IS LAB

MLS 412 Immunology & Serology — High-Yield Exam Reviewer

EVACUATED TUBE SYSTEM (ETS)

Vein Priority: Median Cubital (1st) → Cephalic (2nd) → Basilic (3rd/last resort, near basilic artery)

Key Steps to Remember:

• Tourniquet: <1 min to avoid hemoconcentration (darker blood)

• Release tourniquet when backflow is established

• Bevel facing up on needle insertion

• Mix additive tubes: 3–8 gentle inversions

• Label tubes before leaving bedside

• Discard needle + holder as a unit into sharps container (red)

Order of Draw: Blood cultures → Light Blue (SPS) → Red → SST → PST → Green → Lavender/EDTA → Gray Mnemonic: "Be careful, Stop Light, Red, Stay, Put, Green, LEts, Go"

Special Handling:

• Ammonia → crushed ice

• Cold agglutinin → body temperature

• Bilirubin → protect from light (photosensitive)

Dietary Requirements:

• FBS: 8–10 hrs

• Lipid Profile: 10–12 hrs

• FBS + LP: 10 hrs

SERUM DILUTION

Formula: Dilution = Volume of Solute / Total Volume

Simple Dilution: single step reduction

• Dilution factor = denominator (whole number)

• Example: 1:20 dilution of 2 mL → Serum = 0.1 mL; Diluent = 1.9 mL

Serial Dilution: stepwise, consistent factor each step

• Most common: two-fold (doubling) dilution

• Used to determine titer (strength of antibody)

• Final dilution = product of all individual dilutions

BASIC SEROLOGICAL PRINCIPLES

Serology: identifies/measures antigens, antibodies using body fluids (mainly serum)

Key Terms:

• Affinity: strength of ONE antibody site + ONE epitope

• Avidity: OVERALL strength of multiple binding sites (IgM has highest avidity — 10 binding sites)

• Cross-reactivity: antibody binds structurally similar antigen (e.g., cowpox/smallpox)

• Paratope: antibody determinant (binds antigen)

• Epitope: antigen determinant (binds antibody)

• Sensitization: Ag+Ab bind, NO visible reaction (Step 1)

• Lattice formation: visible agglutination (Step 2)

• Titer: highest dilution showing POSITIVE result

• Seroconversion: antibodies start to APPEAR

• Seroreversion: antibodies start to DISAPPEAR

Factors Influencing Ag-Ab Reactions:

• pH: 6.8–7.0 (near physiological)

• Temperature: IgM → 4–27°C (colder = igMalamig); IgG → 30–37°C (warmer = igGang)

• Concentration: Zone of equivalence = best reaction

  • Prozone: excess antibody → false negative

  • Postzone: excess antigen → false negative

• Centrifugation: catalyst, speeds up lattice formation

Immunoglobulin Abundance: IgG > IgA > IgM > IgD > IgE (GAMDE)

Primary vs. Secondary Immune Response:

PRECIPITATION TECHNIQUES

Precipitation: soluble antigen + soluble antibody = insoluble complex

Turbidimetry: measures REDUCTION in light intensity (detector in line with light)

Nephelometry: measures SCATTERED light at 10–90° angle; amount scattered = index of concentration

Rate Nephelometry: measures rate of increase in light scattering after antibody is added

Radial Immunodiffusion (RID):

• Single diffusion; antibody in gel, antigen diffuses

• Product: precipitation ring

• Bigger diameter = higher antigen concentration

• Mancini (Endpoint): diameter² ∝ concentration (measure after diffusion complete)

• Fahey (Kinetic): diameter ∝ log concentration (measure during diffusion)

• Mnemonic: Fahey = Kinetic; Mancini = Endpoint (James RID FAKME)

Ouchterlony Double Immunodiffusion:

• Both antigen AND antibody diffuse

• Product: precipitation lines

• Identity → smooth arc

• Nonidentity → lines cross

• Partial identity → spur formation

AGGLUTINATION

Agglutination Stages:

