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1

Name 4 applications of flow cytometry (FC).

1. Transplantation
2. Immunophenotyping
3. DNA analysis
4. Immunology

2

Define what transplantation is.

It is the movement of material from one individual to another (related or not).
I.e. donation of a kideny from one individual to another individual.

3

What is HLA?

Human Leucocyte Antigen. They are antigens that are found on human cells, and is a way of self-ID self from non-self. They have two subtypes/classes: HLA type I and HLA type II. Need to be able to detect pre-existing antibodies (Abs)/antigens in recipient's before they get transplants to prevent HAR (hyperactive rejection).

4

How do we test compatibility of a donor with a recipient?

Do cross-match test. This can be done using FC and/or looking at CD34 (a rare marker that is only found in progenitor cells for bone marrow- BM- and the hematopoietic system) analysis (to give understanding on whether the patient will have cells that can re-populate the cells that have been destroyed (I.e. from chemotherapy treatment in cancer patients).

5

HLA Detection.

Use flow methods that use microsphere beads that ate coated in the HLA antigens/HLA typed cells. However, there is a limitation to how many tests can do at once (~4-8 different antigen beads/1 cell). This means that would have to repeat tests over several times which could increase error. If were to use Luminex technology instead, can do batch testing using 1000s of beads. Likewise, could just use cells that recognise which antigens exist in the panel (like in astrology assay using a 96 well plate).

6

HAR caused by HLA Ab IgG.

If having a transplant, and have pre-formed HLA Ab of IgG in the body which is complementary to the HLA antigens on the donated organ tissue, then the organ will be destroyed through complement fixing IgG.

7

IgM presence in relation to HLA- Abs in transplantation.

Having pre-existing allo-IgM Abs against mis-matched antigens of donors can be detrimental (this is because it's associated with naive cytotoxic lymphocytes- CyA sensitive). Patients who have HLA IgM, may also have HLA IgG of the same specificity.

8

Ensure no rejection occurs, what are things we need to ensure the recipient has/shouldn't have?

1. Recipient has NO HLA Abs present
2. Recipient has NO CURRENT HLA Abs present
3. Recipient has HLA Abs present, but they don't react with the antigens that are present on the organ tissue ect.

9

Flow PRA (One Lambda).

Flow PRA = Flow Panel Reacting Antibodies.
Have small set of beads that can be separated based on their varying levels of pychoerythryin (an fluorophore that is tagged on the ends of Abs). Can place HLA antigens on surface of the beads and then take the patients serum and mix the two together. From this, will be able to see whether the patients serum contains the complementary Abs that will bind the antigens on the beads/not. The set of beads are washed in a series of wash steps (to remove unbound Abs), and anti-human IgG tagged with FITC is added. FITC is a powerful fluorophore that will give off a green fluorescent if it binds to the IgG antigens on the beads.

10

What is the limitation with Flow PRA?

There is a low max amount of beads that can be tested at one time, which means that repeats would have to be done, which increases the risk of error each time.

11

Principle of Luminex HLA Ab Detection.

1. Have 3 bead types: HLA A1, HLA A2, HLA A3. They all have different ratio of red:IF dye. These beads are then incubated with patients serum containing anti-HLA A3, at room temperature in the dark with slight agitation. This allows for binding of the Abs (of present) to the complementary bead.
2. The beads then undergo a series of wash steps to remove unbound Abs and reduce background 'noise'.
3. The beads are then incubated, in the same condition, with anti-human IgG tagged with PE.
4. They are then washed again to remove unbound Abs and transferred to the lasers of the Luminex machine.
5. The Luminex has 2 lasers: 1st one is the REPORTER LASER = ID the bead by detecting the IF dye, the 2nd one is the MICROSPHERE ID LASER = detects the presence of the HLA antigen/Ab complex on the bead surface.

12

A more modern version of Complement Dependent Cytotoxic test?

Three Colour Lymphocyte Immunofluoresence (LIFT).

13

What is the aim of LIFT?

To ID T cells (which have only HLA class I antigens on their surface), and B cells (which have both classes of HLA antigens on their surface) in order to see if any action needs to take place.

14

What is the principle behind LIFT?

Takes bloods/blood related sample and run using FC. Need to take sample from known types of cells, and take serum from patient and incubate them together with 3 Abs: anti-CD3-PE (for T cells), anti-CD19-PerCP (for B cells), and anti-IgG-FITC.
Would be able to get forward scatter (FS) and side scatter (SS) and then look at the fluorescent signal that is given off to ID whether it is a T/B cell? Lymphocytes have a low FS and SS (not very big and don't have much cellular content).
Presence of PE signal should indicate the type of lymphocyte that is present.
T CELLS = laser light is one colour, PE is the second colour, and FITC is the third colour = LIFT!
B CELLS = laser light is one colour, PerCP is the second colour, and FITC is the third colour = LIFT!

15

HLA typing using PCR.

Take patient DNA which has HLA locus of interest and let it undergo PCR (to amplify the HLA locus of interest). Can then denature the PCR product and add oligonucleotide probe that is attached to the bead, to re-hybridise the DNA.
ONLY SEQUNECE MATCHING CAN OCCUR TO MAKE DNA RE-HYBRIDISE.

16

CD34 analysis of hematopoietic progenitor/stem cell (HPSC).

