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4.2 Blood Transfusion Science > Leucodepletion of Blood Products > Flashcards

Leucodepletion of Blood Products Flashcards

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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
Q

Why is DMSO not used more often?

A

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

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26
Q

Talk about filtration as a method of leucodepletion

A

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

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27
Q

Why did we start filtrating our products?

A

Variant CJD

Its found mostly in white blood cells hence universal leucodepletion is carried out

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28
Q

Talk about bedside filtration in detail

A

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

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29
Q

Why did we continue to universally leucofiltrate our products

A

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

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30
Q

What products can undergo bedside filtration?

A

Both red cells and platelets

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31
Q

How sensitive is bedside filtration

A

99 to 99.9%

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32
Q

What are the only downsides of bedside filtration

A

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
Q

What is the main benefit of bedside filtration?

A

No excessive filtration, only filtering what you need to

34
Q

How is QA carried out for leucofiltration in the IBTS

A

1 in 100 units are sacrificed for leucofiltration

35
Q

List the five disadvantages of bedside filtration

A

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
Q

In what percentage of CJD patients was CJD found in wbcs

A

In 60% of CJD patients the virus was found in wbc

37
Q

Talk about the effects of storage on white blood cells

A

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
Q

When is pre-storage filtration carried out and how

A

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
Q

What is the main benefit of pre-storage filtration?

A

No cytokines and retains original expiry date (NB!!)

40
Q

What exactly is released by wbcs during storage

A

Cytokines and histamine

41
Q

What can cytokines and histamine do to a recipient

A

Can cause non-specific reactions and alloimmunisation in patients

hence need for removal of wbcs before they fragment and release these

42
Q

What are the two kinds of filters used?

A

A sterile connection device, as with bedside filtration

An in-line filter, as with normal filtration

43
Q

How sensitive is pre-storage filtration

A

96.2% to 99.7%

> 90% of rbcs remain

44
Q

What is the proper name for red cells pre-storage filtrated

A

Red Blood Cells Leucocytes Depleted (RCLD)

include the name of the anticoagulant/preservative used

45
Q

What are the three types of materials used in filters?

A

Cotton wool
Cellulose acetate
Polyester

46
Q

How does polyester work as a filter?

A

WBCS have a charge which attaches to polyester

47
Q

How exactly do filters work

A

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
Q

What can influence rate of filtration

A

Temperature
Speed of flow
Pre-filtration wbc count

49
Q

What % of the unit is lost through filtration

A

10-15% loss of the componentns

50
Q

Talk about rbc filters vs platelet filters

A

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
Q

What can impact LD and cause a failure

A

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
Q

What are the four mechanisms of action of leucodepletion?

A

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
Q

Why is pore size so important in filters

A

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
Q

Describe in your own words how the structures of cellulose acetate differ from polyester and how this is beneficial in filtration

A

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
Q

Talk about the chemistry behind leucofiltration

A

Leukocytes are negatively charged and thus attach to filter material through van der Waals and electrostatic forces

56
Q

What properties affect efficacy of the filter

A

Surface charge of filter material

Hydrophilicity of filter

57
Q

What is a new example of a filter being used

A

Terumo filter

58
Q

How does the Terumo filter work

A

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
Q

When is it ideal to leucodeplete?

A

Within 48 hours of collection
But after 6-8 hours of phlebotomy to allow for phagocytosis of any bacteria in donation

60
Q

Briefly explain how a unit is filtered

A

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
Q

List the advantages of leucodepletion

A

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
Q

Talk about filtration and CMV

A

Patients requiring CMV- blood can be given leucofiltered blood in emergency as LD blood is considered CMV safe in immunocompetent people

63
Q

Talk about febrile reactions

A

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
Q

Talk about the ‘Immunomodulatory’ effect of transfusion

A

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 release cytokines which the recipient wbcs then focus on instead of carrying out their normal surveillance

65
Q

why do we only see GVHD in immunocompromised

A

This is because LD is sufficient to prevent GVHD in immunocompetent

66
Q

Give some examples of cytokines released by wbcs

A

IL-1
IL-6
TNF

67
Q

How many cases of CJD have been transmitted through plasma

A

Zero cases - but potential to happena s 60% found in wbcs

68
Q

Comment on the cost effectiveness of LD

A

0.18 billion cost to do
Between 2.8 and 8 billion

69
Q

Talk about febrile non haemolytic transfusion reaction

A

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
Q

How are FNHTRs treated

A

Usually just treated with paracetemol

71
Q

What are the three mechanisms for FNHTRs?

A

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
Q

Talk about the pathogenesis of FNHTRs in red cell transfusions

A

Red cells - recipient antibodies bind to donor wbc antigens and fix complement - these complexes activate recipient macrophages to release pyrogens

73
Q

Talk about the pathogenesis of FNHTRs in platelet transfusions

A

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
Q

Talk about TRALI

A

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
Q

Why do we not see TRALI in Ireland

A

Universal leucodepletion

Replacement of plasma with additive solution e.g. PAS for platelets

76
Q

How do we make platelets in Ireland

A

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
Q

For who are LD red cells recommended

A

Leukaemias
Immunodeficiency
Stem cell transplants
Bone marrow transplants
Haeoglobinopathy or thalassaemia

78
Q

Who doesnt carry out universal LD and why

A

USA
As it increases cost by 40% => RCC = 400 euro and platelets over 1000

79
Q
A

4.2 Blood Transfusion Science (3 decks)

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