Comprehensive Notes on Serological Testing and Clinical Immunology

Overview of Serology

• Definition: Serology is the branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen-antibody reactions in vitro.

• Serological Tests: These are blood tests that look for antibodies in the blood via antigen-antibody reactions. They involve various laboratory techniques and are used to diagnose numerous disease conditions.

Anti-streptolysin O (ASO or ASLO)

• Definition: ASO is the antibody produced against streptolysin O, which is an immunogenic, oxygen-labile streptococcal hemolytic exotoxin.

• Origin: Produced by most strains of group A and many strains of groups C and G Streptococcus bacteria.

• Terminology: The "O" in the name represents "oxygen-labile." A related toxin, streptolysin-S, is oxygen-stable.

• Primary Function of Streptolysin O: Its main function is to cause hemolysis (the breaking open of red blood cells), specifically beta-hemolysis.

• Clinical Significance:

  • Increased levels of ASO titer in the blood can cause damage to the heart and joints.

  • Elevated levels are typically treated with penicillin.

  • ASO titer (ASOT) assists in the diagnosis of scarlet fever, rheumatic fever, and post-infectious glomerulonephritis.

  • Antibodies are produced as a delayed reaction; thus, presence indicates exposure, but as many people are asymptomatic, presence alone does not automatically indicate disease.

• Test Interpretation:

  • Positive Result: Generally defined as greater than $200\,units/mL$.

  • Normal Ranges: Vary by laboratory and age.

  • Acceptable values (no clinical suspicion):

    • Adults: less than $200\,units$

    • Children: less than $400\,units$

  • Clinical Correlation: Significance is found if the titer is greatly elevated (>200) or if a rise is demonstrated in paired blood samples taken days apart.

  • Timeline: Levels begin to rise after $1-3\,weeks$ of infection, peak in $3-5\,weeks$, and fall to insignificant levels within $6\,months$.

• Limitations:

  • False Negatives: Rate of $20-30\%$. If a false negative is suspected, an anti-DNase B titer should be sought.

  • False Positives: Can result from liver disease and tuberculosis.

• Estimation Methods:

  • Latex agglutination or slide agglutination.

  • ELISA (used for detecting exact titer values).

  • Non-ELISA titer detection requires serial dilution techniques.

• Mechanism of Action: Antibodies produced against the bacteria cross-react with human antigens (mainly collagen), attacking the cellular matrix of the heart, joints, skin, and brain.

• ASO Latex Test:

  • Agglutination occurs when the latex reagent is mixed with serum containing ASO.

  • The sensitivity is adjusted to yield agglutination when ASO levels are greater than $200\,IU/ml$.

• Sample Handling:

  • Only fresh serum specimens should be used.

  • Plasma is prohibited because fibrinogen may cause non-specific agglutination.

  • Samples may be stored at $2-8^\circ C$ for up to $48\,hours$. For longer periods, serum should be stored at $-20^\circ C$.

• Test Components:

  1. ASO Antigen: A stabilized buffered suspension of polystyrene latex particles coated with Streptolysin O ($0.1\%$ sodium azide as preservative).

  2. ASO Positive Control: Human serum with more than $200\,IU/ml$ ASO.

  3. ASO Negative Control: Human serum containing $0.1\%$ sodium azide.

  4. Disposable pipettes and glass test slides.

• ASO Test Procedure:

  1. Bring reagents and samples to room temperature.

  2. Place one drop of undiluted sample into a slide circle using a pipette.

  3. Deliver one drop of positive and negative controls into identified circles.

  4. Mix ASO latex reagent by shaking; add one drop to each control and sample.

  5. Mix each with the reagent using the flat end of a plastic pipette/stirrer across the full area of the circle.

  6. Rock the slide slowly for exactly $2\,minutes$; observe for agglutination under high-intensity light.

  7. Result is considered positive (excess of $200\,IU/ml$) if clumping is observed compared to the negative control.

Rheumatoid Factor (RF)

• Definition: An autoantibody directed against the organism's own tissues. Historically first found in rheumatoid arthritis.

• Target: RF is an antibody against the $Fc$ portion of $IgG$

• Isotypes: Predominantly encountered as $IgM$, but can be $IgA$, $IgG$, $IgM$, $IgE$, or $IgD$.

• Mechanism: RF and $IgG$ join to form immune complexes that contribute to the disease process.

• Testing and Sensitivity:

  • Sensitivity for established rheumatoid arthritis is $60-70\%$.

  • Specificity is $78\%$.

  • $80\%$ of patients who are initially seronegative will eventually undergo seroconversion.

• Interpretation:

  • High levels are defined generally as above $20\,IU/mL$, $1:40$, or over the $95th$ percentile.

