Evaluation of the ability of three laboratory storage conditions to preserve soil contaminated blood for forensic serological analysis

ABSTRACT

Crime scene investigation is an important step in the entire criminal investigation process because this is where evidence is gathered. Blood from the perpetrator or victim of a crime can be left at crime scenes or transferred to other materials such as clothing, knives and guns. Most often, this body fluid is contaminated with soil at outdoor crime scenes but this might be the only or the most important evidence in solving a crime. This study aimed at identifying the most appropriate method of storing crime scene soil-human blood mixed sample prior to Hemastix and Hexagon OBTI serological analysis. Human blood was mixed with soil and stored at three different storage conditions (i.e., Room temperature/25℃, 4℃ and -20℃). Hemastix and Hexagon OBTI serological tests for blood and human blood, respectively were positive for soil-blood mixed samples at all storage conditions throughout a 12 week study period. In conclusion, all storage conditions can be utilized in storing crime scene soil contaminated samples at least for a 12 week period.

INTRODUCTION

All incidence, be it a crime, accident, natural disaster, armed conflict, leaves traces of physical evidence at the scene (UNODC, 2009). Crime scene investigators process and package evidence found at crime scenes and transport them to the Forensic laboratory for scientific analysis. Experts at the laboratory use valid analytical techniques to give a credible forensic report which can help solve a case (National Academy of Sciences, 2009; National Academy of Sciences, 2015). Comparing all other forms of evidence available to an investigator (e.g. confessions, testimonies, and video recordings), physical evidence (body fluids, guns, knives, fingerprints, etc.) plays an important and exceptional role. All other sources of evidence connected to a crime cannot be completely relied upon with the exception of physical evidence when it is recognized and properly collected because it provides objective and most reliable clue about the crime that has been committed (UNODC, 2009).

Serology is used to describe a series of laboratory tests or experiments that makes use of antigen and antibody reactions popularly called immunologic tests (Butler, 2011; Conti and Buel, 2011). Some serological tests also make use of chemical reaction assays, enzyme activity assays, as well as microscopy based assays (Legg, 2013). These tests make use of body fluids and allow the scientist to identify which fluid is it and also confirm the source of the fluid (Williams, 2012; Claridge, 2016). Based on the amount of body fluid left at a scene of crime or based on the material or substrate on which the fluid is deposited, it can be visible to the naked eye or invisible and hence will need enhancement to see it (Eckert, 1996). Serological tests involve an initial preliminary or presumptive colour test, followed by a confirmatory test to determine the species origin of the body fluid in question (Conti and Buel, 2011).

Presumptive tests for blood are fast, safe, inexpensive, very sensitive and easy to perform but do not identify the species origin of the blood in question. Most of these tests detect the presence of hemoglobin molecules which are found in the red blood cells and carry oxygen and carbon dioxide within the body (Shaler, 2002; Spalding, 2003; Tobe et al., 2007; Virkler and Lednev, 2009; Colotelo, 2009; Butler, 2011; Fisher and Fisher, 2012). Though the tests detect the presence of haemoglobin to produce a colour change, it is actually the peroxidase action of the haem group that causes the colour change (William, 2012). A positive presumptive test only indicates the possible presence of blood but it’s not specific for blood because some plant materials that contain peroxidase can also give positive results (Dutelle, 2016). Presumptive tests should use only a small amount of the evidence (Fisher and Fisher, 2012) in order to get enough sample for subsequent confirmatory and DNA Testing. Positive presumptive test in forensic case analysis is usually followed with confirmatory test, which is more specific for a particular species.

Hemastix strips

Hemastix strips are plastic strips that have been treated with a special blood reagent (mixture of 2-methylaniline and hydrogen peroxide). The strips are moistened with distilled water and touched with the suspected blood stain. If blood is present in the substance being tested, the hemoglobin catalyzes the conversion of 2-methylaniline to a green product. In the case of a suspected liquid blood, there is no need to moisten the strip (Newton, 2007; Brown and Davenport, 2015). Hemastix strips were manufactured to detect blood in urine (Fisher and Fisher, 2012; Dutelle, 2016) but are used worldwide by crime scene investigators.

