Immunoserology - Serological Tests (Part 2) PDF

TOPIC: SEROLOGICAL TESTS (PART 2) EVALUATE COMPLEMENT INSTRUCTOR: ZESIL E. GELLE, RMT F. CH50 HEMOLYTIC ASSAY G. AH50 HEMOL...

TOPIC: SEROLOGICAL TESTS (PART 2) EVALUATE COMPLEMENT INSTRUCTOR: ZESIL E. GELLE, RMT F. CH50 HEMOLYTIC ASSAY G. AH50 HEMOLYTIC ASSAY OUTLINE OF THE LESSON H. FUNCTIONAL ASSAY FOR MB LECTIN PATHWAY I. IMMUNOASSAYS I. IMMUNOASSAYS FOR II. LABELED IMMUNOASSAY COMPLEMENT COMPONENT III. TYPES OF LABEL J. COMPLEMENT FIXATION IV. SEPARATION METHOD XIII. FUNCTIONAL ASSAY V. BASIC COMPONENTS OF LABELED A. CELL SEPARATION TECHNIQUE IMMUNOASSAY - DENSITY GRADIENT VI. DEFINITION OF TERMS SEPARATION\ A. RADIOACTIVE LABELS - MAGNETIC SEPARATION B. ENZYME LABELS - PANNING C. FLUOROCHROME LABELS - NYLON WOOL COLUMN D. CHEMILUMINESCENT (STRAW METHOD) VII. METHODS OF COUPLING LABELS XIV. CELL SORTING FACS WITH AG OR AB A. FUNCTIONAL ASSAY VIII. METHODS OF BINDING ABS TO SOLID B. CELL CULTURE PHASE C. LYMPHOCYTE STIMULATION BY IX. CLASSIFICATION AND FORMATS MITOGENS A. COMPETITIVE ASSAYS D. CYTOTOXIC ASSAYS B. NONCOMPETITIVE ASSAYS E. 51Cr RELEASE ASSAY X. ENZYME IMMUNOASSAY XV. EVALUATION OF NEUTROPHIL A. HETEROGENOUS EIA FUNCTION - COMPETITIVE EIA A. NITROBLUE TETRAZOLIUM - NONCOMPETITIVE EIA BLOOD (NBT) TEST - CAPTURE ASSAY B. CHEMOTAXIS ASSAY B. HOMOGENEOUS EIA C. T-CELL ENUMERATION ASSAY - ENZYME MULTIPLIED D. ERYTHROCYTE ROSETTE ASSAY IMMUNOASSAY TECHNIQUE E. T CELL SUBSETS (EMIT) XVI. IDENTIFICATION OF SPECIFIC - DIFA ?? ALLERGENS IN TYPE 1 - Indirect IFAA ?? HYPERSENSITIVITY REACTION - Fluorescence Polarization A. RADIOIMMUNOSORBENT TEST Immunoassay ?? (RIST) C. CHEMILUMINESCENCE B. RADIOALLERGOSORBENT TEST D. RADIOIMMUNOASSAY (RAST) E. COMPETITIVE BINDING XVII. SPECIFIC DISEASE AND ASSOCIATED IMMUNOASSAY LAB TESTS XI. IMMUNOCHROMATOGRAPHY A. HEPATITIS B XII. SPECIAL TECHNIQUES B. HEPATITIS A A. FLOW CYTOMETRY C. HEPATITIS C B. CLINICAL APPLICATION D. HEPATITIS D (DELTA) VIRUS - CELL SORTING E. HIV - IMMUNOPHENOTYPING - EARLY PHASE OF HIV - HLA PHENOTYPING INFECTION C. DNA CONTENT - LATE PHASE OF HIV - DNA PLOIDY INFECTION - DNA CELL CYCLE ANALYSIS - VIRAL REPLICATION - LEUKOCYTE - AIDS CROSSMATCHING - LAB DIAGNOSIS OF HIV D. COMPLEMENT ASSAYS INFECTION E. TWO TYPES OF TEST TO F. ELISA ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 1  G. WESTERN BLOT Aaaaa - Main topic (Main definition) H. DETECTION OF p24 HIV ANTIGEN Aaaaa - Sub-topics ➔ (Sub-definition / I. PCR AAAA - Title of Topic Enumerate) J. NASBA ❖ Additional detail K. VIRUS ISOLATION L. TESTING NEONATES NOTE: (for additional XVIII. SPECIFIC DISEASE STATES information during A. SYPHILIS discussions) B. NON-TREPONEMAL TESTS FOR SYPHILIS - VDRL IMMUNOASSAYS - RPR WHAT ARE IMMUNOASSAYS? - ART ➜ Methods that use antigen-antibody C. USES OF NON-TREPONEMAL reaction to detect and/or quantify antigen, TEST FOR SYPHILIS antibodies, and other molecules D. TREPONEMAL TESTS FOR NOTE: SYPHILIS Direct - Ag - ANTIGENS Indirect - Ab - DARK FIELD MICROSCOPY Sandwich - often times Ag but Ab can be - FTA-Abs sandwiched by 2 Ag. - TPI (T. pallidum Immobilization test) LABELED IMMUNOASSAY - MHA-TP (Microhemagglutination Use specific labels such as enzyme, radioactive Assay for T. pallidum) isotopes, fluorescent, and chemiluminescent E. SENSITIVITY OF TESTS FOR substances in the detection of antigen-antibody SYPHILIS reaction. F. RHEUMATOID ARTHRITIS Develop to measure antigens and antibodies G. COLD AGGLUTININS small in size or present in very low H. ANTISTREPTOLYSIN O TEST concentrations (ASOT) Presence is determined by using a labeled - INDIRECT OR PASSIVE LATEX reactant AGGLUTINATION TEST - ASO HEMOLYTIC TITRATION ANALYTE TEST (NEUTRALIZATION (The substance to be measured) TEST) Antigens I. CRP PROTEIN Hormones J. INFECTIOUS MONONUCLEOSIS Drugs K. SEROLOGIC TESTING Tumor Markers L. PAUL-BUNNELL PRESUMPTIVE Immunoglobulins TEST (CLASSIC) Proteins M. PAUL-BUNNELL SCREENING TEST N. DAVIDSON DIFFERENTIAL NOTE: O. MONOSPOT TEST/ RAPID Labeled Immunoassays makes used of the DIFFERENTIAL SLIDE TEST primary phenomenon of Ag-Ab interaction P. EBV SPECIFIC TESTS where there epitope-paratope interaction. Q. FEBRILE ANTIGEN TESTS Abs can also serve as analyte because an - WIDAL TEST anti-antibody may be used to detect fore the - WEIL-FELIX TEST presence of an antibody. R. LYME DISEASE S. ANAs TYPES OF LABEL Fluorescent Radioactive HIGHLIGHTING BULLET PLACEMENT Chemiluminescent OF TOPICS Enzyme ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 2  ANTIBODIES DETECTION OF LABEL 1. High affinity 1. Radioimmunoassays: a. The higher the affinity of the ab for ag, the = system for counting radioactivity larger the amount of Ag bound to ab and Ex. scintillation counter the more accurately specific binding can 2. Enzymes, Fluorescence, Chemiluminescence: be measured = change in absorbance in a substrate b. Sensitivity is dependent on affinity measured by spectrophotometry 2. Specificity - to monoclonal abs Ex. Fluorescence - spectrofluorometer a. (Ab derived from a single B-cell clone) Chemiluminescence - luminometer REQUIREMENTS SEPARATION METHOD Labels must not alter the reactivity of Means of separating reacted from unreacted molecules. analytes Remain stable fro the shelf life of reagent Solid phase Physical NOTE: ➜ Agitation Vidas enzyme - ALP Wash step Vidas Substrate - 4-methylumbelliferone A combination of an enzyme with a NOTE: fluorescence product. (ELFA juanillo) Aka B/F Separation (bound free) STANDARD OR CALIBRATORS PHYSICAL MEANS Unlabeled analytes of known concentration Decanding Purpose: to establish a relationship between the Centrifugation labeled analyte measured and any unlabeled Filtration analyte that might be present in patient specimens. WASH STEP Determine concentration of unknown analyte. Remove any remaining unbound analyte Source of error NOTE: Point of comparison for the concentration of the SOLID PHASE VEHICLE unknown analyte in the sample. Polystyrene test tubes Standard serves as a basis for the computation Microtiter plates of the unknown. Glass or polystyrene beads Magnetic beads QUALITY CONTROL Cellulose membranes Blank tube: (usually PBS- Phosphate buffered Saline) NOTE: Negative control Solid phase is where either the Ag or Ab is High positive control attached to attach or attract either the Ag or Ab Low positive control that is present in the sample. NOTE: SEPARATION METHOD Control is a solution that may contain several Means of separating reacted from unreacted substances of known concentration. analyte Why is there a need to run a control? To check Solid phase whether you are in control of what you are Physical doing. Wash step CHOOSING A LABEL BASIC COMPONENTS OF LABELED Number of substrate molecules converted per IMMUNOASSAY molecule of enzyme Labeled reagent Ag or Ab Ease and speed of detection Analyte of interest Stability ➜ aka Ligand ➜ Can be either Ag or Ab ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 3  Reactants Appropriate standards Incubation step ➜ Reactants have to be allowed to react with each other Separation step ➜ aka washing or B/F separation Detection of labeled