AUTOIMMUNITY/DISEASES

Conditions in which damage to organs/ tissues results from the presence of autoantibody/autoreactive cells

Autoimmunity

Factors that contribute to development of autoimmune disease:

• loss of self-tolerance, genetics (MHC genes)

• hormonal influence, tissue trauma and release of self antigens

• microbial infections, epigenetic factors (toxins, foods, drugs, aging)

SYSTEMIC LUPUS ERYTHEMATOSUS

• Clinical Manifestations

• fatigue, weight loss, malaise, fever, anorexia

• _ - most frequently reported manifestations

• erthematous skin rash, butterfly rash across nose and cheeks

• _ - major cause of illnes and death

• cardiac - pericarditis, tachycardia, ventricular enlargement

• neuropsychiatric - seizures, mild cognitive dysfunction, psychoses, depression

• hematologic abnormalities - anemia, leukopenia, thrombocytopenia

• fatigue, weight loss, malaise, fever, anorexia

• arthritis - most frequently reported manifestations

• erthematous skin rash, butterfly rash across nose and cheeks

• nephrirtis - major cause of illnes and death

• cardiac - pericarditis, tachycardia, ventricular enlargement

• neuropsychiatric - seizures, mild cognitive dysfunction, psychoses, depression

• hematologic abnormalities - anemia, leukopenia, thrombocytopenia

SYSTEMIC LUPUS ERYTHEMATOSUS

• Lab Features

• Presence of ANAs, circulating immune complexes, decreased complement levels, cryoglobulin, circulating anticoagulant, non-specific elevation of Ig levels

SYSTEMIC LUPUS ERYTHEMATOSUS

Detection of ANA

• Several methods available: IIF, ELISA, RIA, Western blot, Immunoelectrophoresis

• _ - most widely accepted and used test because of high sensitivity

→screening test is commonly performed with _ or _ dilution of parent serum

→ _ - standard substrate

→ positive result is _

→titer of _ is considered clinically significant

→staining patterns: (5)

• Several methods available: IIF, ELISA, RIA, Western blot, Immunoelectrophoresis

• Fluorescent ANtinuclear Antibody (FANA) - most widely accepted and used test because of high sensitivity

→screening test is commonly performed with 1:40 or 1:80 dilution of parent serum

→ HUMAN EPITHELIAL CELL LINE (Hep-2 cells) - standard substrate → Hep-2 cells + patient's serum + FITC-labeled AHG reagent

→ positive result is FLUORESCENCE (under a fluorescent miscroscope using 400x magnification)

→titer of > or equal to 160 is considered clinically significant

→staining patterns: (5)

• HOMOGENOUS/DIFFUSE - uniform staining of the entire nucleus

• PERIPHERAL/RIM - greater staining intensity surrounding the nucleus

• SPECKLED - discrete, fluorescent specks throughout the nuclei

• NUCLEOLAR - prominent staining of nucleoli

• CENTROMERE - numerous discrete speckles (46 speckles) are seen in nuclei

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-dsDNA

→Significance:

• paralles disease activity of _

• Gold standard: _

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-dsDNA

→Significance:

• paralles disease activity of SLE

• Gold standard: IFA staining of Crithidia luciliae

→Staining Pattern (IFA):

• homogenous/diffuse

• peripheral/rim

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-DNP

→Significance:

• give rise to apperance of _

• found in 99% of cases with _

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-DNP

→Significance:

• give rise to apperance of LE cells

• found in 99% of cases with untreated SLE

→Staining Pattern (IFA):

• homogenous/diffuse

• peripheral/rim

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-Smith/Anti-Sm

→Significance:

• found almost exclusively in patients with _

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-Smith/Anti-Sm

→Significance:

• found almost exclusively in patients with SLE

→Staining Pattern (IFA):

• coarsely speckled

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-RNP

→Significance:

• found in cases of _

• _ disease

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-RNP

→Significance:

• found in cases of SLE, RA, Sjogren's syndrome

• Mixed Connective Tissue disease

→Staining Pattern (IFA):

• coarsely speckled

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-histones

→Significance:

• found in cases of _

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-histones

→Significance:

• found in cases of drug-induced lupus

→Staining Pattern (IFA):

• diffuse/homogenous

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-Ro (SS-A)

→Significance:

• found in cases of _

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-Ro (SS-A)

→Significance:

• found in cases of cutaneous/Neonatal SLE

→Staining Pattern (IFA):

• finely speckled

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-La (SS-B)

→Significance:

• associated with _

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-La (SS-B)

→Significance:

• associated with SLE and Sjogren's syndrome

→Staining Pattern (IFA):

• finely speckled

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-nucleolar RNA

→Significance:

• (3)

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-nucleolar RNA

→Significance:

• SLE, Sjogren's syndrome, Systemic Sclerosis

→Staining Pattern (IFA):

• Nucleolar

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-centromere

→Significance:

• (3)

→Staining Pattern (IFA):

