Leucodepletion of Blood Products Flashcards
Talk about how leucodepletion of blood began, origin
Concept of leucodepletion introduced by Fleming in 1920
He used a cotton wool plug - wool packed in a funnel - wbcs bound to cotton
How was the cotton wool filter upgraded?
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
Who designed the first filter for use in blood transfusion and when?
Diepenhorst designed the original leucodepletion filter which contained cottonwool as the filtering agent
His work was published in 1972
After the original work of Diepenhorst in 1972, what upgrade was made to leucodepletion?
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
List four other methods of leucodepletion other than filtration
Cell washing (washing off plasma)
Centrifugation (+separation)
Buffy coat removal
Freezing and de-glycerolising of red cells and apheresis
What is the definition of leucodepletion for rbc packs and platelets?
Each unit must contain less than 1x10^6 leucocytes
For red cells you must also retain over 85% of rbcs
Why is buffy coat removal alone not permitted?
This will not achieve less than 1 x10^6 leucocytes per unit
Doesnt meet criteria for leucodepletion
What are red cells stored in?
Optimal additive solution e.g. SAGM > 2 x10^9
What is genneraly the accepted method of leucodepletion today?
Filtration using third generation filters
What is the main difference between EU and UK/US standards when it comes to leucodepletion?
EU standards require that a minimum of 40g of haemoglobin must be present in each red cell unit after leukocyte depletion
When would freezing de used as a method of leucodepletion?
Freezing/deglycerolisation of rbcs for anyone in need of rare blood
What are the two types of filtration carried out?
Bedside filtration
Pre-storage filtration
Talk about bedside filtration
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
Talk about centrifugation for leucocyte reduction, how is it carried out, what are the pros and cons
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
Talk about saline washing for leucocyte reduction, what are the pros and cons
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
What percentage of people have an IgA deficiency?
1 in 5000 people have some level of deficiency
These are susceptible to anaphylactic reactions when exposed to IgA
Talk about freezing/deglycerolisation for leucocyte reduction
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
How and why do we freeze rbcs with glycerol
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
What are the two different types of cryoprotective agents
Penetrating agents
Non-penetrating agents
Give an example of a penetrating agent and two examples of non-penetrating agents
Penetrating = glycerol
Non-penetrating = HES and DMSO
How do penetrating agents such as glycerol work
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
What does HES stand for?
Hydroxyethyl starch
What does DMSO stand for?
Dimethysulphoxide
How do non-penetrating agents such as DMSO and HES work?
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
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
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
Why did we start filtrating our products?
Variant CJD
Its found mostly in white blood cells hence universal leucodepletion is carried out
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
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
What products can undergo bedside filtration?
Both red cells and platelets
How sensitive is bedside filtration
99 to 99.9%
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
What is the main benefit of bedside filtration?
No excessive filtration, only filtering what you need to
How is QA carried out for leucofiltration in the IBTS
1 in 100 units are sacrificed for leucofiltration
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
In what percentage of CJD patients was CJD found in wbcs
In 60% of CJD patients the virus was found in wbc
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
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
What is the main benefit of pre-storage filtration?
No cytokines and retains original expiry date (NB!!)
What exactly is released in the body caused by wbcs fragmentation during storage
Cytokines and histamine
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
What are the two kinds of filters used?
A sterile connection device, as with bedside filtration
An in-line filter, as with normal filtration
How sensitive is pre-storage filtration
96.2% to 99.7%
> 90% of rbcs remain
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
What are the three types of materials used in filters?
Cotton wool
Cellulose acetate
Polyester
How does polyester work as a filter?
WBCS have a charge which attaches to polyester
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
What can influence rate of filtration
Temperature
Speed of flow
Pre-filtration wbc count
What % of the unit is lost through filtration
10-15% loss of the componentns
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
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
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
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
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
Talk about the chemistry behind leucofiltration
Leukocytes are negatively charged and thus attach to filter material through van der Waals and electrostatic forces
What properties affect efficacy of the filter
Surface charge of filter material
Hydrophilicity of filter
What is a new example of a filter being used
Terumo filter
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
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
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
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
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
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
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
why do we only see GVHD in immunocompromised
This is because LD is sufficient to prevent GVHD in immunocompetent
Give some examples of cytokines released by wbcs
IL-1
IL-6
TNF
How many cases of CJD have been transmitted through plasma
Zero cases - but potential to happena s 60% found in wbcs
Comment on the cost effectiveness of LD
0.18 billion cost to do
Between 2.8 and 8 billion
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
How are FNHTRs treated
Usually just treated with paracetemol
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
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
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
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
Why do we not see TRALI in Ireland
Universal leucodepletion
Replacement of plasma with additive solution e.g. PAS for platelets
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
For who are LD red cells recommended
Leukaemias
Immunodeficiency
Stem cell transplants
Bone marrow transplants
Haeoglobinopathy or thalassaemia
Who doesnt carry out universal LD and why
USA
As it increases cost by 40% => RCC = 400 euro and platelets over 1000