antigen-antibody complexes by appropriate measuring device FLUOROCHROME LABELS DEFINITION OF TERMS These are molecules that absorbs light and in Label - the molecule or an atom aka as a tag the process become transiently excited. signal or marker which is attached to reagent Solid phase : tissue/cell preparations (slides, Ag or Ab or other molecule capable of microbeads, dipstick) producing a signal. Detection: fluorometer, fluorescence microscope Ligand - any molecule (labeled or unlabeled) Examples: FITC, TMR, TRITC that can combine with its complementary Assay: IFA molecule to form an Ag-Ab complex Solid phase - solid support (beads or plastic) NOTE: that is used for immobilization of binder FITC - green molecules (Ag or Ab) during a reaction and TRITC - red separation process. CHEMILUMINESCENT RADIOACTIVE LABELS Chemiluminescent compounds - compound Radioactive Isotopes - are molecules with when oxidized produce light unstable nuclei and therefore emit radiation Examples: luminol, acridinium esters, dioxetane spontaneously phosphate Solid phase: tubes, polystyrene beads, Solid phase: magnetic particles, microtiter microtiter plate plates or gels Examples : 125I, 57CO, 3H Detection: Luminometer Detection: y-scintillation counter Assay: Chemiluminescence Assay (ChemLum Assay: Radioimmunoassays (RIA) or CLA) ENZYME LABELS Enzyme - substances or protein molecules METHODS OF COUPLING LABELS WITH AG OR that catalyze a biochemical reaction by reacting by AB an appropriate substrate to form a breakdown or 1. Tyrosine residues in either Ab or Ag: reaction product. radioisotopes Solid phase: tubes, polystyrene beads, 2. Glutaraldehyde (bifunctional reagent that microtiter plates covalently links amino acid residues) Examples: ALP, Horseradish peroxidase, 3. Biotin-avidin system: enzymes Galactosidase Detection: spectrophotometer picture : biospecific probe and biotin ➜ End product of an enzyme reaction is usually a colored product NOTE: Assay: EIA Biotin is a vitamin Streptavidin is a substance that serves like a ENZYME AND ITS SOURCES paste that can be sourced from streptococcus species or from an egg white. The purpose of the biotin-avidin system is to increase the amount of labels that can be attached to an Ag or Ab. METHODS OF BINDING ABS TO SOLID PHASE ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 4 1. Protein A: binds fc portion of human IgG ENZYMES ASSAY ARE CLASSIFIED AS EITHER subclasses, IgM, IgA, IgE 2. Protein G: binds the fc portion of IgG HETEROGENOUS EIA 3. Protein L: a protein receptor from COMPETITIVE EIA Peptostreptococcus magnus that binds to ENZYME LABELED antigen competes with kappa light chains without interfering with unlabeled patient antigen for a limited number antigen binding of binding sites on antibody molecules that are attached to a solid phase CLASSIFICATION AND FORMATS COMPETITIVE ASSAYS NOTE: Immunoassay in which patient Ag and labeled Heterogenous - a washing step is involved reagent Ag compete for a limited binding sites The relationship involved is INVERSELY on reagent Ab. PROPORTIONAL The amount of label measured is indirectly proportional to the amount of patient antigen NONCOMPETITIVE EIA present When ag is bound to solid phase, px serum with ➜ Antigen in samples unknown ab is added given time to react. After ➜ Antigen with designated label washing, an enzyme labeled antiglobulin is Less bound labeled antigen indicates more added which reacts with any px antibody that is antigen bound to the solid phase. After second washing, the enzyme substrate is added NONCOMPETITIVE ASSAYS Detects: presence of antibody This does not involve competition in binding The amount of labe detected is DIRECTLY sites and allows any patient Ag to be captured. PROPORTIONAL to the amount of antibody in More specific than competitive assay the specimen The amount of label measured is directly Involves 2 incubation and 2 washing steps proportional to the amount of patient antigen present NOTE: AKA ELISA NOTE: Direct, Indirect or Sandwich/Capture INDIRECT LABELED IMMUNOASSAY CAPTURE ASSAY Competitive Non-Competitive aka Sandwich Immunoassays Principle: Excess ab attached to solid phase is Mechanism Competition An excess allowed to combine with the test sample to between amount of an capture any antigen present. After an incubation labeled and antibody is used period, an enzyme labeled antibody is added. unlabelled to capture the This second ab recognizes a different epitope antigen for a analyte from the than the solid phase ab and completes the limited number sample sandwich of binding sites detects : presence of antigen on the Enzymatic activity is DIRECTLY antibody PROPORTIONAL to the amount of antigen in the test sample Relationship Indirect Direct relationship Involves capture Ab-Antigen Analyte-Enzyme between relationship labeled Antibody Label and analyte NOTE: DIRECT LABELED IMMUNOASSAY - analyte is antigen INDIRECT LABELED IMMUNOASSAY - analyte is antibody SANDWICH IMMUNOASSAY - analyte is antigen ENZYME IMMUNOASSAY ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 5 Types of Non Competitive ELISA (TABLE) has high intensity, good photostability and high quantum yield HOMOGENEOUS EIA Tetramethylrhodamine Used in determination of low molecular weight analytes such as hormones, therapeutic drugs NOTE: and drugs of abuse in both serum and urine Red is near the infrared region so red has a Sensitivity is determined by: higher wavelength range compared to green ➜ Detectability of enzymatic activity ➜ Change in the activity when antibody binds DIRECT IFA to antigen Antibody that is conjugated with a ➜ Strength of the antibody’s binding fluorescent tag is added directly to an unknown ➜ Susceptibility antigen that is fixed to a microscope slide. After an incubation ENZYME MULTIPLIED IMMUNOASSAY TECHNIQUE (EMIT) INDIRECT IFA REAGENT antigen is labeled with an enzyme Involves incubation of px serum with a tag and when ab binds to a specific determinant known antigen attached to a solid phase. The slide sites on the antigen, the active site of the is washed, and then an anti human immunoglobulin enzymes is blocked containing fluorescent tag. In the enzyme-mediated immunologic technique method, a drug enzyme complex is NOTE: used as the marker. Indirect IFA is applied in the detection of ANAs When bound to the anti drug antibody, the Direct and indirect IFA belongs to active site of the enzyme (linked to the drug) is HETEROGENEOUS fluorescence blocked. Therefore, when substrate is added, immunoassay no reaction will occur, as shown in Part 1. However, if free drug as in serum is present, FLUORESCENCE POLARIZATION some or most of the enzyme drug complex is IMMUNOASSAY displaced from the anti drug antibody. Homogenous Now the active sites of the liberated Labeled antigens compete with unlabeled ag in enzyme-drug complexes are free, and the the px sample for a limited no. of ab binding substrate undergoes reactions as indicated in sites. The more antigen that is present in the px Part 2. sample, the less fluorescence labeled ag is Negative result (analyte is absent - no color bound and the less the polarization