ABO Typing In Forensic Analysis: to be or not to be in the epoch of genotyping

Keywords

ABO-Typing; Absorption-Inhibition; Absorption-Elution; STR-PCR; Touch DNA Analysis; Epigenomics.

Introduction

Forensic science is an interdisciplinary field that uses scientific knowledge from varied fields like biotechnology, toxicology, chemistry, physics and others to analyze and characterize physical evidence found at the scene of crime. In criminal matters, particularly those involving violence, specimens of blood, semen, and other body fluids or tissues are used as evidence as they provide information that may solve the case. Blood is considered one of the most important biological samples that are frequently obtained from the crime scene. It is regarded as a very important forensic tool since analysis of different aspects of bloodstains can provide valuable information which helps the investigators to have a clear understanding of the circumstances under which a crime has been committed. The use of blood in forensic analysis is a method for identifying individuals suspected of committing a crime, solving disputes in paternity etc [1].

The term Forensic serology has generally been used to refer to the identification and individualization of biological evidence, including all the activities and tests associated with the evaluation and typing of biological evidence in criminal matters. The word serology was derived from serum, the fraction of blood containing antibodies. Blood grouping was long the only means of individualizing biological evidence, and serology classically encompassed blood groups and blood typing [2].

Origin and ABO typing

The importance of blood grouping in medico-legal issues,lies in the fact that blood groups are considered genetic markers since they are strongly inherited following the Mendelian laws, and are unaffected by environmental factors such as nutrition,diet, age, occupation, diseases. It can withstand stringent conditions, such as high heating or drying [3, 4] and thus remain unchanged throughout life [5] Blood grouping is the classification of erythrocytes or red blood cells (RBCs) based on surface markers or antigens like A, B, D, H, etc., present on their cell membrane [6]. There are more than 300 antigens present on the RBC membrane and based on these antigens, 38 blood group systems have been identified by International Society of Blood Transfusion which includes ABO, Rh, P, Kell, MNS, Lewis, Kidd, Diego, etc [7]. Due to complexity, and expense of testing for possible reactions among all known antigens, the simpler ABO and Rh blood typing system remains the primary, cost-effective and conspicuous method in practice for clinical and forensic use [8].

The ABO blood group system, first described in 1901 by Karl Landsteiner is the most basic system which divides blood into four groups, or types: A, B, AB and O [9] Blood group specificity depends upon the inheritance of the ABO and H genes and the subsequent expression of these antigens on the red blood cells. For e.g., an individual having blood group A will expressantigen "A" on the surface of the RBCs and will contain "anti-B" antibody in the plasma.The Rhesus-system is the second most imperative blood group system after ABO[10]. This system is based on the expression of the "D" antigen or Rh factor on the RBC membrane and accordingly, the status of a person is indicated as either Rh-positive (D-antigen present) or Rh-negative (D-antigen absent). Landsteiner's description of blood types gave a new opening to forensic science and based on this, forensic scientists could definitively compare blood evidence left at a crime scene to the blood of a suspect. The Rh factor enabled forensic scientists to better study the blood of suspects and to potentially exclude individuals as the source of blood at crime scenes and narrow down the list of suspects [11]. Therefore blood-typing could be used to help prove innocence, but not toidentify a suspect beyond a reasonable doubt, the standard necessary for a criminal conviction in many criminal courts.

However, accuracy of ABO blood typing is a major concern for the forensic serologist since inception. Small sample quantity, degraded and contaminated blood, hemolysis, putrefaction, mummification and skeletonization of the body during the post mortem [12] often makes the typing method mostly inconclusive either due to confusing observations or due to lack of sufficient stained sample present.

Species identification

Species identification along with ABO- typing of different human samples in Forensic serology has long been one of the well accepted test in criminal investigation [13]. Beside blood, several other evidences, hair, semen and saliva may present in the crime scene, which can be used to link the suspect in accordance with the Locard’s principal.

The amount of biological evidence collected at a crime scene is often extremely small, and thus it becomes particularly important for the forensic scientists to preserve as much of the evidence as possible and select the accurate tests for analysis that require trace amounts of sample.

After detection, the sample requires the test for species identification. Species identification is important prior to ABO-typing because the forensic materials may be contaminated with the animal body fluids. For most of the body fluids the primary analysis for species determination is by ‘precipitin’ test [14] which used the principle of interaction between antigens and the anti-sera raised against the blood cells of the same species. Precipitin tests can be performed in various ways. In every instance, however, the goal is to bring the antigens and antibodies, both in solution, into contact with one another.

