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International Forum
Vox Sang 199;62:5744
Should All Platelet Concentrates
Issued Be Leukocyte-Poor?
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H . W Reesink,
U.E. Nydegger
Alloimunization against HLA antigens
and subsequent refractoriness to platelet
transfusions is one of the major problems in
transfusion medicine today. When leukocytes are not removed from cellular blood
components (red cell concentrates, RBC,
and platelet concentrates, PC), about half of
the multitransfused patients will develop
HLA antibodies [l-3). Refractoriness to platelet transfusion due to HLA antibodies will
lead to a considerable bleeding risk for the
patientsanddoeslimit the possibility for adequate treatment. Blood banks faced with refractory patients are requested to provide
HLA-compatible PC which requires the
maintenance of a large pool of HLA-typed
donors. These donors should consent to, often repeated, cytapheresis within a short
period of time. Besides the emotional stress
for the patients, clinicians, donors and the
blood bank staff, providing HLA-matched
platelets is a costly therapy regarding the labor and the equipment for cytapheresis. It
therefore appears more efficient to prevent
HLA immunization rather than to cope with
problems of refractoriness to PC transfusions.
In several studies it has been demonstrated that the use of leukocyte-poor blood products (leukocyte-depleted RBC and PC) reduces the risk of HLA alloimmunization
from 40-50 to 10-20% [3-51. The majority of
patients who formed HLA antibodies de-
spite the use of leukocyte-poor products had
a history of pregnancies or transfusions of
nonleukocyte-depleted RBC and PC. It is
likely that such patients have a secondary
(booster) immune response to minimal
amounts of HLA antigens, which is unavoidable, not even by leukocyte depletion of
blood products.
The majority of contributors to the Forum do share the opinion that it is worthwhile
to implement leukocyte depletion of PC as a
standard procedure in blood banks. Besides
the prevention of HLA immunization, the
removal of leukocytes also prevents febrile
transfusion reactions and transmission of viruses (such as cytomegalovirus and human T
cell leukemia I and 2viruses) and will provide
better storage conditions.
It is still a matter of debate as to what
extent leukocytes should be removed from
blood products to prevent primary HLA immunization. At present the minimal number
of allogeneic leukocytes in PC to elicit a primary HLA antibody response in patients is
not well known and is estimated to be about
1 x 1O6IeukocytesperdonorunitofPCresulting in a maximum of lo7leukocytes per transfusion [6]. If the amount of leukocytes is reduced to 10-15 X 10' per pool of PC HLA
immunization will drastically be reduced
(3-51. Some authors of the Forum advocate
to remove leucocytes from PC as soon as
possible i.e. within24 hafterthe collectionof
the whole blood to avoid fragmentation of
granulocytes and the release of harmful enzymes from leukocytes.
Several methods to prepare leukocytepoor PC are described in the present Forum
i.e. the use of platelet-rich plasma or buffy
coats derived from units of whole blood as
source for PC with subsequent filtration and
centrifugation for removal of leukocytes.
Our contributors emphasize quality assurance of the final product with regard to the
number of remaining leukocytes, the number and viability of the platelets (swirling effect) and the pH.
A special problem is imposed by the fact
that leukocytes below levels of 108/1cannot be
determined accurately with current methodology (flow cytometers, electronic particle
counters and cell chambers). However, electronicparticle counting isat present probably
the easiest and quickest method to check the
upper limit of leukocytes present in leukocyte-poor blood products.
Finally, althoughcumbersome, studies in
patients should prove that the chosen method of preparation of leukocyte-poor PC indeed was efficient to prevent primary immunization against HLA antigens in recipients
and the cost effectiveness to provide leukocyte-poor PC needs to be determined. One
author states that leukocyte-poor PC prepared from single buffy coats by centrifugation requires trained staff without additional expense, whereas other authors who
prepare these products by filtration are faced
with the extra costs of filters, wafers for the
sterile connection device and special large
bags for storage of the PC. However, most
authors judge that these extra costs are acceptable for the prevention of HLA immunization, compared to the costs and efforts to
provide the refractory patient with HLAmatched PC.
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Q 1992 S. Karger AG.
Basel
0012-9007/92/0621-MI57
$2.75/0
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UV-B irradiation of PC to inactivate
HLA-class I1 bearing cells is regarded to be
not yet applicable by the members of the
Foruni.
It may be concluded from the present
contributors that all patients who may need
platelet transfusions should be transfused
with leukocyte-poor RBC and PC from the
moment they become transfusion-dependent to avoid HLA immunization and subsequent refractoriness to random platelet
transfusions.
H. W. Reesink, MD, PhD
Medical Director Red Cross
Blood Bank Amsterdam
NL-Amsterdam (The Netherlands)
of 10% immunization, mainly in women with
previous pregnancies [4]. Standard platelet
transfusions result in about 50% immunization necessitating HLA-matched platelet
In the past decade several controlled and transfusion. This requires costs for single doalso well-documented uncontrolled studies nor procedures (US$ 30/unit) and platelet
demonstrated a reduction of HLA immuni- cross-matching (US$ 10) and in addition an
organization is necessary to select and call-in
zation by leukocyte depletion of platelet
transfusions [for review, see I]. From these HLA-matched donors anticipating to the instudies the lowest immunizing dose of white dividual needs of a particular patient, often
cells can only be estimated. In a controlled including cito-virology testing. These costs
study, comparing two methods of leukocyte are exclusive the costs to maintain an HLAdepletion, Marwijk Kooy et a]. [ 2 ] found a typed donor file which has to be available
57% incidence of HLA immunization in pa- anyhow independent of the number of altients who received platelet transfusions con- loimmunized patients.
