Immunology-Serology Notes PDF

IMMUNOLOGY-SEROLOGY IMMUNOLOGY HUMORAL IMMUNITY CELL-MEDIATED IMMUNITY Mechanism Antibody-...

IMMUNOLOGY-SEROLOGY IMMUNOLOGY HUMORAL IMMUNITY CELL-MEDIATED IMMUNITY Mechanism Antibody-mediated Cell mediated Immunology – study of a host’s reactions when foreign substances are introduced Cell type B lymphocytes T lymphocytes into the body. Mode of Antibodies in serum Direct cell-to-cell contact or soluble action products excreted by cells Antigen – A foreign substance that induces an immune response Purpose Primary defense against Defense against fungal and viral BODY DEFENSES bacterial infection infections, intracellular organisms, tumor antigens, and graft rejection First line of defense: Structural barriers - Unbroken skin (pH of 5.6)  Lactic acid in sweat CELLS OF THE IMMUNE SYSTEM  Fatty acids from sebaceous glands - Mucosal surfaces Neutrophils  Mucous secretion and motion of cilia in nasopharyngeal passages - 50-70% of total WBCs; has 2-5 lobes  Flushing action of urine and its acidity - Contains neutral staining granules:  Lactic acid production of female genital tract Primary Granules Secondary Granules Tertiary Granules  Hydrochloric acid production in the GIT (pH of 1)  Myeloperoxidase  Collagenase  Gelatinase  Lysozyme in tears and saliva  Elastase  Lactoferrin  Plasminogen  Proteinase 3  Lysozyme activator  Lysozyme  NADPH oxidase Second Line of defense: Natural/Innate Immunity  Defensins - Ability of the individual to resist infection by means of normally present body - Acid hydrolases are found in separate compartments called lysosomes. functions. - No prior exposure is required, and the response does not change with Eosinophils subsequent - 1-3% of total WBCs exposures. - Eccentric and bilobed nucleus; large orange to reddish orange granules - Involved in neutralizing basophil & mast cell products, killing parasites, allergic Third line of defense: Acquired/Adaptive Immunity reactions - Characterized by specificity for each individual pathogen, or microbial agent Basophils - Ability to remember a prior exposure, which results in an increased response - Less than 1% of total WBCs upon repeated exposure - Contain coarse, deep-bluish purple granules that often obscure the nucleus Components of Natural Components of Adaptive - Granules contain histamine, heparin, and ECF-A; granules lack hydrolytic Immune System Immune System enzymes, although peroxidase is present - Binds IgE Cellular Neutrophils T-cells - Exist for only a few hours in the bloodstream Mast cells B-cells Macrophages Plasma Cells Mast Cells Humoral Complement Cytokines - Resemble basophils, but are connective tissue cells and are larger, with a small Lysozyme Antibodies round nucleus and more granules Interferon - Life span: 9 to 18 months - Enzymes: acid phosphatase, alkaline phosphatase, and protease granules - Also binds IgE 1  IMMUNOLOGY-SEROLOGY Monocytes  Margination – from center to periphery of blood vessels - 4-10% of total WBCs; largest cells in the peripheral blood (70 hours)  Diapedesis/Transmigration – migration through endothelium to the tissues - Irregularly folded or horseshoe-shaped nucleus - Cytoplasm stains dull grayish blue with a ground-glass appearance. Inflammation - Granules peroxidase, acid phosphatase, and arylsulfatase, β-glucuronidase, - Overall reaction of the body to injury or invasion by an infectious agent lysozyme, lipase, but no alkaline phosphatase. - The four cardinal signs or clinical symptoms: redness, swelling, heat, pain - Becomes macrophage once migrated in tissues - Major events in inflammation: 1. Increased blood supply to the infected area Tissue Macrophages 2. Increased capillary permeability caused by retraction of endothelial cells - Arise from monocytes; contain no peroxidase 3. Migration of WBCs, mainly neutrophils, from the capillaries to the tissue - Macrophages have specific names according to their location: 4. Migration of macrophages to the injured area  Alveolar macrophage - lungs  Kupffer cells - liver  Microglial cells - brain ACUTE PHASE REACTANTS  Histiocytes - connective tissue. - Monocyte–macrophage system plays an important role in initiating and C-Reactive