Laboratory Techniques

Behind every blood test result, every cancer diagnosis, every drug level, and every infection identified, there is a laboratory. The modern medical laboratory is one of the most powerful diagnostic tools in medicine — a single blood sample can yield dozens of measurements that guide clinical decisions within hours. Medical laboratories use a range of techniques rooted in chemistry and physics. Understanding these techniques helps you: - Interpret test results correctly (knowing their limitations) - Understand why certain conditions are diagnosed in certain ways - Appreciate how new diseases are identified and how treatments are monitored The core techniques include: centrifugation, spectrophotometry, chromatography, electrophoresis, immunoassays, and polymerase chain reaction (PCR). Each exploits a fundamental property of chemistry or physics.

A centrifuge spins samples at very high speed — thousands to hundreds of thousands of revolutions per minute — generating centrifugal forces many times greater than gravity. Denser components migrate to the bottom (pellet); less dense components remain above (supernatant). In clinical medicine: Blood separation: When a blood sample is centrifuged, it separates into three layers: - Bottom (red, dense) — red blood cells - Middle thin layer (white/grey) — the buffy coat, containing white blood cells and platelets - Top (yellow, clear) — plasma (if collected in anticoagulant) or serum (if collected without, after the clot forms and is removed) Most blood chemistry tests (glucose, cholesterol, liver enzymes, creatinine) are performed on serum or plasma — the red blood cells are spun away first. Urine centrifugation: Spinning a urine sample deposits cells, casts, and crystals at the bottom. The pellet is examined microscopically — the presence of red blood cell casts indicates glomerulonephritis; white cell casts indicate pyelonephritis; calcium oxalate crystals suggest kidney stones. High-speed (ultracentrifugation): Used to separate subcellular components, viruses, and large protein complexes. Essential in research to isolate organelles, purify viruses for vaccine production, and study lipoproteins (LDL, HDL cholesterol). Packed Cell Volume (PCV) or Haematocrit: A simple centrifugation test — blood is spun in a thin tube and the proportion occupied by red cells is measured directly. Normal: 0.40–0.52 (men), 0.36–0.47 (women). Reduced in anaemia; elevated in polycythaemia.

Spectrophotometry measures how much light of a specific wavelength is absorbed by a solution. The more of a substance present, the more light it absorbs — a relationship described by the Beer-Lambert Law: Absorbance ∝ Concentration × Path length In practice: the machine shines a beam of light through a sample and measures how much light makes it through to the detector. By comparing to a calibration standard of known concentration, the machine calculates the unknown concentration. Medical applications: Blood glucose measurement: Glucose in a sample reacts with an enzyme (glucose oxidase) to produce hydrogen peroxide, which reacts with a dye to produce a colour. The intensity of the colour — measured spectrophotometrically — is proportional to the glucose concentration. This is how glucometers and laboratory analysers both work. Liver function tests: Bilirubin, albumin, liver enzymes — all measured using colourimetric reactions that produce compounds with characteristic absorbance at specific wavelengths. Haemoglobin measurement: Blood is mixed with a reagent that converts all haemoglobin forms to cyanmethaemoglobin, which absorbs light at 540 nm. The absorbance gives the haemoglobin concentration — the basis of anaemia diagnosis. Pulse oximetry: Uses a special form of spectrophotometry in living tissue. A probe shines red (660 nm) and infrared (940 nm) light through a finger. Oxygenated haemoglobin (OxyHb) and deoxygenated haemoglobin (DeoxyHb) absorb these wavelengths differently. The ratio of absorbances at the two wavelengths gives the oxygen saturation — the familiar SpO₂ percentage. Critically: carbon monoxide poisoning gives falsely normal pulse oximetry because carboxyhaemoglobin absorbs light similarly to oxyhaemoglobin.

