Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Diagnostics
MCD – Diagnostics Introduction to Chemical Pathology 1. List the five common diagnostic tests carried out by the department of chemical pathology Electrolytes – (including Na and K) Urea & Creatinine (U&E) – high levels suggest failure of renal excretion of these substances and hence renal failure Calcium & Phosphate – measure of bone LFT (Liver Function tests) – a measure of liver enzymes o Damage to the liver results in extra liver enzymes leaking into the blood o Particular diseases are associated with particular patterns of enzyme leakage. o Enzymes commonly measured include: Alkaline phosphatase Aspartate amino-transferase (AST) Alanine amino-transferase (ALT) Gamma glutamyl transferase (GGT) Hormone assays – done as a sub-division of chemical pathology department. Hormones commonly measured include thyroxine, TSH and cortisol Glucose – can be repeatedly measured using a glucose sensitive stick on the ward or home. A more sensitive measurement is made within the laboratory. Red cells will consume glucose, even after they are removed from the patient, unless they are poisoned
2. Know how to collect specimens for common tests including electrolytes, urea, glucose and glycosylated haemoglobin Different tubes have different anticoagulants and so have different samples: o Red – none → U&E, TFT o Yellow – gel to speed up clot → U&E, TFT o Purple – potassium EDTA → HbA1c o Green – lithium heparin → Homocysteine o Grey – fluoride oxalate (poison) → Glucose o Turquoise – citrate (anti-coagulant) → more accurate measure of clotting factors No anticoagulant (red/yellow) – for U&E, the blood clots using up all clotting factors, this can then be removed leaving the serum which is needed Anticoagulant – EDTA or heparin, the clotting factors are unused so the blood can be separated into red cells and plasma – the two components which are needed Measuring glucose – red cells consume glucose so the longer this is left out the more glucose is used. Flouride oxalate in the grey topped bottles poisons the cells – preventing glucose use A chemical pathologist in the lab needs to be contacted if: o You want the sample rapidly centrifuged out of hours o To measure labile hormones such as insulin o When CSF (spinal tap) glucose and protein is needed to be urgently measured
3. Describe a typical chemical pathology request form Cardiac enzymes o Are present in the heart muscle o Leak out during MI Troponins Creatine Kinase (CK) Apartate amino transferase (AST) Lactate dehydrogenase (LDH) Normal Ranges: o Na = 135-145mM/L o K = 3.5-5mM/L o Creatinine = 70-150µmol/L o Urea = 2.5-6.7mmol/L o Proteins = 60-80g/L o Calcium = 2.12-2.65mmol/L
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Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Diagnostics
The Virology Laboratory 1. Appreciate the role of the virology laboratory in the diagnosis of infectious disease. When diagnosing a viral illness it is important to: o Take a good history – including vaccinating history, travel (especially in previous 3 weeks), contact with animals, contact with infected persons and occupation o Perform a clinical exam o Diagnosis depends on the clinical findings, the detection of specific antibodies and the detection of a virus Virological tests – the ideal tests should have the following qualities: o High specificity – i.e. have low levels of cross reactivity o Sensitive – detect the virus or antibody at low levels o Rapid – result should be available quickly o Non-invasive – reduces the risk of procedures and allows easy repetition o Cost effective – most only cost a few pounds, some more
2. List the types of specimen that are commonly sent for virological diagnosis. Will depend on the disease being investigated Throat swab – for virus isolation (in virus transport medium, VTM) – useful in diagnosis of enteroviruses and respiratory viruses Stools - for EM and Rotavirus EIA (in sterile pot) - for the diagnosis of enteroviruses and viruses that cause diarrhoea such as rotavirus, astrovirus, adenovirus, noroviruses, etc CSF - PCR for herpes and enteroviruses (in sterile container, VTM (viur transport medium) not required) - for the diagnosis of viruses causing meningitis or encephalitis such as HSV, VZV, enteroviruses, mumps, etc Nasopharyngeal aspirate (NPA) - for respiratory viruses using Immunofluoresence (IF) or PCR, such as RSV, influenza A&B, adenovirus, parainfluenza viruses, SARS etc Urine - virus isolation or PCR depending on which viruses you are interested in (in sterile container), e.g. BK virus, CMV, etc Blood (clotted) - for antibody detection Blood (EDTA) - for PCR. Used for detection and quantification of HIV, HBV and HCV Biopsy samples can be useful in certain circumstances e.g. brain biopsy in encephalitis