Protein regulating the immune response. - Originally thought to be antibody to c-polysaccharide of pneumococci - Functions include microbial killing, tumoricidal activity, intracellular parasite - Most widely used indicator of acute inflammation eradication, phagocytosis, secretion of cell mediators, and antigen presentation. - 1000x increase within 4-6 hours following inflammation - acts somewhat like an antibody, as it is capable of opsonization, agglutination, Dendritic Cells precipitation, and activation of complement by the classical pathway - Most potent phagocyte - 2 mg/L: threshold for high cardiovascular risk - Main function is antigen presentation to T cells - Classified according to tissue location: Serum Amyloid  Langerhans cells - skin and mucous membranes - Associated with HDL-C; plays a role in metabolism of cholesterol  Interstitial dendritic cells - heart, lungs, liver, kidney, GIT - 1000x increase within 24 hours - Increases significantly more in bacterial infections than in viral infections Toll- Like Receptors - Toll: protein found on Drosophila Complement - Highest concentration found on monocytes, macrophages, neutrophils - Main function is mediation of inflammation  TLR-1 – Mycobacteria Mannose-Binding Protein (MBP)  TLR-2 – Gram-positive bacteria - Widely distributed on mucosal surfaces  TLR-4 – Gram-negative bacteria - Lack of MBP is associated with recurrent yeast infections  TLR-5 – Bacterial flagellin Alpha1-Antitrypsin Phagocytosis - Plasma inhibitor of protease 1. Chemotaxis: unidirectional movement of leukocytes to the site of injury - Counteracts the effects of neutrophil invasion during inflammation 2. Adherence: physical contact between the white cell and the foreign particle - A1-AT deficiency  emphysema, juvenile cirrhosis 3. Engulfment: pseudopods extend around the pathogen 4. Phagosome formation Haptoglobin 5. Formation of phagolysosome - Binds to free hemoglobin released during intravascular hemolysis 6. Digestion: release of digestive enzymes to destroy ingested materials - Protects kidney from damaging effects of hemoglobin 2  IMMUNOLOGY-SEROLOGY Fibrinogen  Cortex - Most abundant coagulation factor (200-400 mg/Dl)  Primary follicles: mature & resting B cells, dendritic cells, - Forms the fibrin clot macrophages - Associated with increased risk for coronary artery disease  Secondary follicles: activated B cells  Germinal centers: plasma cells, memory B cells Ceruloplasmin  Paracortex: contains T cells - Binds and transports copper  Medulla - Acts as a ferroxidase, oxidizing iron from Fe2+ to Fe3+ to release iron from ferritin for binding to transferrin Other Secondary Lymphoid Organs - Ceruloplasmin deficiency  Wilson’s disease  MALT (Mucosal-Associated Lymphoid Tissue) - - Found in gastrointestinal, respiratory, and urinary tract - Peyer’s patches: specialized type of MALT located in lower ileum of GIT THE LYMPHOID SYSTEM  Tonsils PRIMARY LYMPHOID ORGANS  Appendix  CALT (Cutaneous-Associated Lymphoid Tissue)  Bone Marrow - Considered as the largest tissues in the body - Fills the long bones; main source of hematopoietic stem cells STAGES IN B-CELL DIFFERENTIATION: - Site of B-cell maturation 1. Pro-B Cell  Thymus - Surface antigens: CD19, CD45R, CD43, CD24, and c-Kit. - Small, flat, bilobed organ located in the thorax/chest cavity - Intracellular proteins: terminal deoxyribonucleotide transferase (TdT) and RAG- - Site of T-cell maturation 1 and RAG-2, which code for enzymes involved in gene rearrangement - Heavy chain rearrangement on chromosome 14 SECONDARY LYMPHOID ORGANS 2. Pre-B Cell  Spleen - Lose CD43, C-kit, and TdT - Largest secondary lymphoid organ - First heavy chain synthesized are μ chains - Acts as a discriminating filter: removes old and damaged cells and foreign - Express μ chains and surrogate light chains antigens from the blood - 2 main types: 3. Immature B Cell  Red pulp – destroys RBCs - Complete surface IgM molecules  White pulp – contains lymphoid tissue arranged around arterioles in - Completion of light chain rearrangement on chromosome 2 and 22 PALS - Surface proteins: CD21, CD40, MHC Class II  PALS: contain T cells  Primary follicles: contain B cells 