DiagnosticsMCD Year 2

Anil Chopra

ContentsDiagnostics 1 – Introduction to Chemical Pathology...........................................................................1Diagnostics 2 – Diagnostic Virology.......................................................................................................2Diagnostics 3 - The Bacteriology Lab....................................................................................................6Diagnostics 4 – Pathology........................................................................................................................7Diagnostics 5 - Use of antibodies as diagnostic tools.............................................................................9

Diagnostics 1 – Introduction to Chemical PathologyAnil Chopra

1. List five common diagnostic tests carried out by the chemical pathology laboratory.

2. Know how to collect specimens for common tests including electrolytes, urea, glucose and glycosylated haemoglobin.

3. Describe a typical chemical pathology request form.

When sending things off to the lab, they need to be properly labelled with the patient details. Each of the collection tubes also has a different coloured top each corresponding to an anticoagulant: Red top have none Yellow top have gel to speed up clot Purple have potassium EDTA Green have lithium heparin Grey have fluoride oxalate

This is because red cells contain glucose which means that glucose level will go down with time. Fluoride oxalate prevents this

Place different samples in different coloured tubes:• U&E : serum in yellow/red top• Glucose: plasma in grey top• HBA1c: plasma in purple top• Thyroid Function Test: serum in yellow/red top• Homocysteine: plasma in green top

NB: serum does not contain clotting factors, plasma does

Results are normally made available on the computer:They give the patients level followed by the normal range.» Thyroid function: thyroid hormones» Adrenal function: electrolytes» Renal function: electrolytes, urea, creatine» Hepatic function: liver enzymes» Cardiac function: Troponins, Creatine kinase (CK), Aspartate amino transferase

(AST), Lactate Dehydrogenase (LDH)

Diagnostics 2 – Diagnostic VirologyAnil Chopra

1. Explain the role of the virology laboratory in the diagnosis of infectious disease.2. List the types of specimen that are commonly sent for virological diagnosis.3. List the laboratory procedures that may be used as part of diagnosis of common viral infections.

There are many new viruses that are constantly manifesting themselves in the population (e.g. HIV, HCV, Hepatitis E, SARS) as well as ones that have been around for a long time (rubella, mumps, herpes simplex).

Whilst there are also a number of different antivirals, the problem with them is that they are specific (not broad spectrum).

The virology lab:• Detect and quantifies viruses (or their components) in clinical samples during infection.• Detects antibody responses to diagnose acute infections.• Detects evidence of past viral infections or responses to vaccines.

Diagnostic Tools used- History- Physical examination- Laboratory tests

o Non specific o Rapido Virological o Cell culture – gold standardo Electron microscopy – viruses are too small to be seen with a light microscope.o Antibody detectiono Antigen detection – immunoflourescence is usedo Genome detectiono Antigen sub-typingo Genome typingo Quantification of antigens and genomes (now essential for diagnosis and monitoring

of HIV, HBV and HCV)- Rubbish in- rubbish out

Typical Samples Taken- Throat swab- for virus isolation

o Used in respiratory diseaseo Enteroviruses

- Stoolso for EM and Rotavirus EIAo Enteroviruses

- CSFo PCR for herpes and enteroviruseso Meningitis/encephalitis.

- Nasopharyngeal aspirate (NPA)- for respiratory viruses using IF.- Broncheo-alveolar lavage (BAL)- Urine - for EM an virus isolation- Blood (clotted)- for antibody detection- Blood (EDTA)- for Polymerise chain reaction- Vomit

o Viruses can be detected by electron microscopy.

Blood Serum Samples are able to detect a Number Of Diseases including:• HIV antibody• HCV antibody• HBV sAg, eAg e-antibody.• Herpes antibodies (HSV, EBV, CMV, VZV etc)• Hepatitis a IgG and IgM• Rubella, parvovirus B19 (erythrovirus)• Measles, mumps serology

Immunoassays: used to detect antibodies and antigens:- EIA: Enzyme immunoassays – varying concentrations of a specific antigen are placed in a

blotting tray. These are then reacted with their specific antibody and agglutination/fluorescence occurs.

- Western Blots – a type of electrophoresis.- RIBAs – Recombinant immunoblot assay- RPHA - reverse passive hemagglutination assay.

Enzyme immunoassays can detect specific antibody classes eg IgM IgG or IgA. They are very sensitive and can quantify amounts of antibody, they are very robust and are adaptable to antibody or antigen detection. Examples include HIV ab, Hepatitis serology etc.Hepatitis C Virus

Part of the genome encodes for the nucleocapsid and envelope proteins, which are structural proteins.

The NS (nonstructural) regions of the genome encode for various nonstructural proteins, which are indicated on the slide.

Several of the NS proteins are enzymes that have functions important to viral replication. Both the RNA helicase and the serine protease are enzymes essential for replication and are

targets for new antiviral strategies based on development of specific inhibitors of these enzymes.

Another target for drug development is the enzyme RNA-dependent RNA polymerase, which is encoded by the NS5B region.