3. List the laboratory procedures that may be used as part of diagnosis of common viral infections. Diagnositic methods o Cell culture
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Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Diagnostics o o o o o
Electron microscopy (EM) Antibody detection e.g. HIV antibody Antigen detection e.g. HBsAg in hepatitis B infection or RSV antigen in respiratory sample Genome detection – e.g. using PCR to detect viral DNA or RNA Quantification of antigens and genomes (now essential for diagnosis and monitoring of HIV, HBV and HCV)
Electron microscopy o Virus structures can be visualised using an electron microscope o Used mostly for stool samples Immunofluorescence (IF) o Useful for the direct detection of viral antigens in clinical samples (eg respiratory viruses) o Can be used for typing and culture confirmation o Relatively quick and inexpensive but subjective and very dependent on the skill of the technician and the quality of the sample Enzyme Immuno assays (EIA’s) o Detection of antibodies and antigens using immunoassays o EIA’s (enzyme immunoassays) o Western blots o RIBA’s (recombinant immunoblot assays useful for eg typing anti-HIV 1 &/or 2) o Specific, sensitive and relatively easy to automate o Can be adapted to detect specific antibody classes e.g. IgM IgG or IgA o Sensitive and can quantify amounts of antibody (e.g. anti –HBs antibody) o Adaptable to antibody or antigen detection o Examples include HIV antibody and antigen, Hepatitis A,B,C serology, rubella, mumps, parvo etc EIA’s for HIV antibody detection o Detection of specific antibody is an indirect method of detecting infection o Non-specific reactions can be a problem, therefore it is important to use multiple formats (generally use 3 different assays) o Interpretation of results must take the clinical circumstances into account Viral gene detection and quantification o Polymerase chain reaction PCR (a target amplification system) o bDNA (signal amplification system) o Both assays used to measure “viral load” in HIV, HCV HBV infection. Polymerase chain reaction o Target amplification to allow detection and quantification over very large dynamic ranges (> 5-8 logs) o Can be very sensitive (as low as 1 genome copy) o Can subtype viruses from PCR products o Problems with contamination - this can be overcome using “Real Time” PCR “Real Time” PCR. Advantages o Quantification during linear phase gives better reproducibility, precision and dynamic range o Readily adapted to multi-plexing i.e. detect multiple viruses in the samples simultaneously o Closed tube monitoring eliminates contamination
The Bacteriological Laboratory 1. Explain the concept of best-guess microbiological diagnosis and the contribution of the laboratory to it. The initial diagnosis of infection is based on the principles of making an informed best-guess clinical diagnosis Present illness: date on onset, symptoms, signs (esp. rashes) Past history: previous infections, hospitalizations, travel, antimicrobial use
2. Describe how the microbiology laboratory works, what investigations it does and how best to use the laboratory. Most microbiology samples are cultured on agar plates, which takes time: o For organisms to multiply sufficiently: usually 24-48 hours o Some need longer incubation: e.g. TB, brucella, actinomycetes o To culture again for antibiotic sensitivities: another 24 hours Microscopy o Direct under the microscope – urine o Various stains (Gram, Zielh-Nielsen (ZN), etc.) – pus, tissue fluids
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Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Diagnostics Fluorescence, with conjugated antibodies to specific antigens Gram stain of CSF, joint fluid, purulent exudates. ZN/auramine stain of e.g. sputum, for TB FTA (fluorescent treponemal antibody) for antibodies to T. pallidum Direct antigen detection (particle agglutination tests, ELISA) Meningococcal antigen in CSF C. difficile toxin in faeces Legionella antigen in urine Molecular probes and amplification (PCR, etc) Chlamydia in genital specimens Rapid PCR for MRSA Serology - looking for antibodies as evidence of infection/immunity o
