produce a false negative result (indicating that blood is not present when in fact blood is present on the surface), particularly if the sample is over-diluted. Therefore we use the dilute Method #2 only in cases where the sampling swab carries an excessive amount of pigment or debris from the artwork.
False Positive Results using Hemastix® Reagent Strips to Test Artworks
Because the Hemastix® method can produce false positives if other oxidates are present, it was important to perform additional blood presence essays using the most-specific Phenolpthalein presumptive blood test method described below.
Test 2 for the Presence of Blood: Using the Phenolpthalein presumptive blood test to test for blood in or on works of art
The dry, folded sterile filter paper was gently rubbed across the surface area to be sampled. Each sample was then treated with in sequence with a drop of ethanol, phenolphthalein , and then with hydrogen peroxide . The treated test samples were immediately observed for a color change to pink, which would indicate that blood is present. The samples must be examined closely and rapidly to watch for the color change, and the test sample should be diluted as little as possible.
Phenolpthalein Presumptive Blood Test and Test Controls Use in Art
Phenolpthalein Presumptive Blood Test Controls for Testing Works of Art
Non-pigmented surface of the same art work: a sterile filter paper was folded into quarters (for convenience) and was then used to collect a small sample of non-pigmented material from the area to be tested. For this project an area of the interior of the wooden box top was selected which was not coated or painted with any visibly detectable paint or pigment.
Known blood reference: a sterile filter paper used to collect a small sample of material from the reference sample of known human blood which had been previously placed on the surface of a sterile wood spatula and dried. This test should produce a positive result in a test for the presence of blood.
Using Luminol® to test for the presence of blood in works of art
The Luminol Blood Test is considered a highly-sensitive widely-used screening test for blood on surfaces and is used [usually at a crime scene] to test for the presence of blood on surfaces even when blood is not visible to the naked eye under normal lighting. The Luminol mixture is prepared and sprayed or applied by sterile swab onto an area or surface to be screened for blood. The area is viewed in total darkness. Luminol will luminescence in total darkness if it detects the presence of even minute amounts of blood - in other words, the preparation will glow in the dark. Luminol is thus a highly sensitive screen for presence of blood on surfaces where blood may not be obviously visible in normal lighting.
However in ordinary forensic use such as at a crime scene, Luminol is made to be sprayed upon a surface containing dilute or old bloodstains. Obviously one should not spray artwork with this or any other reagent mixture since doing so could damage the work. In the case of Luminol, Don Doje explains that the perborate (or hydrogen peroxide) could damage the artwork, depending on the chemistry of the work's composition.
We found that the Phenolpthalein Forensic Presumptive Blood Test was particularly useful in testing the Frida Kahlo memento box inscription because of the concern that the red pigment collected on our filter paper or cotton swab might itself obscure a positive response indicating the presence of blood. Because this test will cause the presence of blood in a sample to be visible in total darkness as the issue of pigment in the sample when we used the Hemastix® Reagent Strip method is overcome. In follow-up testing we used this test to investigate this possibility further. Those results will be published as an update to this article.
Conclusions From Testing the Frida Kahlo Wooden Box for Evidence of Blood
Daniel Friedman is a forensic microscopist, paint failure analyst, and building and environmental investigator residing in the United States and Mexico. His education, credentials, and experience can be read at InspectAPedia.comdanbio.htm and his CV is available at InspectAPedia.com/DJF-CV.htm .
Footnotes to Forensic Test for Blood on a Frida Kahlo Memento Box
The lacquered, hand painted memento box, a traditional keepsake item popular in Mexico and hand painted on its exterior by artisans in Michoacan, also contained a variety of ephemera and documents that by signature and other details, were almost certainly the property of Frida Kahlo and/or Diego Rivera.
The contents have been cataloged separately. The box and its contents are included in a collection of Frida Kahlo/Diego Rivera artworks, letters, mementos, and other ephemera held by Carlos Noyola and Leticia Fernandez, and available for study by invitation at the Centro de Investigación de Arte Mexicano (CIAM) in San Miguel de Allende, Guanajuato, Mexico.
As shown in the photograph above, the inscription and illustration include the following: "Mi sangre, es hoy, sangre Comunista. Estos recuerdos son sangre, de mi corazon, vertida en la copa de desprecio de Diego que me abandono. Frida Kahlo" accompanied by an illustration of a heart pierced by an arrow and dripping blood. A photograph of the box exterior is provided at the end of this article. The memento box was dated 1946. Frida Kahlo died in 1954.
 Hemastix® is produced and marketed by Bayer Health Care, Elkhart IN, and distributed for forensic use by Doje's Forensic Supplies in Ocoee, FL. The swab sampling methods tested here use such a minute volume of fluid that the sensitivity of the test may actually be greater than that promised by the manufacturer.
 Phenolpthalein (C20H14O4 or "HIn") is colorless in acidic solutions and pink in basic solutions. The phenolphthalein used in the presumptive blood test is reduced by two electrons and is pre-dissolved in an alkaline solution. The reduced version of phenolphthalein in the Doje’s test kit is achieved using a zinc powder.
