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From: TSS (216-119-144-16.ipset24.wt.net)
Subject: Effectiveness of leucoreduction for removal of infectivity of transmissible spongiform encephalopathies from blood [FULL TEXT]
Date: August 7, 2004 at 9:26 am PST

-------- Original Message --------
Subject: Effectiveness of leucoreduction for removal of infectivity of transmissible spongiform encephalopathies from blood [FULL TEXT]
Date: Sat, 07 Aug 2004 11:21:24 -0500
From: "Terry S. Singeltary Sr."
To: Bovine Spongiform Encephalopathy
CC: cjdvoice@yahoogroups.com


Lancet 2004; 264: 52931
Veterans Affairs Maryland
Health Care System, VA Medical
Center, University of Maryland
at Baltimore, 10 North Greene
Street, Baltimore, MD 21201,
USA (L Gregori PhD,
R G Rohwer PhD); Health
Canada, Center for Infectious
Disease Prevention and
Control, Ottawa, ON, Canada
(N McCombie RN, A Giulivi MD);
Canadian Blood Services,
Ottawa, ON, Canada
(D Palmer MSc, P Birch MLT);
Pall Corporation, Port
Washington, NY, USA
(S O Sowemimo-Coker PhD)
Correspondence to:
Dr Robert G Rohwer
rrohwer@umaryland.edu
www.thelancet.com Vol 364 August 7, 2004 529

Effectiveness of leucoreduction for removal of infectivity of
transmissible spongiform encephalopathies from blood

Luisa Gregori, Nancy McCombie, Douglas Palmer, Paul Birch, Samuel O
Sowemimo-Coker, Antonio Giulivi, Robert G Rohwer

In 1999, the UK implemented universal leucoreduction as a precaution
against transmission of variant Creutzfeldt-
Jakob disease by transfusion of domestic blood or red blood cells. We
aimed to assess how effectively leucoreduction
reduced infectivity of transmissible spongiform encephalopathies (TSEs)
in blood. 450 mL of whole blood collected
and pooled from scrapie-infected hamsters was leucoreduced with a
commercial filter. Blood cell concentrations
were quantified, and infectivity titres measured. Blood cell recovery
and white blood cell removal complied with
American Association of Blood Banks standards. Leucofiltration removed
42% (SD 12) of the total TSE infectivity in
endogenously infected blood. Leucoreduction is necessary for the removal
of white-cell-associated TSE infectivity
from blood; however, it is not, by itself, sufficient to remove all
blood-borne TSE infectivity.

Transmissible spongiform encephalopathies (TSEs) are
fatal CNS infections that can incubate asymptomatically
for a decade or more in human beings before the
appearance of clinical disease. People in the
asymptomatic phase of variant Creutzfeldt-Jakob disease
(vCJD) appear healthy and donate blood with the same
frequency as any healthy person. Transmission of vCJD
by transfusion was recently recognised in Great Britain.1
To reduce the risk of transfusion transmission of such
diseases in human beings, the UK implemented
universal leucoreduction of donated blood in 1999. This
measure was based on the expectation that infectivity
would be associated with white blood cells.2 However,
findings in blood from infected mice and hamsters
suggested otherwise; at least 40% of the infectivity was
plasma-associated, suggesting that leucoreduction
would not eliminate infectivity (Rohwer laboratory,
unpublished).3 Other investigations showed no loss of
infectivity when small amounts of TSE-infected plasma
were passed through scaled-down filters.4 Similarly, no
significant removal of abnormal prion protein was
detected when units of human whole blood, spiked with
a microsomal fraction from TSE-infected brain, were
passed through leucoreduction filters from any of the
four major suppliers.5 Because of reservations about the
relevance of these experiments, none of these findings
aroused concern.

We investigated the effectiveness of leucoreduction in
removal of TSE infectivity from a human-sized unit of
pooled hamster blood. To ensure that the 150 hamsters
needed for a 450 mL blood pool were at the same
symptomatic stage of disease (wobbling gait and head
bobbing) for each of two separate experiments,
400 weanling golden Syrian hamsters (Harlan, Madison,
WI, USA) were inoculated intracranially with 50 L of
brain homogenate containing about 250 infectious
dose50 (ID50) of hamster-adapted scrapie-strain 263K. A
low dose of infectivity was used to preclude re-isolation
of the inoculum in the blood. This animal protocol was
approved by the University of Maryland Institutional
Animal Care and Use Committee.

We obtained two pools of blood from the hamsters,
one at 106 days and one at 111 days after inoculation.
Under carbon dioxide anaesthesia, 3·5 mL of blood was
drawn from the right ventricle into 0·5 mL of CP2D
anticoagulant. Care was taken not to touch any other
tissue. Only perfect bleeds containing 12·5% CP2D with
no visible clots were pooled.

