Leukocyte reduced blood components

LEUKOCYTE-REDUCED
BLOOD COMPONENTS

Leukocytes (WBCs) in blood components can cause:
• Non-hemolytic febrile transfusion reaction (NHFTR)
• Human leukocyte antigen (HLA) alloimmunizaion
• Transmission of leukotropic viruses (Cytomegalovirus(CMV), Epstein- Barr virus (EBV)
and human T-cell lymphotropic virus type 1 (HTLV-1)
• Transfusion related Graft versus host disease
• Transfusion related acute lung injury (TRALI)
• Transfusion related immunosuppression

◦ Reducing the leukocytes content to less than 5 x 108 in one unit of RBCs prevents most of the
non-hemolytic febrile transfusion reactions.
◦ For other complications such as preventing transmission of CMV or alloimmunization to HLA
antigens, leukocytes content must be reduced to less than 5 x 106C in a unit of RBCs .
◦ Post storage leuko-reduction is not much effective as cytokines generated by leukocytes
duringstorage can cause NHFTR.
◦ Hence leuko-reduction before storage (pre-storage leukoreduction) in blood bank is much better than
the leuko-reduction after storage (post-storage leukoreduction) at the bed side of the patient to
eliminate NHFTR.
◦ Donors lymphocytes engrafting in the recipient and reacting against host antigens may cause
GVHD.

◦ The reaction between leukocyte antigens and antibodies can result in leuko-agglutination,
◦ aggregates of white cells being trapped in the microcirculation of the lungs, causing pulmonary
edema, that is transfusion related acute lung injury (TRALI).
WBC Antigens Antibodies
React
Ag-Ab agglutination
Get trapped in small vessels of lungs
Pulmonary Edema
cause
“Edema"is the medical
term for swelling. Body
parts swell from injury or
inflammation. It can
affect a small area or the
entire body.
TRALI

◦ In most of the cases of TRALI leukocytes antibodies in previously sensitized transfusion
recipient react with leukocytes in transfused blood or plasma, or reverse to it that is, leukocyte
antibodies in blood or plasma react with the recipient's leukocytes forming leukocytes
aggregates which are trapped in the microcirculation of lungs causing TRALI.
Patient’s Antibodies
+
WBCs in transfused blood
Patient’s WBCs
+
WBC Antibodies in transfused
blood
OR

Approximate Residual Leukocytes in Cellular Blood Components
Fresh whole blood 109
Red blood cell concentrate 108 - 109
Buffy cat-depleted red cells 108
Red cells, leukocyte-reduced by filtration <107
Washed red cell concentrate 107
Deglycerolized red 106 – 107
Platelet concentrate Less than or equal to107

Methods of the preparation of
Leucocyte-Reduced Red cells
1. Centrifugation and removing of buffy coat
2. Filtration
3. Washing of red cells with saline
4. Freezing and thawing of red cells

1. Centrifugation and removing of buffy
coat

Centrifugation and removing of buffy coat:
◦ In the centrifugation method for leuokocyte
reduction, the buffy coat layer between the red cells
and the plasma which appear after centrifugation is
drawn off along with plasma and some red cells into
a satellite bag.
◦ A variation of this method is to spin the red cells in an
inverted position so that red cells can be transferred
into a satellite bag, leaving the buffy coat, some red
cells and plasma behind in primary bag.

Method
1. The whole blood unit is centrifuged in an upright position at 5000 x g for 5 minutes at 4°C.
2. The supernatant plasma, buffy coat and some red cells are transferred into a satellite bag.
3. Double seal the tubing between the primary bag and the satellite bag, separate them.
4. Keep the red cells at 4°C.
This is one of the easiest and least expensive method and it can be done in close system, but
it is the least efficient.
◦ It reduces the leukocytes level by 70-80% (less than 1 log) and sacrifices 20% of the red cells.
◦ It does not meet minimum standards for WBC reduction.
◦ Besides its hematocrit is more than > 80% which is difficult to transfuse unless some plasma is returned back or
additive solution is added.
◦ It is also laborious method.
Today this method has been replaced by other more efficient techniques.

◦ Many types of filters are available today that can produce an acceptable leukocyte-reduced product
depending on the requirement.
◦ Microaggregate filters are polyester or plastic screen filters with a pore size of 20-40 micron., which trap
most of the microaggregates composed of white cells, platelets, and fibrin threads that form in blood after
5-6 days of refrigerated storage.
◦ These microaggregates pass through standard blood filters and are trapped in micro-circulation of lung causing
TRALI.
◦ The effectiveness of microaggregate filters of leuko-reduction is increased by cooling the unit at 40C for 3-4 hours
after centrifugation and before filtration.
◦ Filtration of this type usually give a 1 -2 log reduction (90-92%) of leukocytes in the unit (< 5 x 108) and recover most
of the red cells.
◦ Such LR-red cells reduce the incidence of NHFTRs only but does not achieve other goals of leuko-reduction.
◦ Newer leukocyte-reducing filters (third generation) use selective absorption of leukocytes or leukocytes
and platelets.
◦ They are made of polyester or cellulose acetate and will produce a 2 to 4 log (99-99.9%) reduction of the WBCs (< 5
x 106).
◦ There is very little loss of red cells and
◦ the process is quite easy.
◦ They prevent allommunization to HLA antigens, CMV transmission, and NHFTRs.

