Lecture 21 Diagnostic Immunology 2024 PDF

Diagnostic Immunology The clinical immunology laboratory Associate Professor Li Zhang School of Biotechnology and Biomolecular Sciences Room 4106, E26 [email protected] AIMS 1 Understand the principles of the following tests: Nephelometry Immunofluorescence ELISA Immunophenotyping Flow cytometry 2...

Diagnostic Immunology The clinical immunology laboratory Associate Professor Li Zhang School of Biotechnology and Biomolecular Sciences Room 4106, E26 [email protected] AIMS 1 Understand the principles of the following tests: Nephelometry Immunofluorescence ELISA Immunophenotyping Flow cytometry 2 Understand an approach to investigate the following types of diseases with immunology laboratory tests: Autoimmune disease Immunodeficiency FIRST PATIENT 22-year-old woman Over the last 2-3 months has noticed: – Rash on face, especially nose and cheeks Gets worse out in the sun – Sore hands, especially in the wrists and knuckles Hands are stiff in the morning Symptoms have gradually been getting worse Goes to her GP who comments on the rash and finds some tenderness in the wrists BLOOD TESTS Doctor thinks she might have a multi-system autoimmune disease such as systemic lupus erythematosus (SLE) Orders blood tests for – C-Reactive Protein (CRP) – Anti-Nuclear Antibody (ANA) – Anti double-stranded DNA Antibody (dsDNA antibody) C-REACTIVE PROTEIN (CRP) An acute phase protein - part of the innate immune system Synthesized in the liver in response to IL-6 In the presence of inflammation, blood levels rise very rapidly and markedly Binds to damaged tissues, bacteria, complement and phagocytes Can act as opsonin for pathogenic bacteria Widely measured to assess disease activity in inflammatory disorders such as infections and autoimmune diseases NEPHELOMETRY Used to measure more abundant serum proteins (at least 1 mg/L) such as IgG, IgA, IgM, complement components C3 and C4, CRP. Machine adds specific antibodies to serum – In our case, antibodies to CRP are added – When high molecular weight antigenantibody complexes form, machine detects scattering of a light beam Automated, highly reproducible, rapid Not suitable for low abundance serum proteins. https://www.google.com/imgres?imgurl PRINCIPLE OF NEPHELOMETRY The test tube contains patient serum. To measure CRP, the machine adds anti-CRP antibodies which form antigen-antibody complexes with CRP. The high Mr complexes scatter light. The detector measures the rate of increase in the amount of scattered light. CRP RESULT The reference range is less than 10 mg/L – This means 95% of healthy people have a CRP level of less than 10 mg/L Our patient had a CRP result of 85 mg/L – This result tells our doctor that our patient has a significant inflammatory condition – It does not tell which inflammatory condition ANA Good test for multi-system autoimmune diseases Detects serum antibodies that bind to cell nuclei In the patient, – The antibodies form complexes with nuclear antigens – The antigen-antibody complexes deposit in many parts of the body where they cause inflammation The ANA test is done manually Semi-quantitative - positive results are titred Requires operator skill and experience INDIRECT IMMUNOFLUORESCENCE Prepare cell line or animal tissue fixed to slides Add patient’s serum (may contain antibodies) Add antiDetect antibody dye with labelled with fluorescence fluorescent microscope dye wash wash Various autoimmune diseases give different patterns ANA RESULT Our patient has a positive ANA test with: – “Homogeneous” pattern, with uniform staining of nuclei in interphase cells and staining of chromosomes in mitotic figures – High titre ≥ 1:2,560 (reference < 1:80) This is a significant finding and makes the doctor very suspicious that our patient might have SLE. BUT the test is not specific for SLE - other diseases or even healthy people can have a similar result. Common patterns of ANA Antigens of homogenous pattern: dsDNA, histones Kumar et al. Diagnostic Pathology 4, 1 (2009). https://doi.org/10.1186/1746-1596-4-1 Enzyme-linked ImmunoAssay (ELISA) The most sensitive immunological technique (useful when there is too little protein to detect by nephelometry) Enzymes are bound to specific antibodies and produce coloured products Readily automated - quantitative Examples in clinical laboratory: – Serological diagnosis of infectious disease – Autoantibodies – IgE DETECTION OF SPECIFIC SERUM ANTIBODY BY EIA Prepare antigen stuck to plastic well Add patient’s serum (may contain antibody ) wash Add antibody (antiantibody) which is bound to enzyme Add substrate - enzyme produces coloured product wash ANTIBODIES TO DOUBLESTRANDED DNA The test has high specificity for SLE. If antibodies are present, it is highly likely the patient has