The cost-benefit of filtration is only pretaining > 5 x lo7white cells (>8 x 10' leukocytesldonor unit). This incidence of immuni- sent in those patients for whom alloimmunization is also found in patients who received zation increases costs of support such as paunlimited standard blood transfusions. In tients eligible for platelet supportive care,
contrast, patients who were transfused with bone marrow or organ transplantation. Beplatelet concentrates containing < 5 x 107 sides, morbidity and in some instances morleukocytes developed HLA antibodies in tality is reduced when by transfusion of leu8%. The amount of contaminating leuko- kocyte-depleted blood cytomegalovirus
cytes in the platelet transfusions in the latter transmission is prevented, for instance in the
group, however, was in 96% of the trans- case of neonatal transfusions. Although leukocyte depletion also offers other advantagfusions below 5 X lo', still leaving a 'grey'
zone between 5 x 10' and 5 X lo7leukocytes es with regard to reduction of' microaggrefrom which the immunogenicity is not exactly gates and febrile transfusion reactions, less
known, albeit definitely reduced. With the expensive methods such as differential cenavailable knowledge, platelet transfusions trifugation, are effective as well.
An alternative to leukocyte depletion is
should aim at < 5 X 10' leukocytes (< lo6leukocytesldonor unit) and not exceed an upper nor offered by UV-B irradiation inactivating
limit of lo7 above which immunization was HLA class I1 antigens. UV-B irradiation has
incidently shown [3]. To reach this goal by a many drawbacks: Quality control in routine
reproducible procedure, suitable for routine blood banking is impossible, the formationof
blood banking, filtration offers the best ap- microaggregates is enhanced, the metaboproach.
lism of leukocytes is deleterious for an optiFor the moment no studies are available mal environment for platelets during stor(yet) proving that the new generation of cel- age, the transmission of cytomegalovirus is
lulose acetate and polyester filters remove presumably maintained, UV-B induces
white cell fragments effectively with regard DNA damage and the immunosuppressive
to immunization and transmission of cytome- effects of blood transfusions -- a subject of
galovirus, implicating that filtration should concern in oncology - may be potentiated.
not be postponed for several days.
UV-B irradiation may have a place in the
An optimal approach appears to store future in combination with leukocyte deplewhole blood for at least 8 h at 20°C to allow tion in an attempt to further reduce allo-imgranulocytes and monocytes to kill residual munization in high risk recipients with previbacteria which might be present in 2% of all ous pregnancies.
blood withdrawn lege artis. Within 24 h, platelet suspensions should be pooled usingsterA. Brand
ile connection devices, filtered and subseDepartment of Immunohaematology and
quently stored. The shorter the duration of
Blood Bank
storage the better the quality of platelets, but
University Hospital Leiden
when < 10yplatelets/mlare stored in 1,000-ml
Rijnsburgerweg 10
2333 AA Leiden (The Netherlands)
bags providing surface area while rotating,
PVC bags are suitable for several days' storage. The additional costs of such filtration
procedure is approximately fl. 20 (US$ 10)
per random donor unit (using volunteer donors) and this results in an overall percentage
A. Brand
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U.E. Nydegger, MD
Head. Division of Transfusion Medicine.
Central Laboratory of Hematology
University of Berne
CH-3010 Berne (Switzerland)
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References
Howard JE, Perkins HA: The natural history
of alloimmunization to platelets. Transfusion
1978: 18:496S03.
Dutcher JP, Schiffer CA, Aisner J , Wiernik
PH: Alloimmunization following platelet
transfusion: The absence of a dose response
relationship. Blood 1981;57:395-398.
Eernisse JG. Brand A: Prevention of platelet
refractoriness due to HLA antibodies by administration of leukocyte-poor blood components. Exp Hematol 1981:9:77-83.
Murphy MF, Metcalfe P. Thomas H. Eve J,
Ord J. Lister TA, Waters AH: Use of leukocyte-poor blood products and HLA-matched
donors to prevent allo-immunization by platelet transfusions. Br J Haematol 1988529-534.
Brand A. Claas FMJ. Voogt PJ. Wasser
MNJM, Eernisse JG: Alloimmunization after
leukocyte depleted multiple random donor
platelet transfusions. Vox Sang 1988;M: 160166.
Fisher M. Chapman JR, Ting A, Morris PJ:
Alloimmunization to HLA antigens following
transfusion with leukocyte-poor and purified
platelet suspensions. Vox Sang 1985:49:331335.
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58
Reesink/Nydegger/Brand/Pietersz/Andreul International Forum
Gmur/Murphy/Schiffer/Kickler
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References
1 Brand A: White cell depletion. Why and how?
in Nance ST (ed): Blood Transfusion in the
1990's. Arlington, AABB, 1990; pp 35-61.
