1	CLSC320 Principles of Immunology Diagnostic Laboratory Immunology Program for Clinical Laboratory Science Unit - 14 Special Techniques
2	Unit – 14 Guidelines Reading assignment: Pages 290 - 291 of textbook Learning objectives: Those listed on page 291 of textbook Key terms: Those listed on pages 291 & 292 of textbook
3	Chapter Outline Flow Cytometry Complement Assays Functional Assays Histocompatibility Testing e e e
4	OSF – Monoclonal Antibody On pages 293 - 294AOne Step Further@ presents a more in- depth discussion of the production of monoclonal antibodies. This presentation is contained on a separate slide presentation called A One Step Further #16" The student may call up the slide program OSF-16 later or click on the arrow below to view slides now.
5	Clinical Applications of Flow Cytometry 9 identification of cell surface antigens (phenotyping) 9 post-transplantation monitoring 9 monitoring progress of malignant diseases 9 cell sorting 9 DNA analysis
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7	Fluorochromes n Flow cytometry
8	Events Occurring in Flow Cytometry 9 Light Scatter 9 Measurement of fluorescence E Foreward light scatter: E Side light scatter: U measured by in-line detector U measured by detector set at 90^o U measures cell size U measures cell complexity Fluorochrome labeled monoclonal antibodies mixed with sample amount of fluorescence is directly proportional to amount of antibody on cell
9	Methods of Data Presentation 9 Line Plot Histogram method 9 Dot plot Histiogram method
10	Clinical Applications - Summary
11	Clinical Applications - 1 9 Cell sorting cells are tagged with fluorochrome labeled monoclonal antibody E cells can then be sorted (counted) by light emitted Two or three fluorochrome labeled monoclonal antibodies can be mixed with a sample and run simultaneously cells may be physically sorted by using charged plates to deflect cells into different test tubes
12	Clinical Applications - 2 9 Immunophenotyping of cells cells are classified according to surface antigens expressed E some examples of phenotyping: Malignancy cell markers T-cell ALL B-cell ALL B-cell CLL Lymphoma CD3+ CD7+ CD5- CD10/22+ CD19+ CD5/19+ CD20+ CD22+ CD19+ CD20+ CD22+
13	Clinical Applications - 3 9 HLA phenotyping of cells MHC antigens in humans are called HLA antigens A Human Leukocyte Antigens@ E Class I MHC antigens are : U HLA-A U HLA-B U HLA-C E Class II MHC antigens are: U HLA-D U HLA-DR U HLA-DQ E Monoclonal antibodies for specific Class I & II MHC antigens are available
14	Clinical Applications - 4 9 Tumor cell phenotyping E monoclonal antibodies used to specific antigens E Examples of leukemia-associated antigens: U TdT - terminal deoxynucleotidyl transferase U CALLA - common ALL antigen U CD10 - new name for CALLA E T cell ALL has following phenotypes: U CD3 + U CD7 + U CD5 -
15	Clinical Applications - 5 9 Tumor cell phenotyping - continued E B cell ALL has following phenotypes: U CD10/22 + U CD19 + U TdT + in children E B cell CLL has following phenotypes: U CD5/19 + U CD20 + U CD22 + E Lymphoma phenotypes U CD19 + U CD20 + U CD22 +
16	Clinical Applications - 6 9 Diagnosis & Prognosis of Malignancy E DNA content analysis U DNA ploidy \diploidy = 2 copies of each chromosome (all cells except sperm & ova) \monoploidy = 1 copies of each chromosome (sperm & ova) \aneuploidy = cells with <2 sets of chromosome (except sperm & ova) used to monitor effectiveness of L in malignancies
17	Clinical Applications - 7 9 Diagnosis & Prognosis of Malignancy - continued E DNA content analysis - continued U DNA cell cycle analysis \the more DNA synthesized, the more aggressive the tumor
18	Clinical Applications - 8 9 Other applications E Leukocyte Cross-match U Histocompatibility testing - another name \detection of HLA antibodies U Situations that warrant this procedure: \previous graft rejections \living related donors \patients on waiting list for donor organs
19	Complement Assays Specimen requirements: 9 must not be exposed to temperatures above -70⁰C for prolonged periods since complement may breakdown Types of assays: 9 Hemolytic complement activity assay (hemolytic titration) 9 Immunoassays
20	Hemolytic Titration 9 Hemolytic complement activity assay (hemolytic titration) eused to screen for congenital deficiencies of complement components that affect the total hemolytic activity of plasma Utwo functional assays are: \CH₅₀ assay \AH₅₀ assay
21	CH₅₀ Assay U measures entire complement activation sequence (C1 to C9) U determines the dilution of serum (thus complement) needed to lyse 50% of sheep red blood cells that have been coated with rabbit anti-sheep antibodies U standard curve is constructed and used to equate absorbance with concentration of complement
22	AH₅₀ Assay U measures alternative pathway of complement activation U uses a serial dilution of serum (thus complement) and the amount of rabbit red blood cells that have been lyzed is determined using absorbance U standard curve is constructed and used to equate absorbance with concentration of complement
23	Immunoassays for Complement E Specific monoclonal antibodies for various complement components available U only gives a quantification of amount of component available not functionality of component U the following tests can be used: \nephelometry \ELISA