The earliest precipitin test method was the "ring test," where an aqueous solution of antigen is layered over the more dense antiserum solution in a tube. The formation of precipitate formation at the interface between the layers indicates a positive test. This method was largely abandoned when gel-based methods, such as Double Diffusion’ [15] was developed, which was also based on the same principle. Though used globally these methods have some limitations. First, due to unavailability of enough quantity of sample it is always difficult to titrate the required quantity of antigen-antibody to get a visible result and the second, is to eliminate the cross-reactivity of antisera [16] against one species with bloods of evolutionary closed species ( e. g., human &chimpanzee). Both the limitations are some-way related to the particular batch of antisera being used. Therefore, it is always necessary to test the new batch of antisera every time against the homologous blood. The precipitin test can also be done using the antisera, raised against the hemoglobin of the species, which detects the origin of species as well as confirms the presence of blood. Unfortunately, the blood stain often found in the crime scene is dried and is not a true mixture of blood cell and serum. Depending on the environment and clotting time (before drying or after drying) the ratio of cell/serum often varies.The antisera raised against serum or hemoglobin will therefore, produce different results for different cell/serum mixture for the same species.

Nevertheless, the "anti-human" sera raised against human serum proteinsreact with some human physiological fluids, e. g. human semen, saliva, and semen-free vaginal swab extracts although to a lesser extent than they do with serum or blood. Therefore, the antihuman sera can be used to test for human proteins in biological fluids other than serum. It must also sometimes be considered in the interpretation of positive test results when the specimens are or may be mixtures of blood and/or various physiological fluids. There are a number of more involved technical modifications of precipitin tests that have been proposed or used, particularly to help in differentiating closely related species [17].

ABO - Typing

Agglutination is the main criteria to detect antigen-antibody interaction, using the principle of immunochemistry.

The main avenues for ABO typing are (1) Direct method and (2) Indirect technique .The direct method could be classified into two types ; (i) Forward method, depending on the presence/absence of antigen [18] on the cell surface,& (ii) Reverse method depending presence/absence of antibody in the serum part [19]. The indirect method was mainly the antigen-antibody combined technique, for which two basic methods have been reported namely, absorption inhibition [20, 21] and absorption-elution [22, 23]. Both these methods are used to detect the blood group antigens remaining on the ruptured cell membrane. Some other high throughput methods, such as Enzyme linked immune-sorbent (ELISA) assay [24, 25] and Micro-plate method [26] have also been tried. The latter two methods, when used in combination with absorption-elution, helped to increase the efficiency of the result.

Forward method

It is the simplest ABO-typing method, which is mainly used for the fresh blood. The antigen on the cell surface and anti-sera raised against the blood antigen reacts together and a cell-cell attachment occurs through antigen-antibody bridge. This cell-cell attachment (agglutination) can be visualized in the microscope. The efficiency of agglutination depends on the proper cell structure, antibody binding capacity, osmotic fragility etc [27, 28]. In dried, aged stains the antigens are partially or completely destroyed. Sunlight and humidity are also known to be destructive for the antibody stability [29]. Moreover, hemolysis or partial hemolysis has been a common observation during suspension of cells in saline, although, later it was shown that the hemolysis could be partially avoided by using 40% saccharose solution of pH 6 [30].

Lattes method

An alternative reverse approach has been reported by Lattes 19 based on serum antibody. It used the unique property of the ABO system, that an individual’s serum contained antibodies corresponding to the antigen that he or she lacked. This process has its own limitations for the analysing forensic samples as the serum antibodies are far less stable in dried stains. Though some laboratories reported the presence of antigen in the dried stain, they could not opine the antigen type conclusively which may be due to less sensitivity of the method. Mistyping can also occur in the stain having some other unrelated antibodies in the serum, which causes the agglutination of the test cells. The possibility of misinterpretation of result has been a common observation in report using older cells.

Absorption-inhibition

Absorption-inhibitionwas the first combinedmethod reported by Schutze, 1921, where he used the dried blood sample for ABO typing. The method was less sensitive, whichneeded the predeterminedtiter value of the anti-sera to work with. A two-dimensional absorption –inhibition method [21] was proposed, which took best features of inhibition-titration and titration-inhibition at the same time and was shown to be more sensitive than either of them.

