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)
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
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
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
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
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
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
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
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
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
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
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
13
Q
What does PCR-SSO typing stand for?
A
- Polymerase Chain Reaction Sequence Specific Oligonucleotide typing
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
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
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
DNA Analysis
- 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
What is meant by DNA index?
- ratio between the DNA content of a tumour cell and that of a normal diploid cell
27
DNA analysis method
- 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
Clinical DNA analysis in breast cancer
- 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
Cell Cycle Analysis
- 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
Chronic granulomatous disease
- 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
Symptoms of Chronic granulomatous disease
- pneumonia
- abscesses of the skin, tissues, and organs
- suppurative arthritis
- osteomyelitis
- bacteremia/fungemia
- superficial skin infections such as cellulitis or impetigo
