Presenter : Dr. Tousif

Moderator : Dr. Chandrika Rao

LEUCODEPLETION

Introduction

Leucodepletion is defined as a blood

processing step for reducing the

leukocyte content of whole blood,

RBCs, or platelet units down to 5×10 ^6

residual WBCs per unit of component.

ADVERSE EFFECTS OF

CONTAMINATING LEUCOCYTES

HLA alloimmunisation

Platelet refractoriness, graft

rejection

Viral transmission

CMV, EBV, HTLV I and II

Immune suppresion

Post operative infection, cancer

recurrence

Reactions

NHFTR, GVHD

Contaminati

ng

leucocytes

Historical aspects

The concept of removal of leukocytes from the blood was

introduced by Fleming, as early as 1920.

He used a cotton wool plug in a bent glass tube with a

constricted limb, in which blood was placed above the cotton

wool and forced through it.

In 1961 , R.L. Swank while working on a blood viscosity

model, he found that very high pressure was required to force

2-10 days old acid-citrate-dextrose (ACD)-stored blood through

a microfilter, as aggregates of platelets and leukocytes

clogged the filter passes.

The original leucocyte depletion filter contained

cottonwool as the filtering agent and was designed by

Diepenhorst who published his work in 1972

Later, in the 1980s, advancement in technology led to the

development of the first generation cellulose acetate

filters, with a leukocyte removal efficiency of 98 percent

Other methods included cell washing, centrifugation and

buffy coat removal, freezing and deglycerolisation of

red cells and apheresis

LEUCOREDUCTIO

N

PRE-STORAGE

LEUCOREDUCTION

POST-STORAGE

LEUCOREDUCTION

Pre- storage

leucoreduction

Post -storage

leucoreduction

Time of leucoreduction WBCs are removed from

red cells or platelets

soon after the

collection before they

release inflammatory

mediators like cytokines.

WBCs are removed from

red cells or platelets

many days after

collection, often just

before being released to

the patient or the blood

may also be filtered

bedside.

Techniques used Lab side filtration

inline filtration

apheresis

Lab side

bedside filtration

Advantages Reduction in febrile non-

hemolytic transfusion

reaction(FNHTR),bacteri

al and viral

transmissions and HLA

alloimmunization

Prevents viral

transmission and HLA

alloimmunization but

not necessarily FNHTR

Disadvantages Needs proper planning

Inventory management

Need based

Inventory management

Methods of preparation of leucopoor

red cells

Centrifugation

Filtration

Cell Washing

Freezing and Deglycerolization

What is Log reduction?

Log reduction refers to the relative number of

leukocytes that can be eliminated by a

leukoreduction procedure.

Log 1 : the number of wbcs is 10 times less than

original

Log 2 : the number of wbcs is 100 times less than

original

Log 3 : the number of wbcs is 1000 times less than

original

Log 4 : the number of wbcs is 10,000 times less

Many authors and guidelines established Log3 LR as

a minimum requirement to call a product

leukoreduced.

However , Log reduction is dependent on the

original number of white cells in the donor bag and

does not specify the maximum number of white cells it

that bag

Hence , log reduction may not be the correct way of

assessing leukoreduction.

Inverted centrifugation

Collection of blood in double CPD/CPDA blood

collection bag

Centrifuge the bag upside down at 5000 xg for 5 mins

Hang the centrifuged bag on a ring stand or inverted

plasma expressor

Separate the red blood cells into transfer bag, taking a

note that 70% of the red cells are transferred to the

transfer bag

Double seal the tubing

Label the components leucocyte poor red cells by

centrifugation

Double centrifugation

Collect the blood in a triple or quadruple blood

collection bag

Centrifuge at 2000 x g for 3 mins at 22 degree c

Separate the platelet rich plasma PRP

Whole

blood

Separate the bag containing PRP leaving one satellite

bag attached to the primary bag

Centrifuge the red cells by “Inverted Centrifugation”

and separate the leucocyte poor red blood cells

This method reduces leucocytes by

1 Log

Filtration The most effective and efficient method for reducing the

number of leucocytes in a unit is by filtration.

