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4.2 Blood Transfusion Science > Leucodepletion of Blood Products > Flashcards

Leucodepletion of Blood Products Flashcards

1
Q

Talk about how leucodepletion of blood began, origin

A

Concept of leucodepletion introduced by Fleming in 1920

He used a cotton wool plug - wool packed in a funnel - wbcs bound to cotton

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2
Q

How was the cotton wool filter upgraded?

A

We tried to move to using microfilters

But in 1961 R.L Swank found that very high pressure was required to force 2-10 day old acid-citrate-dextrose stored blood through the microfilter, aggregates of platelets and leukocytes clogged the filter

i.e. microfilters got clogged up and prevented even the red cells getting through without high pressure and in a reasonable amount of time

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3
Q

Who designed the first filter for use in blood transfusion and when?

A

Diepenhorst designed the original leucodepletion filter which contained cottonwool as the filtering agent

His work was published in 1972

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4
Q

After the original work of Diepenhorst in 1972, what upgrade was made to leucodepletion?

A

In the 1980s we began using cellulose acetate filters (still used today)

These filters have a leucodepletion rate of 98%

Some filters nowadays also include gels to filter out larger aggregates of cells

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5
Q

List four other methods of leucodepletion other than filtration

A

Cell washing (washing off plasma)

Centrifugation (+separation)

Buffy coat removal

Freezing and de-glycerolising of red cells and apheresis

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6
Q

What is the definition of leucodepletion for rbc packs and platelets?

A

Each unit must contain less than 1x10^6 leucocytes

For red cells you must also retain over 85% of rbcs

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7
Q

Why is buffy coat removal alone not permitted?

A

This will not achieve less than 1 x10^6 leucocytes per unit

Doesnt meet criteria for leucodepletion

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8
Q

What are red cells stored in?

A

Optimal additive solution e.g. SAGM > 2 x10^9

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9
Q

What is genneraly the accepted method of leucodepletion today?

A

Filtration using third generation filters

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10
Q

What is the main difference between EU and UK/US standards when it comes to leucodepletion?

A

EU standards require that a minimum of 40g of haemoglobin must be present in each red cell unit after leukocyte depletion

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11
Q

When would freezing de used as a method of leucodepletion?

A

Freezing/deglycerolisation of rbcs for anyone in need of rare blood

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12
Q

What are the two types of filtration carried out?

A

Bedside filtration
Pre-storage filtration

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13
Q

Talk about bedside filtration

A

Traditional filtration
Was done for anyone who needed CMV negative blood

Filter was sent up with the red cell unit and was done by clinicians

It poses no real issues with contamination of the pack etc as its being immediately used i.e. doesnt matter we have to pierce the pack etc

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14
Q

Talk about centrifugation for leucocyte reduction, how is it carried out, what are the pros and cons

A

Unit is spun and buffy coat is drawn off

Variation of this involves draining rbcs into a satellite bag leaving only leukocytes and buffy coat in primary pack

It is simple and cost-effective however it does not meet criteria for leucodepletion today

20% of the rbcs are also lost in this method

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15
Q

Talk about saline washing for leucocyte reduction, what are the pros and cons

A

Washing of red cells to remove leukocytes, platelets and plasma

An effective but expensive method

Reduces shelf life to only 24 hours

Was once frequently used as a means to prevent febrile reactions

Now only really used for those with IgA deficiency - anaphylactic reactions

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16
Q

What percentage of people have an IgA deficiency?

A

1 in 5000 people have some level of deficiency

These are susceptible to anaphylactic reactions when exposed to IgA

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17
Q

Talk about freezing/deglycerolisation for leucocyte reduction

A

Rbcs are snap frozen with glycerol

Freezing and thawing of red cells can bring about a 95-99% reduction in wbcs

Unfortunately it is expensive, the shelf life is reduced to 24 hours and between 10 and 20% of rbcs are lost

Only used for rare type blood

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18
Q

How and why do we freeze rbcs with glycerol

A

If rbcs were frozen normally any water present would form large ice crystals which would perforate the red blood cells and lyse them

Glycerol does not form ice crystals and thus we can save about 90% of the rbcs

You have to slowly increase the glycerol concentration and then snap freeze the red cells

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19
Q

What are the two different types of cryoprotective agents

A

Penetrating agents

Non-penetrating agents

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20
Q

Give an example of a penetrating agent and two examples of non-penetrating agents

A

Penetrating = glycerol

Non-penetrating = HES and DMSO

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21
Q

How do penetrating agents such as glycerol work

A

These are small molecules which cross the cell membrane into the cytoplasm

This creates an osmotic force which prevents water from migrating outwards

Thus intracellular dehydration is prevented

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22
Q

What does HES stand for?

A

Hydroxyethyl starch

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23
Q

What does DMSO stand for?

A

Dimethysulphoxide

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24
Q

How do non-penetrating agents such as DMSO and HES work?