Examples Microbiological examination of urine o Bedside: Naked eye - clear, cloudy, haemorrhagic. note that although these are NOT microbiological investigations: Dipstick tests for blood, protein, leucocytes, bilirubin, ketones may provide indication of there being an infection, o Microscopy: WBC (pyuria suggests infection), RBC (may also indicate tumour/microemboli/trauma), epithelial cells (suggest the specimen has been contaminated during collection), crystals, casts. o Culture on MacConkey agar (urine should be sterile so any microbial growth is potentially significant) o Quantitative colony count for “significant” bacteruria (>10 5 bacteria/mL) o Antibiotic sensitivity testing of bacteria that grow Microbiological examination of faeces o Naked eye, consistency, blood stained, colour, presence of worms o Microscopy: ova, cysts, parasites o Culture on inhibitory media – deoxycholate-citrate agar (DCA), selenite (Faeces contains 10 12-14 bacteria per gram, so selective media are used to suppress background ‘flora’ organisms) o Toxin detection (Clostridium difficile) o Special stains, e.g. for cryptosporidia
3. Understand how & when to collect specimens, and which investigations to request. In the acute phase of illness and before staring antimicrobials Collection from proper site, avoiding contamination by normal flora Prompt transport to lab since micro-organisms multiply in transit Adequate quantity and appropriate number of specimens Acute sera and convalescent sera (paired), for rising antibody titres
Cell Pathology 1. List three situations where histopathology and cytopathology might commonly be used as a diagnostic method. Pathology Medical science that deals with all aspects of disease Pathogenesis – cause Clinical expression and the lesions produced – the manifestation Disease progression Disease monitoring Histopathology Encompasses surgical and autopsy pathology Histopathologists make diagnoses on tissue – biopsy material, surgical specimens Can make rapid diagnoses, i.e. during an operation of tumours etc using frozen sections Autopsies are performed to determine the cause of death Also used for education and research into disease and treatment Cytopathology Makes diagnoses on cells, sputum and body fluids which contain cells, cervical smears and tissue obtained by fine needle aspirate (FNA) Uses less invasive techniques than biopsy Can be useful in diagnoses of lung tumours – fine brushing via endoscopy Also useful in cervical and breast screening
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Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Diagnostics
2. Describe the nature of specimens sent for histopathology and cytopathology laboratory diagnosis. Biopsies are taken for many reasons – common conditions include inflammatory disease and suspected tumours If a tumour is suspected clinically then it must be removed completely with a margin of normal tissue. Inflammation often only requires a punch biopsy Endoscopy enables the clinician to view the gastrointestinal tract. Biopsies are commonly taken from the stomach to exclude for example cancer, the duodenum to exclude coeliac disease and the large bowel to confirm cancer or diagnose inflammatory bowel disease Bronchoscopy allows a view of the trachea and bronchi and biopsies of suspected tumours or inflammatory lung conditions can be taken Liver biopsies can be performed under imaging guidance for tumours or for diagnosis of liver disease Renal biopsies are taken to determine the nature of glomerulonephritis and other renal disease
3. List two situations where frozen section diagnosis is required. Surgical and autopsy pathology use frozen sections
4. Summarise the main steps involved in processing a specimen for routine histopathology diagnosis and indicate the likely time needed to carry out these steps. Biopsies and whole tissue for histopathology must be fixed in formalin – a preservative that stabilises protein bonds and prevents autolysis. The tissue must then be processed, sections cut and stained and a report written For large specimens allowing overnight fixation or longer is needed (2-3 days sometimes). Small biopsies can be processed in a day and if required in 4-5hrs In surgery for tumours when a diagnoses is needed rapidly tissue may be frozen, a thin section cut on a microme contained in a refrigeration cabinet where it is stained – an answer can be given in 20mins Once set in formalin the specimens are processed to paraffin wax. Before this the sample must have the water removed by alcohol dehydration. Once it has been waxed and cut into sections it is then rehydrated Several sections can be taken from the same tissue block to get maximum information Haematoxylin stains nuclei blue and eosin the cytoplasm pink Most diagnoses can be made using H&E (haematoxylin and eosin) staining Other stains include: o Silver nitrate – melanin pigment, fungi, calcium deposits and certain fibres o Other dyes can be used to show glycogen, mucins and tissue structures o Gram staining for bacteria
5. Explain the additional information available from immunohistochemistry, and give an example of when this technique may be used. For difficult tumours immunocytochemistry techniques are needed – use antibodies to label antigens within tissues Epithelial, mesochyme, nerve and prostate tissues can be specifically identified