Anti-Nuclear Antigens (ANA), their SIFNIFICANCE and STAINING PATTERNS

Anti-centromere

→Significance:

• SLE, mixed connective tissue disease, CREST syndrome

→Staining Pattern (IFA):

• discrete, speckled

OTHER AUTOIMMUNE DISEASE

→RHEUMATOID ARTHRITIS

• Autoantibody:

• Affected Organ/Tissue:

OTHER AUTOIMMUNE DISEASE

RHEUMATOID ARTHRITIS

→Autoantibody:

• Rheumatoid Factor

• Anti-CCP (Cyclic Citrullinated Peptide)

→Affected Organ/Tissue:

• Bones

HASHIMOTO'S THYROIDITIS

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

HASHIMOTO'S THYROIDITIS

→Autoantibody:

• Anti-thyroglobulin

• Anti-TPO

→Affected Organ/Tissue:

• 
  

GRAVE'S DISEASE

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

GRAVE'S DISEASE

→Autoantibody:

• Anti-TSH Receptor

• Anti-TPO

→Affected Organ/Tissue:

• Thyroid gland

TYPE I DM

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

TYPE I DM

→Autoantibody:

• Anti-islet cells

→Affected Organ/Tissue:

• Islet cells of pancreas

MULTIPLE SCLEROSIS

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

MULTIPLE SCLEROSIS

→Autoantibody:

• Anti-MBP (Myelin Basic Protein)

→Affected Organ/Tissue:

• Myelin Sheath of the nerves

MYASTHENIA GRAVIS

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

MYASTHENIA GRAVIS

→Autoantibody:

• Anti-acetylcholine Receptor

→Affected Organ/Tissue:

• Neuromuscular junction

GOODPASTURE'S SYNDROME

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

GOODPASTURE'S SYNDROME

→Autoantibody:

• Anti-Glomerular Basement Membrane

→Affected Organ/Tissue:

• Glomerulus

• Kidney

PRIMARY BILIARY CIRRHOSIS

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

PRIMARY BILIARY CIRRHOSIS

→Autoantibody:

• Anti-mitochondrial

→Affected Organ/Tissue:

• Intrahepatic bile ducts

CHRONIC ACTIVE HEPATITIS

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

CHRONIC ACTIVE HEPATITIS

→Autoantibody:

• Anti-smooth muscle

→Affected Organ/Tissue:

• Liver

PERNICIOUS ANEMIA

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

PERNICIOUS ANEMIA

→Autoantibody:

• Anti-parietal cells

→Affected Organ/Tissue:

• Parietal cells

SJOGREN'S SYNDROME

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

SJOGREN'S SYNDROME

→Autoantibody:

• Anti-salivary and lacrimal glands

→Affected Organ/Tissue:

• exocrine glands

SCLERODERMA / CREST

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

SCLERODERMA / CREST

→Autoantibody:

• Anti-centromere

→Affected Organ/Tissue:

• Skin and connective tissue

GRANULOMATOSIS WITH POLYANGIITIS/WEGENER'S GRANULOMATOSIS

→Autoantibody:

• 
  

→Affected Organ/Tissue:

• 
  

GRANULOMATOSIS WITH POLYANGIITIS/WEGENER'S GRANULOMATOSIS

→Autoantibody:

• Anti-neutrophilic cytoplasmic antibody

→Affected Organ/Tissue:

• Upper respiratory system, lungs, blood vessels

_ - inherited dysfunctions of the immune systems

Primary Immunodeficiencies (PID)

PREDOMINANTLY ANTIBODY DEFICIENCIES

• low levels of immunoglobulins at approximately 5 to 6 months of age; IgG appears to be most affected

Transient Hypogammaglobulinemia of Infancy

PREDOMINANTLY ANTIBODY DEFICIENCIES

• most common congenital immunodeficiency

• susceptible to respiratory GI infections

Selective IgA deficiency

PREDOMINANTLY ANTIBODY DEFICIENCIES

• X-linked inheritance, affect males almost exclusively

• lack circulating mature CD19+ B cells and exhibit a deficiency or lack of immunoglobulins of all classes

BTK deficiency/Bruton's Agammaglobulinemia

PREDOMINANTLY ANTIBODY DEFICIENCIES

• characterized by deficiency of both IgA and IgG antibodies

• associated with spruelike syndrome and malabsorption

Common Variable Immunodeficiency

PREDOMINANTLY ANTIBODY DEFICIENCIES

• the most common subclass deficiency is IgG4, with IgG1 deficiency being the least common

Isolated IgG Subclass deficiency

COMBINED IMMUNODEFICIENCIES

• most serious of the congenital immunodeficiencies

• group of related diseases hat all affect T and B cell function but with differing causes

• present early in infancy with infection by nearly any type of organism

Severe Combined Immunodeficiency

COMBINED IMMUNODEFICIENCIES

• produces a moderate to severe defect in cell-mediated immunity with normal or mildly impaired humoral immunity

• number of T cells decreases due to accumulation of deoxyguanosine triphosphate, a toxic purine metabolite