1. Sensitization (rapid, reversible, no visible reaction)

2. Lattice formation (visible clumping)

Types:

Hemagglutination (Blood Typing):

Forward Typing: packed RBC + known antibody → detect ANTIGEN

• Blue = Anti-A; Yellow = Anti-B; Colorless = Anti-D

Reverse Typing: patient plasma + known RBCs → detect ANTIBODY

Coagglutination: Protein A binds Fc region of IgG; antigen binds FAB region

Agglutination Inhibition:

• Successful inhibition = POSITIVE

• Unsuccessful inhibition = NEGATIVE

Hemagglutination Inhibition: absence of agglutination = antibody present = POSITIVE

COMPLEMENT FIXATION TEST

Stage 1: Antigen + Patient Serum → if antibodies present → complement is FIXED/consumed

Stage 2: Add sensitized sheep RBCs

• Positive (Ab present): complement consumed → NO hemolysis (no lysis of RBCs)

• Negative (No Ab): free complement available → HEMOLYSIS occurs

Counterintuitive: No hemolysis = POSITIVE

Heat inactivation: 56°C for 30 minutes

LABELED IMMUNOASSAYS

Competitive: labeled vs unlabeled antigen compete for limited Ab sites

• More patient antigen → less label bound → LOWER signal

• Inverse relationship: signal ↑ = antigen ↓

Noncompetitive (Sandwich): antibody excess; signal directly proportional to antigen concentration

Heterogeneous: requires separation step Homogeneous: no separation step; faster but less sensitive

ELISA Types:

• Direct: primary antibody with label

• Indirect: secondary labeled antibody

• Sandwich/Capture: antigen between two antibodies

• Competitive: labeled vs unlabeled antigen

Radioimmunoassay (RIA): uses radioactive label (¹²⁵I most popular)

• RIST: measures total serum IgE

• RAST: detects allergen-specific IgE

Immunofluorescence:

• Direct IFA: fluorescent antibody added to unknown antigen on slide; best for antigen detection

• Indirect IFA: patient serum + known antigen → anti-human Ig with fluorescent tag; best for antibody identification

C-REACTIVE PROTEIN (CRP)

Nature: pentraxin family (5 subunits); synthesized by hepatocytes; increases up to 1000-fold in inflammation

Stimulated by: IL-6 (from activated macrophages/monocytes)

Principle: Reverse Passive Agglutination

• Unknown: CRP (antigen)

• Known: latex particles coated with anti-CRP antibody (polystyrene)

• Agglutination = CRP > 6 IU/mL = POSITIVE

CRP Elevated In: viral/bacterial infections, gout, RA, myocardial infarction, acute rheumatic fever

Note: CRP does NOT spike significantly in SLE or RA (autoimmune conditions)

Specimen: red-top or gold-top tube; no fasting required

• Reject: lipemia, hemolysis, bacterial contamination

• Refrigerate: 2–8°C up to 24 hrs; freeze at –20°C longer

Qualitative (Screening): agglutination = CRP > 6 IU/mL → proceed to semi-quantitative

Semi-Quantitative Conversion: titer × 6 = CRP in mg/L

• Example: titer 1:4 → 4 × 6 = 24 mg/L

RHEUMATOID FACTOR (RF)

Disease: Rheumatoid Arthritis (RA) — systemic autoimmune disorder; destroys bone/cartilage

Key Features:

• Pannus formation (invasive granulation tissue in joints)

• Osteoclast overactivation → bone erosion

• TNF-α regulates osteoclast/osteoblast balance

Two Key Antibodies:

• RF (IgM): directed against Fc portion of IgG; not specific (also in SLE, Sjögren's, syphilis, hepatitis)

• Anti-CCP: better and more specific than RF for RA

Principle: Passive Agglutination

• Unknown: Rheumatoid Factor (antibody)

