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correspondence
24-h continuous infusion of platelets for patients with platelet
transfusion refractoriness
Platelet refractoriness (PR) is defined as an inappropriately
low platelet count increment following repeated platelet
transfusions because of immune or non-immune causes. The
transfusion of human leucocyte antigen (HLA) or human
platelet antigen (HPA) compatible platelets is the cornerstone
for patients who are refractory to platelet transfusions
because of the development of alloantibodies against class I
HLA or HPA expressed on platelets (Vassallo, 2013). When
patients with PR have no HLA/HPA-compatible platelet
donors or in the setting of ongoing bleeding, some institu-
tions and published guidance (Hod & Schwartz, 2008) have
suggested employing platelet “drips”. This suggestion is based
on just two original reports of an approach that comprised a
massive prophylactic platelet transfusion (Nagasawa et al,
1978) or slow infusion of platelets (Narvios et al, 2005) in
two and three patients, respectively.
The objective of our study was to review retrospectively our
experience using 24-h continuous infusion of platelets as a pla-
telet transfusion alternative for patients who became refractory
to platelet transfusions. PR was considered when 1-h platelet
count increment (CI) was below 7500 after receiving two con-
secutive, ABO-compatible, <3-day stored platelet concentrates
(PC). The 24-h continuous infusion of platelets was initiated
when HLA- or HPA-compatible platelet donors were not avail-
able or the patient was actively bleeding. Three PCs (ABO-
compatible, stored <3 days) were transfused continuously over
24 h. In order to comply with the standard of expiration of an
open system, i.e., 4 h (European Committee on Blood Trans-
fusion, 2010), one PC was split in to two identical bags using a
sterile connection device. Each split was transfused to the
patient as a 4-h continuous infusion.
Table I shows the clinical and laboratory characteristics of
patients included in this study. Table II shows the character-
istics of the 19 periods of thrombocytopenia for which the
patients received 24-h continuous infusion of platelets. When
starting the 24-continuous infusion of platelets, bleeding
(World Health Organization [WHO] grade ≥2) was present
in 6 (50%) out of 12 periods of 24-h continuous infusion of
platelets in patients with immune PR, and in 3 (43%) out of
7 time periods of 24-h continuous infusion of platelets in
patients with non-immune PR. Haemorrhage disappeared in
all cases during the 24-h continuous infusion. No transfusion
reactions were reported during the 24-h continuous infusion
of platelets.
In the setting of ongoing bleeding or when HLA/HPA-
compatible platelets are not available, only two previous
studies reported that a massive prophylactic platelet transfu-
sion strategy (Nagasawa et al, 1978) or slow infusion of pla-
telets (Narvios et al, 2005) could be a possible platelet
transfusion alternative to manage these patients. However,
Nagasawa et al (1978) reported two patients who received a
massive infusion of platelets, and Narvios et al (2005) slowly
infused (over 6 h) single-donor platelets and random-donor
platelets to three patients. After reviewing the previous two
reports, it is clear that a limited experience in supporting
patients with PR and no available HLA/HPA-compatible pla-
telets has been published. The present study showed that 24-
h continuous infusion of platelets could be a feasible and safe
alternative. Our approach was feasible because we split PCs
in to two units so that each half was infused over 4 h to
comply with the standard of expiration of an open system. It
was also a safe approach because we did not observe any
transfusion reactions, although the number of patients stud-
ied as well as the number of platelet transfusions was low.
Our platelet transfusion approach was efficacious because
severe bleeding (WHO grade 2–4) was present in 9 (47%) out
of 19 transfusion episodes and haemorrhage disappeared in all
cases while receiving 24-h continuous infusion of platelets. The
haemostatic effect after transfusing HLA-incompatible platelet
transfusions that we observed in the present study is supported
by in vitro data reported by our group in the past (Mazzara
et al, 1996). In that study, we reported an increase of deposi-
tion of fibrin on the subendothelial surface after transfusing
HLA-incompatible platelets measured using ex vivo experi-
ments with Baumgarten’s platelet adhesion model.
In our series, 4 patients with PR received 24-h continuous
infusion of platelets as a preventive measure. Our hypothesis
was that our continuous platelet transfusion approach might be
successful in preventing haemorrhage because it was capable of
maintaining the endothelium integrity, as previously reported
by Hanson and Slichter (1985), who estimated that approxi-
mately 7
1 9 109/l platelets per day are required to maintain
the endothelium integrity. An adult patient with a total blood
volume ranging from 5 to 7 l will need a total of 0
36 9 1011 to
0
50 9 1011 platelets per day (Nachman & Rafii, 2008). Accord-
ing to data published by our blood supplier (Blood and Tissue
Bank, 2016), we were transfusing daily about 9 9 1011 platelets,
which was higher than the minimum quantity required for
maintaining the endothelium integrity due to the immune
destruction or consumption occurring in those patients.
Our study has some limitations. First, it was a retrospec-
tive study with a limited number of included patients and
ª 2017 John Wiley & Sons Ltd
British Journal of Haematology, 2018, 181, 386–417
 ª 2017 John Wiley & Sons Ltd
Table I. Clinical and laboratory characteristics of patients receiving 24-h continuous platelet transfusion.

