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AP0609 - Advanced Analytical Techniques > Diagnostic Applications of Flow Cytometry > Flashcards

Diagnostic Applications of Flow Cytometry Flashcards

1
Q

Flow cytometry in transplantation

A
  • Human Leucocyte Antigen (HLA) antibody detection; Flow Cross matching of organs, HLA typing, CD34 analysis of Haemopoietic Stem Cell Transplantations (HSCT)
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2
Q

HLA antibody detection

A
  • Flow Methods utilise beads coated in HLA or HLA typed cells
  • Can be done on Flow Cytometer (beads& cells) or Luminex (beads) platforms
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3
Q

IgG antibodies in transplantation

A
  • Complement fixing antibody –Hyperacute Rejection
  • Non- Complement fixing – early rejection,long term survival may be achieved
  • Antibodies present in historical sera but current negative- 25% graft survival
  • IgG against HLA Class I and II show poor prognosis
  • Non HLA T cell antibodies may be disregarded if not HLA specific
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4
Q

IgM antibodies in transplantation

A
  • IgM presence is associated with reasonable prognosis but pre existing allo IgM antibodies against mismatched antigens of the donor are detrimental
  • IgM antibody is associated with naïve CTL (CyA sensitive)
  • Patients with IgM HLA antibodies may also have IgG antibodies with the same specificity
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5
Q

What is FlowPRA?

A
  • can be used as a first test to detect HLA antibodies and percent PRA (Percent Panel Reactive Antibody) in human sera using flow cytometry.
  • Intended to detect HLA-specific antibodies for pre- and post-transplantation monitoring
  • provides a comprehensive and accurate assay
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6
Q

FlowPRA (One Lambda)

A
  • HLA antigens are bound to beads
  • Beads are separated on the basis PE (FL2)
  • Positive results are determined by the binging of anti human IgG FITC (FL1)
  • Unlike luminex only a maximum of 8 beads can be tested at one time – Multiple tubes
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7
Q

Luminex HLA Ab Detection

A
  • Tepnel (Lifecodes) or One Lambda (LABScreen) commercial kits
  • HLA antigens bound to multiple microspheres
  • Screening tests and ID tests available
  • Up to 100 beads can be used
  • Thousands of beads per well
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8
Q

Principle of Luminex Ab detection

A
  • 5ul beads: HLA A1, A2, A3 mixed with 20ul serum containing anti HLA A3
  • Incubate at 22-25oC in dark with agitation
  • Spin down and wash unbound antibodies
  • Add 100ul anti human IgG-PE
  • Incubate at 22-25oC in dark with agitation
  • If bead is coated with human IgG antibodies, they will bind
  • Reporter laser; detects PE fluorescence signals
  • Microsphere ID laser; detects bead identification signals
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9
Q

Why incubate at 22-25oC with agitation?

A
  • UV light bleaches dyes in beads
  • decent temperature so reaction isn’t slowed by low temp
  • cells have tendency to clump if not agitated
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10
Q

Complement Dependant Cytotoxic Test

A
  • Cells treated with supravital dye, makes them glow green if alive
  • Add them to well containing patient/recipient 0, all done in mineral oil to avoid evaporation.
  • Using 1ul of patient cells, 1ul of sera, 5ul of culture
  • Leave it for 30mins, 22oC for solids to occur
  • If solid occurs, add complement and PI, complement will be activated by FC portion of any bound antibody and start sticking holes in cell.
  • PI is nuclear stain, enters the cell, shows cells off as red
  • Leave for 60mins at 22oC, add EDTA to stop reaction
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11
Q

Three Colour Lymphocyte immunofluoresence (LIFT)

A
  • Typed cells are mixed with patients serum
  • Add 3 diff antibodies; anti CD3/PE (binds to T cells), anti CD19/PerCP (binds to B cells), the 3rd could be anti IgG, anti IgM or combo one which will detect any antibody
  • T and B cells will have same side scatter and forward scatter, relatively low side scatter
  • Analyse results for each cell depending on PE, FITC or PerCP signals
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12
Q

What results can be seen using LIFT?

A
  • If PE signal, it’s a T cell bc it has CD3 on it
  • PE and FITC positive, have an antibody that can recognise T cell
  • PerCP, CD19 B cell and FITC positive, means antibody can detect both T and B cells
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13
Q

What does PCR-SSO typing stand for?

