IMMUNOLOGICAL ASSAY

FLOW CYTOMETRY ANALYSIS

SERUM PROTEIN ELECTROPHORESIS
27072020

Leong PP
 When do we need to test the immune
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response?
Clinical Presentation of Possible Immunodeficiency
 Increased or unexplained susceptibility to infection

 Increased severity of common infections (i.e. unusually

severe systemic reaction to a virus)
 Development of infection with an unusual organism such
as fungus or protozoan
 Unusual reaction to immunization (i.e. systemic reaction
following live virus vaccination)
 Family history of recurrent infections

 Exposure to the human immunodeficiency virus

 Clinical Significance of Cellular Immune
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Testing
 To distinguish the features
 Primary immune deficiency
 Suspected acquired immune deficiency
• HIV infection
• Malignancy
• Traumatic injury (i.e. burns or
accidents resulting in major blood loss
or organ damage)
• Haematologic disease (i.e. Fanconi's
anemia, hemophilia, immune
thrombocytopenia)
• Lymphoproliferative disorders (i.e.
histiocytosis)
• Hemoglobinopathies (i.e. sickle cell
anemia, thalassemia)
• Chromosomal abnormalities (i.e.
DiGeorge syndrome, Down syndrome,
Bloom syndrome, William congenital
dyskeratosis, epidermolysis bullosa,
and Duncan syndrome (X-linked
lymphoproliferative disorder)
• Autoimmune diseases (i.e. rheumatic
disease, mixed connective tissue
disease, type 1 diabetes mellitus,
systemic lupus erythematosus,
amyotrophic lateral sclerosis, multiple
sclerosis, and myasthenia gravis)
 The three phases of the clinical evaluation of
a suspected deficiency of cellular immunity
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•Medical history *
•Family history
•Physical exam
findings *

Riley RS, Henry’s Clinical Diagnosis and Management by Laboratory Methods, 5th Edition, 2017, page 890-912
 Flow Cytometry Immunophenotyping
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 A rapid and convenience technique to analyze

heterogeneous populations of cells for the purpose of
identifying the presence and proportions of the various
populations of interest
 Antibodies are used to identify cells by detecting
specific antigens expressed by these cells, which are
known as markers such as functional membrane/
intracellular proteins involved in cell communication,
adhesion, or metabolism
 Applications : basic research and clinical laboratories
 Step 1: Sample Preparation
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(Immunostaining)
Step 2
Step 1
Lysing buffer

Step 3

Washing
buffer
WBC with antigen
on the surface
Step 4

Analysis
 Flow cytometry – Principle
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1. Particles are traveled in a
stream
2. Light scattering properties of
the particles
3. Energy emitted by the laser
excited fluorescent
markers/compounds

FSC – Forward Scattered

light = cell-surface area
or size of cell
SSC – Side-scattered
light = granularity or
internal complexity of
the cell

https://microbenotes.com/flow-cytometry/
 Flow Cytometer
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https://www.youtube.com/watch?v=EQXPJ7eeesQ
 Scatter Plot of Whole Blood
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SSC

FSC
 Flow cytometry- Applications
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 Clinical immunophenotyping analysis

 Clinical diagnosis of malignancy in body fluids i.e.
leukemia
 Detection of DNA content
 Cell cycle analysis
 Analysis of different stages of cell death and
apoptosis
 To isolated cells of interest
 Flow Cytometry Immunophenotyping -
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Clinical Applications
 Measuring CD4T cells in HIV infection
 Routine immunophenotyping in primary
immunodeficiency diseases
 Management of haematopoietic neoplasm
 Diagnosis and classification of haematopoieric
neoplasm
 Detection of antigens used as therapeutic targets
 Detection of residue neoplastic cells following therapy
 Assessment of biological parameter associated with
prognosis
 BD Multitest 6 Colors TBNK Reagent
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CD3 FITC
CD16/56 PE
CD45 PerCP-Cy5.5
CD4 Pe-Cy7
CD19 APC
CD8 APC-Cy7
 Patient ID

