Diagnostics Flashcards
What are the likely causes of fever, rash and lymphadenopathy with D&V and red patches on lower leg with weight loss?
Viral illness- HIV (BILLY CASE STUDY)
NB. first few symptoms could be glandular fever
What are the likely causes of diarrhoea?
Virus
Bacteria
Parasites
What tests would a GP do if a patient presented with fever, rash, lymphadenopathy and diarrhoea?
FBC, ESR, CRP LFTs U&Es Blood glucose Ix of viral illness Stool culture - bacteriology
What are LFTs?
Liver function tests
What are U&Es?
Urea and electrolytes
What stages are necessary before collecting blood?
Check it’s correct patient
Label CORRECT tube (colour lid is important)
Check if urgent
What is the main difference between different blood collection tubes?
Contain different anticoagulants
What anticoagulants are found in blood collection tubes with the following colours? Red Yellow Purple Grey Blue Green
Red= none Yellow= gel to speed up clotting Purple= potassium EDTA Grey= fluoride oxalate (poison) Blue= citrate Green= heparin
What is the purpose of potassium EDTA in purple topped blood tubes?
Stops blood from clotting and preserves cells
What is the purpose of fluoride oxalate in grey topped blood tubes?
Poison
Red cells will use up glucoses (because they’re living)
So have to poison them to measure glucose)
What blood collection tube is used for U&E?
Serum in yellow/red top
What blood collection tube is used for glucose?
Plasma in grey top
What blood collection tube is used for HBA1c? What is the purpose of HBA1c?
Plasma in purple top
Can measure glucose in the blood over last 3 months
What blood collection tube is used for TFT?
Serum in yellow/red top
What blood collection tube is used for liver function tests?
Yellow/red top
What is the difference between serum and plasma?
Plasma is the blood fluid that carries blood clotting agents
Serum is the water fluid from blood without the clotting factors (appears yellow, at top of centrifuged sample)
If you want to collect serum (in U&E), why do you used a yellow/red topped collecting tube?
Blood clots (no anticoagulant in yellow/red)
Clot can be removed
Leaves serum
What happens if blood is treated EDTA or heparin?
Clotting factors are unused
Blood can’t clot
Blood can be separated into red cells and plasma
In centrifuged blood, describe the layers that would appear
TOP-> BOTTOM
Plasma (yellow)
Lymphocyte and monocyte band (white-ish, opaque)
Density gradient fluid (colourless, clear)
Gel barrier
Erythrocytes and neutrophils
What is found in the blue topped blood tube? What does it do?
Citrate chelates calcium
Need to be careful- need correct quantities= must fill exactly to 4ml
Normally should clot 14 secs
How can glucose from the blood be measured?
Red cells consume glucose (anaerobic glycolysis)
Longer blood is left out-> lower glucose may read
To avoid this, need to treat with fluoride oxalate (poison) which prevents red cells using glucose (so glucose levels stabilise)
GREY-TOPPED TUBE
Why are liver enzymes important to study diagnostically?
Clues from a pattern of enzymes
Extra enzymes leak into blood if liver is damaged-> may cause disease
What liver enzymes (and related substances) are commonly measured?
Alkaline phosphatase
Aspartate amino-transferase (AST)
Alanine amino-transferase (ALT)
Gamma glutamyl transferase (GGT)
Albumin= synthesised in liver Bilirubin= waste product
What hormones are often studied diagnostically in hormone assays?
Thyroxine
TSH
Cortisol
When is a chemical pathologist contacted?
When you want the sample to be rapidly centrifuged out of hours
When you want to measure labile hormones such as insulin
When you urgently need CSF glucose and protein to be measured
If sodium is slightly below the reference range what does this mean?
Often OK
May be due to large volume of water (will affect Na in short term but after passing urine will probably be back to normal)
What could low Na and high K indicated?
Dehydration (e.g. from diarrhoea)
Could also be due to adrenal failure
What are urea and creatine used as a marker of? What does it mean if they are inconsistent?