4. Mature B Cell  Marginal zone: contain dendritic cells - Complete surface IgM and IgD molecule - Known as marginal zone B cells in the spleen and follicular B cells in other  Lymph Nodes secondary lymphoid organs - Filters fluid from tissues - It has a short lifespan of a few days unless activated by antigen - Numerous near joints and where arms and legs join the body - When stimulated, it undergoes transformation to a blast which eventually forms - Lymph fluid flows through spaces called sinuses, which are lined with memory cells and plasma cells macrophages, creating an ideal location for phagocytosis - Layers: 3  IMMUNOLOGY-SEROLOGY 5. Activated B Cells  CD4+: Helper T cells - Exhibit CD25 - Recognize MHC Class II - Occurs when antigens cross-link several surface immunoglobulins on B cells - Secrete cytokines which enhance antibody production by B cells - Orchestrator of effector mechanisms of the immune response 6. Plasma Cells - Subsets: - Ellipsoidal/spherical cells with eccentric nucleus containing heavily clumped,  Th1 – responsible for cell-mediated effector mechanisms; produce dark staining chromatin IFN-γ and TNF-β to protect against intracellular pathogens - Exhibit abundant cytoplasmic immunoglobulins with little to no surface  Th2 – plays a role in regulation of antibody production; protects immunoglobulins against extracellular pathogens - Most differentiated lymphocyte  Treg – immunoregulatory T cell; possess CD4 and CD25; produce - Main function: Antibody production IL-10 and TGF-β 7. Memory Cells 4. Activated T Cells - Progeny of antigen-stimulated B cells capable of responding to antigen with - When antigen is recognized, T cells are transformed into large activated cells increased speed and intensity - Express receptors for IL-2 - T lymphoblasts differentiate into functionally active small lymphocytes that STAGES IN T-CELL DIFFERENTIATION: produce cytokines  Cell-mediated immunity 1. Double Negative Thymocytes Comparison of T Cells and B Cells - Lack CD4 and CD8 T Cell B Cell - Beta chain rearrangement Develop in thymus Develop in bone marrow - Pre-T cell receptor = CD3 + Beta-chain Found in the blood, thoracic duct fluid Found in bone marrow, lymph node, 2. Double Positive T Cell lymph nodes, spleen - Express both CD4 and CD8 End product of activation are cytokines End product of activation are - Alpha chain rearrangement antibodies - When CD3-alpha/beta receptor complex (TCR) is expressed on the cell surface, Antigens: CD2, CD3, CD4, CD8 Antigens: CD19, CD20, CD21, CD40, positive selection takes place MHC Class II  Positive Selection Located in paracortex of lymph nodes Located in cortex of lymph nodes - Allows only double-positive T cells with functional T cell receptors and capable of recognizing and binding self-MHC to survive - Those unable to recognize self-antigens die NATURAL KILLER CELLS  Negative Selection - 3rd population of lymphocytes; small percentage of lymphocytes that do not - Takes place on surviving double-positive T cells express the markers of either T cells or B cells - Those that react to self-antigens undergoes apoptosis - 5-10% of circulating lymphocytes - Bridge between innate and acquired response 3. Mature T Cells - Mediate cytolytic reactions and kill target cells without prior exposure to them - Exhibit either CD4 or CD8 - Surface antigens: - CD8+: Cytotoxic/Killer T cells  CD16: receptor for the fragment crystallizable portion - Recognize MHC Class I  CD56 - Kills virally infected cells  CD94: involved in inhibition of NK cell cytotoxicity 4  IMMUNOLOGY-SEROLOGY Laboratory Identification of Lymphocytes RELATIONSHIP OF ANTIGENS TO THE HOST - Density gradient centrifugation with Ficoll-Hypaque (SG: 1.077 and 1.114)  Autoantigens – antigens belonging to the host - Specimen: diluted defibrinated or heparinized blood  Alloantigens – antigen from other members of the host’s species - Three distinct layers:  Heteroantigens – antigens from other species plasma  Heterophile antigens – heteroantigens that exist in unrelated plants or animals mononuclear cells but are either identical or closely related in structure so that antibody to one will RBCs and WBCs cross react with the antigen