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
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
