Immunologie in der Labormedizin Konteptvorlesung 2 Themenblock 8 Beda M. Stadler Diese Folien stehen auch als PPT Files zum download bereit unter: http://www.iib.unibe.ch/teaching/immed.htm Einige der Folien werden als Illustration verwendet und sind nicht Lernstoff. Sie sind so wie hier entweder orange unterlegt oder umrandet

Lernziele: Immunologie in der Labormedizin KV2 im Überblick Lernziele: Immunologie in der Labormedizin                            Was sind Immunassays? Was versteckt sich hinter den Abkürzungen? Assay Beispiele um immunologische Prinzipien zu vertiefen Aussagekraft von Labordaten Vertrauensgrenzen Knowledge of the concentration of various antigens or antibodies in a patient's blood or other tissues can be very useful in diagnosis and/or in determining the clinical status of a patient. Tests of antigen-antibody interactions fall into two categories, indirect and direct measurements. Precipitation reactions are dependent on the presence of multivalent antibodies and antigens. They are inherently relatively insensitive since there must be enough reactants present to form a viable precipitate. Precipitation reactions include: agglutination, single double diffusion in agar, rocket electrophoresis, and immunoelectrophoresis. Complement fixation is more sensitive than precipitation reactions but is seldom used in clinical labs anymore. Direct measurements of antigen-antibody reactions are very sensitive because they use machines to directly measure the antigen-antibody reactions. Thus, they are not dependent on formation of a visible precipitate. The machines can detect amounts far smaller than one can see. The direct assays include radioimmunoassay, enzyme linked immunosorbant assay, and fluorescent antibody techniques. KV 8.2

Was ankreuzen? KV 8.2

Immunologie in der Labormedizin KV3 im Überblick Immunologie in der Labormedizin Präzipitationsteste Agglutinations Teste Antigen-Antikörper Teste Allgemein Allergie Diagnostik Flowzytometrie HLA Diagnostik Autoimmun Diagnostik Sensitivität und Spezifität von Labortesten Knowledge of the concentration of various antigens or antibodies in a patient's blood or other tissues can be very useful in diagnosis and/or in determining the clinical status of a patient. Tests of antigen-antibody interactions fall into two categories, indirect and direct measurements. Precipitation reactions are dependent on the presence of multivalent antibodies and antigens. They are inherently relatively insensitive since there must be enough reactants present to form a viable precipitate. Precipitation reactions include: agglutination, single double diffusion in agar, rocket electrophoresis, and immunoelectrophoresis. Complement fixation is more sensitive than precipitation reactions but is seldom used in clinical labs anymore. Direct measurements of antigen-antibody reactions are very sensitive because they use machines to directly measure the antigen-antibody reactions. Thus, they are not dependent on formation of a visible precipitate. The machines can detect amounts far smaller than one can see. The direct assays include radioimmunoassay, enzyme linked immunosorbant assay, and fluorescent antibody techniques. KV 8.2

Immunodiffusion (Ouchterlony Gel) Keine Identität 1 A 4 2 3 6 5 Identität 1 A 4 2 3 6 5 Partielle Identität 1 A 4 2 3 6 5 A 1 2 Antigen Kontrollserum Testserum KV 8.2

+ Präzipitinkurve Antigen Antikörper Immunkomplex C B A Anti-B Anti-A Anti-C Antigen Antikörper Immunkomplex Äquivalenz Prozone Postzone Immunkomplex Antigen KV 8.2

Radiale Immunodiffusion AK Konzentration Diameter2 Ag Antikörper im Gel KV 8.2

- + Serum Elektrophorese Globuline gamma beta alpha2 alpha1 Albumin Paraproteinämie-Patient KV 8.2

Immun-Elektrophorese Normales Serum Albumin Transferrin IgM IgA IgG + - Antikörper gegen Serumproteine Patienetenserum mit IgA Mangel Beispiel KV 8.2

Direkter Coombs Test KV 8.2

Der indirekte Coombs Test Step 1 Y +  Y Y Y Patienten Serum Erythrozyten Y Step 2 Y Y Y Y Y Y +  Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagens (Antiglobulin) Y z.B. anti-RhD Antikörper KV 8.2

