Test for the Presence of Blood: Testing a Mexican Memento Box for the Use of Blood in an Inscription Signed by Frida Kahlo
     


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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

Tests were performed at the Centro de Investigación Frida Kahlo to determine whether or not the inscription on a traditional Mexican lacquer painted memento box was written using blood or a mixture of blood and other materials. The author presents the results of those tests and makes recommendations for the use of these procedures for screening other works of art for the possible presence of human or other animal blood. The tests described in this article address possible use of a common practice among artists, mixing body fluids with paint or pigments in creating art works. These tests do not address the age nor authenticity of the object under study. [1]

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:

  1. The Hemastix® Reagent Strip[2],
  2. The Phenolpthalein Forensic Presumptive Blood Test[3] (known as the Kastle-Meyer test), and the
  3. Luminol® test for blood on surfaces[4].

    Also see UV LIGHT BLACK LIGHT USES.

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

Using Hemastix® reagent strips to test for the presence of blood is considered the most sensitive test available.

Hemastix® is a plastic strip to which is affixed a reagent area (Photo 2) that tests for occult blood in urine.[5]The test strip is normally immersed briefly in a urine sample.

The reagent surface area is then observed for any color change (which occurs in less than 30 seconds), and then the color of the reagent surface is compared with a color comparison chart provided by the Hemastix® manufacturer. The resulting color visible on the reagent area ranges from orange through green. Very high levels of blood may cause the color development to continue to blue.

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.[6]

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:

Dampened swab method for surface pigment collection

A swab dampened with sterile water was used to collect pigment and particles from the test surfaces.

In each of the damp swab tests, the dampened swab was touched first to the surface to be tested (Photos 6 & 7 below) and second, it was immediately touched to the rectangular surface area of the Hemastix® test strip where the reagent is contained. The reagent test strip color was then read against the color chart (Photo 2).

It was observed that this method collected an excessive amount of pigment which may have occluded the test result (heavily pigmented swab at right in Photo 3), leading to the decision to continue Hemastix® reagent strip testing using the next two sample preparation methods described below.


Dilution method for surface pigment collection: [7]

A dampened swab was used to collect pigment and particles from the test surface, then it was immersed in 15ml of sterile water (swab in Photo 4). The Hemastix® reagent strips were then dipped briefly into this liquid and read against the color chart.

This method was tried because the damp swab collected excess pigment that may have interfered with a proper reading of the reaction color of the reagent strip. Doje's warns that this method could produce a false negative result so it was used only in the case of excess pigment on an already-collected sample swab.

Dry swab method:[8] Because the damp swab sometimes collected an excessive amount of pigment that may have occluded the test result, the procedure was repeated starting with collection of pigment from the Frida Kahlo box inscription test area using a dry sterile swab. The dry swab was then moistened with a single drop of sterile water and the moistened swab was touched to the directly to the reagent strip and read against the color chart (Photo 2 above).



In the Dry Swab Method (Photo 5), reagent strip #1 was a control that was touched with a swab moistened only with sterile water.

Strip #2 was touched with a swab that had been used dry to sample the red-pigmented inscription area on the box, then moistened slightly with sterile water and touched to the reagent area.

Strip #3 was touched with a swab that had been used dry to sample our known blood source on a clean wood surface, then moistened slightly with sterile water, and touched to the reagent area. Strips #1 and #2 show no evidence of blood. The red visible on swab #2 is pigment from Kahlo’s box inscription. Reagent strip #3 turned green, confirming the detection of blood from the known-source control.

Areas of the Frida Kahlo memento box tested and test controls were selected as follows:

 

Pigmented box inscription: a small section of the Kahlo box top interior red-pigmented inscription was selected for sampling. We selected a heavily-pigmented area and one which would not, if disturbed, obscure the original text.


 

Non-pigmented box surface: an area on the Kahlo box top interior was selected where there was no apparent inscription and no visible paint or pigment.


Known blood reference: a clean wood surface was treated with a known blood-source[9]. This test should produce a positive result in a test for the presence of blood, confirming that the test procedure could detect the presence of blood on a wood surface.

A clean, sterile wood surface (a physician's tongue depressor or spatula): a sterile swab, dampened with sterile water, was used to sample the surface of a sterile wooden spatula. This test should produce a negative result in a test for the presence of blood. The sterile water was also tested with Hemastix® reagent strips before each use of that liquid.

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.