Purine-nucleoside Phosphorylase (PNP) deficiency

COMBINED IMMUNODEFICIENCIES WITH ASSOCIATED SYNDROMIC FEATURES

• triad of thrombocytopenia, eczema, recurrent infections

Wiskott-Aldrich Syndrome

COMBINED IMMUNODEFICIENCIES WITH ASSOCIATED SYNDROMIC FEATURES

• developmental abnormality of the third and fourth pharyngeal pouches that affects thymus development in embryo

• causes quantitative defect in T cells

DiGeorge Syndrome

COMBINED IMMUNODEFICIENCIES WITH ASSOCIATED SYNDROMIC FEATURES

• characterized by cerebellar ataxia (involunatry muscle mobements) and telangiectasias (capillary swelling, red blotched skin )

• abnormal genes produce a defect of both humoral and cellular immunity

Ataxia-Telangiectasia

CONGENITAL DEFECTS OF PHAGOCYTE NUMBER, FUNCTION, OR BOTH

• X-linked or autosomal recessive inheritance that affects neutrophil microbiocidal function, inability to produce superoxide anions required for bacterial/fungal organisms

• defective NADPH oxidase systems

Chronic Granulomatous Disease

CONGENITAL DEFECTS OF PHAGOCYTE NUMBER, FUNCTION, OR BOTH

Chronic Granulomatous Disease - LAB TEST

→ _

• Flow Cytometry - Dihydrorhodamine oxidation (DHR) to rhodamine by respiratory burts of leukocyte is measured

→Nitroblue Tetrazolium Test

• Specimen: _

• Reagent: _

• Positive: _

• Normal: _

• CGD: _

Chronic Granulomatous Disease - LAB TEST

→ NEUTROPHIL OXIDATIVE BURST ASSAY

• Flow Cytometry - Dihydrorhodamine oxidation (DHR) to rhodamine by respiratory burts of leukocyte is measured

→Nitroblue Tetrazolium Test

• Specimen: BUFFY COAT

• Reagent: BACTERIAL SUSPENSION + NBT DYE

• Positive: BLUE FORMAZAN PRECIPITATE

• Normal: 80% - 100% blue ppt

• CGD: <50% blue ppt

CONGENITAL DEFECTS OF PHAGOCYTE NUMBER, FUNCTION, OR BOTH

• deficiency of CD18 - component of adhesion receptors on neutrophils and monocytes (CD11b or CD11c) and on T cells (CD11a)

• leads to abnormal adhesion, motility, aggregation, chemotaxis, and endocytosis by the affected leukocytes

Leukocyte Adhesion Deficiency

characterized by the overproduction of a single immunoglobulin component called a myeloma protein (M protein) or paraprotein by a clone of identical plasma cells

Plasma Cell Dyscrasia

Plasma Cell Dyscrasia

• includes several related syndromes:

• multiple myeloma

• waldenstrom macroglobulinemia

• most serious and mos common of plasma cell dyscrasia

• characterized by excess plasma cells in the bone marrow, a monoclonal immunoglobulin component in the plasma or urine (most common is IgG) and lytic bone lesions

Multiple myeloma

Multiple myeloma

• malignant plasma cells phenotypically express _

• when immunoglobulin levels in blood are sufficiently high, they may cause _ formation of RBCs

• malignant plasma cells phenotypically express CD38, CD56, CD138

• when immunoglobulin levels in blood are sufficiently high, they may cause ROULEAUX formation of RBCs

Multiple myeloma

• Criteria for Diagnosis

• plama cells comprising greater than 10% of bone marrow cells

• evidence of end-organ damage such as bone marrow lesions

• hypercalcemia, renal insufficiency, and anemia

• serum M protein of > or equal to 3g'dL and urinary M protein of > or equal to 200 mg/day

• presence of bence jones protein in urine

• malignant proliferation of IgM-producing plasma cells

• signs and symptoms include weakness, fatigue, anemia, bleeding, plasma hyperviscosity

Waldenstrom Macroglobulinemia

Waldenstrom Macroglobulinemia

• patients have elevated serum monoclonal protein known as _ that migrates in the gamma region during SPE

• Bence jones proteinuria is present in 10% of the case

• Serum b2-macroglobulin levels are generally above the RR limit of _

• in 10% to 20% of patients, IgM paraproteins begave as cryoglobulins

• _ precipitate at cold temperatures and can occlude small blood vesssels in the extremities in cold weather

• patients have elevated serum monoclonal protein known as MACROPROTEIN or IgM PARAPROTEIN that migrates in the gamma region during SPE

• Bence jones proteinuria is present in 10% of the case

• Serum b2-macroglobulin levels are generally above the RR limit of 3.0 mg/dL

• in 10% to 20% of patients, IgM paraproteins begave as cryoglobulins

• CRYOGLOBULIN precipitate at cold temperatures and can occlude small blood vesssels in the extremities in cold weather

SYPHILIS

• causative agent: _

• modes of transmission: (3)

• stages: (4)

