Immunopathology & IHC Markers
1. What this chapter covers, and how NEET PG actually tests it
Immunopathology and immunohistochemistry look like two unrelated lists, and both are usually learned as lists. Neither needs to be.
Each is governed by one rule, and the rule does the work that memorisation otherwise would.
Rule one: timing names the mechanism. Each immune effector arm operates at a fixed characteristic speed, so how long a reaction takes to appear identifies which arm produced it.
Rule two: a tumour keeps the markers of the cell it came from. Malignant transformation changes growth control, not lineage identity, so the marker panel reads backwards to the tissue of origin.
| Area | What the exam asks | The clue given |
|---|---|---|
| Hypersensitivity | Which type | Time from exposure to reaction |
| Autoantibodies | Which disease | A named antibody |
| Immunodeficiency | Which arm failed | Organism type and age at onset |
| Transplant rejection | Which type | Time from transplantation |
| Complement | Which pathway | Which components are low |
| Immunohistochemistry | Tissue of origin | A marker panel |
2. Hypersensitivity: timing names the mechanism
2.1 The four types and their speeds
| Type | Mechanism | Timing | Example |
|---|---|---|---|
| I | IgE on mast cells | Minutes | Anaphylaxis, atopy |
| II | Antibody against fixed tissue antigen | Hours to days | Autoimmune haemolysis, Goodpasture |
| III | Circulating immune complexes deposit | Days to weeks | Serum sickness, lupus nephritis |
| IV | T cell mediated | 48 to 72 hours | Tuberculin test, contact dermatitis |
Type I is measured in minutes because the mediators are preformed. Histamine sits ready in mast cell granules and needs only cross-linking of IgE to be released.
The late phase at four to eight hours reflects newly synthesised leukotrienes and recruited eosinophils, which is why antihistamines control the immediate phase but not the late one.
Type IV takes two to three days because T cells must traffic, recognise antigen and recruit macrophages, and no antibody is involved at all.
That absence of antibody is why type IV reactions cannot be transferred with serum, only with cells.
2.2 Separating type II from type III
Both involve antibody, so timing alone is insufficient. The location of the antigen separates them.
Type II antibody binds an antigen that is already fixed in the tissue; type III antibody binds a soluble antigen in the circulation, and the resulting complex then deposits.
Immunofluorescence shows the difference directly: type II gives a smooth linear pattern along the basement membrane, type III gives a granular or lumpy-bumpy pattern.
Goodpasture syndrome is the standard type II example, with linear staining against the alpha-3 chain of type IV collagen.
Post-streptococcal glomerulonephritis is the standard type III example, with granular subepithelial humps.
Type III damage occurs wherever complexes lodge, which is why serum sickness produces fever, arthralgia, rash and glomerulonephritis together.
2.3 The T-helper subsets that decide which arm responds
Which hypersensitivity type a person develops depends on which T-helper subset dominates, and each subset is defined by the cytokines it makes.
T-helper 1 cells make interferon-gamma and drive macrophage activation, granulomas and type IV reactions.
T-helper 2 cells make interleukin-4, interleukin-5 and interleukin-13, and drive IgE class switching, eosinophils and type I reactions.
The two subsets cross-inhibit each other, which is why a strong T-helper 1 response suppresses allergy and why the balance is central to the hygiene hypothesis.
T-helper 17 cells make interleukin-17 and recruit neutrophils, and their failure explains the chronic mucocutaneous candidiasis seen in hyper-IgE syndrome.
Regulatory T cells express FOXP3 and suppress the others; FOXP3 mutation causes IPEX syndrome, a severe multi-organ autoimmunity of infancy.
Interleukin-12 from macrophages is what drives naive T cells towards the T-helper 1 phenotype, which links this section to granuloma formation.
3. Autoimmunity and the antibodies
3.1 How tolerance fails
Autoimmunity is a failure of tolerance, and there are only a few ways tolerance can break.
Central tolerance deletes self-reactive lymphocytes in the thymus and marrow, and depends on the AIRE gene, which lets thymic epithelium display peripheral tissue antigens.
AIRE mutation causes autoimmune polyendocrine syndrome type 1, which is the cleanest demonstration that central tolerance is an active process rather than a passive absence of self-antigen.
Peripheral tolerance depends on regulatory T cells, on anergy when a T cell meets antigen without costimulation, and on activation-induced cell death.
Molecular mimicry explains rheumatic fever, where antibody against streptococcal M protein cross-reacts with cardiac myosin.
