leucodepletion is the removal of 99% leucocytes from the whole blood, pcv or platelets before transfusing into the donor. this process many infections, transfusion reactions..

1. Presenter : Dr. Tousif
Moderator : Dr. Chandrika R
LEUCODEPLETION

2. 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.

3. 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

6. 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.

7.  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

8. LEUCOREDUCTIO
N
PRE-STORAGE
LEUCOREDUCTION
POST-STORAGE
LEUCOREDUCTION

9. 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 Need based

10. Methods of preparation of leucopoor
red cells
 Centrifugation
 Filtration
 Cell Washing
 Freezing and Deglycerolization

11. 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

12.  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.

13. 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

14. 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

15. 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

16. 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

17. 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.

18. 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.

19.  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.

20. Leucodepletion filtration can be done
 BEDSIDE
During transfusion
 IN-LABORATORY
Before issue from blood bank
 PRE-STORAGE
Inline filtration before component preparation

21. 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

22. 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

23. 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

24. IN LINE FILTRATION

25. blocking bridging
Interception Adhesion
Mechanism of
action of
leucodepletion
filters

26. 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 –

27. 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

29.  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

30. 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

31. 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

32. 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

33.  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 .

34. 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

35. Cryoprotective agents
Penetrating 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

36. Types of Glycerolization
Low 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

37. 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

38. 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.

39.  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.

40. 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

41.  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 % NaCl
0.9 % NaCl + 0.2 %
dextrose
12 % NaCl
0.9 % NaCl + 0.2 %
dextrose

42.  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.

43. methods of leucodepletion

44. 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
Manual methods
Automated
methods

45. 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

46. 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.

47. Clinical benefits of leucocyte
reduction
 Proven relevant clinically
 Likely clinically relevant
 Unproven clinically

48. 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.

49. Likely clinically relevant
 Reduced infectious risk associated with
immunomodulation
 Reduced direct risk of transfusion transmission
bacteria

50. 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)

51. 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

53.  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.

54. 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

55. 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.

56.  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”.

57. 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

58. 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

59. 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,

60.  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%)

61. 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

62.  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

63. Corrected count increment
(CCI)
X
Post
transfusion
platelet
count
Pre
transfusion
platelet
count
Number of platelets
transfused(10 *11)
Body
surface area

64.  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

65.  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

66.  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

67. 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

68.  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

69. 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

70. 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

71.  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,

72.  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

73. 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

74. 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 AML 45% 17% 13% 3%
42.6 % 12.3% 30.6
%
6% 15.6
%
24.7
%

75. 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

76.  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.

77. 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

78. 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

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

80. 6. Kumar H, Gupta PK, Mishra DK, Sarkar RS,
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implementation in developing countries. Asian J
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