• causative agent: Treponema pallidum

• modes of transmission: sexual (primary), parenteral, congenital

• stages: primary, secondary, latent, tertary

SYPHILIS - PRIMARY STAGE

→Clinical manifestations:

→Lab tests:

→Clinical manifestations

• HARD CHANCRE (initial lesion) between 10 to 30 days after infection

→Lab tests

• darkfield examination/DFA

SYPHILIS - SECONDARY STAGE

→Clinical manifestations:

→Lab tests:

SYPHILIS - SECONDARY STAGE

→Clinical manifestations:

• CONDYLOMATA LATA - flat lesions resembling warts in moist areas of the body

• headache, sorethroat, low-grade fever, nasal discharge

→Lab tests:

• Screening: RPR, VDRL

• Confirmatory: TP-PA, FTA-ABS

SYPHILIS - LATENT STAGE

→Clinical manifestations:

→Lab tests:

SYPHILIS - LATENT STAGE

→Clinical manifestations:

• LACK OF CLINICAL SYMPTOMS - non-infectious state in which diagnosis can be made only by serologic methods

→Lab tests:

• Screening: VDRL, RPR

• Confirmatory: TP-PA, FTA-ABS

SYPHILIS - TERTIARY STAGE

→Clinical manifestations:

→Lab tests:

SYPHILIS - TERTIARY STAGE

→Clinical manifestations:

• GUMMAS - localized areas of granulomatous inflammation often found on bones, skin, or subcutaneous tissue

• can lead to NEUROSYPHILIS

→Lab tests:

• RPR, FTA-ABS (serum)

• VDRL (CSF)

• caused by maternal spirochetemia and transplacental transmission of microorganism

• untreated syphilis during pregnancy can lead to stillbirth, neonatal death, or infant disorders such as deafness, neurologic impairment, and deformities

Congenital Syphilis

Congenital Syphilis - CLINICAL MANIFESTATIONS

• Early stage: rash, condylama latum, bone changes, hepatosplenomegaly, jaundice, anemia

• Late stage: eighth nerve deafness, keratitis, Hutchinson's teeth, arthropathy

NON-TREPONEMAL TEST

• used to screen for syphilisbecause of their high sensitivity and ease of performance

• non-treponemal test are POSITIVE within _ after the appearance of primary chancre

• detects presence of _ antibodies = non-specific antibodies directed against cardiolipin constituent of treponemal lipids but usually derived from its host

• tests are based on _ →patient antibody complexes with the cardiolipin antigen

→REACTIVE = medium to large clumps

→WEAKLY REACTIVE = small clumps

→NON-REACTIVE = no clumps or slight roughness

• used to screen for syphilisbecause of their high sensitivity and ease of performance

• non-treponemal test are POSITIVE within 1 to 4 WEEKS after the appearance of primary chancre

• detects presence of REAGIN antibodies = non-specific antibodies directed against cardiolipin constituent of treponemal lipids but usually derived from its host

• tests are based on FLOCCULATION REACTIONS →patient antibody complexes with the cardiolipin antigen

Venereal Disease Laboratory (VDRL)

• both qualitative and quantitative slide flocculation test, can be used on either serum/CSF

• antigen consists of: (3)

• serum is heated at _ degrees celcius for _ minutes to inactivate complement (_ is not heated, it has no native C)

• volume of serum: _

• volume of VDRL antigen: _

• rotation of serum: _

• read MICROSCOPICALLY for FLOCCULATION

• all sera with reactive or weakly reactive results must be testes using the quantitative slide test

• both qualitative and quantitative slide flocculation test, can be used on either serum/CSF

• antigen consists of:

→0.03% cardiolipin (main reacting component)

→0.9% cholesterol (enhances the reacting surface of cardiolipin)

→0.21% lecithin (removes anti-complementary activity of cardiolipin)

• serum is heated at 56 degrees celcius for 30 minutes to inactivate complement (CSF is not heated, it has no native C)

• volume of serum: 0.05 mL pipetted into ceramic ring of slide

• volume of VDRL antigen: 1/60 mL or 1 drop from 18 gauge needle

• rotation of serum: 180 RPM for 4 minutes

• read microscopically for FLOCCULATION

• all sera with reactive or weakly reactive results must be testes using the quantitative slide test

Rapid Plasma Reagin (RPR)

• RPR is a modified VDRL test involving MACROSCOPIC test reaction

• cardiolipin-containing antigen suspension is bound to _ = test is easier to read

• most;y used as screening procedure, MORE SENSITIVE but LESS SPECIFIC than VDRL

• Volume of serum: _

• Volume of RPR antigen: _

• Reagent (Modified VDRL reagent): (4)

• Rotation

• Read MACROSCOPICALLY for FLOCCULATION

• RPR is a modified VDRL test involving MACROSCOPIC test reaction

• cardiolipin-containing antigen suspension is bound to CHARCOAL PARTICLES = test is easier to read

