Establishing A Molecular Diagnostics Laboratory Biology Essay

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

This chapter covers the basic percepts involved in establishing a molecular biology laboratory. Details of equipments and reagents including their selection and use are also given. The basic features involved in quality control in a molecular biology laboratory are also dealt with.

The initial substrate for clinical molecular assays is nucleic acid, which can be either DNA or RNA. There are many advantages of having nucleic acids as the starting point. Nucleic acids are probably one of the most specific markers for genetic diseases and the triplet nucleotide code is probably one of the most precise objects in nature. The DNA sequence of any organism is specific to it. Genetic changes involving changes in the DNA or RNA sequence underlie all genetic and malignant diseases and deciphering these changes can thus provide important diagnostic and prognostic information about these diseases. With what has been covered earlier, it is obvious that analysis of genetic sequences is practical and simple and can be performed in any average pathology laboratory.


The PCR technique uses in vitro amplification of a specific nucleic acid target and creates billions of copies of the target molecule. It thus enables detection of as little as one target molecule in the patient's sample. At the same time, this specificity also leads to exquisite sensitivity.

Contamination of the workbench can occur every time a centrifuge is opened. This can be due to nucleic acids which are carried forward to the next reaction and create a false amplification. Cross contamination can also occur when the patient's samples are carried over by the pipette or through operator's hands. Thus, it is essential to provide adequate space in the molecular laboratory to ensure that the problem of sample contamination is reduced to minimum.

In the laboratory, it is essential to use barrier containment through the use of physically separate work areas for reagent preparation, specimen accession, nucleic acid extraction and analysis of amplified material. The laboratory should have three independent rooms or areas. Two of these rooms are considered clean rooms. All the procedures that are done before starting the PCR are performed in these rooms. These rooms are, therefore, called preamplification rooms. The third room is used for the PCR itself and for running the gels. This room is, therefore, called the post amplification room.

Different activities are carried out in these areas. The first two rooms are used for reagent preparation and specimen preparation respectively. It is important to prepare the reagents and samples in a laminar flow cabinet. The cabinet should have an ultraviolet light source and the surface of the cabinet should be wiped with 10% hypochlorite and 75% ethanol before and after each procedure. The equipments and instruments in each area should not be shared with any other area of the laboratory. In particular, it is important to remember that the equipments used for DNA isolation should never be used for the PCR and vice versa. It is also preferable that gloves and laboratory wear be used in this area. If both DNA and RNA extraction are carried out in the same laboratory, the areas where the extractions are carried out should be different and a dedicated set of instruments should be used for each extraction.

The third room contains the thermocyclers used for in vitro nucleic acid amplification. The thermocyclers should be plugged into dedicated power lines with their own circuit breakers. The use of a sine wave UPS is advisable. Gel electrophoresis is performed in the same room and it requires a considerable amount of space. The gel documentation system should be set up at considerable distance from the thermocyclers and the gel running area. Sometimes, the operator would like to view the gel without taking photographs. Under such circumstances, it is important to make sure that the lighting in the room is reduced to minimum.

It is preferable to keep the air handling systems of these rooms completely independent of each other. Also, the pre amplification rooms should be kept at positive air pressure, while the post amplification room should be under negative air pressure. This is to ensure that nothing leaks out of the laboratory and contaminate the environment. The easiest way to ensure what appears to be a complicated proposition is to construct an anteroom in each laboratory. Construction of a double door system around the anterooms is important to avoid contamination. It is also important that the doors be perfectly sealed to further limit contamination.

Real Time technology has recently been introduced into the clinical laboratory. The real time thermal cyclers perform amplification and simultaneously detect the product. Thus contamination is reduced to a minimum. It is, therefore, far easier to work with real time technology as compared to conventional PCR. However, it is unlikely that real time PCRs will replace conventional PCRs for routine diagnostics. Hence, the problems associated with contamination cannot be wished away.

Decontamination can be easily performed by cleaning workbenches with 10% hypochlorite and then with a 75% solution of ethanol. Ultraviolet irradiation of work surfaces is also effective. DNA degrades in the presence of UV light. It cross links the two strands of DNA by forming thymidine dimmers. This cross linked DNA can no longer serve as an effective template. The UV light is most effective against sequences over 700 bases in length and hence it is most effective against genomic DNA. It may not be as effective against PCR products.