that will be change detected. Positive reaction - color change Inversely proportional Therefor, the intensity of the reaction produced Negative increased polarization and the serum drug concentration have a direct Positive decreased polarization relationship Uses fluorometer NOTE: CHEMILUMINESCENCE IMMUNOASSAY HOMOGENEOUS COMPETITIVE The emission of light caused by a chemical IMMUNOASSAY reaction (typically oxidation) producing an The relationship involved is DIRECTLY excited molecule that decays back to its original PROPORTIONAL state Light Quenching - inhibits the light emission Spectrophotometer Measurement of light in a narrow wavelength Advantages: range; wavelength Excellent sensitivity Relationship stable FLUORESCENCE IMMUNOASSAY Relatively non-toxic Fluorphore/fluorochrome - absorbs energy from Expensive to perform an incident Faster turn-around time Fluorescein - absorbs maximally at 490 -495 nm and emits green color at 517 - 520 nm. It ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 6 Disadvantages: NOTE: Lack of precision in injection of hydrogen Typical example is Competitive RIA peroxide or some biological materials like plasma or urine causes quenching of light emission Advantages Disadvantages Excellent False results from sensitivity lack of precision in (between injection of attamoles 10-18 to hydrogen peroxide zeptomoles 10-21) or if some Reagents are biological materials stable and like plasma or relatively non toxic urine cause Inexpensive to quenching of light NOTE: perform emission First, there is a solid phase antibody Faster turnaround Then, there is competition between labeled time antigen and patient’s antigen NOTE (for table): NOTE: Quenching means inhibiting or stopping light Radioactivity is measured in the first one at reaction in both chemiluminescence and 100%, when there is no antigen from the fluorescence. This interferes with light patient. With increasing concentration of the patient’s RADIOIMMUNOASSAY antigen there would be competition, thus The technique in which a radioisotope is used decreasing the radiation emitted. as a tag or label for the detection of ag-ab The more the antigen in a patient's serum, the complex less the radiation emitted from the test. Hence, Radioisotopes are covalently attached to there is INVERSE RELATIONSHIP between antigens or antibodies analyte concentration and percent radioactivity Radiation emitted from the reaction can be detected by gamma rays or scintillation counter Yalow and Berson are persons behind RIA Rosalind Yalow got a Nobel Peace Prize in NOTE: Medicine (1977) for this. Competitive binding is classified as COMPETITIVE BINDING IMMUNOASSAY Heterogeneous due to the presence of b/f The analytes being detected competes with a separation. radio-labeled analyte for a limited number of binding sites on a high affinity ab The concentration of the radioactive analyte is in excess so all binding sites in the antibody will be occupied. If patient antigen is present, some antibody binding sites will be bound with the unlabeled analyte. The concentration of the analyte is INVERSELY PROPORTIONAL to the radioactivity emitted ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 7  NOTE: Sensitivity is measure in antibody per microgram per mL In the highlighted rectangular portion, the most sensitive is Radioimmunoassay (0.0006-0.006) as compared to ELISA. Radioimmunoassay - overall highly sensitive but it is still a health hazard Agglutination is sensitive than precipitation Labeled IA > Agglutination > Precipitation IMMUNOCHROMATOGRAPHY Principle: the analyte is applied at one end of the strip and migrates towards the distal end, where there is an absorbent pad to maintain a NOTE: constant capillary flow rate. Solid phase contains antigen that detects a The sample is loaded and it constitutes the patient’s analyte like IgE antibody. Then, after labeled antibody making an antigen-antibody washing a secondary anti-antibody with a label complex. is added. The antigen-antibody is immobilized in the Antigen - Antibody - Anti-antibody Complex detection zone. Formation This would form a complex and present as a Concentration of analyte is DIRECTLY positive result PROPORTIONAL with radioactivity NOTE: Conjugate - meaning something labeled ➜ Antigen-conjugate - it is an antigen labeled ➜ Antibody-conjugate - it is an antibody labeled NOTE: Colloidal gold - most common label SANDWICH RIA - concentration is directly Absorbent Pad - facilitates capillary movement proportional to the radioactivity of the sample For example: ➜ Sample: HbSAg SUMMARY: ➜ Conjugate Pad will have: anti-HbSAg with Indirect RIA - directly proportional colloidal gold Sandwich RIA - directly proportional ➜ Capture in Test line: antibody specific for HOMOGENOUS (EMIT) - directly proportional another epitope of the antigen. FPIA - inversely proportional ❖ Principle: Sandwich complex ❖ It only gives a QUALITATIVE INSTRUMENT: RESULTS Scintillation Counter - Radioimmunoassay ➜ Control line: Antibody or antigen bound to instrument. Not used in the laboratory since it is the Fc region of Antibody (based on a health hazard product insert. ❖ Procedural control - tells you what you are doing is okay. Not positive or negative control. ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 8  Cells of a specific size have their own place in SPECIAL TECHNIQUES the graph. FLOW CYTOMETRY Histogram quantifies the fluorescence intensity, Flow Cytometer meaning, the more cells there are, the more ➜ Used to quantitate component or structural intense is the fluorescence and the dot plot features of cells by optical means and also quantifies the percentage and fluorescence display data graphically intensity of the cells. Principle ➜ Cells suspended in liquid medium flow past CLINICAL APPLICATION a laser beam one at a time. Then the laser CELL SORTING beam transiently activates fluorochromes Flow cytometer with cell sorting capabilities can and the light is emitted as fluorochrome separate cells by size or by an antibody marker returns to its stable state. How does it work? NOTE: ➜ The single cell suspension is introduced There are CD markers for cell identification, cell into the flow cytometer chamber that lineage identification, and cell population contains a cell free buffer (sheath fluid). identification that’s why this is called cluster of ➜ Then the buffer surrounds the sample in differentiation. The more CD markers there are, such a way the cells flow in a single file in the more specific and the more specific these the center of a flowing stream. As a result CD markers are, then you can identify what the cells pass through the laser beam one particular cell. cell at a time. For example you have harvested a population ➜ The laser light transiently activates the of lymphocytes and you want to know which of fluorochrome, and when it returns to its these are CD4+ T cells or CD8+ T cells. The stable state, light energy is emitted. principle of flow cytometry can be used, and ❖ FITC = Green; TRITC = Red with the use of specific dyes, especially with the ➜ This emitted light is picked up by a use of monoclonal