Serology Quiz 2

SLE (Systemic Lupus Erythematosus)

Chronic inflammatory disease; multi-organ involvement (skin, joints, kidney, lungs, CNS)

Tissue damage is due to ab/ag complexes in the renal glomeruli, skin, and choroid plexus of the brain

Anemia: 50% leukopenia

Decrease in 25-50% platelet count

Elevated ESR

Negative ANA, rules out SLE

Specific for SLE: anti-dsDNA and anti-Sm: titers will rise in active disease and fall in remission, useful in monitoring disease

AntiSSA Ags are associated with the neonatal SLE syndrome

Drug induced SLE

Associated with long term ingestion of several drugs: procainamide, hydralazine, anticonvulsants, chlorpromazine

Antibodies to histones are present

Milder form of Lupus: homogenous ANA pattern due to anti-histone antibodies and absence of anti-dsDNA

Raynaud's Syndrome

Vasoconstriction on extremities

Associated with the antinuclear antibody (RNP)

Ribonucleoprotein (anti-RNP)

Sjorgren's Syndrome

Chronic inflammatory disease

Strong association with HLA-C, HLA-DR3

Middle aged to elderly women

Autoantibodies: 90% rheumatoid factor

ANA: anti-LA

Definitive diagnosis: biopsy of labial salivary gland

Scleroderma

Cyanosis, puffy face, hard skin, atrophy, GI symptoms, lung, heart arrhythmias

2 forms of the disease: Progressive diffuse and systemic CREST

Calcinosis: bone formation

Raynaud: vasoconstriction of hands/feet

Esophageal involvement

Sclerodactyly: skin on fingers harden

Telangiectasia: spider veins

ANA

Centromere pattern due to ACA (Anticentromere antibody)

Nucleolar pattern can also be seen in diffuse type of organ involvement

Insulin-dependent Diabetes Mellitus (IDDM)

Deficient insulin production due to immune destruction of the B cells of the pancreas

Direct result of congenital Rubella

Juvenile onset

Immunoglobulin binds to tissue receptor for insulin - prevent biological action of insulin

HLA-DR3 and - DR4, HLA-DQw8 Ags: genetic susceptibility

CD4 T-lymphocyte

Mixed connective tissue disease

Diffuse tissue disease that doesn’t fall into one disease category

Joint pain, stiffness, esophageal dysfunction, progressively worsening.

75% leukopenia; deforming arthritis

ANA: 50% low titer RF/high titer of anti-RNP, anti-ssDNA

Distinguish from SLE: absence of multiple anti-SM and anti-ds-DNA

Ankylosing spondylitis

Males have a more progressively severe form than females who have a greater involvement of peripheral joints and cervical spine

Fibrolysing and ossifying process of the ligaments and synovial capsules in bone

Lumbar pain and limited lumbar motion

Prostatitis, conjunctivitis, aortic valve disease, upper lobe pulmonary fibrosis

Rheumatoid Arthritis

Affects the synovium and articular surface of multiple joints with varying degrees of systemic involvement

High incidence in elderly and females 20-40 years old

Joint swelling, morning stiffness, weight loss, fatigue, low grade fever

RF: rhematoid factor (also found in infections)

Circulating immune complexes consist of immunoglobulins and complement, and RF

ANA: 14-28% of pts

RA latex agglutination detects mostly IgM RF

Felty's syndrome

RA associated with splenomegaly and leukopenia, prone to bacterial infections

HLA-DR4 found in 95% of patients

High titer of RF assay

+ANA

Juvenile RA and HLA typing

Joint swelling, Still's disease: +HLA-DR5

40%: +HLA-DRw6

Girls <6 years: +HLA-Dw5 and +HLA-DR5

Boys <6 years: +HLA-B27

20%: +HLA-DR4

ANA: positive only in few cases

Multiple Sclerosis

Autoimmune disorder of the CNS

Formation of lesions (plasques) in the white matter of the brain and spinal cord → progressive destruction of the myelin sheath of axons

Closely associated with inheritance of a particular HLA molecule coding for the beta chain DR(DRB1*1501)

Oligoclonal bands seen on CSF Electrophoresis

Myasthenia gravis (MG)

Autoimmune disease that affects the neuromuscular junction, characterized by weakness and fragility of skeletal muscles

Antibody-mediated damage to acetylcholine receptors in skeletal muscle → progressive muscular weakness

Goodpasture's Syndrome

Presence of an autoantibody to the glomerular basement membranes, resulting in injury to the glomerulus that progresses rapidly to renal failure