Release of sequestered antigen explains sympathetic ophthalmia, in which trauma to one eye exposes antigens the immune system never learned to tolerate and the other eye is attacked.
3.2 Sensitivity and specificity are the real question
Autoantibody questions almost always turn on whether an antibody is a screening test or a confirmatory one.
Antinuclear antibody is sensitive but not specific, so a negative result largely excludes lupus while a positive result proves little.
Anti-double-stranded DNA and anti-Smith are specific for lupus; anti-double-stranded DNA also tracks disease activity and nephritis.
Anti-histone antibody indicates drug-induced lupus, classically from hydralazine, procainamide or isoniazid, which characteristically spares the kidney.
3.3 The antibody table worth knowing cold
| Antibody | Disease |
|---|---|
| Anti-dsDNA, anti-Smith | Systemic lupus erythematosus |
| Anti-histone | Drug-induced lupus |
| Anti-centromere | Limited scleroderma (CREST) |
| Anti-Scl-70 (topoisomerase I) | Diffuse scleroderma |
| Anti-Ro and anti-La | Sjogren syndrome, neonatal lupus |
| Anti-Jo-1 | Polymyositis with interstitial lung disease |
| Anti-mitochondrial | Primary biliary cholangitis |
| Anti-smooth muscle | Autoimmune hepatitis type 1 |
| Anti-transglutaminase, anti-endomysial | Coeliac disease |
| Anti-cyclic citrullinated peptide | Rheumatoid arthritis, specific |
| c-ANCA (anti-proteinase 3) | Granulomatosis with polyangiitis |
| p-ANCA (anti-myeloperoxidase) | Microscopic polyangiitis, eosinophilic granulomatosis |
Anti-Ro crosses the placenta and causes congenital heart block, which is why it is checked in pregnancy planning for connective tissue disease.
Anti-cyclic citrullinated peptide is more specific than rheumatoid factor and appears earlier, so it is preferred for both diagnosis and prognosis.
3.4 HLA associations
Human leukocyte antigen associations are pure recall, but they are cheap marks and appear reliably.
| HLA allele | Association |
|---|---|
| B27 | Ankylosing spondylitis, reactive arthritis, psoriatic arthritis, inflammatory bowel disease arthropathy |
| B57:01 | Abacavir hypersensitivity |
| B15:02 | Carbamazepine-induced Stevens-Johnson syndrome in South Asians |
| DQ2 and DQ8 | Coeliac disease |
| DR3 and DR4 | Type 1 diabetes mellitus |
| DR2 | Multiple sclerosis, Goodpasture syndrome |
| DR4 | Rheumatoid arthritis |
| A3 | Hereditary haemochromatosis |
Class I molecules are HLA-A, B and C and present to CD8 cells; class II are DP, DQ and DR and present to CD4 cells.
The B57:01 and B15:02 associations are the two with direct prescribing consequences, since testing before abacavir and before carbamazepine is now standard practice in the relevant populations.
4. Immunodeficiency: the organism names the arm
4.1 Reading the infection pattern backwards
Which organisms cause trouble tells you which arm has failed, because each arm defends against a different class of pathogen.
Antibody deficiency gives recurrent encapsulated bacterial infection — pneumococcus, Haemophilus, Neisseria — beginning around six months as maternal IgG wanes.
T cell deficiency gives viral, fungal and opportunistic infection from birth, since maternal antibody offers no protection against these.
Phagocyte defects give catalase-positive bacterial and fungal infection, with abscesses and poor wound healing.
Complement deficiency of the terminal components gives recurrent neisserial infection specifically, because the membrane attack complex is what kills Neisseria.
4.2 The named conditions
Bruton agammaglobulinaemia is X-linked, caused by a BTK tyrosine kinase defect, with absent B cells and absent tonsils.
Selective IgA deficiency is the commonest primary immunodeficiency, often asymptomatic, and matters chiefly because of anaphylaxis to blood products containing IgA.
DiGeorge syndrome is a 22q11 deletion with thymic and parathyroid aplasia, giving T cell deficiency with hypocalcaemic tetany and conotruncal cardiac anomalies.
Severe combined immunodeficiency involves both arms, presents in infancy with failure to thrive and persistent candidiasis, and is fatal without transplantation.
Wiskott-Aldrich syndrome is X-linked with eczema, thrombocytopenia with small platelets, and recurrent infection.
Ataxia-telangiectasia combines a DNA repair defect with cerebellar ataxia, oculocutaneous telangiectasia and raised alpha-fetoprotein.
