Molecular diagnostics


Quantitative personalized medicine can detect changes in a variety of measurable body parameters not only as disease becomes apparent by clinical symptoms but much earlier. Measurements require material that can be analyzed and there are several types of body samples available for molecular diagnostic analyses.


The six major body samples used in molecular diagnostics

These six samples are listed below ordered by the difficulty to obtain them, from 1. easiest to 6. hardest.


  1. Hair follicles
  2. Saliva
  3. Urine/Feces
  4. Blood
  5. Cerebrospinal fluid
  6. Tissue biopsies



As everybody knows saliva samples are used for quick genotyping in screenings for suitable bone marrow donors for leukemia patients, or on the forensic side to identify potential offenders in crimes. Hair follicles serve a similar purpose plus the hair shaft doubles as a long-term recording of many substances that enters the body, e.g. drugs that can be traced many months after the were used. Everybody has been asked numerous times to provide a urine sample during a visit to the surgery of a MD. This also quite infamous with athletes as it is the sample of choice to check for doping. 


All of these three types of body samples are rather easily obtained and do not require any invasive technique. Due to the fact that those samples usually contain only substances that are excreted from the body (with the exception of hair follicles), quite a number of conditions cannot be detected with those samples alone. This is where the most used and most universal tissue sample comes in: blood. Although usually not readily available (unless somebody is bleeding) the little prick of the needle required is usually regarded as minimally invasive and usually well tolerated (although not well-liked). The benefit is huge since the blood is the “window to the body” as already stated earlier in this blog. Quite an number of conditions including the presence and growth of tumors can be detected in blood samples, which is why they are also often referred to as “liquid biopsy”. Quite a number of molecular diagnostic texts are based on blood samples. 


Cerebrospinal fluid is usually taken from the neck part of the spine and requires a rather delicate handling of the needle by the MD as this is in dangerous vicinity of the major nerve connections of the spinal cord. Nothing one would do without a pretty good reason and corresponding other diagnosis justifying such an effort. However, since the brain and the spinal cord are separated from the blood system by the blood-brain barrier, many processes and substances important for the differential diagnosis of brain related diseases cannot be obtained any other way. 


Last not least tissue samples taken by surgery or needle biopsy provide the ultimate source of molecular information. In biopsies all tissue-specific details become accessible, from particular tumor biology to disease-associated change in RNA or protein expression. However, there is a price to pay for such deep-reaching information. Tissue samples are usually only obtainable by invasive procedures from deep needle biopsies all the way to major surgery.  


Figure 6: Molecular diagnostics 

Principle analysis techniques in molecular diagnostics


Genetics / DNA sequencing 


In case of hereditary diseases or predisposition for diseases (such as Huntington’s disease or breast cancer) risk predictions can be derived from the genome. This works already in newborns where it is now routinely used to scan for a selected number of hereditary conditions that might cause health problems in the baby or later on. This is usually not done by actual DNA-sequencing but by testing the DNA with prefabricated microarrays that contain specific DNA-probes which will give a signal when they find a counterpart in the DNA of the sample. However, direct sequencing of the sample DNA is already being used in some cases and can be expected to be the method of choice in the future. The advantage being that not just a few preselected conditions would be screened but the whole genome of each newborn. The disadvantage is that we still lack the knowledge and the ability to provide a comprehensive full genome analysis with interpretations of all differences to the standard (whatever that standard will be defined to be). 


A clear advantage of this DNA-screening ist that it only needs to be done once in a healthy individual since the DNA sequence is not supposed to change over life-time. All markers for potential trouble later on will faithfully remain a part of the individual for his/her lifetime. 


But there is an important exception to that rule, unfortunately on the down-side. We all can and do acquire additional mutations in the DNA of our cells during our life. Usually this affect only cells that are part of our body but not germ cells, which will transmit such mutations to our children. The other cells that stay with us are called somatic cells. Mutations in somatic cells can induce tumors, if we are lucky benign tumors, that are no more than an extra lump of tissue. If we are less lucky, these tumors turn malign, i.e. we develop cancer.  In many cases the presence of tumor cells can also be determined by genetic / DNA sequence analysis of blood samples long time before any manifest tumor becomes detectable allowing early warning scenarios (liquid biopsies). 


However, often no such early warning happens because there is no general monitoring of apparently healthy people as this is presently neither logistically nor economically feasible. Therefore, many patients are diagnosed only when they already became patients, i.e. suffering from a disease already causing clinically relevant symptoms. In these cases diagnostics employing personalized medicine measurements can be used to select the best possible therapeutic approach for a particular patient group or even individual patient.


Expression /RNA-testing


Especially in biopsy material the status and levels of many or even almost all mRNAs (these are the ones coding for proteins) can be determined either via expression microarrays (similar to the DNA testing just with probes for the RNAs instead of mutations) or by direct sequencing with the Next Generation Sequencing (NGS) method RNA-seq. NGS is a truly disruptive technology that brought the effort and cost of nucleotide sequencing (DNA and RNA) into the three to four digit range paving the way to diagnostic application of such methods. Monitoring the changes brought about in the composition of the cellular RNA affords a much deeper resolution and fine diagnosis than the cruder genetic methods. It also provides an actual picture of what is going on at the time of sample taking. 


Protein testing 


Checking for the presence of particular proteins is a long-time routine in any ordinary MDs surgery. The CRP (C-Reactive Protein) level can be determined in a quick test and is usually taken as an indicator for the presence of a bacterial infection. Today, molecular testing for a whole buch of proteins allows far more detailed analyses of medical conditions than such a quick test for only one protein. We can check for a whole panel of indicators for inflammation, immune status, or other proteins that show up in places they are not supposed to be. For example, proteinuria is a condition indicating severe kidney-damage, where proteins appear in urine samples which a healthy kidney would retain in the body rather than allowing their escape into the urine. 


Metabolite screening


Here again the principle is very old but modern molecular medicine has added quantities of new metabolites to be assessed in body samples. One of the best known examples for metabolite screening is the ubiquitous finger prick assay diabetes patients routinely have to carry out checking their blood glucose levels. Levels of many other metabolites can be determined in order to detect aberrant metabolization of drugs, presence of disease-indicating/causing metabolites (e.g. phenylketonurea), high levels of triglycerides in blood (e.g. as part of the metabolic syndrome) or toxic substances that should not be there. Metabolite screening is routinely done mostly in urine and blood samples but can sometimes complement other assays in biopsies. 


What’s coming up next?


Next week I will discuss several examples of actually used molecular tests in order to elucidate their potential as well as their limitations in more detail.

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