In BM transplants, need to harvest cells from the bloodstream of the individual. To do this, would have to 'pump' the individual with Granulocyte Macrophage Colony Stimulating Factor- GMCSF. This drug encourages the BM to go into overdrive and continuously produce these cells to higher than normal levels, so that the BM can push them out into the bloodstream for easier collection.
CD34 = doses based and adjusted on body length. Safe zone for transplant = 3.5 x 10^6 mill cells/kg body weight (2.5 is absolute min!)

17

The principle behind CD34 enumeration.

This method allows for you to be able to make an ABSOLUTE CELL COUNT (cells/microlitre), as opposed to just % of the population. In this method, fluorophores of a known concentration are added. They are designed to be out of the range of the cells looking for and the Abs looking at. To obtain the no. cells in the collection, the absolute cell count x dilution factor.
i.e. 1000 spheres = 1 microlitre, therefore if using 3000 spheres = know that 3 microlitres been used so can calculate the conc value.

18

Transfusion Related Acute Lung Injury (TRALI)

V. severe, but slef-limiting condition. It's not a haemolytic disease. Presentation usually follows platelet/plasma rich transfusions. Patient would find it diff to breathe after having transfusion, need be ventilated (would therefore likely be taken to ICU for recovery). Can return to norm ward after couple days.
Some evidence that is mediated by anti-HLA/anti-HNA (anti-granulocyte Abs) w/in the donor plasma. The Abs circulate and excite the neutrophils in lung (which go into overdrive = increase in oxidative stress) = Respiratory Stress. The self-lim part = material breaks down so rxn begins to subside.

19

What is the basic principle of modded LIFT?

Essentially, allows for us to understand if the patient already has Abs on the surface of their cells, and if they do, which cells are they on the surface of?
i.e. HLA = surface of lymphocytes, HNA = surface of granulocytes.

20

What are platelets?

Small fragments that are broken off from a larger cell (megakarocyte). They are necessary in clotting and wound healing.

21

Do platelets carry antigens on their surface? If they do, what kinds?

Platelets carry ABO group antigens as well as HLA class I antigens on their surface. They also carry their own antigen types: HPA (human platelet antigens). There are 16 main antigens (HPA1-16), however, these antigens are unique as they are BI-ALLELE (have 2 versions of the allele coding for the antigen) = a form and b form. The a form = most common and higher in frequency. The b form = less common. The diff btwn the all the antigens for HPA is that of a POINT MUTATION (change in base pair gives rise to a form and b form).

22

Neonatal Alloimmune Thrombocytopenia (NATIP).

Platelet equivalent to Haemolytic disease of the newborn (HDN).
Mum = low freq antigen: HPA1b1b, Dad = HPA1a1b/HPA1a1a (high freq antigen). Baby = if gets HPA1a, it produces a platelet Abs against this antigen that the mum's IS would not have seen. Since platelets are small, can diffuse through the placenta easily. The platelets would be recognised as foreign by the mum's IS. This would lead to destruction of the baby's platelets by the mum's IS, resulting in the baby being born w/ this Petichae rash/brusing/intercranial haemorrhages.
Major concern as, unlike HDN, can happen on 1st pregnancy- so need to know before baby is born to put procedures in place to aid w/ delivery and for further pregnancies.

23

Platelet Immunofluorescence Test (PIFT).

Simple screening test. Take father platelets and mum's serum, incubate them together w/ anti-IgG-FITC. Mum's Abs detect dad's platelets and bind = green fluo! Can run through FC, gate on basis of size (small platelets = v. FS and SS), and look at the levels of immunofluo present.

24

What is immunophenotyping mainly used for?

Identifying and treating cancers and blood cancers (i.e. leukaemias). Can be used to ID the type of leukaemia the patient has by: ID the specific cells involved. Can then target treatment and Abs therapies accordingly.

25

What do we look for in FC DNA analysis?

Cell cycle (CC) and ploidy. Normally have 46 chromosomes (23 pairs), but in disease such as cancer, would have an abnormal ploidy which would be indicated by the extra DNA present. Gives indication of how aggressive tumour is.

26

i.e. of breast cancer DNA analysis using FC:

Take extracted cels and permeate them to allow them to take up nuclear dye. On graph, if there is a single pair present = 1 peak. Will sometimes see on graph, a much smaller peak next this main peak = shows the cell is transitioning through the cc (diploid--> haploid). Start to see additional peaks = more cellular content = more DNA = worse outcome. Knowing this can result in more effective treatments (giving clinicians more info to be able to mod therapies/treatments for the patient, to increase success rate in cancer removal.

27

Cell cycle analysis (CCA).

Used to develop new chemos/working out what issue is w/in the malignant cell.

28

G1 phase, S phase, G2 phase?

G1 phase = 1st big peak in cc analysis graph. Shows normal diploid cell content.
S phase = this is prep for the production of more DNA for mitosis.
G2 phase = little peak after S phase. Have double the DNA here as will undergo mitosis and split the DNA into 2 daughter cells (back to normal haploid content).

29

What can CCA show us?

How the drug is working to slow down the rate of tumour turnover and what is happening under certain conditions to the cell.

30

Immunology: i.e. Chronic Granulomatous Disease (CGD).

Suffer from range of symptoms and other diseases (i.e. abscesses of skin and organs and pneumonia ect). CGD is due to recurrent bacterial infections, that are not effectively dealt w/ by body's IS. W/ CGD = phagocytic cells engulf the bacterial infected cells and spray them w/ free radicals. However, the oxidative burst pathway becomes ineffective as enzy NADPH oxidase becomes inactive (due to genetic mutation). Can't do anything w/ the bac infec cells. Patient's undergo lifetime of transfusions.