Fundamentals of clinical laboratory techniques Flashcards
define pathology
The science of the causes and effects of diseases
What does Clinical Biochemistry study?
The measurement of chemicals (natural & unnatural) in blood, urine, and other body fluids.
What is the focus of Haematology?
The study of blood cells and the tissues that make them.
What does Blood Bank Transfusion Medicine involve?
Analyzing a patient’s blood group and providing safe and appropriate blood or blood components.
What does Immunology study?
The quantity and function of components of the immune system.
What does Microbiology focus on?
Infection (bacteria, viruses, parasites) and how to treat them, including the measurement of antibiotic levels.
What does Histopathology (Cell Pathology) examine?
The nature of cells and their organization into tissues.
What does Molecular Genetics detect?
Human, tumor, bacterial, or viral genes.
What does Clinical Biochemistry investigate?
The biochemical investigation of bodily fluids such as blood, urine, and CSF.
Why are analytes measured in the biochemistry laboratory?
To diagnose and monitor various medical conditions.
How many routine assays are performed in the biochemistry department?
More than 80 different routine assays.
How many tests are referred to other laboratories?
More than 300 different tests.
Why is Clinical Biochemistry important?
Many illnesses are reflected in disturbances in the body’s chemistry.
How does Chemical Pathology connect science and medicine?
By analyzing the chemistry of bodily fluids to assess organ function, diagnose diseases, and recommend treatments.
What is an example of Clinical Biochemistry in diagnosing disease?
High blood glucose levels may indicate diabetes.
What regulates blood glucose levels?
The hormone insulin.
What happens if the body doesn’t produce enough insulin?
Diabetes may develop.
What are the two main approaches to diagnosis in Clinical Biochemistry?
Classical medical processes and screening/preventative medicine.
What is an example of a screening program in preventative medicine?
Newborn blood spot screening.
What does aetiology focus on in Clinical Biochemistry?
Identifying the underlying defect causing a disease.
What are the 3 key questions in understanding aetiology?
- How does the defect lead to pathology?
- What secondary pathology might result?
- What is the appropriate treatment strategy?
What is guided therapy in Clinical Biochemistry?
Treatment decisions based on genetic susceptibility.
What does ISE stand for?
Ion Selective Electrode.
What does the ISE method measure?
The concentration of sodium, potassium, and chloride.
What type of measurement does ISE use?
Potentiometric measurement.
What sample types are used in ISE testing?
Serum, plasma, urine, and other fluids.
How long does an ISE test take?
Only 30 seconds.
What is the function of the Cobas ISE module?
It is dedicated to electrolyte measurement.
How many tests can the ISE module process per hour?
Up to 1800 tests per hour.
What are the two main components of the ISE system?
An ion selective electrode and a reference electrode.
What happens to the sample before measurement in ISE?
The sample is diluted, and the electrodes are placed within it.
What is the function of the ion selective electrode’s membrane?
It allows only the ion of interest to pass through.
What occurs across the membrane in the ISE method?
Ion exchange, leading to the generation of an electrical potential.
What does the generated electrical potential represent?
The composition of the sample.
what are 3 examples of things that can cause low sodium?
- Heart failure
- Head injury
- Medications eg diuretics
what are 3 examples of things that can cause low potassium?
- Inadequate nutrition
- Vomiting / diarrhoea
- Medications eg. diuretics
what are 3 examples of things that can cause high potassium?
- Spurious (old or haemolysed sample)
- Kidney dysfunction
- Addison’s disease
what are 3 examples of things that can cause high sodium?
- Inadequate water intake
- Diabetes insipidus
- Water loss eg.diarrhoea, vomiting
What type of ISE method does the Cobas ISE use?
Indirect ISE.
How does indirect ISE differ from direct ISE?
Indirect ISE dilutes the sample in a buffer before measurement, while direct ISE does not.
Where is direct ISE commonly used?
In most point-of-care analyzers.
What are the advantages of indirect ISE?
- Requires only a small sample.
- Less expensive than direct ISE.
- Has a large dynamic range.
What is a disadvantage of indirect ISE?
It can produce erroneous results if the patient has high levels of proteins or lipids.
What effect does an increased solid phase have on sodium measurement in indirect ISE?
It can cause a falsely low sodium concentration.
Why does an increased solid phase lead to false low sodium readings?
There is less water in the sample, reducing the apparent sodium concentration after dilution.
What is an example of a condition that can increase the solid phase in blood?
Multiple myeloma (due to paraprotein production).
What is the term for falsely low sodium results due to high solid phase levels?
Pseudohyponatremia.
What types of assays does the Roche c702 module measure?
General chemistry assays such as urea, creatinine, LFTs, calcium, magnesium, etc.
