The Case of the Vasectomized Rapist
By Arthur W. Young
One of the many advantages of working at a private laboratory is the ability to assist other crime labs which lack the technology or expertise with which to bring a case to a close. Here, technology and expertise go in two directions: into the future… and, sometimes, into the past. Consider a recent rape case of a teenaged girl, where the middle-aged suspect was vasectomized: How do you analyze DNA when there are no sperm cells?
That’s the question a colleague of mine asked me when this case came to her. She had positively identified semen on the victim's underwear, but despite two attempts at DNA analysis, the most that she could find was a small signal indicating the presence of a tiny amount of Y chromosome. The problem was that the DNA analysis was targeting on the girl, due to her overwhelming contribution to each of the samples.
The first thing that the reader should know is that no vasectomy is ever complete. On numerous occasions, it has been observed that semen from vasectomized men still contain sperm. That's because the medical definition of “sterile” isn’t the same as the forensic one. In an entire ejaculate, there may only be a few thousand spermatozoa. Theoretically, it’s just a matter of finding and isolating them. Practically, however, it's like finding a needle in a haystack.
The first old method that was used was known as “AP mapping.” AP is short for acid phosphatase, which is an enzyme that is present in very large quantities in semen. However, it isn’t unique to semen; it can be found elsewhere, including bacteria. For this reason, it’s a good preliminary test for semen, but not a confirmatory one. It’s not a commonly-used method in today’s DNA age, and that’s a shame: just because something isn’t new doesn’t mean that it’s bad. The technique begins by laying a moistened piece of filter paper on the crotch of the girl’s underwear. Acid phosphatase is soluble in water, so it will transfer to the moist filter paper quite readily. However, the transfer can only be as good as the contact between the two surfaces, and the underwear’s irregular seams tended to trap air bubbles. Normally, this method calls for the use of a weight (like a book) but I opted for a large plastic bag full of sand: the sand will conform to the uneven surface of the underwear while still applying weight. A piece of clear plastic wrap between the filter paper and sandbag protects the filter paper from contamination caused by the sandbag, but it also lets me use the sandbag again.
The next step involves treating the filter paper with alpha-naphthyl acid phosphate, a light brown chemical that turns purple in the presence of acid phosphatase. Like a treasure map, the treated filter paper reveals the location of probable semen stains for further investigation. My hope: to find a place where the male is present in higher quantities than the female.
Spermatozoa have a singular function, for which, they are highly specialized: fertilize an ovum. To do this, spermatozoa must endure an arduous trip through the female reproductive system, which is fraught with changes in pH, bacteria, and her own immune system. As a defense, spermatozoa have an extremely durable outer shell, which is rich in disulfide bonds (a type of molecular bond that gives tremendous strength to proteins and can be found most commonly in hair and fingernails). That difference in durability can be exploited, and DNA analysts can separate sperm cells from all other types of cells, in a method known as a differential DNA extraction.
Having identified the most likely places where semen was deposited on the underwear, a differential DNA extraction was performed on three likely samples. I am hopeful, but I am also concerned: the AP map suggested that the semen may not be localized enough to be able to collect a sufficient number of spermatozoa to get a DNA profile. Surely enough, the sperm fraction is clean. Time to go to Plan B.
"Plan B" is a relatively new technology called Y-STR analysis that analyzes small segments of DNA on the Y chromosome. Since the Y chromosome is what determines whether a person is male or female, this specialized DNA analysis is male-specific. In fact, these sequences are so unique that, in the entire animal kingdom, they can only be found in the male primate. Y-STR analysis can ignore contributions of DNA from a female and home in on the smallest contribution from a male, even if the male contribution is less than 0.1%. However, these segments are transmitted from father to son with no changes (unless there is a mutation, which is an exceedingly rare event). As such, this analysis can’t differentiate between a father and his son, because they share the same Y chromosome and will therefore share the same Y-STR profile. As a corollary to that fact, it can’t differentiate between two sons of the same father… nor the sons of those sons. In short, any two males who have a direct paternal lineage can’t be distinguished with this analysis. Despite its limitations, it is successful: one of the three samples produced a profile, and it was the same as that of the suspect.