“specific leucocyte depleting filters” are made up of

» cellulose acetate (Erypur-G, Organon Technika)

» polyester ( sepa cell R-500, Asaki )

» tightly packed fibres of combed cotton(Imugard, Terumo)

» Pall RC 50 and RC 100

Cotton wool and cellulose acetate filters are more effective.

Filters are less expensive as compared to other methods as

automated cell washing, freezing and deglycerolization.

Blood filters First generation filters

» 170 to 240 µm

» part of all red cell transfusions sets

» remove large clots and particulate debris

Second generation filters

» 40 µm

» remove microaggregates of fibrin, platelets and

leucocyte

from red cell concentrates.

» reduce the number of leucocytes by 1

magnitude of log

to 5 x 10^8 per unit.

Third generation filters

» designed specifically for the removal of ‘free’

leucocytes.

» they retain both microaggregate and free cells

» reduce the number of leucocytes by 3

magnitude of log

to 5 x 10^6 per unit.

Leucodepletion filtration can be done

BEDSIDE

During transfusion

IN-LABORATORY

Before issue from blood bank

PRE-STORAGE

Inline filtration before component preparation

BEDSIDE FILTRATION Disadvantages

» reduced efficacy since slow filtration of warmed blood

» cannot assess product quality

» control of factors difficult

» lack of consistency

» ineffective in preventing effects due to storage

changes

Advantages

reduces cost as used only for selected patients

IN-LABORATORY FILTRATION Disadvantages

» additional delay to filter before issue

» adverse effects due to storage related changes

cannot be prevented

Advantages

» easy to standardize

» quality can be assessed

» reduces cost as used only for selected patients

PRE-STORAGE/INLINE

FILTRATION Disadvantages

» cost escalation unless universal leucodepletion

Advantages

» easy to standardize and convenient

» quality can be assessed

» decreased NHFTR

» decreases alloimmunization and platelet refractoriness

IN LINE FILTRATION

blocking bridging

Interception Adhesion

Mechanism of

action of

leucodepletion

filters

Apheresis in leucodepletion

Current day cell separators are able to collect

leucoreduced red cell or leucoreduced platelet

concentrates.

It serves as a good alternative to the expensive

filters.

For this reason , nowadays Single Donor Platelets

(SDP) collected by apheresis is preferred over Random

Donor Platelets(RDP).

Ongoing quality control program –

to assess the effectiveness of cell separators

Cell washing

Saline washing of red blood cells results in the

removal of majority of leucocytes and platelets.

Either manually or by using machines e.g

haemonetics cell washing machine or

Cobe 2991 blood cell processor

Procedure

1. Red blood cells are mixed with large volumes of

physiologic saline.

2.The red cell- saline mixture is then centrifuged and the

supernatant is removed.

The procedure removes

- about 99 % of the plasma proteins

- upto 20 % of the RBCs

- between 70% to 95% of the WBCs , depending on

the methodology

Washing of RBCs with saline

Collect the blood in a double collection bag

Centrifuge the bag at 2000 x g for 3 mins at 2-6

degree C

Separate the plasma along with buffy coat into a

transfer bag

Double seal the tubing and separate the transfer bag

Connect the blood bag with sterile cannula to 250 ml

saline container

Drain the saline into the blood bag

Remove the cannula from saline container and

replace the cannula’s original plastic cover

Mix the blood thoroughly with saline

Centrifuge the bag at 5000 x g for 5 mins

Place the bag carefully on expressor and release the taped cannula into empty saline bag and express the

saline residual buffy coat into the empty container

Repeat the washing procedure thrice, seal the tubing close to bag and separate the cannula

It is an open process

Shelf life of 24 hrs from the time of entry.

With the advent of filtration methods for leucocyte

reduction, the popularity of washed red blood cells

have declined.

The primary use of washed products remains the IgA

deficient patients with anti- IgA .

Freezing and Deglycerolization

This process also results in a product with a decreased

concentration of leucocytes.