New drug targets include protease, helicase, RNA-dependent RNA polymerase.

3'UTR3'UTR

Nucleocapsid

Envelope

5'UTR5'UTR

Transmembrane Cofactor

MetalloproteaseSerine proteaseRNA helicase

NS3NS2C E2E1 NS4A NS4B NS5BNS5A

IFN-PKRrepressor

RNA-dependentRNA polymerase

Human Immunodeficiency Virus – HIVWe can detect a number of things in the HIV virus: Detect the infectious virus (virus isolation). Detect protein components of the virus (p24 antigen in HIV, surface antigen in HBV etc) Detect genetic components of the virus (cDNA or RNA) quantitative or qualitative tests are

available. Detect the host response e.g. antibody or cell responses.

o IgM is detectable in the first few months after infection.o Followed by IgG – these are detectable for life. o In the UK there are 3 different test using 3 different assays.

Polymerise Chain Reaction: these use DNA polymerase to create large amounts of DNA.

Branched-Chain DNA (bDNA): uses a phosphorescent chemical that binds to the RNA in the suspect DNA. The brighter the test sample is, the more virus particles are in the blood.

Tissue Microarray (TMA): a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin embedded tissues such as clinical biopsies or tumor samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced, array pattern. Sections from this block are cut using a microtome, mounted on a microscope slide and then analyzed.

Nucleic Acid Sequence Based Amplification: 1. RNA template is given to the reaction mixture, the first primer attaches to its complementary site

at the 3' end of the template 2. Reverse transcriptase synthesizes the opposite, complementary DNA strand 3. RNAse H destroys the RNA template (RNAse H only destroys RNA in RNA-DNA hybrids, but

not single-stranded RNA) 4. the second primer attaches to the 5' end of the DNA strand 5. T7 RNA polymerase produces a

complementary RNA strand which can be used again in step 1, so this reaction is cyclic.

Diagnostics 3 - The Bacteriology LabAnil Chopra

1. Explain the concept of best-guess microbiological diagnosis and the contribution of the laboratory to it.

2. Describe how the microbiology laboratory works, what investigations it does and how best to use the laboratory.

3. Understand how & when to collect specimens, and which investigations to request.4. Describe the limitations of microbiology laboratory investigations.5. To be familiar with the turn-around times of different investigations, particularly the delays

inherent in making cultural diagnoses.6. To know how to interpret laboratory results of the commonly used tests.

There are 4 main ways in which can identify the infectious agents:- direct visualisation under a microscope- cell cultures- direct detection using

o antigens: agglutination of particles covered in antibodies or ELISAo nucleic acids: probe detection or polymerise chain reaction.

- indirect detection using antibodies: plate covered with antigen and exposed to sample.

Other things done in bacteriology lab: Checking of antibiotic susceptibilities Blood levels of antibiotics Recording numbers of isolates for Infection Control purposesMicrobiology labs are send samples such as swabs, aspirates, faeces, tissues, sputum. The stain them, and place them onto various agars. They use enrichment methods where they are expected to be sterile and use selective methods amongst specific pathogens.

Limitations of Microbiology Lab: Culturing parasites, various awkward bacteria, most viruses Antibodies to Listeria, Shigella, gonococci, coag-neg staphs etc Antigen or nucleic acid Contamination Adequate quantity and appropriate number of specimens Unlabeled specimens.

Microbes need time to grow in cultures: Culture: usually 24-48 hours Sensitivity: another 24 hours

Some need longer incubation: e.g. TB, Brucella

Diagnostics 4 – PathologyAnil Chopra

1. List three 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 two 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.

Cellular Pathology (Histopathology)Histopathologists:

- Examine surgical Biopsyo Biopsies and operative specimens sent to lab ranging in size.

- MDT’s on cancer and inflammation patients- Perform autopsies- Teach- Do research

BiopsiesA biopsy is the surgical removal of part of a tissue which is then analysed by a histopatholgist

A biopsy can tell us: Diagnosis Determination of the severity of disease Need for treatment Progression monitoring

The risks of a biopsy include Pain (1/5) Bleeding (1/500) Bleeding needing transfusion or surgery (1/2000) Puncture of the lung, gallbladder, kidney or intestine (1/ 3000) Death (1/9000) Obtaining Sections Identify specimen with unique number “Cut up” take representative sections to cassettes Process to paraffin wax - dehydrate Cut thin section – rehydrate – stain (H&E) Stains – Gram, ZN, Silver, Mucin, Immunoperoxidase

Immunocytochemistry: For difficult tumours - antibody systems to label antigens within tissues. Different types of tissue can be correctly identified.Frozen Sections: the tissue is frozen to -20ºC and then a small part of it is cut off, put onto a glass slide and then stained. Can assess: Tumour Spread of tumour to lymph nodes Margins (is it all out)

Autopsies Coroners post mortems

o Used to determine natural or unnatural cause of deatho What was the terminal event?