We didn't have problems with vasectomized rapists in the old days. I am one of the few practitioners of the old methods left, known as forensic serology. Admittedly, the methods look like alchemy compared to today’s computer-driven DNA analysis, but there were distinct advantages. However, we were never bothered by vasectomized individuals then, because the markers we were analyzing were biochemical ones, and those could be found in the seminal fluid--not the sperm.
The entire human population can be divided into two groups: the “secretors” and the “non-secretors.” About 75% of people are secretors, or people who secrete water-soluble molecules of their blood type in their non-blood fluids like saliva, semen, and vaginal fluid. Hence, a secretor who has ABO Type A blood will produce detectable levels of ABO Type A antigens in his/her non-blood fluids, like saliva. The remaining 25% are non-secretors. Regardless of whether you’re a secretor or a non-secretor, one thing is certain: it’s a genetically-inherited trait.
Once again, I’m likely to face problems with contributions from the girl, where her ABO type could interfere with my ability to find information about him. Worst case scenario: they’re both secretors and also share the same blood type. Best case scenario: she’s a non-secretor and he’s a secretor with ABO Type AB blood, the rarest of blood types. Given that there are only four possible ABO types (A, B, O, and AB), the chances that they are both secretors and the same blood type is pretty high, especially since ABO Type A and ABO Type O blood comprise about 85% of the human population.
Another old system is phosphoglucomutase (PGM), an enzyme involved in converting glycogen (an intermediate fuel source for your body similar to starch) to fat, and back. Like ABO type, everyone has it, but not everyone has the same type. Hence, systems like PGM were known as isomeric enzymes, or isozymes. Unlike blood type, however, there are ten possible types, thus reducing the probability of any two people sharing a single type. There’s a distinct advantage here, in that PGM levels in semen are significantly higher than those found in vaginal fluid, so the girl’s type won’t be much of an issue.
The third and final polymorphic isoenzyme system that can be found in semen is peptidase A (PepA). This one wasn't terribly useful, however, as variants are fairly rare. In fact, peptidase A type 1 (PepA:1) occurs in over 98% of Caucasians. Peptidase A type 2 (PepA:2) and the heterozygous form, type 2-1 (PepA:2-1) occur most commonly among those of African descent.
That’s the question a colleague of mine asked me when this case came to her. She had positively identified semen on the victim's underwear, but despite two attempts at DNA analysis, the most that she could find was a small signal indicating the presence of a tiny amount of Y chromosome. The problem was that the DNA analysis was targeting on the girl, due to her overwhelming contribution to each of the samples.
The first thing that the reader should know is that no vasectomy is ever complete. On numerous occasions, it has been observed that semen from vasectomized men still contain sperm. That's because the medical definition of “sterile” isn’t the same as the forensic one. In an entire ejaculate, there may only be a few thousand spermatozoa. Theoretically, it’s just a matter of finding and isolating them. Practically, however, it's like finding a needle in a haystack.
The first old method that was used was known as “AP mapping.” AP is short for acid phosphatase, which is an enzyme that is present in very large quantities in semen. However, it isn’t unique to semen; it can be found elsewhere, including bacteria. For this reason, it’s a good preliminary test for semen, but not a confirmatory one. It’s not a commonly-used method in today’s DNA age, and that’s a shame: just because something isn’t new doesn’t mean that it’s bad. The technique begins by laying a moistened piece of filter paper on the crotch of the girl’s underwear. Acid phosphatase is soluble in water, so it will transfer to the moist filter paper quite readily. However, the transfer can only be as good as the contact between the two surfaces, and the underwear’s irregular seams tended to trap air bubbles. Normally, this method calls for the use of a weight (like a book) but I opted for a large plastic bag full of sand: the sand will conform to the uneven surface of the underwear while still applying weight. A piece of clear plastic wrap between the filter paper and sandbag protects the filter paper from contamination caused by the sandbag, but it also lets me use the sandbag again.
The next step involves treating the filter paper with alpha-naphthyl acid phosphate, a light brown chemical that turns purple in the presence of acid phosphatase. Like a treasure map, the treated filter paper reveals the location of probable semen stains for further investigation. My hope: to find a place where the male is present in higher quantities than the female.
