Disadvantages of blood typing in forensics

"DNA typing" is a catch-all term for a wide range of methods for studying genetic variations. Each method has its own advantages and limitations, and each is at a different state of technical development. Each DNA typing method involves three steps:

Laboratory analysis of samples to determine their genetic-marker types at multiple sites of potential variation.

Comparison of the genetic-marker types of the samples to determine whether the types match and thus whether the samples could have come from the same source.

If the types match, statistical analysis of the population frequency of the types to determine the probability that such a match might have been observed by chance in a comparison of samples from different persons.

Before any particular DNA typing method is used for forensic purposes, it is essential that precise and scientifically reliable procedures be established for performing all three steps. This chapter discusses the first two—laboratory analysis and pattern comparison—and Chapter 3 focuses on statistical analysis.

There is no scientific dispute about the validity of the general principles underlying DNA typing: scientists agree that DNA varies substantially among humans, that variation can be detected in the laboratory, and that DNA comparison can provide a basis for distinguishing samples from different persons. However, a given DNA typing method might or might not be scientifically appropriate for forensic use. Before a method can be accepted as valid for forensic use, it must be rigorously characterized in both research and forensic settings to determine the circumstances under which it will and will not yield reliable results. It is meaningless to speak of the reliability of DNA typing in general—i.e., without specifying a particular method. Some states have adopted vaguely worded statutes regarding admissibility of DNA typing results without specifying the methods intended to be covered. Such laws obviously were intended to cover only conventional RFLP analysis of single-locus probes on Southern blots—the only method in common use at the time of passage of the legislation. We trust that courts will recognize the limitations inherent in such statutes.

Forensic DNA analysis should be governed by the highest standards of scientific rigor in analysis and interpretation. Such high standards are appropriate for two reasons: the probative power of DNA typing can be so great that it can outweigh all other evidence in a trial; and the procedures for DNA typing are complex, and judges and juries cannot properly weigh and evaluate conclusions based on differing standards of rigor.

The committee cannot provide comprehensive technical descriptions for DNA typing in this report: too many methods exist or are planned, and too many issues must be addressed in detail for each method. Instead, our main goal is to provide a general framework for the evaluation of any DNA typing method.

Essentials Of A Forensic DNA Typing Procedure

Scientific Foundations

The forensic use of DNA typing is an outgrowth of its medical diagnostic use—analysis of disease-causing genes based on comparison of a patient's DNA with that of family members to study inheritance patterns of genes or with reference standards to detect mutations. To understand the challenges involved in such technology transfer, it is instructive to compare forensic DNA typing with DNA diagnostics.

DNA diagnostics usually involves clean tissue samples from known sources. It can usually be repeated to resolve ambiguities. It involves comparison of discrete alternatives (e.g., which of two alleles did a child inherit from a parent?) and thus includes built-in consistency checks against artifacts. It requires no knowledge of the distribution of patterns in the general population.

Forensic DNA typing often involves samples that are degraded, contaminated, or from multiple unknown sources. It sometimes cannot be repeated, because there is too little sample. It often involves matching of samples from a wide range of alternatives present in the population and thus lacks built-in consistency checks. Except in cases where the DNA evidence excludes a suspect, assessing the significance of a result requires statistical analysis of population frequencies.

Despite the challenges of forensic DNA typing, we believe that it is possible to develop reliable forensic DNA typing systems, provided that adequate scientific care is taken to define and characterize the methods. We outline below the principal issues that must be addressed for each DNA typing procedure.

Written Laboratory Protocol

An essential element of any clinical or forensic DNA typing method is a detailed written laboratory protocol. Such a protocol should not only specify steps and reagents, but also provide precise instructions for interpreting results, which is crucial for evaluating the reliability of a method. Moreover, the complete protocol should be made freely available so that it can be subjected to scientific scrutiny.