Passive Hämagglutination (Beispiel: Anti-Syphilis Antikörper) In Passive Hemagglutination, sheep red blood cells (SRBC) are coated with a purified antigen (in this case from T. pallidum, the bacteria which causes syphilis).  Allowed to settle in a well of a 96-well plate, such cells well settle into a small button, as seen in the last six wells of the top row (vairous negative controls).  In the presence of antibody, such red cells will agglutinate, yielding the pattern seen at the top left (a 1:160 dilution of a control positive serum).  Each of the sixteen patients' sera (numbered 5-19) is added to such "sensitized" SRBC (top of each pair of wells) as well as normal, unsensitized cells (bottom).   In this example, those patients' sera numbered 10, 12, 16, 17 and 18 show a positive reaction for the presence of anti-syphilis antibodies, all the others are negative. KV 8.2

Latex Agglutination KV 8.2

Blutgruppenbestimmung: Geltechnik monoklonaler Antikörper gegen Blutgruppen A Antigen in der oberen Gelmatrix Resultat: Blutgruppe A RhD neg KV 8.2

z.B. Schwangerschaftstest rapid determination of human chorionic gonadotropin (hCG) in urine specimens Primary antibodies (anti-hCG labelled or on solid phase) Secondary antibodies (anti-Ig on solid phase) KV 8.2

Allergieserologie: welcher Test? Anti-b2-Glycoprotein 1 (IgG) ELISA U/ml <15 Cardiolipin-Antikörper (IgG) SYNELISA GPL-U/ml <12 ECP ("eosinophil cationic protein") FEIA g/L 2.5-15 Tryptase 1-13.5 Methylhistamin RIA µg/g Kreatinin 70-160 Total IgE kIU/L (altersabhängig) Allergen-spezifisches IgE (RAST) Rastklassen 0-6 Allergen-spezifisches IgG mg/L < 50 mg/L Präzipitine Immundiffusion qualitativ negativ Analyse Messprinzip Masseinheit Referenz-bereich KV 8.2

KV 8.2

Immun Assay: RIA & ELISA Radio Immuno Assay Enzyme Linked Immuno Sorbent Assay Isotop Enzyme markierter Antikörper Antikörper Antigen Messung der Radioaktivität oder Enzymaktivität KV 8.2

ELISA Markierter Antikörper Substrat Enzym Anti-A Produkt A KV 8.2

Sandwich ELISA Anti-Antikörper Substrat Enzym                                      Anti-A Produkt A KV 8.2

Standardisierung KV 8.2

Beispiel: Flowzytometrie   Beispiel: Flowzytometrie Analyse Messprinzip Masseinheit Referenzbereich HLA-B27 Zytofluorometrie qualitativ positiv/negativ Lymphozyten Zytofluorometrie Zellen/l 1200-2800 CD3 absolut Zytofluorometrie Berechnung Zellen/l % der Lymphozyten 690-2540 55-84 CD4 absolut Zytofluorometrie Berechnung Zellen/l % der Lymphozyten 410-1590 31-60 CD8 absolut Zytofluorometrie Berechnung Zellen/l % der Lymphozyten 190-1140 13-41 NK-Zellen Zytofluorometrie Berechnung Zellen/l % der Lymphozyten 90-590 5-27 B-Zellen Zytofluorometrie Berechnung Zellen/l % der Lymphozyten 90-660 6-25 KV 8.2

Fluorescence Activated Cell Sorter FACS sample sorted cells nozzle red fluorescence sheet flow prism green fluorescence laser right angle scatter forward scatter sorted cells KV 8.2

Zellpopulationen Link alle CD Marker ? KV 8.2

CD-Antikörper Funktion Vorkommen CD Antigen CD 2 CD 3 CD 4 CD 8 CD 14 Rezeptor für CD 58 (LFA-3), alternativer Aktivierungsweg von T-Lymphozyten unreife und reife T-lymphozyten CD 3 T-Zell-Rezeptor-assoziierte polypeptidketten, Signaltransduktion reife T-Lymphozyten CD 4 assoziative Erkennungsstruktur (Bindung an MHC-Klasse II Moleküle), HIV-Rezeptor T-Helferzellen, Monozyten, Makrophagen CD 8 Bindung an MHC-Klasse I Moleküle zytotoxische T-Lymphozyten CD 14 Rezeptor für bakterielles Endotoxin (LPS=Lipopolysacharid) Monozyten, Makrophagen CD 16 Fc-gamma Rezeptor III NK-Zellen, Monozyten-Subpopulationen, Makrophagen CD 23 niedrig affiner (bindender) Rezeptor für IgE B-Lymphos, follikuläre dendritische Zellen (=FDC) CD 25 Interleukin-2-Rezeptor aktivierte T-Lymphozyten IgM Antigenrezeptor unreife + reife B-Lymphos IgD reife B-Lymphos HLA-DR MHC-Klasse II Moleküle, Bindung von Antigenpeptiden AG-präsentierende Zellen (dendritische Zellen, Monozyten, B-Zellen), aktivierte T-Lymphos KV 8.2