• Known: latex particles coated with human IgG (antigen)

• Agglutination = RF present

Strongest environmental risk: cigarette smoking (doubles risk)

Specimen: same as CRP (red-top, no fasting)

• Reagent color: WHITE (vs CRP = Blue; ASO = Yellow)

Semi-Quantitative: titer × 12 = RF in IU/mL (some kits use factor 20)

• Example: 1:4 → 4 × 12 = 48 IU/mL; OR 4 × 20 = 80 IU/mL

ANTISTREPTOLYSIN O (ASO)

Causative Agent: Group A Streptococcus (Streptococcus pyogenes)

Virulence Factors:

• Protein F → attachment

• Hyaluronic acid capsule → inhibits phagocytosis (mimics host)

• M protein → major virulence factor; antiphagocytic (board exam answer)

• Streptolysin O → oxygen-labile; subsurface hemolysis; more destructive

• Streptolysin S → oxygen-stable; surface hemolysis

• Streptokinase → activates plasminogen → plasmin → dissolves clots

• DNase → degrades DNA

• Hyaluronidase → "spreading factor"; breaks down connective tissue

• C5a peptidase → destroys complement chemotactic factors

• Streptococcal pyogenic exotoxins → superantigens → cytokine storm

Sequelae (complications, NOT primary infections):

• Rheumatic Fever (M protein mimics heart tissue → molecular mimicry)

• Acute Post-Streptococcal Glomerulonephritis (APSGN) → antigen-antibody complex trapped in glomeruli → hematuria

ASO Test Purpose: detect PAST streptococcal infection (not current); evidence of recent Group A infection

Titer rises: 1–2 weeks post infection; peaks 3–6 weeks after symptoms

Principle: Passive Agglutination

• Unknown: ASO antibodies (patient serum)

• Known: latex coated with stabilized Streptolysin O

• Agglutination = ASO present = POSITIVE

Qualitative: agglutination = ASO > 200 IU/mL Semi-Quantitative: titer × 200 = ASO in IU/mL

• Example: 1:4 → 4 × 200 = 800 IU/mL

False negative: hemolyzed sample (free hemoglobin neutralizes streptolysin O)

• Reagent color: YELLOW

RAPID PLASMA REAGIN (RPR) — SYPHILIS

Causative Agent: Treponema pallidum subspecies pallidum (hard chancre)

• Haemophilus ducreyi → soft chancre (painful)

Stages of Syphilis:

• Primary: single painless hard chancre

• Secondary: skin rash, mucous membrane lesions, Condyloma lata

• Latent: no symptoms (Early <12 months; Late >12 months)

• Tertiary: rare; affects brain (Neurosyphilis), eyes (Ocular), ears (Otosyphilis)

Tests for Syphilis:

Reagin: IgM and IgG antibodies against cardiolipin (non-specific)

RPR Principle: flocculation (larger than precipitation)

• Antigen: cardiolipin + charcoal particles + cholesterol + lecithin

• Unknown: reagin antibody

• Positive: black clumps (charcoal) on white card background

Choline chloride in RPR antigen → inactivates complement → no heating needed

Report as: Reactive / Non-reactive (NOT positive/negative)

• Any clumping = REACTIVE; smooth gray = NON-REACTIVE

Rotation: 8 minutes at 100 rpm (mechanical) + 3–4 to-and-fro motions (manual)

• Read in "wet" state under bright light

• Do NOT read after 20 minutes

False reactive causes: malaria, TB, pregnancy, autoimmune disorders, recent vaccination

Specimen storage: 2–8°C up to 72 hours

WIDAL TEST (TYPHOID FEVER)

Causative Agent: Salmonella enterica serotypes Typhi and Paratyphi A, B

Key Antigens:

• O antigen (Ohne): somatic/cell wall; thermostable; shared between Typhi and Paratyphi

• H antigen (Hauch): flagellar; thermolabile; species specific

• Vi antigen: capsular (unique to Salmonella — NO K antigen)

