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MCD Diagnostics Alexandra Burke-‐Smith
1. Introduction to Chemical Pathology Professor Karim Meeran
1. List five common diagnostic tests carried out by the department of chemical pathology
• Electrolytes – (including Na and K) • Urea & Creatinine – high levels suggest renal failure • Calcium & Phosphate – measure of bone • LFT (Liver Function tests)
o 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 (ALP) § Aspartate amino-‐transferase (AST) § Alanine amino-‐transferase (ALT) § Gamma glutamyl transferase (GGT)
• Hormone assays – done as a sub-‐division of chemical path. Hormones commonly measured include thyroxine, TSH and cortisol
• Glucose – can be reapidly measured using a glucose sensitive stick on the ward or home. A more sensitive measurement is made in the lab.
o Red cells will consume glucose (anaerobic glycolysis), even after it is out of the patient, unless they are poisoned:
o Fluoride oxalate is used for this purpose. • Cardiac enzymes (present in heart muscle, leak out during MI)
o Troponins o Creatine Kinase (CK) o Apartate amino transferase (AST) o Lactate dehydrogenase (LDH)
2. Know how to collect specimens for common tests including electrolytes, urea, glucose and
glycosylated haemoglobin When collecting blood…
• Make sure you use the right bottle • If you get it wrong throw it away and start again • Make sure you get the correct patient • Label the tube correctly with the patients name • If it is urgent ensure it gets to the lab in time
Different tests require the use of different anticoagulants, therefore samples must be put in different bottles:
Bottle Colour Contains Test Red None U&E, TFT, LFTs
Yellow (orange) Gel to speed up clot U&E, TFT, LFTs Purple Potassium EDTA HbA1c
Green Lithium Heparin Homocysteine Grey Fluoride oxalate (poison) Glucose
Turquoise Citrate (anti-‐coag) Clotting factors
Normal Ranges: • Na = 135-‐145mM/L • K = 3.5-‐5mM/L • Creatinine = 70-‐150µmol/L • Urea = 2.5-‐6.7mmol/L • Proteins = 60-‐80g/L • Calcium = 2.12-‐2.65mmol/L
MCD Diagnostics Alexandra Burke-‐Smith
• No anticoagulant is added to the red or yellow bottles, as the blood naturally clots using clotting factors. The clotted blood can then be removed leaving the serum which is what is needed for the test
• When anticoagulants are used (EDTA or heparin), the blood will use the clotting factors to clot, therefore it can be separated into red cells and plasma (what is needed for the test)
• Measuring glucose – red cells consume glucose therefore the longer the sample is left before testing, the more glucose is used and thus the lower its concentration. Fluoride oxalate in the grey bottle kills the red cells, therefore preventing them from using up the glucose
A chemical pathologist in the lab needs to be contacted if:
• You want the sample rapidly centrifuged out of hours • To measure labile hormones such as insulin • When CSF (spinal tap) glucose and protein is needed to be urgently measured
3. Describe a typical chemical pathology request form
MCD Diagnostics Alexandra Burke-‐Smith
2. The Diagnosis of Infection and the Use of the Virology Laboratory Dr Mark Atkins
1. Understand what tests are available for diagnosing viral infections When diagnosing a viral illness, it is important to:
• Take a good history, including vaccination history, travel (especially in previous 3 weeks), contact with animals, contact with infected persons and occupation
• Perform a clinical exam Diagnosis depends on the clinical findings, the detection of specific antibodies and/or the detection of a virus in the appropriate clinical sample Ideal qualities of a virological test:
• High specificity i.e. have a low level of cross reactivity. • Sensitive-‐ detect the virus or the antibody at very low levels • Rapid-‐ results should be available in a timely fashion. • Non-‐invasive. This reduces the risks of the procedure and makes then easier to repeat if
necessary. • Cost effective. Most virology tests only cost a few pounds each but some of the molecular
tests are significantly more expensive, so use them wisely. Diagnostic Methods
• Cell culture – the gold standard but time consuming. Some viruses such as Hep C will not grow in cell culture. Cell culture needed in forensic cases
o Main method of herpes simplex and respiratory virus detection • Electron Microscopy (EM) – virus structures can be visualized
o Mostly used for stool samples • Antibody detection e.g. in HIV • Antigen detection, e.g. HBsAg in Hep B • Genome detection – using PCR to detect viral DNA or RNA • Quantification of antigens and genomes including antigen sub-‐typing and genome typing –
essential for diagnosis and monitoring of things such as HIV, HBV and HCV Immunofluorescence (IF) -‐ Useful for the direct detection of viral antigens in clinical samples (eg respiratory viruses)
• Can be used for typing and culture confirmation • 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) -‐ Detection of antibodies and antigens using immunoassays.