Hemastix doesn’t interfere with subsequent DNA analysis (Dutelle, 2016).

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Figure 1: Hemastix strips showing positive (dark blue) and negative (yellow) for blood (Source: Ghana Police Forensic Science Lab)

Hexagon OBTI Kit

The hexagon OBTI is an immunochromatographic test kit for confirming the presence of human blood at a crime scene or the laboratory (Hermon et al., 2003). The kit can detect minute quantities of hemoglobin. A test pouch contains a test cassette and a liquid collection medium (Tris buffer, pH 7.5). The test device comprises a conjugate consisting of blue particles and antibodies (anti-human Hb). When a blood sample is mixed with the Tris buffer and applied to the test cassette, human hemoglobin in the sample reacts with reagent comprising of blue materials and monoclonal anti-human hemoglobin antibodies. This immunocomplex moves in the cassette to the T region where it is captured by an immobilized second antibody directed at human Haemoglobin forming a blue line at the T region on the cassette. This indicates the substance tested is human blood. Unreacted reagents migrate further and are bound in a second line by immobilized IgG antibodies. This is the control line on the test cassette and indicates correct function of the test kit (http://www.bluestar-forensic.com/pdf/en/instructions_hexagon_obti.pdf, accessed 21st February 2017). A visible blue line at the control region alone indicates a negative result for human blood. The Hexagon OBTI test is useful with aged and degraded material (James et al., 2002).

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Figure 2: Hexagon OBTI test kit for detecting human blood (Source: Ghana Police Forensic Science Lab)

At outdoor violent-related crime scenes, many situations may cause human biological evidence from the suspect or victim to be deposited in the soil (Kasu and Shires, 2015). This evidence mixed with soil at outdoor crime scenes might be the only source of evidence available to the investigator. In forensic biological fluid analysis, it is always advisable to make sure the crime scene sample is actually from a particular species being human or animal.

Evidence submitted to the laboratory from crime scenes are usually not processed immediately due to large number of pending cases, unavailability of analysts or unavailability of reagents. There is thus the need to investigate the proper way of storing these evidence at the laboratory to prevent destruction prior to their processing.

The primary objective of this work was to do a comparative evaluation of three laboratory storage conditions on soil-blood mixed samples found at crime scenes using serological analysis.

METHODS

Ethical clearance

Ethical clearance for this study was obtained from the Committee of Human Research, Publications and Ethics of the Komfo Anokye Teaching Hospital and the School of Medical Sciences, Kwame Nkrumah University of Science and Technology (KNUST).

Blood sample

Fresh adult male whole blood sample from a single person in a tube with Ethylenediaminetetraacetic acid (EDTA) anticoagulant was obtained from Jubilee Hospital, an accredited private Hospital at Akim-Oda, Eastern Region of Ghana and transported on ice to the Forensic lab, Accra. Freshly collected whole blood from same source was also obtained from the same facility on same day without anticoagulant and sample preparation prior to storage done before been transported. Identity of donor isn’t known and sample collection was done by a staff of the facility.

Soil

Dried black soil was collected in paper envelope from a garden at the compound of the Ghana Police Forensic Lab and brought to the lab.

Storage facilities

Room with an air condition of 25 °C temperature, fridge with temperature set to 4 °C and a freezer with -20 °C temperature served as the three sample storage conditions for this study.

Kits for serological tests

Hemastix test strips and Hexagon OBTI test cassettes were used for the serological aspect of this study.

Study time

The study was conducted at Jubilee Hospital, Akim-Oda and the Ghana Police Forensic Science Laboratory, Accra, over a period of 12 weeks.