Renal failure (urea and creatinine concentration rise)
Creatine can cope for a few days, urea struggles
Creatine probably indicates permanent damage to GFR
Could have high urea with normal creatine
Why do you need to be aware of haemolysis in a blood sample? How can you know if blood has been haemolysed?
Red cells lyse and this releases the Ca inside red cells
High K often due to haemolysed sample
So when centrifuged-> haemolysed blood serum appears pink
If the potassium is raised, what does the lab need to check?
If the blood is haemolysed (pink serum)
High K often due to haemolysed sample
How does creatinine show renal function?
Marker of GFR (normal creatinine means normal GFR)
Very little is absorbed or secreted by the tubules
How does urea show renal function?
Levels rise when a patient is dehydrated
GFR stays the same
When studying liver enzymes, what else can be useful to measure?
Measure AST and GGT in a patient with jaundice
What cardiac enzymes are studied in diagnostic tests by the GP?
Troponins (rapid test for abnormal cardiac function) Creatine kinase (CK) Aspartate amino transferase (AST) Lactate Dehydrogenase (LDH)
What are cardiac enzymes? What do they indicate?
During a heart attack, heart muscle is damaged
Enzymes leak into blood in large amounts
Indicate if someone has had a heart attack
What types of laboratory tests are used in diagnostic virology?
Non-specific
Virological
Outline what is seen on a patient with measles virus?
Blotchy wine stain rash
Red all over
Outline what is seen on a patient with shingles virus?
Dermatomal distribution
Red blotches
What features/components of a virus can be detected? (With e.g.s)
Infectious virus (virus isolation and EM)
Protein components (antigens) of the virus (p24 antigen in HIV, surface antigen in HBV etc.)
Genetic components of the virus (DNA or RNA)
Host response (e.g. antibody or cell response)
What are the main diagnostic methods for viruses?
Cell culture
Electron microscopy
Antibody detection (serology - EIA)
Antigen detection (Immunofluorescence - IF enzyme immunoassay - EIA)
Genome detection (PCR)
Quantification of antibody/antigens
Serotyping (e.g. HIV)
Quantification of genomes (viral load)
Genome sequencing (genotyping, antiviral resistance testing)
What are the limitations of lab tests?
Give rise to false negative and false positive results
What is sensitivity?
Test’s ability to correctly id positive samples
What is specificity?
Test’s ability to correctly id negative samples
What is quantification of genomes essential for?
Quantification of genomes-> identifies viral load
Essential for diagnosis and monitoring of HIV, HBV and HCV, and also for CMV and EBV in the immunocompromised
What samples are usually used in virus investigations?
Throat swab, Nasopharyngeal aspirate (NPA), bronchoalveolar lavage (BAL), ET secretions
Stools
Urine
CSF
Blood (clotted or EDTA)
Saliva
When are throat swaps used in viral detection?
Detection of respiratory viruses (IF or EIA) = PCR
When are stools used in viral detection?
For rotavirus, adenovirus and norovirus antigen detection (EIA)
PCR
When is urine used in viral detection?
For BK virus and adenovirus
PCR
When is CSF used in viral detection?
For herpes viruses and enteroviruses
PCR
When is clotted blood used in viral detection?
For serology (ab detection)
When is blood (with EDTA) used in viral detection?
For PCR
Viral load testing
When is saliva used in viral detection?
For serology and/or PCR (e.g. measles)
Compare IgM and IgG (relating to viral diagnostics)
IgM= lower specificity (see more false positives)
Found in acute phase of disease
Duration= 3 months
IgG= higher specificity
Found in acute phase of disease
Duration= lifelong
What can avidity tests distinguish between?
Acute, recurrent or past infection by avidity of marker-specific IgG
For rubella, CMV, HIV, hepatitis viruses, EBV etc.
What can 4th generation EIAs of HIV detect? Why is this useful?
Ab + p24 Ag
Allows us to identify infection at an earlier level
Before abs have time to work in the blood
Why do all reaction samples from an HIV EIA undergo confirmatory testing in a second assay? What happens to confirmed positives next?