• Examples include EIA’s (enzyme immunoassays), Western blots, RIBA’s (recombinant immunoblot assays useful for eg typing anti-‐HIV 1 &/or 2)
• Specific, sensitive and relatively easy to automate. • Can be adapted to detect specific antibody classes (e.g. IgM IgG or IgA) or antigens • Sensitive and can quantify amounts of antibody (e.g. anti –HBs antibody) • Examples include HIV antibody and antigen, Hepatitis A,B,C serology, rubella, mumps,
parvo etc o EIA’s for HIV antibody detection is an indirect method of detecting infection. Non-‐
specific reactions may be a problem, therefore interpretation of results must take the clinical circumstances into account
MCD Diagnostics Alexandra Burke-‐Smith Viral gene detection and quantification
• Examples include Polymerase chain reaction PCR (a target amplification system), and bDNA (signal amplification system)
• Both assays used to measure “viral load” in HIV, HCV HBV infection. • Polymerase chain reaction uses 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 quantification during linear phase gives better reproducibility, precision and dynamic range.
o Readily adapted to detect multiple viruses in the samples simultaneously. o Closed tube monitoring eliminates contamination
2. Appreciate the range of viruses that can cause human disease 3. Know what clinical samples to take to enable you to make the correct diagnosis
A range of viruses can cause various different types of infection. These are detected using different methods of sampling. Respiratory infection:
• Nasopharyngeal aspirate (NPA) • Throat swab • Broncheo-‐alveolar lavage (BAL) • Stools for SARS • Blood for PCR if disseminated infection suspected • Acute and convalescent serology
CNS disease – meningitis/encephalitis:
• CSF for PCR • Stools and throat swab for enterovirus detection • Serology for West Nile infection and other arboviruses-‐ travel history important
Diarrhoea and vomiting:
• Stool • Vomit – can be used to detect viruses via EM but stool better • PCR or antigen detection assays for noroviruses best used for stool samples
HIV:
• Detect specific Ab as an indirect method of testing for virus • Antibodies may become detectable within weeks to months of infection • IgM is first to appear followed by IgG • Ab’s are detectable for life
Summary Sampling methods will depend on the disease being investigated:
• Throat swab -‐ for virus isolation (in virus transport medium, VTM) -‐ useful in the 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.
MCD Diagnostics Alexandra Burke-‐Smith
• 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.
Remember: the results of any tests need to be interpreted in the clinical context of the patient.
MCD Diagnostics Alexandra Burke-‐Smith
3. The Diagnosis of Infection and the Use of the Bacteriology Laboratory Dr Hugo Donaldson
1. Explain the concept of best-‐guess microbiological diagnosis and the contribution of the laboratory to it.
2. Describe the investigations the microbiology laboratory does and their limitations 3. Describe the limitations of microbiology laboratory investigations. 4. Be aware of the turn-‐around times of different investigations, particularly the delays inherent
in making cultural diagnoses. 5. To know how to interpret laboratory results of the commonly used tests.
