New Appro

NEW APPROACHES TO IMPROVE THE CORD BLOOD UNIT
HEMATOPOIETIC PROGENITOR CELL CONTENT
Sari Juutistenaho
Finnish Red Cross Blood Service

Helsinki, Finland
Department of Obstetrics and Gynecology

Helsinki University Central Hospital
University of Helsinki
Helsinki, Finland
ACADEMIC DISSERTATION
To be publicly discussed, with the permission of the Faculty of Medicine,

University of Helsinki, in the Nevanlinna Auditorium of the Finnish Red Cross
Blood Service, Kivihaantie 7, Helsinki, on November 12th, 2010, at 12 noon.
Helsinki 2010
ACADEMIC DISSERTATIONS FROM
THE FINNISH RED CROSS BLOOD SERVICE
NUMBER 55
SUPERVISOR
Riitta Kekomki, M.D., Ph.D.

Docent
Finnish Red Cross Blood Service
Helsinki, Finland
REVIEWERS
Pekka Riikonen, M.D., Ph.D.

Docent
Department of Pediatrics
Kuopio University Hospital
Kuopio, Finland
Mervi Taskinen, M.D., Ph.D.

Docent
Hospital for Children and Adolescents
Helsinki, Finland
OPPONENT
Freja Ebeling, M.D., Ph.D.

Docent
Department of Medicine
Helsinki, Finland
ISBN 978-952-5457-23-0 (print)

ISBN 978-952-5457-24-7 (pdf)
ISSN 1236-0341
http://ethesis.helsinki.fi
Helsinki 2010
Yliopistopaino
Things should be made as simple as possible,
but not any simpler.
Albert Einstein
1 ABSTRACT
Recent efforts have been focused on finding ways to increase the hematopoietic
progenitor cell (HPC) content of cord blood (CB) units. The aim of this study was
to identify factors that may improve the selection and quality of CB collections for
banking and transplantation.
In 167 CB units collected and processed at a national CB bank, mean platelet

volume (MPV) correlated with CB unit CD34+ cells and colony-forming units
(CFUs); this is a novel finding. MPV can be thought to represent general
hematopoietic activity, as newly formed platelets have been reported to be large.
Stress during delivery is hypothesized to lead to mobilization of HPCs through

cytokine stimulation. Accordingly, low-normal umbilical arterial pH,
hypothesized to be associated with perinatal stress, correlated with high CB unit
CD34+ cell and total CFU (CFU-TOT) numbers. The associations were closer in
vaginal deliveries than in Cesarean sections. Vaginal delivery entails specific
physiological changes, which may also affect the hematopoietic system. Thus,
different factors may predict CB HPCs in the two modes of delivery. In addition,
placental weight correlated with CB unit HPCs; hematopoietic activity has
previously been observed in the placenta.
Theoretical models were created to enable the use of platelet characteristics

(MPV) and perinatal factors (umbilical arterial pH and placental weight) in the
selection of CB units with high HPC counts. These observations could thus be
implemented as a part of the evaluation of CB collections for banking.
Hemostasis activation during CB collection was assessed with prothrombin

activation fragment 1+2 (F1+2), a direct indicator of thrombin generation, and
platelet factor 4 (PF4), indicating platelet activation. Altogether three sample
series were collected during the set-up of the CB bank as well as after changes in
personnel and collection equipment. The activation decreased from the first to
the subsequent series, which were collected with the bank fully in operation and
following international standards. As hemostasis activation may have unwanted
effects on CB cell contents, it should be monitored and minimized.
Culture assays provide information about the hematopoietic potential of the CB

unit. However, these assays are often labor-intensive. In this study, in processed
CB units prior to freezing, megakaryocytic colony growth was evaluated in
semisolid cultures with a novel, simple scoring system. Three investigators
analyzed the colony assays, and the scores were highly concordant. In addition,
4
ABSTRACT
erythroid cells were observed in liquid cultures of cryostored and thawed,

unseparated CB units without exogenous erythropoietin (EPO). This was
hypothesized to be due to the erythropoietic effects of thrombopoietin (TPO),
endogenous EPO production, and diverse cell-cell interactions in the culture. This
observation underscores the complex interactions of cytokines and supporting
cells in the heterogeneous cell population of the thawed CB unit.
5
TABLE OF CONTENTS
1 Abstract .......................................................................................... 4
2 Abbreviations ................................................................................... 8
3 List of original publications .................................................................. 9
4 Introduction ................................................................................... 10
5 Review of the literature ..................................................................... 11