To exclude non-specific reactivity (false positives)
Confirmed positives undergo typing (HIV 1 vs 2)
Repeat blood sample and EDTA blood for HIV viral load from all new positives
What is point-of-care testing?
POCT is laboratorytestingor analyses performed in the clinical setting by non-laboratory healthcare professionals
Performed close to the patient so the results are available more quickly than when samples are sent off
What is the benefit of RSV point of care testing?
Positive reaction-> infection control protocols can be put into place before patient enters ward
E.g. isolation
Why is virus isolation in cell culture rarely used? What is it still useful for?
Reference lab only
Slow, time consuming (hence expensive)
Still useful for phenotypic antiviral susceptibility testing (HSV)
Why is microscopy of viruses rarely used? When can it be used?
Viruses are too small to be seen by light microscopy
They can be visualised DIRECTLY using an electron microscope
Sample types: stool and vesicle fluids
How does an infant usually present with respiratory synctial virus (RSV)?
with respiratory distress in winter
Hyperinflation of chest
Bronchilitis (probably caused by respiratory syncytial virus (RSV)
What is immunofluorescence (IF) used for in virus detection? What are the limitations?
Useful for the direct detection of viral antigens in clinical samples (DIF)
(e.g. respiratory viruses)
Can be used for typing and cell culture confirmation
Rapid and inexpensive but subjective and very dependent on the skill of the technician and the quality of the sample
What kind of samples can be used for respiratory tract infections?
Throat swab +/- nose swab Nasopharyngeal swab Nasopharyngeal aspirate (NPA) Bronchoalveolar lavage (BAL) Endotracheal tube (ET) secretions
-> for respiratory virus PCR
What is multiplex PCR?
Test for >1 virus per tube
Quicker and more cost effective than normal pCR
Technical reasons- limited to 3 (maybe 4) viruses
List examples of respiratory tract infections caused by viruses
Influenza Parainfluenza RSV Rhinovirus Human metapneumovirus (HMPV) Adenovirus Bocavirus Coronavirus
Give two examples of viruses affecting the CNS?
Meningitis
Encephalitis
How can CNS viruses be detected?
CSF for PCR (HSV, VZV, enterovirus)
Stools and throat swab for enterovirus detection (i.e. by PCR)
Blood for serology and/or PCR for West Nile and/or Japanese Encephalitis virus infection and other arboviruses
In the clinical history below, what CNS viral disease tests would be considered?
Meningitis or encephalitis
Young child with febrile fits
Immunocompromised
Recent travel to endemic region
In context of an outbreak
SSPE
Meningitis or encephalitis: HSV, VZV and enterovirus
Young child with febrile fits: add HHV-6 and parechovirus
Immunocompromised (eg HIV): add CMV, EBV and JC virus
Recent travel to endemic region: consider Japanese Encephalitis, West Nile virus, equine encephalitides, tick borne encephalitis
In context of an outbreak: e.g. mumps
SSPE: measles antibody index
When a patient has diarrhoea and vomiting, what is used to diagnose a viral cause?
Stool (preferred)
Vomit- lower yield
PCR multiplex or antigen detection assays (EIA)
What viruses commonly cause D&V?
Norovirus Rotavirus Adenovirus, Sapovirus Astrovirus
What is PCR?
Polymerase chain reaction
Method for amplifying specific RNA (RT-PCR) or DNA sequences
Cycle ( x 30)
Denature
Primer annealing
Chain elongation (with Taq polymerase)
… cyclical with dsDNA
What is dsDNA?
Double stranded DNA
Made from RNA with reverse transcriptase
Need to make copy of virus you’re looking for
What types of serological tests are used in viral diagnosis?
IgM vs IgG
Antibody avidity
What types of PCR are used in viral diagnosis?
Reverse transcription PCR Real Time PCR Multiplex PCR Viral load testing Sequencing (antiviral drug resistance testing)
What does it mean if a patient is CMV IgG positive but IgM negative?
They had CMV in the past but not currently
What does enlarged glands mean? What does enlarged lymph nodes mean?