2 Manvijk Kooy M. van Prooijen HC, Moes M,
Bosman-Stants I, Akkerman JWN: The use of
leukocyte-depleted platelet concentrates for
the prevention of refractoriness and primary
HLA allo-immunization: A prospective randomized trial. Blood, in press.
3 Fisher M, Chapman JR, Ting A, Morris PJ:
Alloimmunization against MHC antigens after
platelet transfusions is due to contaminating
leukocytes in the platelet suspension. Vox
Sang 1985;49:331-335.
4 Brand A, Claas FMJ. Voogt PJ, et al: Alloimmunization after leukocyte depleted multiple
random donor platelet transfusions. Vox Sang
1988;54:16Ck166.
R. N . I. Pietersz
Transfusion of leukocyte-depleted blood
products will reduce HLA sensitization resulting in fewer febrile transfusion reactions
and less immune-mediated refractoriness to
platelet therapy [l]. Furthermore, transmission of leukocyte-associated viruses will be
diminished. The immunogenic dose of leukocytes, however, is still unknown and dependson the type of leukocytes, the immunogenicity of the HLA antigens and the responsiveness of the recipient. Moreover, the
commonly available methods for counting
leukocytes do not have a great accuracy if the
leukocyte concentration drops below 1 cell
per PI. If we assume that the immunogenic
dose is 50 x lo6 leukocytes as recently suggested by van Manvijk Kooy et al. [2], how
can this be achieved in blood bank routine.
For the filtration of red cell concentrates
various filters are available to reduce the leukocyte number to below 50 x 10' and even
10 x 10'. However, most filters have a maximum capacity to retain leukocytes and perform better if the initial leukocyte number is
diminished to approximately 1,000x lo',
which can be accomplished by first removing
the buffy coat from the red cells (quality control data from 1990in Amsterdam; Cellselect
filter, NPBI; n = 14.113, leukocytes < 2 x lo6
in 100% of the filtered unit).
For platelet concentrates (PC) it is not
easy to meet a criterion of 50 x 10' leukocytes
in a pool of 4-6 donor units.
Very careful preparation of PC with the
platelet-rich plasma method results in leukocyte contaminations of 20 x lo6or higher per
donor unit. In PCpreparedfrom singledonor
buffy coats routinely fewer than 10 x 10' leukocytes are present and in more than 90% of
these PC fewer than 5 x 10' (quality control
data from 1990 in Amsterdam: n=5.216
pools of 6 PC, leukocytes 5 k 4, mean k SD,
per donor unit). However, this low leukocyte
contamination can only be achieved at the
cost of a decrease in platelet number to 4045 x 10' per donor unit [3], (quality control
data from 1990 in Amsterdam: n=4.178
pools of 6 PC: platelets 47 k 9, mean t SD,
per donor units).
In another method of PC preparation
from buffy coats the buffy coats are pooled
and the pool is centrifuged for a second time.
A platelet-rich plasma with a high platelet
count (75 X 10') and a low leukocyte contamination, i.e. below 50 x 106per 4 donor units
could be obtained routinely [4].
For PC obtained by cytapheresis it depends on the machine used, how high the
leukocyte contamination will be. With some
machines leukocyte numbers per PC are below 50 x lo6, while with others the PC may
contain 1,OOO x 10' or more leukocytes.
To remove leukocytes from PC an extra
centrifugation can be applied or the PC can
befiltered. Bothmethods will induce platelet
loss ranging from 10 to 20%. Another drawback of the leukocyte depletion filters for
platelets is the same as for red cell filters: the
capacity of the filter to remove leukocytes
is not always known. Since the initial number
of leukocytes in the PC may vary from approximately 50 to more than 1,000 x lo6,the
leukocytes in every unit of filtered PC should
be checked. An initially low number of leukocytes in the PC will improve the final result.
For every method used a final check before issuing the leukocyte-depleted PC
should include: (1) the swirling effect in the
PC; (2) a leukocyte count with an upper limit
of 50 X 10' leukocytes per PC, and (3) a platelet count. Considering the possibilities for
preparation and the costs, leukocyte-poor
PC from single buffy coats can be manufactured routinely only requiring trained staff
and special inserts for centrifugation [3].
Pooling of buffy coats is easy to perform but
requires the use of asterile connection device
(SCD) and special large platelet storage
bags, which increases the costs [4]. Cytapheresis is expensive in donor time and software.
Leukocyte depletion by filtration of the PC
will increase the costs with the price of the
filter(US$30), andifthefilteredPCaretobe
stored with the price of the wafers (US$1.5
each) for the SCD connections and special
large platelet storage bag (US$ 15). Moreover, storage of nonfiltered PC has been assessed more thoroughly than of filtered PC
which are often mixed with solutions used to
prime or rinse the filter.
There is sufficient evidence that if PC
contain < 50 x lo6leukocytes, cytomegalovirus transmission will be prevented. UV-B irradiation may alter the function of class I1
bearing cells but as yet there is too little evidence that the method is safe for clinical use
to prevent alloimmunization. UV-B may induce unknown late effects on DNA synthesis, the effect of irradiation on platelet function and survival is not yet known, there are
still practical problems to be solved concerning the irradiation and type of plastic for the
bags. Finally, UV-B irradiation will not prevent transmission of leukocyte-associated viruses.