5.1 Hematopoiesis ........................................................................ 11
5.1.1 Theories of hematopoiesis ................................................ 11
5.1.2 Hematopoietic progenitors ............................................... 12
5.1.3 The human placenta ........................................................ 13
5.1.4 Hematopoiesis in the fetus ................................................ 14
5.2 Normal hemostasis in the neonate .............................................. 15
5.2.1 Primary hemostasis ......................................................... 15
5.2.2 Secondary hemostasis ..................................................... 16
5.2.3 Markers of hemostasis activation ....................................... 18
5.3 Methods for analyzing cord blood cells ......................................... 20
5.3.1 Cell counting .................................................................. 20
5.3.2 Hematopoietic cytokines in cell cultures .............................. 21
5.3.3 Semisolid progenitor cell cultures ....................................... 22
5.3.4 Liquid cultures ................................................................ 23
5.3.5 Uncertainties of the analyses ............................................. 23
5.4 Cord blood as a hematopoietic stem cell source ............................. 24
5.4.1 Hematopoietic progenitors in cord blood .............................. 24
5.4.2 Cord blood transplantation ................................................ 26
5.5 Cord blood collection and banking ............................................... 31
5.5.1 Obstetric and perinatal factors ........................................... 32
5.5.2 Collection methods .......................................................... 36
5.5.3 Cord blood processing ...................................................... 39
5.5.4 Cryopreservation ............................................................ 40
5.5.5 International standards, accreditation, and registries ............ 41
6 Aims of the study ............................................................................. 44
7 Materials and methods ..................................................................... 45

7.1 Materials of the original studies .................................................. 45
7.2 Cord blood banking .................................................................. 45
6
TABLE OF CONTENTS
7.2.1 Donor recruitment ........................................................... 45

7.2.2 Cord blood collection ........................................................ 46
7.2.3 Cord blood processing and cryopreservation ......................... 48
7.3 Laboratory assays ................................................................... 48
7.3.1 Cell counting with an automated analyzer............................. 48
7.3.2 Semisolid progenitor cell assays ......................................... 49
7.3.3 Liquid cultures of thawed cord blood units ............................ 50
7.3.4 Flow cytometry ............................................................... 51
7.3.5 Cytocentrifugation .......................................................... 51
7.3.6 ELISA assays for F1+2 and PF4 .......................................... 52
7.4 Ethical considerations .............................................................. 53
7.5 Statistics ............................................................................... 53
8 Results .......................................................................................... 54
8.1 Cord blood progenitor cell content (I - II) ..................................... 54
8.2 Platelet characteristics (I) ......................................................... 55
8.3 Perinatal characteristics (II) ...................................................... 56
8.4 Models for the estimation of hematopoietic progenitor cells (I - II) .... 57
8.5 Hemostasis activation in cord blood collection (III) ........................ 58
8.6 The hematopoietic potential of cord blood units in vitro ................... 61
8.6.1 Megakaryocytic cells in a semisolid assay (I) ......................... 61
8.6.2 Erythroid cells in liquid cultures of thawed cord blood (IV) ....... 61
9 Discussion ...................................................................................... 64
9.1 The cell content of cord blood units .............................................. 64
9.2 Platelet characteristics as determinants of hematopoietic progenitors 65
9.3 Perinatal stress factors for the selection of cord blood collections ...... 66
9.4 Hemostasis activation during cord blood collection ......................... 67
9.5 Culture assays of cord blood units ............................................... 69
9.6 Methodological aspects of the study ............................................ 70
9.7 The future of cord blood banking ................................................. 71
10 Conclusions .................................................................................. 72
11 Acknowledgements ........................................................................ 74
12 References ................................................................................... 76
7
2 ABBREVIATIONS
BFU-E Burst-forming unit - erythroid