Glands being up= mumps
Enlarged lymph node=cervical lymph adenopathy
What are the main parts of sequencing applications for viral diagnosis?
Genotyping
Antiviral resistance testing
Phylogenetic analysis
What microbiological diseases can cause diarrhoea and vomiting?
Infectious Diarrhoea Endocarditis (SBE) Syphilis Toxoplasma Tuberculosis Brucellosis Melliodosis
What are the common diagnostic techniques in bateriology?
Culture (sterile sites= blood/CSF or non-sterile sites)
Serology
Molecular techniques
Antimicrobial susceptibility testing
What microbiological disease can’t be cultured? How can it be diagnosed?
Syphilis can’t be cultured
Rely on serology to diagnose
Is the skin a sterile site?
No
Approx 50 mil bacteria on average square cm of human skin
What are the non-sterile sites in the body? What does this mean?
Nasopharynx Skin Upper bowel Lower bowel Vagina
Organisms may not be affecting them
Presence of organism isn’t diagnostic
What does a positive blood culture show?
Indicator (change in colour) says blood culture is positive
Use non-selective medium (designed to grow any/as many as possible because shouldn’t be any bacteria in blood)
Don’t have growth suppressors etc.
Why should blood/pus be taken before giving antibiotics? When shouldn’t you wait?
If there is small growth on agar, antibiotic could hide it-> false negative
Shouldn’t wait if you suspect meningitis (very septic)
What can gram stain cultures show?
Show type of cell (based on cell wall)
+VE= deep purple colour stain
- Thick peptidoglycan layer
- 1 membrane (cytoplasmic)
- VE= pinky colour stain
- Thin peptidoglycan layer
- 2 membranes (cytoplasmic and outer)
What is coagulase? What is it used for?
Coagulaseis a protein enzyme produced by several microorganisms that enables the conversion of fibrinogen to fibrin
Used to distinguish between different types of Staphylococcus isolates
What are the types of staphylococci? What do they cause?
Bacteria
- S. aureas= e.g. MRSA
- > severe infections e.g. skin/soft tissue, endocarditis, osteomyelitis - Coagulase negative staphylococci
- > skin commensals of low pathogenic potential
- > can infect prosthetic material causing pacemaker infections and endocarditis
What is an alpha result in haemolytic streptococci?
Alpha is incomplete haemolysis
E.g. pneumococcus and group of viridile streptococci
What is a beta result in haemolytic streptococci?
Beta is complete haemolysis
E.g.
Strep group A= streptococcus pyogenes
Strep group B
Meningitis and sepsis
What can cause diarrhoea?
Bacteria e.g. salmonella, shigella, campylobacter, e coli, c diff, cholera
Parasites e.g. amoeba, giardia, cryptosporidium
Viruses
What are the 3 most common causes of food poisoning?
Salmonella (inc S. typhi )
Shigella
Campylobacter
Where are giardia and cryptosporidium parasites more common?
Rural areas
Giardia= unclean water Cryptosporidium= contact with animals, spread by soil, water, food or surfaces contaminated by infected faeces
What investigations from stool samples are there for bacterial diseases?
Culture on agar plates
Only Salmonella, Shigella and Campylobacter
looked for routinely
Different pathogens have different culture
requirements
Clostridium difficile- toxin detection or PCR for
toxin gene
What investigations from stool samples are there for parasites?
Concentration, special stains
Look for ova sometimes
Why are different kinds of agar used in bacteriology?
Suppress commensural bacteria (particularly important for non-sterile sites)
e.g. XLD= selects for salmonella
TCBS= vibrio cholerae
One type selects for campylobacter and can be incubated at hotter temp (campylobater can survive)
What is MIC?
Minimum Inhibitory Concentrations
Lowest conc of an drug that will inhibit the visible growth of a microorganism aftantimicrobial er overnight incubation
What can you tell from gradient MIC studies?
See diameter of bacterial resistance
Large= sensitive Small= resistant
What can you tell from disc diffusion studies of MIC?