Absorption-elution

The absorption-inhibition method was subsequently replaced by Absorption-Elution method [22] and was consequently refined by the method developed by Kind (1960), which was predominately used by most of the laboratoriesfor its high sensitivity. Though ABH antigen detection was reported even for 42 weeks aged samples by absorption –elution method [4], some discrepancies were also observed [12]. In this context, a comparative study between direct agglutination and absorption-elution methods was performed, which showed some cross reactivity in absorptionelution method for the blood stored at room temperature for 60 days [28]. That result could not comprehensively eliminate the possibility of mistyping for the aged, dried stains [31] even using absorption-elution method. Haemolysis might also produce negative results [32]. Though, ABH antigens were shown to be stable [3] at high heat, activity changes was reported for the dried blood stains on standing [4]. Surface type (cotton, synthetic fabrics, wooden surface) was also shown to affect the efficiency of this method with age [33]. High sensitivity of this method can also be a disadvantage as some other body secretions like, semen, saliva which are also the source of ABH antigen might interfere with the result.

However, all ABO typing methods for dried, aged blood stains, were complex, tedious, time-consuming and low throughput, which were difficult to automate. As it involved, extensive sample handling, and complex techniques, which were coupled with human skill and experience, a lot of discrepancies and false positive/ negative results were also found [12]. The method also became vulnerable while determining the blood group of wet stains and/ or decayed bodies [12] and possibilities of mistyping occurred in aged blood sample [4]. The failure might be attributed to the loss of antigenicity or to the acquired antigenicity by bacterial contamination [34] and/or partial digestion of erythrocyte membrane by proteolytic enzymes [35]. All these limitation made most of the methods unreliable to work with.

ABO typing in other body fluids

In the 1930s, it was found that the blood group antigens, were not confined to erythrocytes but were also present in various other body secretions and tissue fluids, such as saliva, semen, sweat, urine, vaginal secretions, etc., from which blood groups could be determined [36], provided the subject had the secretor status [37, 38]. The status of an individual as secretor or non-secretor is determined by the presence of Lewis antigens, Lea and Leb, which are not intrinsic to the RBC membrane but are synthesized by intestinal epithelial cells and circulate in plasma [39, 40]. The ABO group specific substances are typically present in high concentrations in body fluids of secretors and the secreted substances can withstand drying and retain their antigenic activity over a prolonged period. The forensic serologists took advantage of these characteristics to group stains of body fluids such as semen [41] and saliva [42] that assisted in the investigation of crimes where there was lack of blood sample. Studies on the secretor status of blood group antigens had extensively been studied for saliva [37, 38, 43] vaginal sample [44], sweat stains [45] and semen [46]. ABH antigens were also expressed in some indoor pets also [46], so the secretion from the saliva of rabbit, cat, dog interacted with the antibodies raised against ABH antigen [47]. It is therefore important to exclude false positive reaction and mis-judgement of species identification in the blood group determination.

Due to unavailability of adequate techniques, analysis of anti-A and anti-B haem-agglutinins in saliva or other body fluids were not utilized as evidence in the medico legal cases initially [48]. Later, a lot of modifications were done to develop highly precise techniques to determine blood group antigens in body fluids. At present, two methods are used to type blood and body fluids for ABO grouping, the absorption-inhibition (AI) and the absorption-elution (AE) methods. Absorption-inhibition showed a 100% positive correlation for ABO blood grouping in dried salivary samples and was found to be a more effective technique for recognizable proof and secretor status determination in different populations [49]. A different study carried out the estimation of blood group antigens on cigarette butt [50] also showed the Absorption- inhibition method to be more fruitful. But in a comparative study [42] absorption-elution technique was demonstrated to be more sensitive, specific, and effective in determination of ABO blood grouping in dried saliva stain.

ABO-typing from other tissue

ABH antigen is expressed in human cells including dermal epithelium, endothelial cells of blood vessel Hassall’s corpuscle [51], renal tubules, secretary cell of respiratory tract [52], and male reproductive organs [53]. Indirect typing method were mostly tried for hair [54, 55] tooth [56-59] and bone tissue [60, 61]. Some histochemical techniques have also been used for hair [52, 62] and other tissue [53]. However, the histochemical method is very time consuming which requires a lot of skill dependent steps to reach to a conclusive result.

Advent of DNA technology

Unfortunately, the limitations of ABO-typing methods (Table 1) for forensic samples along with variable reports by different laboratory, made the results inconclusive in many cases. The effect of autolysis, dehydration, loss of antigens by temperature, humidity, and aging, or contamination by microbes or animals may haveled to variations in the study. Moreover, even though, properly done, it did not always have the potential to produce concrete evidence to individualize the offender and therefore may be proven futile in the court of law.