• most;y used as screening procedure, MORE SENSITIVE but LESS SPECIFIC than VDRL

• Volume of serum: 0.05 mL pipetted into 18-mm circle on plastic-coated disposable card

• Volume of RPR antigen: one free-falling drop using calibrated 20-gauge needle

• Reagent (Modified VDRL reagent): (4)

→cardiolipin, cholesterol, lecithin

→charcoal ( for easy visualization of results)

→choline chloride (inactivates complement)

→EDTA (prevent oxidation of lipid)

• Rotation

• Read MACROSCOPICALLY for FLOCCULATION

BIOLOGIC FALSE POSITIVE FOR VDRL/RPR

• Systemic Lupus Erythematosus

• Rheumatoid arthritis

• Rheumatic fever

• Infectious hepatitis

• Infectious mononucleiosis

• Leprosy

• Malaria

• Pneumococcal pneumonia

• Pregnancy

• Old age

• detect antibody directed against the T. pallidum organism or against specific treponemal antigens

• more difficult to perform and more time-consuming

• utilized as CONFIRMATORY test

• Ex: FTA-ABS, TP-PA

TREPONEMAL TESTS

FTA-ABS (Fluorescent Treponemal Antibody Absorption Test)

• Principle: _

• highly sensitive and specific but more time-consuming to perform

• sample preparation: _

• antigen used: _

→antigen fixed into slides + px serum + FITC labeled AHG = (+) FLUORESCENCE

• slides are read under a fluorescent microscope, intensity of _ color is reported on a scale of 1 to 4

• no fluorescence indicates a negative test and a result of _ or above is considered reactive

• Principle: INDIRECT IMMUNOFLUORESCENCE

• highly sensitive and specific but more time-consuming to perform

• sample preparation:

→patient serum is heated at 56 degrees celcius for 30 minutes

→pre-incubation with non-pathogenic treponemes (retiter strain) acts as a sorbent to remove cross-reactovotu with other non-pathogenic spirochtes

• antigen used: NICHOL'S STAIN (virulent strain)

→antigen fixed into slides + px serum + FITC labeled AHG = (+) FLUORESCENCE

• slides are read under a fluorescent microscope, intensity of GREEN color is reported on a scale of 1 to 4

• no fluorescence indicates a negative test and a result of 2 or above is considered reactive

TREPONEMA PALLIDUM - PARTICLE AGGLUTINATION (TP-PA)

• this assay is excellent for resolving INCONCLUSIVE FTA-ABS results

• particle agglutination test used _

• presence of T. pallidum antibodies is indicated by _ of the sensitized gel particles which form lattice-like structure that spread to produce a smooth mat that covers the well's surface

• this assay is excellent for resolving INCONCLUSIVE FTA-ABS results

• particle agglutination test used COLORED GELATIN PARTICLES COATED WITH TREPONEMAL ANTIFENS

• presence of T. pallidum antibodies is indicated by AGGLUTINATION of the sensitized gel particles which form lattice-like structure that spread to produce a smooth mat that covers the well's surface

Traditional and Reverse Testing Algorithm for Syphilis

HEPATITIS A

• family: _

• other names: _

• MOT: _

• incubation: _

• infection does not progress to chronic state abd is usually SEL_LIMITING

• Diagnostic markers:

→_ - shed in feces of infected individuals during early acute stage of infection

→_

→_

1. _ - marker for acute hepatitis A (peaks during first month of illness)

2. _ - produced as a result of natural infection or immunization

• family: Picornaviridae

• other names: infectious hepatitis/short-incubation hepatitis

• MOT: fecal-oral

• Incubation: 28 days

• infection does not progress to chronic state abd is usually SEL_LIMITING

• Diagnostic markers:

→HAV Ag- shed in feces of infected individuals during early acute stage of infection

→HAV RNA (RT-PCR)

→ANTI-HAV ANTIBODIES

1. IgM Anti-HAV - marker for acute hepatitis A (peaks during first month of illness)

2. IgG Anti-HAV - produced as a result of natural infection or immunization

HEPATITIS B

→ family: _

→other names: _

→MOT: _

→incubation: _

→development of chronic HBV infection occurs in 10% of infected adults

→ _ = VIRION = present in blood, semen, urine, colostrum, other body fluids

→ family: Hepadnaviridae (dsDNA)

→other names: serum hepatitis/ long-incubation hepatitis

→MOT: parenteral, sexual, perinatal

→incubation: 30 - 180 days

→development of chronic HBV infection occurs in 10% of infected adults

→ DANE PARTICLE = VIRION = present in blood, semen, urine, colostrum, other body fluids

SEROLOGIC MARKERS FOR HBV

→ANTIGENS

• _ - first detectable marker found in serum; marker of infection (acute or chronic)

• _ - indicates active viral replication; marker of high degree of infectivity

• _ - not detected in serum but present in liver cells

• HBsAg - first detectable marker found in serum; marker of infection (acute or chronic)

• HBeAg - indicates active viral replication; marker of high degree of infectivity