6. Describe the benefits of the autopsy Autopsies are performed by the authority of the coroner or by permission of the relatives Questions that can be answered include: o What was the terminal event? o Where their complications? o Where did the cancer originate? o Was the diagnosis correct? o Education for health car professionals
7. List 3 benefits of cytology screening. Screening only has value if these (and other) criteria can be met: o Test is easy and non invasive o High take up in the population o A significant number of cases can be detected o Something can be done about the disease However there are problems with for example, cervical screening: o Failure to obtain a satisfactory sample (smear) o Failure to make a proper smear o Poor staining o Poor interpretation
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Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Diagnostics
Antibodies as Diagnostic Tools 1. Explain how antibodies can be generated (experimentally or commercially) for diagnostic purposes. Antibodies can be raised to any antigen or protein Can be used therapeutically, diagnostically and for immunodiagnosis All due to their ability to attack specific antigens Antibodies are produced via immunisation – whether it be in cows, horses, mice humans etc Immunised with a particular antigen; adjuvant also used = act to boost the immune system and so increase Ab production. An example are aluminium derivatives Normally 2 booster injections after initial one Collect serum for use - produces a polyclonal antibody response Cloned myeloma tumour cells, that have been fused with spleen cells from an immunised animal using PEG to allow membrane fusion, can also be used in the lab to produce monoclonal Ab’s for the Ag that was used to immunise the animal with Polyclonal = normal antibody response to antigen invasion forming a heterogeneous mixture of antibodies. Antibodies are directed to several determinants on the antigen and the antibodies to a particular determinant will be of different affinity. So these are polyclonal antibodies, they derive from many different clonal B cells. A heterogeneous mixture against many epitopes of Ag Monoclonal = Homogeneous colony on antibodies derived from a single B-cell clone. Normally made artificially by fusing one antibody producing cell with a tumour cell that will form a clone of cells which will divide and produce the same antibody for a long period of time. Used as reagents for detecting cell markers Genetically engineered Abs = the cloning and expression of antibody genes is used in order to create antibody libraries. These can then be transferred into mammalian cells in order to complete glycosylated antibodies. Engineered chimeric antibodies are also possible
2. Understand the therapeutic and diagnostics use of manufactured antibodies. Therapeutic Prophylactic protection against microbial infection Anti-cancer therapy Removal of T-cells from bone marrow grafts Block cytokine activity Diagnostic Tissue typing Blood group serology Immunoassays o Hormones o Antibodies o Antigens Immunodiagnosis o Infectious diseases
3. Explain the basic principles of detection methods. Detection methods: o Rely upon Ab-Ag interaction o Known Ab’s to detect specific Ag’s – e.g. detection of streptococci in throat swabs o Known Ag’s to detect specific Ab’s – e.g. HIV tests to test for anti-HIV Ab’s Immuno-precipitation: o Ag-Ab interaction o Ab excess and Ag excess forms small soluble complexes o Ab and Ag equivalence leads to extensive cross linking and the formation of large insoluble complexes which precipitate out – this is what is wanted Immuno-agglutination: o E.g. haemagglutination used in blood type determination. o Qualitative assay to look for blood agglutination and presence of Ag o Semi-quantitative assay to get approx concentration of Ag Labelled Ab o Ab’s can be labelled with radioactive isotopes – e.g. 125I, 14C, 35S Radioimmunoassays (RIA) Autoradiography o Can also be labelled with enzymes which convert an added substrate to a coloured product – e.g.
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Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Diagnostics
o o o
alkalinephosphatase. E.g. immunoperoxidase staining ELISA = enzyme linked immunsorbent assay – enzymes added to antibodies causing a coloured reaction – the amount of colour is proportional to the amount of antigen Can also be labelled with fluorescent compounds such as fluorescein which are excited under polarised light under a microscope. E.g. for looking at anti-neutrophil cytoplasmic antibodies Magnetic beads – cell separation Coloured beads – luminex assays
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