DNA Fingerprinting

Forensic science is nothing but the art of piecing together a crime scene in order to determine how the crime was committed and who was responsible. DNA evidence is one of the most prominent pieces of evidence, which is lacking in ABO typing, method to individualize the perpetrator.

Restriction Fragment Length Polymorphism ( RFLP) Restriction fragment length polymorphisms, or RFLPs as they are commonly known, were the first type of DNA fingerprinting which came onto the scene in the mid-1980’s [63]. that focus on the size differences of certain genetic locations. Some other methods, utilizing the RFLP principle were used, such as, amplified fragment length polymorphism [64], and terminal restriction fragment length polymorphism [65].

Variable Number Tandem repeats VNTR

Variable number tandem repeats, or VNTRs represent specific locations on a chromosome in which tandem repeats of 9-80 or more bases repeat a different number of times between individuals. These regions of DNA are readily analyzed using the RFLP approach and a probe specific to a VNTR locus. The fragments are a little shorter than RFLPs (about 1-2 kilo base pairs), but are created through the exact same process.

STR-PCR analysis

After the invention of Polymerase Chain Reaction by Mullis [66], the DNA fingerprinting is largely being done by STR marker analysis [67-69]. Unlike VNTRs which analyze mini-satellites that have repeat of 9-80 base pairs STRs use microsatellites which have repeat sequences of only 2-5 base pairs [70], tandemly repeated at a specific locus, mostly in the non-coding regions , introducing the “less is more” philosophy to the world of DNA fingerprinting. This was a big step forward in forensic science since the length of DNA fragment being analyzed is short enough to be amplified by polymerase chain reaction (PCR), which enabled the analysis of a very small sample of DNA. This method is quicker and easier than any previously known method and match it to a person’s identity. The STR typing method is nowadays mostly performed by the robotics starting from, DNA extraction, amplification (PCR) and all through DNA profiling. That is why, a skilled or trained personnel may not be needed to perform the analysis, though the interpretation part would still require some knowledgeable person in this field.

Next Generation Sequencing (NGS)

Next generation sequencing (NGS) was another landmark in DNA fingerprint analysis, which is capable of sequencing thousands of genomic regions of a species simultaneously to get idea about the phenotype, age of the unknown source retrieved from the crime scene [71]. A promising NGS approach was reported with simultaneous analysis of 10 STRs, 386 ancestory-specific SNP s, and the complete mtDNA [72]. Other two studies [73, 74] has been reported, where they analysed 27 autosomal, 7-X chromosomal, 24-Y chromosomal, 94 identity specific SNPs and 56 ancestory informative SNPs in a single sequencing. The method has been shown to be applicable in degraded and low template samples [75] as well.

Rapid-Hit technology

This is also a high-throughput method which does not require any human intervention in whole process. It takes help of the simple “sample in-profile out’ principle and analysis is done in a single step, including extraction, amplification and sequencing within 2 hrs [76]. The minimum time taken for genotyping makes it very useful for the criminal justice system and has a tremendous potential to use it at the crime-spot, to include/exclude the suspect. This method is currently under validation for law enforcement use.

Merits & demerits of DNA- typing

The crime rate around the globe is increasing alarmingly and the need for fast and effective methods of crime and criminal detection is also increasing. As the criminal justice system comes of age, it really requires individualization of a suspect from a crime scene. ABO-typing could be considered as the evidence which was able to place an individual in a general class but failed to identify the offender conclusively. For example, blood typing can be used to establish whether someone has type A, B, AB, or O blood, which can be useful in helping to investigate a crime by either including or excluding an individual from the list of suspects but cannot pin down the actual culprit. On the contrary, individual evidence, such as fingerprints and DNA, can be used to unravel the identity of an individual. DNA analysis has made possible the accurate typing of very small traces of body fluid evidence to a very high level of individuality in many cases. A laboratory worker might be virtually certain, therefore, that a biological trace has originated from a particular person.

Apart from the advantages over mere ABO typing (Table. 2) the DNA typing has a vast potential in the criminal justice system in future. Touch DNA is one of them. It was a break-through technology developed [77-79], which did not require any biological fluids to type DNA, instead it could isolate the DNA from the touch site of the object having the skin epithelial cells of the accused remain at the crime scene. When a crime is committed, the offender often may touch items, like weapon, clothing, victims body, etc [80], which were used as a DNA source of the perpetrator. It helped massively in investigating “cold cases” which were closed due to lack of evidence.