• HBcAg - not detected in serum but present in liver cells

SEROLOGIC MARKERS FOR HBV

→ANTIBODIES

• _ - (total) - elevated levels in acute/chronic hepatitis B

• _ - useful marker during window phase of infection; acute hepatitis B infection

• _ - persists in the lifetime of infected individual, indicates past infection

• _ - first serologic evidence of convalescent phase, indicates low degree of infectivity

• _ - marker of recovery and immunity, marker of vaccination (positive titer: 10 mIU/mL)

• ANTI-HBC - (total) - elevated levels in acute/chronic hepatitis B

• ANTI-HBC IgM - useful marker during window phase of infection; acute hepatitis B infection

• ANTI-HBC IgG - persists in the lifetime of infected individual, indicates past infection

• ANTI-HBe - first serologic evidence of convalescent phase, indicates low degree of infectivity

• ANTI-HBS - marker of recovery and immunity, marker of vaccination (positive titer: 10 mIU/mL)

SEROLOGIC MARKERS FOR HBV

• done thru PCR, useful adjunct in detecting early acute HBV infection

• used to evaluate the effctiveness id antiviral theaphy in patients with chronic hepatitis B

HBV DNA

Reaction with SEROLOGIC MARKERS and their INDICATIONS

HEPATITIS C

• family: _

• other name: _

• MOT: _

• incubation: _

• majority of infections are asymptomatic, symptoms occuring only in 20% of the population

• 85% develop _ with increased risk of cirrhosis and hepatocellular carcinoma

• used to be the leading cause of _

• diagnostic tests: (2):

• family: Flaviviridae (ssRNA)

• other name: Non-A Non-B hepatitis

• MOT: parenteral, sexual, perinatal

• incubation: 7 weeks

• majority of infections are asymptomatic, symptoms occuring only in 20% of the population

• 85% develop CHRONIC HEPATITIS with increased risk of cirrhosis and hepatocellular carcinoma

• used to be the leading cause of TRANSFUSION-ASSOCIATED HEPATITIS

• diagnostic tests: (2):

→ANTI-HCV

→HCV RNA or VIRAL LOAD TESTING

HEPATITIS D

• family: _

• requires _ for its replication > _

• primarily transmitted through _ means and is commonly severe

• Coinfection: -

• Superinfection: _

• Diagnostic markers:

1. _ - elevated in acute phase, but persist for only a short period of time

2. _ - signifies an acute, chronic, or past hepatitis D infection

3. _ - used to confirm a positive HDV antibody screen

HEPATITIS D

• family: ssRNA, genus Deltavirus

• requires HBV for its replication > HDV utilizes HBsAg as its own envelope

• primarily transmitted through PARENTERAL means and is commonly severe

• Coinfection: simultaneous infection of HBV and HDV

• Superinfection: additional HDV infection in patients with chronic hepatitis B infection

• Diagnostic markers:

1. Anti-HDV IgM - elevated in acute phase, but persist for only a short period of time

2. Anti-HDV IgG - signifies an acute, chronic, or past hepatitis D infection

3. HDV RNA testing - used to confirm a positive HDV antibody screen

HEPATITIS E

• family: _

• MOT: _

• presents as acute, SEL-LIMITING HEPATITIS without progression to chronic state

• incubation: _

• _ infected with HEV1 or HEV2 have a mortality rate of 20% to 25% becuase of obstetric complications or development of fulminant hepatitis

• Diagnostic markers:

1. _ - present during acute infection but dcelines in early recovery period

2. _ - detect patients in later stages of infection, determines past exposure

3. _ identified thru PCR, performed on blood or stool samples

• family: Hepeviridae (ssRNA)

• MOT: fecal-oral, mostly transmitted through contaminated drinking water

• presents as acute, SEL-LIMITING HEPATITIS without progression to chronic state

• incubation: 2 - 6 weeks

• PREGNANT WOMEN infected with HEV1 or HEV2 have a mortality rate of 20% to 25% becuase of obstetric complications or development of fulminant hepatitis

• Diagnostic markers:

1. ANTI-HEV IgM - present during acute infection but dcelines in early recovery period

2. ANTI-HEV IgG - detect patients in later stages of infection, determines past exposure

3. HEV RNA - identified thru PCR, performed on blood or stool samples

INFECTIOUS MONONUCLEOSIS

• acute infectious disease of reticuloendothelial system caused by _

• target cells: _

• other names: _

• MOT: _

• Infectious process:

• acute infectious disease of reticuloendothelial system caused by EPSTEIN-BARR VIRUS

• target cells: B CELLS (CD21)

• other names: GLANDULAR FEVER, KISSING DISEASE

• MOT: INTIMATE CONTACT WITH SALIVARY SECRETION from infected individual

• Infectious process:

1. EBV infects the epithelium of the oropharynx and salivary glands

2. Lyphocytes in tonsillar crypts are directly infected

3. Infected B cells and activated T cells proliferate and expand

4. Polyclonal B cells produce antibodies to host and viral proteins

INFECTIOUS MONONUCLEOSIS - Manifestations

→HEMATOLOGICAL

• absolute _ of greater than 50% of total WBCs and at least 20% are _

• absolute KYMPHOCYTOSIS of greater than 50% of total WBCs and at least 20% are DOWNEY CELLS