Given the critical importance of the distance and the energy of irradiation for decontamination, pull-down UV lights (254 nm wavelength) that can be adjusted to within 60-90 cm above the workbenches maybe used after the work in the work area has been finished. Also, because of the large size of a few hundred base pairs and the fact that dried aerosols are less susceptible to UV damage than 'wet' aerosols, PCR fragments must be irradiated for extended periods- optimally, overnight.

Prevention of cross contamination can be performed in several ways. Movement of personnel within the laboratory should be minimized. The laboratory staff should clean the area themselves and put all the trash into trash bags, so that the housekeeping staff need not enter the laboratory. The laboratory staff should also use laboratory coats and caps while working and the use of gloves should be made compulsory. To further prevent cross contamination, disposable pipette tips containing a hydrophobic barrier should be used.

The maintenance of stock reagents is critical. Delivery of stock reagents and material for sample preparation is best done directly to the reagent storage and set-up area. Vessels containing reaction mixtures should always be centrifuged briefly before opening. After the stock solutions have been checked for suitability, they should be divided into aliquots for storage and further use. This reduces the danger of contamination through frequent opening of reaction vessels and pipetting. Vessels containing reaction mixtures should always be centrifuged briefly before freezing. In general, most solutions used for PCR are stored in frozen state. Frequent use of freeze/thaw cycles of master stock solutions must be avoided; therefore, stock solutions should be frozen in small aliquots so that they can be easily used for individual reactions. Because the required volume for these stock solutions is determined by the number of PCR reactions usually carried out in one assay run in the laboratory, no specific volume recommendations are given here. Aliquot stocks of suitable sizes are predispensed into micro reaction vessels and are subsequently frozen for storage.


The basic equipments required in a molecular laboratory are detailed in Table 1. Additional specialized equipments, such as automated DNA sequencer, real time thermocyclers and high performance liquid chromatography machines, may be required to develop the laboratory further. Sky is the limit when it comes to setting up a molecular biology laboratory and it would not be unusual to desire to set up an advanced laboratory by incorporating microarray technique. However, only the basic equipment required is presented below.

Table 1 - Equipments required for setting up a basic molecular biology laboratory




DNA Thermal Cyclers


Details provided later

Laminar Flow Chambers


Details provided later

Freezers/ Refrigerators


Any standard company

-20°C deep freezer


Details provided later

-70°C deep freezer


Details provided later

Electronic balance


Any standard company

pH meter


Any standard company

Heating/ stirring plate


Any standard company

Water bath


Any standard company

Electrophoresis power supply


Details provided later

Mini PAGE electrophoresis apparatus


Any standard company

Medium sized PAGE electrophoresis apparatus


Any standard company

Photodocumentation system


Details provided later

Agarose gel electrophoresis apparatus


Any standard company

Micropipettes 0.5 - 10 µL


Any standard company

Micropipettes 10 - 100 µL


Any standard company

Micropipettes 100 - 1000 µL


Any standard company

Vortex mixer


Any standard company

Dry bath


Any standard company

Table top refrigerated centrifuge


Details provided later



Any standard company

Angle head microcentrifuge


Details provided later

<H2>DNA Thermal Cyclers

It is important to invest in a good thermal cycler. The temperatures in the reaction are critical and a small change can make the world of difference. Several companies manufacture thermal cyclers; Eppendorf, Bio Rad and Perkin Elmer manufacture excellent machines although the price may be on the higher side. However, it is better to spend money on a good quality machine rather than regretting later. The criteria which would be useful in selecting a machine are as follows:

It should have a 96-well single block.

Alternately, it may have a dual block capacity, comprising of two 48 well blocks

The tube capacity should be between 0.2ml and 0.5 ml

It should be operative at 220 V/ 50-60 Hz.

It should have an average ramp rate of 3°C / second.

It should have a temperature accuracy of +/- 0.2°C.

It should have a storage capacity of at least 100 programs

It should have a high resolution graphic display

It should have an auto restart option.

It should have a lid temperature of at least 100°C

It should be provided with a gradient heating capacity for gradient PCR

It is important to purchase a thermal cycler with a capacity for holding 96 tubes. This is because, it allows processing of a large number of samples in a single reaction. Often, it may so happen that two tests may be required to run simultaneously. Let us assume that the number of tests to be run is 35 in the first reaction and 39 in the second reaction. Under such circumstances, the value of a dual block comes into play. 35 reactions can be run in one block and 39 reactions can be run in the second block. Several companies manufacture thermal cyclers where it is possible to use two blocks simultaneously. The two blocks hold 48 tubes each and can be programmed separately. In effect, it would be possible to run two entirely different programs within the same time frame.