However, STR profile data was found to be ineffective, where the suspect is unknown and any genetic data was unavailable to compare with. In those cases, DNA profile was compared with those in data banks, kept in many state and nationally for the offender, which could link the perpetrator to the crime scene. For nonmatching DNA profiles, a statistical approach [81] was proposed. The ancestry-informative SNP marker PCR multiplexing was also an effective method to infer the geographic background of the donor by using five major sub-continental population groups [82]. New investigative lead was also provided using uni-parentally inherited markers (mitochondrial DNA and non-recombinant Ychromosome) to infer the ancestries of the maternal/paternal background of a DNA sample and thus, helped to narrow down the suspect list.

Another, limitation what the forensic scientists often encounter is the inability to determine the age from the skeletal remain, since a lot depends on the age of person in question. Though the morphological analysis of the skeleton was the only way [83] using mostly hard tissues, such as,tooth, bone etc, the method have been shown to produce ambiguous result [84]. A promising method for more accurate age prediction arose from the field of epigenomics, where epigenetics and the DNA-methylation markers have been proposed to estimate age, tissue type and even differentiate between monozygotic twins [85]. A significant change of age-related DNA methylation level at CpG dinucleotide islands was shown to be associated with increasing age in epigenome-wide association study [86-88]. Gene expression had also been reported to correlate with human age [89]. But both these method required relatively higher amount of DNA to work with, which is normally absent in the typical forensic specimen [90].

Though omnipotent, DNA typing has some inherent limitation in forensic cases. As it is unable to establish the nature of the biological material which may or may not be important in a particular case. The importance of identification versus typing tests with an evidence item has to be considered in the context of the case. A successful DNA typing on a licked envelope flap or on a swabbing from a bite mark, was likely to be informative regardless of whether saliva could be identified or not. On the other hand, the identification and species-determination aspects of a forensic examination can sometimes be more important for a suspected hit-and-run case. The suspect may be absolved of suspicion by a finding that bloodstains on his vehicle were of nonhuman origin.

In addition, a virtual match between evidence and a person might have little meaning if there is an innocent explanation for the finding. This would be true, for example, when a victim's genetic profile is matched to bloodstains on a suspect's clothing in a case in which the victim and suspect lived in the same household or the suspect is able to offer a plausible explanation for the stains.

Not withstanding the DNA typing revolution, some activities long associated with "forensic serology" remain important and continue to be a vital part of forensic biological evidence analysis. Blood and physiological fluid stains and traces still require identification. More importantly, those aspects of "forensic serology" most characteristic of its association with criminal sciences remain critical if biological evidence analysis is going to help to unravel a case. Recognizing the crucial physical evidence in a given case, using experience and judgment to select the most important and informative specimens for typing in terms of the case, and interpreting stain patterns are essential skills that cannot be replaced by any DNA typing technique.

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Table 1:

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Table 1. Limitation of different ABO-typing methods.

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Table 2:

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Table 2. Advantage of genotyping over ABO-typing.

Conclusions

Degraded samples, loss of antigenecity due to effect of environmental factors, lack of ability to individualize the perpetrator, made the ABO-typing methods unfruitful to the judiciary and rapidly being replaced by high-throughput genotyping. Sex determination and age estimation is often required for the human or skeletal remains found in the crime scene to get a preliminary idea about the crime. Limitation to discriminate between male and female is another feature that makes the traditional. ABO- typing methods inapplicable in various cases. On the contrary, while the STR-PCR can determine the sex, new approaches like, epigenomics can infer about the approximate age of the skeletal remains. Paternity dispute is another area often encountered in both criminal and civil cases, such as, guardianship, inheritance, adultery and fornication, where, ABO–typing is of no use as a conclusive evidence as the A, B, AB and O blood groups are never unique. Genotyping is the only option toconfirm paternity of the disputed off-spring in such cases.Non-secretory status of human subject can also make the other body fluids, other than blood, unsuitable for ABO typing. The problem can be addressed by genotyping where saliva, semen, or other body fluids may be used to individualize a person. In addition, in absence of any detectable biological fluid also, touch-DNA can be handy to get results in presence of skin cell remains in the crime scene. Thus, genotyping, in its present form, mostly fulfils the requirements for the forensic experts, to unravel a case conclusively to court of law. However, in spite of being eclipsed by genotyping, ABO typing is still being used and may be continued to be in use as a routine-tool for forensic analysis in developing countriesbecause of its cost-effectiveness.It is only a matter of time when forensic experts will rely on genotyping methods and the ABO-typing will either be discarded or confined only to a primary screening.

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