Immunology and serology tests

In short

Immunology is the study of the immune system - the cells, antibodies and complement proteins that recognise and remove foreign antigens - and serology is the laboratory use of antigen-antibody reactions in serum to diagnose infection, autoimmune disease and immune status. An MLT student must know both the biology (immunoglobulin structure, immunity types, hypersensitivity) and the bench technique (agglutination, precipitation, ELISA, immunofluorescence, rapid cards, CLIA, flow cytometry), plus the titre arithmetic examiners love. In India these tests run in every NABL-accredited pathology lab, ICTC/ART centres, IDSP and NCDC surveillance networks, blood centres and diagnostic chains (Dr Lal PathLabs, SRL, Metropolis, Thyrocare, Agilus, Apollo, AIIMS/PGI/CMC labs). It is examined heavily in B.Sc MLT university papers, in AIIMS/PGI/NIMHAN technician recruitment and in placement technical rounds on ELISA plate work, HIV testing strategy and kit quality control.

Explained

Immune system basics

• Antigen (immunogen): a substance recognised by the immune system; epitope is the small part the antibody actually binds. Hapten is too small to be immunogenic alone but becomes so when coupled to a carrier (penicillin reactions).
• Innate immunity: present from birth, non-specific, no memory - skin and mucosa, pH, lysozyme, neutrophils, macrophages, NK cells, complement, interferons, acute-phase proteins (CRP, ferritin).
• Adaptive immunity: specific, with memory - humoral (B cells → plasma cells → antibodies) and cell-mediated (T cells). CD4 helper T cells direct the response; CD8 cytotoxic T cells kill infected cells; Treg cells restrain it.
• Active immunity: produced by the host - natural (infection) or artificial (vaccine); slow onset, long-lasting. Passive immunity: ready-made antibody transferred - natural (maternal IgG across placenta, IgA in breast milk) or artificial (anti-tetanus immunoglobulin, anti-rabies immunoglobulin, anti-snake venom); immediate but temporary.
• Immunological memory: the secondary response is faster, higher in titre and IgG-dominated - the basis of paired-sera testing and of booster doses.
• MHC / HLA: class I (HLA-A, B, C) on all nucleated cells presents to CD8; class II (HLA-DR, DQ, DP) on antigen-presenting cells presents to CD4. HLA typing is used in transplant matching (ORGAN - the Transplantation of Human Organs and Tissues Act, 1994, amended 2011, with NOTTO/ROTTO/SOTTO registries).
• Complement: classical pathway (triggered by antigen-antibody complexes, C1q), alternative and lectin pathways; all converge on C3 → C5-9 membrane attack complex. Functions: lysis, opsonisation (C3b), chemotaxis (C5a), immune-complex clearance. C3 and C4 fall in active SLE and post-streptococcal nephritis.

Immunoglobulins and antigen-antibody reaction

• Structure: Y-shaped glycoprotein of two identical heavy and two identical light chains joined by disulphide bonds; Fab fragments carry the two antigen-binding sites (variable regions), Fc carries the biological functions (complement fixation, Fc-receptor binding, placental transfer). Papain cleaves into 2 Fab + 1 Fc; pepsin gives F(ab')₂.
• IgG - about 75-80% of serum immunoglobulin, monomer, crosses the placenta, fixes complement, main antibody of secondary response and of past/convalescent infection; 4 subclasses.
• IgM - pentamer, largest, cannot cross the placenta, best complement fixer and agglutinator, first antibody formed; IgM in a neonate or in a single acute sample indicates recent/current infection or congenital infection.
• IgA - monomer in serum, dimer with secretory piece in secretions (tears, saliva, colostrum, gut); mucosal defence; tissue-transglutaminase IgA is the coeliac screen.
• IgE - least in serum, binds mast cells and basophils via Fc receptors, mediates type I allergy; measured as total IgE and allergen-specific IgE.
• IgD - mainly a B-cell surface receptor; little diagnostic use.
• Antibody types used as reagents: polyclonal (immunised animal serum - many epitopes, cheaper, batch variation) and monoclonal (hybridoma; Köhler and Milstein - single epitope, highly reproducible, basis of modern kits and blood-grouping antisera).
• Antigen-antibody bonding: non-covalent and reversible - hydrogen bonds, ionic, hydrophobic and van der Waals forces. Affinity = strength of one Fab-epitope bond; avidity = total strength of multivalent binding (IgM has low affinity but high avidity).
• Specificity and cross-reactivity: shared epitopes cause false positives - the classical example is heterophile antibody in the Paul-Bunnell test, and malaria/leptospirosis/typhus cross-reactions in dengue serology.
• Lattice hypothesis (Marrack): visible agglutination/precipitation needs an optimal antigen-to-antibody ratio (zone of equivalence). Excess antibody = prozone, excess antigen = postzone; both give false negatives, corrected by diluting the serum.
• Titre: the reciprocal of the highest serum dilution still giving a visible reaction. A fourfold (two-tube) rise between acute and convalescent sera taken 10-14 days apart is diagnostic of recent infection.