Two in-line leucofiltration systems from Pall
Corporation (Port Washington, NY, USA) were
evaluated. We selected the Leukotrap WB collection set
for the infectivity study because filtration and
component separation of hamster blood was fully
compliant with American Association of Blood Banks
(AABB)6 specifications, and required only two titrations
for interpretation. The Leukotrap RC-PL system

Volume White blood cells Red blood cells, Platelets,
(mL)* total (% of total) total (% of total)
Total (% of total)Log 10reduction
Whole blood 448·5 2·1109 (100%)0 3·71012 (100%)1·4 1011 (100%)
Leucoreduced blood 424·2 3·0106(0·15%)2·9 3·61012 (100%)1·5 1011 (100%)
Plasma 179 3·0105 (0·02%)3·8 0 (0%) 1·11010 (8%)
Red blood cells + AS3 305·9 2·0106 (0·15%)3 3·11012(86%)1 1011 (71%)
*Volume measurements were obtained by weight using experimentally
determined densities of whole hamster blood,
1·04 g/mL. Values are average of at least three separate microscopic
determinations using a haemocytometer and by flow
cytometric measurements with white cells stained with propidium iodide.
AS3 is a preservative and stabiliser.
Table 1: Blood component cell numbers and volumes before and after
leucoreduction

For personal use. Only reproduce with permission from Elsevier Ltd
Research Letters

approached, but did not fully achieve all specifications;
furthermore, because more than one filter is involved,
more titrations would have been required to evaluate the
removal of infectivity.

For the infectivity study, 448·5 mL of CP2Danticoagulated
whole hamster blood was pooled into the
whole-blood receiving bag of a Leukotrap WB collection
set and processed within the 8-h time limit specified by
the AABB. Filtration was done at room temperature
under gravity with a 60-inch pressure head on the in-line
WBF2 filter, and was completed in 30 min. After
removal of a 19 mL sample of the leucoreduced whole
blood for subsequent testing, the remainder was
centrifuged at 4150 rpm (about 5000 g) for 8 min at
room temperature in a Sorvall RC-3C centrifuge. The
plasma fraction was expressed into a satellite in-line bag.
A preservative and stabiliser, AS3, was added to the red
blood cells. Samples of the pre-filtration whole blood,
post-filtration whole blood, red blood cells, and plasma
were removed for analysis of cell composition and for
titration in animals.

Cellular composition of the blood was assessed with a
HemaVet five-part differential cell counter calibrated for
hamster blood cells (Drew Scientific, Oxford, CT, USA).
The residual white blood cell concentrations in the
leucoreduced samples were measured by manual count
and flow cytometry.

Infectivity of whole and leucoreduced blood was
quantified by limiting dilution titration, a method
developed in the Rohwer laboratory. The two samples
were processed and inoculated separately and
sequentially. Each sample of blood was sonicated with a
separate sterile probe to lyse cells and disperse
infectivity. It was then immediately inoculated
intracranially, 50 µl at a time, into about 100 weanling
golden Syrian hamsters that were deeply anaesthetised
with pentobarbital. Animals were maintained for
566 days; those that contracted scrapie were killed when
the clinical diagnosis was conclusive, and animals still
alive at the end of the study were killed. All brains were
tested for the presence of the proteinase K-resistant
form of prion protein by western blot using 3F4
antibody.

The limiting dilution of an endpoint dilution titration
is that at which not all of the inoculated animals
become infected. At limiting dilution, the distribution
of infectivity into individual inoculations is described by
the Poisson distribution, where P(0)=probability of no
infections at that dilution and inoculation volume, or
(1probability of infection). From the Poisson
distribution P(0)=e(titre)
, and titre=ln(P[0]) expressed as
ID/(inoculation volume). SD of the limiting dilution
titre is the square root of the titre in ID/mL divided by
the total volume inoculated in mL.

Table 1 shows the distribution of cells in
each component of the scrapie-infected blood.
Leucofiltration reduced the number of white blood cells
by 2·9 log, thereby meeting the AABB standard. White
cell contamination of the red blood cell fraction and red
blood cell recovery were within AABB specifications of
less than 5106 and greater than 85%, respectively.
Hamster platelets are not removed by the WBF2 filter,
and partition with the red cells during centrifugation.

The incubation times of infections in each
measurement are shown in the figure. At limiting
dilution, incubation times begin at the end of the
predictable dose response seen in endpoint dilution
titrations (about 140 days) and rarely extend beyond
500 days. All clinical and western blot results were
consistent.

The limiting dilution titre of the whole blood pool
(table 2) was close to the values from titrations of
similar pools of whole blood by this method
(unpublished data). Leucofiltration of whole blood
removed only 42% (SD 12) of the initial TSE infectivity
(table 2); of the 5900 ID present in the original unit of
blood, 3400 ID were recovered in the leucofiltered
blood.

