Clinical Chemistry: Concepts & Techniques

Clinical Chemistry: Basic Concepts & Techniques in Chemical Pathology

• Obtain lecture notes and written assignments on the OLE platform
• Attendance will be taken twice per class
• The first attendance mark will be taken during the first 30 minutes
• Students arriving after 30 minutes will not be marked for the first attendance
• The second attendance mark will be taken at the end of the class
• Notify instructor via email for approval if unable to attend lecture, 80% attendance is the minimum
• Due to large material coverage, lectures will cover most of the time
• Lecture notes are posted on OLE one day before or on the same day of the lecture
• Written assignments help recognise, memorise, and understand knowledge taught in lectures.
• Submit written assignments one week after each lecture
• Pay attention to folders and due dates on OLE, late submissions will result in mark deductions proportional to days elapsed

Highlights

• Includes Introduction, Specimen Reception & Processing, General Equipment & Laboratory Techniques, and Spectrophotometric Measurement

Term & Definition

• Pathology is the study of the cause and mechanisms of development
• Pathology looks at structural alterations of cells, physiological changes like pathophysiology and morphologic changes like histopathology
• Pathology studies the clinical manifestations like signs & symptoms
• Clinical pathology is a medical specialty for diagnosing disease using lab analysis of body fluids such as blood, urine & tissue
• Clinical pathology utilises Clinical Chemistry, Hematology, Microbiology and Histopathology etc.
• Chemical pathology/Clinical Chemistry/Clinical Biochemistry utilises chemical reaction tests/chemical analysis for biomarkers in body fluids
• Biomarkers are in fluids such as blood and urine for disease diagnosis/monitoring
• Analytical techniques and specialised instruments aid chemical pathology
• Physiology + Biochemistry + Analytical Chemistry combine for analyses

Biochemistry

• Biochemistry is a sub-discipline of chemistry and biology
• Biochemistry involves study of chemical processes within and relating to living organisms
• It is divided into structural biology, enzymology, and metabolism.
• Others include Gene/DNA structure.

Analytical Chemistry

• Analytical Chemistry studies and applies instruments and methods/technologies to separate, identify, and quantify substances
• Analytical Chemistry methods result in analysis of substance identity and concentration
• Qualitative or quantitative analysis includes chromatography, electrophoresis, electrochemistry, Immunoassay, spectroscopy, and mass spectrometry

Other related terms

• At the clinical or pathology level, a key role is the Chemical Pathologist, who is a clinical doctor
• At the technical level, there is a Clinical Biochemist (scientist), Scientific Officer, and Medical Laboratory Scientist/Technician

Lab Techniques & Concepts

• Definition & concept involves preanalytical/analytical and laboratory automation
• Biochemical techniques involve weighing using a balance, where accuracy considerations include accuracy to decimal places, tareing the balance and skills in weighing different materials
• Pipetting involves autopipettes
• Other techniques are performing dilutions and preparation of working solutions via stock solutions
• Equipment used includes centrifuges and pH meters
• Spectrophotometric measurement includes wavelength scans and setting up calibration curves

Clinical Chemistry Features

• Auto-analyzers are used for daily, high-throughput lab results in routine benches doing thousands of tests per day, except for specialised benches
• Specialized benches do urgent labs and metabolic screening labs.
• Quantitative measurement involves various methodologies.
• Stringent quality assurance measures occur from sample collection to reporting, spanning pre-analytical, analytical, and post-analytical phases
• Comprehensive case results interpretations include multiple testings such as Albumin, liver enzymes- ALT, AST and ALP for validating diagnosis and aiding in treatment

Specimen Reception & Processing

• Specimen Reception & Processing includes laboratory workflow, importance of specimen reception & processing and pre-analytical assessments
• Preanalytical assessment involves assessment of types of specimen such as plasma versus serum, urine and CSF
• Involves looking at Types of containers used, transport, storage and to prevent interference

Summary of laboratory work cycle

• Work cycle involves consideration of clinical question, request form and specimen collection
• Requires transport to the laboratory, and specimen reception/accessioning using a barcode for processing.
• Analysis can be chemical, immunological using chromatography and electrophoresis with calibration and quality control
• Post-analysis includes calculations and compilation of data and interpretation before reporting with a standard format

Specimen Reception: Reception Area

• Includes samples being sorted and checked against request forms and patient ID details
• Includes requested tests entered into LIS or laboratory information system
• Includes the use of a unique barcode and check digit that gives sample ID and test information
• Important steps are centrifuge of specimens such as blood, urine and separation if required with labelled stored aliquots

The importance of specimen reception & processing

• Sample reception and processing is a key element of the analytical process
• Is an important area for medical technologist training in the laboratory
• Failures in process can lead to risk in integrity of analyte/analytical process, traceability of the sample, quality of the result, health of the patient and safety of staff

Specimen Types

• Specimen types include blood, where serum, plasma, whole blood, and red cells may be used
• Plasma is separated when an anticoagulant is used.
• Serum requires blood to clot before spinning.
• Haemolysis which is the rupturing of red blood cells needs to be checked for.
• Blood Gas Analysis can be done
• A key specimen type is Urine, using spot/timed and 24h urine