• AH antigen → specific to Paratyphi A

• BH antigen → specific to Paratyphi B

Principle: Direct Agglutination

• Known: killed bacterial suspension of Salmonella (antigen)

• Unknown: patient antibodies

Widal Kit Colors:

• Blue stained = O antigen (somatic)

• Red stained = H antigen (flagellar)

Interpretation:

Significant titer: >1:80

• Normal individuals may show agglutination up to 1:40

O antibody: early to come, early to leave H antibody: late to come, late to leave

Four-fold rise (2 tube difference): indicates ACTIVE infection

Tube Agglutination Incubation:

• O antigen: 48–50°C for 4 hours

• H antigen: 48–50°C for 2 hours

• OR 37°C for 18–20 hours

Typhidot: more specific and faster than Widal; less cross-reactivity

INFECTIOUS MONONUCLEOSIS (Monospot)

Causative Agent: Epstein-Barr Virus (EBV) = Human Herpesvirus Type 4 (HHV-4)

• "Kissing disease" — transmitted mainly through saliva

Pathology:

• EBV infects B lymphocytes

• Atypical lymphocytes seen = Downey cells (reactive T cells, NOT monocytes)

• Lymphocytes >50% WBC; >10% atypical lymphocytes

Heterophile Antibodies:

• IgM class; non-specific

• React with horse, ox, goat, sheep RBCs

• Titer of 1:56 is significant

• NOT specific to EBV

Paul-Bunnell-Davidsohn Test:

Mnemonic: GFBM — Guinea pig removes Forssman; Beef removes Mono

Principle of Monospot: Passive Agglutination

• Latex coated with Paul-Bunnell antigen (from bovine RBC membranes)

• Detects IM heterophile antibodies

• Agglutination = POSITIVE

Forssman Antigen: glycolipid heterophile antigen (NOT found normally in humans); found in guinea pig, dog, cat, mouse, horse, fowl RBCs

Semi-quantitative dilutions: 1:2, 1:4, 1:8, 1:16, 1:32

DENGUE DETECTION

Causative Agent: Dengue virus (ssRNA Flaviviridae); 4 serotypes (DENV 1–4)

• Vector: Aedes aegypti (urban; 2 silvery lines); Aedes albopictus (rural; 1 silvery line)

NS1 Antigen:

• First viral marker to appear; detectable days 1–9 after fever onset

• Indicates early dengue infection; does NOT identify serotype

Antibody Timeline:

• IgM: detectable 5–10 days after onset (primary); 4–5 days (secondary)

• IgG: appears day 14 in primary; rises 1–2 days after onset in secondary; persists for life

Result Interpretation:

Test: Immunochromatographic assay (Bioline Dengue Duo)

• NS1 Ag: 3 drops (100 µL) specimen

• IgG/IgM: 10 µL specimen + 4 drops assay diluent

• Read at 15–20 minutes (do NOT read after 20 min)

• Any line, no matter how faint = POSITIVE

Cross-reactivity: with other Flaviviruses (West Nile, Zika, Yellow Fever, Japanese Encephalitis)

HEPATITIS

Transmission Summary:

• A & E: fecal-oral (contaminated water) — "tAE"

• B & C: blood/body fluids

• D: requires HBV (defective virus)

Hepatitis B Serological Markers:

HBsAg: detectable 2 weeks–2 months before symptoms; persists 2–3 months

Immunized with HBsAg: HBsAg(–), Anti-HBs(+), Anti-HBc(–) Natural immunity: HBsAg(–), Anti-HBs(+), Anti-HBc(+)

Hepatitis C: "Non-A/Non-B"; Gold standard = RT-PCR; Anti-HCV detectable 8–10 weeks post-exposure

Hepatitis D:

• Co-infection: (+) IgM anti-HBc

• Superinfection: (+) IgG anti-HBc

HBsAg Test Principle: Immunochromatographic Assay (sandwich)