Procedure For Identifying Patterns

There must be an objective and quantitative procedure for identifying the pattern of a sample. Although the popular press sometimes likens DNA patterns to bar codes, laboratory results from most methods of DNA testing are not discrete data, but rather continuous data. Typically, such results consist of an image—such as an autoradiogram, a photograph, spots on a strip, or the fluorometric tracings of a DNA sequence—and the image must be quantitatively analyzed to determine the genotype or genotypes represented in the sample. Quantitation is especially important in forensic applications, because of the ever-present possibility of mixed samples.

Patterns must be identified separately and independently in suspect and evidence samples. It is not permissible to decide which features of an evidence sample to count and which to discount on the basis of a comparison with a suspect sample, because this can bias one's interpretation.

Procedure For Declaring a Match

When individual patterns of DNA in evidence sample and suspect sample have been identified, it is time to make comparisons to determine whether they match. Whether this step is easy or difficult depends on the resolving power of the system to distinguish alleles. Some DNA typing methods involve small collections of alleles that can be perfectly distinguished from one another—e.g., a two-allele RFLP system based on a polymorphism at a single locus. Other methods involve large collections of similar alleles that are imperfectly distinguished from one another—e.g., the hypervariable VNTR systems in common forensic use, in which a single sample might yield somewhat different allele sizes on repeat measurements.1 It is easy to determine whether two samples match in the former case (assuming that the patterns have been correctly identified), but the latter case requires a match criterion—i.e., an objective and quantitative rule for deciding whether two samples match. For example, a match criterion for VNTR systems might declare a match between two samples if the restriction-fragment sizes lie within 3% of one another.

The match criterion must be based on the actual variability in measurement observed in appropriate test experiments conducted in each testing laboratory. The criterion must be objective, precise, and uniformly applied. If two samples lie outside the matching rule, they must be declared to be either ''inconclusive" or a "nonmatch." Considerable controversy arose in early cases over the use of subjective matching rules (e.g., comparison by eye) and the failure to adhere to a stated matching rule.

Identification of Potential Artifacts

All laboratory procedures are subject to potential artifacts, which can lead to incorrect interpretation if not recognized. Accordingly, each DNA typing method must be rigorously characterized with respect to the types of possible artifacts, the conditions under which they are likely to occur, the scientific controls for detecting their occurrence, and the steps to be taken when they occur, which can range from reinterpreting results to correcting for the presence of artifacts, repeating some portion of the experiment, or deciding that samples can be reliably used.

Regardless of the particular DNA typing method, artifacts can alter a pattern in three ways: Pattern A can be transformed into Pattern B, Pattern A can be transformed into Pattern A + B; and Pattern A + B can be transformed into Pattern B. It is important to identify the circumstances under which each transformation can occur, because only then can controls and corrections be devised. For example, RFLP analysis is subject to such artifacts as band shifting, in which DNA samples migrate at different speeds and yield shifted patterns (A→B), and incomplete digestion, in which the failure of a restriction enzyme to cleave at all restriction sites results in additional bands (A→A + B).

Some potential problems can be identified on the basis of the chemistry of DNA and the mechanism of detection in the genetic-typing system. Anticipation of potential sources of DNA typing error allows systematic empirical investigation to determine whether a problem exists in practice. If so, the range of conditions in which an assay is subject to artifact must be characterized. In either case, the results of testing for artifacts should be documented. Empirical testing is necessary, whether one is considering a new method, a new locus, a new set of reagents (probe or enzyme) for a pre-existing locus, or a new device. Under some circumstances, even small changes in procedure can change the pattern of artifacts.

Once potential artifacts have been identified, it is necessary to design scientific controls to serve as internal checks in each experiment to test whether the artifacts have occurred. Once the appropriate controls are identified, analysts must use them consistently when interpreting test results. If the appropriate control has not been performed, no result should be reported. When a control indicates irregularities in an experiment, the results in question must be considered inconclusive; if possible, the experiment should be repeated. A well-designed DNA typing test should be a matter of standardized, objective analysis.

