J Forensic Sci, March 2014, Vol. 59, No. 2 doi: 10.1111/1556-4029.12352 Available online at: onlinelibrary.wiley.com
TECHNICAL NOTE CRIMINALISTICS Daniel Petersen,1 Ph.D. and Frank Kovacs,2 Ph.D.
Phenolphthalein False-Positive Reactions from Legume Root Nodules*
ABSTRACT: Presumptive tests for blood play a critical role in the examination of physical evidence and in the determination of subsequent
analysis. The catalytic power of hemoglobin allows colorimetric reactions employing phenolphthalein (Kastle-Meyer test) to indicate “whether” blood is present. Consequently, DNA profiles extracted from phenolphthalein-positive stains are presumed to be from blood on the evidentiary item and can lead to the identification of “whose” blood is present. Crushed nodules from a variety of legumes yielded phenolphthalein falsepositive reactions that were indistinguishable from true bloodstains both in color quality and in developmental time frame. Clothing and other materials stained by nodules also yielded phenolphthalein false-positive reactivity for several years after nodule exposure. Nodules from leguminous plants contain a protein (leghemoglobin) which is structurally and functionally similar to hemoglobin. Testing of purified leghemoglobin confirmed this protein as a source of phenolphthalein reactivity. A scenario is presented showing how the presence of leghemoglobin from nodule staining can mislead investigators.
KEYWORDS: forensic science, blood test, phenolphthalein, bloodstain, hemoglobin, leghemoglobin, DNA typing, DNA extraction, Hemas-
tix, Kastle-Meyer, presumptive, legume, false-positive, polymerase chain reaction, short tandem repeats, D3S1358, D5S818, vWA, D13S317, FGA, D7S820, amelogenin, D16S539, D8S1179, TH01, D21S11, TPOX, D18S51, CSF1PO, D2S1338, D19S433, capillary electrophoresis
The presence of bloodstains on items of physical evidence is often critical to how a criminal investigation proceeds. A negative presumptive test for blood can terminate interest in an evidentiary item, thus precluding pursuit of further analysis of biological evidence. Conversely, a positive presumptive test for blood can be the decisive factor in prioritizing evidence for further analytical steps such as DNA typing. DNA profiles extracted from phenolphthalein-positive stains are presumed to be from blood on the evidentiary item and can thus lead to the identification of whose blood (e.g., suspect or victim) was deposited upon the crime scene evidence. Traditional catalytic tests such as phenolphthalein (1) or more recently developed immunological tests such as HemaTraceâ (Abacus Diagnostics, West Hills, CA) (2,3) are routinely used to screen suspected stains for the presence of blood. These colorimetric tests are inexpensive and rapid, allowing screening of potential bloodstains even while on-site at the scene of a crime. The phenolphthalein test is based on the catalytic activity of the heme-group component of hemoglobin and involves the oxidation of a reduced phenolphthalein substrate to produce a rapid color change after the addition of hydrogen peroxide (4,5). Previously, certain heavy metal substrates or vegetable extracts 1 Oregon State Police Forensic Services Division, Portland Metro Forensic Laboratory, 13309 SE 84th Avenue, Suite 200, Clackamas, OR 97015. 2 Chemistry Department, University of Nebraska at Kearney, 905 W 25th Street, Kearney, NE 68959. *Presented partially at the 63rd Annual Scientific Meeting of the American Academy of Forensic Sciences, February 21–26, 2011, Chicago, IL. Co-author Frank Kovacs supported by University of Nebraska at Kearney Research Services Council. Received 8 Nov. 2012; and in revised form 16 Jan. 2013; accepted 9 Feb. 2013.
© 2013 American Academy of Forensic Sciences
have been shown to occasionally yield false-positive reactions with the phenolphthalein test (3,4). We report here that root nodules from commonly found members of the legume family (Leguminosae) can also produce falsepositive phenolphthalein reactions. Such root nodules (Fig. 1) are known to contain leghemoglobins, which are small hemecontaining proteins with structures similar to those of the human oxygen transporters hemoglobin and myoglobin (6). The falsepositive phenolphthalein reactions can be attributed to the presence of leghemoglobin. A sample scenario and mock evidence is presented which shows how the presence of leghemoglobin from nodule staining can mislead investigators regarding the origin of DNA profiles obtained from evidentiary items. Materials and Methods Samples A variety of common red fruit samples and root nodules were obtained from freshly harvested garden- or greenhouse-grown plants. Stains were created by crushing root nodules to release contents onto paper, cloth, and cotton swabs. Stained materials were stored at room temperature and at 20°C. Bean plant tissue areas from three separate plants were combined and crushed (mortar and pestle), and the resulting extract was collected onto cotton swabs. Phenolphthalein (Kastle-Meyer) Test Phenolphthalein reagents were prepared and used in accordance with the standard methods (5,7). Tests were performed by consecutive addition of reduced phenolphthalein stock solution and 3% hydrogen peroxide. A deep pink color developing within 481
482
JOURNAL OF FORENSIC SCIENCES
DNA Extraction and Concentration Nodule-stained samples were incubated for 30 min at 56°C in 500 lL of lysis buffer (0.01 M Tris, pH 8, 0.01 M EDTA, 0.1 M NaCl, 2% SDS) with 0.75 mg of Proteinase K. Cotton material was removed from the lysate using a spin-basket (5 min/15,500 9 g) prior to purification using the Qiagen Biorobotâ EZ1 and EZ1â DNA Investigator Kit (Valencia, CA). After quantification, DNA samples estimated to be
TECHNICAL NOTE CRIMINALISTICS Daniel Petersen,1 Ph.D. and Frank Kovacs,2 Ph.D.