How long does each test take on the Roche c702 module?
Each test takes about 10 minutes.
How many tests can the Roche c702 module process per hour?
Up to 2000 tests per hour.
What types of samples can be used on the Roche c702 module?
Serum, plasma, urine, CSF, stool samples, and fluids.
What technology does the Roche c702 module use to measure analytes?
Photometric technology.
How are organic and inorganic compounds measured in photometric assays?
By determining the absorbance of wavelengths of light.
What happens when light enters the photometer in a photometric assay?
The light hits a diffraction grating, which splits it into different wavelengths.
How are the wavelengths of light detected in a photometric assay?
The wavelengths are reflected onto a fixed array of 12 photodiodes, each detecting light at an individual wavelength.
What type of analytes are measured using photometric assays?
Creatinine (kidney function), bilirubin (liver function), glucose, magnesium.
What wavelength change occurs in the glucose hexokinase reaction?
340 nm.
How is glucose concentration determined in the hexokinase reaction?
By measuring the change in absorption at 340 nm, which is proportional to glucose concentration.
What are the main limitations in photometric assays?
Interferents that cause changes in wavelength.
What factors can affect sample integrity in photometric assays?
Drugs, dyes, high levels of proteins, lipids, or cells in the sample.
What other factors can affect measurements in photometric assays?
Clots or air bubbles.
How is sample integrity monitored in the chemistry module?
Detection system for clots, bubbles, and liquid level; measurement of serum indices.
What are serum indices?
Measurements performed on every sample analyzed on the chemistry module.
How are icteric, haemolysed, and lipaemic samples identified?
By absorbance measurements at different wavelengths of light.
How do interferents affect analyte measurements?
They have variable effects on the measurement of different analytes.
What happens if a serum index measurement breaches a threshold for an assay?
The IT system removes the result as it is not accurate.
Why might a CRP result be removed in a newborn baby?
Difficulty in drawing blood can cause haemolysis, and if the haemolysis index exceeds 500 for CRP, the result is automatically removed.
What is the Roche e801 module also known as?
The immunoassay unit.
How do immunoassays work?
They quantify an analyte based on the reaction between an antigen (analyte) and an antibody.
What types of samples are primarily used in the e801 module?
Serum and plasma.
How long do assays on the e801 module take?
Between 9 and 32 minutes.
How many tests can the e801 module process per hour?
Up to 2000 tests per hour.
What technology does the e801 module use?
Electrochemiluminescence (ECL) technology.
What are the two types of immunoassays?
Sandwich immunoassay and competitive immunoassay.
How many antibodies are used in a sandwich immunoassay?
Two antibodies.
How does a sandwich immunoassay work?
A capture antibody binds to a solid surface, the sample is added, and a second antibody binds to a different epitope on the antigen, forming a “sandwich.”
What role does the second antibody play in a sandwich immunoassay?
It has a molecular label that forms part of an ECL reaction.
How is the signal related to analyte concentration in a sandwich immunoassay?
The signal is proportional to the analyte concentration.
What type of analytes typically use sandwich assays?
Larger analytes, such as TSH (thyroid-stimulating hormone).
How many antibodies are used in a competitive immunoassay?
One antibody.
What type of analytes typically use competitive assays?
Smaller analytes, such as testosterone.
How does a competitive immunoassay work?
The sample is mixed with a labelled analogue, and both the labelled analogue and the analyte compete for binding to the capture antibody.
How is the signal related to analyte concentration in a competitive immunoassay?
The signal is inversely related to analyte concentration.
What two electrochemically active substances are involved in the ECL reaction?
Tris(2,2’-bipyridyl) ruthenium (II) complex [Ru(bpy)₃]²⁺ and Tripropylamine (TPA).
Where does the ECL reaction occur?
At the surface of a platinum electrode when a voltage is applied.
What happens to TPA when voltage is applied?
TPA is oxidized at the electrode, forming an intermediate radical-cation, which then releases a proton (H⁺) to produce a TPA-radical.
What happens to ruthenium in the ECL reaction?
Ruthenium releases an electron and is oxidized to form a [Ru(bpy)₃]³⁺ cation.
How does the ECL reaction produce light?
The TPA-radical transfers energy to [Ru(bpy)₃]³⁺, causing it to return to [Ru(bpy)₃]²⁺ and enter an excited state, which emits light at 620 nm when it decays.
When does the ECL reaction cycle restart?
The cycle restarts when [Ru(bpy)₃]²⁺ is regenerated, continuing until all TPA in the electrical field is depleted.
How is light emission used in ECL-based immunoassays?
Under an electrical current, TPA reacts with ruthenium complexes (bound to biotinylated antibody) to emit light. The emitted light is measured to determine the concentration of the analyte.