Other specimen types 

• Cerebrospinal fluid (CSF) which is collected from the thecal sac
• Another specimen type is Pleural fluid

Anticoagulant tubes

• Tubes contain agents that prevent the blood from clotting
• Different colour tubes are used for different tests
• Light-blue tubes use 3.2% sodium citrate and prevent blood from clotting by binding calcium for Coagulation.
• Red or gold tubes contain clot activator which promotes blood clotting to separate serum
• Green tubes have heparin for inhibiting thrombin and thromboplastin for stat tests
• Lavender tubes have EDTA which bind calcium for Hematology and blood bank
• Gray tubes have fluoride & oxalate which inhibits glycolysis for glucose analysis
• The order of draw for anticoagulant bottles is from Citrate, plain, heparin and EDTA to Fluoride

Transport specimens

• Special care is required to transport specimens inside hospitals and to external laboratories
• Pneumatic tube systems can be used to urgent samples and require quality control
• Transport distances depend on wards on site, local hospital and field for general practice
• Need to be transported in heat blocks for cryoglobulin
• Safety measures must be implemented as all samples are infectious

Sample Storage

• For longer term storage, or unstable analytes, the serum must be stored frozen to avoid compromise

Factors affecting sample quality

• Factors affect sample quality/integrity during time and at differing temperatures
• Serum/plasma should be separated from blood centrifuge as soon as possible
• For longer storage the serum sample must be frozen

Reasons for specimen rejection

• The specimen maybe rejected for no sample availability at all
• Presence of a needle in the sample
• Insufficient blood volume which is an inadequate quantity compared to the anticoagulant ratio

Interferences

• Other issues affecting sample quality involve the interference that can come from patient conditions and sample conditions
• Patient variation is seen in issues such as fasting, time of day, exercise, posture, age, gender and menopause
• Sample variation is seen due to issues with wrong anticoagulation, wrong temperature, transport and swapped tests

Tests and required tubes

• A range of tests are implemented and are very sensitive to pre- analytical variations
• For example tests such as U&E,LFT,B12 and haemoglobin all have specific tubes and conditions such as temperature which they have to adhere to

Urine test

• Samples could be rejected if not handled correctly, and require the right analysis

Pre-analytical Considerations

• Pre-analytical steps are the highest source of errors in the lab.
• Proper Patient preparation needs to take place, considerations are fasting, time of day, exercise, gender and stage of life
• In sample collection, steps must occur that look into swapping or lack of tubes, incorrect tubes, lack of labels, incorrect storage or transport

Timing sample collection

• Drug estimations require the time since the last dose
• Fasting samples must be measured for levels of lipid and analysis
• Also some analytes must be tested as they go under durinal level and sexual hormones must be monitored during the menstrual phases

General Equipment & Laboratory Techniques

• Balance uses and differences between Analytical balance and Top Loading balance
• A 'top-loading balance', as they are often referred to as, is a balance that does not have a draft shield
• An analytical balance is a class of a balance that has a readability of 0.1 mg or better
• The analytical balance depends on the accuracy

Weighing rules

• A bubble should centralise on weighing machine
• Never use bare hands, always use safety gloves
• Never weigh any chemical or hot surface
• Don’t spill or overload any chemical inside equipment, clean quickly
• Direct weighing, set zero
• Indirect, weight difference by a clean formula

Solutions

• Solutions must be measured in percentage
• By volume weight and mass
• Solutions expressed in % can be prepared by a)mass percents (m/m), b) volume percents (v/v) or c) mass/volume (m/v)
• Pipetting techniques use forward and reverse systems dilution and dilution techniques are performed

Centrifugation

• Centrifugations need to seperate particles from various sources
• RCF, is depended on g force related to rpm, and danger can occur so it must meet specific conditions

Centrifuge Safety

• The centrifuge should be level and must be balanced or significant damage + personal injury will occur
• Always wait for rotor to stop moving and take care if there a breakage occurs which requires safety procedure
• If the spill is contained within a closed cup, bucket or rotor, spray the exterior with disinfectant and allow at least 10 minutes of contact time.

Blood Products

• Plasma products are produced via anticoagulation
• Serum produced by removing blood at room temperature at 15 - 30 minutes,
• The resulting supernatant is designated serum.

Anticoagulation tubes

• Tubes must be handled in specific order to prevent errors
• The order is to collect blood in a specific way, citrates need to be placed at the top and fluoride oxalate needs to be placed at the bottom

Ph Meter

• Ph is measure in water,
• Ph Meter works by the power and hydrogen concentration in a source

Calibrations

• The are a variety of methods that can be undertaken to calibrate
• 2 points and automatic endpoint must be set

spectrophotometry

• Is the most effective in lab, that measures chemicals and spectrum
• Measures absorption against concentration
• Uses photometry with absorbance

Spec Alignment to law

• Law measures linear lines by transmission and measures with with Beer's Law
• Measures with the substance under high and low concentration
• Needs Wavelength for certain analyte