of the other Adjuvants Automated Method: Cell Flow Cytometry - substance administered with an immunogen that increases immune response - Identifying cells based on the scattering of light as cells flow in single file through (ex. Aluminum salts, Freund’s complete adjuvant) a beam layer - Freund’s complete adjuvant: mineral oil, emulsifier, killed mycobacteria - Fluorescent antibodies are used to screen for subpopulations, such as B cells, T helper cells, and T cytotoxic cells. Mitogens – substance the stimulate cell division  Phytohemagglutinin – T cell Manual Method: Rosette Technique  Lipopolysaccharide – B cell 1. Lymphocytes are separated from whole blood and then mixed with a suspension  Pokeweed mitogen – T and B cell of sheep RBCs. If three or more RBCs are attached to a lymphocyte, it is considered a rosette. 2. Sheep cells attach to the CD2 antigen, found only on T cells. MAJOR HISTOCOMPATIBILITY COMPLEX 3. Count 200 cells using a counting chamber and the percent forming rosettes is - Also referred to as Human Leukocyte Antigen (HLA) calculated - Main function: bring antigen to the cell surface for recognition by T cells - Most polymorphic system found in humans NATURE OF ANTIGENS MHC Genes The ability of an immunogen to stimulate a host response depends on the ff - Found on short arm of chromosome 6 characteristics: - Inherited as haplotypes; expressed codominantly 1. Macromolecular size (MW: 10,000- 100,000) - Three categories/classes: 2. Chemical composition and molecular complexity  MHC Class 1 – coded at 3 different loci: A, B, C  Proteins  MHC Class 2 – situated in the D region (DP, DQ, DR)  Carbohydrates  MHC Class 3 – located between class I and II regions; codes for  Lipids complement and cytokines (TNF); secreted proteins but not expressed on  Nucleic acids cell surfaces 3. Foreignness 4. Ability to be processed and presented with MHC molecules MHC CLASS I MOLECULES Structure: Haptens - Expressed on all nucleated cells - nonimmunogenic materials that, when combined with a carrier, create new - Highest on lymphocytes; and low or undetected on hepatocytes, neural cells, antigenic determinants (ex. Poison ivy) muscle cells, and sperm - Therapeutic drugs and hormones can function as haptens - Made up of α-chain (α1, α2, α3) and β2-microglobulin - Nonclassical class I antigens: E, F, G (G antigens are expressed on trophoblast cells during pregnancy 5  IMMUNOLOGY-SEROLOGY Processing: Nature of Light Chains - Presents endogenously processed antigens (viral or cytosolic proteins) to - 2 main types of light chains: kappa and lambda CD8+ T cells - Bence Jones Proteins - Both class I and class II molecules are synthesized in the rough endoplasmic  L chains secreted by malignant plasma cells found in the urine of patients reticulum, but only class I molecules bind peptides in the endoplasmic reticulum. with multiple myeloma Upon combining with MHC class I, the peptide-MHC complex is transported  Precipitate at 60°C, redissolves at 80°C through the Golgi complex to the cell surface. Heavy Chain Sequencing MHC CLASS II MOLECULES - Isotype – unique amino acid sequence in the heavy chain that is common to Structure: all immunoglobulin molecule of a given class in a given species - Expressed primarily on APCs (B cells, monocytes, macrophages, and dendritic - Allotype – minor variations in the constant region in some individuals cells) - Idiotype – variations in the variable region of each chain unique to a specific - Made up of α-chain and β-chain antibody molecule - Nonclassical class II antigens: DM, DN, DO Hinge Region Processing: - Located between CH1 and CH2 - Presents exogenously processed antigens (bacterial proteins) to CD4+ T cells - Has a high content of proline and hydrophobic residues allowing for flexibility - The binding site of MHC class II molecules are first occupied by an invariant - Only delta, gamma, alpha chains have a hinge region, mu and epsilon do not. chain (Ii). Invariant chain is degraded by a protease, leaving just a small fragment called class II invariant chain peptide (CLIP) attached to the peptide-binding IgG cleft. CLIP is then exchanged for exogenous peptides. The exogenous peptide- - Predominant