• Specimen: 100 µL serum/plasma

• Read at 20 minutes

HIV

Causative Agent: RNA retrovirus; Family Retroviridae, Genus Lentivirinae

Structural Genes:

• gag gene: encodes p24 (capsid), p17 (matrix)

• pol gene: encodes reverse transcriptase (p66/p51), integrase (p31), protease (p10)

• env gene: encodes gp160 → gp120 (docking/attachment) + gp41 (fusion)

Key Proteins:

• gp120 = attachment to CD4 receptor

• gp41 = membrane fusion

• p24 = key early marker of infection; forms capsid

• p17 = matrix protein (between core and envelope)

Co-receptors:

• CXCR4 → T lymphocytes (X4 or T-tropic strains)

• CCR5 → Macrophages (R5 or M-tropic strains)

Tip: X in CXCR4 = T cells; CC in CCR5 = Macrophages

Replication Cycle:

1. Attachment (gp120 + CD4) → 2. Fusion (gp41) → 3. Reverse transcription (RNA→DNA) → 4. Integration (provirus) → 5. Replication → 6. Assembly → 7. Budding + Protease cleavage → mature virion

Stages of HIV:

Normal CD4:CD8 ratio = 2:1; In AIDS = <0.5:1 (inverted)

Transmission (most to least common): Sexual contact → Parenteral → Vertical (perinatal)

• NOT transmitted by saliva

ART Drug Classes:

• Entry inhibitors (block CCR5 binding)

• Fusion inhibitors (block gp41 fusion)

• Reverse transcriptase inhibitors (block RNA→DNA)

• Integrase inhibitors (block provirus integration)

• Protease inhibitors (block protein cleavage)

Lab Diagnosis Algorithm (CDC 2014):

1. Screening: 4th-generation ELISA (detects HIV-1/2 Ab + p24 Ag simultaneously)

2. Differentiation: Geenius HIV 1/2 Supplemental Assay (replaces Western Blot)

3. Confirmatory/Early: Nucleic Acid Testing (NAT/HIV RNA) — detectable by day 10

First markers to appear (in order): HIV RNA → p24 antigen → HIV IgM → HIV IgG → Western Blot bands

Western Blot Positive: ≥2 of these 3 bands: p24, gp41, gp120/160

• p24 + gp41 = POSITIVE

• gp41 alone = INDETERMINATE

Geenius HIV 1/2:

• HIV-1 positive: reactivity to gp160 + gp41 OR 1 envelope + p24 or p31

• HIV-2 positive: reactivity to BOTH gp36 AND gp140

Philippine rHIVda: T1 (screening) → T2 → T3 (Geenius); all 3 positive = HIV confirmed

Disease Monitoring:

• CD4 count: best indicator of immune function; done by flow cytometry

• Viral load (HIV RNA copies/mL): monitors ART effectiveness; goal = undetectable

U = U: Undetectable = Untransmittable

ACUTE PHASE REACTANTS (CRP topic)

Positive APR (increase): CRP, Fibrinogen, Haptoglobin, Ceruloplasmin, Complement C3/C4, Serum Amyloid A, Anti-chymotrypsin

Negative APR (decrease): Albumin (most abundant blood protein), Transferrin, Alpha-fetoprotein

Regulated by: IL-6, IL-1, TNF-α (pro-inflammatory cytokines) Produced by: Liver (hepatocytes)

Systemic Response Categories:

• Neuroendocrine: fever, HPA axis activation (CRH→ACTH→cortisol)

• Metabolic: muscle breakdown, ↑CPK

• Hematologic: leukocytosis, thrombocytosis, anemia

• Biochemical: ↓Fe, ↓Zn, ↑Cu

QUICK COMPARISON TABLE: CRP vs RF vs ASO

AGGLUTINATION INHIBITION vs HEMAGGLUTINATION INHIBITION

Good luck on your exams! Fighting and Faithing!