Hashimoto's Thyroiditis

Diffuse non-tender enlargement of the thyroid gland, increased TSH, normal T3/T4, eventually becomes hypothyroid

94% have titer of 1:100 of the anti-thyroid microsomal Ab : Microsomal >1:6400

Graves Disease

Diffuse enlargement of thyroid (hyperplasia), tachycardia, characteristic eye symptoms include: infiltrative ophthalmopathy (exophthalmos)

TSI Thyroid stimulating immunoglobulins bind to the thyroid cells and stimulate thyroid activity (hyperthyroid)

GI Tract disease

86% have Abs against gastric parietal cells lipoprotein cytoplasmic component

Type I: B12 blockin, prevents IF binding

Type II: IF binds, but precluded by intestinal

Liver disease

Non-infectious inflammation in the bile ducts, disease manifests initially as a painless jaundice with itching

70% have ANA mixture of speckled, homogenous, anticentromic, nuclear membrane patterns

Inflammatory Bowel Disease (IBD)

Chron’s disease and chronic ulcerative colitis are distinct forms

ANCA (anti-neutrophilic cytoplasmic antibody) are autoantibodies directed against the intracellular components of PMNs

Straining of ANCA with proteinase 3 generates fluorescent patterns: c-ANCA, p-ANCA

Organ-Specific

Insulin-dependent Diabetes Mellitus (IDDM)

Multiple Sclerosis: CNS

Myasthenia gravis: neuromuscular junctions

Goodpasture's Syndrome: kidneys and lungs

Hasimoto's Thyroiditis: thyroid

Graves Disease: thyroid

Liver disease: liver and bile ducts

Autoimmune Liver Disease: liver and bile ducts

Inflammatory Bowel Disease (IBD)

GI Tract Disease: Celiac, Autoimmune gastritis - organ specific, IBD - systemic

Systemic Autoimmune

SLE

Drug induced SLE

Sjogren's Syndrome

Scleroderma

Mixed connective tissue disease

Ankylosing spondylitis

Rheumatoid Arthritis

Felty's syndrome: complication of RA

Raynaud's Syndrome: Primary - not autoimmune, Secondary - systemic diseases

Juvenile RA: depends on subtype

Cause of tissue injury in autoimmune disorders

Autoimmunity: Breakdown of the immune system in its ability to discriminate between self and non-self

Autoantibodies complex with patient's own DNA

Immune complexes are deposited in vascular systems in organs and tissues

Immune-mediated tissue injury from action of complement

Latex Agglutination (Agglutination)

Ex. Assays for rheumatoid factor (RA), Rubella, CRP

Particulate test Ags that have been absorbed onto latex beads react with Abs. When Ag/Ab/Latex particle complex forms, agglutination is seen as visible clumps

Haemagglutination (Agglutination)

Using RBCs in viral testing

-Ex: Rubella

Hemagglutination inhibition (HAI):

Pt serum with Rubella Ab + incubation with Rubella Ag = agglutination

When chick RBC is added, no agglutination occurs (pos. reaction)

Passive hemagglutination (PHA): human RBC coated with soluble Rubella virus Ag will agglutinate in the presence of Rubella Ab

After dilution with phosphate buffer and adding Ag-RBC, incubate at RT for 2 hours

RBC button at bottom of well = neg

Agglutination = pos

Flocculation tests (Agglutination)

VDRL or RPR (Non-treponemal tests)

Precipitate of fine particles that is microscopic (VDRL) or macroscopic (RPR-uses charcoal)

Reactive = med to large clumps

Weak Reactive = small clumps

Nonreactive = no clumps; tail when swirling

Double immunodiffusion (Ouchterlony formation)

(1) Fusion of the lines at their junction to form an arc represents serological identity or the presence of a common epitope

Looks like a sad face ☹️

(2) a pattern of crossed lines demonstrates

two separate reactions and indicates that the compared antigens share no common epitopes

Looks like ❌

(3) fusion of two lines with a spur indicates partial identity. In this last case, the two antigens share a common epitope, but some antibody molecules are not captured by antigen and travel through the initial precipitin line to combine with additional epitopes found in the more complex antigen. Therefore, the spur always points to the simpler antigen

Electrophoretic immunodiffusion

Radial immunodiffusion (RID):

-Pt IgG diffuses across agar with Ab incorporated with Anti-IgG/M/A

-Outside the zone of equivalence, precipitin ring forms (diameter is proportional to Pt Ig concentration)

Immunoelectrophoresis (IEP): separation of proteins into bands

Immunofixation electrophoresis (IFE): separation of proteins as Ab placed in trough running parallel to electrophoresis