BM Bone marrow
CB Cord blood
CD Cluster of differentiation
CFU Colony-forming unit
CFU-GM Colony-forming unit - granulocyte / macrophage
CFU-Mixed Colony-forming unit - granulocyte / erythrocyte / macrophage /
megakaryocyte; equivalent to CFU-GEMM
CFU-TOT Total colony-forming units; sum of CFU-GM, BFU-E, and CFU-
Mixed
ELISA Enzyme-linked immunosorbent assay
EPO Erythropoietin
F1+2 Prothrombin activation fragment 1 + 2
FACT Foundation for the Accreditation of Cellular Therapy
GlyA Glycophorin A (CD235a)
GVHD Graft-versus-host disease
HLA Human leukocyte antigen
HPC Hematopoietic progenitor cell
IL Interleukin
IMDM Iscoves modified Dulbeccos medium
ISHAGE International Society of Hematotherapy and Graft Engineering
MPV Mean platelet volume
NC Nucleated cell (concentration)
PF4 Platelet factor 4
RBC Red blood cell
SCF Stem cell factor
SD Standard deviation
TNC Total nucleated cell (count)
TPO Thrombopoietin
WBC White blood cell
8
3 LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications, which are referred to in
the text by their Roman numerals (I - IV). The articles are reproduced with the
permission of the copyright holders.
I Eskola M*, Rekunen S*, Aroviita P, Mttnen S, Hiilesmaa V, Sainio S &

Kekomki R. Association of cord blood platelet characteristics and hematopoietic
progenitor cells. Transfusion 48:884-92, 2008.
II Juutistenaho S, Eskola M, Sainio S, Aranko K & Kekomki R. Association of

stress-related perinatal factors and cord blood unit hematopoietic progenitors is
dependent on delivery mode. Transfusion 50:663-71, 2010.
III Juutistenaho S, Vahtera E, Aranko K & Kekomki R. Prothrombin activation

fragment 1 + 2 as a marker of coagulation activation in cord blood collection for
banking. Transfusion Medicine 20:250-7, 2010.
IV Juutistenaho S*, Mttnen S*, Eskola M, Aranko K & Kekomki R. Erythroid

cells from thawed unseparated cord blood in vitro without exogenous
erythropoietin. Submitted.
*These authors contributed equally to the study.
In addition, some unpublished data, in particular on multiple regression models

for the estimation of CB unit HPCs, are presented.
9
4 INTRODUCTION
The first successful bone marrow (BM) transplantations to reconstitute the

hematopoietic system were performed in the 1960s (Gatti RA et al., 1968;
Thomas ED, 1999). BM transplantation became an established procedure in the
following decades (Thomas E et al., 1975). In addition to BM, HPCs can be
collected from peripheral blood after growth factor stimulation (Russell N et al.,
1996). To date, over 14 million potential BM donors have been listed in
international registries (www.bmdw.org; September 2010).
HPCs are also present in CB (Knudtzon S, 1974; Nakahata T & Ogawa M, 1982;
Broxmeyer HE et al., 1989). In 1972, transient hematopoietic engraftment was
reported following multiple CB transfusions for one patient with acute
lymphoblastic leukemia (Ende M & Ende N, 1972). The first sibling-donor CB
transplantation with successful long-term hematopoietic reconstitution was
reported in a boy with Fanconi anemia in 1989 (Gluckman E et al., 1989). In the
1990s, the feasibility of CB hematopoietic stem cell transplantation from both
related (Wagner JE et al., 1995; Rocha V et al., 1998) and unrelated (Kurtzberg J
et al., 1996; Wagner JE et al., 1996) donors was confirmed. Brunstein and
colleagues have reviewed the early experiences in unrelated CB transplantation
(Brunstein CG et al., 2007a). To this day, over 14 000 unrelated-donor CB
transplants have been performed (Kurtzberg J, 2009).
CB banks have been established to enable unrelated CB transplantation

(Rubinstein P et al., 1994; Harris DT, 1996; Lazzari L et al., 1996; Armitage S et
al., 1999a; Donaldson C et al., 2000; Aroviita P et al., 2003). Attempts have been
made to improve the quality of CB units through e.g. standardization of bank
procedures (Gluckman E et al., 1998; Rebulla P et al., 1999), of stem cell
enumeration (Dzik W et al., 1999; Gratama JW et al., 2003; Moroff G et al.,
2006) and of the assessment of HPC viability (Brand A et al., 2008). Over 400
000 CB units are currently available in international registries (www.bmdw.org;
September 2010). However, a significant proportion of patients is still left
without a suitable donor, necessitating further development of CB banking
processes.
10
5 REVIEW OF THE LITERATURE
5.1 Hematopoiesis
5.1.1 Theories of hematopoiesis
Several models of hematopoietic stem cell renewal and differentiation have been
proposed. In the traditional, hierarchical model of hematopoiesis, pluripotent
hematopoietic stem cells have the capacity to self-replicate to secure long-term
repopulating capacity, and to differentiate along a lineage-specific path
(Migliaccio G et al., 1986). According to the model, the differentiation proceeds
from stem cells into primitive HPCs, further into lineage-committed HPCs and
finally into mature blood cells (Ogawa M et al., 1983).
Whether the differentiation of stem cells to more committed progenitors is a