  • Found in $80\%$ of rheumatoid arthritis cases and $70\%$ of Sjögren's syndrome cases.

  • Higher levels correlate with greater destructive articular disease and disability.

  • Can be found in $5-10\%$ of healthy persons, especially the elderly.

• Other Associated Conditions: Systemic lupus erythematosus (SLE), interstitial pulmonary fibrosis, Hepatitis B, chronic liver disease, Essential mixed cryoglobulinemia, Primary biliary cirrhosis, Infectious mononucleosis, Bacterial endocarditis, Leprosy, Sarcoidosis, Tuberculosis, Syphilis, Visceral leishmaniasis, Malaria, Leukemia, Dermatomyositis, Systemic sclerosis, and post-vaccination/transfusion in healthy individuals.

Widal Test

• History: Developed in 1896 by Georges-Fernand Widal.

• Purpose: A presumptive serological test for enteric fever or undulant fever (Typhoid fever).

• Mechanism: Bacteria causing typhoid fever are mixed with serum containing specific antibodies from an infected individual.

• Diagnostic Timeline: Antibody levels take $7-14\,days$ to rise, limiting early diagnosis applicability.

• Alternative Diagnostics: Cultures of blood, urine, and feces. Salmonella typhi and paratyphi produce $H_2S$ from thiosulfate and can be identified on bismuth sulfite agar.

• Widal Test Kit Antigens:

  • $S. enterica$ serotype Typhi $O$ antigen.

  • $S. enterica$ serotype Typhi $H$ antigen.

  • $S. enterica$ serotype Paratyphi $AH$ antigen.

  • $S. enterica$ serotype Paratyphi $BH$ antigen.

• Interpretation Guidelines:

  • Active Infection: $TO$ antigen titer is more than $1:160$.

  • Past Infection/Immunization: $TH$ antigen titer is more than $1:160$.

  • Diagnostic Requirement: A fourfold increase in titer (e.g., $1:40$ to $1:640$) during infection or conversion from $IgM$ to $IgG$ is consistent with infection.

• Agglutinin Response Timeline:

  • O-antibody: Earliest response; appears $6-8\,days$ after onset. Concentrations fall $6\,months$ after exposure.

  • H-antibody: Develops slower ($10-12\,days$ after onset) but persists longer. Some studies find $O$ titers of greater diagnostic significance as $H$ agglutinins can rise in response to other infections.

Rose Bengal Plate Test (RBT) for Brucella

• Types of Brucella causing human infection:

  1. B. melitensis: Found in sheep/goats; causes most human cases (Middle East, Spain, Greece, Latin America, India).

  2. B. suis: Found in wild pigs; common in the United States, Europe, and South America.

  3. B. canis: Spread from dogs; found in the Americas, Japan, and Central Europe.

  4. B. abortus: From cattle; occurs worldwide (wiped out in parts of Europe, Japan, Canada, Australia).

• Methodology: A rapid slide-type agglutination assay using a stained $B. abortus$ suspension at $pH\,3.6-3.7$ and plain serum.

• Procedure:

  1. Mix $0.03\,ml$ of test serum with an equal volume of antigen on a white tile to produce a $2\,cm$ diameter zone.

  2. Agitate for $4\,minutes$ at ambient temperature.

  3. Any visible reaction is positive.

• Limitations:

  • Low sensitivity in chronic cases and low specificity in endemic areas.

  • Strongly positive sera may appear negative due to the Prozone phenomenon.

  • Positive results should be confirmed via Complement fixation test (CFT) or ELISA.

Prozone Phenomenon

• Definition: A zone of relatively high antibody concentrations within which no agglutination or precipitation occurs.

• Reasoning: Caused by antibody excess, blocking antibodies, or non-specific inhibitors in the serum.

• Resolution: Reaction occurs as the antibody concentration is lowered below the prozone.

C-reactive Protein (CRP)

• Definition: A protein produced by the liver in response to inflammatory cytokines like Interleukin-6 ($IL-6$). Classified as an acute phase reactant.

• Timeline: Levels rise within a few hours of tissue injury, infection, or inflammation.

• Biological Role: Interaction with the complement system.

• Clinical Utility:

  • Post-surgery: Levels normally increase in $2-6\,hours$ and return to normal by the third day. Elevated levels on day 3 indicate infection.

  • Monitoring: Used for Inflammatory Bowel Disease (IBD), lymphoma, SLE, rheumatoid arthritis, and osteomyelitis.

  • Treatment efficacy: Levels drop quickly if treatment (e.g., for cancer or infection) is successful.

  • Differentiation: Extremely elevated CRP suggests bacterial rather than viral infection.