Level of leucocyte reduction : 95% to 99%

Red cell loss : upto 20%

Expensive

FREEZING RBCs WITH CRYOPROTECTIVE

AGENTS

THAWING OR DEGLYCEROLIZATION

Cryoprotective agentsPenetrating agents Non penetrating agents

• small molecules

• Cross the cell

membrane into the

cytoplasm

•Creates osmotic force

which prevents water

from migrating outward

•Hence , it prevents

intracellular dehydration

• Glycerol

• large molecules

• do not enter the cell

• instead, form a shell

around it

• prevents loss of water

and subsequent

dehydration

• hydroxyethyl starch

(HES)

dimethylsulphoxide

Types of GlycerolizationLow glycerol (20% w/v) High glycerol (40% w/v)

• very rapid

• more controlled freezing

process required

• the cryoprotection of the

glycerol is minimal

• liquid nitrogen is routinely

used for this method

•Storage temperature is -120

degree celcius

•Because of the minimal

amount of protection by the

glycerol, temperature

fluctuation during storage can

cause RBCs destruction.

•Slow

•Uncontrolled freezing

process

• the cryoprotection of the

glycerol is more

• mechanical freezer

• storage temperature is -80

degree celsius

• most widely used• fairly simple

Preparation

The RBCs and glycerol( 40% w/v) are warmed to atleast

» 25 degree C by using a dry warming chamber for 10

to 15 mins

OR

» at room temperature for 1 to 2 hours.

Set the glycerol bottles in a water bath for 15 mins at 25-

37 degree c

label the freezing bag

Process of glycerolization

100 ml of glycerol is added to RBCs as they are

agitated on a shaker.

Then the shaker is turned off, allowing for gradual

equilibration between the glycerol and RBCs.

After about 5 to 30 mins, these glycerolized cells are

transported to the freezing bag.

The remaining 300 ml of glycerol is added slowly and

in a stepwise fashion with gentle mixing.

The glycerolization process should be performed at

temperature between 25 to 32 degree celcius.

The process should be completed within 4 hours

The bag containing the glycerolised cells is placed in

a protective canister and frozen in a -80 degree

celcius freezer.

The frozen red blood cells are then stored at -65

degree celcius or colder for upto 10 years from the

date of the original phlebotomy.

Thawing / deglycerolization Reverse of glycerolization or freezing process

The cryoprotective glycerol must be slowly removed

and replaced with isotonic solution before transfusing

the cells to the patient.

3 basic stepsEquilibration of thawed RBCs with hypertonic

solution

Washing with solutions of gradually decreasing

hypertonic strengths

Resuspension in an isotonic electrolyte solution

containing glucose

The thawing process takes approximately 30 mins

The unit is first allowed to thaw at 37 or 42 degree

celcius depending on whether or not units are frozen

Washing the RBCs with solutions of decreasing

osmolarity

Exception: donor with sickle trait

12 % NaCl 1.6 % NaCl0.9 % NaCl + 0.2 %

dextrose

12 % NaCl0.9 % NaCl + 0.2 %

dextrose

RBCs , frozen, once thawed and deglycerolized, are

stored at 1 to 6 degree celcius and have a shelf life of

24 hours(open system) from the time deglycerolization

was begun.

The deglycerolized RBCs should have

» a recovery of 80% or more of the original RBCs

» a viability of 70% of the transfused RBCs 24 hours

after

transfusion.

methods of leucodepletion

Methods for counting of residual

leucocytes

The number of residual leucocytes in leucodepleted

blood components is very low

automated cell counters are not accurate in counting

them

- Nageotte counting chamber is used

- sensitive and accurate in counting less than 5

leucocyte per µl

- Flow cytometry, using propidium iodine alone or

combined with thiazole orange

Manual methods

Automated

methods

Quality control of leucoreduction

Leukoreduced

platelets

Apheresis (SDP)

Total platelet count : > 3 x 10*11

pH : > 6.2

Residual leucocytes : < 5 x 10*6

Random donor

platelets

(pooled)

Total platelet count : > 4.5 – 5.5 x

10*10

pH : > 6.2

Residual leucocytes : < 5 x 10*6

Leukocyte

reduced , red

cell concentrate

(RCC)

Residual leukocytes : < 5 x 10*6

Red cell loss < 15 % of original

Quality assurance in leucodepletion

Quality assurance and standard operating procedure

for leucodepletion has to be formulated

The staff needs to be properly and adequetly trained in

methods of leucodepletion.