Sensitivity to Quantity, Mixture, and Contamination

Evidence samples might contain very little DNA, might contain a mixture of DNA from multiple sources, and might be contaminated with chemicals that can interfere with analysis. It is essential to understand the limits of each DNA typing method under such circumstances.

Experiential Foundation

Before a new DNA typing method can be used, it requires not only a solid scientific foundation, but also a solid base of experience in forensic application. Traditionally, forensic scientists have applied five steps to the implementation of genetic marker systems:2,3

Gain familiarity with a system by using fresh samples.

Test marker survival in dried stains (e.g., bloodstains).

Test the system on simulated evidence samples that have been exposed to a variety of environmental conditions.

Establish basic competence in using the system through blind trials.

Test the system on nonprobative evidence samples whose origin is known, as a check on reliability.

When a technique is initially developed, all five steps should be carefully followed. As laboratories adopt the technique, it will not always be necessary for them to repeat all the steps, but they must demonstrate familiarity and competence by following steps 1, 4, and 5.4

Most important, there is no substitute for rigorous external proficiency testing via blind trials. Such proficiency testing constitutes scientific confirmation that a laboratory's implementation of a method is valid not only in theory, but also in practice. No laboratory should let its results with a new DNA typing method be used in court, unless it has undergone such proficiency testing via blind trials. (See Chapter 4 for discussion of proficiency testing.)

Publication and Scientific Scrutiny

If a new DNA typing method (or a substantial variation on an existing one) is to be used in court, publication and scientific scrutiny are very important. Extensive empirical characterization must be undertaken. Results must be published in appropriate scientific journals. Publication is the mechanism that initiates the process of scientific confirmation and eventual acceptance or rejection of a method.

Some of the controversy concerning the forensic use of DNA typing can be traced to the failure to publish a detailed explanation and justification of methods. Without the benefit of open scientific scrutiny, some testing laboratories initially used methods (for such fundamental steps as identifying patterns, declaring matches, making comparison with a databank, and correcting for band shifting) that they later agreed were not experimentally supported. In some cases, those errors resulted in exclusion of DNA evidence or dismissal of charges.

Technical Issues In RFLP Analysis

Choice of Probes

A DNA probe used in forensic applications should have the following properties:

• It should recognize a single human locus (or site), preferably one whose chromosomal location has been determined.

• It should detect a constant number of bands per allele in most humans.

• It should be characterized in the published literature, including its typical range of alleles, and its tendency to recognize DNA from other species.

• It should be readily available for scientific study by any interested person.

The committee recommends against forensic use of multilocus probes, which detect many fragments per person. Because such probes might detect fragments with quite different intensities, it is difficult to know whether one has detected all fragments in a sample—particularly with small and degraded forensic samples—and difficult to recognize artifacts and mixtures. Such problems increase the difficulty of pattern interpretation. Multilocus probes increase the risk of incorrect interpretation, and numerous single-locus probes, which do not pose such problems, are available. The use of enough single-locus probes gains the advantages of the single multilocus probes without the problems of interpretation.

Southern Blot Preparation

The basic protocol for preparing Southern blots is fairly standard, but testing laboratories vary in such matters as choice of restriction enzyme, gel length and composition, and electrophoresis conditions. Such differences do not fundamentally affect the reliability of the general method, but some enzymes might require characterization (e.g., each restriction enzyme must be characterized for sensitivity to inhibitors, for tendency to cut at anomalous recognition sites under some conditions—often called "star activity"—and for tendency to produce partial digestions), and differences in gels and electrophoresis conditions will affect resolution of fragments and retention of small fragments.

Questions have arisen concerning the use of ethidium bromide, a fluorescent dye that binds to DNA and so allows it to be visualized. Some laboratories incorporate ethidium bromide into analytical gels before electrophoresis; others stain gels with ethidium bromide after electrophoresis. The committee strongly recommends the establishment of a National Committee on Forensic DNA Typing under the auspices of an appropriate government agency, such as NIH or NIST, to provide expert advice primarily on scientific and technical issues concerning forensic DNA typing.

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