immunoglobulin in humans and has the longest half life MHC class II complex is then transported to the cell surface. - Secondary response antibody - The four subclasses mainly differ in the number & position of disulfide bridges  Complement fixation: IgG3 IgG1 IgG2 ANTIBODY STRUCTURE AND FUNCTION  Crossing placenta: IgG1 IgG3 Ig4 IgG2 - Immunoglobulins are end products of B cells found in the serum - Major functions: - Appear in the gamma band when subjected to electrophoresis at pH 8.6  Providing immunity for the newborn  Complement fixation, opsonization, neutralizing toxins and viruses, STRUCTURE precipitation and agglutination - Antibody structure was elucidated by Gerald Edelman and Rodney Porter - Tetrapeptide consisting of 2 heavy chain & 2 light chains linked by disulfide IgM bonds. - Known as macroglobulin; found mainly in the intravascular pool because of its - Variable region = amino terminal end; constant region = carboxy terminal end large size - Papain – cleaves immunoglobulin into 3 fragments - Pentamer form is found in secretions; 5 monomeric units held by J chain  1 Fc (fragment crystallizable) – for effector functions including - Monomer form is found on the surface of B cells opsonization and complement activation. - Assumes a starlike shape with 10 functional antigen-binding sites  2 Fab (fragment antigen-binding) – for antigen binding; consists of 1 light - Primary response antibody chain and ½ heavy chain - Functions of IgM: - Pepsin – cleaves immunoglobulin at the carboxy-terminal side yielding F(ab)2  Complement fixation, agglutination, opsonization, toxin neutralization and Fc’ 6  IMMUNOLOGY-SEROLOGY IgA Primary Antibody Response: - Two subclasses of IgA: 1. Lag Phase – no detectable antibody  IgA1 – predominant form found as a dimer joined by a J chain; found in serum 2. Log Phase – antibody titer increases logarithmically  IgA2 – mainly found in secretions 3. Plateau Phase – antibody titer stabilizes - Secretory component (SC) facilitates transcytosis of IgA across mucosal 4. Decline Phase – antibody is catabolized surfaces and protects Fc portion from enzymatic digestion - Main function: patrol mucosal surfaces and act as a first line of defense Secondary (Anamnestic) Antibody Response - Aggregation of IgA immune complexes trigger alternate complement pathway - response differs from a primary response: - Capable of acting as opsonins 1. Shorter lag phase, longer plateau, and more gradual decline. 2. IgG is the predominant type formed IgD 3. Higher antibody titer (typically 10-fold or greater than the primary response) - Found on the surface of immunocompetent but unstimulated B lymphocytes - 2nd type of immunoglobulin to appear (IgM being the first) - Plays a role in B-cell activation, maturation and differentiation MONOCLONAL ANTIBODY - Very specific antibody rising from a single plasma cell that has been cloned or IgE duplicated - Least abundant Ig in the serum - Discovered by Georges Kohler and Cesar Milstein - Most heat labile (heating to 56°C for 30 minutes to 3 hours results in - Fusion an activated B cell with a myeloma cell that can be grown indefinitely in conformational changes and loss of ability to bind to target cells) the laboratory. Myeloma cells are cancerous plasma cells. - Attaches to basophils and tissue mast cells by means of high affinity receptors - Mediates antihelminthic responses and induces type I hypersensitivity Hybridoma Production 1. Mouse is immunized with a specific antigen, then the spleen is harvested 2. Spleen cells are fused with myeloma cells in the presence of polyethylene glycol (PEG), producing hybridoma IgG IgM IgA IgD IgE 3. After fusion, cells are placed in culture containing hypoxanthine, Percent of total 70-75% 10% 10-15% 200 IU/mL - Reagent: cardiolipin, cholesterol, lecithin 2. AntiDNase B Testing 2. Rapid Plasma Reagin (RPR) - useful in patients suspected of having glomerulonephritis preceded by - Uses unheated serum, result is read macroscopically streptococcal skin infections - Principle: flocculation - Principle: Neutralization - Reagent: colorless alcoholic solution containing cardiolipin, lecithin, - Presence of DNase is measured by its effect on a DNA-methyl-green charcoal, choline chloride and thimerosal conjugate TREPONEMAL TESTS - 4+ = intensity of color is unchanged; 0 = total loss of color. - detect antibody directed against the T. pallidum organism or against specific treponemal antigens. 