Lecture 6. Diagnostic Techniques Year 2023

Lecture 6 – Diagnostic techniques
What are we going to study in this lecture?
• Diagnostic tests run in a real NHS Immunology laboratory
• Common techniques run in an Immunology research lab
Complete this lecture with Immunological techniques lecture from MADD module in Term 1
Diagnostic tests run in a real NHS Immunology lab
https://www.ouh.nhs.uk/
immunology/diagnostic-tests/
Diagnostic tests run in a real NHS Immunology lab
1. Allergy serology
2. Autoimmunity
3. Immunodeficiency
4. Immunochemistry
5. Infectious immunology
6. Transplant immunology
Allergy serology
Allergy serology
• Detection of specific IgE abs to a range of antigens – Immunocap IgE
https://www.thermofisher.com/phadia/wo/en/our-solutions/immunocap-allergy-solutions.html
Allergy serology
Allergy serology
https://www.ouh.nhs.uk/immunology/diagnostic-tests/documents/sige-list.pdf
Autoimmunity
Autoimmunity
• Coeliac screening – IgA tissue transglutaminase abs (TTGA)
Confirmation: IgA endomysial abs (EMA)
TTGA - Immunoblot or Dot-blot
Serum of the patients
in each dot
Anti-IgA secondary ab Transglutaminase

conjugated with enzyme protein
Addition of enzyme
substrate
Autoimmunity
• Coeliac screening – IgA tissue transglutaminase antibody (TTGA)
Confirmation: IgA endomysial antibody (EMA)
TTGA – Immunoblot or Dot-blot
EMA – Indirect Immunofluorescence
Autoimmunity
Autoimmunity
• Systemic autoimmune investigations – IgG anti-nuclear abs (ANA)
Confirmation: Immunocap Extractable nuclear IgG abs (ENA)
ANA – Indirect Immunofluorescence in Hep2000 cells
Autoimmunity
Autoimmunity
• Organ-specific autoimmune investigations – Detection of autoantibodies (IgG)
Indirect Immunofluorescence in organ-specific cells
ELISA
Immunoblot or Dot-blot
Immunodeficiency
Immunodeficiency
• Presence of immune cells: Flow cytometry
Cellular and Molecular Immunology

Abbas, Lichtman, Pillai. 8th Edition
Immunodeficiency
• Function of immune cells: Ability to proliferate to mitogens (CFSE assay)
Peripheral Blood Mononuclear Cells (PBMCs)

+
Carboxyfluorescein succinimidyl ester (CFSE)
+
Mitogens
Immunodeficiency

+
Carboxyfluorescein succinimidyl ester (CFSE) incubation Proliferation?
+ (Flow cytometry)
Mitogens
Immunodeficiency

+
Carboxyfluorescein succinimidyl ester (CFSE) incubation Proliferation?
+ (Flow cytometry)
Mitogens
2nd generation
Unlabelled
1st generation
3rd generation
Immunochemistry
Infectious immunology
Detection of T reactive cells against specific pathogens - ELISpot
Detection of T reactive cells against specific pathogens - ELISpot
Detection of T reactive cells against specific pathogens – FluoroSpot
Common techniques run in an Immunology research lab
Let’s take a break…

Transplant immunology (Organ transplantation)

HLA Class I
Major antigens Mum’s haplotype Dad’s haplotype Your haplotype
HLA-A HLA-A*01:03 HLA-A*03:04 HLA-A*01:04
HLA-B HLA-B*07:25 HLA-B*08:10 HLA-B*07:08
HLA-C HLA-C*03:16 HLA-C*09:21 HLA-C*09:16
Minor antigens
HLA Class II
Major antigens Mum’s haplotype Dad’s haplotype Your haplotype
HLA-DR HLA-DR*01:15 HLA-DR*01:03 HLA-DR*01:01
HLA-DQ HLA-DQ*06:09 HLA-DQ*04:08 HLA-DQ*04:06
HLA-DP HLA-DP*01:15 HLA-DP*02:04 HLA-DP*04:15
Minor antigens
Early detection of
preformed abs
against donor blood type
and HLA molecules

