Sample Source

There are, essentially, two types of tissue available for nucleic acid analysis: fresh and preserved. The ideal source of nucleic acid is, naturally, fresh tissue. If extraction is not possible immediately, it is critical to rapidly limit the damaging action of tissue endonucleases. Prompt flash-freezing of solid tissue with liquid nitrogen preserves nucleic acids and can facilitate subsequent tissue and cell disruption. Timely freezing is especially important to extract RNA successfully. For RNA purification, fresh tissue can also be placed directly in commercially available reagents that preserve cellular RNA for up to a week at room temperature (Ambion, Austin, TX). RNase and DNase are rapidly denatured in the presence of chaotropic agents like guanidium isothyocyanate (GITC). A minimum concentration of GITC of 5 mol/L is necessary for effective RNA preservation, and GITC-preserved tissue can also be stored at room temperature for almost a week without significant RNA loss (2).

2.1. FRESH TISSUE The utility of fresh tissue is enhanced by the powerful sensitivity of PCR testing. Exfoliated cells obtained by swabbing or rinsing mucous membanes provide enough nucleic acid to allow PCR-based testing of genomic or foreign (infectious) DNA (3,4). Buccal cells, for example, can be obtained noninvasively by swab and air-dried onto a glass slide. Although the cells are unfixed, the DNA is preserved well enough over short periods for transport, extraction, and PCR analysis (3,5). As an alternative, buccal cells are collected with a saline mouthwash and pelleted for immediate analysis.

Cervical cells obtained by swab or brush can also be used for PCR or other DNA testing. This is especially useful in the detection of cervical human papilloma virus (HPV), subtypes of which are associated with increased risk of cervical neoplasia. A commercial system for the detection of HPV DNA has been developed that employs specific RNA probes and chemilumi-nescent antibody (Digene Corporation, Gaithersburg, MD).

From: Molecular Diagnostics: For the Clinical Laboratorian, Second Edition Edited by: W. B. Coleman and G. J. Tsongalis © Humana Press Inc., Totowa, NJ

Table 1

The Effect of Tissue Fixatives on the Purification of Nucleic Acid


Active contents

Tissue effect

Nucleic acid purification

Neutral-buffered formalin

B-5 fixative

Bouin's fixative

Zenker's fixative Alcohol


Mercuric chloride Usually mixed with formaldehyde

Picric acid Acetic acid Formaldehyde Potassium dichromate Mercuric chloride Ethanol or methanol

Nucleic acid base hydroxymethylation Crosslinking of DNA and protein

Mercury-protein complexes reduce DNA extraction yields

Acidic DNA Depurination Formaldehyde effects

Metal-protein complexes

Reduced high-molecular-weight nucleic acid with increased fixation time Suitable for most testing Low molecular weight or no extractable nucleic acid Occasionally nucleic acid sufficient for PCR testing Low molecular weight or no extractable nucleic acid

Low molecular weight or no extractable nucleic acid Good or excellent nucleic acid yields, including high molecular weight Fixation time has no effect

Cervical swabs collected with this kit are useful for up to 2 wk at room temperature, and longer with refrigeration. Fixed cytologic preparations, such as Papanicolaou-stained cervical smears, can also provide useful nucleic acid for PCR testing after many years of storage (5).

Fresh DNA is also available from the hair root. Again, this tissue source combines the advantages of fresh DNA with easy transport and procurement. The robust nature of the sample and PCR assay allow hair root DNA to be used in testing after proteinase K treatment, but without formal nucleic acid extraction (3). This source of DNA is especially useful in forensic testing, when little other tissue might be available.

2.2. FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE Although certainly preferable, fresh tissue is not always available for diagnostic molecular studies. For many clinical and research laboratories, the logistical limits of specimen collection make fixed solid tissue or blood more likely sources of nucleic acid.

The archival banks of formalin-fixed/paraffin-embedded tissue accumulated by most pathology departments provide a potentially vast source of tissue for both diagnostic and research analysis. Unfortunately, nucleic acid derived from fixed tissue can be less than ideal. There are many tissue fixatives currently employed by health care and research facilities. The most common is neutral-buffered formalin. Exposure of nucleic acid to formalin results in the formylation of free nucleotide amino groups, methylene bridging of bases, and crosslinking of nucleic acid with available protein. The net result is increased nucleic acid fragmentation (6-9). With increased fixation time, the amount of available high-molecular-weight nucleic acid is markedly reduced. Tissue fixation in formalin for longer than 24 h will likely reduce the yield of high molecular-weight nucleic acid.

Even more problematic are fixatives containing mercuric chloride. Such fixatives include B-5 and Zenker's fixatives (both used in hematopathology). Many groups have documented limited success extracting nucleic acid from tissues fixed with these agents (6,8-10). There is evidence that mercury complexes with available protein and speculation that these large complexes inhibit extraction techniques (8-11). This phenomenon can be compounded with Zenker's fixative, which also contains the heavy metal chromium. Acid-containing fixatives (Bouin's fixative and Zenker's pH 2.0 fixative) could cause nucleic acid depurination. By contrast, alcohol-based fixatives allow the purification of high-quality nucleic acid. Alcohol fixation, however, is not routinely used in most applications and might not be appropriate for other clinical uses of tissue. A summary of fixative effect on nucleic extraction is presented in Table 1.