Eine Immunantwort verfolgen KV 8.2

CD-Marker bei der Diagnose von lymphohämopoietischen Erkrankungen   Stammzelle Prä-B. frühe B. mittlere B. reife B. plasmozytoide B. Plasmazelle    ALL PLL, Burkitt Lymphom CLL, foll. L. Haarzell-L. Waldenström Myelom CD19 CD 20 CD 34 CD 52 CD138 KV 8.2

Beispiel: HLA Typisierung Analyse Messprinzip Masseinheit Referenzbereich HLA-B27 Zelllyse qualitativ positiv/negativ HLA-Typ I (A, B, C) (serologisch) Phänotypisierung HLA-Typ I (A, B) PCR-SSP HLA-Typ II (DR) HLA-Typ II (DQ) HLA-Typ II (DP) HLA-Typ Organempfänger T-Zell Cross-match negativ B-Zell Cross-match Leukozytenantikörper KV 8.2

HLA Typisierung einst... Serologically defined antigens. Technically, tissue typing means determining the particular alleles of membrane antigens possessed by the cells of an individual. Blood typing is the most common tissue typing done and the most important one for all tissue transplants. However, in practice tissue typing means determining the particular alleles of the human HLA (figure below). Currently, most typing is done by mixing the lymphocytes of the person to be typed with complement and anti-serum to specific HLA alleles. Some antisera is obtained from women who have had several children, so they have become sensitized to HLA antigens contributed by their husbands. Other antisera comes from people who have had transfusions and become sensitized to HLA antigens in the donated blood. Finally some is obtained from volunteers who have been deliberately exposed to HLA antigens. If the lymphocytes are killed by the antisera and complement, they must have had one or more antigens recognized by the antiserum. Since most of the antisera used recognize more than one allele, overlapping specificities must be used, and a computer matches which alleles the different antisera have in common. Efforts to obtain monoclonal antibodies specific for only one antigen have been disappointing for several technical reasons. Most research efforts now are to adapt molecular biological techniques (restriction fragment length polymorphism with or without amplification via polymerase chain reaction) to the identification of HLA alleles, particular class II since they are harder to identify serologically. KV 8.2

Überlebensrate Nierentransplantate Number of HLA mismatches A & B DQ & DR 0 0 A & B DQ & DR 3 or 4 1 or 2 The three class I genes (HLA-A, -B, & -C) serve as the main targets of graft rejection. Of these, HLA.-B is strong-er than HLA-A which is much stronger than HLA-C. Therefore, the surgeon will always choose to match HLA-B if possible. (In fact, HLA-C is so weak that labs do not usually try to match it.) We used to think that HLA-B was the critical antigen to match. However, as can be seen from the figure at the left, D region products are even more important. On reflection, this is not surprising, since class II genes (HLA- DP, - DQ, & - DR) are involved in control of the immune response. A mismatch in these genes stimulates production of the cytokines, which in turn, enhance all immune responses in the area including the ones to HLA-B and -A. There is a strong synergy between mismatches in both class II and class I genes. KV 8.2

HLA-B27 Typisierung mittels FACS KV 8.2

HLA Typisierung serologisch KV 8.2

PCR KV 8.2

HLA: Molekulare Diagnostik KV 8.2

Array Technologie                                                                                                                                                                                                                                                                                                                                                                                                                                                             „DNA Chips“ KV 8.2

Western Blot: Prinzip KV 8.2

HIV: Western Blot Lane 1, HIV+ serum (positive control) Lane 2, HIV- serum (negative control) Lane A, Patient A Lane B, Patient B Lane C, Patient C KV 8.2

Beispiel: Autoimmunserologie Analyse Messprinzip Masseinheit Referenzbereich Rheumafaktor Nephelometrie IU/ml < 20 Zellkerne (ANA) IIF Titer < 1:80 Thyreoidea Thyroglobulin Agglutination < 1:400 C1-Esteraseinhibitor (funktionell) Fotometrie % des Normwertes 70-130% C1q-tragende Immunkomplexe ELISA g/ml < 35 Hautbiopsie DIF qualitativ negativ Schleimhautbiopsie KV 8.2