• Method: Based on the 1957 Singer latex agglutination method. Uses latex particles coated with goat $IgG$ anti-human CRP.

• Qualitative Procedure:

  1. Bring reagents/serum to room temperature.

  2. Place 1 drop of serum, positive control, and negative control on separate circles.

  3. Add 1 drop of CRP latex reagent each.

  4. Mix and spread over the cell area and tilt the slide for $2\,minutes$.

• Semi-quantitative Procedure: Perform serial two-fold dilutions of sample in $9\,g/L$ saline. The titer is the highest dilution showing a positive result.

• References and Notes:

  • Reference range: $0-10\,mg/L$.

  • High concentrations may yield false negatives (antigen excess); negative sera should be retested at a $1:10$ dilution.

  • Reaction time is critical; exceeding $2\,minutes$ causes drying and false positives.

  • Freezing the reagent causes spontaneous agglutination.

Human Chorionic Gonadotropin (hCG)

• Definition: A glycoprotein hormone secreted by the developing placenta.

• Timeline: Detected as early as $6-7\,days$ after conception. At the first missed period, concentration is roughly $100\,IU/ml$, peaking at the end of the first trimester.

• Biological Function: Maintains fetal viability, prevents disintegration of the ovary's corpus luteum, maintains progesterone production, and affects immune tolerance.

• Tumor Marker: hCG is produced by seminoma, choriocarcinoma, germ cell tumors, teratoma, and islet cell tumors.

• Latex Slide Test:

  • Detects hCG at levels of $0.3\,IU/ml$ and higher.

  • Uses monoclonal antibodies.

  • Specimen: First morning urine contains the highest concentration.

• hCG Procedure:

  1. Place 1 drop of urine on a slide circle.

  2. Add 1 drop of Latex Reagent (anti-hCG monoclonal antibody-coated particles).

  3. Stir and rock for $2\,minutes$.

  4. Agglutination within $2\,minutes$ is positive. Do not interpret after $3\,minutes$.

• Limitations: Specimens must be urine only; concentrations below $0.3\,IU/ml$ are negative. Trophoblastic neoplasms (hydatidiform mole) must be ruled out.

Syphilis Serological Testing

Treponema pallidum Hemagglutination Assay (TPHA)

• Purpose: Treponemal test for syphilis caused by the spirochete $Treponema pallidum$.

• Principle: Passive hemagglutination using avian erythrocytes sensitized with $T. pallidum$ antigen (Nichols strain).

• Mechanism: Serum is diluted in absorbing diluent to remove cross-reacting heterophile/nonpathogenic antibodies. Positive reaction forms a smooth mat of agglutinated cells; negative reaction forms a compact button.

• Similar Tests:

  • TP-PA: Uses gelatin particles; higher sensitivity in primary stage.

  • MHA-TP: Confirmatory test (less common now).

Venereal Disease Research Laboratory (VDRL) Test

• Purpose: Non-specific flocculation test for population screening.

• Specimen: Serum (must be heat-inactivated to destroy complement) or CSF. Plasma cannot be used.

• Antigen: Cardiolipin-Cholesterol-Lecithin detects "reagin antibodies" ($IgM$ and $IgG$ against lipoidal material from damaged host cells and cardiolipin from treponemes).

• Observation: Using a microscope with $10x$ objective and $10x$ eyepiece.

• False Results:

  • False Positives: Leprosy, Hepatitis B, Infectious Mononucleosis, autoimmune diseases.

  • False Negatives: Prozone phenomenon (antibody excess).

Rapid Plasma Reagin (RPR) Test

• Terminology: "Rapid" (done in minutes), "Plasma" (the sample type, though serum is also usable), "Reagin" (antibodies against substances from damaged cells).

• Antigen Composition: Modified VDRL antigen with choline chloride and $EDTA$ (for stability) and charcoal particles (as visualizing agents).

• Mechanism: Charcoal co-agglutinates with antibodies forming black clumps on white cards. Negative result is a uniform gray color.

• Qualitative Procedure:

  1. Place $50\,\mu l$ of specimen on an $18\,mm$ card circle.

  2. Add $17\,\mu l$ of antigen suspension.

  3. Rotate card for $4-8\,min$ and observe for flocculation.

Questions & Discussion

• Is plasma usable in VDRL? No, only serum or CSF is used.

• What defines the titer in semi-quantitative CRP tests? The highest dilution showing a positive result.

• Why is the acidity for Rose Bengal specified? The test is performed with antigen at $pH\,3.6-3.7$.

• What happens if CRP reagent is frozen? It results in spontaneous agglutination.

• What is the significance of the "O" in ASO? It stands for oxygen-labile, differentiating it from the oxygen-stable streptolysin-S.