Ideally, the tube capacity should be 0.2 ml, that being the standard size of a PCR tube. However, it is advisable to have a provision to use 0.5 ml tubes also. The ramp temperature is the speed with which the machine attains the desired temperature. A ramp temperature of 3°C/sec is adequate. It is important to store programs. Some thermal cyclers can store programs on external storage devices. However, it is good if the programs can be stored on the machine itself. The display is an important feature although it needn't be stressed upon. Most standard machines come equipped with high resolution graphic display. The auto restart option is important since it is difficult to restart a program once it goes off. The lid temperature, though important, is of relatively less concern. The lid temperature is the temperature of the cover of the thermal cycler. Ideally it should be at least 5°C higher than the temperature of the block. Most thermal cyclers have lid temperatures that far exceed 100°C.

The point about the gradient heating is important. A thermal cycler which has this option can be programmed to carry out gradient PCRs so that standardization of the PCRs can be done with ease. It is not an essential item on the list but at least one of the two thermal cyclers in the laboratory should have this feature.

<H2>Laminar Flow Chambers

Besides the provision of basic features, as already discussed, in the molecular biology laboratory, it is important to have a UV light source attached to the body of the laminar flow chamber. This is to ensure degradation of DNA after use and prevention of cross contamination. A class 2 laminar airflow system usually suffices.

<H2>-20°C deep freezer

This is an extremely important part of the armamentarium of a molecular biologist. The criteria for selecting a deep freezer are as follows:

It should be operative at 220 V/ 50-60 Hz.

It should have a capacity of at least 170 litres

It should be able to maintain a temperature of -20°C

It should be provided with a digital display for indicating temperature with an accuracy of ± 1°C

It should have an audio visual alarm that runs when the temperature deviates from the pre set temperatures.

The audio visual alarm should be battery controlled. The battery should be rechargeable

It should have a power failure alarm as well as a door open alarm

It should be provided with stainless steel trays

It should be provided with adequate lighting inside

It should be easily accessible and washable

It should have a double door system. Each of the inner doors should give access to one shelf only. This is important and should be emphasized so that the contents of the freezer are exposed to the external environment as little as possible

<H2>-80°C deep freezer

The criteria for its selection are as follows:

It should be operative at 220 V/ 50-60 Hz.

It should have a capacity of at least 170 litres

It should be able to maintain a temperature of -80°C

It should have a digital display for indicating temperature with ± 2°C accuracy

It should have an audio visual alarm that runs when the temperature deviates from the pre set temperatures

It should be provided with stainless steel trays

It should be equipped with wheels for easy mobility. This is desirable because these refrigerators are usually very heavy and provision of wheels immensely aids in their mobility.

<H2>Electrophoresis power supply

Many companies manufacture power supply equipments. For DNA and RNA based PCRs, a voltage of up to 250 V is more than adequate. The current also need not be very high; up to 50 mA usually suffices. However, it is always good to plan for future. If one decides to increase the scope of the laboratory for Western Blotting at some point in future, it would be necessary to invest in a power pack with a capacity of at least 3 amperes.

<H2>Photo documentation system

Many companies produce photo documentation systems. When purchasing such a system, it is important to remember that the CCD camera should have zoom lens included with it. UV filters should be available in the camera and a pivoting LCD screen is very much desirable. It is necessary to have a memory card to store the images in a JPEG or TIFF format. The memory card should be so chosen that a large number of images can be stored in it.and hence the memory card should be chosen accordingly.

The transilluminator should have a wavelength of 302 nm with a filter size of about 20 x 20 cm. The system should be such that the operator is protected from the effects of UV light.

The photo documentation system should be so configured that it is compatible with the computer system. Buying a printer to print the images is optional.

<H2>Table top refrigerated centrifuge

This is another extremely important piece of equipment that is required in a molecular biology laboratory. The criteria which would be useful in selecting a centrifuge machine are as follows:

The machine should be operative at 220 V/ 50-60 Hz.

It should have a capacity to hold at least 24 tubes

It should have the ability to hold tubes of a capacity between 0.5 and 2 ml. The ability to hold at least 24 tubes, each with a capacity of up to 2 ml is important because that is the size of the Eppendorf tubes usually used for DNA extraction.

The maximum speed should be at least 14000 rpm. Higher speeds are preferable.