Nephelometry This technique is widely used in clinical laboratories because it is relatively easily automated. It is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (usually about 70 or 75 degrees). Antibody and the antigen are mixed in concentrations such that only small aggregates are formed that do not quickly settle to the bottom. The amount of light scatter is measured and compared to the amount of scatter from known mixtures. The amount of the unknown is determined from a standard curve. KV 8.2

Antinukleäre Antikörper (ANA) Immunfluoreszenz Antinukleäre Antikörper (ANA) Fluorochrom markierter AK Y Ag Gewebe KV 8.2

Maus Lymphknoten gefärbt mit einem anti-T-Zell Antikörper Antibodies can be used to localize particular antigens within histological sections.  In this case, a frozen section of a mouse lymph node is stained with a fluorescent antibody specific for an antigen expressed by T-cells.  This image demonstrates the fact that T-cells are located only within specific regions of lymphoid tissue, the Diffuse Cortex (DC) of the lymph node, in this case.  Another region within the node, the Primary Follicle (PF) contains very few T-cells.  KV 8.2

In situ Hybridisierung KV 8.2

FISH KV 8.2

FISH Beispiel Fluorescent in situ hybridisation of a small intestinal biopsy in a case of Whipple's disease (confocal laser scanning microscopy). Tropheryma whipplei rRNA is blue, nuclei of human cells are green and the intracellular cytoskeletal protein vimentin is red. Magnification approximately 200 x. KV 8.2

Immunhistochemie Immunohistochemistry is a technique for identifying cellular or tissue constituents (antigens) by means of antigen-antibody interactions, the site of antibody binding being identified either by direct labelling of the antibody, or by use of a secondary labelling method. In Situ Hybridization techniques allow the demonstration of specific nucleic acid sequences (genes) in their cellular environment.   Above: Human Papillomavirus DNA demonstrated by In Situ Hybridisation (pink) in epithelial cells identified by indirect immunofluorescence using antibody against cytokeratin (green) Human Papillomavirus DNA demonstrated by In Situ Hybridisation (pink) in epithelial cells identified by indirect immunofluorescence using antibody against cytokeratin (green) KV 8.2

Sensitivität und Spezifität Sensitivität (epidemiologisch) Sensitivität (analytisch) niedriger Titer Serokonversion Verdünnung KV 8.2

oder... Sensitivität = Wahrscheinlichkeit, dass Test bei kranken Personen positiv ausfällt Spezifität = Wahrscheinlichkeit, dass Test bei gesunden Personen negativ ausfällt KV 8.2

Genauigkeit der Labordiagnostik echt Positive Sensitivität = X 100% echt Positive + falsch Negative echt Negative Spezifität = X 100% echt Negative + falsch Positive KV 8.2

Aussagekraft der Labordiagnostik positiver prädiktiver Wert = echt Positive X 100% echt Positive + falsch Positive negativer prädiktiver Wert = echt Negative X 100% echt Negative + falsch Negative KV 8.2

Prädiktive Werte PPV = 45/ 45+5 = 90% Annahme: Test Sensitivität = 100% / Spezifität = 99.5% Population #1, die Prävalenz der Infektion ist hoch Population: 1000 Seren getestet 50 Seren von infizierten Individuen 950 Seren von nicht infizierten Individuen Test Results: 50 positive: 45 aus der infizierten Gruppe 5 falsch positive aus der nicht infizierten Gruppe Der positive prädiktive Wert ist: PPV = 45/ 45+5 = 90% 9 von 10 positiven Resultaten sind von infizierten Individuen KV 8.2

Prädiktive Werte PPV = 2/ 2+5 = 28.6% Annahme: Test Sensitivität = 100% / Spezifität = 99.5% Population #2, die Prävalenz der Infektion ist tief Population: 1000 Seren getestet 7 Seren von infizierten Individuen 993 Seren von nicht infizierten Individuen Test Results: 7 positive: 2 aus der infizierten Gruppe 5 falsch positive aus der nicht infizierten Gruppe Der positive prädiktive Wert ist: PPV = 2/ 2+5 = 28.6% KV 8.2

Immunologie in der Labormedizin KV2 im Rückblick Immunologie in der Labormedizin Traditionelle Serologie Moderne immunologische Teste Molekularbiologische Methoden Outlook: Point-of-care testing ? Immunologie Wahlpraktikum 3. Studienjahr 2004 „Ein Einblick in die Labormedizin“ http://www.immunology.unibe.ch/teaching/Praktikum/praktikum.htm KV 8.2