Sample preparation for storage

1 g of soil was mixed with 1 ml of whole blood sample containing Ethylenediaminetetraacetic acid (EDTA). In all, six soil-blood mixed samples containing EDTA were stored at each storage condition (Room temperature/25 °C, 4 °C and -20 °C). 1 g of soil was also mixed with 1 ml of whole blood sample without EDTA. In all, three soil-blood mixed samples without EDTA were stored at each storage condition. Soil-blood mixed samples with EDTA were stored for 2, 4, 6, 8, 10 and 12 week periods. Soil-blood mixed samples without EDTA were stored for 4, 8 and 12 week periods. The samples were stored in capped tubes at 4 °C and -20 °C and in dried state on paper druggist folds at room temperature/25 °C.

Serological Analysis of samples

Sample preparation prior to analysis

The soil-blood mixed sample stored under each condition was placed in appropriate tube and 4 ml of DNAse/RNAse free water added to it. It was allowed to stand for 30 minutes at room temperature for the blood to come into solution after which it was vortexed thoroughly to mix soil particles and blood. The tube was then centrifuged with Eppendorf centrifuge at 14000 rpm for 30 seconds. The supernatant was pippeted and used for serological tests.

Serological tests controls for the study

Prior to soil-blood mixed samples storage at the three conditions, Hemastix and Hexagon OBTI test controls using clean blood with EDTA, clean blood without EDTA, soil-blood mixed sample with EDTA and soil-blood mixed sample without EDTA all from same blood collected from Jubilee Hospital were done. Hexagon OBTI tests controls were done following the procedure described below.

Holding the tube upwards, the red cap was unscrewed. 5ul of the supernatant was added to the buffer of the test kit and two drops of the buffer and blood mixture applied to the cassette. It was allowed to stand for 5 minutes and reading was taken.

Hemastix test controls were done by applying the tip of the strip to the supernatant and reading taken after a minute.

Effect of Ethylenediaminetetraacetic acid (EDTA) on the study

Serological tests results from soil-blood mixed samples with EDTA and soil-blood mixed samples without EDTA were compared in terms of positive and negative results from each sample type, storage time and storage condition.

RESULTS

Hemastix and Hexagon OBTI control tests for whole blood with EDTA, whole blood without EDTA, soil-blood mixed sample with EDTA and soil-blood mixed sample without EDTA all gave positive results. Throughout the study, the Hemastix and Hexagon OBTI kits tested positive for soil-blood mixed samples stored at all three conditions as seen in Table 4.1.

Table 1: Serological tests results for soil-blood mixed samples stored at various conditions overtime.

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RT= Room temperature/25; 4= Fridge; -20= Freezer; += positive result; EDTA= Soil-blood mixed sample with EDTA; No EDTA= Soil-blood mixed sample without EDTA

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Fig 3: Results for the Hexagon OBTI Control tests

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Fig 4: Results from Hexagon OBTI tests on samples stored at the three storage conditions

Cassettes with ‘No EDTA’ written on them means the blood samples were devoid of EDTA. ‘RT’ means room temperature or 25.

DISCUSSION

Throughout the study, both Hemastix and Hexagon OBTI tests were positive for soil-blood mixed samples stored at all three storage conditions. This suggests that hemoglobin was present in the samples throughout the study and its concentration was enough for detection by the kits. This implies that all the three storage conditions were able to preserve blood for serological tests.

All samples containing EDTA anticoagulant and those without EDTA tested positive for both Hemastix and Hexagon OBTI tests. This means EDTA did not have any effect on the hemoglobin in the blood so far as Hemastix and Hexagon OBTI serological tests were concerned. This agrees with work by Matheson and Veall in 2014 where EDTA didn’t had effect on blood in Hemastix testing.

CONCLUSIONS

The three Storage conditions (Room temperature/25 °C, 4 °C and -20 °C) had no effect on the samples in terms of performing Hemastix and Hexagon OBTI serological tests.

Soil-blood mixed samples with and without EDTA all tested positive for Hemastix and Hexagon OBTI serological tests. Thus, EDTA had no effect on serological tests in this study.

RECOMMENDATIONS

The study time should be extended to know whether storage condition and storage time will have effect on the results of the Hemastix and OBTI serological tests.

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