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59
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In conclusion, for patients with malignant
hematological disorders andor chemotherapy. patients on hemodialysis and organ
transplant candidates, PC should be leukocyte-depleted. Since in most large centers the
majority of patients belong to this group all
PC should be leukocyte-depleted.
G. Andreu
The French standards for leukocyte-poor
red cell and platelet concentrates have been
recently modified. For platelet concentrates,
three different productsare to beconsidered:
(1) platelet concentrates prepared from
whole blood donation (= standard platelet
concentrates): they contain at least SO x 10'
plateletsinavolumeof40-70 ml. Leukocytepoor platelet concentrates contain less than
5 x 10" leukocytes; (2) platelet concentrates
prepared by apheresis along with plasma collection: they are defined as multiple of standardplatelet concentrates, accordingto their
platelet and leukocyte content; (3) platelet
concentrates prepared by apheresis (= single
donor platelet concentrates); they contain at
least 400 X 10' platelets in less than 500 ml.
Leukocyte-poor single donor platelet concentrates contain less than 5 x 10" leukocytes. Actually, it is already possible to produce leukocyte-poor platelet concentrates
containing less than 0.1x lo6 leukocytes in
platelet concentrates prepared from whole
blood, and 10'Ieukocytes in single donor platelet Concentrates. However, standards must
take account of all the techniques and the
materials in use in the country, and the values
finally adopted should be accessible for every
blood center in France.
The fact that leukocyte contamination is
harmful to platelets during storage has been
studies in experiments involving add-back of
controlled quantities of leukocytes in otherwise identical platelet preparations [l].In addition, spontaneous lysis of leukocytes during storage is not negligible. The major part
of cytoplasmicas well as membrane materials
released cannot be blocked by the filters used
for leukodepletion. Moreover, it has been
shown in an animal experiment [2] that filtration after storage was less efficient than
early filtration to prevent alloimmunization
against major histocompatibility complex
antigens. These three arguments are in favor
of anearly leukocyte depletion, at the time of
preparation of platelet concentrates. Platelet
integrity immediately after the preparation
of platelet concentrates is highly dependent
on the method used: if we consider only standard platelet concentrates, the buffy coat
methods are less detrimental to platelets than
the conventional platelet-rich plasma procedure [3].
All the above information leads to the
conclusion that the best way to prepare and
store leukocyte-poor concentrates is the
combination of the buffy coat methodology
and an early filtration before storage. This
association has already been developed in
somecentres[4]. TheriskofHLAimmunization in patients multitransfused with red cell
and platelet concentrates when standard
products are used varies from 28 to 71%
(mean = 41%) as analyzed in 19published and
personal data from 1979 to 1989, involving
overall 1,393 patients. Standard blood components used in these studies were almost
all prepared with the conventional plateletrich method, therefore highly contaminated
with leukocytes. In contrast, 0-28%
(mean = lY%)ofpatientstransfusedwith leukocyte-poor blood components form antiHLA antibodies, in 11 studies involving 663
patients. In almost all these studies, leukocyte-poor blood components were prepared
at the time of transfusion, and the number of
leukocytes remaining in the final products
was higher than the values we can obtain
today with the association of the buffy coat
methodology and filtration. However, leukocyte-poor blood components do not seem
to be able to completely prevent HLA alloimmunization: they are an efficient means
to prevent primary HLA immunization,
while secondary response i n previously
HLA-sensitized patients, either by transfusion or pregnancy is poorly prevented. In our
recent experience at the HBtel-Dieu in Paris.
10 out of 54 patients transfused with leukocyte-poor blood components had HLA antibodies, a secondary response being most
probable for 8 patients.
UV-B irradiation is an interesting means
to expect a further reduction of transfusioninduced HLA immunization: there is a body
of experiments suggesting that the failure of
mononuclear cells to stimulate allogeneic
cells in vitro and in vivo after UV-B irradiation is not a simple absence of response, but
rather the development of an immune tolerance in the recipient. The difficulties to organize a UV-B trial today are less technical [S]specific blood bags and irradiator are needed
-than conceptual: as UV-B irradiation cannot be performed on red cell concentrates,
patients must receive the best leukocytepoor red cell concentrates available. In the
French trial, we decided as acceptable a maximumof2 x 1O'leukocytecontent perredcell
concentrate, which needs the systematic
combination of buffy coat methodology and
filtration, and a rigourous quality control.
The comparison with a group of patients receiving leukocyte-poor blood components is
mandatory. More questionable is the necessity of a control group receiving standard pla-
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R.N. I. Pietersz, MD PhD
Red Cross Blood Bank Amsterdam
Plesmanlaan 125
1066 CX Amsterdam (The Netherlands)
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References
Brand A, Claas FMJ, Voogt PJ, Wasser
MNJM, Eernisse JG: Alloimmunization after
leukocyte depleted multiple random donor
platelet transfusions. Vox Sang 1988;54:160166.
Van Marwijk Kooy M, van Prooijen HC, Moes
M, Bosma-Stants I , Akkerman JWN: The use
of leukocyte-depleted platelet concentrates
for the prevention of refractoriness and primary HLA alloimmunization: A prospective
randomized trial. Blood 1991:77:1-15.