Phenolphthalein False-Positive Reactions from Legume Root Nodules*
ABSTRACT: Presumptive tests for blood play a critical role in the examination of physical evidence and in the determination of subsequent
analysis. The catalytic power of hemoglobin allows colorimetric reactions employing phenolphthalein (Kastle-Meyer test) to indicate “whether” blood is present. Consequently, DNA profiles extracted from phenolphthalein-positive stains are presumed to be from blood on the evidentiary item and can lead to the identification of “whose” blood is present. Crushed nodules from a variety of legumes yielded phenolphthalein falsepositive reactions that were indistinguishable from true bloodstains both in color quality and in developmental time frame. Clothing and other materials stained by nodules also yielded phenolphthalein false-positive reactivity for several years after nodule exposure. Nodules from leguminous plants contain a protein (leghemoglobin) which is structurally and functionally similar to hemoglobin. Testing of purified leghemoglobin confirmed this protein as a source of phenolphthalein reactivity. A scenario is presented showing how the presence of leghemoglobin from nodule staining can mislead investigators.
KEYWORDS: forensic science, blood test, phenolphthalein, bloodstain, hemoglobin, leghemoglobin, DNA typing, DNA extraction, Hemas-
tix, Kastle-Meyer, presumptive, legume, false-positive, polymerase chain reaction, short tandem repeats, D3S1358, D5S818, vWA, D13S317, FGA, D7S820, amelogenin, D16S539, D8S1179, TH01, D21S11, TPOX, D18S51, CSF1PO, D2S1338, D19S433, capillary electrophoresis
The presence of bloodstains on items of physical evidence is often critical to how a criminal investigation proceeds. A negative presumptive test for blood can terminate interest in an evidentiary item, thus precluding pursuit of further analysis of biological evidence. Conversely, a positive presumptive test for blood can be the decisive factor in prioritizing evidence for further analytical steps such as DNA typing. DNA profiles extracted from phenolphthalein-positive stains are presumed to be from blood on the evidentiary item and can thus lead to the identification of whose blood (e.g., suspect or victim) was deposited upon the crime scene evidence. Traditional catalytic tests such as phenolphthalein (1) or more recently developed immunological tests such as HemaTraceâ (Abacus Diagnostics, West Hills, CA) (2,3) are routinely used to screen suspected stains for the presence of blood. These colorimetric tests are inexpensive and rapid, allowing screening of potential bloodstains even while on-site at the scene of a crime. The phenolphthalein test is based on the catalytic activity of the heme-group component of hemoglobin and involves the oxidation of a reduced phenolphthalein substrate to produce a rapid color change after the addition of hydrogen peroxide (4,5). Previously, certain heavy metal substrates or vegetable extracts 1 Oregon State Police Forensic Services Division, Portland Metro Forensic Laboratory, 13309 SE 84th Avenue, Suite 200, Clackamas, OR 97015. 2 Chemistry Department, University of Nebraska at Kearney, 905 W 25th Street, Kearney, NE 68959. *Presented partially at the 63rd Annual Scientific Meeting of the American Academy of Forensic Sciences, February 21–26, 2011, Chicago, IL. Co-author Frank Kovacs supported by University of Nebraska at Kearney Research Services Council. Received 8 Nov. 2012; and in revised form 16 Jan. 2013; accepted 9 Feb. 2013.
© 2013 American Academy of Forensic Sciences
have been shown to occasionally yield false-positive reactions with the phenolphthalein test (3,4). We report here that root nodules from commonly found members of the legume family (Leguminosae) can also produce falsepositive phenolphthalein reactions. Such root nodules (Fig. 1) are known to contain leghemoglobins, which are small hemecontaining proteins with structures similar to those of the human oxygen transporters hemoglobin and myoglobin (6). The falsepositive phenolphthalein reactions can be attributed to the presence of leghemoglobin. A sample scenario and mock evidence is presented which shows how the presence of leghemoglobin from nodule staining can mislead investigators regarding the origin of DNA profiles obtained from evidentiary items. Materials and Methods Samples A variety of common red fruit samples and root nodules were obtained from freshly harvested garden- or greenhouse-grown plants. Stains were created by crushing root nodules to release contents onto paper, cloth, and cotton swabs. Stained materials were stored at room temperature and at 20°C. Bean plant tissue areas from three separate plants were combined and crushed (mortar and pestle), and the resulting extract was collected onto cotton swabs. Phenolphthalein (Kastle-Meyer) Test Phenolphthalein reagents were prepared and used in accordance with the standard methods (5,7). Tests were performed by consecutive addition of reduced phenolphthalein stock solution and 3% hydrogen peroxide. A deep pink color developing within 481
482
JOURNAL OF FORENSIC SCIENCES
DNA Extraction and Concentration Nodule-stained samples were incubated for 30 min at 56°C in 500 lL of lysis buffer (0.01 M Tris, pH 8, 0.01 M EDTA, 0.1 M NaCl, 2% SDS) with 0.75 mg of Proteinase K. Cotton material was removed from the lysate using a spin-basket (5 min/15,500 9 g) prior to purification using the Qiagen Biorobotâ EZ1 and EZ1â DNA Investigator Kit (Valencia, CA). After quantification, DNA samples estimated to be