The maximum centrifugal force should be at least 25000g

The acceleration time to reach maximum speed should be less than 15 seconds. The acceleration and deceleration time are important since time is at a premium.

The machine should have a fixed angle rotor with an aluminum cover.

The sound level should be < 60 dB. A loud centrifuge can irritate the nerves of even the most patient worker.

The machine should have a digital display and control which should display the rotor speed, value of g* and the temperature of centrifuge.

The machine should be able to maintain a sample temperature of ≤ 4°C at maximum speed. Higher temperatures melt the plastic used in the Eppendorf tubes.

The machine should be light and portable.

Finally, the centrifuge should be refrigerated. The centrifuge machine should be able to maintain the samples at 4°C or less at maximum speed. Higher temperatures melt the plastic used in the Eppendorf tubes.

<FN>*Centrifugal force of acceleration</FN>


There are basically two test formats used in molecular biology laboratories. One type of test format is the kind that is developed in-house by each laboratory. The second kind of test format is one in which the reagents are provided in a kit by the kit manufacturers.

<H2>Tests developed in-house

This is also called a home brewed assay. This term refers to those assays which are fully developed and validated by the laboratories performing them. These assays use a combination of reagents that are purchased from a variety of manufacturers. Each laboratory determines the performance characteristics of the assays of a clinical condition for a particular test population. The analytical and clinical validation of the entire testing process is the responsibility of the laboratory.

<H2>Kit based manual methods

There are two categories of tests. In the first category of the tests, the manufacturer provides quality control reagents for every step required to carry out the test. Several tests such as those used for quantification of viral load in HIV patients follow this methodology. To start with the molecular test, the kit has reagents for nucleic acid isolation, amplification and detection. The sensitivity, specificity and tolerance limits are also specified in the kit.

In the second category of tests, the reagents are provided for only a particular step in the testing. For example, this step may be any of nucleic acid extraction, PCR amplification or nucleic acid detection. These tests maybe combined together by using different kits for different stages of the entire process. The validation is then done separately for each step of the process.


The primary goal of the molecular diagnostic laboratory is to provide reliable and timely clinical investigations that produce results which are important in patient care. Tests selection is critical. The tests should provide a more effective method for the diagnosis and management of the patient. The molecular test may also replace an existing laboratory test if it is more cost effective. The molecular tests maybe more sensitive, specific or may have a reduced turn around time which would lead to an improvement in patient care.

Once it has been decided to incorporate molecular testing into the protocol, it is important to decide the form of these tests and the technique to be utilized. The methodologies have already been enumerated previously. It is important to keep in mind the clinical question and the advantages and disadvantages of each methodology.


Essentially, validation includes assessment of the sensitivity and specificity of a test. It is a complex process and can be divided into two phases; analytical and clinical validation. The parameters that must be assessed during analytical validation are sensitivity, specificity, accuracy, precision and assay linearity for quantitative assays. Sensitivity refers to the lower limit of detection. Essentially this means that a sensitive test can pick up an abnormality even if the abnormality is minimal. This ultimately decides if the test will be useful for its intended purpose. Specificity mentions if there is a chance of a cross reactivity with other nucleic acids other than the target sequence. In the clinical validation, the clinical utility of the test is assessed with regard to its intended use.

If the test being developed is an in-house molecular assay, it is essential to optimize each step of the analytical process. These steps include nucleic acid extraction, amplification, detection, calculation and result interpretation. When all the steps have been validated, it is necessary to put all the steps together and reoptimise the entire test again. After the test per se has been validated, the pre analytical variables also need to be validated. These variables include the specimen type, transport and storage handling requirements. After optimization, the laboratories must perform and document analytic validation for the assay's intended use. Finally, it is necessary to look at the clinical validity of the test.

Clinical validation requires evaluation of the clinical sensitivity of the test by testing an appropriate number of samples from patients who have been diagnosed with the disease. The test must also be performed on patients who do not have the disease to analyse the positive and negative predictive values of the test. It is also necessary to compare the sensitivity and specificity with a gold standard method.

It is important to realize that genetic testing in molecular biology may have several constraints. Firstly, a test may not detect all the possible mutations that could be present in a particular gene causing the disease. A single gene may have a host of mutations and it may not be possible to test for all these mutations. Secondly, the importance of genetic testing in a single gene disorder cannot be denied. However, there are several other variables like modifier genes and gene-gene interactions which would produce a phenotype which may not be entirely predicted by the genotype. Therefore, genetic testing needs to be viewed in its complete perspective.