Approximate MIC
If certain size= sensitive
Cheaper than other MIC studies
How long can infectious diarrhoea persist?
Usually short term
What do histopathologists study?
Tissues
From: Biopsies (mostly) Resection specimens Frozen sections (can be very quick) Post-mortems
What do cytopathologists study?
Cells
From:
Smears (cervical screening)
Fine need aspirations
What are the main questions to ask when studying a histopathological biopsy?
Is it normal?
Is it inflamed? (If so, what is the cause?)
Is it cancer? (If so, what type is it?)
What are the main questions to ask when studying a histopathological resection specimen?
How far has the cancer spread? (Is this affecting drainage)
Is it all out? (Judge by margins)
What are the main questions to ask when studying a histopathological frozen section?
Is it cancer?
Is it all out?
RAPID- can use quickly in middle of surgery
What post-mortems are more common; hospital or coroner?
Coroner’s
Why are coroner’s post-mortems used? Why are they more common than hospital post-mortems?
When they don’t know or other factors e.g. poisoning, self-harm, industrial
Don’t need consent- they are legal post mortem
NB. Can’t take any tissue
How are sections obtained for histopathology?
Specimen must be properly labelled
Fix in formalin
Embed in paraffin wax
Cut sections
What processes can happen to histopathological sections?
Stain e.g. gram
ID specific antigens using antibodies (immunohistochemistry)
Carry out molecular tests
How long does a histopathology result take to reach the clinician for the following processes?
Frozen section
Biopsies
Resection specimens
Frozen section= 30 mins
Biopsies= 2-3 days
Resection specimens= 5-7 days
What does Kaposi’s sarcoma indicate?
HIV AIDs
What test would you use to visualise a vascular tumour infiltrating collagen bundle?
Immunocytochemistry for CD31
What does it mean if fine needle aspiration of one of the enlarged nodes reveals a mixed cell population?
Reactive lymphadenopathy
Describe an antibody (structure)
Y shaped molecule
Has an antigen binding region at end of tips of Y (have variable regions)
Hinge region in middle
Why are antibodies used as the basis of many diagnostic tests?
Specificity of abs for their target ags
Antibodies can be raised against almost any antigen (including immunoglobulins for other species= anti-antibodies)
What is indirect labelling using an anti-antibody?
Antigen binds to antibody Reporter used (on antibody)
When are antibodies produced by the patient?
In autoimmune disease
For defence against infection
List manufactured antibodies
Antisera from immunised animals (polyclonal)
Monoclonal antibodies
“Genetically engineered” antibodies
How are monoclonal antibodies generated?
Mouse challenged with antigen
Spleen cells (limited cell division, produce Ab, HGPRT +ve) Myeloma cells (immortal, no ab production, HPRT -ve)
SPLEEN CELLS AND MYELOMA CELLS FUSE
Hybridomas
Culture in HAT medium select for positive cells (immortal, produce Ab, HGPRT +ve)
Clone by limiting dilution
Harvest monoclonal antibodies
How are antibodies produced using recombinant DNA technology?
Isolate population of genes encoding ab variable regions
Construct fusion protein at V region with a bacteriophage coat protein
–
Clone random population of variable regions gives rise to a mixture of bacteriophages (phage-display library)
Select phage with desired V regions by specific binding to antigen
What are the therapeutic uses of manufactured antibodies?
Prophylactic protection against microbial infection
E.g. IVIG, synagis (anti-RSV)
Anti-cancer therapy
E.g. anti-HER2
Removal of T-cells from bone marrow grafts
E.g. Anti-CD3
Block cytokine activity
E.g. Anti-TNFa
Outline the nomenclature for therapeutic monoclonal antibodies
- omab
- imab
- umab
-OMAB
Mouse monoclonal e.g. anti-CD3 muronomab
-IMAB
Chimeric or partly humanised e.g. anti-TNFa infliximab
-UMAB
Human e.g. anti-RSV palivizumab
What are the diagnostic uses of manufactured antibodies?