Classification of serological techniques

1. Precipitation (soluble antigen + antibody): ring/tube test, Ouchterlony double diffusion (identity, non-identity, partial identity patterns), single radial immunodiffusion (Mancini) - area of ring ∝ antigen concentration, immunoelectrophoresis, counter-current immunoelectrophoresis (CIE), rocket electrophoresis (Laurell), nephelometry/turbidimetry (light scatter - today's route for CRP, ASO, RF, complement, immunoglobulin levels).
2. Agglutination (particulate antigen): direct/active - Widal, Weil-Felix, brucella tube agglutination, blood grouping; passive/indirect - antigen coated on carrier particles (latex, treated RBCs): RA (RF) latex, CRP latex, RPR carbon particles; reverse passive - antibody coated on the particle to detect antigen (HBsAg latex); antiglobulin (Coombs) - detects non-agglutinating IgG using anti-human globulin; agglutination inhibition - used in pregnancy and some drug tests; haemagglutination inhibition (HAI) for influenza antibody; TPHA for syphilis.
3. Complement-dependent: complement fixation test (CFT) - haemolysis means NO patient antibody (negative); immune adherence; complement-mediated lysis in Wassermann-type tests. Largely historical but examined.
4. Labelled immunoassays: radioimmunoassay (RIA, ¹²⁵I - Yalow and Berson; now rare because of radiation waste), ELISA/EIA (enzyme), immunofluorescence (IFA/DFA), chemiluminescence (CLIA/ECLIA), fluorescence immunoassay, immunochromatographic rapid cards/lateral flow, western blot/immunoblot, line immunoassay (LIA).
5. Cell-based: flow cytometry (CD4/CD8 counting, leukaemia-lymphoma immunophenotyping), ELISPOT and interferon-gamma release assay (IGRA - TB Gold), lymphocyte transformation tests, skin tests (Mantoux - type IV).