The concept of quality control can be understood in three distinct steps. These are as follows:

Quality control of the testing process

Quality control of equipments (already discussed under 'equipments' in this chapter)

Quality assurance

<H2>Quality Control of the Testing Process

It must be emphasized that every step in the testing process starting from reagent preparation to performance of the actual assay needs to be validated. Essentially, since a molecular laboratory deals predominantly with the PCR, the quality control steps associated with PCR will be elaborated upon.

Although every amplification assay is prone to contamination, the technical efforts associated with different types of nucleic analyses vary widely. For example, genotyping patients usually do not require optimization of the amplification conditions for improved detection limits. On the other hand, for detection of minimal residual disease or for virus detection, setting a very low detection limit is a prerequisite. Accordingly, contamination risk may not be a major problem in one application but obviously can be critical in another. Also, if different enzymes have to be used in subsequent steps of a given test, as in reverse transcriptase (RT) PCR, the handling of the additionally required material must be considered a potential contamination hazard.

<H3>Controls related to preparation of test material

For control of DNA preparation, agarose gel electrophoresis is most commonly used. The average length of the DNA is 100 kb in routine manual preparation methods. In DNA preparation kits suitable for PCR, the average range is between 30 and 40 kb. Even degraded DNA emits fluorescence after electrophoresis. The presence of potential inhibitors is usually assessed by photometry at 260 and 280 nm. In a good DNA preparation, the A260/A280 ratio should be in the range of 1.75-2.0; otherwise, the contamination (e.g., with residual protein or phenol) may be too high.

The fastest method for controlling the quality of the total RNA preparation is agarose gel electrophoresis under non denaturing conditions, as was applied in the separation of DNA. In case of doubt, however, the RNA should be run on an agarose gel under denaturing conditions to check its integrity. The three major ribosomal RNA species (28S, 18S, and 5S) will be detectable as relatively sharp bands. Smearing of bands to lower molecular masses or absence of the bands strongly indicates decay of RNA. In addition, the banding pattern in agarose gel electrophoresis will indicate the degree of DNA contamination in the RNA preparation.

<H3>Controls for cDNA synthesis and amplification

Use of an internal reaction control is crucial for monitoring the performance of cDNA synthesis. cDNA synthesis can start either from mRNA transcripts, which are present in each RNA preparation (i.e., mRNA of ubiquitously expressed, so-called housekeeping genes such as transcripts for ribosomal proteins), or from RNA, which is added to the sample as an internal standard at the time of preparation. Internal controls for synthesis are positive controls that lead to a defined product; if the amplification is not successful, the controls indicate degradation of RNA, faulty priming during cDNA synthesis, or absence of enzyme activity. Thus, it is not possible to verify if the conversion has been successful until a PCR has been run on the sample.

<H3>PCR reaction

Internal reaction controls are positive controls and are particularly important in cases where the presence or absence of an amplification product is diagnostically relevant, i.e., with gene deletions or Y-chromosomal sequences. One way would be to amplify a gene that is essential for the organism to survive like the gene for the vitamin D-binding plasma protein; no homozygous loss of gene activity has ever been identified. Therefore, coamplification of a segment of this gene by the PCR will be in all cases successful and will demonstrate the successful amplification reaction. If the internal control is negative, a problem with the reaction is most likely.

External positive controls of appropriate DNA and appropriate dilutions allow the quality of the reaction solution to be checked. The same master mix solutions used for the diagnostic test (i.e., patient's material) must be used in the external positive controls. Amplification controls should be performed with each reaction. Controls of this type increase the contamination risk in the test series, if the nucleic acid used corresponds to a positive control sample. Vector-cloned target sequences or a genomic DNA of known copy number is suitable.

An external negative control (contamination control) must be performed in each PCR test. A blank reaction vessel taken through the entire course of the sample preparation comes into contact with all solutions used in the preparation, but contains no amplifiable material (so-called mock preparation). If necessary, different mock preparations can be integrated at various preparation stages in the course of nucleic acid preparation. In this manner, the individual steps at which contamination can occur, can be identified. Mock controls allow assessment of the overall quality of the PCR test.

<H2>Quality assurance

Quality assurance can be essentially divided into quality control within the laboratory and that outside the laboratory (external quality control).

<H3>Internal quality control

This occurs at several stages, which are discussed next..