Table I - Blood Presence Test Results Using Hemastix® reagent strips

Test Sample

Dampened Swab

Dilute Method

Dry Swab

Red Pigmented Inscription on wooden box lid

Negative, possibly occluded by excessive pigment

Negative

Negative

Non-pigmented surface on wooden box lid

Faint green, weak positive, possible blood/contaminant[10]

Faint green, weak positive, possible blood/contaminant

Faint green, weak positive, possible blood/contaminant

Known blood reference on wood

Positive

Positive

Positive

Clean, sterile wood surface

Negative

Negative

Negative

Sterile water source

Negative

Negative

Negative

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.

Photograph 9 shows four Hemastix® test strips containing various amounts of red-pigmented material from Kahlo’s inscription inside the box lid. Three results showing only red pigment may have obscured a faint positive reaction (causing a false negative conclusion).

One test strip (center of three strips at right) contains a faint green (positive for blood) reaction - the same reaction obtained when sampling an area of the box top interior that contained no pigment at all. This could be a false positive reaction and it is discussed next.

False Positive 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 positive result (indicating that blood is present when in fact it is not), particularly in the presence of a contaminant containing an oxidant.

The sample labeled #2 shown in this photograph was collected from an un-pigmented "plain" wood area of the interior of the box top – an area where a negative result would be expected (no blood detected).

But the sample produced a light green reaction on the Hemastix® test strip as shown in this photo. Obtaining a "positive" Hemastix® reaction to the control sample from an un-pigmented (no red inscription) area of wooden surface appeared to represent a false positive.

Since the box being tested is at roughly 60 years old, was stored in unknown conditions, and because the artist (or time or storage conditions) could have applied other materials to the work, there may be several explanations including the presence of superficial debris containing other oxidants.

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 Phenolpthalein Forensic Presumptive Blood Test (shown here) is considered the most specific test for the presence of blood on a surface.

This bloodstain evidence test kit was designed for forensic use and includes sterile collection media with three reagent chemicals used in this order: ethanol, phenolphthalein reagent, and hydrogen peroxide.

The sample turns pink in the presence of blood from humans or other animals. This is a non-destructive test, which is intended to leave the sample intact.

The reaction of heme in hemoglobin (in blood) with the hydrogen peroxide is a catalytic reaction, which makes this test very sensitive to very small quantities of blood in the sample. [11] [12]


The phenolphthalein presumptive blood test is a highly specific test used to assess potential false positive results of the other blood presence tests on works of art (and in crime scene forensic investigative work). [13]

The photograph shows our dry wipe samples of (#6) a non-pigmented area of the Kahlo box top interior and (#7) a pigmented area before treatment of these samples with the reagent chemicals. Using test controls similar to those described for the Hemastix® procedure, the phenolphthalein presumptive blood test involves using an alcohol-dampened sterile swab (using ethanol) or using a dry sterile swab (which will collect less pigment from the test surface) or a sterile filter paper can be used to wipe-sample the surface to be tested.

Since in our test of the Kahlo box lid interior there was light pigment visible on the dry folded filter paper, we did not need to use alcohol to moisten the filter paper before applying the three chemicals as catalytic reagents.

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 [14], and then with hydrogen peroxide [15]. 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

Pigmented inscription on art work to be tested for blood: a sterile filter paper was folded into quarters (for convenience) and is then used to collect a small sample of pigmented material from the area to be tested for presence of blood. The photo at left shows the red pigment collected by this sample after the ethanol step. This is pigment from the inscription, not a positive blood-reaction.



The phenolphthalein presumptive blood test results showed a negative result on this sample of the pigmented area of the Kahlo memento box, confirming that the pigmented area did not contain blood. The slight pink color at the tip of the folded filter paper shown in the photo at left was slightly diluted but otherwise unchanged from the red pigment collected when the box inscription was dry wiped with the paper.

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.

The phenolphthalein presumptive blood test results showed a negative result on the sample of the non-pigmented area of the Kahlo memento box, confirming that the earlier modest positive reaction when using the Hemastix method was a false positive. (Photo 15 shows sample after reagents were applied.)

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.

The phenolphthalein presumptive blood test results showed a positive result when performed on a dry-wipe filter paper sample of the known human blood source (a drop of blood placed on a sterile wood spatula and dried), confirming that the chemistry of this test was functioning normally.

 

Photograph 16 shows the characteristic pink stain appearing after the sequential application of ethanol, phenolphthalein, and hydrogen peroxide to this sample.


 

Bare filter paper: A sterile filter paper was used with no sample material, to confirm the normal behavior of the reagent chemicals when they are applied in proper sequence.

This test should (and did) produce a negative result in a test for the presence of blood.[16]

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.[17] 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.