ELISA, rapid tests and automated platforms - how they work

• Direct ELISA: antigen coated on the well, enzyme-labelled antibody binds it. Fast, fewer steps, less sensitive.
• Indirect ELISA: antigen coated, patient serum antibody binds, then enzyme-labelled anti-human IgG/IgM + substrate. The standard antibody-detection format (HIV screening, anti-HCV, measles IgG, dengue IgM MAC-ELISA).
• Sandwich (capture) ELISA: antibody coated on the well captures the antigen from serum; a second labelled antibody detects it. Needs antigen with ≥2 epitopes - used for HBsAg, dengue NS1, hormones, troponin.
• Competitive ELISA: sample antigen competes with labelled antigen - colour is inversely proportional to analyte; used for small molecules (T3/T4, cortisol, anti-HBs, anti-HBc total).
• Fourth-generation HIV ELISA/CLIA detects p24 antigen plus HIV-1/2 antibody, shortening the window period to roughly 2-3 weeks against 3-4 weeks for third-generation antibody-only tests.
• Enzymes and substrates: horseradish peroxidase with TMB (blue → yellow at 450 nm after H₂SO₄ stop) or OPD; alkaline phosphatase with pNPP (405 nm). Read on an ELISA plate reader; wash steps with PBS-Tween remove unbound reagent - inadequate washing is the commonest cause of false positives.
• Cut-off and interpretation: cut-off OD is computed from kit controls (for example mean negative control OD + a stated factor, or by the formula in the kit insert); S/CO (sample OD ÷ cut-off) ≥1 is reactive. Every run must have negative, positive and sometimes a low-positive control, and blanks.
• Lateral flow / immunochromatography: serum wicks along nitrocellulose past colloidal-gold-labelled antibody to a test line and a procedural control line; no control line = invalid test, repeat. Basis of HIV, HBsAg, HCV, malaria RDT (HRP-2/pLDH), dengue NS1/IgM/IgG, syphilis, COVID-19 and pregnancy (hCG) kits.
• CLIA/ECLIA: acridinium ester or ruthenium labels measured as light in relative light units; wide dynamic range, quantitative, fully automated (Architect, Cobas, Vitros, Maglumi) - now the backbone of large Indian labs for HBsAg, anti-HCV, HIV, TSH, vitamin D.
• Immunofluorescence: direct (labelled antibody onto tissue/smear - rabies Negri antigen, respiratory virus DFA, skin biopsy in pemphigus); indirect (patient serum on substrate, then FITC-anti-human IgG - ANA on HEp-2 cells, ANCA on ethanol-fixed neutrophils, anti-dsDNA on Crithidia). Reported with pattern and titre.
• Western blot: antigens separated by SDS-PAGE, transferred to nitrocellulose, probed with patient serum; bands identify the specific protein (gp120/gp41/p24 in HIV) - a confirmatory, not screening, test.