24	Interpretation of Tests Results - 1 9 Reduced levels of complement components E complement consumption during in vivo formation of ag-ab complexes E reduced synthesis of complement E increased catabolism of complement E presence of an inhibitor to complement 9 Reduced levels of complement activity E seen in diseases such as: U lymphoma U allograft rejection U infectious hepatitis with arthritis U severe combined immunodeficiency U Systemic lupus with glomerularnephritis U immune complex disease
25	Interpretation of Test Results - 2 9 Increased levels of complement components E increased levels of complement occurs in certain diseases but significance is unknown E seen in diseases such as: U gout U diabetes U thyroiditis U acute rheumatic fever U rheumatoid arthritis
26	Complement Dependent Assays Complement Fixation tests Hemolysis-Inhibition tests
27	Complement Fixation Tests - 1 ecomplement acts as an indicator (production of hemolysis) in detecting ag-ab reactions. ecomplement in patient sample is destroyed by: U heating at 56⁰C for 30 minutes U ag + ab + complement in a series of dilutions are incubated together E stage 2 - indicator system U sheep RBC=s + anti-sheep antibodies (hemolysin) are added and incubated
28	Complement Fixations Tests - 2 E Interpretation of results: U positive results U negative results \ highest tube showing no hemolysis \ hemolysis in all tubes \ lack of hemolysis indicates ag-ab complex formed in stage 1 and bound the complement \ hemolysis indicates ag-ab complex did not form in stage 1 and so there is free complement to react in stage 2
29	Hemolysis Inhibition Tests - 1 E Detection of anti-toxin antibodies to streptoccal infections includes measurement of : U anti-streptolysin O (ASO) U anti-DNase B - for skin infections U anti-NADase U anti-hyaluronidase E Diseases associated with streptococcal infections include: U acute rheumatic fever U post-streptococcal glomerulonephritis
30	Hemolysis Inhibition Tests - 2 E Principle for ASO titer assay: The procedure is based on the fact that anti- streptolysin antibodies will neutralize or inhibit streptolysin toxin from hemolyzing red blood cells. E Procedure for ASO titer assay: U serial dilution of patient serum (ASO) made U commercially prepared Streptolysin O is added to each tube then incubated U human type O red blood cells are added U tubes centrifuged and checked for hemolysis
31	Interpretation of ASO Titer Assay U the reciprocal of the highest dilution showing no hemolysis is the ASO titer (Todd units) U the test should be repeated in 2 weeks and if there is a 4-fold increased titer (>two tubes) then it indicates a positive test U negative test (low titer) does not rule out possible disease U normal range = < 166 Todd units
32	Functional Assays of Immune Competence Specimen for assay: lymphocytes are the preferred cell for performing functional studies Cell separation techniques: Density gradient separation Magnetic separation Panning or plating Nylon wool column (straw method) Cell sorting using flow cytometry Cell cultures
33	Density Gradient Separation neutrophils and erythrocytes are more dense than mononuclear cells such as lymphocytes so when anticoagulated blood is layered over a ficoll solution (greater density than mononuclear but less dense than neutrophils & RBC=s) and centrifuged, the monuclear cells will be on top of the ficoll layer neutrophils & erythrocytes ficoll layer mononuclear cells serum
34	Magnetic Separation magnetic beads are coated with cell marker specific monoclonal antibodies (i.e. CD19 for B-cells) and once cells have attached to magnetic beads the beads are separated with a magnetic field Bead beads in tube blood added beads separated
35	Cell Separation Techniques - 1 Panning or plating a plastic plate is coated with monoclonal antibodies with cell marker specificity and sample is added to plate. Cells with specific cell markers are bound to antibodies while those lacking cell markers are washed off Nylon wool column (Straw method) B-cells will adhere to nylon wool packed in a straw while T-cells are flushed out. Cell sorting with flow cytometry fluorochrome labeled antibodies bind with specific cell markers and the resulting tagged cells are separated by flow cytometer
36	Cell Separation Techniques - 2 Cell cultures U patient lymphocytes are stimulated using various substances to proliferate U a labeled DNA precursor - ³H-thymidine is added to culture and amount of radiocativity remaining in harvested cells is an indication of lymphocyte function U Mixed lymphocyte reaction: \ used to evaluate a patients lymphocyte response to foreign HLA antigens \ one-way protocol: \ two-way protocol: lymphocytes exposed to foreign HLA on lymphocytes treated so they don=t respond both lymphocyte populations allowed to respond
37	Cytotoxicity Assays Three stages involved in lymphocyte-mediated cytotoxicity: 9 conjugate formation 9 triggering of membrane and cellular events 9 pore formation in target cell membrane Udirect membrane-to-membrane contact between cytotoxic cell and target cell to form a conjugate Uchanges occur in cytotoxic cell with resulting producion of various cytokines and receptors Uperforin may cause pore formation Ucytotoxic cell may degranulate