Because it is less important to have high-molecular-weight nucleic acid for most PCR applications, formalin-fixed tissue can be an excellent source of diagnostic material for these assays. Additionally, as with fresh hair or buccal samples, some PCR assays can be performed by direct amplification from formalin-fixed, paraffin-embedded tissue without prior DNA extraction (12).

The problems presented by overfixation of tissue are complicated by the need for tissue without autolysis, necrosis, or inadequate fixation. With proper care and buffered formalin, most tissue specimens can be completely fixed in 24 h, and this amount of time allows for good yields of high-molecular-weight DNA (10). RNA can also be extracted successfully from formalin-fixed tissue, although formalin might modify the bases enough to inhibit subsequent RT-PCR. Heating the purified sample prior to PCR might remove the offending mono-methylol groups (11).

2.3. BLOOD Many times, the best available DNA source is blood. Blood samples, however, present a unique problem in that the specimen is mixed with an agent to inhibit coagulation. Heparin, ethylenediaminetetraacetic acid (EDTA), and acid citrate dextrose (ACD) are all used to prevent in vitro blood clot formation. Generally, both EDTA and ACD specimen tubes provide good yields of nucleic acid appropriate for PCR and other assays. Greater than 70% of the original high-molecular-weight DNA (>25 kb) can be recovered from blood stored for 3 d in either of these preservatives, even when stored at room temperature (13). Yields are even better when samples are refrigerated.

Heparin, on the other hand, is a problem when mixed with samples intended for nucleic acid extraction. Heparin adsorbs to nucleic acid and is not completely removed by standard extraction techniques. Residual heparin in a DNA or RNA sample can inhibit restriction digests, PCR, and other enzyme-based molecular biology assays. The inhibition of PCR depends, to some extent, on the relative concentrations of template and heparin. Heparin concentrations as low as 0.05 U per reaction volume might prevent amplification (2,14). The sensitivity of various commercial polymerases does appear to vary, however, with some functioning normally at higher heparin levels (14).

Ideally, blood samples will be obtained in either ACD or EDTA. Nonetheless, occasionally, a heparinized sample might be the only source of nucleic acid available. Attempts to remove heparin with repeated ethanol precipitation, boiling and filtering, pH modification with gel filtration, or titration with protamine sulfate do not appear to eliminate subsequent heparin assay inhibition (15,16). For PCR requiring only minimal sensitivity, sample dilution might overcome this inhibition. Obviously, if amplification of a low-copy-number template is desired (e.g., infectious disease testing), sample dilution might compromise assay sensitivity. Serial washing of the buffy coat with saline prior to DNA extraction might also prove useful if white blood cells are the source of template DNA (15).

In the event that a heparinized sample must be used and dilution or washes are inadequate or inappropriate, a few options remain. Heparinase treatment of the extracted DNA might allow subsequent use of the sample for high-sensitivity PCR or other testing. Heparinase is costly, however, and the heparinase preparation might be contaminated with small amounts of RNase. The presence of RNase precludes the use of heparinase in RNA purification protocols (2). Alternately, heparin-free RNA can be precipitated out with lithium chloride. The addition of lithium chloride (final concentration 1.8 M) to a nucleic acid solution precipitates RNA, leaving inhibiting lipopolysaccharide or heparin in solution (16). This technique is inexpensive and effective.

2.4. FORENSIC SAMPLES Special consideration must be given to the forensic tissue specimen. Often, these samples are neither fresh nor preserved. In general, the quantity and quality of nucleic acid decreases with specimen age. Bloodstains might provide better DNA than bone samples, especially when the specimens are old and poorly preserved (17). Success has also been reported with tooth pulp, various soft tissues, and hair roots.

Complicating forensic nucleic acid degradation is environmental contamination, the unavoidable repercussion of specimen collection from an uncontrolled environment. Bloodstains, for example, can be seen on an essentially infinite variety of surfaces. Certain surface types present specific problems. Fabric dyes, especially indigo dye used in denim, could contaminate nucleic acid extractions and inhibit PCR. Using capillary action, dye can be removed by drawing saline through the fabric. Nucleic acid is transferred by this solution to a nylon membrane while dye remains in the fabric (18). Other surfaces, like varnished wood, could also reduce the quality and quantity of forensic DNA (19). Even on an ideal surface, the stain or sample might have been washed prior to discovery or exposed to forensic reagents like 3-aminophthalhydrazide (known as Luminol; it fluoresces in the presence of heme and is used to detect bloodstains during field investigation). Although Luminol does not appear to affect subsequent PCR analysis of extracted DNA, surface cleaning can destroy DNA evidence (19). A more detailed discussion of forensic specimen collection is beyond the scope of this chapter, but it should be apparent that this is a challenging and interesting endeavor.

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