Histological biochemical analysis

The aim of laser micro-isolating applications is to isolate specific nucleated cells and extract DNA for STR typing in order to determine a DNA profile (Plate 10.7). The first step is to make a cell smear on a microscope glass in order to visualize the specimen under the microscope lens. The method for specimen preparation depends on the nature of the biological material to be analysed (Di Martino et al., 2004a, 2004b). In any case, the material of interest must be collected on a supporting synthetic polymeric membrane - polyethylene tereph-thalate (PET) or polyethylene naphthalate (PEN) - that may be mounted onto a glass slide or a metallic frame. In this way, during the laser cutting of an area containing a distinct cell type, a fragment of supporting membrane is collected together with the sample of interest into the microtube. The presence of the membrane does not interfere with the analytical procedures. Prior to specimen mounting, in order to avoid nucleic acid contaminations of the membrane, all procedures take place in a dedicated DNA-free area, and the membrane is sterilized by autoclaving and/or UV treatment. The latter is particularly useful because it eliminates electrostatic charges from the membrane, thus avoiding adhesive effects on the laser-cut fragment that could interfere with harvesting the cells.

In forensic analysis, a wide range of material, such as blood, organic fluids, hair or a mixture, may be collected on the membrane and subsequently subjected to morphological identification by laser-based methods. Specific identification of cell type, such as spermatozoa, epithelial cells from mucosae or skin, leucocytes or cells from hair follicles, frequently requires the use of several microscopic procedures.

The cell smear can be stained or left unstained. In the former case the operator must employ a stain that will not damage nuclear DNA, whereas in the latter case phase-contrast microscopy is used. It is often preferable to work on stained samples if the forensic laboratory is supported by a histological service that can perform the most appropriate cytological/histological staining. Good slide preparation and cellular morphology is essential to correctly distinguish different cell types. It will also ensure that the quality of DNA is sufficient to produce an unambiguous result. Many staining procedures have been investigated, leading to the identification of Papanicolau, Haematoxylin/Eosin and Giemsa as the basis of the best staining protocols for forensic applications. For the great majority of specimens, the optimal balance between morphology and DNA integrity is usually obtained by air fixation and Giemsa staining (Di Martino et al., 2004a). The above-mentioned stains do not intercalate DNA nor are they able to fragment the DNA backbone. Importantly they do not cause polymerase chain reaction (PCR) inhibition either. Other chemical staining agents, such as Nuclear Fast Red-Picroindigocarmine, may influence DNA preservation in spermatozoa and result in poor single random repeat (STR) DNA profiles. No matter which stain is used, in order to avoid contamination all the staining steps must be performed by dispensing the reagents on the surface of the specimen, which is then placed horizontally into an incubation chamber. Before starting the microdissection procedure, it is very important to verify that the specimen is dry, especially when membrane-coated slides are utilized, because any moisture present between the glass surface and the membrane may interfere with the detachment of the cut portion. An important step shared by all microdissection procedures is inspection of the cap of the microtube to confirm that the cut fragment has been collected. To facilitate the identification of membrane fragments, it is useful to leave a margin around the cell, drawing an irregular distinguishable outline.

Extraction of DNA from cell samples collected by laser microdissection is another delicate step. Different extraction protocols will be required according to the kind of specimen the operator is dealing with. Cell types differ in their biochemical and ultrastuctural properties: for example, amounts of fatty acids, phospholipids and proteins, the presence or absence of a cell wall, etc. Certain types of cell are more resistant to lysis, which is the primary step in DNA extraction. Lysis/DNA extraction takes place in a pH-controlled aqueous buffer with a hydrophobic environment useful for membrane disruption and in the presence of a protease.

Because forensic specimens are often typified by very low concentrations of potentially degraded DNA, consideration must be given to different extraction protocols at the outset of each case so that the best method can be employed (Di Martino et al., 2004b; Giuffre et al., 2004).

A wide range of extraction kits/products are commercially available. Not all are suited to laser microdissected samples because of the small physical dimensions of the samples, the small amounts of DNA and the presence of PCR inhibitors, etc. Three types of reagents have emerged as being most suited to DNA extraction from laser microdissected samples (Chelex®, ion exchange chromatography and magnetic resin) and modified protocols have been developed in all cases.

1. Chelex®: a resin capable of chelating bivalent ions present in the cell lysate; such ions are potential PCR inhibitors and nuclease cofactors. The advantage of this method is that the whole lysate may be treated without further cleavage, although this is not a purification system. The concentration of the resin solution depends on the kind of cell sample under investigation, while the amount of resin required is usually limited in the case of laser microdissected samples. The extraction volume is related to the number of microdissected cells and can be decreased to a few microlitres in the case of DNA extraction from a single cell (Staiti et al., 2005).

2. Ion exchange chromatography: this is a good purification system that can be used for some difficult samples. It is based on alkaline lysis and the separation of cellular debris from DNA using an ion-exchange silica column. Its application to laser microdissected samples is dependent on the number of cells available (down to 8-10 haploid cells), but it is not so useful when DNA extraction is performed on five cells or less. A potential drawback is that DNA damage could occur as a result of the alkaline environment, which is very important when working in low copy number conditions. DNA extracted from 1-10 microdissected cells needs to be dissolved in very small volumes of buffer solution because it will greatly influence the quality of the subsequent PCR.

3. Magnetic resin: a purification system based upon the separation of cell debris from DNA by magnetic beads suspended in the same solution as the cell lysate. The chemistry of the system is most suited to laser microdissected spermatozoa and hair bulbs. As a consequence of the buffer composition, complete recovery when working with a very restricted number of cells (one or two) is seldom possible. Moreover, it is a very challenging method that requires great operating skill, otherwise DNA may become degraded. This has been noticed especially when dealing with telogen laser microdissected hair bulbs, where old and totally keratinized cells are present and their DNA is already partially fragmented.

Laser microdissection allows operators to isolate specific cells from minute samples to determine DNA profiles, gender and species of origin (Di Martino et al., 2005). As laser microdissection generates DNA templates at the lower limits of concentration, it is essential to consider the stoichiometry of the PCR.

There are two PCR strategies in general use. First, DNA can be separated into several aliquots and individually amplified. This is the traditional method of nucleic acid amplification, which can be applied to laser microdissected samples. This approach allows the detection of allelic drop-out since multiple aliquots are available for analysis. Second, the operator performs a single reaction of amplification, using all the DNA solution extracted from the forensic specimen. In principle any allelic drop-out observed is likely to be a biologically related phenomenon rather than an artefact of the PCR (Plate 10.8).

An intermediate situation between a complete STR-typed allelic profile and one affected by allelic drop-out can occur as a consequence of the reduced amount of DNA template in the PCR solution. When examining genotype peak heights it is important to discriminate, within an apparent heterozygous genotype, whether unbalanced alleles are due to the presence of a stutter product at that locus. Only a precise determination of allele peak areas or, even better, an initial design of a duplicate reaction set for each sample can assist in this situation.

In summary, operators aiming to perform analysis of low copy number DNA by the laser microdissection technique should take into account the following guidelines:

1. Adjust the protocols in order to work on low volumes.

2. Reach the maximum grade of purity of the extracted DNA; additionally evaluate the possibility to increase the injection time during capillary elec-trophoresis in order to reduce the influence of low-molecular-weight particles.

3. Duplicate the PCR assay on the same sample in order to potentially minimize any stochastic effect.

4. Avoid unnecessarily increasing the number of PCR cycles; the Taq polymer-ase concentration can be increased within the PCR reaction mix.

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