Serologic Procedures and Test Results

The lattice principles behind precipitin lines, reaction zones, and titer interpretation are covered in Antigen-Antibody Interactions and Complement.

Precipitation forms a visible lattice from soluble antigen and antibody. Ouchterlony double immunodiffusion places antigen and antisera in wells cut into an agarose gel and lets each diffuse toward the other. Precipitin lines form where concentrations reach equivalence, and comparing control and unknown wells gives three patterns: fused, continuous lines show identity, crossing lines show nonidentity, and a fused line with a spur shows partial identity, where one antigen shares an epitope with a second and has an additional one. Anti-Sm and anti-U1 RNP can produce partial identity because their targets occur in related small nuclear ribonucleoprotein complexes but are not identical. Immunodiffusion remains a reference method for resolving which extractable nuclear antigen an antibody recognizes, with high specificity and low throughput.^{1,21 Abbas AK, et al. Cellular and Molecular Immunology. 2025.2 Benito-Garcia E, et al. Guidelines for immunologic laboratory testing in the rheumatic… 2004.}

Flocculation is precipitation confined to a narrow antigen-concentration window. The VDRL and RPR flocculation assays apply it to lipoidal antigens as described in Syphilis, Lyme, and Tuberculosis Serology.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Agglutination applies the same cross-linking to particulate antigen, including whole cells and inert particles coated in advance with antigen or antibody.

In the inhibition formats, no agglutination is the positive result. Cryptococcal antigen testing by latex agglutination uses the reverse-passive format. Lateral-flow cryptococcal antigen assays are generally preferred where available because they are rapid and simple.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

On an RPR card, antilipoidal antibody in the specimen binds the cardiolipin antigen into a lattice that traps the charcoal particles. The visible clumps are called floccules.^{1515 Papp JR, et al. CDC laboratory recommendations for syphilis testing, United… 2024.} A reactive specimen shows black floccules against the white card. A nonreactive specimen shows a uniform light-gray color. Reactive, minimally reactive, and nonreactive controls are tested in each run, and patient results are reported only when every control gives its expected pattern.^{1616 Remel. RPR Card Test Instructions for Use. 2009.}

Hemagglutination is read in a microtiter well. Agglutinated red cells do not settle and coat the well as a diffuse film. Red cells that do not agglutinate settle into a compact button. In hemagglutination inhibition, a button in a patient well means antibody inhibited viral hemagglutination at that dilution. That reading is valid only when the cell control without virus settles into a button and the positive-control antiserum inhibits as expected.^{1717 Kaufmann L, et al. An optimized hemagglutination inhibition (HI) assay to quantify… 2017.}

Labeled immunoassay formats differ in two design features: how bound and free label are separated, and whether patient analyte competes with the reagent or is captured between two antibodies.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Separation. Heterogeneous assays physically separate bound from free label with a wash, magnetic capture, or centrifugation before reading. Homogeneous assays are designed so that bound and free label give different signals in the same tube. This design sacrifices some analytical sensitivity for speed and full automation.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Binding format. Competitive assays give labeled and patient analyte a fixed, limited number of antibody sites, so more patient analyte leaves less bound label: when bound label is measured, its signal decreases as patient analyte increases. Homogeneous competitive formats such as the enzyme multiplied immunoassay technique (EMIT) and the cloned enzyme donor immunoassay (CEDIA) measure an enzyme signal that rises with analyte. The design suits small haptens that cannot bind two antibodies at once. Sandwich (capture) assays use capture antibody in excess across the validated measuring interval, allowing a large fraction of patient analyte to bind before a labeled second antibody against a different epitope completes the sandwich. The signal rises directly with analyte within that interval, and the format requires a large enough analyte to present two distinct epitopes.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

In an idealized competition model, labeled and patient antigen have equal affinity and access to the same binding sites. If binding does not substantially deplete either free pool, the labeled share of bound antigen follows their relative abundance. With 90 units of labeled antigen, the model predicts:

These percentages describe the labeled share of the bound antigen. They do not show what fraction of all labeled antigen was captured. Actual assays use calibrators at known concentrations and an assay-specific curve fit to account for binding and signal behavior.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Historical and current formats

In direct immunofluorescence, the fluorochrome is conjugated to the antibody that recognizes the target. Antigen fixed to a slide, or present on cells, is incubated with the labeled antibody, washed, and read under ultraviolet illumination. Bright fluorescence on a dark field is positive. The format identifies microbial antigen directly in specimens, with Legionella pneumophila and Chlamydia trachomatis as classical applications, and, read by flow cytometry, it is the basis of leukemia and lymphoma immunophenotyping with CD-marker panels.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Indirect immunofluorescence (IIF) adds an amplifying second layer: fixed antigen or cells capture patient antibody, then a fluorescent anti-human-immunoglobulin conjugate binds it. Several labeled secondary molecules can bind each captured primary antibody, so IIF amplifies signal beyond direct labeling, and one anti-human conjugate can be used with every antigen substrate in the laboratory. For antigen detection, IIF uses an unlabeled primary antibody first. For antibody detection, patient antibody acts as the primary reagent. It remains the reference method for HEp-2 ANA screening, Crithidia luciliae anti-dsDNA confirmation, the historical fluorescent treponemal antibody absorption (FTA-ABS) test, varicella-zoster virus (VZV) FAMA testing, and Epstein-Barr virus (EBV)-specific serology. It is slow, requires a trained reader, and varies with buffer, conjugate, and microscope conditions among laboratories.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Multiplex bead immunoassays dye polystyrene beads with unique ratios of red and infrared fluorescent dyes, giving 100 to 500 distinct identities in commercial platforms, one antigen or one antibody per bead. Flow cytometry reads each bead's color identity and bound-antibody fluorescence simultaneously, so single-antigen HLA antibody testing, autoantibody panels, donor-specific antibody screening, and cytokine profiling run from one small sample volume.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Antinuclear antibodies