A
  • Polymerase Chain Reaction Sequence Specific Oligonucleotide typing
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14
Q

PCR-SSO typing

A
  • Amplification of HLA locus of interest with biotinlyated primers
  • After amplification, you add short oligonucleotide (24-40 bp long) designed to match specifically to one HLA gene
  • Denaturation of amplified product
  • Hybridisation of amplified product with Oligonucleotide probes bound to beads
  • Addition of Streptavidin/PE reporter
  • Computer analysis of positive results leads to assignments
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15
Q

CD34 Analysis of HPSC

A
  • CD34 is present on 2-4% of all normal marrow mononuclear cells
  • Studies by Berenson Antigen (1988) showed that CD34-positive marrow cells rescue lethally irradiated baboons
  • Flow methods utilise a mix of anti CD34, anti CD45 and fluorspheres to generate an absolute count
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16
Q

Principle of CD34 Enumeration

A
  • Absolute Count (cells/μl) = Number of CD34 cells (R4) + Fluorospheres Conc / Number of Fluorospheres counted (R7)
  • To obtain the number of cells in a collection the absolute count is multiplied by the dilution factor, the volume (l) of the pack and 10^6
17
Q

Transfusion Related Lung Injury (TRALI)

A
  • Severe type of non-haemolytic transfusion reaction; donor antibodies reacting with patient
  • Etiology unclear but associated with antibodies to white cells; suspected biggest culprit is anti-granulocyte antibodies, donor antibodies against neutrophil
  • Patients HNA A1, donor HNA A1 antibody. Mixture causes a reaction which drags patients neutrophils to lungs, winds them up and causes them to degranulate, causes respiratory distress
18
Q

Principle of TRALI

A
  • Modification of LIFT
  • Gate on Lymphocytes and Granulocytes
  • Incubate with anti human IgG/FITC
  • Analyse lymphocyte and granulocyte populations for FITC fluorescence
19
Q

Most important Antigens for the Blood Transfusion laboratory

A
  • ABO
  • HLA class I
  • Platelet specific (Human Platelet Antigens-HPA)
20
Q

Human Platelet Antigen Nomenclature

A
  • At present, there are 16 documented antigen systems
  • Bi-allellic co-dominant
  • Numbered 1-16 with an a form and b form (HPA-1a,HPA-1b)
  • Single point mutation
21
Q

Neonatal AlloImmune ThrombocytoPeinia (NAITP)

A
  • Similar to HDN
  • Reduced platelet count at birth
  • Petichae rash/Bruising/Intracranial haemorrhage
  • Affects 1 in 1000 pregnancies
  • Can happen in first pregnancy
  • 85% of cases caused by anti HPA-1a
  • HLA DRB3*0101 association (1:3 chance of forming ab)
  • Usually unexpected- no ante natal screening programs
  • Not assoc. with anti-HLA
22
Q

Platelet ImmunoFlorescence Test (PIFT)

A
  • Use Fathers platelets and Mothers serum
  • Gate on platelets based on low Forward Scatter and low Side Scatter
  • Analyse gated region for FITC fluorescence
23
Q

Steps in PIFT

A
  • Platelets from father, serum from mother are mixed
  • Incubate at 37oC for 30mins and wash
  • Add FITC conjugated anti IgG or IgM
  • Incubate at 4oC for 30mins
  • Analyse on flow cytometer
24
Q

Immunophenotyping

A
  • Help the diagnosis of Leukaemia’s by the presence or absence of cell surface markers
  • Each leukaemia and cancer has diff vulnerabilities and diff weak points
  • Need better understanding of which leukaemia or cancer they have in order to better tailor therapy, whether its going to be more or less susceptible to diff drugs
25
Q

DNA Analysis

A
  • one of the first applications for flow cytometry
  • Malignant cells are frequently aneuploid (an abnormal number of chromosomes) and can have prognostic significance
  • The DNA content of a tumour may be expressed as the DNA index
26
Q

What is meant by DNA index?

A
  • ratio between the DNA content of a tumour cell and that of a normal diploid cell
27
Q

DNA analysis method

A
  • Propidium iodide binds stoichiometrically - thus the number of molecules of probe bound is equivalent to number of molecules of DNA
  • Cant enter cell through an intact membrane
  • Membrane must be disrupted by a detergent (Triton-X)
  • Then measure cell counts against PI fluorescent signal
28
Q

Clinical DNA analysis in breast cancer

A
  • 200 patients with Stage I and II breast cancer were studied
  • Full clinical details with 10 year follow up was available
  • Surgery consisted of mastectomy with axillary node sampling
  • DNA content was determined on paraffin blocks
  • Outcome: Patients with aneuploidy showed earlier relapse and shorter survival times
29
Q

Cell Cycle Analysis

A
  • One of the earliest applications of flow cytometry was the analysis of cell cycle position by quantitation of cellular DNA.
  • Flow cytometry is still the method of choice for fast, accurate determination of cell cycle distributions
30
Q

Chronic granulomatous disease

A
  • Phagocytes cannot form the oxidative burst
  • Phagocyte NADPH oxidase is responsible for the generation of the oxidative burst and is inactivated by genetic mutations
31
Q

Symptoms of Chronic granulomatous disease

A
  • pneumonia
  • abscesses of the skin, tissues, and organs
  • suppurative arthritis
  • osteomyelitis
  • bacteremia/fungemia
  • superficial skin infections such as cellulitis or impetigo

AP0609 - Advanced Analytical Techniques (10 decks)

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