Pietersz RNI, Reesink HW, Dekker WJA. Fijen FJ: Preparation of leukocyte-poor platelet
concentrates from buffy coats. I Special inserts
for centrifuge cups. Vox Sang 1987:53:203207.
Eriksson L, Hogman CF: Platelet concentrates in an additive solution prepared from
buffy coats. Vox Sang 1990;59:140-1-15.
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60
Reesink/Nydegger/Brand/Pie tersz/Andreu/
Gmiir/Murphy/Schiffer/Kickler
International Forum
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telet concentrates. We found this group useful, since the only one randomized clinical
trial comparing leukocyte-poor and standard
platelet concentrates, while all patients receive leukocyte-poor red cell concentrates,
was not conclusive [ 6 ] .
In case UV-B irradiation efficiency is
demonstrated to reduce secondary response,
it could not replace the use of leukocyte-poor
blood components: leukodepletion has
many other advantages apart from HLA immunization prevention. It would be an additional technique. If the efficiency is well established in the next few years by clinical
trials, it will be a benefit for the patients at
medical as well as economical point of vue:
patients receive always less platelet concentrates when they have no HLA immunization, nor refractoriness to platelet transfusion.
ml. Neutrophils (constituting up to 10%)nor-
Jurgl? Gmiir
Leukocyte depletion of blood products by
use of *thirdgeneration’ (adsorption) filters is
being used with increasing frequency in an
effort to prevent HLA alloimmunization.
Several clinical studies suggest that there is a
clear trend with the incidence of reported alloimmunization reaching zero when the number of leukocytes falls below 5 X lo6per transfusion [I. 21. To obtain such ‘clean’ products
the use of ‘third generation’ filters is inevitable which adds considerable costs and additional work to the preparation of blood components and leads to some cell loss. Therefore. the question arises wether such ‘clean’
products are of proven clinical advantage
compared to red blood cell (RBC) or platelet
transfusions (PLT) leukocyte depleted by a
simple centrifugation step and containing 1-2
log higher leukocyte counts. This question is
not easy to resolve. It is well accepted that
modern conventional counting techniques by
electronic inpedance (Coulter Counter S Plus
1V)or light scatter (TechniconH*l)cannotbe
utilized to monitor white cell counts below
0.1 x 10’A corresponding to 2-4 x 10’ per single donor apheresis product (SD-PC) or
5 x loh per standard platelet concentrate
(PC). A modified microscopic counting
method(1:2(PC)andl:lO(RBC)ratherthan
1:lO and 1:lOO dilution, respectively: 36 rather than 9 squares counted) seems to increase
the sensitivity by 2 log (PC) and 1log (RBC),
respectively. However, a comparison of
counting methods and fluorescein-activated
flow cytometric techniques has revealed that
> 2 log leukocyte depletion cannot be accurately monitored by the former [3]. At our
institution analyses of serially diluted PC by
FACS showed that the correlation of predicted and measured cell counts are no longer
linear below 0.02 X 10’11 [unpubl. observations]. This corresponds to 4 X 10‘ per SD-PC
or 1 x 10’per PC. Thus. for the present leukocyte-poor blood components have to be defined by an upper limit of leukocyte content
which seems to be in the order of 5 X lo6 per
platelet transfusion and lo7 per RBC transfusion. Due to these technical limitations the
term ’leukocyte-free’blood components is inappropriate. Moreover, clinical studies comparing standard and ‘virtually leukocyte-free’
RBC and/or PLT transfusions should be carefully checked for their leukocyte counting
methodology [l, 21.
Leukocytes are usually present in PC in
numbers varying from 1x 10’ to 3 x 10’ per
mally disintegrate during the first 2 days of
storage and thereby may release hydrolytic
enzymes from their granules into the plasma.
It has been shown that these enzymes affect
membrane composition and platelet function [4]. These observations point to the importance of removing leukocytes from PC
before storage. On the other hand, caution is
indicated because there is no published experience documenting that storage life is not
altered by preceding filtration.
For more than 10 years it has been our
policy to use exclusively random SD-PC in
patients who will need platelet support for
more than 2-3 weeks. This policy is substantiated by the low incidence of alloimmunization observed after random SD-PC in leukemiapatientswithoutpriorsensitization[5].In
our hands, the rateof antibody formation and
refractoriness is comparably low whether leukocyte-rich (-4 x 10y/PLT) or leukocytepoor (-3 x 10s/PLT)random SD-PCare used
[5]. Hence, it would be difficult to prove that
an additional filtration of SD-PC would add
any advantage to the already low incidence of
alloimmunization (2/49) in nonpresensitized
recipients of standard SD apheresis platelets.
By which measure the higher incidence in patients with prior pregnancies could be reduced is not yet established.
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G. Andreu, MD
Poste de Transfusion Sanguine
HBtel-Dieu de Paris
1 place du Parvis Notre-Dame
75181 Paris Cedex 01 (France)
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References
I Pietersz RNI. de Korte D, Reesink HW, van
Den Ende A, Dekker WJA. Roos D : Preparation of leukocyte-poor platelet concentrates from buffy coats. Vox Sang 1988:55:
11-20.
2 Blajchman M: Communication presented at
the Research and Progress session on leukocyte-poor blood components. ISBTlAABB
meeting. Los Angeles. November 10-15.