Important diagnostic serology tests and interpretation

• Widal: tube agglutination for *S. Typhi/Paratyphi* O and H antibodies. Significant titres are usually taken as O ≥ 1:160 and H ≥ 1:160 in Indian endemic settings, but local baselines vary - a rising paired titre is far better evidence. Limitations: positive after vaccination or past infection, negative in the first week, false positives in malaria and other fevers; blood culture is the gold standard and slide Widal kits are unreliable. Typhidot (IgM/IgG) and IgM lateral-flow tests are alternatives.
• Syphilis: non-treponemal VDRL and RPR detect reagin antibody against cardiolipin - cheap, titrable, used to monitor treatment (a fourfold fall indicates response); biological false positives in pregnancy, malaria, leprosy, SLE, and the prozone in secondary syphilis. Treponemal TPHA, TP-PA, FTA-ABS, and treponemal rapid tests are confirmatory and stay positive for life. ICMR/NACO antenatal screening uses a rapid or RPR test with confirmation.
• HIV: NACO national strategy screens with a highly sensitive test and confirms with two more kits of different antigen/principle - Strategy III (three different kits) for diagnosis of an asymptomatic person at an ICTC, with the whole-blood/serum rapid tests validated by NIB Noida. Infants under 18 months need DNA/RNA PCR (early infant diagnosis) because maternal IgG persists. Viral load and CD4 count by flow cytometry monitor therapy.
• Hepatitis B panel: HBsAg = current infection; anti-HBs = immunity (vaccine or recovery); IgM anti-HBc = acute; total anti-HBc = ever infected; HBeAg = high infectivity. Hepatitis C: anti-HCV screen then HCV RNA; the National Viral Hepatitis Control Programme funds this.
• Dengue: NS1 antigen positive from day 1 to about day 5-7; IgM from day 5 onward (MAC-ELISA is the NVBDCP/NCVBDC reference); IgG rises in secondary infection. Only NIV-Pune-evaluated kits are to be used in the national programme.
• Rheumatology: RF latex/nephelometry (also positive in elderly, chronic infection, hepatitis C), anti-CCP (more specific for rheumatoid arthritis), ANA by IFA on HEp-2 as the SLE screen with patterns (homogeneous, speckled, nucleolar, centromere), then specific anti-dsDNA, anti-Sm, anti-SSA/SSB, anti-Scl-70, anti-Jo-1 by ELISA or line assay; ANCA (c-ANCA/PR3, p-ANCA/MPO) in vasculitis; ASO titre and anti-DNase B after streptococcal infection.
• Acute-phase and other markers: CRP (turbidimetry; hs-CRP for cardiac risk), procalcitonin for bacterial sepsis, ferritin, serum protein electrophoresis with immunofixation for myeloma M-band, cryoglobulins, cold agglutinins.
• Other named tests examined: Weil-Felix (rickettsia, OX-19/OX-2/OX-K), Paul-Bunnell/Monospot (infectious mononucleosis heterophile antibody), Rose-Bengal and standard tube agglutination for brucellosis, lepromin and Mantoux skin tests, Casoni test (hydatid), Elek test (diphtheria toxigenicity by precipitation), Ascoli and Coombs tests, ASO, cysticercosis and leptospira IgM ELISA, scrub typhus IgM ELISA.
• Hypersensitivity (Gell and Coombs): Type I immediate, IgE, mast cells - anaphylaxis, asthma, urticaria (tested by skin prick, total/specific IgE, tryptase); Type II antibody-mediated cytotoxic - haemolytic disease of newborn, autoimmune haemolytic anaemia, Goodpasture; Type III immune complex - serum sickness, SLE, post-streptococcal glomerulonephritis, Arthus reaction; Type IV delayed cell-mediated - Mantoux, contact dermatitis, graft rejection. Type V (stimulatory) - Graves disease, in some classifications.
• Immunodeficiency and tolerance: primary (X-linked agammaglobulinaemia, SCID, DiGeorge, chronic granulomatous disease - tested by NBT/DHR), secondary (HIV, malnutrition, steroids, malignancy); autoimmunity results from loss of self-tolerance, molecular mimicry and epitope spreading.

Specimen, quality and regulation in Indian practice

• Specimen: 3-5 mL clotted blood in a plain/SST tube; separate serum within 2 hours; haemolysed, lipaemic or icteric samples interfere with optical reads. Store serum at 2-8 °C up to 48-72 hours, at −20 °C (or −70 °C for long-term) for longer; avoid repeated freeze-thaw. Heat-inactivation of serum at 56 °C for 30 min destroys complement for CFT. Plasma/EDTA is used only where the kit insert allows it.
• Validity and performance: sensitivity, specificity, PPV, NPV, likelihood ratio and prevalence effect; screening tests are chosen for sensitivity, confirmatory tests for specificity. Report interferences - rheumatoid factor and heterophile antibodies cause false positives in sandwich assays; biotin and HAMA interfere with some CLIA assays; hook effect (antigen excess) can lower a very high result.
• Quality control: run kit controls each batch; plot internal QC on Levey-Jennings charts and apply Westgard rules (1-3s, 2-2s, R-4s, 4-1s, 10x) for quantitative assays; participate in EQAS/PT (CMC Vellore, AIIMS, NABL-approved providers); lot-to-lot verification; record reader calibration, incubator and fridge temperatures.
• Regulation and standards: labs are accredited to ISO 15189 (NABL-112 / MC-2024 criteria); kits are regulated as in-vitro diagnostic medical devices under the Medical Devices Rules, 2017 framed under the Drugs and Cosmetics Act, 1940 (CDSCO licensing; IVD manufacture and import need CDSCO approval, and HIV/HBV/HCV kits additionally need batch release/validation by the National Institute of Biologicals, Noida). The Clinical Establishments (Registration and Regulation) Act, 2010 applies in adopting states.
• Biosafety and waste: work per the kit insert and NACO/ICMR laboratory biosafety guidance; most serology is BSL-2. Discard sera, tips and used plates as yellow or red category waste under the Bio-Medical Waste Management Rules, 2016 (as amended); sharps in white puncture-proof containers; 1% sodium hypochlorite for spills.
• Consent, confidentiality and reporting: HIV testing requires informed consent and pre/post-test counselling and results are confidential under the HIV and AIDS (Prevention and Control) Act, 2017; notifiable diseases must be reported to IDSP/IHIP; MTP and PCPNDT rules apply to related testing contexts. The ICMR National Essential Diagnostics List specifies which serology tests each level of facility must offer.