Blood group serology
Immunoassays
- Hormones
- Antibodies
- Antigens
Immunodiagnosis
- Infectious diseases
- Autoimmunity
- Allergy (IgE)
- Malignancy (myeloma)
What is ELISA?
Enzyme linked immunosorbent assay
Add anti-A ab covalently linked to enzyme (to 2 samples, one with antigen A and one with antigen B)
Wash away unbound antibody
Enzyme makes coloured products from added colourless substrate
Measure absorbance of light by coloured product
(Sample with ag A= coloured)
What is lateral flow assay architecture?
Rapid testing
Simple devices intended to detect the presence (or absence) of a target analyte in sample (matrix) without the need for specialized and costly equipment
Where could serum antibodies against HIV be from?
Mother
What immunological conditions can cause the following?
Vague aches and pains
Loss of appetite and weight loss
“Glands” up in his neck (lymph adenopathy)
Fever, rash, small red patches, some lumpy
Vague aches and pains= immune complexes
Loss of appetite and weight loss= poor nutrition (effect on bone marrow cells)
“Glands” up in his neck (lymph adenopathy)= immune activation
Fever, rash, small red patches, some lumpy= acute phase, activation, immune complexes
What does it mean if you are concerned about immune complexes?
Concerned about:
Inflammation / complement activation
Serum sickness (immune complexes in circulation)
Immune complex glomerulonephritis
Immune complex deposition at other sites (skin, joints, lungs)
What is immunodeficiency?
State in which the immune system’s ability to fight infectious disease and cancer is compromised or entirely absent
Most are acquired (“secondary”) due to extrinsic factors that affect the patient’s immune system
How can immunodeficiency be studied?
Serum immunoglobulin levels (serum electrophoresis, ELISA, nephelometry)
Specific antibodies (ELISA)
Lymphocyte subsets (flow cytometry)
What is serum electrophoresis used for?
Measures specific proteins in the blood to help ID some diseases
I.e. Monoclonal expansion of B cells
? B cell malignancy
Investigate for myeloma
If there is an active immune response-> not immunodeficient
What are lymphocyte subpopulations (ID’d by flow cytometry)
CD3+= T cells- pan T cell marker CD4+= T cells- T helper/cells CD8+= T cells- cytotoxic T cells CD19+= B cells CD56+= Natural Killer (NK) cells
What do you need for flow cytometry?
Continuous power supplies
Complex IT systems
Standardised sampling
High cost, highly specialised precision equipment
Advanced, reliable infrastructure
Mixture of cells is labeled with fluorescent antibody
What is the natural history of HIV infection?
0-6 weeks
Primary infection leads to decrease CD4+ T lymphocyte count (1050->500)
3-9 weeks Acute HIV syndrome Wide dissemination of virus Seeding of lymphoid organs Increase and then decrease of HIV RNA copies per ml plasma
Clinical latency (until approx 8/10 years)
Gradual increase in HIV RNA copies per ml plasma
Gradual decrease in CD4+ count
Around 8 years
Constitutional symptoms
(Trajectory continues for HIV RNA copies per ml plasma
and CD4+ count)
Around 10 years
Opportunistic diseases and death
Steep increase in HIV RNA copies per ml plasma
CD4+ count around 0
How is a patient treated and monitored for HIV?
Patient tested for abs to HIV
If HIV+ perform CD4 count-> low CD4 count
If HIV+ perform viral load-> high viral load
Commence ART (1st line therapy)
Monitor CD4 count and viral load (every 3 months)
If CD4 count decreases and/or viral load increases then try ART (2nd line therapy)
What defines the extent of immune damage and predicts short term outlook in ART naive HIV-1 patients?
CD4 T cell count
As CD4 declines, what symptoms of HIV appear?
700= Lymphadenopathy and thrombocytopenia 500= bacterial skin infection, herpes simplex, zoster, oral, skin fungal infections 400= Kaposi's sarcoma 300= Hairy leukoplakia and TB 200= PCP, cryptococcis, toxoplasmosis 100= CMV, lymphoma