A

These are large molecules which cannot enter the cell

Instead they form a shell around the rbcs and prevent loss of water and subsequent dehydration

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25
Why is DMSO not used more often?
There is a toxicity associated with DMSO Instead it is moreso used for freezing white blood cell especially wbc cell lines e.g. for MABs DMSO will have to be removed if these cells were to be used on someone due to toxic effects
26
Talk about filtration as a method of leucodepletion
Most effective and efficient method Can be done either concurrently with transfusion or immediately after collection Current filtration reduces the number of wbcs by at least x1000
27
Why did we start filtrating our products?
Variant CJD Its found mostly in white blood cells hence universal leucodepletion is carried out
28
Talk about bedside filtration in detail
Leucocyte-reduction filters used during infusion instead of standard blood filter This allows for filtration regardless of the age of the product but really has to be done within 2 days of donation in order to get any benefits of leucodepletion i.e. to prevent febrile reactions No special handling required of the unit before transfusion
29
Why did we continue to universally leucofiltrate our products
Wbcs will release cytokines if left in red cell packs When transfused these cytokines will act on the recipient white blood cells etc This causes febrile haemolytic reactions etc Thus by removing these we prevent cytokine effects etc
30
What products can undergo bedside filtration?
Both red cells and platelets
31
How sensitive is bedside filtration
99 to 99.9%
32
What are the only downsides of bedside filtration
You cannot do any kind of quality assurance on it i.e. you cannot actually determine if leucodepleted or not More expensive filter than standard blood and platelet filters
33
What is the main benefit of bedside filtration?
No excessive filtration, only filtering what you need to
34
How is QA carried out for leucofiltration in the IBTS
1 in 100 units are sacrificed for leucofiltration
35
List the five disadvantages of bedside filtration
Reduced efficacy due to slow filtration of warmed blood - less effective filtration Cannot assess quality Control of factors difficult Lack of consistency - every clinician will be different Ineffective in preventing effects due to storage changes - i.e. if old blood used cytokines will be present - think about having to do a blood film on old blood
36
In what percentage of CJD patients was CJD found in wbcs
In 60% of CJD patients the virus was found in wbc
37
Talk about the effects of storage on white blood cells
Leucocytes begin to disintegrate quickly when stored at 2-6 degrees White cell fragments etc may be capable of initiating an immune response and may carry viral activity
38
When is pre-storage filtration carried out and how
Donation is filtered within 48 hours of phlebotomy using a sterile filter No external docking for rbcs but there is for platelets i.e. red cell pack has inbuilt filter
39
What is the main benefit of pre-storage filtration?
No cytokines and retains original expiry date (NB!!)
40
What exactly is released in the body caused by wbcs fragmentation during storage
Cytokines and histamine
41
What can cytokines and histamine do to a recipient
Can cause non-specific reactions and alloimmunisation in patients hence need for removal of wbcs before they fragment and release these
42
What are the two kinds of filters used?
A sterile connection device, as with bedside filtration An in-line filter, as with normal filtration
43
How sensitive is pre-storage filtration
96.2% to 99.7% >90% of rbcs remain
44
What is the proper name for red cells pre-storage filtrated
Red Blood Cells Leucocytes Depleted (RCLD) include the name of the anticoagulant/preservative used
45
What are the three types of materials used in filters?
Cotton wool Cellulose acetate Polyester
46
How does polyester work as a filter?
WBCS have a charge which attaches to polyester
47
How exactly do filters work
Lymphocytes, monocytes and granulocytes are trapped in the small pores and by adherence e.g. to polyester LD also includes platelet activation causing secondary adhesion of granulocytes and monocytes Direct adhesion and physical trapping of the more rigid lymphoid cells in the fibre of the filter
48
What can influence rate of filtration
Temperature Speed of flow Pre-filtration wbc count
49
What % of the unit is lost through filtration
10-15% loss of the componentns
50
Talk about rbc filters vs platelet filters
Different filters are used As platelets will also adhere to polyester in rbc filters Instead we use a trapping mechanism for platelets instead of an adhesion based LD method
51
What can impact LD and cause a failure
The capacity or capability of the LD system - wcc of donor might be too high - some wbcs get through Potential manufacturing defects in the filter or pack system The proportion of components that are tested for residual leucocytes Donor-related causes e.g. HbS trait (sickles get stuck in filter) or clotted samples etc
52
What are the four mechanisms of action of leucodepletion?
Blocking -> filter blocks nuclei of wbcs Bridging -> two nucleated cells cannot get through filter - form a bridge only allowing rbcs to get by Interception -> heavier nucleated cells fall into side wells in filter, rbcs get pushed over these and down through filter Adhesion -> normal wbc filter -> wbcs stick to filter
53
Why is pore size so important in filters
You want the wbcs to be blocked but dont want the filter to be blocked as no rbcs would get through Hence why we use decreasing pore size to ensure flow of cells through a series of filters i.e. filter one catches most, filter 2 catches more, filter 3 catches last few cells etc The first layer in the filter is often a gel to catch the largest cells This prevents the need for high pressure to force the cells through the filter
