**Abstract**

The field of Forensic diagnostics is evolving very rapidly keeping in pace with the emerging technology in the various fields. Several biomarkers up to the molecular level have been discovered which aid in solving cases. Pregnancy diagnosis from traces of blood could aid in solving cases of finding a missing pregnant lady or illegal abortions. But the challenge posed could possibly be the minimal amount of blood obtained for diagnosis. Here comes in the role of RT PCR diagnosing mRNA which is pregnancy specific, i.e., for hPL and beta hCG. The additional advantage would be that a small quantity suffices. Even if the blood stain is dried and degraded, the detection rate is good. This could add weightage to the investigation as a vital clue or change the course of investigation**.** The other areas of application of obstetric biomarkers are sexual assault, maternal substance abuse and paternity testing.

**Keywords:** Crime scene, mRNA, bloodstains, RT-PCR, noninvasive paternity testing, STR, SNP

### **1. Introduction**

Forensic diagnostics is one area which is developing in an extremely fast rate and has changed the way in which investigations are handled. The principles of the various disciplines -immunology, biotechnology, biochemistry, molecular biology etc. is integrated into the various diagnostic modalities developed to solve a case scientifically in order to achieve the final result [1]. But pregnancy diagnostics is one area in forensics which has a lot of gray areas.

The possible cases could be sexual assault of a pregnant woman, criminal miscarriages, feticides, drug and alcohol abuse, paternity detection. Crime scenes often yield samples – biological human body fluids – vaginal fluid, saliva, skin and liquid tissues- semen and blood. These provide the necessary genetic material which could help in firstly, establishing the identity of the concerned individual and also aids in establishing the cellular origin of the concerned material. Hence, the investigators need appropriate and effective tools to aid in detection of cellular origin of the sample [2].

Bloodstains from pregnant women can be diagnosed using obstetric markers and aid in solving cases related to criminal miscarriages, feticides, infanticides and identification of missing pregnant women. The earlier markers worked on the principle of immunodetection of protein specific for pregnancy needing large amount of bloodstains for detection with lower sensitivity rates. The possibility of utilization of placental derived mRNA in plasma of the mother using RT-PCR in detection of blood stains proved to be promising [3].

Alcohol intake during early pregnancy causes a teratogenic effect on the fetus is a known fact. But unfortunately there is no well documented screening method in pregnancy to detect alcohol use. One marker that is sensitive and specific which is gaining importance is Phosphatidylethanol 16:0/18:1 [4]. The other markers being evaluated are non-oxidative direct ethanol metabolites such as ethyl glucuronide (EtG), ethyl sulphate (EtS) and fatty acid ethyl esters (FAEEs) [5].

Likewise, use of obstetric markers in assistance to solve the various forensic cases have been studied for some time now. Earlier the use of these markers were downplayed, but now they are having an emerging role in aiding to solve crimes.

## **2. Obstetric markers for bloodstains**

#### **2.1 History of the various biomarkers**

Bloodstains at the site of crime are often obtained. But is there a way to detect if the bloodstains belong to a pregnant woman? The answer is yes. But, of course a lot of research has been done in order to find the "ideal" detection method. The base for the research is the use of methods to detect hormones or associated proteins specific for pregnancy and puerperium.

#### *2.1.1 Pregnancy Hormones*

The earliest attempts for detection of **pregnancy hormones** in bloodstains started in the 1900s. The first hormone researched was choriogonadotropins hormones as it was one of the ways to establish that the bloodstain belonged to a pregnant lady. In 1932, Goroncy invented a test based on the Aschheim-Zondek test, which detected pregnancy. He made modifications to it and thought that it could prove valuable as an investigation. Although the drawback of this test was frequent false negative results. So it was not a very effective method to detect if bloodstains belonged to a pregnant woman.

After that many techniques were employed for the same- Hemagglutination Inhibition test, crossed electroimmunodiffusion procedure. In the Hemagglutination test, the test cells are hCG sensitized RBCs. Suspected stain extract with the control was incubated with antihCG titres and then addition of test cells was done. But this method was cumbersome and was discarded.

#### *2.1.2 Pregnancy Specific Proteins*

The detection of **pregnancy specific proteins** in bloodstains to determine the pregnancy status was also attempted. The 4 proteins detected were – PAPP A, HPL, SP1 and Pregnancy associated alpha 2 glycoprotein. In 1971, Bohn reported that by Immunodiffusion, four proteins could be detected in pregnant serum. The first component was identical to hPL, one another was an a2-glycoprotein, while the remaining two were glycoproteins. In the pregnancy serum, four antigens were detected by Gall and Halbert in 1972. They named them as pregnancy associated plasma proteins A, B, C and D, or PAPP-A, -B,-C and –D. In 1973, Bohn coined the term SP1 for the pregnancy specific 8 – glycoprotein [6]. A lot of research was done to establish which of the hormones or proteins were the best indicator of pregnancy with high specificity [7].

Strejc et al. published a paper in 1989 wherein he detected SP 1 by the process of Immunoprecipitation using self-produced antiserum and it was considered a reliable marker, though, the drawback was that the detection was after 4th month of pregnancy and the reliability was 91–95%.

**125**

**Figure 1.**

*Human placental lactogen mRNA nucleotide (source: [15]).*

*Obstetric Markers as a Diagnostic Forensic Tool DOI: http://dx.doi.org/10.5772/intechopen.97670*

*2.1.3 Placental Messenger RNA (mRNA)*

beta hCG mRNA which is detected in maternal plasma.

tests of pregnancy which have high chances of false positivity [13].

reliable and utilized [9].

determine which is ideal.

