Basic Concepts & Techniques in Chemical Pathology

Basic Concepts in Chemical Pathology

• Lecture notes and written assignments are available on the OLE platform
• Attendance will be taken twice for each class, first 30 minutes and at the end
• Special circumstances for absence require notification via email, and 80% attendance is mandatory
• Lecture material will be posted on OLE one day before the lecture
• Written assignments will further help students recognize, understand, and memorize lecture content
• Assignments must be submitted one week after each lecture to avoid mark deductions

Accreditation and Graduation Requirements

• Attendance is a crucial accreditation requirement
• Students must exceed 80% attendance in each course to be eligible for registration as a Medical Laboratory Technologist with the MLT Board, otherwise course retake is required
• 100% attendance is mandatory for practical lab classes
• BSCHMLS program requires at least a grade of C in all core courses for graduation as a Medical Laboratory Technologist with the MLT Board
• A retake of the corresponding core course is necessary upon receiving a failing grade (F) or marginal grade (C-/D)

Clinical Chemistry: Key Concepts

• Introduction to Pathology and Clinical Pathology
• Specimen Reception and Processing, which are vital to lab work
• Essential equipment and laboratory techniques
• Spectrophotometric Measurement for analysis

Pathology

• Pathology is the study of disease that includes:
  • The cause
  • Mechanisms of development (pathogenesis)
  • Structural alterations of cells
  • Consequences of changes (signs and symptoms)

Clinical Pathology

• Clinical Pathology is a medical specialty focused on diagnosing disease through laboratory analysis of body fluids like blood and urine
• Clinical Chemistry, Hematology, Microbiology, and Histopathology are relevant tools

Chemical Pathology/Clinical Chemistry/Clinical Biochemistry

• Exploys chemical reaction tests, using chemical analysis, for various biomarkers in bodily fluids like blood and urine
• Goal is to aid in disease diagnosis and monitoring using analytical techniques and specialized instruments
• It combines Physiology, Biochemistry, and Analytical Chemistry

Biochemistry

• Biochemistry is a sub-discipline of both chemistry and biology
• It studies the chemical processes within and relating to living organisms at a cellular level
• Biochemistry is traditionally divided into structural biology, enzymology, and metabolism

Analytical Chemistry

• Analytical Chemistry studies and uses instruments and methods/technologies to separate, identify, and quantify substance
• Methods include separation, identification, or quantification to analyze substance identity and concentration
• Qualitative or quantitative techniques include chromatography, electrophoresis, electrochemistry, Immunoassay, spectroscopy, and mass spectrometry

Terms Related to Chemical Pathology

• Clinical/Pathology Level:
  • Chemical Pathologist (Clinical doctor)
• Technical Level:
  • Clinical biochemist (Scientist at technical level)
  • Scientific Officer
  • Medical Laboratory Scientist/Technician

Laboratory Techniques and Concepts

• The biochemical techniques used in clinical chemistry laboratories include analytical and pre-analytical techniques alongside laboratory automation

Technique Considerations

• Weighing accuracy to decimal places, using balance
• Pipetting correctly with autopipettes and sample processing with transfer pipettes
• Dilution with direct and indirect methods
• Preparation of working solution from stock solution (v/v%) or from solid(w/v%)
• Proper operation of equipment like centrifuges and pH meters
• Spectrophotometric measurements

Clinical Chemistry Key Features

• Clinical Chemistry uses auto-analyzers for high throughput of laboratory results (biomarkers)
• Analysis generally thousand of tests can be produced daily but specialized equipment is needed for urgent labs
• Tests use quantitative measurement with various methodologies
• Strict quality assurance is maintained from sample collection to result reporting, covering pre-analytical, analytical, and post-analytical phases
• Interpretation of case results with multiple tests, like Albumin and liver enzymes (ALT, AST, ALP), validates diagnosis and aids treatment

Specimen Reception and Processing

• The workflow and importance guide these steps
• Preanalytical considerations:
  • Types of specimens: plasma, serum, urine, CSF
  • Types of containers: blood, bile, CSF, urine and Transport & Storage

Lab Workflow Cycle

• The lab workflow cycle includes
  • Clinical question
  • Request form
  • Specimen collection
  • Transport to laboratory
  • Specimen reception/accessioning (e.g. barcode) /processing (aliquoting
  • Analysis (chemical, immunological, chromatography, electrophoresis with calibration and quality control
  • Post-analysis (e.g. calculation, compilation with data collected)
  • Result interpretation and Reporting with standard format

Specimen Reception

• Samples sorted and quality checked against request forms
• Correct patient identification details and tests are entered into LIS, laboratory information system
• Specimens are given unique barcodes, check digit (sample ID + test information contained) and aliquots are stored
• Samples are centrifuged (e.g. whole blood, urine)

