- Olga Timofeeeva, PhD
The primary aim of the molecular pathology laboratory is to provide clinical diagnostic/prognostic services for the molecular analysis of clinical material submitted to the anatomical and clinical laboratories of the Temple University Hospital. The goal of the molecular pathology rotation is to provide a comprehensive understanding of the role of molecular pathology in clinical and anatomic pathology. Residents rotating through the molecular pathology laboratory are given extensive handouts on the theoretical basis underlying the principle of the polymerase chain reaction (PCR). In addition, these handouts also include topics such as real-time PCR, FRET (fluorescence resonance energy transfer) analysis, cDNA microarrays, etc. They are trained to perform as well as interpret the molecular diagnostic test results. The primary testing in the molecular diagnostics laboratory are viral loads and genotyping for HIV-1 and HCV and HPV genotyping. Other assays include hypercoagulation assays for Factor V Leiden and prothrombin gene mutation analysis. In this one calendar year, the molecular pathology laboratory will perform approximately 1,500 HIV-1 and HCV viral loads, 200 HIV-1 and HCV genotyping, 2,000 HPV genotyping and 300 Factor V Leiden and Prothrombin (Factor II) gene mutation analysis.
During the first week, the residents will spend time reading material describing the theoretical basis of the amplification techniques that are used in the Molecular Pathology Laboratory for diagnostic purposes. This will include:
- The principle underlying the methods used for the extraction and isolation of nucleic acids (DNA and RNA) from various tissue sources;
- The various constituents of the amplification reaction mixture and their importance to a successful assay. This part will include information about how to design primers for PCR, the criteria necessary for optimizing the PCR reaction, the importance of the concentration of deoxynucleotides, magnesium, etc.
- The choice of detection systems (e.g., gel electrophoresis, DNA sequencing, hybridization based Elisa assays, etc.) for identifying the target nucleic acid molecule.
- Interpretation of the results.
- QA/QC issues with regards to the prevention of contamination of the specimens.
During this week, the resident will be given ample opportunity to observe the techniques being utilized in the molecular pathology laboratory to enable them to understand and correlate the theoretical with the practical. During the second week, the resident will be trained in the various methods of extraction of nucleic acids from various tissue sources. They will extract and isolate RNA and DNA from tissues. During the third week, the residents will set up PCR and RT-PCR reactions using the nucleic acid isolated in week two.
During this week, emphasis will be placed on understanding the various criteria used for selection of the primers for amplification, concentration of the various components of the PCR reaction and the method used at the back end for identification of the amplified template. During the fourth week, the residents will spend time on interpretation of the results obtained by amplification. They will also be exposed to some unusual cases that have been obtained in the laboratory during the past years. Future assays for consideration of development include CMV quantitation as well as EGFR, Kras, Jak2 and Bcr/Abl gene mutation analysis.