Every leucodepleted product has to be labelled as

“leucodepleted product”

Food and Drug administration(FDA) guidelines

recommend testing of 1% of leucodepleted units which

should contain less than

5 x 10*6 WBCs per unit.

Clinical benefits of leucocyte

reduction

Proven relevant clinically

Likely clinically relevant

Unproven clinically

Proven relevant clinically

Reduced frequency and severity of Non-

Hemolytic Febrile Transfusions Reaction

(NHFTR)

Reduced risk of Cytomegalovirus transmission

Reduced risk of HLA- alloimmunization and

platelet refractoriness

Reduced mortality and organ dysfunction in

cardiovascular surgery patients.

Likely clinically relevant

Reduced infectious risk associated with

immunomodulation

Reduced direct risk of transfusion transmission

bacteria

Unproven clinically

Avoidance of variant Creutzfeldt - Jacob disease

(vCJD)

Avoidance of HTLV I/II , EBV etc.

Reduced risk of Graft Versus Host Disease

(GVHD)

Reduced risk of Transfusion Associated Acute

Lung Injury

(TRALI)

Non- hemolytic febrile transfusions

reactions (NHFTR) and

leucodepletion

Most common

90 % of transfusion reactions

Benign and self limiting

Clinical signs and symptoms

» temperature elevation of more than 1 degree C or 2

degree F

occurring during or shortly after transfusion.

» chills ,nausea, vomiting ,headache, backpain

This reactions mimic Acute Hemolytic Transfusion Reaction

Incidence

» 6.8 % after red cell transfusion

» 37.5 % after platelet transfusion

causes

» HLA alloimmunizaton

» IL 1 , IL 6 , TNF

» cytokine accumulation during storage released from

leucocytes

Older the component , Greater the likelihood of the reaction.

The rate of NHFTR has been shown to be reduced

significantly by transfusing pre-storage leucodepletion.

Laboratory tests AHTR FNHTR

Pink or red coloured

discolouration in post

transfusion plasma

present absent

Yellow or brown discoloration

of the sample drawn 6-8 hrs

after transfusion

present absent

Direct antiglobulin test (DAT) Positive Negative

Haemoglobinuria Present Negative

CMV transmission and

leucodepletion

Cytomegalovirus (CMV) also called HHV 5

It is transmitted by the leucocytes present in

transfused blood components.

A blood transfusion recipient must be exposed to donor WBCs that

are latently infected

with CMV

CMV then must be reactivated

the cell must survive long enough in the host to

release infectious virus

particles.

Several patient populations are at risk for serious

morbidity as a result of transfusion-associated CMV

infection (TA-CMV)

» low birth weight infants

» oncology patients

» allogenic bone marrow transplant recipients

» immunosuppressed patients

The American Association of Blood Banks (AABB)

has thus suggested that residual leukocyte levels less

than 5 x 10*6 make a blood component “CMV-safe”.

Transmission of other viruses

Human Herpes Virus 8 ( HHV 8)

Epstein-Barr virus ( EBV)

Human T-cell Lymphotrophic Virus

HTLV I

HTLV II

Strong evidence by

recent studies

no conclusive studies

Some experimental

in vitro studies

indicate a significant

reduction

Yersinia enterocolitica and

leucoreduction

Y. entercolitica is the most commonly encountered

serious bacterial contaminant associated with RBC

transfusion

Growth favours by

» low temperature

» iron-rich environment such as in stored RBC

components

Several studies have demonstrated that Y

enterocolitica inoculated into blood components

under experimental conditions is diminished or

prevented by pre-storage leukoreduction, after a

Variant Crutzfeldt Jacob Disease

transmission (vCJD) and

leucoreduction

It is a degenerative neurological disorder that is

incurable and invariably fatal.

CJD is at times called a human form of mad cow

disease (bovine spongiform encephalopathy or

BSE).

caused by an agent called a prion which are

misfolded proteins.

The disease leads to rapid neurodegeneration,

causing the brain tissue to develop holes and take a

Infectivity can be detected in the lymphoreticular

system as well as in blood.