Key points

• Antigen vs antibody test - antigen (NS1, HBsAg, p24, HRP-2) shows current presence of the organism; antibody shows the host response, which lags behind.
• IgM vs IgG - IgM means recent/acute (and in a newborn, congenital) infection; IgG means past infection, immunity or maternal transfer.
• Titre - reciprocal of the highest dilution still reactive; a fourfold rise or fall is the diagnostic/therapeutic criterion.
• Prozone - false negative from antibody excess (seen in secondary syphilis VDRL); postzone from antigen excess; both fixed by dilution.
• Zone of equivalence - optimal Ag:Ab ratio where lattice formation and visible reaction are maximal.
• Affinity vs avidity - single-site bond strength vs total multivalent strength; IgM has high avidity, and IgG avidity testing dates an infection in pregnancy (toxoplasma, rubella).
• Indirect ELISA detects antibody; sandwich ELISA detects antigen; competitive ELISA gives colour inversely proportional to analyte.
• Fourth-generation HIV assay = p24 antigen + antibody, window about 2-3 weeks; infants under 18 months need nucleic acid testing.
• NACO Strategy III - three different kits (different antigen or principle) to diagnose HIV in an asymptomatic person.
• VDRL/RPR are non-treponemal, titrable, used for monitoring; TPHA/FTA-ABS are treponemal and stay reactive for life.
• Widal needs paired sera; single titres are interpreted against the local endemic baseline, with blood culture as the gold standard.
• CFT - haemolysis = negative (complement free); no haemolysis = antibody present.
• Coombs/antiglobulin test - anti-human globulin bridges non-agglutinating IgG-coated cells; direct for in-vivo coating, indirect for serum antibody.
• Complement - C3b opsonises, C5a attracts, C5-9 lyses; low C3/C4 indicate active consumption in SLE.
• Hypersensitivity I-IV - IgE, cytotoxic antibody, immune complex, delayed T-cell; Mantoux is type IV.
• Control line on a rapid card is procedural; no control line means an invalid test, not a negative one.
• Screening vs confirmation - choose high sensitivity to screen, high specificity to confirm; PPV depends on disease prevalence.
• Quality and law - ISO 15189/NABL accreditation, Levey-Jennings with Westgard rules, EQAS participation, CDSCO/MDR 2017 for kits, BMW Rules 2016 for waste, HIV Act 2017 for consent and confidentiality.

Formulas and examples

Example 1 - serial dilution and titre. A Widal tube row is set up by adding 0.1 mL serum to 0.9 mL saline (tube 1), then transferring 0.5 mL serially into 0.5 mL saline.

• Tube 1 dilution = (0.1 + 0.9)/0.1 = 1:10. Each later transfer doubles it: 1:20, 1:40, 1:80, 1:160, 1:320.
• Agglutination with O antigen is visible up to tube 5 → O titre = 160; H agglutination up to tube 4 → H titre = 80.
• Interpretation: O 160 is at/above the usual significant cut-off in Indian practice; H 80 alone is not. Advise a convalescent sample after 10-14 days - a rise to O 640 (two tubes = fourfold) would confirm recent enteric fever. Blood culture remains the confirmatory test.

Example 2 - fourfold change in RPR on treatment. Pre-treatment RPR reactive to 1:32; three months after benzathine penicillin, reactive to 1:8.