antibodies specific to CD4 or photomultiplier tube CD8 markers. For example, CD4+ T cells can ❖ Just like the principle of be identified by FITC-labeled antibodies while spectrophotometry where there is a CD8+ T cells can be identified by photomultiplier tube that converts TRITC-labeled antibodies. They can be sorted radiant energy into an electric and then eventually separated into either CD4+ current that is subsequently T cells or CD8+ T cells depending on the translated into numbers or values. fluorescent tags that are carried by their specific ➜ The signal is converted to one that can be monoclonal antibodies. So they separate read by the computer and the information various populations of cells. is then displayed in the form of histograms or dot plots. IMMUNOPHENOTYPING ❖ The histogram quantitates the Provides information that identifies specific cell fluorescence intensity whereas the lineage and the cell’s maturation stage dot plots quantifies both Cells expressing a specific marker are exposed fluorescence intensity and the to fluorochrome-labeled monoclonal antibodies percentage of fluorescence cells The fluorescent antigen-antibody complexes will hence you get the cell populations form on the surface of these cells. depending where their group Fluorescence is detected by the flow cytometry appears in the histogram. NOTE: NOTE: CD3 is common to all but CD4 is for helper T Components include a signal detector and cells and CD8 is for cytotoxic T cells. converters, computer system for data collection, processing and storage It absorbs light to the shorter wavelengths and HLA PHENOTYPING when it goes back to its ground state it emits Specific fluorochrome-labeled antibodies to light of a longer wavelength. each HLA specificity are used and allowed to It analyzes cell populations one at a time. react with either Class I and II HLA ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 9  Monoclonal antibodies used are presence of HLA antibodies in the patient fluorochrome-labeled and antigen specific serum with specificity for Class I HLA or donor EX. Antibodies to the TDT (terminal T cells are evidenced by a positive peak deoxynucleotidyl transferase), CALLA (common acute lymphoblastic leukemia NOTE: antigen) Flow cytometry can be used also for leukocyte cross matching especially for matching of an NOTE: organ donor and an organ recipient. If you have a population of cells or sample Principle: Donor T cell is reacted with the which is suspected to be a tumor cell, then you patient’s serum. If there are specific HLA just make use of the monoclonal antibodies antibodies in the patient serum reacting to the specific to these antigens. Then if there is donor T cells, there will be formation of specificity between a monoclonal antibody that cell-antibody complexes. How will you is labeled and the antigen that you are looking determine that there is now the incompatibility for, and if there is fluorescence, it means that it of the cell-antibody complex? A secondary is positive. fluorescent-labeled anti-antibody is added. The complex formed here is an indirect complex. DNA CONTENT This indirect complex between that of the donor DNA PLOIDY cell, the antibody of the recipient, and the Used during chemotherapy to monitor fluorochrome-labeled antibody is examined effectiveness of treatment in eliminating the under the use of a flow cytometer. If there is aneuploid clone and in predicting the outcome fluorescence, there is incompatibility between of certain malignancies the donor and the recipient. DNA CELL CYCLE ANALYSIS COMPLEMENT ASSAYS Used to measure DNA content at various cell Functional assay require immediate separation cycle phases during replication of serum from clotted blood and storage at -70C The more DNA that has been synthesized the or lower to preserve maximal activity integrity more aggressive is the cancer May serve as an important tool for detection of complement deficiency that may be associated NOTE: with conditions, such as induction, autoimmune Still making use of monoclonal antibodies or inflammatory disease. specific to the DNA. If there are many Why is there a need for us to know if there is monoclonal antibodies bound to the proliferating complement deficiency? DNA (such as of cancer cells), it means that ➜ Since complements function on the innate there is an aggressive cancer. immune response. Once you identify what particular antigen/matter ➜ MBL pathway and Alternative pathway are you're looking for in a cell, to be able to identify under innate immune response while that particular cell and a monoclonal antibody Classical pathway is involved in both can be produced against that cell and that innate and adaptive immune response antigen, using a monoclonal antibody since it can’t be activated unless there are specifically labeled with a fluorescent dye, then antibodies specific to the antigen. you can identify whatever condition you wish to Follow up: Which complement pathway is most identify in a population of cell or in a cell. active in an infant? MBL Pathway LEUKOCYTE CROSSMATCHING TWO TYPES OF TEST TO EVALUATE Donor T cell showing fluorescence because of COMPLEMENT the presence of donor HLA antigen/patient HLA Hemolytic Complement Activity Assay Ab/goat anti-human (FITC-labeled) complex on Immunoassays their surface, are detected as a separate characteristic peak on the histogram Donor T cell + Patient’s serum = Cell Ab II. HEMOLYTIC COMPLEMENT ACTIVITY ASSAY complex HEMOLYTIC TITRATION The cross match is considered positive or Used to detect complement activity incompatible when T cell histogram indicates ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 10  used as a screening test for congenital ➜ Alternative pathway is activated by deficiency of complement components that spontaneous hydrolysis of C3 and C3d, affect the total hemolytic activity of the serum. and subsequently when it attaches and is needed, then the pathway is activated. CH50 HEMOLYTIC ASSAY MBL Pathway - uses chicken RBCs Total hemolytic activity required for production ➜ It uses chicken RBCs because it probably of lysis of antibody-coated cells is quantified by has lots of mannose on its intestine determining the dilution of serum needed to Interpretations of the Complement Assays: lyze 50% of sheep red blood cells (SRBCs) that have been sensitized with rabbit anti-sheep 1 Test Result 2 Test Result antiserum. ➜ It measures the activity of the CLASSICAL CH50 very low or 0 Missing C1q, C1r, C1s, Pathway. It involves sheep RBCs that are (AH50 normal) C2, or C4 sensitized, then a patient's serum which (Components of may contain the complement is added, and Classical Pathway) through several dilutions to the serum, we will know if the CH50 is reached, or the AH50 very low or 0 Missing properdin, or dilution needed to lyze 50% of the (CH50 normal) (very rarely) Factor B or sensitized SRBCs. Factor D ➜ End product is HEMOLYSIS of sensitized (Alternative Pathway SRBCs, determined using a proteins) spectrophotometer, and standards and control are plotted. CH50 and AH50 are Missing C3, C5, C6, It is quantified by determining