54
Describe in your own words how the structures of cellulose acetate differ from polyester and how this is beneficial in filtration
In cellulose acetate - fibres overlap randomly In polyester fibres cross link in a uniform fashion -> form a lattice -> areas of cross over will have the best adhesion -> increased the binding sites for wbcs -> the tighter these fibres the less wbcs will get through but the longer the filtration will take etc
55
Talk about the chemistry behind leucofiltration
Leukocytes are negatively charged and thus attach to filter material through van der Waals and electrostatic forces
56
What properties affect efficacy of the filter
Surface charge of filter material Hydrophilicity of filter
57
What is a new example of a filter being used
Terumo filter
58
How does the Terumo filter work
Uses 'coral-like' polyurethane Mechanically traps majority of leukocytes in small pores or dimples in the material There is very limited cell material interaction and the absence of cellular activation
59
When is it ideal to leucodeplete?
Within 48 hours of collection But after 6-8 hours of phlebotomy to allow for phagocytosis of any bacteria in donation
60
Briefly explain how a unit is filtered
Whole blood undergoes a hard spin to separate into plasma, buffy coat and rbcs The plasma is LD'd to form FFP (not yet done in Ireland) Additive solution is added to the RBCS and are LD'd to form RCCs The buffy coat is combined with other buffy coats and PAS and then soft spun and LD'd to form pooled platelets
61
List the advantages of leucodepletion
Prevention of FNHTRs Prevention of transmission of viruses such as CMV, HTLV1/2, HHV-8 and EBV Reduces the 'immunomodulatory' effect of transfusion (TRIM?) Avoid sensitisation to HLA (important in transplantation) Required for neonatal transfusions to prevent TRGVHD Minimises GVHD in bone marrow patients etc ->irradiation is gold standard but it all helps Platelet refractoriness due to HLA alloimmunisation is reduced Depletion of cytokines which can cause fever Possible prevention of CJD Increases glucose availability for rbcs There is no good reason not to filter
62
Talk about filtration and CMV
Patients requiring CMV- blood can be given leucofiltered blood in emergency as LD blood is considered CMV safe in immunocompetent people
63
Talk about febrile reactions
Used to be very common before leucofiltration Rise in temp of 1.5 degrees Most were due to white blood cells Used to get one every single week
64
Talk about the 'Immunomodulatory' effect of transfusion
Known as TRIM - transfusion related immune modulation Whereby red cell transfusion can suppress the immune system of the recipient This can lead to recurrence of cancer and infections in certain patients It happens when wbcs left in the donation break down and when transfused these fragments release cytokines which the recipient wbcs then focus on instead of carrying out their normal surveillance
65
why do we only see GVHD in immunocompromised
This is because LD is sufficient to prevent GVHD in immunocompetent
66
Give some examples of cytokines released by wbcs
IL-1 IL-6 TNF
67
How many cases of CJD have been transmitted through plasma
Zero cases - but potential to happena s 60% found in wbcs
68
Comment on the cost effectiveness of LD
0.18 billion cost to do Between 2.8 and 8 billion
69
Talk about febrile non haemolytic transfusion reaction
Febrile episodes where there is a temperature rise greater than 1.5 degrees Incidence post 6-7% of red cell and 35% of platelet transfusions receiving standard non-leucocyte transfuision Symptoms of flushing, chills/rigors, fever, tachycardia within 30 mins t o2 hours of transfusin Influenced by number of wbcs quantity of cytokines, titre of anti-leucocyte antibodies etc
70
How are FNHTRs treated
Usually just treated with paracetemol
71
What are the three mechanisms for FNHTRs?
Donor cells react with recipient leukocyte antibodies and cause a release of interleukin of donor origin Donor cells react with recipient antibody and form antigen-antibody complexes that react with recipient monocytes to result in the release of recipient interleukins Residual donor leukocytes present in platelet concentrates during storage release interleukin passively transfused to the reci[ient
72
Talk about the pathogenesis of FNHTRs in red cell transfusions
Red cells - recipient antibodies bind to donor wbc antigens and fix complement - these complexes activate recipient macrophages to release pyrogens
73
Talk about the pathogenesis of FNHTRs in platelet transfusions
Reactions are due to release of pyrogenic cytokines IL-1, IL-6, TNF from leucocytes into plasma during 5 day platelet storage Case for an alternative platelet storage medium Anti-HLA, HPA and granulocyte Abs have all been seen to cause FNHTR
74
Talk about TRALI
Transfusion related acute lung injury Acute non-cardiogenic pulmonary oedema i.e. plasma leaks into lungs Antibodies, particularly donor anti-HLA bind to recipient granulocytes and degranulation occurs Complement fixing and activation Second highest cause of transfusion related fatalities
75
Why do we not see TRALI in Ireland
Universal leucodepletion Replacement of plasma with additive solution e.g. PAS for platelets
76
How do we make platelets in Ireland
Plasma removed from pooled platelets Replacement with PAS 25% plasma, 75% PAS => no antibodies Apheresis is from just one donor - must be anti-HLA negative
77
For who are LD red cells recommended
Leukaemias Immunodeficiency Stem cell transplants Bone marrow transplants Haeoglobinopathy or thalassaemia
78
Who doesnt carry out universal LD and why
USA As it increases cost by 40% => RCC = 400 euro and platelets over 1000
79

4.2 Blood Transfusion Science (21 decks)

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