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 Truecount Beads

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 TBNK Analysis
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 Multicolour flow cytometric analysis of lymphocyte

subsets
 CD3, CD3+CD4+, Cd3+CD8+, CD19+ and CD16/56+
 CD45 gating is used to accurately define the leucocytes
 CD4/CD8 ratio = 2.0 (normal)
 Higher than 2.0 – strong immune response
 <2.0 – Low CD4 count (DiGeorge syndrome, benign
thymoma, the early phase of hepatitis C virus infection,
Kawasaki disease, protein-calorie malnutrition, and
malignancy)
 <1.0 – may associated with HIV infection

User manual for BD Multitest 6-Color TBNK Reagent

 Reference
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 BD Multitest 6-Colour TBNK Reagent Application Note

 Purdue University Cytometry Laboratories website:
http://www.cyto.purdue.edu/
 The Scripps Research Institute Flow Cytometry Core
Facility: http://facs.scripps.edu/
 Review Question
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 What do you expect on the CD4 cell count,

CD4:CD8 ratio and CD4 percentage of a patient
with progressive HIV infection?
 What is the implication of increased CD16/46 cell
count or percentage?
20 Serum Protein Electrophoresis
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 An easy method of separating serum proteins

based on the physical properties of the proteins
including net charge, size and shape
 Plasma protein levels change in response to acute
inflammation, malignancy, trauma, necrosis,
infarction, burns and chemical injury
 Also known as ‘acute reaction protein pattern’
 To diagnose or monitor multiple myeloma or
investigate underlying cause of low albumin but a
relatively high total protein
 Indications for requesting SPEP
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1st indication based on clinical finding:

 Suspected multiple myeloma, Waldenström’s macroglobulinemia,

primary amyloidosis or other related disorders
 Unexplained bone pain or fracture
 Recurrent infections
 Unexplained peripheral neuropathy (not able to be attributed to
another condition, e.g. type 2 diabetes, chemotherapy)
 Unexplained proteinuria
 To monitor treatment of multiple myeloma to see if the monoclonal
band is reduced in quantity or disappears completely with treatment
 Indications for requesting SPEP
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2nd indication based on Lab findings:

 High (or low) total serum globulin or immunoglobulin

 Extremely high percentage of lymphocytes
 Unexplained anaemia (multiple myeloma is a recognised cause of non-iron
deficiency anaemia) or other persisting cytopaenias for which there is no
other explanation
 Unexplained high ESR (>50) with a normal CRP
 Unexplained hypercalcaemia or renal impairment
 Red cell rouleaux formations noted on the peripheral blood smear
 Presence of urine free light chains (Bence-Jones proteinuria)
 As a follow up to abnormal findings on other laboratory tests, such as total
protein and/or albumin level, elevated urine protein levels, elevated
calcium levels, or low white or red blood cell counts
 Specimen
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 Serum (rather than plasma)

 Container : Red-top tube, serum separator tube
(SST)A fasting specimen is not needed.
 Interferences : presence of lipemia; haemolysis
falsely elevates the total protein result because
of the release of RBC proteins into the serum
 Routine SPEP
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 Serum electrophoresis
 Analysis using densitometer

https://www.youtube.com/watch?v=IcjREcjIpAY
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Fraction Example
Albumin • Largest protein component in serum
Alpha-1 globulins • Globular proteins - highly mobile in alkaline &
Alpha-2 globulins electrically charged solutions – express inhibitory
function on certain blood proteases and other
biological activities
• Alpha1-protein fraction -alpha1-antitrypsin, thyroid-
binding globulin, and transcortin
• Alpha2-protein - ceruloplasmin, alpha2-macroglobulin,
and haptoglobin
Beta 1& 2 globulin • Beta1- transferrin
• Beta2 - beta-lipoprotein. complement proteins
Gamma globulins • Immunoglobulin
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 Monoclonal vs Polyclonal Gammopathies
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 Monoclonal gammopathies – associated with

malignant or potential malignant conditions
M protein – narrow spike in gamma, beta or alpha2-
regions – associated with multiple myeloma (if M
protein >3g/dL)

 Polyclonal gammopathies – associated with

reactive or inflammation conditions – usually non-
malignant
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Kyle RA. Sequence of

testing for monoclonal
gammopathies. Arch Pathol
Lab Med. 1999;123:114–8.
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Thank You