Immunoelectrophoresis is a double-diffusion technique that incorporates electrophoresis to enhance results

Complement inactivation (Complement fixation)

Complement fixation: complement is used as a reagent: complement is "fixed" within Ag/Ab complex formed

Uptake of complement is an indicator of the Ag/Ab formation

Lack of hemolysis indicates complement has reacted with the test Ag/Ab complex

Hemolysis of indicator SRBC coated with hemolysin, indicates that complement is not fixed into Ag/Ab complex

RIA (Labeling)

Radioactive labels with known emission properties

Uses: mostly used for trace elements in blood like hormones and serum proteins or elements too small to be detected otherwise

One reactant (Ag or Ab) is radio labeled

Competitive: usually Ag is labeled – “tracer”

Less signal = more concentrated

Non-competitive: Ab is labeled

Directly proportional

EIA/ELISA (Labeling)

Ex. horseradish peroxidase, alkaline phosphatase, glucose-6-PD, beta-galactosidase

Common substrate: orthophenylene diamine (OPD), NADH → NAD

Controls must be included in assay: 3 negative + 2 positive

Absorbance greater than or equal to cutoff of the negative control is reactive

If positive, repeat the EIA and send for confirmatory test (Western Blot, IFA)

Enzyme catalyzes substrate molecules and amplifies the signal

Enzyme activity may be monitored directly or coupled with co-enzyme action → colored product, measured photometrically

FIA (Labeling)

Common labels: Fluorescein isothiocyanate (FITC), rhodamine isothiocyanate

Uses: DFA assays for bacterial or viral detection

Compounds that absorb radiant energy at higher wavelength and emit radiant energy at a lower/longer wavelength

MEIA: microparticle EIA

Antinuclear antibodies (ANA test)

Fluorescent Treponemal Antibody (FTA-ABS): fluorescence detected on microscopic slides, visual evaluation of Ag/Ab complex formation

Fluorescence polarization (FPIA): fluorescent labelled antigen competes with patient antigen for limited number of antibody-binding sites; inverse ratio between patient antigen and amount of polarization – homogenous assay

Chemiluminescent Labels

Common labels: Acridinium esters, luminol, ruthenium derivatives, nitrophenyl oxalates

Uses: cardiac markers, vitamin D level, total IgE

Organic compounds emit a photon of light in response to a chemical reaction, as they revert to their ground state

PCR (Molecular diagnostic)

Extracted DNA from an organism such as Chlamydia allows for amplification by PCR

Low levels of specific DNA sequences are amplified using oligonucleotides or primers (small portions of a single DNA strand) and enzymes

Western Blot (immunoblot)

EIA reaction is used to detect the bang (HIV testing)

Ag material is electrophoresed for separation and blotted to a nitrocellulose membrane

Membrane is incubated with specific Ab, Ag/Ab complex is a band

Southern Blot (immunoblot)

Fragments detected using radioisotope (single stranded DNA fragments and radiography)

Specimen DNA is fragmented using restriction enzymes and separated by using restriction enzymes and separated by electrophoresis and blotted onto nitrocellulose membrane

Nephelometry

Quantitation of immunoglobulins

Light scattering application used to detect Ag/Ab complexes

Distribution of Rayleigh light scatter which is symmetrical in forward and backward direction

More light scatter = higher concentration of Ag/Ab complex

Competitive assay

Higher specificity

Competition occurs between a labeled Ag and unlabeled Ag for a limited number of binding Ab sites

Labeled Ag + unlabeled Ag + limited Ab = Ag/Ab labeled + Ag/Ab unlabeled + free labeled Ag

Amount of Ag is indirectly related to the amount of label signal

Less bound labeled Ag indicates more antigen present in sample reacting with the limited Ab sites

EMIT (Enzyme Multiplied Immunoassay Technique)

Competitive Assay

Automated method commonly utilized for drug detection

Patient's urine sample contains the unlabeled drug metabolite that competes with a labeled drug metabolite. Only the unbound enzyme-label drug is left to react with the substrate.

Measure drug level which is directly proportional to enzyme activity detected.

Non-competitive assay

higher sensitivity and specificity

Sandwich assay: Analyte is sandwiched between two highly specific antibody reagents

Two step process, utilizes wash steps to isolate the sandwich complex and remove excess unbound labels

Concentration of labeled Ag is directly proportional to bound Ab

Homogenous and Heterogenous Immuno assay

Homogenous and Heterogenous Immuno assay

Homogenous: do not require separation, limited washings

Heterogenous: require separation or washing of bound Ag/Ab complexes on solid phase

Solid phase: fixed receptor sites, immobilized on a surface such as wells or beads