Spermatozoa have a singular function, for which, they are highly specialized: fertilize an ovum. To do this, spermatozoa must endure an arduous trip through the female reproductive system, which is fraught with changes in pH, bacteria, and her own immune system. As a defense, spermatozoa have an extremely durable outer shell, which is rich in disulfide bonds (a type of molecular bond that gives tremendous strength to proteins and can be found most commonly in hair and fingernails). That difference in durability can be exploited, and DNA analysts can separate sperm cells from all other types of cells, in a method known as a differential DNA extraction.
Having identified the most likely places where semen was deposited on the underwear, a differential DNA extraction was performed on three likely samples. I am hopeful, but I am also concerned: the AP map suggested that the semen may not be localized enough to be able to collect a sufficient number of spermatozoa to get a DNA profile. Surely enough, the sperm fraction is clean. Time to go to Plan B.
"Plan B" is a relatively new technology called Y-STR analysis that analyzes small segments of DNA on the Y chromosome. Since the Y chromosome is what determines whether a person is male or female, this specialized DNA analysis is male-specific. In fact, these sequences are so unique that, in the entire animal kingdom, they can only be found in the male primate. Y-STR analysis can ignore contributions of DNA from a female and home in on the smallest contribution from a male, even if the male contribution is less than 0.1%. However, these segments are transmitted from father to son with no changes (unless there is a mutation, which is an exceedingly rare event). As such, this analysis can’t differentiate between a father and his son, because they share the same Y chromosome and will therefore share the same Y-STR profile. As a corollary to that fact, it can’t differentiate between two sons of the same father… nor the sons of those sons. In short, any two males who have a direct paternal lineage can’t be distinguished with this analysis. Despite its limitations, it is successful: one of the three samples produced a profile, and it was the same as that of the suspect.
We didn't have problems with vasectomized rapists in the old days. I am one of the few practitioners of the old methods left, known as forensic serology. Admittedly, the methods look like alchemy compared to today’s computer-driven DNA analysis, but there were distinct advantages. However, we were never bothered by vasectomized individuals then, because the markers we were analyzing were biochemical ones, and those could be found in the seminal fluid--not the sperm.
The entire human population can be divided into two groups: the “secretors” and the “non-secretors.” About 75% of people are secretors, or people who secrete water-soluble molecules of their blood type in their non-blood fluids like saliva, semen, and vaginal fluid. Hence, a secretor who has ABO Type A blood will produce detectable levels of ABO Type A antigens in his/her non-blood fluids, like saliva. The remaining 25% are non-secretors. Regardless of whether you’re a secretor or a non-secretor, one thing is certain: it’s a genetically-inherited trait.
Once again, I’m likely to face problems with contributions from the girl, where her ABO type could interfere with my ability to find information about him. Worst case scenario: they’re both secretors and also share the same blood type. Best case scenario: she’s a non-secretor and he’s a secretor with ABO Type AB blood, the rarest of blood types. Given that there are only four possible ABO types (A, B, O, and AB), the chances that they are both secretors and the same blood type is pretty high, especially since ABO Type A and ABO Type O blood comprise about 85% of the human population.
Another old system is phosphoglucomutase (PGM), an enzyme involved in converting glycogen (an intermediate fuel source for your body similar to starch) to fat, and back. Like ABO type, everyone has it, but not everyone has the same type. Hence, systems like PGM were known as isomeric enzymes, or isozymes. Unlike blood type, however, there are ten possible types, thus reducing the probability of any two people sharing a single type. There’s a distinct advantage here, in that PGM levels in semen are significantly higher than those found in vaginal fluid, so the girl’s type won’t be much of an issue.
The third and final polymorphic isoenzyme system that can be found in semen is peptidase A (PepA). This one wasn't terribly useful, however, as variants are fairly rare. In fact, peptidase A type 1 (PepA:1) occurs in over 98% of Caucasians. Peptidase A type 2 (PepA:2) and the heterozygous form, type 2-1 (PepA:2-1) occur most commonly among those of African descent.