Hospital post mortems

o Used to detect cancer siteo Reassurance for family and doctors. o Education for health care professionals.o Explains why treatment was unsuccessful.

CytopathologyNon invasive method of diagnosis Fine Needle Aspirates – diagnosis of tumours Endoscopy when brushings of the tumour can be taken (Brochio alveolar lavage). Fluids (fluids in abdominal cavity, ascites) Cervical and breast screening (cervical smears) Material for special stains to diagnose infection. Sputum (cough up)

Screening Screening Criteria

o Test must me easy and non-invasiveo Simple and not unpleasanto High percentage of cases detected.o Treatment should be available

Problems with Screeningo Diffuculty in obtaining satisfactory sampleso Labs fail in making a proper smear.o Poor stainingo Poor interpretation by mistake.

BiopsiesDone when fine needle aspiration is unable to be done e.g.:

- liver- renal- skin: diagnoses include…

o rasheso tumouro lichen planus – inflammation of skin and oral mucosa. o Pemphigoid – autoimmune blistering of the skin (IgG binds to basement membrane).

- endoscopieso gastrointestinal tracto bronchoscopy

Assessment of Breast Pathology Clinical – e.g. history and examinations. Imaging

Mammogram Ultrasound

Fine needle aspirate. Trucut biopsy

Skin lumpsWhen they are sent off for a biopsy, surgeons need to know what it is, where is it and if it is to be excised or biopsied.

Abcesses Hot – vasodilatation Red - vasodilatation Discharging pus- neutrophils Swelling – oedema

Length of Time Needed to Reach Clinician Large specimens - 2-3 days. Small biopsies: 1 day.

Frozen section - 20 minutes.

Diagnostics 5 - Use of antibodies as diagnostic toolsAnil Chopra

1. Summarise the structure of an antibody (IgG) and the nature of the immune response.

2. Explain how antibodies can be generated (experimentally or commercially) for diagnostic purposes.

3. Give examples of types of substance that are typically identified diagnostically by means of antibodies.

4. Explain the use of secondary antibodies in detection.5. Describe the advantages in different situations of the following types of detection

system: enzyme-linked antibodies, radioactively labelled antibodies, fluorescently-labelled antibodies.

The basic structure of antibodies contains:• Immunoglobulins• 4 polypeptide chains• 2 heavy & 2 light chains• variable & constant regions• Variable regions

– antigen binding sites• Constant region

– major effector functions• Hinge region

– flexibility maximises chances of binding 2 Antigens

Antibodies are produced in response to antigens by B-cell clones, either through monoclonal or polyclonal expansion of B-cells.

Antibodies are antigen specific and hence they are used in vaccinations to develop high levels of protective antibodies, or in passive immunity to give protection for a certain amount of time e.g. maternal protection, or intravenous immunoglobulins (IVIG).

Immunisation:- A weakened or inactive form of the antigen is given to the patient together

with an adjuvant (increases immune response).- Booster injections are often given (usually 2).- This produces a polyclonal antibody response

o heterogeneous mixture of antibodieso against many different epitopes

Antibodies can be produced against almost any antigen including antibodies themselves (anti-antigens/anti-Ig).

Monoclonal antibodies can be produced by using animal organs:

Recently, antibodies are synthesised using genetic engineering. Antibody genes are cloned and expressed and antibody libraries are produced. These can be used in a number of ways:Theraputic:

• 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

– hormones– antibodies– Antigens

• Immunodiagnosis– infectious diseases

The detection methods used rely on the interaction between antibodies and antigens. They use known antibodies to identify specific antigens and known antigens to detect specific antibodies.Immunoprecipitation: this is where antibodies specific to the antigen we want to find are placed into the sample. These will then bind to the antibodies and cause agglutination (sometimes a protein is then added to make the antibodies insoluble). These are then centrifuged and precipitated off.

Haemagglutination: this is used in both blood typing and in viral assays. In blood typing, the different anti-a and anti-b antibodies are added to the samples to conclude whether they are certain blood types. It can also be used to find out the concentrations of certain antigens or antibodies in blood samples.

Labelled Antibodies: Radioactive isotopes are added to the samples (125I, 35S, 14C, 3H) and they are detected by autoradiography (photographic film) or radioactive counters. Enzymes (e.g. alkaline phosphatase) can also be use convert added substrate to coloured product – spectrophotometer - ELISA. Fluorescent compounds (e.g. fluorescein, rhodamine) are also used, as they become excited by polarised light.

ELISA: Enzyme- linked immunosorbent assay:

Antibodies can be used in clinical practice in a number of different ways:

Clinical Immunology:-- Immunodeficiency

o Serum immunoglobulin levels are checked by serum electrophoresis, ELISA, Nephelometry (using light and measuring the amount of scattered light).

- Malignancy- Autoimmunity- Inflammation- Tissue typing and Transplantation

Pathology:-- Clinical Chemistry- Haematology and Blood Transfusion- Medical Microbiology- Histopathology