9 - semen identification in a forensic sample - part 3

ABO typing of semen in secretors is conventionally conducted using the technique of absorption-inhibition. The principle behind the technique is simple, and can be illustrated with reference to an A secretor. If anti-A is added to a sample (semen, saliva or a semen stain extract), the antibody will complex with the antigen in the sample. If a suspension of A cells is now added, there will be no agglutination since there is no free antibody in solution. A matrix can be set up to cover A, B, and H. There are some important factors to remember when conducting the test:

• The indicator cells are best prepared as a weak suspension in a saline-albumin solution.
• The antisera should be titrated against the indicator cells and used at the weakest dilution that will give a reliable result.
• The titration is conducted using serial dilutions.
• Negative results in inhibitions may be due to small sample size or a weak expression of the Se gene.
• Negative results should be reported as “No ABH activity detected.”
• Absolute definition of secretor status requires Lewis typing of RBCs and confirmation of the presence of Lewis b substance.

Some laboratories do not perform inhibitions but go straight to absorption-elution. The principle behind this approach is that it will detect ABH activity in all cases, not just secretors. However, certain issues must be noted:

• Invoking conclusions about secretor status from absorbtion-elution results is not reliable since the difference is quantitative.
• High levels of antigen can result in false negative in absorption-elution as the Ab-Ag complex dissolves in the excess Ag.
• It is always best to prepare at least a 1:10 dilution of extract in absorption-elution to try to overcome these problems.

8 - semen identification in forensic sample - part 2

The ability to draw inferences as to the origin of semen in a sexual assault case is obvious. The power to do this in traditional serology was limited, and depended mainly on ABO and PGM typing. The PGM₁ locus is expressed in semen and vaginal secretions, and the methodology and interpretation used in its typing are exactly the same as for blood.

ABO typing is somewhat different. Almost everyone has at least trace levels of antigen in their body secretions that correspond to their ABO blood type. However, about 80% of the population has very high levels of these antigens in body secretions. These persons are described as secretors. The remaining 20% of the population are described as non-secretors and have concentrations of the antigens that are too low for normal detection. The quantitative difference is genetically determined. The gene responsible has two alleles, a dominant form, Se, and a recessive one, se. Thus SeSe and Sese persons are secretors and sese persons are non-secretors.

The A, B, and H antigens are polysaccharides. They are found on RBC surfaces as lipo-polysaccharides and in secretions as glycoproteins. The biochemical genetics involves four genes as shown in the table below.

Biochemical Genetics
Gene	Expression	RBC genotype	Secretions genotype
Le, H and secretor	Dominant H allele in presence of dominant Se allele converts Lea substance to mixture of H, Lea and Leb substances	Depends on A and B alleles	Leaand Leb substances present together with A and/or B depending on A and B alleles
Le, ABO A allele together with H and Se	A blood group, Lewis a and b	A, H, Le(a-b+)	Lea, Leb , A, H
Le, ABO B allele together with H and Se	B blood group, Lewis a and b	B, H, Le(a-b+)	Lea, Leb , B, H
Le, ABO O (silent) allele together with H and Se	O blood group (No A or B blood group), Lewis a and b	H, Le(a-b+)	Lea, Leb , H
Le, H and sese	A, B or H depending on ABO gene	A. B. H depending on ABO gene, Le(a+b-)	Lea only
Le, hh and Se	no ABH or Lewis	No ABO activity (Bombay phenotype)	Lea only
lele, H and Se	ABO depending on ABO gene alleles, no Lewis	A, B, H (depending on ABO alleles), Le(a-b-)	A, B, H (depending on ABO alleles), no Lewis
lele, H and sese	A, B, or H depending on ABO gene	A, B, H depending on ABO gene, Le(a-b-)	No ABH, no Lewis
lele, hh and Se	No ABH or Lewis	No ABO activity (Bombay phenotype) Le (a-b-)	No ABH, no Lewis
Note: Secretors all have an Le allele, an H allele and an Se allele. For simplicity, AB heterozygotes are not listed in the Table – they will have A and B.

7 - semen identification in a forensic sample - part 1

Human semen contains unusually high concentrations of acid phosphatase, which can therefore be the basis of a screening test. The reaction is based on the hydrolysis of phosphate esters and detection of the liberated organic moiety by production of a color complex. For example, the reaction of acid phosphatase with sodium alphanapthylphosphate and fast blue B to produce a purple-blue coloration. As with the screening test for blood, a positive result is the rapid formation of the intensely colored product – less than 20 seconds or so, or 30 seconds at most.

The best identification of semen is from its microscopy. The morphology and dimensions of the human spermatozoon are unique. The small sperm, particularly if they have lost their tails, can be difficult to locate microscopically especially in samples which have bacterial or yeast infection. Detection is simplified by histopathological staining. The most usual stain is popularly known as Christmas tree stain because of the bright colors. It utilizes nuclear fast red that differentially stains the DNA-containing head bright crimson, and a counter-stain of picric acid - indigocarmine (PIC) that stains the tails green-blue-gray. The traditional histological staining of hematoxylin and eosin (H&E) is also used, as is Giemsa stain.

Problems may be encountered if the seminal fluid is from a man who has a low sperm count (oligospermia) or who has no spermatozoa present in his seminal fluid (aspermia). In situations where the presumptive alkaline phosphate test indicates the presence of semen, but the microscopical analysis yields no detectable spermatozoa, tests are carried out to determine the presence of a protein, P30, or prostate specific antigen (PSA), which is only found in high concentration in human semen. Some laboratories even use P30 testing in place of microscopical examination for semen identification. It can be detected by precipitin reaction with a specific antiserum using the Ouchterlony process. There is also a quantitative immunological test utilizing an enzyme-linked reaction (ELISA).

However, the currently accepted method of choice for identification of semen in all circumstances is detection of p30 using the ABAcard^® test strips manufactured by Abacus Diagnostics, Inc. The strips work in the same way as described above for confirmation of blood, except that they use anti-p30 monoclonal and polyclonal antisera, and a pink dye.