Hyper-IgM syndrome is a CD40 ligand defect, so class switching fails: IgM is normal or high while IgG, IgA and IgE are low.
The pattern of immunoglobulin classes therefore identifies hyper-IgM syndrome without any genetic test.
4.3 Complement
The classical pathway is activated by antibody, the alternative pathway by microbial surfaces, and the lectin pathway by mannose.
C3 is the convergence point of all three, so a low C3 with a normal C4 suggests alternative pathway activation.
Hereditary angioedema is C1 esterase inhibitor deficiency, with non-pruritic angioedema and no urticaria, and does not respond to antihistamines or adrenaline in the way an allergic reaction does.
Paroxysmal nocturnal haemoglobinuria is a GPI anchor defect leaving cells without CD55 and CD59, so complement lyses them; flow cytometry has replaced the Ham test.
5. Transplant rejection: timing again
| Type | Timing | Mechanism |
|---|---|---|
| Hyperacute | Minutes | Preformed recipient antibodies |
| Acute cellular | Days to months | T cell mediated |
| Acute humoral | Days to months | Donor-specific antibodies, C4d staining |
| Chronic | Months to years | Vascular intimal fibrosis |
Hyperacute rejection occurs on the operating table because the antibodies are already present and need no time to be generated, and cross-matching exists specifically to prevent it.
Acute cellular rejection shows a lymphocytic infiltrate and responds to increased immunosuppression.
Acute humoral rejection is identified by C4d deposition in peritubular capillaries, which is a footprint of complement activation by donor-specific antibody.
Chronic rejection is the one form that does not respond to immunosuppression, because the damage is established fibrous narrowing rather than active inflammation.
Graft-versus-host disease is the reverse situation, in which donor T cells attack the recipient, and classically targets skin, gut and liver.
It requires an immunocompetent graft and an immunocompromised host, which is why it complicates bone marrow rather than solid organ transplantation.
Irradiating blood products before transfusing severely immunosuppressed patients exists specifically to prevent transfusion-associated graft-versus-host disease.
6. Immunohistochemistry: lineage is retained
6.1 Why the panel works
A malignant cell loses growth control but keeps the differentiation programme of its parent tissue.
So the marker profile of a metastasis of unknown origin points back to where it started, which is often what determines treatment.
| Marker | Indicates |
|---|---|
| Cytokeratin | Epithelial (carcinoma) |
| Vimentin | Mesenchymal (sarcoma) |
| Desmin | Muscle |
| Smooth muscle actin | Smooth muscle |
| S-100 | Melanoma, Schwannoma, Langerhans cells |
| HMB-45, Melan-A | Melanoma, more specific than S-100 |
| Leukocyte common antigen (CD45) | Lymphoma |
| Chromogranin, synaptophysin | Neuroendocrine |
| Thyroid transcription factor 1 | Lung adenocarcinoma, thyroid |
| CDX2 | Colorectal |
| PSA | Prostate |
| GFAP | Glial |
| CD117 (c-KIT) | Gastrointestinal stromal tumour |
| CD31, factor VIII | Vascular endothelium |
| Calretinin | Mesothelioma |
| Alpha-fetoprotein | Hepatocellular, yolk sac tumour |
6.2 The panels that decide management
Breast cancer is classified by oestrogen receptor, progesterone receptor and HER2, which determine whether endocrine therapy, trastuzumab or neither is used.
Triple-negative disease lacks all three and therefore has no targeted option, which is why it carries a worse prognosis.
CD117 positivity in a gastric submucosal tumour identifies a gastrointestinal stromal tumour and makes imatinib the treatment, a direct link from an immunohistochemical stain to a drug.
Calretinin distinguishes mesothelioma from adenocarcinoma in a pleural biopsy, a distinction with major medicolegal as well as clinical consequences.
The lymphoma markers follow B and T lineage: CD19 and CD20 for B cells, CD3 for T cells, CD15 and CD30 for Reed-Sternberg cells.
6.3 Serum tumour markers, and what they are actually for
Serum markers are examined alongside immunohistochemistry, and the recurring point is that almost none of them is a diagnostic test.