1990.
3 Fijnheer R, Pietersz RNI. de Korte D, Gouwerok CWN. Dekker WJA, Reesink HW,
Roos D: Platelet activation during preparation
of platelet concentrates: A comparison of the
platelet-rich plasma and the buffy coat methods. Transfusion 1990:30:634-638.
4 Angue M. Chatelain P. Domy M. Guignier F,
Richaud P: Preparation de concentres des plaquettes humaines deleucocytees par centrifugation et filtration d’un pool de buffy-coats
connectes stkrilement. Revue Fr Transfus Hemobiol, in press.
5 Andreu G. Boccaccio C. Lecrubier Ch, Fretault J. Coursaget J, Leguen JP, Oleggini M,
Fournel JJ, Samama M: Ultra violet irradiation of platelet concentrates: Feasibility in
transfusion practice. Transfusion 1990:30:401406.
6 Schiffer CA, Dutcher JP, Aisner J. Hogge D.
Wiernik PH, Reilly JP: A randomized trial of
leukocyte-depleted platelet transfusion to
modify alloimmunisation in patients with leukemia. Blood 1983:62:815.
Jurg P. Gmur, MD
Universitatsspital Zurich
Departement fiir Innere Medizin
Abteilung fiir Hamatologie
Ramistrasse 100
8091 Zurich (Switzerland)
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References
1 Brand A, Claas F, Voogt P, et al: Alloimmunization after leukocyte-depleted multiple random donor platelet transfusions. Vox Sang
198854: 160.
2 Saarinen U, Kekomaki R, Siimes M, Myllyla
G: Effective prophylaxis against platelet refractoriness in multitransfused patients by use
of leukocyte-free blood components. Blood
1990;75:512.
3 Bodensteiner DC: Leukocyte depletion filters: a comparison of efficiency. Am J Hemato1 1990:35:184.
4 Sloand EM, Klein HG: Effects of white cells
on platelets during storage. Transfusion 1990:
30:333.
5 Gmur J, Burger J, Sauter Chr, et al: Alloimmunization by leukocyte-rich or leukocytepoor random single donor platelets. Progr Clin
Biol Res 1990;337:45.
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61
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Scott Murphy
Several studies strongly suggest that leukodepletion of blood products prior to infusion reduces the incidence of subsequent
alloimmunization in recipients [I]. Alternative approaches such as ultraviolet irradiation show promise but have not yet been adequately tested clinically. There are no established disadvantages of leukodepletion of
platelet concentrates other than the increased cost and the loss of 10% of the platelets processed. Therefore, current data support leukodepletion of all platelet concentrates issued to patients with diagnoses in
which the need for long-term plateletsupport
can be predicted. In fact, some of the most
difficult patients to support are those who
have been alloimmunized by transfusion priortotheestablishmentofsuchadiagnosis. In
such presensitized individuals, the dose of
leukocytes necessary to elicit a secondaryimmune response may be very small and beyond the capacity of any leukodepletion procedure. Therefore, in principle, all blood
products issued should be leukopoor. Like all
clinical recommendations, this recommendation is a best estimate as optimal management for an individual patient at a point in
time when the data available is still incomplete.
It has been calcualted that the number of
patients who will benefit significantly from
leukodepletion is rather small [ 2 ) . Therefore, it is particularly important that the cost
of leukodepletion be low. The extent of leukodepletion required is not yet known. It
seems obvious that the requirement will vary
frompatienttopatient. Forexample,inacute
leukemia, some patients have very stormy
courses requiring frequent infusions of many
units of platelets while others go through induction rather easily with relatively few infusions. We assume that the risk for alloimmunization varies with the total number of
contaminating leukocytes infused within a
given time period. Current data suggests that
we must achieve at least a 2 log reduction
from 1IeukocytellO' platelets, which is characteristic for random donor concentrates
prepared from platelet-rich plasma to 1leukocytell0' platelets. Perhaps another one or
two logs of reduction will ultimately be required.
In the long run. it will probably be preferable to develop methods which will render
platelet concentrates relatively free of leukocytes at the time of their preparation. Some
devices for plateletpheresis [3] and the buffy
coat method [4] of processing donations of
whole blood represent steps in this direction.
However, a second step, probably a filtration
step, will be required to achieve optimal leukodepletion in most cases. Filtration at the
bedside duringinfusion iseffective, at least in
theory IS]. However, it is difficult to assure
use of consistent techniques and establish adequate quality control in this setting. Furthermore, there may be some disintegration
of leukocytes during storage prior to infusion. Leukocyte fragments may be immunogenic but not filtrable [6]. Therefore it seems
preferable to complete leukodepletion in the
blood center at some point during the first
24 h of storage. This step has to be compatible with maintenance of platelet integrity
during subsequent storage for 4-5 days.
There is not yet adequate data showing that
any leukodepletion procedure meets this requirement. However, it seems highly likely
that such procedures will be developed and
verified in the near future.
Scott Murphy, MD
Professor of Medicine
Associate Director for Clinical Programs
Cardeza Foundation for Hematologic Research
1015 Walnut Street
Philadelphia, PA 19107 (USA)
..............................................
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References
1 Meryman HT: Transfusion-induced alloimmunization and immunosuppression and the effects of leukocyte depletion. Transfusion Med
Rev 1989;3:180.