What is the Hook Effect?
A phenomenon in sandwich assays where very high analyte levels lead to falsely low results.
How can the Hook Effect be detected?
By diluting the sample and remeasuring.
Why is antibody production challenging?
It is expensive and prone to batch variability.
What is cross-reactivity in immunoassays?
When similar analytes (e.g., steroids) interfere with test results.
How does biotin interfere with some immunoassays?
Biotin supplements can disrupt biotin-streptavidin binding, leading to inaccurate results.
How do competitive immunoassays work?
The sample analyte competes with a labeled analogue for antibody binding; signal is inversely proportional to analyte concentration.
What type of analytes use sandwich immunoassays?
Larger molecules like TSH.
What type of analytes use competitive immunoassays?
Smaller molecules like testosterone.
What is osmolality?
The concentration of dissolved particles in a solution.
When is serum osmolality measured?
To investigate hyponatremia, hypernatremia, abnormal urine output, excessive thirst, or toxic alcohol ingestion.
What principle does an osmometer use?
Freezing point depression.
What toxic alcohols can affect serum osmolality?
Methanol and ethylene glycol.
What can urine osmolality indicate?
The cause of high or low urine output.
What is freezing point depression?
The freezing point of a liquid is lowered when another substance is dissolved in it.
How is the sample prepared in an osmometer?
It is injected, supercooled, and agitated to cause crystallization.
What does an osmometer measure?
The osmolality of a sample based on freezing point depression.
What happens after crystallization in an osmometer?
The resulting heat of fusion raises the sample temperature to a plateau equilibrium.
How is osmolality determined in an osmometer?
A high-precision thermistor measures the plateau temperature, which is used to calculate osmolality.
What is the purpose of measuring chloride in sweat?
It is used to diagnose cystic fibrosis.
How does cystic fibrosis affect sweat chloride levels?
Patients with cystic fibrosis have higher levels of chloride in their sweat than unaffected individuals.
Is sweat chloride concentration affected by genotype?
No, this feature is independent of the genotype.
How is localised sweating induced for sweat chloride measurement?
By using electrical stimulation on the surface of the arm.
What device is used to collect sweat for analysis?
A macroduct sweat collector
How does the sweat chloride analyser work?
It works by Coulometric titration.
What is in the working solution of the sweat chloride analyser?
An acid buffer plus stabiliser, with no silver ions.
What happens when a sweat sample is added to the solution?
The indicator current drops, and silver ions are released by the anode.
What occurs after the silver ions are released in the sweat chloride analyser?
All chloride ions are precipitated out as silver chloride, restoring the original silver chloride concentration.
What does the period of current flow represent in the sweat chloride analyser?
It is proportional to the sweat chloride ion concentration.
What can cause borderline results in sweat testing?
Newborn screening or genotype positive but inconclusive sweat test results.
What are some limitations of the sweat chloride testing method?
It is manual, requiring accurate pipetting and manual transcription.
How long can sweat collection take in some cases?
It can take around 30 minutes.
Why can sweat collection be difficult in newborns?
It can be difficult to obtain sufficient sweat from newborns.
What is the Uvikon XL Spectrophotometer used for?
It is used to run the CSF Xanthochromia assay.
What causes blood to leak into the cerebral ventricles and CSF after a subarachnoid haemorrhage (SAH)?
Blood leaks into the cerebral ventricles and CSF after a subarachnoid haemorrhage (SAH).
What is Xanthochromia?
Xanthochromia is the yellow discolouration of CSF caused by the release of oxyhaemoglobin from disintegrating red blood cells and its conversion to bilirubin.
How is CSF collected for Xanthochromia testing?
CSF is collected via a lumbar puncture.
How does the spectrophotometer analyze the CSF sample?
The spectrophotometer scans the CSF sample using a range of wavelengths to determine absorbance peaks.
What does the spectrophotometer detect for Xanthochromia?
It detects absorbance peaks showing the presence of oxyhaemoglobin and bilirubin, the breakdown products of blood.
What can cause interfering peaks in Xanthochromia testing?
Certain drugs, such as the antibiotic doxycycline, can cause interfering peaks.
What is required for the manual method of Xanthochromia testing?
The manual method requires specialist training
What can introduce traces of blood in CSF testing?
Previous lumbar punctures performed within the last 2 weeks may introduce traces of blood.
Why is it important to protect the CSF sample from light?
Bilirubin breaks down in the presence of light, which can affect the test results.
When is the Xanthochromia test valid after a potential event?
The test is not valid until 12 hours after the possible event, as it takes 12 hours for blood to break down into bilirubin and oxyhaemoglobin.