the dilution of very low or 0 C7, C8, and C9 serum needed to lyse 50% of sheep’s RBCs (Common pathway that have been sensitized with rabbit anti-sheep proteins) antiserum AH50 HEMOLYTIC ASSAY There is continuous attachment of C9 in the Is performed to evaluate alternative complex since if there is only one C9 attached, pathway-dependent lysis, using unsensitized it can’t form a porin channel, which could cause rabbit red blood cells (RRBCs) and human influx of water into the cell, which makes the cell complement. hypotonic, and leads to lysis. ➜ The serum is serially diluted in the amount ➜ In some books, 9n is written to indicate of hemoglobin released through lysis is how many C9 attaches to the complex. measured. ➜ It uses unsensitized RABBIT RBCs. II. IMMUNOASSAYS FOR COMPLEMENT Serial dilution of patient serum is prepared and COMPONENT incubated with RRBCs Results provide molecular concentration particular complement in serum FUNCTIONAL ASSAY FOR MBLECTIN PATHWAY These sets of tests don’t give information of the Uses chicken erythrocytes to assess functional integrity of the complement proteins, mannose-binding lectin in serum because they only determine the concentration ➜ Same thing happens where there is of the particular complement protein. dilution of the serum and the ➜ This is like in determining C3 using radial concentration/dilution capable of lysing immunodiffusion, wherein the bigger the 50% of the RBCs are determined, but this diameter, the higher the concentration, but time, it uses CHICKEN RBCs. it doesn’t tell you the activity but only gives the molecular concentration. As a summary: Immunoassays for Complement Component: CH50 - uses sensitized sheep RBCs. ➜ Nephelometry ➜ It must be sensitized since the activator of ➜ ELISA the Classical Pathway is the Ag-Ab ➜ Radial Immunodiffusion complex. Interpretation of Results: AH50 - uses rabbit RBCs ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 11  ➜ We know when complement levels go ❖ Neutrophils and RBCs - found at the down: bottom ➜ Complement consumption ❖ Ficoll titrate solution - is used to ➜ Increased Catabolism separate neutrophils and RBCs and mononuclear cells REDUCTION IN COMPLEMENT ACTIVITY ❖ Mononuclear cells - found on top Lymphoma Cell separated can be used for cell-mediated Allograft rejection cytotoxicity and tissue typing Infective hepatitis with Arthritis SCID MAGNETIC SEPARATION SLE Separation can be performed by employing Immune complex disease commercially prepared magnetic beads coated with monoclonal Abs with specificity for CD NOTE: markers using flow cytometry. Why are complement activity reduced in ➜ This time, it uses magnets so mononuclear autoimmune diseases? Due to immune complex cells specific to a particular CD marker of a formation just like in SLE. That’s why C3 levels are cell when bound to a magnet, are made to decreased in chronic SLE, unless the patient's SLE react with a cell population and is controlled using therapy. If uncontrolled, there will automatically, it will separate attached be LOW C3 levels, HIGH ESR, and presence of cells, and the opposite pole of the magnet LE Cells. is allowed to react, and will be separated using magnetic beads. INCREASE LEVEL OF COMPLEMENT Thyroiditis PANNING Gout Used to separate cell subpopulation from Diabetes total cell population Plastic plate coated w/ monoclonal Ab + Test NOTE:There is increased complement levels cells = Cells w/ Antigen specificity binds to the especially in acute conditions. antibodies on the plate and the rest can be washed off. COMPLEMENT FIXATION ➜ Whatever is bound to the monoclonal Abs This detects for presence of antibodies on a on the surface of the plate/microtiter well particular antigen, and follows the principle of will be diluted and separated to get the cell the Classical Pathway. subpopulation that we are after. Interpretations: ➜ POSITIVE - absence of cell lysis NYLON WOOL COLUMN (STRAW METHOD) ➜ NEGATIVE - presence of cell lysis Based on the principle that B lymphocytes adhere to nylon wool. FUNCTIONAL ASSAY Immune cells’ functional assay are being tested, NOTE: Cells are separated from the blood of the which could be lymphocytes. patient using: But before a functional assay can be performed, Leukocyte crossmatching Phenotyping separation of cells must be done. Functional Assays CELL SEPARATION TECHNIQUE CELL SORTING FACS DENSITY GRADIENT SEPARATION Fluorescence-activated Cell Sorting Lymphocyte separation It also follows the principle of flow cytometry Based on the fact that neutrophils and RBCs ➜ Those that are labeled with FITC with are denser than mononuclear cells specificity for example to CD4+ T cells, ➜ This uses a solution of greater density than and those labeled with TRITC, with mononuclear cells but lesser than specificity for CD8+ T cells will be neutrophils and RBCs. separated from each other due to ➜ Layers formed: monoclonal Abs. ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 12  51 After cells have been sorted, they can now be Cr RELEASE ASSAY tested for functional assays. A specific test commonly used in vitro lymphocyte-mediated cytotoxicity assay FUNCTIONAL ASSAY Used as an indicator of efficiency of CTL in CELL CULTURE lysing such target cells as tumor cells, Can be used to evaluate immune competence allogeneic tissue cells and virally-infected cells. of function and capacity of T and B lymphocytes ➜ The radioactive Chromium that is added to by using various stimuli on allogeneic cells to the target cells will be released once lysis activate lymphocytes and trigger their occurs. proliferation ❖ Radioactive Chromium is used due Stimuli on allogeneic cells to cultivate to its ability to bind to intracellular lymphocytes and trigger their proliferation proteins in target cells. ❖ Increased radioactivity means NOTE: CTL is working. Why do we need to test cells if they grow or not? EVALUATION OF NEUTROPHIL FUNCTION ○ To know if they are functioning or NITROBLUE TETRAZOLIUM BLOOD (NBT) TEST not. Growth in culture means cells Used to determine the phagocytic function of are functional. If there is no growth neutrophils in the presence of a mitogen or When neutrophils go through an oxidative burst, stimuli, then cells are non-functional. they reduce the yellow NBT dye to dark blue formazan. LYMPHOCYTE STIMULATION BY MITOGENS ➜ If neutrophils are able to phagocytize as In-vitro techniques used to evaluate patient’s stimulated for example by a latex, then immune response in microtiter plates they are 100% FORMAZAN POSITIVE Amount of cell proliferation is measured by which turns yellow NBT dye to blue. incorporation of a radioactive marker such as ❖ If it remains yellow, it means 3H thymidine into a newly synthesized DNA neutrophils are not phagocytizing. and detecting the limited radioactivity in counts Failure to produce hydrogen peroxide to reduce per minute. the nitroblue dye is of diagnostic significance in ➜ The amount of radioactivity is DIRECTLY determining overall reduction and phagocytic PROPORTIONAL to the efficiency or function of neutrophils. functional integrity of the immune response, since the cells