Problems in identification of muscle origin.

In July 2000 D.M. (born in 1986), E:S (born in 1940), and I.S.(born in 1964) disappeared. During repairing of the draining pipes connected with the living place of these missing persons, pieces of meat were found. The possible human origin of these pieces of meat has to be identified. These pieces of meat were preserved for several weeks in unbuffered formalin.

In August 2000 man and female corpses were found and they were identified by police as D.M. and E.S. The dead body of I.S. has not been found yet.

The research of tissues suspected of human origin.

Pieces of muscle tissue with skin and underskin layer preserved in unbuffered formalin were presented to the research.

The material (tissue held in unbuffered formalin) was analysed with next methods:

1. outer examination

2. microscopy, during which skin of human and pig origin was compared with the skin from the pieces preserved in formalin

3. immuno-histchemical research during which next antibodies for human proteins were used: Anti-Human Cytokeratin, Anti-Vimetin, Anti-Human Desmin, Anti-human Von Willebrand Factor (F VIII), Anti-HLA-2.

Immuno-histochemical research was carried out on the material of possible human origin and the pig tissues brought from the slaughterhouse. Possible cross-reactions of antibodies Anti-F VIII and Anti-HLA-2 with the material of animal origin was tested with the immunoblot method using the material from a pig, hen, sheep, horse and cow.

4. DNA analyses

1. isolation of DNA with phenol-chloroform method;

2. DNA examination using the test-system SGM Plus, which is ment to analyse material of human origin. The result of the analyse was forced with capillarysequenator ABI PRISM 310.

3. DNA analyse using the system for examination HAL gene of pigs;

4. Analyse of the mitochondrial DNA. The first hypervariable region of the mitochondrial DNA was amplified with the help of polymerase chain reaction.

Results of the research.

1. Outer examination

During the outer examination of the pieces of tissue of the possible human origin it came out, that the skin on several samples was covered with thin hair and reminded so the skin of human or pig origin. The thickness of the underskin fat layer was some centimetres - that excludes the possibility that the pieces of tissue belong to some small animal. The colour and the structure of the fat reminded more that of a human being than a pig

2. Comparing microscopy

Next objects were analysed:

1. human skin (from the autopsy material)

2. skin of the pig (from the slaughteryhouse right after animal's execution, no mechanical nor thermic manipulations followed)

3. skin from the tissue pieces (preserved in formalin) of possible human origin.

The structure of the unharmed skin of human or pig origin is microscopically so similar that it is impossible to separate them trustworthily.

3)Immuno-histochemical research

The monoclonal antibodies Anti-Human Cytokeratin, Anti-Vimetin, Anti-Human Desmin gave immunohistochemical reaction with both materials - those from pig and from the possible human origin.

The test with human F VIII and HLA-2 antibodies gave positive reaction only with the material of possible human origin (there was no positive reaction with the material from the pig). Controlling possible cross-reactions of the last two antibodies they gave a weak positive reaction with the material of animal origin, too.

1. DNA analyse

It was not possible to get any result while analysing DNA from the tissue preserved in formalin using test system SGM Plus for genome DNA, amplifying the first hypervariable region of the mitochondrial DNA and searching the HAL gene of the pig.

The possible human origin of the tissues preserved in formalin stays without a sure conclusion. The result of the research is:

a) The thin hair indicates to the skin of possible human or pig origin.

b) The thickness (couples of centimetres) of the underskin layer excludes the possibility that the material belongs to a small animal.

c) During the microcopy the antibodies against human proteins were used; clearly positive result with the material of possible human origin came out using two antibodies (anti-F VIII, anti-HLA-2) and there was negative result with the material from pig. This gives us the information that it can be human material. But at the same time with immunoblot method it came out that these antibodies can give cross-reaction with animal tissues depending on different conditions. Due to this cross-reaction we cannot consider the result of this test with antibodies to be a correct proof that the material of possible human origin is human indeed.

d) During DNA analyse it was impossible to separate any suitable DNA from the material preserved in formalin. Obviously it's due to conditions in which the material was held.

In this case there is no sure proof if the material preserved in formalin is of human or animal origin. So it isn't possible to make one clear conclusion and this case remains only on the level of description of the findings.

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