Animal studies have shown that B lymphocytes

might play a key role in disease transmission

A recent study by Gregory et al, in a hamster model

of blood-born transmissible spongiform

encephalopathy (TSE) transmission, showed that

filtration leukoreduction was associated with a

reduced risk of TSE infection (from 48.1% to

31.5%)

Platelet refractoriness and

leucodepletion

Definition : It is defined as a poor increment

following platelet

transfusion on more than 2 occasions.

Each unit of platelets from whole blood must contain

at least 5.5 × 10*10 platelets and should increase

the platelet count by 5,000 to 10,000/µL in a 70-kg

human

Common problem in patients receiving multiple

transfusions.

The efficiency of a platelet transfusion can be

assessed either by doing platelet count at 1 hr and

24 hr after transfusion or by clinical assessment of

patient.

A corrected count increment (CCI) can provide

valuable information about patient response to a

Corrected count increment

(CCI)

X

Post

transfusion

platelet

count

Pre

transfusion

platelet

count

Number of platelets

transfused(10 *11)

Body

surface area

CCI < 7.5 X 10*9/L measured at 10 minutes to 1 hr

after platelet transfusion

= UNACCEPTABLE RESPONSE.

Causes of platelet refractoriness :

immune mechanism

non immune mechanism

HLA alloimmunization

Alloimmunization is defined as a recipient immune

response against antigens on tissue from a

genetically dissimilar donor

The presence of antibodies against class I HLA

antigens is the most common immune cause of

platelet transfusion refractoriness.

These antibodies form after exposure to

corresponding antigen expressed on contaminating

WBCs in transfused blood components

Immune

mechanism

Non immune mechanism

- associated with fever, septicemia, DIC and

splenomegaly

The reduction in alloimmunization is a well

documented beneficial effect of using leukoreduced

blood component transfusions, and is especially

important for repeatedly transfused patients and

Transfusion related

immunomodulation and

leucodepletion

The immune system may undergo transient depression

following the administration of blood and blood products.

evidence from a variety of sources indicates that allogenic

blood transfusion (ABT)

» enhances the survival of renal allografts

» increase the recurrence rate of resected

malignancies

» increase the incidence of postoperative bacterial

infections

» reduce the recurrence rate of Crohn’s disease

Immune suppression can be due certain cytokines,

including interleukins and some growth factors.

Some constituents of cellular blood products may be

responsible for TRIM which include allogenic

mononuclear cells and soluble HLA peptides I that

circulate in plasma.

T and B cells that recognize host cells and might

destroy them must be removed before they can

develop into mature lymphocytes

randomized clinical trials demonstrate that

leukodepletion can reduce the immunomodulatory

Transfusion associated graft-versus-

host-disease ( TA-GVHD) and

leucodepletion

It is the proliferation of T-cell lymphocytes derived

from the donor

Patients with cell-mediated immunodeficiency are

at particular risk for TA-GVHD.

A very recent report describes a reduction in reports

of

TA-GVHD to the United Kingdom Serious Hazards

Of Transfusion Program (UK SHOT) since the

introduction of Universal Leucoreduction (ULR) in the

UK

i.e.11 reports of GVHD associated with non-

leukoreduced blood components, against only 2 with

the use of leukoreduced blood components

Transfusion related acute lung injury

(TRALI) and leucodepletion

severe, often fatal, and complex complication of

transfusion

the most common cause of transfusion-associated

mortality reported to both the United States Food and

Drug Administration (US FDA) and UK SHOT

several mechanisms for TRALI have been postulated

» Popovsky’s immune-mediated model

» Silliman’s two-hit model

Popovsky’s immune-mediated model

» donor antibodies and less frequently recipient

antibodies

causing an immune reaction targeting leukocyte

antigens

Silliman’s two-hit model

»first hit : physiological insult that activates

pulmonary endothelium and promotes priming,

resulting in the adhesion of neutrophils,(sepsis and

trauma)

»second hit : activation of the neutrophils,

causing the release of biological active mediators

present in the blood components

If Silliman’s model is valid,

leukocyte filtration and the use of younger blood

components

in at-risk patients should reduce the incidence of

TRALI.

Leukoreduction reduces biologically active mediators

associated with prolonged storage

Author Patient

group

Standar

d

Leucode

pleted

Standar

d

leuodepl

eted

Red

cells

platelets

Van

Marwijk

Kooy et

al. 1991

Leukaem

ic

42 % 7 % 46 % 11% - -

Al

Momen

et al.