stochastic or deterministic process has been debated. In stochastic models, a
hematopoietic stem cell commits to one of two pathways, either self-renewal to
sustain the progenitor pool or differentiation into a mature blood cell, in a
random fashion (Till JE et al., 1964; Nakahata T et al., 1982). Deterministic
models propose that any one cell at any stage of development has only one,
predetermined pathway of progeny and differentiation (Nicola NA & Johnson GR,
1982; Novak JP & Stewart CC, 1991).
The hierarchical model of hematopoiesis has been challenged with modern

interpretations of the stem cell. In these models, the stem cell may differentiate
along various paths, exceeding the classical lineage boundaries. In the so-called
chiaroscuro stem cell model, a fluctuating continuum exists between the
primitive stem cells and more differentiated progenitors (Quesenberry PJ et al.,
2002). In the phase space model of hematopoiesis, the differentiation of the cell
into more mature progeny or its de-differentiation into a primitive cell type is
dependent on the growth factors, growth inhibitors, and cell-cell interactions of
its environment (Kirkland MA, 2004). These modern theories allow for stem cell
plasticity, the totipotency of stem cells and the potential use of CB HPCs for a
wide range of indications not restricted to hematological conditions.
11
REVIEW OF THE LITERATURE
5.1.2 Hematopoietic progenitors
In CB banking, HPCs are recognized based on their surface markers (cluster of

differentiation, or CD, antigens) and their properties in culture. As no single
antigen unequivocally defines the hematopoietic stem cell, all these markers are
indirect and permit the isolation of a more or less heterogeneous cell population.
In addition to HPCs, the CD34 antigen has been described on embryonic
fibroblasts and the endothelial cells of small vessels (Krause DS et al., 1996).
CD34 is a cell surface glycoprotein with possible roles in stem cell homing and the
maintenance of an immature cell phenotype (Gangenahalli GU et al., 2006). The
adult BM CD34+ cell population has been shown to give rise to all the different
hematopoietic CFUs seen in semisolid cultures (Sutherland DR & Keating A,
1992) and to provide long-term hematopoietic reconstitution in humans after
myeloablative therapy (Krause DS et al., 1996).
CD34+ cells constitute a heterogeneous population, and more specific markers

for HPCs have been sought. Distinct CD34+ cell populations have been identified
in flow cytometric analyses. Immature HPCs are thought to be enriched in the cell
population expressing high levels of CD34 (Sutherland HJ et al., 1989). Within
the CD34+ cell population, CD38- cells have been suggested to be more primitive
progenitors than CD38+ cells (Terstappen LW et al., 1991). The CD34+CD38-
cell population may be responsible for long-term engraftment after
hematopoietic stem cell transplantation, while the CD34+CD38+ cell population
is capable of short-term engraftment (Hogan CJ et al., 2002). Glycoprotein
CD133 has been reported to be expressed on primitive CD34+ stem cells (Yin AH
et al., 1997). HPCs have also been distinguished by their high expression of the
aldehyde dehydrogenase enzyme (Storms RW et al., 1999; Gentry T et al.,
2007). Despite these and several other studies on the immunophenotype of stem
and progenitor cells, CD34 is still commonly used as an HPC marker in
hematopoietic stem cell grafts.
In 1974, Knudtzon reported the growth of granulocytic progenitor cell colonies in

CB semisolid cultures (Knudtzon S, 1974). The presence of both primitive and
more committed HPCs in CB has since been confirmed (Nakahata T & Ogawa M,
1982; Broxmeyer HE et al., 1989; Broxmeyer HE et al., 1992). In semisolid
cultures, multipotent hematopoietic progenitors are recognized as CFU-Mixed
(colony forming unit - granulocyte / erythrocyte / macrophage / megakaryocyte;
also called CFU-GEMM). Committed progenitors differentiating along various cell
lineages include the granulocyte-macrophage CFUs (CFU-GM) and erythroid
burst-forming units (BFU-E). The CFU-TOT number of CB refers to its entire CFU
content, and it is assessed in CB banks to evaluate the hematopoietic potential
and viability of CB units. However, despite ongoing efforts, neither the CFU
12
assays nor the immunophenotyping of CB HPCs have been standardized.
CB is also reported to contain primitive HPCs with high replating efficiency