Initial diagnosis of infection is based on the principle of making an informed, best-‐guess clinical diagnosis based upon: History taking
• Present illness: time and onset of infection, symptoms, signs (esp. rashes), pattern of infection (acute or chronic)
• Other associated illnesses • Past history: previous infections particularly with resistant organisms eg MRSA,
hospitalizations, travel, antimicrobial use • Family and social history • Other contributory factors • Drug and antimicrobial history
Types of investigations made by the microbiology lab:
• Agar plate culture. Types of culture include: o In air – for aerobic and facultative anaerobes o In CO2, eg for pneumococcus o In reduced O2 tension, eg ampylobacters o In absence of O2, eg anaerobes such as clostridia o At different temperatures:
§ 37oC = mesophils § 42 oC = thermophils § 22 oC = cryophils
• Microscopy – either direct under microscope, using stains or fluorescence • Direct antigen detection • Molecular probes and amplification • Serology
Collection of a sample The optimum time for collection of a specimen is:
• Before starting anti-‐microbial treatment • During the acute phase of illness • Collection of acute, convalescent and paired sera
o Acute sera: serum from a patient suffering from a particular infection o Convalescent sera: Serum from a person who has recuperated o Convalescent sera which may be of use in treating a person with the same infection;
while acute-‐phase serum has ↑ IgM antibodies, CS has ↓ IgM, and ↑ IgG antibodies • Collection of clinically relevant specimen • Collection from proper site with minimum contamination or normal flora • Adequate quantity and appropriate number of specimens • Clearly labelled • Transport – make sure it reaches the lab on time (portering problems) An urgent specimen
will only be dealt with, with prior arrangement
MCD Diagnostics Alexandra Burke-‐Smith Procedure for making a diagnosis:
• Naked eye examination of the specimen • Microscopy – e.g. light, fluorescent etc – quick and simple, valuble in some situations,
limited in others o Most microbiology samples are cultured on agar plates, which takes time:
§ For organisms to multiply sufficiently: usually 24-‐48 hours § To culture again for antibiotic sensitivities – another 24 hours.
• Culture – for detection of pathogen, sensitivity testing and epidemiological typing • Serology – used when looking for antibodies as evidence of infection/immunity. • DNA/RNA – PCR analysis • Direct antigen detection (particle agglutination tests, ELISA)
NB: Specimens that are processed in the microbiology labs without further consultation include:
• CSF • Urgent auramines • Blood cultures • Fluids – pleural, joint, ascetic • Rapid antigen screen • Gastric aspirates • Corneal scrape • Swabs taken following urgent operations
Specimens that are referred to the on call medical microbiologist include: • Antibiotic assays – done as batches to make cost effective performed twice a day • Pregnancy tests • Serology • Faeces • Routine sputa • HVS
Examples of microbiological examinations Urine:
• Bedside: naked eye – clear, cloudy, haemorrhagic, bile stained • Dip stick (NB: not microbiological investigations) – blood, protein, leucocytes, bilirubin,
ketones • Microscopy – WBC (pyuria suggests infection), RBC (may also indicate
tumour/microemboli/trauma), crystals, casts, renal epithelial and squamous epithelial cells • Culture on MacConkey agar (urine should be sterile so any microbial growth is potentially
significant in an appropriately taken sample) • Quantitative colony count for significant bacteria (more than 105/ml) • Antibiotic sensitivity testing of bacteria that grow
Sputum:
• Naked eye examination – mucoid, purulent, blood stained, caseous • Microscopy – gram stain for polymorphs associated with bacteria • Culture – to look for specific pathogens like pneumococcus • Special culture:
o TB o Legionella o Nocardia o aspergillus
• Broncho-‐alveolar lavage • Protected bronchial brushing • Open lung biopsy • Needle aspirate of pleural effusion
MCD Diagnostics Alexandra Burke-‐Smith Cerebrospinal Fluid (CSF):
• Naked eye examination – clear, cloudy, haemorrhagic, xanthochromic • Gram stain – to look for type e.g. ploymorph lymphocyte and proportion of cells • ZN/auramine staining • Antigen detection • Culture and sensitivities • PCR
Faeces
• Naked eye – consistency, blood stained, colour, presence of worms • Microscopy – ova, crysts, parasites • Culture on inhibitory media – e.g. DCA, selenite (faeces contains 1012-‐14 bacteria per gram,
so selective media are used to suppress background “flora” organisms) • Toxin detection (Clostridium difficile) • Special staining – e.g. Cryptosporidia
Routine blood cultures
• Aerobic cultures, anaerobic cultures, microaerophilic, CO2 culture • Sensitivity testing • Extended cultures – e.g. TB, fungal cultres, brucella, bacterial endocarditis • Antigen detection – meningococcus, pneumococcus, legionella, haemophilus, Cryptococcus
etc Other important investigations/tests performed in microbiology labs:
• Infection control: o Screening and monitoring o Mandatory for national surveillance – e.g. MRSA o Antibiotic resistance screening
• Animal inoculation tests • Identification of difficult organisms • Testing for bacterial toxins • Typing and finger printing bacteria to trace cross infections and outbreaks • Organise national quality control for individual labs
