STEPTRACKERIMMUNOLOGY + LABORATORY MEDICINE | IMMUNE TESTS | HIGH-YIELD REVIEW

Immunologic Tests

How the lab measures antibodies, antigens, cells and complement — and how to read each result without being fooled by its limits.

FOCUS ON
ELISA: antigen or antibody captured on a plate, detected by an enzyme-linked antibody (color change) — HIV 4th-gen, hepatitis serology, many autoantibodies. Western blot: proteins separated by size then probed (historic HIV confirmation, Lyme confirmation). Immunofluorescence: direct (tissue-bound antibody — linear in Goodpasture/pemphigoid, granular in lupus/PSGN, net-like in pemphigus) vs indirect (patient serum on substrate — ANA, ANCA patterns). Flow cytometry: fluorescent antibodies count cell types by CD markers (CD4 count, leukemia/lymphoma immunophenotyping, PNH, LAD, DHR test). Coombs: direct (antibody on patient RBCs) vs indirect (antibody in serum). Complement: C3, C4, CH50/AH50. Protein electrophoresis + immunofixation: M spike. IGRA / PPD: T-cell memory to TB.
TEST
PRINCIPLE
USES
PITFALL
ELISA
Antigen–antibody binding on a plate, enzyme-linked detection antibody → color
HIV, hepatitis, Lyme screen, autoantibodies, hCG
High sensitivity; confirm positives with a specific test
Western blot
Electrophoresis by size → transfer → antibody probe
Lyme confirmation (after ELISA)
Replaced for HIV by differentiation assay
Direct immunofluorescence
Labeled anti-human Ig/C3 applied to patient tissue
Glomerulonephritis, bullous disease, vasculitis
Linear (anti-GBM, pemphigoid) · granular (immune complex) · intercellular net (pemphigus)
Indirect immunofluorescence
Patient serum on cells/tissue, then labeled anti-human Ig
ANA (HEp-2), ANCA (c- cytoplasmic, p- perinuclear)
ANA pattern hints at target (homogeneous → dsDNA/histone; centromere)
Flow cytometry
Cells tagged with fluorescent anti-CD antibodies pass a laser
CD4 count, lymphoma/leukemia typing, PNH (CD55/59, FLAER), LAD (CD18), CGD (DHR)
Measures surface (and some intracellular) markers
Direct Coombs (DAT)
Anti-human globulin added to patient RBCs → agglutination if coated
AIHA, hemolytic transfusion reaction, HDFN
Warm (IgG) vs cold (C3) pattern
Indirect Coombs
Patient serum + reagent RBCs → detect free antibodies
Antibody screen before transfusion, prenatal Rh testing
—
Complement levels
C3, C4 (consumption); CH50 (classical), AH50 (alternative) function
SLE activity, cryoglobulinemia, complement deficiency, HAE (↓ C4)
Acute-phase rise can mask consumption
SPEP · immunofixation · free light chains
Separate serum proteins; identify monoclonal heavy/light chain
Myeloma, MGUS, Waldenström, AL amyloid
Abnormal κ:λ ratio = clonal
IGRA · tuberculin skin test
IFN-γ release by T cells to TB antigens (ESAT-6, CFP-10) · DTH reaction
Latent TB screening
IGRA not affected by BCG · both falsely negative in immunosuppression
Agglutination · precipitation
Antibody cross-links particles · soluble complexes
Blood typing, latex tests (Cryptococcus antigen), Monospot
Prozone effect with antibody excess
PCR / NAAT
Amplifies nucleic acid
HIV viral load, chlamydia/gonorrhea, HSV in CSF
Detects nucleic acid, not viability

Mechanism — sandwich ELISA

Capture antibody on plate
→
Patient antigen binds
→
Enzyme-linked detection antibody
→
Substrate → color ∝ amount

Indirect ELISA flips it: antigen on the plate catches the patient's antibody.

Immunofluorescence patterns

PATTERN
DISEASE
TYPE
Linear IgG along GBM
Goodpasture
II
Granular IgG + C3
Lupus nephritis, PSGN, MPGN
III
Mesangial IgA
IgA nephropathy, IgA vasculitis
III
Net-like intercellular IgG
Pemphigus vulgaris
II
Linear at basement membrane
Bullous pemphigoid
II
Granular IgA at dermal papillae
Dermatitis herpetiformis
Celiac

How it's tested

COMMON CONFUSIONDirect Coombs (antibody already on the patient's RBCs → hemolysis) vs indirect Coombs (free antibody in serum → crossmatch, prenatal screen).
THE TRAPA negative TB test (PPD or IGRA) in a patient with advanced HIV or on steroids doesn't exclude infection — T cells can't respond.
YOUR APPROACHWhat is being detected (antibody, antigen, cell, complement, nucleic acid) → method → interpretation → known false results.

If you see… think…

Test counting CD4⁺ cells → flow cytometry
Antibody coating a patient's own RBCs → direct Coombs ⁺
Prenatal antibody screen in an Rh ⁻ mother → indirect Coombs
Linear IgG on glomerular basement membrane → anti-GBM disease
"Lumpy-bumpy" granular deposits → immune-complex GN
Confirmatory Lyme test after ELISA → Western blot
TB test unaffected by prior BCG → IGRA
Monoclonal spike on SPEP → plasma cell dyscrasia
Absent CD55/CD59 on flow → PNH
Cryptococcal antigen detection in CSF → latex agglutination / lateral flow