For the tests discussed in this article, Doje recommended a modified procedure for using Luminol to screen works of art for blood while minimizing the chances of damage to the artwork itself. Used as recommended by Doje, this test procedure should not damage the work. For testing works of art, the Luminol is mixed with water while omitting the perborate until ready for use. [18]

A sterile swab, dampened with the Luminol/water mixture, is touched to the test area on the surface of the artwork. After sampling the artwork's surface, a catalyzing agent, hydrogen peroxide, is applied directly to the test swab (rather than mixing the Luminol-perborate combination and applying that mixture directly to the artwork).

Use of the Luminol Test for Blood when Overcoming Sampling Limitations of the Hemastix® Reagent Strip or Phenolpthalein reagent method:

In examining the Kahlo box top interior inscription, the author did not make initial use of the Luminol blood screening procedure because prior tests using both the Hemastix® reagent test strip for blood and the Phenolpthalein presumptive blood test procedure both indicated that no blood was detected. However we were concerned that the use of a small amount of blood mixed with paint pigments and solvents might have been obscured using the two forensic methods discu8ssed above.

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

  • The Kahlo box inscription does not appear to contain blood. Two forensic procedures indicated that the sampled area of red inscription on the interior of the lid of the Frida Kahlo memento box, while it refers to Kahlo’s blood, does not in fact contain human nor animal blood.
  • The Hemastix® reagent strip test method worked well as a sensitive screen of artwork for the presence of blood. Even when a dry swab of a known blood source contained no material visible to the naked eye, the reagent detected blood from the sample with no ambiguity. In three variations of sample preparation, the Hemastix® test produced a negative result (no blood detected) for the pigmented area of the box lid, but it produced a weak positive result (possible blood detected) for an un-pigmented, un-painted area inside the same box top, probably because of non-blood contaminants on the test surface.

    False positives are possible with this method if there are surface contaminants. False negatives can also occur: weak but valid positive results might be obscured by excessive sample pigment which happens to be the same color as a reaction-color of the reagent strip. Therefore thoughtful sample preparation is essential, obtaining "enough" but not "too much" material. The reagent is so sensitive to blood that very little sample material is needed to produce a reliable result.
  • The phenolphthalein presumptive blood test method was sensitive to and easily demonstrated the presence of blood in the control sample. Importantly, this procedure worked well to eliminate an apparent false positive test result with the Hemastix® reagent.[19]

    Because of the wide variety of possible contaminants that can be present on the surface or within pigmented materials on works of art, especially old objects, it is important to have a false positive screen available, such as the Phenolpthalein presumptive blood test procedure. This test is an important compliment to the Hemastix® test but it must be performed properly and with an understanding of the test’s catalytic chemistry since the phenolphthalein + perborate-wet sample will turn pink when it dries. A novice could mistake this end-reaction as a positive indication of blood.
  • It is important that any sampling and tests performed on works of art be conducted so as to protect the integrity of the art work, to avoid damaging it, and at the same time, the test must remain chemically and technically valid. Because it removes the least amount of material, is least likely to affect the surface of a work of art, and because it avoids overloading the swab with pigment or other surface debris, the author prefers the dry-swab Hemastix® method as a starting point for testing works of art for the presence of blood, and dry filter paper wipes when using the phenolphthalein presumptive blood test.
  • It is also important that the experimental design of the test procedure include controls of a known blood sample, controls for background contamination, and that other controls are used to identify false positive reactions.

Author

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 .

References

  • Doje's Forensic Supplies, P.O. Box 500, Ocoee, FL 34761 Tel: 407.880.8149 Fax: 407.880.8150 http://www.dojes.com, Judy and Don Doje assisted in specification of the forensic procedures used in this study and were our supplier of forensic supplies for the tests performed.
  • "Hemastix® Reagent Strips for Urinalysis, Test for Blood in Urine", Bayer Health Care LLC, Elkhart, IN 46514, product literature AN07016C, Rev. 5/06 USA (C) 1992, 2003 Bayer Health Care LLC

 

InspectAPedia.com/Mexico/FridaKahloSangre.htm - 29 Sept 2007

© Daniel Friedman, all rights reserved

http://InspectAPedia.com/Mexico/FridaKahloSangre.htm

Footnotes to Forensic Test for Blood on a Frida Kahlo Memento Box

[1]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.

[2] 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.

[3] 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.

[4] 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.

[5] 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.

[6] "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.

[7] 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.

[8] The Dry Swab Method:

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.

[13] 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).

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