• 1:32 → 1:8 is a drop of two doubling dilutions = fourfold fall → satisfactory serological response.
• A rise from 1:8 to 1:32 instead would be a fourfold rise = relapse or reinfection. TPHA would remain reactive throughout and must not be used for monitoring.

Example 3 - ELISA cut-off and S/CO. A fourth-generation HIV ELISA gives negative control ODs 0.040, 0.046, 0.052 and the insert says cut-off = mean NC + 0.100. Patient ODs: A = 0.085, B = 0.320, C = 0.148.

• Mean NC = (0.040 + 0.046 + 0.052)/3 = 0.138/3 = 0.046. Cut-off = 0.046 + 0.100 = 0.146.
• A: S/CO = 0.085/0.146 = 0.58 → non-reactive. B: 0.320/0.146 = 2.19 → reactive. C: 0.148/0.146 = 1.01 → reactive, but borderline (within about 10% of cut-off) - repeat in duplicate, then follow NACO Strategy III with two more kits of different principle before any result is given.

Example 4 - sensitivity, specificity and predictive values. A dengue NS1 rapid kit is evaluated against RT-PCR in 500 fever cases: TP = 180, FN = 20, FP = 30, TN = 270.

• Sensitivity = 180/(180+20) = 180/200 = 90.0%. Specificity = 270/(270+30) = 270/300 = 90.0%.
• PPV = 180/(180+30) = 180/210 = 85.7%. NPV = 270/(270+20) = 270/290 = 93.1%.
• Prevalence = 200/500 = 40%; accuracy = (180+270)/500 = 90.0%.
• If the same kit is used in a low-season population with 5% prevalence: of 1,000 people, 50 diseased → TP = 45, FN = 5; 950 healthy → FP = 95, TN = 855. PPV = 45/140 = 32.1% - the same kit, far poorer positive predictive value. This is why confirmation is mandatory in low-prevalence screening.

Example 5 - QC on a quantitative CRP assay. The control lot mean is 12.0 mg/L with SD 0.8 mg/L. Today's value is 14.0 mg/L.

• z = (14.0 − 12.0)/0.8 = +2.5 SD → breaches the 1-2s warning and the 1-3s limit is not crossed, but a 2.5 SD value is outside ±2 SD, so the run is flagged; check for the 2-2s rule against the second control before accepting.
• CV% at this level = (0.8/12.0) × 100 = 6.7%, acceptable for a turbidimetric CRP (usually ≤10%).

Where it is used

• Diagnostic lab chains and hospital serology sections: Dr Lal PathLabs, SRL, Metropolis, Agilus, Thyrocare, Apollo, Fortis, AIIMS, PGIMER, CMC Vellore - roles as Serology/Immunoassay Technician, Lab Technologist and QC/EQAS coordinator, running ELISA plates, CLIA analysers and rapid tests.
• National programmes: ICTC/ART centres and SACS under NACO (HIV testing, CD4 counts), NCVBDC sentinel sites for dengue and chikungunya IgM, National Viral Hepatitis Control Programme, NTEP labs (IGRA and TB serology restrictions - serology for active TB is not recommended), IDSP/IHIP outbreak serosurveys and ICMR national serosurveys.
• Blood centres and transplant immunology: mandatory TTI screening (HIV, HBV, HCV, syphilis, malaria) by ELISA/CLIA with NAT in larger centres; HLA typing and crossmatching for renal and marrow transplants at NOTTO-linked centres.
• IVD manufacturing and regulatory QC: J Mitra, Span Diagnostics, Meril, Mylab, Transasia/Erba, Oscar Medicare - kit development, batch QC, stability and CDSCO/NIB validation under the Medical Devices Rules, 2017.
• Veterinary, food and export labs: brucellosis and FMD ELISA at ICAR institutes, allergen and antibiotic-residue ELISA in FSSAI-notified food labs.
• Research and vaccine work: ICMR-NIV Pune, NII, THSTI, CDSA, Serum Institute and Bharat Biotech - neutralisation assays, antibody titration for immunogenicity trials, hybridoma and monoclonal antibody production.