3. Streptozyme test - measures antibodies against five extracellular streptococcal antigens 1. FTA-ABS (Fluorescent Treponemal Antibody Absorption Test)  anti-streptolysin (ASO), anti-hyaluronidase (AHase), anti-streptokinase - one of the earliest confirmatory tests (ASKase), anti-nicotinamide-adenine dinucleotide (anti- NAD), anti- - Principle: Indirect Fluorescent immunoassay DNAse B antibodies. - Reagent antigen: Nichol’s strain dried and fixed on slide - positive in 95% of patients with acute poststreptococcal glomerulonephritis - Absorbent: Reiter treponemes – removes cross reactivity to other caused by GAS pharyngitis. treponemes 20  IMMUNOLOGY-SEROLOGY 2. MHA – TP (micro-hemagglutination T. pallidum Test) Acute Hepatitis B infection - Principle: Hemagglutination - Reagent antigen: tanned formalin sheep RBC coated w/ treponemal antigen 3. T pallidum particle agglutination (TP-PA) test - uses colored gelatin particles coated with treponemal antigens and are more sensitive in detecting primary syphilis VIRAL DISEASES Hepatitis Viruses Hepatitis Serologic & Family Transmission Chronic Hepatitis B infection Virus Molecular Markers IgM anti-HAV Fecal-oral A Picornaviridae Total anti-HAV Blood transfusion (rare) HAV RNA HBsAg HBeAg IgM anti-HBc Parenteral, sexual, B Hepadnaviridae Total anti-HBc perinatal Anti-HBe Anti-HBs HBV DNA Parenteral, sexual, Anti-HCV C Flaviviridae perinatal HCV RNA Laboratory Detection Hepatitis Virus Methods Mostly parenteral, but IgManti- HDV D Deltavirus also sexual, perinatal; IgGanti- HDV Anti-HAV – chemiluminescent microparticle immunoassays A HBV infection required HDV RNA HAV RNA – RT-PCR Anti-HBV – EIA, CLIA IgM anti-HEV B Fecal-oral HBV DNA – qPCR, bDNA signal amplification E Hepaviridae IgG anti-HEV Blood transfusion Anti-HCV – EIA, CLIA, rapid immunoblot assays HEV RNA C HCV RNA – RT-PCR, qPCR, NAT, Anti- HDV – Immunoassays D HDV RNA – RT-PCR Anti- HEV – ELISA E HEV RNA – RT-PCR 21  IMMUNOLOGY-SEROLOGY Human Immunodeficiency Virus Laboratory Tests - Genus: Lentivirinae Serological Tests: - Family: Retroviridae 1. ELISA - Mode of Transmission: intimate sexual contact, contact with blood or other body - Standard screening method for HIV antibody fluids, or perinatally (from infected mother to infant). Generation Principle Detects Structure of HIV: 1 st Indirect ELISA using viral lysate The genome of HIV includes three main structural genes—gag, env, and pol—and a HIV-1 antibodies antigens number of regulatory genes Indirect ELISA using highly 2nd purified recombinant from HIV-1 HIV-1 & HIV-2 antibodies and HIV-2 Sandwich ELISA using 3rd HIV-1 & HIV-2 antibodies recombinant HIV-1 and HIV-2 Sandwich ELISA using recombinant HIV-1 & HIV-2 HIV-1 & HIV-2 antibodies, 4th antigens, and monoclonal p24 antigen antibody to HIV-1 p24 2. Western Blot - Was the standard confirmatory test - prepared commercially as nitrocellulose or nylon strips containing individual HIV proteins that have been separated by polyacrylamide gel electrophoresis and blotted onto the test membrane. Gene Product Function gag p17 Inner surface of envelope P24 Core coat for nucleic acids reported as positive if at least 2 of the following 3 bands are P9 Core-binding protein present: P6 Binds to genomic RNA p24, gp41, and gp120/gp160 env gp120 Binds to CD4 on T cells gp41 Transmembrane protein associated with gp120 pol p66 Subunit of reverse transcriptase; degrades original HIV RNA p51 Subunit of reverse transcriptase p31 Integrase; mediates integration of HIV DNA into host genome p10 Protease that cleaves gag precursor 22  IMMUNOLOGY-SEROLOGY Disease Monitoring 1. CD4 T cell - Flow cytometry is the gold standard for enumeration of CD4 T cells - Lymphocytes are selected for analysis (or “gated”) on the basis of their low- side scatter and ability to stain brightly with CD45 antibody Stages CD4 T cell count (cells/μL) 1: Acute HIV ≥500 2: Chronic HIV 200-499 3: AIDS