acted on the CHEMOTAXIS ASSAY mitogens. Chemotaxis - migration of neutrophils from the circulation to the site of infection. CYTOTOXIC ASSAYS Measures the ability of neutrophil to move in Tests the cytotoxic ability of cytotoxic T a directed, migratory pattern towards a lymphocytes (CTL).. stimulus. Three stages of lymphocyte-mediated ➜ Example in an enlarged chamber, cytotoxicity chemotactic factors are placed at the ➜ Conjugate formation - direct bottom and neutrophils are placed on top. membrane-to-membrane contact between When neutrophils react with chemotactic the cytolytic cell and target cell to form factors, they will migrate from top to bottom conjugate of the chamber. ❖ CTL will form a conjugate with a ➜ If there is no migration, these are termed target cell. as LAZY NEUTROPHILS (Lazy ➜ Triggering various membrane and cellular Neutrophil/Leukocyte Syndrome). metabolic events ➜ Then, pore formation happens on the T-CELL ENUMERATION ASSAY target cells, resulting in the release of I. ERYTHROCYTE ROSETTE ASSAY cytoplasmic granules. aka E-rosette assay Sheep RBCs (not sensitized) + peripheral lymphocyte → MULBERRY SHAPED CELLS ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 13  T CELL SUBSETS NOTE: Fluorescent antibody assays utilizing Analyte: Antibody monoclonal antibodies Conjugated: Anti-antibody What is found on the solid phase: Antigen NOTE: This is just like Fluorescent Activated Cell Sorting (FACS) which is an application of the SPECIFIC DISEASE AND ASSOCIATED LAB principle of flow cytometry. TESTS NOTE: IDENTIFICATION OF SPECIFIC ALLERGENS IN Hepatitis virus classifications- 6 (A,B,C,D,E TYPE 1 HYPERSENSITIVITY REACTION and G) RADIOIMMUNOSORBENT TEST (RIST) HEPATITIS B Used to measure total IgE concentration NOTE: Procedure: Has envelope, nucleocapsid, HBcAg, lipid ➔ Serum is added to a paper disk which has bilayer, HBsAg been coupled with sheep or rabbit Only Hepatitis virus that is DNA- double antibodies to human IgE stranded. ➔ The IgE in serum reacts specifically with After incubation there is increase of HBsAg the coated disk. Core antigen is not found only Anti-HBc ➔ After washing, radiolabelled anti-human Clue that it is a recent resolving infection- Anti IgE is added to the disk which couples with HBc IgM IgE in the patient's serum. Core window phase- Anti HBc total or Anti ➔ After rewashing, the radioactivity of the HBc IgM complex on the disk is measured. 2 laboratory tests to identify that the px has The amount of radioactivity is directly Hepa B- HB Ag and Anti HBc proportional to the amount of IgE in the serum. Active chronic- HBe Ag (infectivity marker / This assay follows a sandwich format - replicating virus) heterogenous. HBe Ag- extractable fragment of the core Chronic HBV- look for HBsAg (carrier and no NOTE: active replication) You will not have a type 1 hypersensitivity Sequence of Ab production: Anti-HBc, Anti- reaction to an allergen you were not previously HBe and Anti- HBs exposed to. The one being sandwiched is an antibody RADIOALLERGOSORBENT TEST (RAST) Used to measure specific IgE levels Procedure: ➔ A specific allergen is coupled to a solid phase. ➔ Serum to be examined is then reacted with the solid phase-allergen complex. ➔ IgE, if specific to the allergen, will fix to the solid phase. ➔ Unbound IgE is removed by washing after radiolabeled Ab to human IgE is reacted to the solid phase. ➔ After rewashing, the radioactivity of the particles is measured to give the serum level of the IgE specific to the allergen. The amount of radioactivity is directly proportional to the amount of IgE in the serum. This assay follows an indirect format - heterogenous. ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 14  HEPATITIS A HIV NOTE: Etiologic agent of AIDS Infection: Fecal-oral route Discovered by Luc Montagnier and Robert Increased ALP- liver infection (jaundice) Gallo HEPATITIS A LAB DIAGNOSIS EARLY PHASE OF HIV INFECTION Past infection i.e. immunity determined by In early phase HIV infection, initial viruses are the detection of HAV-IgG by EIA M-tropic. Their envelope glycoprotein gp120 is IgM coupled with high ALP levels capable of binding to CD4 molecules and chemokine receptors (CCR5) found on HEPATITIS C macrophages. HCV-RNA - various techniques are available e.g. PCR and branched DNA. May be used NOTE: to diagnose HCV infection in the acute M - tropic - Monocyte/Macrophage tropism. It phase. However, its main use is in would infect monocyte/macrophage during early monitoring the response to antiviral therapy phase and not CD4+ cells. NOTE: LATE PHASE OF HIV INFECTION RIBA- Radio Immuno Blot Assay In the late phase of HIV infection, most of the viruses are T-tropic, having gp120 capable of binding to CD4. NOTE: T-Tropic - T cell tropism. It will now infect T-cells (CD4+ T-cells). VIRAL REPLICATION CD4 cells may be destroyed in the process, body attempt to replace lost CD4 cells, but over the course of many years body is unable to keep the count at a safe level AIDS CD4 count is less than 200, this indicates advanced HIV disease. LAB DIAGNOSIS OF HIV INFECTION HEPATITIS D (DELTA) VIRUS Antibodies Superinfection Antigens ➜ Develop chronic HDV infection Nucleic Acid ➜ High risk of severe chronic liver disease Virus in Culture ➜ May present as an acute hepatitis ELISA HEPA D PREVENTION First serological testing developed to detect HIV HBV-HDV superinfection infection ➜ education to reduce risk behaviors among ➔ Easy to perform persons with chronic HBV infection ➔ Easily adapted to batch testing ➜ “Delta virus” ➔ Highly sensitive and specific ➜ Needs Hepa B envelope for it to thrive Useful for: ➔ Screening blood products NOTE: ➔ Diagnosing and monitoring patients INFECTIONS IN HEPA D: ➔ Research ➜ Coinfection ➜ Superinfection- develop chronic Hepa D WESTERN BLOT ➜ HEPA A and E- has vaccines Most popular confirmatory test ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 15  Utilized lysate prepared from HIV virus Difficult due to presence of maternal IgG The lysate is electrophoresed to separate out antibody the HIV proteins (Ag) Detection of IgM and IgA antibodies P17, p24, p31, gp41, p51, p55, p66, gp120, Measurement of p24 antigen gp160 Antibodies p24 and p55 appear earliest but SPECIFIC DISEASE STATES decrease or remain undetectable SYPHILIS Result Interpretation Causative agent: T. pallidum ➔ No bands = negative Course of disease is divided into 4: primary, ➔ Presence of 3 bands (p24, p31, gp41, secondary, latent, tertiary gp120/160) = positive PRIMARY ➔ CDC states that any 2 bands present ➜ Characterized by appearance of transient (p24, gp41, or gp120/160 = positive painless, firm lesion at the site of infection about three weeks after initial infection DETECTION OF p24 HIV ANTIGEN SECONDARY Early detection in seronegative patient ➜ Multiple widespread lesion Newborns CSF ➜ Two months after primary infection highly Monitoring disease progress infectious stage ➜ Formation of anti-T. pallidum Abs NOTE: ➜ Lesions heal after a month During viral replication, p24 is the first to be ➜ Absence of medical intervention, disease assembled. enters a latent stage Why is detecting