Interview questions

• Q: What is the difference between affinity and avidity? A: Affinity is the binding strength between one antigen-binding site and one epitope; avidity is the overall strength of a multivalent antibody-antigen interaction, so pentameric IgM has high avidity despite low per-site affinity.
• Q: A VDRL is negative in a patient with obvious secondary syphilis. Why? A: Prozone phenomenon - antibody excess prevents lattice formation; repeating the test on diluted serum reveals a high titre.
• Q: How do you distinguish an indirect from a sandwich ELISA? A: Indirect ELISA coats antigen on the well and detects patient antibody with labelled anti-human immunoglobulin; sandwich ELISA coats a capture antibody to trap antigen from the sample and detects it with a second labelled antibody.
• Q: Why does a fourth-generation HIV test detect infection earlier? A: It detects p24 antigen as well as antibody, so it becomes reactive during the antigenaemic phase about 2-3 weeks after exposure, before seroconversion.
• Q: Why can't you use antibody tests to diagnose HIV in a 6-month-old infant? A: Maternal IgG crosses the placenta and persists up to 18 months, so DNA/RNA PCR (early infant diagnosis) is required.
• Q: A rapid test card shows a test line but no control line. What do you report? A: Nothing - the test is invalid; repeat with a fresh device, since an absent control line means reagent or procedural failure.
• Q: What does haemolysis mean in a complement fixation test? A: Haemolysis means complement was free to lyse the indicator cells, so no antigen-antibody complex formed - the test is negative.
• Q: Why is a single high Widal titre unreliable in India? A: Because past infection, vaccination and cross-reacting febrile illness raise baseline titres in endemic areas; paired sera showing a fourfold rise, and ideally blood culture, are needed.
• Q: What is the hook effect and where does it matter? A: At very high analyte concentrations a sandwich assay gives a falsely low result because excess antigen saturates both antibodies separately; suspect it when the clinical picture does not match, and retest on dilution (HBsAg, hCG, ferritin).
• Q: Which is more important for a screening test - sensitivity or specificity, and why? A: Sensitivity, so that few true cases are missed; specificity is then supplied by a confirmatory test, because positive predictive value falls sharply at low prevalence.
• Q: Which Indian rules govern diagnostic kits and the disposal of used sera and ELISA plates? A: Kits are IVD medical devices under the Medical Devices Rules, 2017 (CDSCO; HIV/HBV/HCV kits also need National Institute of Biologicals batch validation); waste goes in yellow/red categories under the Bio-Medical Waste Management Rules, 2016 as amended.
• Q: Why is serum heated at 56 °C for 30 minutes in some serological tests? A: To inactivate complement and some non-specific inhibitors, which would otherwise interfere - essential before complement fixation tests.

Common mistakes

• Treating a reactive screening test as a diagnosis - skipping the confirmatory kit or the NACO three-kit strategy, and reporting an HIV result from a single test.
• Reporting a prozone or hook-effect result as negative or low instead of repeating on dilution.
• Confusing titre direction - calling 1:320 a lower titre than 1:80, or treating a two-fold (one-tube) change as significant when four-fold is required.
• Muddling the ELISA formats, especially forgetting that colour is inversely proportional to analyte in a competitive assay.
• Poor technique at the bench: incomplete washing, using reagents straight from the fridge instead of bringing them to room temperature, cross-contaminating wells, mismatching kit lots, reading at the wrong wavelength or after the stated time, and ignoring expired or temperature-abused kits.
• Mis-reading IgM/IgG - calling IgG in a neonate an active infection, or expecting IgM positivity in the first two days of dengue fever when only NS1 is detectable.
• Skipping controls and QC documentation, not plotting Levey-Jennings charts, and not acting on Westgard rule violations or EQAS outliers.

Related topics

Blood banking and transfusion · Medical microbiology: infections and diagnosis · Clinical pathology · PCR and DNA sequencing · Haematology · Clinical biochemistry: organ function tests