Internship - FMRI, Gurgaon (Dec '18 - Jan '19)

Internship - FMRI, Gurgaon (Dec '18 - Jan '19)

• 1.
  Internship at Fortis MemorialResearch Institute, Gurgaon 27th December 2018 – 9th Jan 2018 A 2-week observational training in the Clinical Laboratory An H&E-stained brain tissue under a microscope.
• 2.
  The Department The clinicallaboratory has a fundamental role in the diagnosis and treatment of diseases and disorders. The lab at Fortis is divided into 4 sub-departments: 1) Microbiology 2) Histopathology and cytology 3) Biochemistry 4) Haematology
• 3.
  Microbiology - Deals withthe accurate diagnosis of bacterial, viral and fungal viruses. (microorganism-caused diseases) - It has distinct protocols for the categories of disease. For viral diseases, there are specific test kits (HbsAg for Hep. B, VDRL for syphilis, HIV for human immunodeficiency virus, HCV for Hep. C, Typhoid tests). This is serology testing. For Bacterial and fungal diseases, there is a longer process. It depends on the sample received, but it is largely similar: 1) The sample is received and some amount is inoculated into cell-growth media (there are different types for growth of different microorganisms) 2) The culture plate is left in an incubator for 12-24 hours. 3) It is then taken out, and if colonial growth is present, it is kept. Otherwise, if colonial growth is absent, a negative is marked for the patient. If growth is present, it is differentiated by color into LF (lactose fermenting), NLF (non-LF) and GPC (gram-positive cocci). If NLF or GNB, oxidase test is performed. 4) For bacterial growth, the procedure is to either prepare slides for microscopy, conduct the bacterial ID and antibody-sensitivity test (AST), or both. For slides, staining is often done (either gram-stain or AFB) 5) AST is conducted using a machine, while microscopy is performed manually. Test reports are thereafter generated.
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  Pictures AST machine (left)and result page (up). A plate with bacterial growth Serology tests for viruses. Making the inoculated plates.
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  Histopathology (and Cytology) •This is the study of tissue (histopathology) and fluids (cytology) for detection and diagnosis of cancer. • There are two major processes involved in this laboratory: H&E (haemotoxylin and eosin) staining, and IHC (immunohistochemistry) Before either of these processes are done, grossing is the first step. This is where a specialist cuts small, representative specimens from received tissue samples (small, medium, large and radical sizes). These specimens are then put into formalin, and then processed in a tissue processor overnight. Thereafter, the processed tissue is embedded and heated (to room temperature) in paraffin. This makes the frozen blocks that are used for section cutting. H&E – Haemotoxylin and Eosin This is the standard stain for all tissue samples. First, a rotatory microtome is used to slice thin cross-sections from the sample block. This is put in a floating water path, and stuck to a microscope slide (protein in tissue causes the adhesion). Then, the slides are dried and put into the auto-staining machine. This machine is entirely automatic and performs various kinds of stains (PAP, H&E for ex.). Once this is done, the slides are mounted, labelled and sent to the laboratory doctors for analysis. IHC – Immunohistochemistry (Immuno = immunity = antigen-antibody reactions, Histo = tissue) This gives insight into the stage and kind of cancer. Cancer cells have specific surface antigens that bind to certain antibodies. If this binding happens, a brown color is produced and it is interpreted that the slide has cancerous tissue. This process requires: 1) Endogenous blocking – blocking the nonspecific antigens. 2) Buffering – this is used several times! 3) Diluted antibodies – solution of conc. antibodies in water 4) Primary and secondary antibodies – primary antibodies do the reaction, secondary antibody attaches to the primary antibody (both antibodies are from entirely different species to prevent non-specific antigen binding) 5) DAB – a chemical that leads to the (brown) color change (attaches to the secondary antibody) There is also FS (Frozen Sample) Analysis. These samples come directly from the OT, are quickly cut into specimens and made blocks of using cryostat (freezing microtome), manually stained with H&E, and sent off.
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  Pictures IHC procedure. Slides stainedwith H&E. A kidney in a formalin container (it has cancerous tissue) Extracting ‘representative’ samples and putting them in small trays.