2 Schiffer CA, Dutcher JP, Aisner J, Hogge D.
Wiernik PH, Reilly JP: A randomized trial of
leukocyte depleted platelet transfusion to
modify alloimmunization in patients with leukemia. Blood 1983;62:815.
3 Hester JP, Ventura GJ, Bouzher T: Platelet
concentrate collection in a dud-stage channel
using computer-generated algorithms for collection and prediction of yield. Plas Ther
Transf Techno1 1987;8:377.
4 Pietersz RNI, Reesink HW, Dekker WJA. Fijen FJ: Preparation of leukocyte-poor platelet
concentrates from buffy coats. Vox Sang 1987;
S3:203.
5 Kickier TS, Bell W, Ness PM, Drew H. Pall D:
Depletion of white cells from platelet concentrates with a new absorption filter. Transfusion
1989;29:411.
6 Engelfriet CP, Diepenhorst P. Vande Giessen
M, Von Riesz E: Removal of leukocytes from
whole blood and erythrocyte suspensions by
filtration through cottonwool. Vox Sang 1975:
28:81.
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62
Reesink/N ydeggerlBrandlPieterszl Andreul
GmiirMurphylSchifferlKickler
International Forum
Charles A . Schifler
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The development of antibody against
class I HLA antigens is the major cause of
immune-mediated refractoriness in recipients of multiple platelet transfusions. Alloimmunization develops in approximately
one third of patients with acute myeloid leukemia (AML) undergoing induction chemotherapy, in a smaller fraction of patients with
acute lymphocytic leukemia and in a higher
proportion of individuals not receiving concurrent cytotoxic chemotherapy [I]. Although most attention is appropriately focused on the clinical problem of the management of alloimmunized patients, perhaps the
biologically most interesting point is that the
majority of individuals exposed to HLA antigens from multiple donors do not form antibody against these antigens and indeed behave as if they were immune ’tolerant’.
There is compelling preclinical evidence
to suggest that it is the leukocytes suspended
in platelet transfusions which are responsible
for the initial recognition by the recipient’s
immune system with the subsequent development of alloantibody [reviewed in 21. In
addition. there is evidence that even modest
leukocyte depletion by avariety of meanscan
attenuate or eliminate febrile transfusion reactions in patients who have had repeated
reactionsof this type [3].In recent years more
effective blood filters have become available
which can results in a > 3 3 log reduction of
leukocytes from platelet suspension and
packed red blood cells to a level of contamination which may be ‘nonimmunogenic’. A
number of clinical trials have been performed, most of which have been interpreted as
demonstrating a reduction in alloimmunization when filtered blood products are utilized
[reviewedin4].There aremanymethodologic issues, however, which prevent general extrapolation of these results. There are also
sufficient differences amongst the trials to
preclude the application of the now fashionable ‘metaanalysis’technique. Some of these
problems include: (1) small numbers of patients (largest trial includes 69 evaluable patients); (2) variability in patient diagnoses
and treatment: (3) exclusion of potentially
’presensitized’ patients with prior pregnancies or transfusions from some studies; (4)
inconsistencies in the frequency ofalloimmunization noted with comparable amounts of
leucocyte contamination; ( 5 ) variable quality control of leucocyte numbers in transfused products.
Table 1. Potential
effect of leukocytedepleted platelets
for 100 patients with
Initial total
AM1
Alloimmunized on admission or
anamnestic response
Alloimmunization rate
CR rate
Intensive post .CR treatment
‘Effectiveness’ of leukocyte depletion
Granulocyte transfusions
Protocol errors
Patients,
%
Patients
‘benefitting’
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100
10
3040
75
15
67
5
10
90
36
27
21
14
13
12
CR = Complete remission.
The available filters are quite expensive
and represent a considerable increase in
blood banking administrative and technologist costs, particularly since all of the red
blood cell transfusions must also be leukocyte-depleted. In addition, all filters produce
appreciable (10-20%) loss of platelets, potentially increasing the number of units
which must be transfused. There is little experience with storage of filtered platelets
(most, if not all studies filtered immediately
before administration), and limited information about the reproducibility of leukocyte
depletion when these techniques are used in
blood centers of different size and sophistication or at the bedside. The latter point is of
particular relevance because of data inferring that transfusions should contain < lo6
leukocytes in order to reduce immunogenic-
isof particular interest because it is potentially extremely simple to administer. Because of
the cost considerations, the inconclusive (albeit very tantalizing) results of the available
clinical trials, and our current ability to successfully support the fraction of patients who
do become alloimmunized, platelet leukocyte filtration to prevent alloimmunization in
multiple transfused recipients cannot be recommended for routine use at this time.
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ence of a variety of factors suggest that only
10-15% of patients might actually ‘benefit’
ry using standard platelet transfusions and
the approximate 10% of patients alloimmunized at oresentation would not derive benefit from leukocyte-depleted blood products
(table 1).
Because of these considerations, any
technique employed to reduce alloimmunization must be inexpensive and produce sufficient and reproducible leukocyte depletion
or modification. A large, randomized multiinstitutional study (Tryal to Reduce Alloimmunization to Platelets), which should address many of the questions raised by the
studies which have been done to date, has
been begun in the United States. Leukocyte
filtration, as well as UV-B irradiation of platelets, will be evaluated. The latter approach
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References
zyxw
munized patients. Blood 1987;70:1727-1729.