Antinuclear antibodies target nuclear components, including dsDNA, histones, nucleosomes, and centromere proteins. Extractable nuclear antigen panels include Sm, RNP, SS-A/Ro, SS-B/La, and other named specificities. Some panels also include anti-Jo-1, whose target is cytoplasmic. ANA is present in most patients with systemic lupus erythematosus and also occurs in other autoimmune diseases, chronic infections, some cancers, pregnancy, and healthy people. Prevalence in healthy populations rises with age and with lower screening dilutions, so a percentage applies only to the stated method, cutoff, and population. A positive ANA is therefore a sensitive screen with limited specificity.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Three assay families are used for clinical cytokine profiling for autoimmune disease, infection, allergy, and inflammatory states. Multiplexed ELISAs and microarrays measure several cytokines from one sample, each in its own well or array spot with paired controls. Microbead assays measure up to about 100 analytes per tube with colored, antigen- or antibody-coated beads read by flow cytometry, using the multiplex principle described above. Measured analytes include acute-phase reactants such as CRP, Th1 and Th2 discriminators such as IFN-gamma, IL-2, IL-4, IL-5, and IL-10, and colony-stimulating factors.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

ELISpot counts individual cytokine-secreting cells. The other two families measure total cytokine mass. Peripheral blood mononuclear cells isolated by gradient centrifugation are plated on capture-antibody-coated wells and stimulated with antigen. A sandwich-format detection step then leaves a countable spot of captured cytokine directly beneath each responding cell. T-SPOT.TB applies this principle to M. tuberculosis exposure, as described in Syphilis, Lyme, and Tuberculosis Serology.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Confirmatory testing after screening

Screening and supplemental assays have different purposes, so the laboratory follows the validated algorithm for that analyte. Several infectious-disease algorithms use a different method or antigen set after a reactive screen: HIV screening reflexes to antibody differentiation, syphilis reflexes across the nontreponemal (lipoidal antigen) and treponemal divide, and Lyme reflexes from a first-tier immunoassay to an immunoblot or paired EIA. A solid-phase anti-dsDNA result may be followed by Crithidia luciliae immunofluorescence when the laboratory's algorithm calls for greater specificity.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Complement interpretation

CH50 and AH50 pathway screens, their preanalytic requirements, and the follow-up of abnormal screens with component-specific antigenic and functional testing are covered in Antigen-Antibody Interactions and Complement. Loss of one C1 subcomponent can leave the other two antigenically present with no C1 function, and C2 antigen can be present without function, so antigenic measurement alone cannot exclude a functional defect.^{11 Abbas AK, et al. Cellular and Molecular Immunology. 2025.}

Immunodeficiency confirmatory testing

When screening or clinical findings suggest immunodeficiency, confirmatory tests narrow the defect to a compartment: humoral defects prompt B-cell counts by flow cytometry and in vitro B-cell proliferation testing; cell-mediated defects prompt T-cell subset counts, lymphocyte proliferation to mitogens and recall antigens, and adenosine deaminase and purine-nucleoside phosphorylase enzyme assays; phagocyte defects prompt adhesion-molecule analysis (CD11a/b/c, CD18), phagocytosis and killing assays, chemotaxis, and myeloperoxidase, G6PD, and NADPH-oxidase component assays; complement defects are resolved with the component assays described in the Antigen-Antibody Interactions and Complement lesson.^{1111 Orange JS, et al. 2025 inborn errors of immunity practice parameter. 2026.}

Flow cytometry is central to that workup: fluorescent antibodies against CD markers on peripheral blood mononuclear cells after red-cell lysis identify the reduced CD3 population of partial DiGeorge syndrome, the profound T-cell deficiency of complete athymia, and markedly reduced B cells in classic BTK deficiency. Secondary immunodeficiency complicates interpretation. An absent B-cell result occurs after rituximab, an anti-CD20 monoclonal that depletes CD19-positive and CD20-positive B cells, so review the medication history before genetic testing is pursued. Interferon-gamma release assay (IGRA) platforms measure M. tuberculosis-specific responses and are not general cellular-immunity tests, and commercial ATP-release assays used in some transplant settings are not validated substitutes for standard inborn-error-of-immunity functional testing. Molecular testing is recommended for suspected SCID and many inborn errors of immunity. The laboratory reports urgent screening findings and identifies molecular confirmation as pending so the clinical immunology service can act under its protocol.^{1111 Orange JS, et al. 2025 inborn errors of immunity practice parameter. 2026.}

General principles of serologic diagnosis

Serology detects the host response, so timing and interpretation are specific to the infection and assay. No single IgM timeline or paired-titer rule applies to all infections. For Lyme disease, early antibody testing can be negative and IgM results after more than 30 days of illness are disregarded. For EBV, viral capsid antigen (VCA) IgM with absent Epstein-Barr nuclear antigen (EBNA) antibody supports primary infection. Paired acute and convalescent sera usually add little. For hepatitis C, a reactive antibody result requires RNA testing to establish current infection.^{12-1412 Centers for Disease Control and Prevention. Clinical testing and diagnosis for Lyme disease.14 Centers for Disease Control and Prevention. Clinical screening and diagnosis for hepatitis C.}

Each report identifies the test performed, its result, any incomplete supplemental testing, and the relevant timing limitation, so the reader knows the limits of the result. The infection-specific sequences are covered in Syphilis, Lyme, and Tuberculosis Serology, Viral Infectious Disease Serology, and HIV Testing and Monitoring.