Leucoreduction can be done at three different point.
(1) Prestorage leuko-reduction
(2) After storage leuko-reduction in blood bank, before issue
(3) Bed side filtration

Prestorage leuko-reduction :
◦ Leukocytes begin to disintegrate quickly when stored at 1-6°C.
◦ These white cells fragments may initiate an immune response to HLA antigens and carry viral activity.
◦ White cells in stored blood may also produce cytokines which may cause NHFTRs.
◦ In order to prevent these effects it may be desirable to remove white cells prior to storage.
This can be done by using one of two technologies, the sterile tube connection device or the inline
filter.
1. Using the sterile tube connecting device, a bedside LR-filter can be connected to a unit of blood prior
to storage and to another sterile bag.
◦ The cells can be filtered from the primary bag through attached filter into the attached bag.
◦ This retains the original expiry date of the initial product and all red cells, platelets, and plasma.

2. The second technology is possible with specially designed blood collection bags system with
Integral - in line filters incorporated between primary collection bag and a satellite bag.
◦ Within 8 hours after phlebotomy, the blood is passed through this filter into an attached
collection bag.
◦ The filtered red cells in the bag are leukocyte reduced and retain the original expiry date.
◦ Leukocytes removal with this system is 99-99.9 per cent (3 log), with > 90 percent red cells
remaining.

Impact of Prestorage Leukoreduction
Potential patient benefits
◦ Decrease in febrile non-hemolytic
transfusion reactions.
◦ Decrease alloimmunization.
◦ Reduce exposure to intracellular
Viruses (e.g. CMV).
◦ Prevent immunomodulation
◦ Reduce tumor spread
Results from Prestorage
leukoreduction
◦ Cytokins poroduction is reduced
or eliminated.
◦ White cells are removed before
fragmentation.
◦ Tumor metastases are reduced in
animals

After storage leukoredction:
◦ The blood or red cells are filtered to
reduce leukocytes in the blood bank
before issue.
◦ In laboratory filtration of blood /red
cell can be properly standardize and
adapted to QC program.
◦ However red cells may have
disintegrated leukocytes and
cytokins.
Bedside filtration:
◦ Bedside filtration commonly being
practiced has been shown to be
quite effective to prevent NHFTRs
but they may also have
disintegrated leukocytes and
cytokines which may cause
FNHTRs.
◦ They are not much effective for
prevention of HLA
alloimmunization.

3. Washing of red cells with saline

◦ Washing of red cells removes leukocytes, platelets and plasma, It can be done manually or
using machine e.g. Haemonetic cell washing machine.
1. Manual Method
(1) Collect the blood in a double bag and store at 2-4°C, till it is processed.
(2) Centrifuge the bag at 5000 x g (heavy spin) for 5 minutes at 2-6°C.
(3) Separate the plasma along with buffy coat into a satellite bag/transfer bag.
(4) Double seal the tube between primary and satellite bags.
(5) Connect the bag with red cells to I.V. saline bottle with bag to bottle connector under
laminar flow. Transfer about 250 ml saline in the red cell bag.
(6) Clamp the tube of bottle connector (transfer set) and seal the tube of the bag distal to the
spike or needle of the transfer set by di-electric sealer. Remove the needle from the tube of the
bag and cover the spike/needle with its plastic cover.

(7) Mix well red cells and saline. Centrifuge the bag at heavy spin (5000 x g for 5 minutes).
(8) Place the bag on the expresser and remove the saline in waste receptacle.
(9) Repeat the washing three times (steps 5 to 8).
(10) After the final wash add 60-70 ml saline in the red cells and mix.
(11) Seal the tubing close to the bag and separate the connector.
(12) Keep washed red cells at 2-6°C.
Whole process is done under laminar flow.
 Shelf-life of the washed red cells is 24 hours.
 All aseptic precautions should be taken.

4. Freezing and thawing of red cells

REMOVING OF LEUKOCYTES BY FREEZING AND
DEGLYCEROROLIZATION.
Low Glycerol Solution (20% W/V cencentration)
◦ The glycerolizing solution consists of 35.0 gm% glycerol, 2.88% mannitol, and 0.65g sodium
chloride.
◦ Whole blood in CPD is centrifuged at 3000x g for seven minutes.
◦ Plasma is taken off and freezed.
◦ Low glycerol solution equal in volume to the red cells (e.g. 250 ml of solution for 250 ml of
red cells) is added with vigorous shaking.
◦ The glycerolized red blood cells are transferred to a polyolefin plastic bag and kept in
aluminium container which is then placed upright in a bath of liquid nitrogen at -196°C and
then stored at -120°C in liquid nitrogen vapour.

High glycerol solution (40% W/V concentration).
• The glycerolizing solution consists of 6.2 M glycerol solution that contains 57 gm% glycerol, 1.6
gm% Na lactate, 0.03 gm% KCl and a total of 25 mEq/1 of monobasic and disodium phosphates to
produce a pH of approximately 6.8
◦ Prior of glycerolization, whole blood in CPD solution, fresh or stored at 4°C for no more than 3-4
days, is centrifuged at 3000x g for 7 min;
◦ supernatant plasma is expressed into satellite bag and used for preparation of components or
freezed.
◦ Appropriate volume of 6.2 M glycerol solution is added in two stages e.g. 300 ml when the weight
of the packed red cells is 150-230 g.
◦ First 100 ml of glycerolizing solution is added to the cells in the collecting bag with vigorous
shaking.
◦ After at least two minutes of equilibration, the remainder glycerol solution and the partially
glycerolized cells are transferred to a 850 ml polyolefin bag (Hebia blood bag).
◦ The bag is centrifuged, the supernatant is expressed and the red cells are frozen at -80°C using a
deep freezer.
◦ They can then be stored at -60 to -65(‘C.

Leukocyte reduced blood components

Leukocyte reduced blood components

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