Then came the detection of hCG by the Enzyme technique, which proved

aminopeptidase(CAP) and leucine aminopeptidase (LAP), which were detected only after fourth month of gestation. In 1973, Oya et al. demonstrated that alkaline phosphatase which is heat stable was detected in pregnancy after fourth month. Only disadvantage was the large quantity of blood required. But this method was

The big breakthrough came with the promising technique of detection of **Placental mRNA** for beta -hCG & hPL by RT-PCR in the bloodstains from pregnant women [10]. A lot of research was done in the 21st century with respect to this. **What is mRNA?** mRNA is the step in between protein-encoding DNA translation and the proteins production by ribosomes [11]. Placenta expressed genes based mRNA transcripts easily detected in maternal plasma prove that the source of fetal nucleic acid release is placenta [12]. Only placental trophoblasts express hPL and

**Method of Detection of mRNA**: In order to detect presence of these mRNA, RT-PCR is the method of choice. Advantages of the RT-PCR technique is manifold. First it is highly specific and sensitive. The second advantage is that it can be designed to be human specific, proving to be advantageous over the immunological

The controversy was that mRNA was thought to be highly unstable and was considered to be degraded quickly making its use as a possible detection tool in old stains questionable. But few studies have refuted this dogma and there are instances

**Human Placental Lactogen**: The various parts of the hPL mRNA is depicted in **Figure 1**. As per the demonstration, RT-PCR detection of hPL was noted throughout pregnancy increasing until delivery and it was undetectable within 24 hours following delivery, which makes it an ideal test [13]. The added advantage is that, when there is a mixed sample, it can be designed to be human specific [10].

wherein the mRNA has been detected in bloodstains as old as 16 years [14]. An ideal mRNA-based test should be in detectable quantities early in pregnancy as well as throughout pregnancy with rapid clearance after delivery. Out of the known 11 genes which were reported to have pregnancy-specific expression patterns, only 2 genes have been thoroughly studied - β-subunit of the hCG and human placental lactogen [13]. Let us look at both the molecules closely to

to be faster and sensitive and specific [8]. Initially, the Polyacrylamide disc gel Electrophoresis was employed by Oya et al., to examine cysteine

#### *Obstetric Markers as a Diagnostic Forensic Tool DOI: http://dx.doi.org/10.5772/intechopen.97670*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

(EtG), ethyl sulphate (EtS) and fatty acid ethyl esters (FAEEs) [5].

**2. Obstetric markers for bloodstains**

**2.1 History of the various biomarkers**

specific for pregnancy and puerperium.

*2.1.1 Pregnancy Hormones*

belonged to a pregnant woman.

*2.1.2 Pregnancy Specific Proteins*

of pregnancy and the reliability was 91–95%.

Alcohol intake during early pregnancy causes a teratogenic effect on the fetus is a known fact. But unfortunately there is no well documented screening method in pregnancy to detect alcohol use. One marker that is sensitive and specific which is gaining importance is Phosphatidylethanol 16:0/18:1 [4]. The other markers being evaluated are non-oxidative direct ethanol metabolites such as ethyl glucuronide

Likewise, use of obstetric markers in assistance to solve the various forensic cases have been studied for some time now. Earlier the use of these markers were downplayed, but now they are having an emerging role in aiding to solve crimes.

Bloodstains at the site of crime are often obtained. But is there a way to detect if the bloodstains belong to a pregnant woman? The answer is yes. But, of course a lot of research has been done in order to find the "ideal" detection method. The base for the research is the use of methods to detect hormones or associated proteins

The earliest attempts for detection of **pregnancy hormones** in bloodstains started in the 1900s. The first hormone researched was choriogonadotropins hormones as it was one of the ways to establish that the bloodstain belonged to a pregnant lady. In 1932, Goroncy invented a test based on the Aschheim-Zondek test, which detected pregnancy. He made modifications to it and thought that it could prove valuable as an investigation. Although the drawback of this test was frequent false negative results. So it was not a very effective method to detect if bloodstains

After that many techniques were employed for the same- Hemagglutination

The detection of **pregnancy specific proteins** in bloodstains to determine the pregnancy status was also attempted. The 4 proteins detected were – PAPP A, HPL, SP1 and Pregnancy associated alpha 2 glycoprotein. In 1971, Bohn reported that by Immunodiffusion, four proteins could be detected in pregnant serum. The first component was identical to hPL, one another was an a2-glycoprotein, while the remaining two were glycoproteins. In the pregnancy serum, four antigens were detected by Gall and Halbert in 1972. They named them as pregnancy associated plasma proteins A, B, C and D, or PAPP-A, -B,-C and –D. In 1973, Bohn coined the term SP1 for the pregnancy specific 8 – glycoprotein [6]. A lot of research was done to establish which of the hormones or proteins were the best indicator of pregnancy with high specificity [7]. Strejc et al. published a paper in 1989 wherein he detected SP 1 by the process of Immunoprecipitation using self-produced antiserum and it was considered a reliable marker, though, the drawback was that the detection was after 4th month

Hemagglutination test, the test cells are hCG sensitized RBCs. Suspected stain extract with the control was incubated with antihCG titres and then addition of test

Inhibition test, crossed electroimmunodiffusion procedure. In the

cells was done. But this method was cumbersome and was discarded.

**124**

Then came the detection of hCG by the Enzyme technique, which proved to be faster and sensitive and specific [8]. Initially, the Polyacrylamide disc gel Electrophoresis was employed by Oya et al., to examine cysteine aminopeptidase(CAP) and leucine aminopeptidase (LAP), which were detected only after fourth month of gestation. In 1973, Oya et al. demonstrated that alkaline phosphatase which is heat stable was detected in pregnancy after fourth month. Only disadvantage was the large quantity of blood required. But this method was reliable and utilized [9].