INFECTIOUS MONONUCLEOSIS - Manifestations

→SEROLOGICAL

• increased titer of _

1. IgM antibodies produced as result of polyclonal B cell actibvation and reacts with horse, sheep, and bovine red cells

2. Produced by 40% of patients during first week and by 80% to 90% of patients on third week

3. a titer of _ is clinically significant in patients with suspected infectious mononucleiosis

SEROLOGICAL

• increased titer of HETEROPHILE ANTIBODIES

1. IgM antibodies produced as result of polyclonal B cell actibvation and reacts with horse, sheep, and bovine red cells

2. Produced by 40% of patients during first week and by 80% to 90% of patients on third week

3. a titer of 1:56 or GREATER is clinically significant in patients with suspected infectious mononucleiosis

INFECTIOUS MONONUCLEOSIS - Manifestations

→SEROLOGICAL

• Markers of acute infection (2)

• Markers of past infection and convalescent phase: (1)

• Markers of acute infection (2)

1. Anti-VCA IgM (viral capsid antigen)

2. Anti-EAD (early antigen diffuse)

• Markers of past infection and convalescent phase: (1)

1. Anti-EBNA IgG (Epstein Barr Nuclear Antigen)

Serological Tests for Heterophile Antibodies

• routine presumptive test

• used SHEEP RBCs as reagent

• agglutination - indicates presence of heterophile antibodies

• no agglutination - indicates absence of heterophile antibodies

Paul-Bunnel Test

Serological Tests for Heterophile Antibodies

• uses technique of absorption using Guinea pig kidney cells and beef erythrocytes

• differentiates IM from other heterophile antibodies

Davidsohn Differential Test

*any absorbed antibodies are removed by centrifugation and supernatant fluid is tested with sheep RBCs

Serological Tests for Heterophile Antibodies

• uses horse RBCs as reagent to agglutinate IM heterophile antibodies

MONOSPOT TEST

HIV

• causative agent of _

• family: _

• it has a unique enzyme called _ that transcribes RNA to DNA

• MOT: _

• body fluids considered to be infectious: _

• causative agent of Acquired Immunodeficiency Syndrome (AIDS)

• family: Retroviridae

• it has a unique enzyme called Reverse Transcriptase that transcribes RNA to DNA

• MOT: sexual contact, parenteral, perinatal route (mother to infant)

• body fluids considered to be infectious: CSF, synovial fluid, pleural fluid, peritoneal fluid pericardial fluid, amniotic fluid, saliva from dental procedures, other fluids containing visible blood

HIV - 2 TYPES

1. HIV-1

• identified by _

• _ are responsible for majority of HIV-1 infections worldwide

• Former names:

• identified by Luc Montagnier of FRance and Robert Gallo and Jay Levy of US

• GROUP M are responsible for majority of HIV-1 infections worldwide

• Former names:

→Human T-cell Lymphotropic Virus III (HTLV-III)

→Lymphadenopathy-associated Virus (LAV)

→AIDS-associated Retrovirus (ARV)

HIV - 2 TYPES

2. HIV-2

• it is related but distinct virus from HIV-1

• endemic in _

• less pathogenic and has lower rates of transmission

• it is related but distinct virus from HIV-1

• endemic in West Africa

• less pathogenic and has lower rates of transmission

Structural Genes of HIV (3)

• Env (envelope antigen)

• Gag (group antigen)

• Pol (polymerase)

Structural Genes of HIV

1. Env (envelope antigen)

• found in _

• codes for _, has 2 subunits:

→_ - knobs/ spikes - for attachementy to CD4

→_ - spans inner and outer membrane, fuse viral envelope with host cell membrane

→involved in the fusion and attachemnt of HIV to CD4+ cells

1. Env (envelope antigen)

• found in VIRAL ENVELOPE

• codes for gp160 has 2 subunits:

→gp120 - knobs/ spikes - for attachementy to CD4

→gp41- spans inner and outer membrane, fuse viral envelope with host cell membrane

→involved in the fusion and attachemnt of HIV to CD4+ cells

Structural Genes of HIV

2. Gag (group antigen)

• located in _

• codes for _

• located in nucleocapsid

• codes for p55 (structural proteins: p6, p9, p17, p24)

Structural Genes of HIV

3. Pol (polymerase)

• codes for enzymes necessary for replication (_ and _)

→ _ - transcribe RNA to DNA

→ _ - degradation of HIV RNA

→_ - inserts viral DNA into genome of infected host cells

→_ - cleaves structural proteins used to make mature virions

• codes for enzymes necessary for replication (p66 and p51)