38	Chromium (⁵¹Cr) Release Assay 9 used to evaluate the in vitro efficiency of T_cytotoxic cell in lysing such target cells as tumor cells, allogenic cells, or viral infected cells 9 when target cells are lyzed the chromium-labeled proteins are released and amount of chromium released (in the supernatant) is proportional to the quantity of lyzed cells
39	Evaluation of Neutrophil Function Nitroblue tetrazolium dye reduction test (NBT test) U neutrophils can reduce NBT dye via respiratory burst producing H₂O₂ as a result U failure to reduce the NBT dye (colorless to blue- black deposits called Aformazan@) or produce H₂O₂ indicates metabolic defects which can lead to: chronic granulomatous disease Flow cytometry - now method of choice Chemiluminescence U neutrophils emit small amounts of energy (Chemiluminescence) during respiratory burst U luminol+latex particles phagocytized by neutrophil and amount of light emitted can be measured using a luminator
40	ANCA Anti-neutrophil cytoplasmic autoantibodies (ANCA) U detection of autoantibodies associated with small vessel vasculitis U these autoantibodies are directed against: \ proteinase 3 \ myeloperoxidase U these autoantibodies are detected using indirect immunofluorescence techniques U positive tests are further evaluated using ELISA technique
41	Histocompatibility Testing Phenotyping: 9 Also called: U tissue typing U serologic tissue typing U HLA typing 9 Used for the detection of various cell markers that include HLA present on cell surface of potential donor and/or recipient mononuclear cells 9 Class I MHC antigens consist of: U HLA-A, HLA-B, and HLA-C 9 Class II MHC antigens consist of: U HLA-DR, HLA-DQ, and HLA-DP
42	Histocompatibility Testing – Procedure -1 Microcytotoxicity test for class I HLA: U isolated blood cells are reacted with a reagent panel of antisera (known HLA antigen specificity) U complement is added to test system U hemolysis indicates antigen-antibody complex has formed (>50% of cell lysed) U no hemolysis indicates no antigen-antibody complex has formed or U a dye may be added (eosin or trypan blue) which the target cell will uptake if ag-ab has activated complement (>50% uptake dye)
43	Histocompatibility Testing – Procedure -2 Microcytotoxicity test for class II HLA: U similar to that used for class I HLA except: \ specificity of antisera is for HLA-DR, HLA-DQ \ B cells are isolated from T cells and enriched since 80% of circulating lymphocytes are resting T cells which lack class II HLA.
44	Histocompatibility Testing – Procedure - 3 Microcytotoxicity test for HLA antibodies: U similar to that used for class I HLA except: \ HLA containing cells are used to react with serum (antibodies) of recipient U the following antibodies are tested for: \ HLA cytotoxic antibodies \ HLA autoantibodies \ Donor specific alloantibodies donor serum + recipient cells crossmatched to ensure compatibility
45	Cross-matching Procedures Mixed Lymphocyte Reaction Flow cytometry Lymphocytotoxicity via chromium-release assay \Donor + recipient lymphocytes are combined \strength of reaction is proportional to the differences in HLA \donor T cells + patient serum (ab=s) + tagged anti-Human globulin allowed to flow through flow cytometer \crossmatch is positive when T cells show class I HLA incompatibility
46	End of Special Techniques Press the ESC key to end program
47	One Step Further Production of Monoclonal Antibodies OSF - 16 Pages 293 & 294 7click arrow to return to main program
48	Monoclonal Abs - 2 Definition: emonoclonal antibodies are antibodies that possess a single specificity (idiotype) for a specific antigenic determinant (epitope) Alternative name: ehomogeneous antibodies Techniques used to produce monoclonal antibodies: esomatic cell hybridization (cell fusion) erecombinant DNA technique Principle of monoclonal production by somatic cell hybridization: emonoclonal antibody production is based on the fact that each member of a B lymphocyte clone (daughter cells) will express the same idiotype throughout their life span thus maintaining the identical epitope specificity
49	Monoclonal Abs - 3 Somatic cell hybridization technique: Mouse-enzyme deficient myeloma cell + B lymphocyte from mouse that has been injected with specific antigen Cell fusion Hybridoma cell Cultured in media that allows only hybridomal cells to survive Monoclonal Abs with ag specificity
50	Monoclonal Abs - 4 Recombinant DNA technique: emRNA is isolated from lymphoid tissue of donor who has been sensitized with a specific antigen eisolated mRNA is converted to dsDNA using reverse transcriptase + DNA polymerase enzymes epolymerase chain reaction (PCR) is used to amplify the gene fragments of V_H and V_L ethe amplified fragments of V_H and V_L are then injected into E. coli bacteriophage via a plasmid vector and gene fragments fuse with bacteriophage protein eeach recombinant bacteriophage contains a different combination of V_H and V_L genes thus producing antibodies of differing specificities. ethese specificities are then identified by reacting with different antigens and are then cataloged into a monoclonal antibody library
51	Monoclonal Abs - 5 7click arrow to return to main program