1992

Leukaem

ic

- - - - 21.5 % -

Myllyla e

al. 1993

Renal

Leukaem

ic

AML

67 %

-

-

19 %

-

-

-

52%

18%

-

0%

2.9%

-

-

-

-

-

-

Bedford-

Russel

et al.

1993

Neonate

s

30.4 % 0% - - - -

Alloimmunisation Refractoriness Blood product

reduction

Saarinen

et al.

1993

leukaemics 30 % 0% 10% 0% 13.6

%

28.6%

Oksanen

et al.

1994

AML 38% 17% 21% 3% 14.8

%

26.5%

Williamso

n et al.

1994

Cancer

AML

37.5%

63%

22.4%

31%

31%

-

24%

-

-

-

-

-

Blumberg

et al.

1995

AML

Lymphoma

-

-

-

-

-

-

-

-

0%

43.7

%

43.2%

50.3%

Adamzik

et al.

1995

Cancer

AML

27%

-

0%

-

27%

-

0%

-

-

-

8.3%

15.7%

Novotney

et al.

1995

Aplastic

Thrombocytopenics

- 12% - 5% - -

Killick et

al. 1997

Aplastic

Anaemics

50% 12% - 0% - -

T.R.A.P

Study

AML 45% 17% 13% 3%

42.6 % 12.3% 30.6

%

6% 15.6

%

24.7

%

Adverse effect of leukoreduction

The major disadvantages associated with leukocyte

reduction is cost

Severe hypotensive reactions due to leukocyte

reduced blood components have been associated

mainly with the transfusion of blood components

filtered at the bedside.

However, recent reports show that hypotensive

reactions can occur even with the prestorage

leukoreduction of blood products in which a defect in

the metabolism of kinins may be a risk factor

Recently a number of cases of “red eye syndrome”

were noted to occur in transfusion recipients who had

received filtered red cells through one particular brand

of filter.

While the precise cause of this adverse reaction is not

known, it is speculated that cellulose acetate,

employed in that particular filter, may have had a

causal role.

Aside from this, there are no other documented

adverse effects of leukoreduction.

Universal leucoreduction(ULR)

Leucoreduction of all blood components prepared in

the blood centre is called universal leucoreduction

As a routine procedure

Selective leucoreduction : leucoreduction only for a

specified set

of conditions.

Internationally, 19 countries have implemented

universal leukocyte reduction (ULR) as part of their

blood safety policy.

The main reason for not implementing ULR other

Need for Leukoreduction in India

As per the National AIDS Control Organization, there is a

requirement of 8.5-9 million units of blood in our country

annually.

Thalassemic population requiring regular transfusions

and hemato-oncology patients requiring different types

of blood component support.

A majority of them become alloimmune to various red

blood cell, platelet, and HLA antigens which leads to

various immunohematological problems

References 1. Rudmann SV. Textbook of blood banking and

transfusion medicine. 2nd ed. Elsevier;2005.

2. Blaney KD, Howard PR. Basic and applied concepts

of blood banking and transfusion practices. 3rd ed.

St. Louis:Elsevier;2013

3. Harmering D. Modern blood banking and transfusion

practices. 6th ed. Philadelphia:F.A.Davis

Company;2012.

4. Murphy MF, Pamphilon DH, Heddle NM. Practical

transfusion medicine. 4th ed. John Wiley and Sons

Ltd;2013.

5. Bassuni WF, Blajchman MA, Al-Moshary MA. Why

implement universal leukoreduction?. Hematol Oncol

Stem Cell Ther. 2008 April;1(2):106-19.

6. Kumar H, Gupta PK, Mishra DK, Sarkar RS,

Jaiprakash M. Leucodepletion and blood products.

MJAFI. 2006;62:174-7.

7. Sharma RR, Marwaha N. Leukoreduced blood

components:advantages and strategies for its

implementation in developing countries. Asian J

Transfus Sci. Jan 2010;4(1):3-8.

8. Pietersz RN, Meer PF, Seghatchian MJ. Update on

leucocyte depletion of blood components by

filtration. Transfus Sci. 1998;19(4):321-8.