Importance of Specimen Handling

• Specimen reception and processing is vital to the laboratory's training
• Negligence leads to:
  • compromised sample integrity
  • traceability, result quality, and patient health
  • risks to staff

Types of Specimens

• Blood
  • Serum, plasma, whole blood, and red cells are relevant
  • Must check for haemolysis
• Urine
  • Spot, timed, and 24-h urine samples
• Pleural fluid
• Cerebrospinal fluid (CSF):

Anticoagulant Tubes

• ORDER OF DRAW - Extremely Important for accuracy, with common mistake is to draw K3EDTA tube first
• Tube additives and functions include:

Tube Examples

• Light Blue:
  • 3.2% Sodium citrate
  • Prevents blood from clotting by binding calcium
  • Used for: Coagulation
• Red or Gold tiger top:
• Serum without
• Clot activator promotes blood clotting with glass or silica particles
• Used for: Chemistry, serology, immunology
• Green:
  • Sodium or lithium heparin
  • Prevents clotting by inhibiting thrombin and thromboplastin
  • Used for: Stat and routine chemistry
• Lavender or Pink:
  • Potassium EDTA Prevents
  • Prevents clotting by binding calcium
  • Used forr: Hematology and blood bank
• Gray:
  • Sodium fluoride, and sodium or potassium oxalate
  • Fluoride inhibits glycolysis, and oxalate prevents clotting by precipitating calcium.
  • Used for: Glucose (especially when testing will be delayed), blood alcohol, lactic acid

Proper Transport

• Transportation of specimens inside the hospital/lab and outside /inside depends of needs

Transport Considerations

Distances of transport

• From wards on same site
• Other hospitals, General Practitioner (GP) surgeries, and Other remote sites Special considerations
• Taxis for urgent specimens, ice for unstable analytes, and heat blocks for certain proteins are considerations
• Safety involves handling all potentially infectious samples

Devices for Sample Storage

• There are different devises for short/long term sample storage

Keys to Sample Quality and Integrity

• Serum or plasma should be separated from blood cells with centrifuge as soon as possible and then stored in a refrigerator
• Serum of unstable for further processing should be frozen at (-20°C)
• Key factors are time and temperature of sample

Rejected samples

• Reasons include samples with: no sample available, presence of a needle, or inadequate blood
• Inadequate samples can be incorrect as this throws of the anti-coagulant ratio

Interference

• Another important factor is Interference of samples

Errors Contributing to Sample Rejection

• Errors are of two variation Patient variation and Sample variation:
• Examples of patient variation could include;
• The test was taken at the wrong time of day as diurnal rhythm affects cortisol
• The test was taken at the wrong posture eg renin-aldosterone
• Examples of sample variation:
• The test was taken with Swap sample or Wrong tube, or poor Temperature control

Patient, Sample Preparation and Timing of Samples: Pre-analytical impacts

• Patient sample has greatest impact in the pre analytical stage

Patient Preparation

• Includes fasting for triglycerides
• Sampling at appropriate time of day
• Exercise considerations and patients
• Patient posture/position

Sample

• Swapped samples, issues with aliquot, poor anti coagulants, poor temperature and transport can all lead to issues

Sample Tubes

• Vary depending in required tests
• Blood, gases CSF have specialised collection protocols

Colored Urine Example

• Pre analytical variations can have massive impact
• Spot urine or acid preservation issues can have adverse affects

Urine Color Indications

• Clear- to much water intake, to little electrolyte balance
• Yellow normal
• Orange- potential drugs are in use

Sample Equipment and Storage

• A analytical chemistry lab should have:
• Top loading balance or analytical balance

Balances

• Top balances are less accurate only use if accuracy requirements are low: general use only
• Milligram requires and analytical balance (+-0.00001)

Rules for Weighing

• Ensure accurate balance and horizontal surface level
• Never touch chemicals with bare hands
• No chemical to make contact with balance
• Do not weigh hot or cold substances

Weighing Techniques

• Direct: with the use of zeroed scales and direct addition of substance
• Indirect: measure with a container what is added for reference

Solutions

• Solution Concentrations % measurements include, Mass over mass, volume over volume or mas over volume

Pipetting

• Forward and reverse actions

Dilution

• Direct and indirect techniques are avalible
• The golden formula being M1V1=M2V2

Centrifugation

• Based on density centrifugation can be used to break liquids or mixtures

Centrifugal Force

• RCF relative centrifugal force depends on speed and relative orientation

Centrifugation Speeds

• The higher RCF the faster separation occurs however over high RPMS can be dangerous