<H4>Control of restriction digestion - Digestion of genomic DNA can be reduced by inhibition of the activity of enzymes present in the preparation, inappropriate reaction conditions, and low enzymatic activity. The digestibility of genomic DNA can be assessed through agarose gel electrophoresis. The following criteria serve for assessment of successful restriction:

After restriction digestion with most enzymes, a continuous, mostly smeared band is observed between high- and low-molecular-mass ranges. This reflects the size heterogeneity of the restricted genomic fragments. Where digestion is not successful, a pronounced high-molecular mass fraction persists in the molecular range corresponding to the uncut DNA. With some enzymes, a high-molecular-mass fraction may be observed even after extended digestion incubation times. Thus, although disappearance of the high-molecular-mass fraction indicates full enzymatic activity, persistence of this fraction does not necessarily indicate insufficient digestion.

An easy method to assess the presence of inhibitors in the sample is to take an aliquot of the digestion mix, and then add to this, a known amount of high-molecular-mass molecular marker. The restriction enzyme should digest the molecular weight marker. The suitability of the enzyme is assessed by checking the digested DNA fragments and comparing the same with the DNA marker. It is also possible to extend the digestion times of the genomic DNA, if required.

The human genome contains repetitive sequences, e.g., mitochondrial DNA, which may appear as distinct sharp bands (satellite bands) within the background smear of heterogeneous fragments. Satellites, therefore, do indicate successful restriction; however, not all enzymes generate a satellite band pattern.

Restriction digestion of PCR products is often applied to identify mutations on the basis of various restriction fragment lengths. Compromised enzymatic activity in quality assessment of molecular amplification methods can lead to false interpretation of results. Complete digestion of PCR products can be controlled only by a second invariant restriction site of the same enzyme in the PCR product to be tested. This must be strictly observed in the construction of the diagnostic fragment, to assure correct interpretation of results.

<H4>Control of electrophoresis - Use of calibrators to check the length of the PCR products helps in quality control of electrophoresis. Many manufacturers offer good calibrators for measuring length in different molecular mass ranges; these are known as DNA ladders. These often permit exact size determination of the electrophoretically separated fragments.

The second feature that helps in quality control of electrophoresis is using defined concentrations of a product. This helps us to determine detection limit of the visualization process used. For example, from a known DNA quantity added into digestion, one can calculate DNA quantity for each fragment as a fraction of the uncut DNA amount. Because the ethidium bromide fluorescence is proportional to the DNA quantity, and also because the relative quantity of the individual bands of the calibrator is known, the concentration of PCR fragments can be judged in comparison with the fluorescence intensity of the bands of the calibrators.

It is important to emphasise that controls must be subjected to the same sample preparation procedures as the diagnostic specimens.

<H4>Control of sequencing

Manufacturers usually supply suitable DNA amplification templates, and matching primers, as controls for their sequencing reagents. Moreover, this sequencing reaction can help control the quality of the sequencing gel electrophoresis. For analysis of mutations, the wild-type allele and samples from family members should be sequenced in parallel.

<H3>External quality control

Considering the multitude of methodological variants and diagnostic approaches, it does not appear feasible to set up external quality-assessment trials for every diagnostic problem, especially if the diseases considered are rare. There are two major objectives of external quality control testing: methodological proficiency testing and application-based proficiency testing.

Methodological proficiency testing is intended to control the quality of the elementary analytical steps in molecular genetic diagnosis: the DNA/RNA preparation, the performance of the PCR method with supplied "standard primers," and the agarose gel electrophoresis.

Application-based proficiency testing is suitable for relatively frequent diagnostic questions, e.g., the factor V Leiden mutation in thrombophilia, caused by the resistance of clotting factor V to activated protein C.

Continuous cell lines obtained either from diseased individuals or those stably transfected with genes coding for the respective gene products, could be used as standardized template sources for proficiency testing in molecular diagnostics laboratories. For rare genetic diseases, international trials make more effective use of resources and expertise.

In conclusion, during recent years laboratory science has faced an increasing interest in molecular diagnostics and a corresponding demand for routine genetic testing. Expectations are high for two reasons: First, much is expected from molecular testing. There are several expectations nurtured by scientific progress. This is well illustrated by the human genome project. Second, physicians have become used to high quality of test results from the routine clinical laboratory through their day-to-day use of more conventional laboratory markers. These two expectations are likely to govern the future of pathologists in the coming years.