MCD Diagnostics Alexandra Burke-‐Smith
4. Cellular Pathology Dr Marjorie Walker
1. List 3 situations where histopathology and cytopathology might commonly be used as a diagnostic method.
2. Describe the nature of specimens sent for histopathology and cytopathology laboratory diagnosis.
3. List 2 situations where frozen section diagnosis is required 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. 5. Explain the additional information available from immunohistochemistry, and give an
example of when this technique may be used 6. Describe the benefits of the autopsy 7. List 3 benefits of cytology screening
Terminology Pathology: the medical science and speciality practice classified as the study of disease. It deals with all aspects of disease, including:
• Nature • Cause • Development • Consequences • Pathogenesis • Manifestation • Monitoring disease progression
Histopathology: encompasses surgical and autopsy pathology to make diagnoses on tissues
• This includes biopsy material, surgical specimens • Histopathologists can make rapid diagnoses when necessary (use of frozen sections) • Autopsy is used to determine cause of disease, explain unsuccessful treatment, spread of
disease o Also used for education and research o Autopsies may be performed by the authority of the coroner (suspicious/unknown
COD), or by permission of the relatives or the deceased
Cytopathology: diagnoses on cells • Uses cellular specimens eg sputum, cervical smears • Less invasive technique than obtaining tissue for histopathology
Biopsies Biopsies are taken for numerous reasons: skin lesions, suspected tumours (must be excised completely with margin of normal tissue), rash (only may require punch biopsy) There are various types of biopsies:
• 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
MCD Diagnostics Alexandra Burke-‐Smith Cytopathology Uses
• diagnosis of lung tumours (non invasive) • aid diagnosis in endoscopy when brushings of the tumour can be taken • cervical and breast screening. • Fluids such as urine and ascities can be examined for cells,
Procedure for suspected tumours Surgical specimens removed at operation are all sent to Histopathology for diagnosis and to determine if a tumour is completely excised.
• The histopathologists and cytopathogists will generate a report of the examination of the specimen, which is sent to the clinician responsible for the patient.
• It is important that the report gives a conclusive diagnosis and a statement of tumour excision and prognostic features – for example if surrounding lymph nodes are involved or the tumour spread on to the peritoneum.
Use in screening Cervical smears are prepared preserved (fixed) with alcohol, stained by a special stain called Papanicolaou and permanently preserved.
1. Who carries out the procedure? a. Sample obtained: nurse/Dr b. Preparation methods in lab = automated (mostly) or well-‐trained/experienced
MLSEs (medical laboratory scientific officers) c. Slide reading = trained screening technical staff d. Check results = consultant
2. Cervical smears are performed to pick up cervical cancer as part of the screening programme
3. Screening criteria a. Test is easy and non invasive b. High take up in the population c. A significant number of cases can be detected. d. Something can be done about the disease.
4. Problems with cervical screening: a. Failure to obtain a satisfactory sample (smear). b. Failure to make a proper smear. c. Poor staining. d. Poor interpretation
Specimen Procedures Specimens for histopathology (biopsies and whole tissue) must be “fixed” in formalin, a preservative that stabilises protein bonds and prevents autolysis.
• The tissue then must be processed, sections cut and stained and the histopathologist must write the report.
o For large specimens, allowing for overnight fixation or longer if the specimen is large or fatty, this process should take 2-‐3 days.
o Small biopsies can be processed in a day and if a rapid diagnosis is required then rapid processing takes 4-‐5 hours.
• A very rapid diagnosis may be required during an operation – is this a tumour? Is the margin of excision adequate? Is this lymph node involved? Have I got the parathyroid?
o Frozen section tissue is received in the laboratory without any fixative. § A small sample is selected and frozen rapidly. § A thin section is cut on a microtome contained in a refrigeration cabinet,
fixed rapidly and then stained with haematoxylin and eosin. § An answer can be given by phone within 20 minutes.