Their real value is monitoring treatment response and detecting recurrence, because a marker that falls and then rises again is far more informative than a single value.
| Marker | Tumour | Note |
|---|---|---|
| Alpha-fetoprotein | Hepatocellular, yolk sac, non-seminomatous germ cell | Also raised in neural tube defects |
| Beta-hCG | Choriocarcinoma, germ cell tumours | Raised in normal pregnancy |
| CA-125 | Ovarian epithelial | Raised in endometriosis, pregnancy, cirrhosis |
| CA 19-9 | Pancreatic | Negative in Lewis antigen-negative individuals |
| CEA | Colorectal | Used for recurrence, not screening |
| PSA | Prostate | Raised in benign hyperplasia and prostatitis |
| Calcitonin | Medullary thyroid carcinoma | Also a screening test in MEN 2 families |
| Chromogranin A | Neuroendocrine tumours | Falsely raised by proton pump inhibitors |
A pure seminoma does not raise alpha-fetoprotein, so a raised value in a testicular tumour reported as seminoma means a non-seminomatous element has been missed, and management changes accordingly.
Calcitonin is the exception to the monitoring rule, since it is genuinely used to screen at-risk relatives in multiple endocrine neoplasia type 2.
7. Worked examples
Example 1
A patient develops fever, urticaria, arthralgia and haematuria ten days after receiving antivenom.
The timing is the first thing to use. Ten days is far too slow for a type I reaction and too fast for a chronic process.
Ten to fourteen days is precisely the interval needed to mount a primary antibody response against a foreign protein, after which antigen is still present and complexes form.
Multi-system involvement — joints, skin and kidney together — is the signature of circulating complexes depositing wherever they lodge.
The diagnosis is serum sickness, a type III hypersensitivity reaction, and complement levels will be low from consumption.
Example 2
A biopsy from a supraclavicular node shows a metastatic carcinoma. It stains positive for cytokeratin and thyroid transcription factor 1, and negative for CDX2 and PSA.
Cytokeratin positivity establishes that this is a carcinoma rather than a sarcoma or lymphoma.
Thyroid transcription factor 1 narrows the origin to lung or thyroid, and the negative CDX2 and PSA exclude colorectal and prostate primaries.
In a supraclavicular node the practical answer is lung adenocarcinoma, and thyroglobulin staining would be added to exclude a thyroid primary.
The panel has converted an undifferentiated deposit into a treatable diagnosis without any imaging.
Example 3
A 9-month-old boy has had three episodes of pneumococcal pneumonia. Tonsils are absent. Serum immunoglobulins of all classes are very low, and B cells are absent from the blood.
Encapsulated organisms and onset at around six months point to an antibody problem, since maternal IgG protects until then.
Absent tonsils indicate absent B cell follicles, meaning the defect is in B cell development rather than in antibody production by existing cells.
The diagnosis is Bruton X-linked agammaglobulinaemia, caused by a Bruton tyrosine kinase defect that arrests B cell maturation.
Hyper-IgM syndrome would show normal or raised IgM with present B cells, and common variable immunodeficiency presents far later.
8. Traps the exam sets repeatedly
Treating a positive antinuclear antibody as diagnostic of lupus. It is a sensitive screening test with poor specificity, and anti-double-stranded DNA or anti-Smith is needed to confirm.
Confusing linear with granular immunofluorescence. Linear means antibody against a fixed tissue antigen and therefore type II; granular means deposited immune complexes and therefore type III.
Giving adrenaline and antihistamines for hereditary angioedema. The mechanism is bradykinin from C1 esterase inhibitor deficiency, not histamine, so the response is poor and specific therapy is required.
Assuming S-100 confirms melanoma. S-100 also marks Schwannoma and Langerhans cell histiocytosis, so HMB-45 or Melan-A is needed for specificity.
Forgetting that IgA deficiency matters for transfusion. Patients with anti-IgA antibodies can have anaphylaxis to plasma-containing products, and require washed cells.
Summary
Two rules carry the chapter: timing names the immune mechanism, and a tumour keeps the markers of its parent cell.
Type I hypersensitivity takes minutes because mediators are preformed; type IV takes two to three days because T cells must traffic and recruit.
Type II and type III both involve antibody and are separated by whether the antigen is fixed in tissue or circulating, seen directly as linear versus granular immunofluorescence.
Antinuclear antibody screens for lupus; anti-double-stranded DNA and anti-Smith confirm it, and anti-histone indicates the drug-induced form.
The organism pattern identifies the failed arm: encapsulated bacteria for antibody, opportunists for T cells, catalase-positive organisms for phagocytes, Neisseria for terminal complement.
Hyper-IgM syndrome is recognised from the immunoglobulin pattern alone, since class switching fails while IgM production does not.
Transplant rejection is classified by timing, with hyperacute rejection occurring within minutes because the antibodies already exist.
Immunohistochemistry reads a metastasis backwards to its origin, and some markers decide treatment directly, as CD117 does for gastrointestinal stromal tumours.