Ideally, leucoreduction would be validated by
measuring infectivity concentrations before and after
leucoreduction of full units of vCJD-infected human
blood. However, it is not currently possible to assay

530 www.thelancet.com Vol 364 August 7, 2004
Days after inoculation
0 100 200 300 400 500 600 700
Number of animals
Leucoreduced whole blood
Whole blood
Volume Total animals Total animals Titre in ID/mL Fractional distribution
inoculated (mL) inoculated infected (SD) of infectivity
Whole blood 5·2 104 50 13·1 (1·6)1
Leucoreduced blood 5·4 108 34 7·6 (1·2)0·58
Titre and SD calculated from the Poisson distribution as described in
the text.
Table 2: Concentration of TSE infectivity in whole and leucoreduced blood
Figure: Incubation times of infections from whole and leucoreduced blood
Results of inoculations of whole blood are represented by data above the
horizontal line; those from inoculations
of leucoreduced blood are shown below the line. Circles represent
infected animals. Squares represent uninfected
animals that survived to the end of the experiment. Triangles represent
animals that died intercurrently of causes
other than the inoculum.
For personal use. Only reproduce with permission from Elsevier Ltd
Research Letters

either infectivity or the infection-specific form of the
prion protein in human blood. By contrast, limiting
dilution titration of rodent blood can detect less than
1 ID/mL of TSE infectivity and can readily show a
difference of less 20% between samples. With this
technique we did a study that: avoided the issue of
spikes by using endogenously infected blood; avoided
the question of scale by using a human-sized unit of
fresh hamster blood obtained within the time limits
specified for human blood; minimised the possibility of
artefact by using a commercial blood collection set with
integral filtration unit and a blood centre centrifuge and
expressor; and achieved precision in the infectivity
measurements by limiting dilution inoculation of 5 mL
of each fraction. We assessed the performance of the
filter by measuring the level of white blood cell
reduction obtained and the cell recoveries of each
component. The leucoreduction met or exceeded AABB
specifications for all relevant variables.

Leucoreduction removed only 42% of the initial TSE
infectivity from whole blood. This distribution is
consistent with that obtained in a centrifugal separation
of TSE-infected hamster whole blood, in which the
buffy coat contained 70% of the total white cells but
only 45% of the total whole blood infectivity
(unpublished data). Both methods showed that a
substantial proportion of the TSE infectivity was not
associated with white cells. We have shown previously7
that TSE infectivity is not associated with highly
purified platelets, and we are currently testing purified
red blood cells. We presume that the majority of bloodborne
infectivity is plasma-associated.

Although leucoreduction is a necessary step for
removing white-cell-associated TSE infectivity from
blood, this process is insufficient to remove the risk
from an infected transfusion unit. Due to the low
concentration of TSE infectivity in blood and the
absence of screening or inactivation alternatives,
removal is an attractive strategy. However, the
feasibility of removal depends upon the actual
associations and distributions of TSE infectivity in
blood itself, which can only be ascertained by
assessment of endogenous blood-borne infectivity.

www.thelancet.com Vol 364 August 7, 2004 531

Contributors
The overall design and execution of the experiment, including
management of the logistics and all the infectivity work, was by
L Gregori and R G Rohwer with the assistance of the staff of the
Molecular Neurovirology Laboratory. A Giulivi, N McCombie,
D Palmer, and P Birch supplied expertise on blood centre operations,
blood collection, component separation, leucoreduction, and
quantitation of white blood cells. D Palmer and P Birch undertook and
interpreted flow cytometry. S Coker supplied expertise on the use of the
collection set and leucofilter.
Conflict of interest statement
R G Rohwer is a cofounder and part owner of Pathogen Removal and
Diagnostics Technologies, which is developing technologies for the
removal of TSE infectivity from blood and other materials. L Gregori
receives contract support from Pathogen Removal and Diagnostics
Technologies for studies on TSE removal. S Coker is an employee of
Pall Corporation, which produces leucofilters and is developing TSE
removal strategies for blood. The remaining authors declare that they
have no competing financial interests.
Acknowledgments
We thank the staff of the BSL-3 animal facility at the VA Medical
Center, Baltimore for their excellent animal care. This study was funded
by Health Canada and by the US National Heart, Lung and Blood
Institute Award # HL-63930. Health Canada participated in the study
design, assisted with leucofiltration, and facilitated flow cytometry
analysis. They had no role in the infectivity measurements, their
analysis, or interpretation. Health Canada reviewed and approved the
final submission without changes. The National Heart Lung and Blood
Institute participated only as a source of funding. Pall Corporation
supplied a blood centrifuge, plasma expressor, and tube sealer, and
served as consultants on the use of their collection sets and filters.
References
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3 Brown P, Rohwer RG, Dunston BC, MacAuley C, Gajdusek DC,
Drohan WN. The distribution of infectivity in blood components
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Articles

http://www.thelancet.com Vol 364 August 7, 2004

TSS





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