Chromatography — Separating Mixtures Chromatography separates the components of a mixture by exploiting differences in how strongly each component binds to a stationary material vs how readily it moves with a mobile phase (liquid or gas). High-Performance Liquid Chromatography (HPLC): The gold standard for measuring drug levels in blood. A sample is pumped under high pressure through a column packed with tiny particles. Different drug molecules travel through at different speeds and emerge separately, each detected and quantified. Used to monitor drugs like tacrolimus (transplant rejection prevention), methotrexate (cancer and autoimmune), and many antibiotics. Gas Chromatography-Mass Spectrometry (GC-MS): The most accurate method for drug and toxicology testing. Volatile compounds are separated by gas chromatography, then each component is fragmented in a mass spectrometer and identified by its unique mass spectrum — like a molecular fingerprint. Used in forensic toxicology, doping testing in sport, and confirming immunoassay results. Thin-Layer Chromatography (TLC): The simplest form — a sample is spotted on a coated plate and a solvent travels up by capillary action. Different compounds travel different distances depending on their polarity. Used in pharmacies to verify drug identity and purity. Electrophoresis — Separating by Charge Electrophoresis applies an electric field to a gel or solution. Charged molecules migrate toward the oppositely charged electrode — larger molecules more slowly, smaller ones more quickly. Serum protein electrophoresis: Separates blood proteins into bands: albumin, alpha-1, alpha-2, beta, and gamma globulins. An abnormal spike in the gamma region (M-band or paraprotein) is characteristic of multiple myeloma — a plasma cell cancer producing large amounts of a single abnormal antibody. DNA electrophoresis: DNA fragments produced by PCR or restriction enzymes are separated by size on an agarose gel. Used to confirm pathogen identification, diagnose genetic conditions, and forensic DNA profiling.

Immunoassays — Using Antibodies to Detect Anything Immunoassays exploit the exquisitely specific binding of antibodies to their target antigens to detect and quantify almost any substance in blood, urine, or tissue. Enzyme-Linked Immunosorbent Assay (ELISA): The most widely used immunoassay. The antigen (or antibody) of interest is captured on a plate by a specific antibody. A second antibody linked to an enzyme is added. When the enzyme's substrate is added, it produces a colour change proportional to the amount of antigen present — measured spectrophotometrically. Used for: - HIV diagnosis (detecting antibodies to HIV proteins) - Pregnancy testing (detecting hCG) - Hepatitis B surface antigen detection - Thyroid hormone (TSH, T4) measurement - Troponin measurement (heart attack diagnosis) - COVID-19 antibody testing Lateral flow assay (rapid antigen test): The technology behind home pregnancy tests and COVID-19 rapid tests. Works like a miniaturised ELISA — a sample flows along a strip by capillary action, picks up antibody-conjugated coloured particles, and the complex is captured at a test line, producing a visible band. Simple, fast (minutes), no equipment needed — but less sensitive than laboratory ELISA. Polymerase Chain Reaction (PCR) PCR is one of the most revolutionary techniques in modern medicine. It amplifies tiny amounts of DNA or RNA millions of times, allowing detection of even a single copy of a pathogen's genetic material. How it works: 1. Denaturation — heat to 95°C separates the double DNA strand 2. Annealing — cool to 50–65°C; short DNA sequences (primers) bind specifically to the target sequence 3. Extension — heat to 72°C; DNA polymerase copies the target sequence 4. Repeat 30–40 cycles — each cycle doubles the amount of DNA After 30 cycles: 2³⁰ = over 1 billion copies from a single original molecule. RT-PCR (reverse transcription PCR) first converts RNA to DNA — allowing detection of RNA viruses like SARS-CoV-2, influenza, and HIV. Medical uses of PCR: - COVID-19, influenza, RSV diagnosis - HIV viral load measurement (monitoring treatment) - Tuberculosis diagnosis - STI testing (chlamydia, gonorrhoea) - Cancer mutation testing (e.g. KRAS, BRCA) - Prenatal genetic diagnosis