2 Meryman HT Transfusion-induced alloimmunization and immunosuppression and the effects of leukocyte depletion. Transfusion Med
Rev 1989;3:18&193.
3 Schiffer CA, Patten E., Reilly J , Patel S: Effective leukocyte removal from platelet preparations by centrifugation in a new pooling
bag. Transfusion 1987;27:162-164.
4 Schiffer CA: Prevention of alloimmunization
against platelets. Blood. in press.
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63
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Thomas S. Kickler
Clinical and experimental data suggest
that leukocyte depletion from units of platelets may serve two main purposes first prevention of alloimmunization and second improvement in the function and viability of
stored platelets [l,21. If it can be shown that
there is significant improvement of platelet
quality by leukocyte depleton prior to storage, prestorage leukocyte depletion should
become a standard procedure for all platelet
concentrates. If the prevention of alloimmunization is the only concern, not all patients
require leukocyte-depleted platelet and leukocyte depletion would only be required for
selected patients.
The definition of leukocyte-poor platelets is dependent upon the purpose of the
leukocyte depletion. If one wishes to reduce
alloimmunization, several clinical studies
suggest that residual leukocyte contamination of 10' may be sufficient [l].However it
should be noted that these studies were performed in immunosuppressed patients. It is
unknown whether this degree of leukodepletion is sufficient for other patient groups not
heavily immunosuppressed such as those
with aplastic anemia.
If the goal of leukocyte depletion is the
improvement of platelet quality and viability, it isnot known whether reducing residual
leukocytes to 10'will be sufficient to enhance
the quality of stored platelets [2]. Little is
known about the number of leukocytes that
are important in mediating injury to the platelet during storage. From personal observations, removal of 3-4 logsof leukocytes prior
to storage may be sufficient to improve the in
vivo and in vitro recovery of platelets after 5
days of storage.
If leukodepletion is to be performed it
should be done prior to storage to gain the
benefit of improving the quality of stored
platelets as well as reducing the risk of alloimmunization. Contaminating leukocytes may
cause impaired platelet viability and function
independent of their influencing any drop in
pH. Neutrophils may become disrupted during centrifugation or during storage leading
to the release of hydrolytic enzymes. These
enzymes, specifically Cathepsin G and elastase, can alter glycoprotein IB/IX expression
and possibly other important membrane receptors. Lymphocytes, the predominant
contaminating leukocyte in platelets, mayalso inhibit platelet function [3].
Leukocyte depletion may be efficiently
performed by filtration leading to 2-3 log
reduction in leukocyte contamination. If one
prepares platelets by the buffy coat method,
very low levels of leukocyte contamination
will also result [4].It may even be possible to
combine filtration with platelet preparation
by the buffy coat method to make a more
leukocyte-poor product.
It is not known whether ultraviolet irradiation will prove superior to leukocyte depletion in preventing alloimmunication to platelet transfusions. The simplicity of ultraviolet
irradiation makes it an appealing alternative
to filtration. However, there are only limited
data on the efficacy of ultraviolet irradiation
in preventing the alloimmune response to
platelet transfusions in humans [ 5 ] .
Numerous clinical studies have been performed using leukocyte removal as a modality in preventing or delaying alloimmunization to HLA antigens. Although there is considerably more experience with removal of
leukocytes than with ultraviolet irradiation,
the studies are relatively small and the criteria of refractoriness or alloimmunization inadequate. Only patients requiring relatively
long-term platelet transfusions are at risk for
alloimmunization. Surgical patients would
not benefit from the expense of using leukocyte-depleted platelets. It should also be
noted that up to 10% of platelets may be lost
by filtration. This may mean that if routine
filtration removal of leukocytes is instituted,
more platelets may need to be prepared to
supply the platelet needs of patients.
................................................
References
1 Kickler TS: Platelet alloimmunization. Transfusion Med Rev 1990;4:8-18.
2 Sloand EM. Klein HG: Effect o f white cells on
platelets during storage. Transfusion 1990;30:
333-338.
3 Nicolini FA, Wilson AC, Metha P, Metha JL:
Comparative platelet inhibitory effects of human neutrophils and lymphocytes. J Lab Clin
Med 1990:116:147-152.
4 Fijnheer R, Pietersz RNI, Korte D . Gouwerok CWN, Dekker WJA, Ree\ink HW. Roos
D: Platelet activation during preparation of
platelet concentrates: A comparison of the
platelet rich plasma and the buffy coat methods. Transfusion 1990;30:634438.
5 Menitove JE, Kagen LR, Aster RH, et al: Alloimmunization is decreased in patients receiving UV-B irradiation platelet concentrates
and leucocyte depleted red cells. Blood 1990;
761404.
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64
Thomas S. Kickler MD
Associate Director
The Johns Hopkins Hospital
Department of Laboratory Medicine
Immunohematology Division
600 North Wolfe Street
Baltimore, MD 21205 (USA)
Reesink/Nydegger/Brand/Pietersz/Andreu/ International Forum
Gmiir/Murphy/Schiffer/Kickler