SLE. Performed with purified double-stranded DNA in ELISA. Our patient’s result is 59 IU/mL Reference range is < 7 IU/mL This is strong evidence for a diagnosis of SLE Summary: Taking the laboratory results together with the patient’s symptoms, the doctor makes a diagnosis of SLE. PATIENT 2 6-month-old boy, referred to a children’s hospital because of repeated infections since he was about 3 months old Has had severe pneumonia and a few middle ear infections Gets better after treatment with antibiotics, then gets sick again Hospital doctors are suspicious of immunodeficiency and take blood Two of the most important tests are serum immunoglobulins and lymphocyte immunophenotyping SERUM IgG 20 Serum IgG (g/L) Measured by nephelometry There are different reference ranges for children – Ig is slow to develop so levels are lower in children High levels at birth from transplacental transfer of maternal IgG. IgG falls in the first few months until infants produce their own. 16 12 8 4 0 IMMUNOGLOBULIN RESULTS Patient results (g/L) IgG IgA IgM 0.2 < 0.05 < 0.05 Reference ranges (g/L) At age 6 months 2.2-7.0 0.08-0.88 0.35-1.0 These results show that the baby has very low IgG and undetectable IgA and IgM, even allowing for the low amounts expected at this age. LYMPHOCYTES STAINED BY ANTIBODY In a traditional blood smear, CD4 T, CD8 T and B cells look similar or identical. They can easily be distinguished by “lymphocyte immunophenotyping”. The subsets have different surface markers These are detected by adding monoclonal antibodies that have been pre-labelled with different fluorescent dyes. The cells are then passed through a flow cytometer, where laser beams strike the cell and measure size (forward scatter) and granularity (side scatter) The lasers also excite the fluorescent dyes which emit at characteristic wavelengths. The flow cytometer measures the properties of each cell. Cells are injected into a shear fluid to create a passage of cells in single file in front of a laser (cells usually are stained with fluorescence labelled antibodies). The forward scatter detectors opposite the laser, and a side aperture at 90 degrees to the laser. Some laser light is refracted by the edge of the cell, picked up by the forward scatter detectors, with larger cells scattering more light (FSC intensity is proportional to the size of the cell). Some laser light is deflected by the internal features like the nucleus and granulation, the greater the complexity the greater the deflection into the side aperture (SSC intensity is proportional to the cell internal complexity). Other laser light is absorbed by the fluorochrome then emitted at a different wavelength of lights. Optics and electronics: optical filters and dichroic mirrors to filter and move light to the detectors. 20 Flow cytometry technique https://melodyreports.com/wp-content/uploads/2019/07/Flow-Cytometry.jpg Flow cytometry technique Cell sorting Fluorescence activated cell sorter (FACS). Specialized type of flow cytometry. The fluorescent character of interest of each cell is measured. An electrical charging is placed just at the point where the stream breaks into droplets. The charged droplets are directed into different containers based on their charge. abcam.com 21 LYMPHOCYTE GATING Red cells are lysed before putting the sample in the flow cytometer. The instrument is set to ignore the small platelets. This leaves all the white cells, but in this case we are only interested in the lymphocytes. All the white cells are analyzed, then lymphocytes are then selected or “gated” by analysis of: forward scatter versus side scatter OR CD45 versus side scatter LYMPHOCYTE GATING Lymphocytes (coloured) Monocytes Granulocytes BLOOD LYMPHOCYTES STAINED WITH CD3 AND CD19 antibodies lymphocytes T Anti-CD3 Anti-CD19 B NK + B NK T Yellow dye = CD19 BLOOD LYMPHOCYTES STAINED WITH CD3 AND CD19 - LYMPHOCYTE GATE ONLY B cells NK cells No cells T cells Blue dye = CD3 CD3 AND CD19 STAINING B cells (CD19 pos) are purple T cells (CD3 pos) are red NK cells (CD3-CD19-) are green Patient CD19 CD19 Healthy control CD3 CD3 No B cells in this patient IMMUNOPHENOTYPING RESULTS Patient results (X 109/L) CD3 CD19 CD16 3.74 0.00 0.46 Reference ranges (X 109/L) At age 6 months 1.7-3.6 0.5-1.5 0.3-0.7 This baby’s total blood lymphocytes are 4.2 x 109/L (reference range for this age 2.2-6.0). Summary: These results show that the baby has undetectable B cells in the blood. The results are consistent with the very low levels of antibodies. Why does the baby have low but detectable IgG? QUESTIONS How many cells do we analyse in the flow cytometer for each marker: A tens of thousands B hundreds of thousands C millions How many dollars does a flow cytometer cost? A tens of thousands B hundreds of thousands C millions