|
|
|
|
|
|
Tetramethylbenzidine (Probus) Leucomalachite green (Merck) Phenolphthalein (Panreac) Ethanol (Panreac) Potassium hydroxide (Panreac) Powdered zinc (d'Hemio) Hydrogen peroxide (Prolabo) Glacial acetic acid (Probus) Ascorbic acid (Merck) Distilled water |
Preparation and study of samples Blood extraction Also: Analytical balance Centrifuge Oven Refrigerator Observation chamber Pressure roller |
2. Reagent preparation: We added 1.6 g of o-tolidine base to 40 mL of absolute ethanol. To this we added 30 mL of glacial acid and 30 mL of distilled water.
3. Specimen preparation: We prepared blood solutions. Concentration is expressed in milliliters of blood per total volume in milliliters. We added 1 mL of the blood sample to a test tube, followed by 9 mL of distilled water, for a final solution concentration of 1:10. We diluted 1 mL of 1:10 solution to 10 mL to yield a corresponding 1:100-concentration solution. We used the same procedure, in turn, to obtain sample concentrations of 1:1000, 1:10000, and 1:100000.
4. Acid solution preparation: Using the normal concentration of lemon juice in ascorbic acid, 0.38 mg/mL (Jimenez et al. 1994), we prepared a base aqueous solution to test the bloodstain and to obtain diluted solutions with a smaller concentration of the contaminant, from 2.e-3 M to 2.e-7 M.
5. Blood-specimen preparation with the addition of the contaminant: Each test tube sample contained 1 mL of the blood solution and 1 mL of the different ascorbic acid solutions.
6. Bloodstain preparation on semiporous filter paper: We applied five drops of the sample on filter paper.
7. Study of the presumptive test: We added two drops of reagent to the samples (fresh solution [0.1 mL], dried stain, and print), and then if no color change was observed, we added two drops of a 3 percent solution of hydrogen peroxide.
7.1. Control test: We ensured that no positive reactions were caused by the contaminant.
7.2. Analysis of liquid, stain, and print samples: We obtained prints by dampening filter paper with distilled water and applying pressure over the stain. The test was performed on the print leaving a stain on the paper.
Tests Using Tetramethylbenzidine,
Leucomalachite Green, and Phenolphthalein:
We repeated the
procedure previously described using the reagents tetramethylbenzidine,
leucomalachite green, and phenolphthalein, but the tests were
only performed on stains on filter paper. We prepared the reagents
as described by Eckert and James (1989).
Tests Using Tolidine:
The results we obtained are shown in Table
1. This table was used to construct Figures
1A, 1B, and 1C.
Tests Using Tetramethylbenzidine,
Leucomalachite Green, and Phenolphthalein:
The results we obtained are shown in Table
2. Figure 2 provides a comparison
of the effectiveness of the reagents in samples contaminated
by ascorbic acid.
When added to a blood sample, ascorbic acid gives rise to a false negative upon application of the presumptive test using tolidine as a reagent. The effect of the contaminator may vary as follows:
The second experiment we performed demonstrates the possibility of obtaining false negatives with reagents frequently used in presumptive tests. Just as false positives are sometimes encountered, our tests affirm that false-negative results may be obtained as a result of sample contamination.
We proved that there are differences in sensitivity with respect to the action of the contaminant among different reagents. Following analysis of the tests performed on stains on paper, we found that false negatives were detected using tetramethylbenzidine with low-concentration blood samples (1:400000), whereas o-tolidine and leucomalachite green produced the same with samples that were slightly more concentrated (1:200000) and phenolphthalein gave false negatives in samples concentrated at 1:2000.
One possible explanation is that the reducer competes for oxygen in the reagent and prevents oxidation. Alternately, the ascorbic acid may also reduce the reagent after it has been oxidized by the hydrogen peroxide, thus preventing the characteristic color from appearing because of the breakdown of the reagent and its rapid disappearance.
The presence of ascorbic acid may not be the only cause of false results. It is possible for a bloodstain to go unnoticed if it has had sufficient contact with a product with a high reduction strength (detergents and foods). Evidence examiners should also consider that a variety of circumstances, such as washing, rain, heat, and time, may reduce the concentration of blood in the sample, with small amounts of contaminant being sufficient to give rise to a false negative in the presumptive test. Examiners may, therefore, encounter stains that look like blood, but these stains would not be recognized as such in a presumptive test. This would give rise to a false negative in an otherwise reliable presumptive test, with the result that important evidence may be lost.
The frequency with which contamination-related problems are encountered is unknown, but such problems do exist. Therefore, it is impossible to enumerate the specific circumstances in which special care should be taken to avoid such problems. As we noted in the introduction, the important point is that presumptive tests performed on a bloodstain may give rise to a negative result, thereby truncating subsequent tests.
We studied false-negative results in bloodstain presumptive tests using blood samples to which contaminating substances were added. As a result, we found that a reduction compound (ascorbic acid) added to the blood sample may give rise to a false-negative result for the test. This occurred regardless of the type of sample used (liquid, stain, or print), although different degrees of sensitivity were observed.
Castelló, P. A. Critical review of presumptive tests in bloodstain investigations: False negatives in Adler's testAn application of forensic chemistry. Doctoral thesis, University of Valencia, Spain, 1997.
Cox, M. A study of the sensitivity and specificity of four presumptive tests for blood, Journal of Forensic Sciences (1991) 36:1503-1511.
Culliford, B. J. and Nickols, L. C. The Benzedrine test: A critical review, Journal of Forensic Sciences (1964) 1:175-191.
Eckert, W. G. and James, S. H. Interpretation of bloodstains evidence at crime scenes. Elsevier, New York, 1989.
Gisbert, J. A. Forensic medicine and toxicology (5th ed.). Salvat, Barcelona, Spain, 1998.
Jimenez, A., Cervera, P., and Bacardi, M. Food composition table. Sandoz Nutrition, Barcelona, Spain, 1994.
Verdú Pascual, F. A. and Gisbert Grifo, M. S. Investigation of bloodstains: False negative results of the Benzedrine test. Forensic Science International (1995) 71:85-86.
Windholz, M., Budavari, S., Stroumtsos, L. Y., and Fertig, M. N. The Merck index (9th ed.). Elsevier, Rahway, New Jersey, 1996.
FORENSIC SCIENCE COMMUNICATIONS JULY 1999 VOLUME 1 NUMBER 2
|