→ Reverse transcriptase (p51) - transcribe RNA to DNA

→ Ribonuclease (p66) - degradation of HIV RNA

→Integrase (p31) - inserts viral DNA into genome of infected host cells

→Protease (p10) - cleaves structural proteins used to make mature virions

Effects of HIV Infection on Immune System

• HIV receptor: _

• HIV co-receptor: _

• prime target cells are _ - HALLMARK OF HIV INFECTION

• decresaed in cytotoxic T cell activity

• decrease monocyte/macrophage chemotaxis and decrease NK cells activity

• HIV downregulate the production of _ on surface of host cell it infects

• initial viral replication results to increased levels of _

• B lymphocytes are stimulated to produce antibodies to HIV which can usually be detected in host serum by _ weeks after primary infection

→the first antibodies to be detected are directed against _ followed by antibodies against gag proteins like p24

• HIV receptor: CD4

• HIV co-receptor: CXCR4 and CCR5

• prime target cells are T helper - HALLMARK OF HIV INFECTION

• decresaed in cytotoxic T cell activity

• decrease monocyte/macrophage chemotaxis and decrease NK cells activity

• HIV downregulate the production of CLASS I MHC on surface of host cell it infects

• initial viral replication results to increased levels of p24

• B lymphocytes are stimulated to produce antibodies to HIV which can usually be detected in host serum by 6 weeks after primary infection

→the first antibodies to be detected are directed against gp41 followed by antibodies against gag proteins like p24

HIV INFECTION

1. PRIMARY INFECTION

• there is high levels of virus (viremia)

• flu-like symptoms, fever, lympadenopathy, myalgia, sore throat, arthralgia, weight loss, fatigue

• symtoms usually appear _ after infection

3 to 6 weeks

HIV INFECTION

2. LATENT INFECTION

• there is decreased viremia

• virus is stillmpresent, only at lower levels

• absence of clinical symptoms, median length of _

10 years

HIV INFECTION

3. AIDS

• profound immunosuppression

• CD4+ count of less than

less than 200 cells /uL

LAB WORK-UP FOR HIV

1. CD4+ T CELL ENUMERATION

• Normal: _

• ratio of CD4 to CD8 should be _

• gold standard for enumerating CD4 count is _

• Normal: 450 - 1500 cells/uL

• ratio of CD4 to CD8 should be 2:1

• gold standard for enumerating CD4 count is FLOW CYTOMETRY

LAB WORK-UP FOR HIV

2. Testing Algorithm by CDC

• Screening: _

• Confirmatory: _

• Discrepant results: _

• Screening: Rapid Combination Immunoassay (ELISA/CLIA)

• Confirmatory: Rapid Differentiation Immunoassay (ELISA/CLIA)

• Discrepant results: Nucleic Acid Amplification Test

LAB WORK-UP FOR HIV

3. Western Blot

• prepared commercially by _ or _ containing individual HIV proteins

• any HIV antibodies present will bind their corresponding antigen on test membrane

• result should be reported as POSITIVE if at least _ out of 3 bands are present: (3)

• prepared commercially by NITROCELLULOSE or NYLON STRIPS containing individual HIV proteins

• any HIV antibodies present will bind their corresponding antigen on test membrane

• result should be reported as POSITIVE if at least 2 out of 3 bands are present: (3)

→p24

→gp41

→gp120/gp160

LAB WORK-UP FOR HIV

4. HIV ANTIGEN DETECTION

• _ antigen testing through caoture EIA

• levels of this antigen in circulation are thought to correlate with amount of HIV replication

p24

LAB WORK-UP FOR HIV

5. Rapid HIV Diagnostic Algorithm (rHIVda)

• shall be done in sequence of _

• HIV POSITIVE result shall be released if _

• shall be done in sequence of three HIV RDTs

• HIV POSITIVE result shall be released if three rHIVda RDT result are all reactive (T1+, T2+, T3+)

LAB WORK-UP FOR HIV

6. NUCLEIC ACID TESTING

→Qualitative

• used to determine whether or not detectable HIV nucleic acid is present in human plasma

• beneficial in cases where serological results are or in diagnosis of

→Quantitative / Viral Load Test

• thru _ - amplifies complimentary DNA generated from HIV RNA

• viral load tests areused routinely to monitor effectiveness of antiretroviral theraphy among patients

→Qualitative

• used to determine whether or not detectable HIV nucleic acid is present in human plasma

• beneficial in cases where serological results are inconclusive or in diagnosis of infants less than 18 months

→Quantitative / Viral Load Test

• thru Reverse Transcriptase - Polymerase Chain Reaction - amplifies complimentary DNA generated from HIV RNA

• viral load tests areused routinely to monitor effectiveness of antiretroviral theraphy among patients

_ are biological substances that are increased in blood, body fluids, or tissues of patients with a particular type of cancer

tumor markers

Tumor markers have four major clinical applications:

• Screening

• Diagnosis

• Prognosis

• Monitoring

Tumors possess antigens that are recognized as foreign by immune system. They can be broadly classified into two groups:

• TSA (Tumor Specific Antigen)

• TAA (Tumor Associated Antigen)