Reduction reduces the normally intense pink color of the cationic form of phenolphthalein to the faint yellow color used in the Kastle-Meyer test. The reduced phenolphthalein is oxidized back to its colored form when the hemoglobin (in human or animal blood) reacts with the hydrogen peroxide to form a hydroxyl radical that in turn oxidizes the reduced phenolphthalein back to its colored form.
 The Luminol® test is distributed by Doje's Forensic Supplies in Ocoee, FL and by other crime scene forensic material suppliers as well. The Doje’s Luminol test kit is a patented chemical that is shipped as dry powder and prepared by mixing with sterile water and hydrogen peroxide.
 The chemistry of the Hemastix® test is based on the peroxidase-like activity of hemoglobin that catalyzes the reaction of diisopropylbenzene dihydroperoxide and 3,3',5,5'-tetramethylbenzidine.
 "Hemastix® Reagent Strips for Urinalysis, Test for Blood in Urine", Bayer Health Care, Elkhart IN, USA, AN07016C, Rev. 5/06 USA, product literature included in the product package.
 The Dilution Method: A surface swab which picks up too much pigment or surface debris from the artwork being examined could damage the work, and it may result in an excessively dark coloration on the Hemastix® reagent strip, possibly obscuring the color change of the reagent. In our tests of the Hemastix® Damp Swab Method the dampened swab in fact did collect an excessive amount of pigment from the inscription test area (Photo 3).
Using the "Dilution method" of sample preparation (Photo 4), after stirring the sterile water with the sample swab, a Hemastix® test strip was dipped momentarily into container of water, removed by dragging the edge of the test strip across the lip of the container (to remove excess liquid). Then the test strip was compared with the color comparison chart provided by the manufacturer.
Photograph 4 ("Dilution Method") shows both the original Hemastix® reagent strip (red blood visible on the overloaded reagent strip labeled "3" collected by the wet swab method and the strong green Hemastix® reagent strip (after dipping in the container of water+swab), indicating the pronounced presence of blood in this sample.
 The Dry Swab Method: Because the dampened swab collected excessive pigment from the inscription test area, we repeated the Hemastix® reagent test method for presence of blood using instead, a dry sterile cotton swab and rubbing the test surface very gently to remove only a minimal amount of material.
Other than using a dry swab to collect the surface sample,followed by slightly moistening the swab with sterile water, the test procedures and test controls were identical to those used in Hemastix® test #1 (dampened swab method). Three dry swab sample method reagent strip results are shown at photograph 5.
 A blood sample was volunteered and donated by Diego Noyola; a drop of his blood was applied to the surface of a sterile wooden spatula and then dried to simulate the material of the interior of the lid of the Kahlo wooden memento box being studied.
 The test result for this area provided a weak positive indication for blood. Because the pigmented area provided an essentially negative result, it was posed that the un-pigmented area of the box lid or the entire lid surface may contain debris including oxidates. To address the possibility of a "false positive" detection of the presence of blood, this area was tested further using the Phenolpthalein presumptive blood test which is described below
 Paraphrasing Winkled, "Kastle-Meyer Test", which refers to Cox, M. (1991). "A Study of the Sensitivity and Specificity of Four Presumptive Tests for Blood". J. Foren. Sci. 36(5).
 The test kit was produced and distributed by Doje's Forensic Supplies in Ocoee, FL.
 The phenolphthalein presumptive blood test kit (Doje's Cat.# 302) contains three bottles of reagents: Phenolpthalein, Hydrogen peroxide, and alcohol, plus filter paper and sterile swabs (for sample collection). The kit also includes a reference blood sample (cloth square stapled to a card).
 Phenolpthalein step: First waste a few drops of phenolphthalein aside in order to clean any old oxidized residue from the dropper and to avoid contaminating the test. Then add one drop of phenolphthalein reagent to the sampled area on the [swab or] filter paper.
IF a PINK stain appears within a few seconds of applying the phenolphthalein and before the hydrogen peroxide has been applied, THEN this is a FALSE POSITIVE indication of presence of blood and it means that a contaminant is present and the test for blood using this method is invalid - repeat the procedure with a new sample.
 Hydrogen Peroxide step: IF NO REACTION occurred at the phenolphthalein step, THEN add one drop of Hydrogen Peroxide to the sample. IF a PINK indication is observed within a few seconds, this is a CORRECT POSITIVE indication of presence of blood. The sample has detected blood. IF NO REACTION occurs at this step this is a CORRECT NEGATIVE indication of the presence of blood. The sample has not detected blood.
 Judy Doje points out that the phenolphthalein test will eventually turn pink in all cases since this is a catalytic reaction. That is why the reaction has to be evaluated immediately on application of the reagents in order to evaluate it as an indicator for the presence of blood.
 Luminol permits the visualization of bloodstains where blood has been washed or wiped away and has been used to visualize previously invisible bloody footwear impressions on crime scenes.
Normally the completely-mixed Luminol and perborate solution has a short shelf life and is intended to be used promptly. However the Luminol/water mixture (omitting the perborate) should last several weeks-months, though it will eventually oxidize out. This modified mixture may have application if multiple works of art are to be examined for blood.
 This test is considered the most-specific test for blood and thus was able to rule in or out possible false positives that can occur with the Hemastix® procedures.
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