antigen more reliable in LATENT testing than antibody in newborns? Because newborns have not developed antibodies and ➜ Starts after the disappearance of the there is a possibility that the antibody tested secondary lesions came from the mother. ➜ Asymptomatic but serological response is activated PCR TERTIARY Looks for HIV RNA in the white blood cells of a ➜ Two to 50 years after infection person ➜ Benign syphilis (gumma), NASBA cardiovascular or neurosyphilis Amplifies HIV RNA NON-TREPONEMAL TESTS FOR SYPHILIS NOTE: VDRL Nucleic acid-sequence-based amplification Flocculation VIRUS ISOLATION Antigen: Used to definitely diagnose HIV ➜ Cardiolipin - phospholipid derived from Best sample is peripheral blood, but CSF, beef heart; responsible for reactivity. saliva, cervical secretions, semen, tears, or Reacts with reagin antibody material from organ biopsy are acceptable ➜ Lecithin -Produces standard reactivity ➜ Cholesterol - serves as center for absorption of tissue lipids to increase Ag VIRAL LOAD TESTING size Antibody: IgM or IgG to damaged tissue or NOTE: organism Used to monitor the patient’s response to anti-retroviral drugs. Preferred test for CSF specimen Serum requires heat inactivation TESTING NEONATES ➜ Inactivate complement ➜ 56 degrees Celsius for 30 mins ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 16  180 rpm Use to visualize mobile organisms from primary Result reported as: or secondary lesions ➜ Reactive: medium to large aggregates ➜ Weakly Reactive: small clumps (insert pic of different Spirochetes) ➜ Non Reactive: no clumping or slight roughness as compared with Ag control NOTE: Borrelia - loosely coiled RPR Legionella - tightly coiled with a hook Treponema - regularly coiled Flocculation More sensitive but less specific than VDRL FTA-Abs Antigen: added with charcoal for visualization Indirect immunofluorescence No heat inactivation necessary —- choline Patient’s ab attaches to adsorbed and chloride immobilized T. pallidum (Nichols Strain) False positive: malaria, SLE, RA, hepatitis, Observed under fluorescence microscope pneumonia, aging and IM ➜ Similar to ANA’s principle ➜ Conditions that lead to inflammation and ➜ FITC - green fluorescence tissue destruction 100 rpm for 8 minutes TPI (T. pallidum Immobilization test) ART Antigen is suspension of motile T. Pallidum Automated Reagin Test First developed to detect presence of Flocculation anti-treponemal Abs Result is generated from the machine if abs are present treponemes are immobilized A. USES OF NON-TREPONEMAL TEST FOR Utilizes darkfield microscopy SYPHILIS Too expensive and cumbersome Population screening test MHA-TP (Microhemagglutination Assay for T. Following serologic response to therapy pallidum) since antibody titer reverts to negative within: ➜ One year in primary syphilis Simple passive hemagglutination ➜ Two years in secondary syphilis Involves red cells sensitized with T palladium + Reactive tests diagnostic of syphilis when: patient’s serum ➜ Titer very high or increasing compatible If Ab is present in serum, there is with primary or secondary syphilis hemagglutination ➜ Clinical History Used as substitute to FTA-Abs VDRL only test employed for evaluation of SENSITIVITY OF TESTS FOR SYPHILIS CSF Primary Stage: B. TREPONEMAL TESTS FOR SYPHILIS ➜ Increased sensitivity: FTA-Abs, RPR, ANTIGENS VDRL Nichol’s strain of T. pallidum (pathogenic) Secondary Stage: Reiter strain of treponemes (non - T. pallidum ➜ All stage equally sensitive (non-pathogenic) Late Stage: ➜ Equal sensitivity: FTA-Abs, MHA-TP, TPI DARK FIELD MICROSCOPY ➜ Poor sensitivity: Reagin tests ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 17 NOTE: ➜ Increased ESR FTA-Abs is the gold standard diagnostic test for ➜ Increased CRP syphilis During Late Stages, it is sure that there is COLD AGGLUTININS production of Antibody. Reagin is decreased. Antibody agglutination below 25 degrees Sensitivities of Serologic Tests celsius IgM antibodies : usually anti-I or anti-i Test Primar Secon Latent Late Marked rise indicated Mycoplasma y dary pneumonia infection Low titer elevations: influenza or adenovirus VDRL 78 100 95 71 infection (74-87) (88-100 (37-94) ) Do not refrigerate specimen (Ab will bind to RBCs leaving serum fee of Abs and result in RPR 86 100 98 73 false negative or decreased cold agglutinin titer) (77-100 (95-100 ) ) ANTISTREPTOLYSIN O TEST (ASOT) FTA- 84 100 100 96 Tests employed to screen Streptococcus ABS (70-100 pyogenes infection ) Streptolysin-O is an oxygen labile hemolysin MHA - 76 100 97 94 which can lyse RBCs TP (69-90) (97-100 SEROLOGIC TESTS: Indirect or passive ) latex agglutination test ➜ Qualitative or screening test: Serum Abs + latex coated with Strep O = agglutination ➜ Semi-quantitative method: involves RHEUMATOID ARTHRITIS double serial dilution of patients serum; Affects the joints and periarticular tissues last dilution showing agglutination is used Autoantibodies (IgM or IgG) (rheumatoid to determine the titer factors) to Fc portion of Ig molecules RF Latex Slide Test NOTE: ➜ latex with human igG + diluted serum = AGN- Acute Glomerulonephritis ARF- Acute Rheumatic Fever agglutination RF latex tube test A. INDIRECT OR PASSIVE LATEX ➜ Latex with adsorbed IgG + diluted serum = AGGLUTINATION TEST agglutination Increases number of positive test when synovial Qualitative or screening test: serum abs + fluid is used latex coated with Strep-O = agglutination Latex test more sensitive, but sheep cell Semi-quantitative method: involves double agglutination test (Rose and Waaler) more serial dilution of patient’s serum; last dilution specific showing agglutination is used to determine the ➜ Stabilize sheep erythrocytes sensitized titer (two-fold serial dilution) with rabbit IgG anti-sheep erythrocytes + serum of RA patient = NOTE: HEMAGGLUTINATION (TITER = >8 IU/mL) Titer - the amount of antibody that is present in Increased C3 and C4 levels the circulation towards a particular antigen. Increased levels of APPs: Amount of antibody to antigen ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 18  Two fold dilution: 1:2, 1:4, 1:8, 1:16 etc ➜ Post operative surveillance (fall within normal 7-10 days post op) B. ASO HEMOLYTIC TITRATION TEST ➜ MI - increase correlates with size of infarct (NEUTRALIZATION TEST) ➜ Renal transplantation - increase = graft Dilute serum with buffer (1:10; 12; 50; 100; 166; rejection 250; 333; 500; 625; 833; 1250; 2500) ➜ Burns = severity of burn Add strep-O antigen Incubate NOTE: Add Group O red cells Commonly used acute phase protein that Incubate, read Todd units (reciprocal of highest increases when there is inflammation. dilution showing no hemolysis CRP immediately increases after tissue insult RESULT: and decreases upon recovery ➜ RBC control: RBC + buffer = no hsCRP- Analyte better index for predicting hemolysis inflammatory conditions such as MI ➜ SLO control: SLO + buffer + RBC = hemolysis INFECTIOUS MONONUCLEOSIS If ab is absent, streptomycin is not neutralized Caused by Epstein-Barr Virus (Herpes virus) and will lyze RBC Disease of the RES Normal: less than 166 Todd units/mL Symptoms range from asymptomatic