How can our immune system generate preformed abs against different
HLA molecules?
Three circumstances:
HLA molecules?
Previous transplantations
HLA molecules?
Blood transfusion
HLA molecules?
Blood transfusion
Pregnancies
Detection of pre-formed abs:
Complement-dependent cytotoxicity (CDC) – IgG1 and IgG3 (activators of complement system)
Kumar, Jayant et al. (2018)

Polish Journal of Surgery. 90. 41.46
Virtual crossmatch - Luminex
Kumar, Jayant et al. (2018)

Polish Journal of Surgery. 90. 41.46
CDC vs. Luminex
Isolation of PBMCs and/or splenocytes (animal studies)

Western-blot
ELISA
Immunohistochemistry
Immunoflourescence
Flow cytometry
ELISpot
Western-blot:
Identification of proteins – Gel loaded with an unknown protein extract and
transferred to a membrane. Membrane incubated with a known
primary ab. Only qualitative test
Identification of abs in serum from patients – Gel loaded with a known set of
proteins and transferred to a membrane. Membrane incubated
with serum from patients to detect protein-specific abs. Only
qualitative test
Western-blot:
Identification of proteins – Gel loaded with an unknown protein extract and
transferred to a membrane. Membrane incubated with a known
primary ab. Only qualitative test
Identification of abs in serum from patients – Gel loaded with a known set of
proteins and transferred to a membrane. Membrane incubated
with serum from patients to detect protein-specific abs. Only
qualitative test
ELISA:
Identification of abs in serum from patients – Plate coated with a known
protein and incubated with serum samples from patients to
detect protein-specific abs. Titration is possible so it is a
qualitative and quantitative test
Immunohistochemistry:
Identification of proteins – Tissue from biopsy is incubated with a known
primary ab. Qualitative and quantitative test
Identification of abs in serum from patients – Tissue from biopsy is incubated
with serum from patients to detect tissue-specific abs.
Only qualitative test
Immunohistochemistry:
Identification of proteins – Tissue from biopsy is incubated with a known
primary ab. Qualitative and quantitative test
Identification of abs in serum from patients – Tissue from biopsy is incubated
with serum from patients to detect protein-specific abs.
Only qualitative test
Immunofluorescence:
Identification of proteins – Cells are incubated with a known primary ab. Only
qualitative test
Identification of abs in serum from patients – Cells are incubated with serum
from patients to detect protein-specific abs. Only qualitative test
Flow cytometry:
Cell phenotype – Extracellular and intracellular markers. Qualitative and
quantitative test
Wille-Reece et al. (2006)

The Journal of experimental He, Xiaoning et al. (2013). PloS
medicine. 203. 1249-58. one. 8. e60473..
Flow cytometry:
Cell phenotype – Use of magnetic beads isolation
Flow cytometry:
Miltenyi Biotec
https://www.miltenyibiotec.com
/GB-en/
Flow cytometry:
Flow cytometry:
Cell phenotype – Use of tetramers
https://www.mblbio.com
/tetramer/
Flow cytometry:
Cell phenotype – Use of tetramers
https://www.mblbio.com
/tetramer/
Flow cytometry:
Cell phenotype – Extracellular and intracellular markers
Use of magnetic beads
Use of tetramers
Cell proliferation – CFSE
Flow cytometry:
Cell phenotype – Extracellular and intracellular markers
Use of magnetic beads
Use of tetramers
Cell proliferation – CFSE
ELISpot:
Cell activity – Plate coated with a specific-cytokine ab to detect the cytokine
release from cells incubated with certain stimuli. Qualitative and
quantitative test
Lecture 6 – Diagnostic techniques
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Lecture 6. Diagnostic Techniques Year 2023

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Lecture 6 – Diagnostic techniques

What are we going to study in this lecture?

• Diagnostic tests run in a real NHS Immunology laboratory

• Common techniques run in an Immunology research lab

Anti-IgA secondary ab Transglutaminase

Cellular and Molecular Immunology

Peripheral Blood Mononuclear Cells (PBMCs)

Peripheral Blood Mononuclear Cells (PBMCs)

Peripheral Blood Mononuclear Cells (PBMCs)

Let’s take a break…

Cellular and Molecular Immunology

against donor blood type

and HLA molecules

Cellular and Molecular Immunology

Kumar, Jayant et al. (2018)

Kumar, Jayant et al. (2018)

Isolation of PBMCs and/or splenocytes (animal studies)

Wille-Reece et al. (2006)

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