Module 1: Topic 1: Scope of Clinical Biochemistry

Reasons for Requesting Tests

• to establish/confirm a diagnosis

• to reassure a patient

• to avoid litigation

• to monitor disease state

• monitor therapy progress

• for population screening

Clinical Specimens: Bodily fluids/excreta

• saliva e.g. micro, drugs

• urine e.g. amylase

• blood e.g. troponins, cholesterol

• cerebrospinal fluid e.g. multiple sclerosis

• faeces e.g. micro
  

Clinical Specimens: Tissue samples

biopsy e.g. surgery, needle biopsy

Approximately what % of diagnostic errors occur in which diagnostic testing cycle phase?

pre-analytical phase

Pre-analytical errors are detected in ____ phases

all three phases: pre-, analytical and post-

Types of Patient ID

• critical and unambiguous patient ID

  • name

  • DOB

  • gender

  • UR or barcode

• Location

• name of medical prac

• time, date + collectors signature on test request

Reducing Sample Variability in Pre-analytical Errors:

• patient ID checks

• quality management

Reducing Sample Variability in Reagents, instrumentation, use of standards?:

standards used to monitor performance regularly

Reducing Sample Variability in Labelling and data handling?:

appropriate protocols in place

Reducing Sample Variability in Lab accreditation:

comparison of standard samples between labs

What are some errors in Blood Sampling and Explain?

1. blood sampling technique

   1. haemolysis w release of RBC Contents (K+, proteins, enzymes)

2. stasis during venipuncture

   1. water diffuses away from plasma falsely increasing plasma content lvl

3. not enough specimen taken

   1. insufficient test material

4. errors in timing

   1. cholesterol measurement from unfasted patient

5. container not appropriate for specimen

   1. anticoag use

6. inappropriate sampling site

   1. downstream of intravenous line

7. incorrect specimen storage

   1. prolonged blood storage at 4 degrees C or freezing of speciment harvested for blood count

What are some sources of variation prior to analysis?

Patient related:
- cyclical biological changes
- medication and smoking
- exercise
- stress
- posture

Collection:
- preservation
- anticoagulation
- haemolysis
- contamination
- Patient ID

After collection:
- sample transport and storage

Types of Testing Errors: random errors affecting Precision
What is the cause?
What type of distribution is seen?

unknown and unpredictable sources
Normal distribution

What is Precision and how is it determined?

a measure of how closely measurements cluster together and is limited by random errors.
determined by repeating measurements

Types of Testing Errors: systematic errors affecting Accuracy
What is the cause?

operator error or from measuring instruments

Systematic errors can show ____: alteration from standard values with pattern of divergence

Trend

Systematic errors can show _____: abrupt change in measurements

Shift

What is Accuracy and what reduces it?

Measure of how closely measurements is to the true value of the quantity being assayed and is often reduced by systematic errors

What type of systematic error is a constant error?

specific difference between new/test and reference methods

What type of systematic error is a proportional error?

variable difference between new/test and reference methods

What are some causes of errors?

• input data req - such as standards used, calibration values

• instruments used - accuracy, repeatability

• observer fallibility - reading errors, blunders, equipment selection, analysis and computation errors

• environment - any external influences affecting measurement

what information is required to interpret results and what questions can we ask about the lab data and tests?

req info on N pop variability
what is the reproducibility of test results?
what is the test sensitivity/specificity?

what is the term for random changes that reduce agreement between replicate measurements?

imprecision

what type of imprecision defines the difference between results for the same specimen when assayed repeatedly at the same time?

Within-assay Imprecision

what type of imprecision defines the difference between results for the same specimen when assayed repeatedly at different times?

Day-to-day imprecision

What does coefficient of variation express?

expresses precision and should be as close to zero as possible

what is the coefficient of variation equation?

What is Sensitivity?

a measure of positive results
a measure of freq of a POSITIVE test result when a particular disease is present
sensitivity represents the percentage of TP results out of TP + FN results

What is Specificity?

a measure of negative results
a measure of freq of NEGATIVE test results when a particular disease is present
specificity represents the percentage of TN results out of TN + FP results

What is the equation for test sensitivity?

What is the equation for test specificity?

How is the usefulness of tests expressed visually?

as receiver operating chracteristic curves (ROC)
the greater the area under the curve = the more useful the test

What types of instruments are available for biochemical analysis?

• Spectrophotometry

• Atomic absorption

• Nephelometry

• Electrodes

• Electrophoresis

• Immunoassay

• HPLC

What is spectrophotemtry? and what is it most used to measure?

• the technique is defined as: “measurement of light by a specific molecule”

• used most freq to measure the amount of purity of molecule in solution

What is the principle of atomic absorption spectroscopy?

• principle: flame dissociates metal from chemical bonds > unexcited (ground) state

• light from hollow cathode lamp (0.01 nm bandwidth) enters flame and is absorbed by ground state atoms > decreasing intensity

what does nephelometry measure?

measurement of precipitation of antibody:antigen complex from solution

What are Ion-Selective electrodes used to measure?

measure the concentration of specific ions in a solution.

What is the principle of electrophoresis

• principle: Zone electrophoresis is migration of charged particles in a support medium within an electric field

• proteins are zwitterionic - can be negatively or positively charged depending on solution pH

What is migration dependent on in electrophoresis?

• electric charge of molecule

• size and shape of molecule

• electric field strength

• properties of support

• temperature

what does changes in reletive band intensity represent in serum protein electrophoresis?

imformative of pathological changes

How do immunoassays work?

use antibodies to bind specific molecules:

• selective purification of target molecules

• highly sensitive detection

• quantitation by measurement of signal intensity

What three types of immunoassays are mentioned? and how do these differ

1. Two-site immunometric assay

   • Antigen binds between two antibodies (solid phase and labeled antibody). The activity of the label bound is proportional to the amount of antigen.

2. Limited reagent or competitive immunoassay standard curve

   • Limited reagent: Labeled antigen competes with unlabeled antigen for a limited amount of antibody binding sites.

3. Immunolite automated immunoassay

What is HPLC

Application: Separating and quantifying a wide range of compounds.

Principle: Components in a sample are separated based on their interactions with a stationary phase and a mobile phase.