to severe High titer associated with: Streptococcal self-limiting infections, AGN (Type 3), ARF (Type 2) Most common age 15-25 POSITIVE - no hemolysis More in women than men NEGATIVE - hemolysis Children often show no antibodies May be reactivated later in life (chronic fatigue CRP PROTEIN syndrome) One of the several “Acute phase reactants” Transmitted by saliva (kissing disease) or proteins in response to inflammation and/or blood also necrosis Causes Burkitt’s lymphoma in Africa and It derived its name from the facts that it reacted New Guinea with the c-polysaccharide of pneumococcal cell Found in children walls Also associated with a rare form of squamous Elevated CRP levels have been associated with cell carcinoma of nasopharynx virtually all acute bacterial infections, some Symptoms: tumors and various types of tissue destruction ➜ 4-7 weeks incubation such as myocardial infarction and ➜ Fever post-operative necrosis ➜ Sore throat As an indicator —- after the tissues insult and ➜ Lymphadenopathy decreases at a faster rate upon recovery than ➜ Malaise other acute phase proteins ➜ Headache Normal values: 3.0 mg/dL Causes Burkitt’s lymphoma Can reach as high as 1000 times its normal 4-7 week incubation: fever, sore throats amount Correlations: ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 19 NOTE: Also picks-up forssman and serum sickness antibodies Herpesvirus- 8; clinical latency= recurring infections in herpes virus PAUL-BUNNELL SCREENING TEST SEROLOGIC TESTING General screening test for Heterophile Abs Two types of antibody testing: ➜ Sbs to Forssman Ag Antibodies to EBV ➜ Abs in Serum Sickness ➜ Four Abs ➜ Abs in IM ❖ IgM anti-viral capsid antigen (VCA) shows a current infection (best marker), All agglutinate sheep’s RBCs lasts about 12 weeks ❖ Anti-early antigen (EA) - recent DAVIDSON DIFFERENTIAL infection Tells which heterophil antibody is present ❖ Anti-nuclear antigen (EBNA) - old based on the fact that antibodies can be infection absorbed out of sera Heterophiles Antibody Tests Then Paul-Bunnell is run again with absorbed ➜ Antibodies stimulated by one antigen bt serum can react to antigens of other species ➜ Mono Abs are absorbed by beef ➜ Mono: Formed in response to antigens on ➜ Forssman Abs are absorbed by guinea infected cells pig ➜ React with Sheep, horse, and beef RBCS ➜ Serum sickness by both Forssman: formed after exposure to certain bacteria (salmonella, shigella, strep DAVIDSOHN DIFFERENTIAL SLIDE TEST pneumoniae) ➜ Reacts with sheep RBCs and guinea pig To be considered adsorbed there must be kidney greater than three tube difference between the Serum sickness: formed in response to presumptive titer and the differential titer injection of horse serum (found in some vaccines before) Heterophile Kidney Beef Reacts with: horse serum; reacts sheep antibody Extract Erythrocyte horse, beef, and guinea pig kidney Infectious Not absorbed Adsorbed Mono Sheep Horse Beef Guinea Forssman Adsorbed Not absorbed IM / / / Serum Adsorbed Adsorbed Forssman / / Serum / / / / PAUL-BUNNELL PRESUMPTIVE TEST (CLASSIC) Inactivate sera to destroy complement Dilute sera 1:7 to 1:7168 Add fresh sheep cells (not older than 1wk) Incubate for 2 hours centrifuge and read Highest tube to show agglutination 1:224 or greater significant ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 20  ➜ Past infection: anti-EBNA, IgG anti-VCA Heterophile Agglutinatio AGGLUTINATI antibody n after ON AFTER without IGM anti-VCA Absorption ABSORPTION with Guinea WITH BEEF FEBRILE ANTIGEN TESTS Pig Kidneys ERYTHROCYT WIDAL TEST E Direct agglutination methods Infectious Agglutination No Antigens Mono agglutination ➜ O Ag (somatic) (ABCDE) ➜ H flagellar Forssman No Agglutination agglutination ➜ Vi (capsular) - Abs identify carriers Widal agglutination test detects titer H and O Serum No No agglutinins agglutination agglutination ➜ 4-fold rise in titer of O agglutinins plus clinical symptoms for diagnosis of typhoid fever DAVIDSOHN DIFFERENTIAL SLIDE TEST NOTE: 1:80 is the significant titer in Widal test Let’s say for example, how are we going to ADVANTAGES DISADVANTAGES report? After running a dilution and after running the test, you are able to observe 4+ at 1:20 the When properly The Davidsohn 4+ at 1:80, 2+ 1:160 and no more agglutination performed, this test is differential is very time at 1:320. Which titer are you going to report as specific for IM and consuming and positive: false-positive results burdensome. ➜ We report 4+ at 1:80 titer are rare. ➜ Just remember to interpret the Widal test result the dilution should be atleast 1:80 na MONOSPOT TEST/ RAPID DIFFERENTIAL SLIDE mag positive then the grading of the TEST agglutination is atleast 2+ Horse RBCs are used - more sensitive Another example, 1:20 2+, 1:40 2+, 1:80 2+, indicators of Abs found in IM 1:320 negative ➜ If stronger with sera adsorbed with beef ➜ We report it as 1:80 2+. Since dira nakita red cell stroma, the test is NEGATIVE ang 2+ sa 1:80 ➜ If agglutination is absent in both mixtures, Another example, 1:20 4+, 1:40 2+, 1:80 the test is NEGATIVE negative , 1:160 negative EBV SPECIFIC TESTS ➜ We don’t report this as positive because Immunofluorescent tests for IgM or IgG the significant titer is 1:80 antiviral capsid antigen (VCA), anti-early We don’t report the result below 1:80 and less antigen (EA), and anti-nuclear antigen than 2+ agglutination (EBNA) Antibodies to O antigen are IgM Appearance and denaturation of EBV specific Antibodies to H antigen are IgGDISEASE antibodies differentiate acute from past infection IgG or IgM anti-VCA in absence of anti-EBNA WEIL-FELIX TEST supports diagnosis of IM EBV Specific Serology Test is based on cross - reaction between ➜ Recent or current infection: IgM Rickettsial Abs and Abs to O antigen of anti-VCA, anti-EA, IgG anti-VCA without certain strains of Proteus anti-EBNA OX 19 or OX 2: RMSF ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 21  OXK: antigen for scrub typhus Titer >1:160 or fourfold rise significant in absence of concurrent Proteus infection LYME DISEASE Cause by B. burgdorferi Transmitted by Tick Laboratory tests: ➔ ELISA to detect Abs to OsPC NOTE: OsPC - outer surface protein C Justin Bieber and Bella Hadid both have Lyme disease In the peripheral pattern, you can notice a marked increase intensity at the periphery of the nucleus of the cell In the homogenous pattern, the entire nucleus of the cell is stained homogenous and smooth In the nucleolar pattern, ma count mo na ang mga dots In the centromere pattern, you can count the dots, usually 40-80. The dots represent the ANAs kinetochore. In the speckled pattern, the nucleus is grainy ANA Predominant Disease In the mitochondrial pattern, this is associated Antigen with biliary cirrhosis. The staining is on the cytoplasm because the mitochondria are found Peripheral nDNA SLE in the cytoplasm Homogenous nDNA, SLE histones Nucleolar Nuclear RNA SSc, SLE Centromere Kinetochore CREST Speckled Various SLE, SSc, ribo-nucleo-pro Sjoren’s teins Syndrome END OF TRANS NOTE: Indirect IFA ISBB: IMMUNOSEROLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 22