Cytocentrifugation to Detect Cellular Material

After verifying the integrity of the adhesive tape placed around the box containing the tubes in which the fragments of the swabs performed on 03/22/2011 were inserted, we proceeded with further laboratory tests.

Analysis to detect cellular material was conducted via the technique of cytocentrifugation, by means of the Cytospin 3 apparatus produced by Shandon. A cytocentrifuge is an instrument which employs centrifugal force to isolate and prepare a cellular monostrate on appropriate slides, and at the same time, allows cellular integrity to be preserved. From each sample for DNA extraction, a small cotton fragment was removed, on which analysis to detect any cellular material present was performed.

Before proceeding with the analysis of the samples, all the working surfaces were treated with disinfecting and antifungal solutions, left to act for about 30 minutes before cleaning. Furthermore, to limit any contamination as far as possible, an appropriate support was used to prevent contact between the plate containing the samples and the working surfaces.

All the operations were carried out using disposable gloves, which were regularly changed between one sample and the next.

Each sample was washed by means of repeated pipetting, in a 90-μL volume of extraction swab. The entire volume was then loaded onto a sample holder with a centrifuge and a sterile, disposable funnel, and subjected to sedimentation using the following parameters: time 5 minutes, velocity 800 rotations per minute (rpm), average acceleration. The entire operation was performed at room temperature.

After cytocentrifugation, the samples were immediately observed with an optical microscope to determine the presence of material deposited on the slide.

With prior notice to the parties, on May 30, 2011, at 11:30 am, at the Histology and Medical Embryology Section of the University of Rome — La Sapienza, we proceeded with the coloration of the cytocentrifugation slides, obtained on April 5, 2011, and the acquisition of images of the slides themselves.

On this date, no consultants for the parties were present.

The following memorandum was drafted: “We note that none of the consultants for the parties, regularly informed of the testing session, were present. Consequently, the Appointed Experts performed what was announced (photographic documentation of cytocentrifugation results). The present memorandum ends at 1:30 pm.”

In order to determine the presence of any cellular material, each sample was colored with hematoxylin (Hematoxylin, Vector Laboratories, CA. Cat. H-3401). Hematoxylin is a common basic colorant used to highlight acidic cellular structures such as endoplasmic reticulum and nucleic acids contained in the cell nucleus. The structures colored with hematoxylin appear blue in color.

Excess colorant was removed with water and each sample was analyzed with an optical microscope with 20X magnification. A ZEISS Axioskop 2 plus microscope was used for this purpose, and the slides were individually photographed.

Descriptions and images of the material present in the individual slides are reported in the following table:

[table part 1]

[table part 2]

[table part 3]

[translations of descriptions:

A: Sample presents various debris of inorganic nature. Granules with circular/hexagonal morphology with a radial and/or linear structure are evident. No cellular material is present.

B: Sample presents debris of inorganic nature. No cellular material is present.

C: Sample presents various debris of inorganic nature. No cellular material is present.

D: Sample presents various debris of inorganic nature. No cellular material is present.

E: Sample presents a few pieces of debris and inorganic crystals. A very few granules with circular/hexagonal morphology and central linear structure are present. Cellular material is not evident.

F: Sample presents debris and a very few granules with circular/hexagonal morphology with central radial structure. Cellular material is not present.

G: Sample presents debris of inorganic nature. Cellular material is not present.

H: Sample presents numerous granules with circular/hexagonal morphology with central radial structure, identical to those observed in other samples. Sample H, however, displays a decisively higher number of these structures compared to the samples already observed. Cellular material is not present.

I: Sample presents various debris of inorganic nature. Certain granules with central radial structure identical to those observed in previous samples are present. Cellular material is not present.

L: Sample presents certain precipitates of intense color but certainly not of cellular nature. Probably oxidized precipitates.

M: Sample M appears similar to sample L. The density of the fragments is decisively higher. Granules are not in evidence. Cellular material is not present.

C.N.: Sample presents rare debris of inorganic nature.]

Analysis and photographic examination of various images present in the literature allowed us to establish that the structures present in certain slides (granules with circular/hexagonal morphology with central radial structure) present a morphology attributable to that of granules of amide, a polysaccaridic carbohydrate of vegetable nature. The sample which presents the greatest concentration of these structures is sample H. By means of microscopic analysis, moreover, it was possible to determine that the dimensions of the individual granules are between 3-10 μm approximately.

Next: Conclusions (1)