Antibody testing
Age ABO & Rh(D) Transfusion Previous
Patient Gender (years) Diagnosis group history pregnancies Non-immune causes PRA ELISA Luminex HPA PR

1* F 48 AML A Pos Yes Yes No 93 50 Pos Pos Immune

2 M 38 NHL O Neg No NA Fever, amphotericin B ND Neg ND Neg Non immune

British Journal of Haematology, 2018, 181, 386–417

3 F 54 ALL A Pos Yes Yes No 9 ND ND ND Non immune
4 M 50 NHL O Pos No NA Fever 81 Neg ND Neg Non immune
5 F 65 ALL A Neg Yes Yes Fever, active infection 34 ND Pos Neg Immune
6* F 69 AML A Pos Yes Yes Fever 43 44 Pos Neg Immune
7* F 59 AML A Neg Yes Yes Fever, active infection, 95 76 Pos Neg Immune
haemorrhage
8* M 61 AML A Neg Yes NA Fever 3 Neg ND Neg Non immune
9* F 63 AML A Pos Yes Yes Fever, active infection 51 80 Pos Neg Immune
10 M 36 NHL A Pos Yes NA Fever, active infection, Neg Neg ND Neg Non immune
amphotericin B
11 F 68 AML A Pos Yes Yes No 40 17 Pos Neg Immune
12 F 63 AML A Neg Yes Yes No 70 89 Pos Neg Immune
13 F 44 AML A Pos No Yes No 90 Neg ND ND Non immune
14 F 51 AML O Pos Yes Yes No 87 100 Pos Pos Immune

ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; ELISA, Enzyme-linked immunosorbent assay; F, female; HPA, Human platelet antigen antibodies; M, male; NA, Not applicable;
ND, Not done; Neg, negative; NHL, non-Hodgkin lymphoma; Pos, positive; PR, Platelet refractoriness; PRA, panel-reactive antibody.
*These patients received 24-h continuous infusion of platelets at two different periods of time.
Correspondence

387
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Correspondence

Table II. Characteristics of 24-h continuous infusion of platelets.

Platelet refractoriness

Characteristic Immune Non immune P All

Patients, n 8 6 NA 14
Periods of 24-h infusion of platelets, n 12 7 NA 19
Duration of thrombocytopenia, days 21 (10–27) 21 (5–60) 0
6* 21 (5–60)
Median platelet count at morning before 24-h platelet infusion, 9109/l 8 (4–17
5) 17 (13–19) 0
035* 13 (4–19)
Duration of 24-h infusion of platelets, days 6
5 (1–18) 5 (3–8) 0
1* 6 (1–18)
Platelet concentrates transfused during 24-h platelet infusion, n 14 (3–54) 11 (6–21) 0
3* 14 (3–54)
Median platelet count at morning during 24-h platelet infusion, 9109/l 11 (3–39) 20
5 (10–35) 0
2* 12 (3–39)
Bleeding (WHO grade)
None 2 2 1
0† 4
Grade 1 4 2 6
Grade 2 2 2 4
Grade 3 2 0 2
Grade 4 2 1 3

Values are median (range).

NA, Not applicable; WHO, World Health Organization.
*t-student test.
†Chi-squared test.

the assessment of bleeding events was not as accurate and

Author contributions
purposeful as would have been in a prospective study. Sec-
ond, we could not analyse the potential increase in the JC designed the study, collected and analysed data, wrote the
alloimmunization rate in patients with PR after receiving manuscript, and approved the final version. FG and GC col-
pooled PCs, as some authors argue (Stanworth et al, 2015). lected and analysed data. ML revised the manuscript and
However, to our knowledge, we here reported the largest ser- approved the final version.
ies of patients with PR that received 24-h continuous infu-
sion of platelets as a transfusion intervention aimed to
Disclosure of conflicts of interest
prevent or treat haemorrhagic episodes. We encourage other
authors to report their experience with this approach or The authors declare no conflicts of interest.
other similar platelet transfusion strategies to overcome the
Joan Cid
serious therapeutic challenge when patients become refrac-
Francisca Guijarro
tory to platelet transfusions.
Gloria Carbass
e
In conclusion, a 24-h continuous infusion of platelets to
Miguel Lozano
patients with PR might be a feasible and safe platelet transfu-
Apheresis Unit, Department of Haemotherapy and Haemostasis,
sion strategy when HLA/HPA-compatible platelets are not
IDIBAPS, Hospital Cl
ınic, University de Barcelona, Barcelona, Spain.
available, as well as efficacious in treating and preventing
E-mail: jcid@clinic.ub.es
haemorrhage in patients with haematological diseases and
chemotherapy-induced thrombocytopenia.
Keywords: platelet refractoriness, platelet transfusion, haemorrhage

Acknowledgements First published online 14 March 2017

doi: 10.1111/bjh.14572
We acknowledge all the patients and nurses that participated
in the study.