MCD Diagnostics Alexandra Burke-‐Smith How are sections of specimens obtained? All specimens are allocated an identity number and logged in to the computer. • During the daily cut up of specimens the pathologist or biomedical scientist selects and describes the tissue samples to be examined. • Some specimens (for example biopsies) are processed whole, while larger specimens (e.g. mastectomies, colonic specimens) have a few selected pieces removed. • Each selected piece of tissue is placed in a small perforated plastic container with a lid. This plastic cassette receives the laboratory number. • These tissue blocks are processed to paraffin wax. • Once it has been waxed and cut into sections it is then rehydrated • The waxed sample can then be cut into thin sections and mounted on slides (normally 5 microns thick) • 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:
Ø Silver nitrate – melanin pigment, fungi, calcium deposits and certain fibres Ø Other dyes can be used to show glycogen, mucins and tissue structures Ø Gram staining for bacteria Ø For difficult tumours immunocytochemistry techniques are needed – using labelled antibodies
MCD Diagnostics Alexandra Burke-‐Smith
5. Antibodies as Diagnostic Tools Dr Keith Gould
Immune basis Antibodies can be raised to any antigen or protein. This concept can be used therapeutically, diagnostically and for immunodiagnosis The unique specificity of antibodies for their target antigens is the basis of many diagnostic tests. The immune response relies upon 2 exposures.
Primary Exposure Secondary Exposure Mainly IgM Ab at 5-‐10 days in serum Levels rise over next 10-‐20 days Slow decline, never completely disappears
Ab response more rapid Higher levels, both peak and baseline Mainly IgG with a higher affinity for antigen
All Abs from one B-‐cell clone have the same Fab-‐Ag specificity
• Abs from different B-‐cells may have the same Fc portion but will have different Fab portions • B-‐cells can produce both membrane and secreted forms of the same Ab • B-‐cells can switch production from one Ig type to another while maintaining the same Fab
Types of immunity
• Active immunity = vaccination to develop large quantities of Abs • Passive immunity = maternal protection, anti-‐toxin and anti-‐sera antibodies • IVIG = (Intravenous IG) = normal herd immunity, e.g. tetanus, hepatitis, rabies, varicella-‐
zoster. Given to immuno suppressed patients Types of antibody responses 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. 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
1. Understand the therapeutic and diagnostic use of manufactured antibodies 2. Give examples of types of substances that are typically identified diagnostically by means of
antibodies Therapeutic use
• Prophylactic protection against microbial infection • Anti-‐cancer therapy • Removal of T-‐cells from bone marrow grafts • Block cytokine activity & regulation
Diagnostic use • Tissue typing • Blood group serology • Immunoassays
o hormones o antibodies o antigens
• Immunodiagnosis
MCD Diagnostics Alexandra Burke-‐Smith
o Infectious diseases o Autoimmunity o Allergy o Malignancy
3. Explain the basic principles of detection methods 4. Describe the advantages in different situations of the types of detection system; for example,
enzyme-‐linked antibodies, radioactively labeled antibodies Detection methods These rely upon antibody-‐antigen interactions:
• Known antibodies can be used to detect the presence of specific antigens, eg detection of streptococci in throat swabs
• Known antigens can then be used to detect specific antibodies, eg HIV tests to test for anti-‐HIV antibodies
Immuno-‐precipitation
• Excess Ag and Ab form small soluble complexes, but Ab and Ag equivalence > extensive cross-‐linking and the formation of large insoluble complexes which precipitate
• This can be used to isolate and concentrate a particular antigen Immuno-‐agglutination
• Semi-‐quantitative assay to get approximate concentration of Ag • Clinical example is haemagglutination used in blood type determination; used to determine
the presence of particular antigens Labeled Antibodies
• Ab’s can be labelled with radioactive isotopes – e.g. 125I, 14C, 35S • Can also be labelled with enzymes which convert an added substrate to a coloured product
– e.g. alkalinephosphatase. E.g. immunoperoxidase staining o ELISA (enzyme-‐linked immunosorbent 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
5. Understand how antibodies may be used in clinical practice
Use in clinical immunology:
• Immunodeficiency o Serum immunoglobulin levels are measured using serum electrophoresis, ELISA, or
nephleometry (IgG, IgM, IgA only) o Specific antibodies can then be measured using ELISA
• Malignancy • Autoimmunity
o Autoantibodies can be investigated with biopsy, serology and immunofluorescence • Inflammation • Tissue typing and transplantation
Use in Pathology:
• Clinical chemistry • Haematology and blood transfusion • Medical microbiology • Histopatholgy
MCD Diagnostics Alexandra Burke-‐Smith
6. Explain how antibodies can be generated (experimentally or commercially) for diagnostic purposes
Antibodies are produced via immunisation – whether it be in cows, horses, mice humans etc
• Immunised with a particular antigen + adjuvant o Adjuvants act to boost the immune system and so increase Ab production. An
example are aluminium derivatives • Normally 2 booster injections are administered after initial one • This 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
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