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  Flow Cytometry - Theflow cytometer is a modern innovation used to identify and measure physical and chemical characteristics of a cell population. It has several applications, but the laboratory uses it for diagnosing blood cancer (T-ALL, B-ALL, AML etc. ) and stem cell enumeration. How it works (blood cancer diagnosis) – immunophenotyping of leukemia A blood sample is first received. It is often peripheral blood or bone-marrow blood samples (first mobilized). It is then mixed with certain fluorescent markers known as fluorophores. The blood has different lymphocytes – B, T and NK (natural killer). Each has a different set of markers (B has CD1,2,3 and T has CD19 and NK has CD16,56 for example). The sample is put in the cytometer. The cytometer mixes the sample with a saline, and this saline is put into a narrow tube. One cell (a singlet) passes through the laser at a time, and as it does so, the light of the laser is scattered. There are two kinds of scatterings – front scattering (FSC) and side scattering (SSC). FSC is characteristic of the size (both are directly proportional) while SSC is characteristic of the inner complexity of the cell. Along with the scattering, the machine also measures the fluorescent activity of different markers. Depending on antigen-antibody binding, the graph produced shows the singlet points on a scatter plot; whether the population is positive for that marker is thus measured. Machine used is BD FACSCanto 2 It is also used for stem cell enumeration. CD34+ marker is used for this, and this marker is highly specific to stem cells. Stem cell viability is also measured.
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  This picture demonstratesthe presence of T-cell lymphoma. Polymorphs are neutrophils. Reading the graph Axis label = the marker used The plotted dots = individual readings (Scale used is logarithmic for everything but FSC and SSC – for them, it’s linear) The graph tells you of the extent to which a certain marker is present. The presence of this marker informs you of the abnormalities. In the graphs with the ‘red’ color, some are positive and some aren’t for the specific marker. Since some markers are native to abnormal cells, whenever this marker tests positive, that certain cell is cancerous.
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  Biochemistry - This isa largely automated department. The few people present here only operate the machines, generate the test reports, and perform blood-grouping. - (Not too much insight was gained here because the doctors didn’t distill much information) Blood-grouping Done through agglutination of blood. There are different blood-type samples (A, B and O). A bit is taken of each, and mixed with the blood to be tested in a test tube. This is put in a centrifuge. Once it is taken out, the agglutination is highly evident. It happens due to antigen-antibody reactions. Biochemistry There are two machines, each of which has a backup (so, total of 4 machines). Machines are Vitros 4600, Abbott i1000SR Architect Plus. The chemistry system utilizes the principle of reflectance spectrophotometry and dry slide chemistry. It tests for biochemical levels, such as CRP (C-reactive protein) and KFT and LFT (kidney/liver function test). The reading also measures the hemolysis, icteric (ness?), and turbidity of the blood sample. The immunoassay analyzer is used for things such as viral markers and cancer. It operates on the principle of CMIA. The machines require daily check-ups. This can be done automatically
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  Pictures A results screen Chemistryanalyzer (up) and immunoassay analyzer (left)
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  Haematology- This departmentis for blood and urine composition tests. Tests such as CBC are conducted here. Tests here - ESR (erythrocyte sedimentation rate) This test is performed on the blood plasma. Light passes through the plasma and its transmission is analyzed as it does. As everything sediments, the light transmission increases. High sedimentation rate can be a sign of low hemoglobin. - Urine routine test Three aspects – physical (appearance), chemical (done using a strip with different colors for different levels) and microscopic (without stains). - Blood slides and microscopy Staining for these slides is done with Leishman’s stain. It stains all the blood cells. - CBC (complete blood count) Works on the principle of fluorescence. There is a complex machine that can analyse one blood sample in less than 1 minute. Reports are printed on the screen with a graph, and the levels of different cells. When something is abnormal, the report will say ‘positive’ on the top. - Coagulation analyzer (PT and APTT) - Blood grouping Here, blood grouping is done in a similar way as biochemistry. However, instead of actual blood, they use antibodies (Anti-A, Anti-B and Anti-D, for RH factor) The laboratory seeks to conduct a complete analysis of their blood samples within 90 minutes of receiving them.
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  Pictures Staining procedure. A report(positive for abnormal levels)
• 13.
  Reflection The internship wasmy first exposure to a biological laboratory, and it was a rich exposure since every sub-department played a crucial and specific role within the whole diagnostic system. From creating bacterial cultures in Microbiology to using human tissue for cancer diagnosis in Histopathology and employing modern machines for CBC in Haematology, there was an abundance of knowledge to acquire. Fortunately, the doctors and trainees were of great help in helping me understand what was going on. Personal observations can only get you so far, and the questions that arise from them need to be addressed by experts; fortunately, I had sources for that. Mr. Feeroj, in the department of histopathology, and the other doctors and trainees, as well as the Flow Cytometry lab technicians, were of immense help, answering all questions that I could come up with. It was a great experience.