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sion of fresh, stored, or lyophilized platelets.
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(2005) Slow infusion of platelets: a possible
alternative in the management of refractory
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Marsh, J. (2015) Platelet refractoriness–practical
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Assessment of the T cell receptor repertoire in long-term

platelet donors by next generation sequencing

Platelet transfusions are a key element of supportive care for
patients with haematological malignancies. The safety of vol-
unteer donors is of high priority. However, it has been
reported that platelet donors have decreased lymphocyte
counts correlating with the number of donations (Richa
et al, 2008). While there is no evidence for any clinical sig-
nificance or immune dysfunction in long-term platelet
donors and a competent immune system is assumed (Richa
et al, 2008; Strauss, 2008), no estimation of the immune
repertoire has been performed so far.
Functional T cells that recognize foreign antigens via their
T cell receptors (TCRs) are important for an effective
immune response. The TCR repertoire changes in response
to immunological challenge. Naive T cells have a more
diverse TCR repertoire than memory T cells (Arstila et al,
1999). In this study, next generation sequencing (NGS) of
the TCR alpha chain (TRA) was performed on CD8+ T cells
to assess the TCR repertoire of long-term platelet donors in
order to determine whether T cell diversity is compromised
in these individuals.
Five male long-term platelet donors (age 20–40 years)
with a history of at least 30 platelet donations and regular
donation activity during the last 12 months were recruited
by the Department of Transfusion Medicine at the Univer-
sity Hospital of the TU Dresden. Five non-donor samples
were taken from healthy male non-blood-donors aged
between 20 and 40 years (Ethical Review Board approval
EK-279072013). Only male donors were used to avoid sex-
specific differences as described previously (Richa et al,
2008).
For immunophenotyping and sorting, T cell subsets were
characterized by flow cytometry using the markers CCR7,
CD45RA, CD27 and CD28 as previously published (Link
et al, 2016), (Data S1). RNA isolation, cDNA synthesis and
library preparation as well as NGS, with minor changes to
protocol, were performed as previously described (Link et al,
2016), (Data S1). TCR repertoire diversity was estimated
using Simpson’s diversity index (Ds) (Venturi et al, 2007)
where Ds ranges from 0 (low diversity) to 1 (high diversity).
The Ds was also expressed as the inverse of the
complementary probability Dsinv = 1/(1 Ds). The Wilcoxon
signed rank test was used to compare Ds.
Four of the five platelet donors were lymphopenic, as
defined by a peripheral blood lymphocyte count <1
5 9 109/l.
None of the non-donors were lymphopenic. The relative
frequencies of CD8+ and CD4+ T cells was not different
between the two groups and no differences between the
differentiation stages for both CD8+ and CD4+ T cells were
apparent.
For each sample, the number of TRA reads and number
of TRA clonotypes were determined per 500 000 CD8+ naive
and CD8+ memory T cells analysed. The naive repertoire
contained a large number of very low frequent clonotypes. In
contrast, the memory repertoire was composed of fewer but
more frequent clonotypes (Table I, Fig 1).
Mean Ds for TRA diversity was calculated as 0
999961 for
the naive repertoire and 0
974946 for the memory repertoire.
Using the Dsinv, this results in a mean Dsinv of 27 361 and 69
for the naive and memory compartment, respectively, indi-
cating a 665-fold (range 144–1680) higher diversity for the
naive TRA repertoire (P = 0
002, Table I). Platelet donors
and non-donors had similar numbers of frequent clonotypes
in their memory compartment and a comparable high num-
ber of low frequent clonotypes within the naive repertoire
(Fig 1, Fig S1). In addition, no significant differences in
diversity indices were observed between platelet donors and
non-donors for the naive TRA repertoire (mean Ds, 0
999965
vs. 0
999957; mean Dsinv, 29 140 vs. 25 581) and the memory
repertoire (mean Ds, 0
983024 vs. 0
966867; mean Dsinv, 77
vs. 62).
A large proportion of the reads within the memory CD8+
T cells were from clonotypes that were also present in the
naive CD8+ T cells (overlapping clonotypes) with no signifi-
cant differences between platelet donors and non-donors
(mean 72
17% vs. 78
10%). The majority of reads within the
naive CD8+ T cells were from clonotypes that were only pre-
sent in the naive compartment. Nevertheless, a higher pro-
portion of overlapping clonotypes in the naive repertoire was
seen for the platelet donors compared to the non-donors
(13
34% vs. 9
36%, P = 0
006).

ª 2017 John Wiley & Sons Ltd 389

British Journal of Haematology, 2018, 181, 386–417