(Nakahata T & Ogawa M, 1982). These primitive cells can be recognized in long-
term cultures, and include the high proliferative potential - colony-forming cells
(Lu L et al., 1993) and the long-term culture-initiating cells (Pettengell R et al.,
1994). These cell types are thought to represent the potential of CB for long-term
hematopoietic reconstitution. However, as the results are only obtained after
several weeks, they are not suitable for routine analyses in CB banks.
Figure 1. The fetomaternal interface of the placenta.
5.1.3 The human placenta
The placenta has recently been suggested to serve as a hematopoietic organ

during fetal development (Mikkola HK et al., 2005). The placenta plays a vital role
in fetal nutrition and has been described to provide hormones and cytokines that
affect both the mother and the fetus (Pasca AM & Penn AA, 2010). The placenta
develops through the gestation, regulated by physical and chemical signals, to
accommodate the increasing needs of the fetus (Charnock-Jones DS & Burton GJ,
2000).
13
The fetomaternal interface of the placenta consists of the trophoblast and stroma
with fetal capillaries (the fetal chorion) penetrating the decidua (maternal
structures; Figure 1). Maternal blood is described to be in direct contact with
fetal structures (Cross JC et al., 2003). With increasing gestational age, the
trophoblast layer and the endothelial cells in fetal capillaries thin to allow for
more efficient diffusional exchange of gases (Charnock-Jones DS & Burton GJ,
2000). Normally, two umbilical arteries and one umbilical vein in the umbilical
cord connect the fetus to the placenta (Benirschke K, 1998).
In a mixed population with 81% African Americans, the mean weight of the
normal full-term placenta has been reported to be 678 grams, with a standard
deviation (SD) of 144 grams (Dombrowski MP et al., 1994). Thus, the theoretical
reference range ( 2 SD) of placental weight would be 390 - 966 grams. The
weight of the placenta depends, e.g., on the weight of the neonate and on ethnic
background. Thus, the reference ranges differ slightly in different populations.
The blood volume in the fetoplacental circulation has been reported to be

approximately 110 mL/kg fetal weight (Wardrop CA & Holland BM, 1995). At
term, a mean 67% of the total blood volume is in the fetal circulation, and the
transfusion of placental blood to the fetus continues for approximately three
minutes after delivery unless the umbilical cord is clamped (Yao AC et al., 1969).
The estimated blood volume of the full-term newborn infant is 82 - 86 mL/kg
(Brugnara C, 2009). In addition to cord clamping, the extent of placental-to-fetal
transfusion depends on the positioning of the infant relative to the placenta.
Naturally, the same things - that is, the management of the infant after birth and
the cord clamping practices of the maternity unit - affect the blood volume
available for collection in the placenta and umbilical cord.
5.1.4 Hematopoiesis in the fetus
The circulation is essential for the sustained development of the fetus; thus, the
stages of fetal hematopoiesis parallel that of general fetal growth. In humans,
erythroblasts and CD34+ cells have been observed in the extraembryonic yolk
sac during the third week of gestation (Galloway JL & Zon LI, 2003).
Erythroblasts have been detected in the circulation from four weeks onward
(Migliaccio G et al., 1986).
Stem cells have been described to colonize the fetal liver at about five to six
weeks of gestation (Migliaccio G et al., 1986), and by the seventh week, the liver
has become the main hematopoietic organ (Galloway JL & Zon LI, 2003).
Definitive erythropoiesis is thought to become dominant from eight weeks of
14
gestation (Migliaccio G et al., 1986). Whether definitive, intraembryonic

hematopoiesis originates from extraembryonic or intraembryonic sources is
controversial. Both the yolk sac (Migliaccio G et al., 1986) and the
intraembryonic aorta-gonad-mesonephros region (Orkin SH & Zon LI, 2002)
have been implicated. Hematopoietic stem cell-supporting activity and HPCs
have also been discovered in the placenta (Mikkola HK et al., 2005; Robin C et al.,
2009).
In the BM, HPCs have been observed from the 11th week of gestation (Galloway
JL & Zon LI, 2003). In addition, hematopoiesis in the spleen, thymus, and lymph
nodes begins in the third month of gestation. From six months onward, the BM is
the principal site of hematopoiesis; however, hematopoietic activity can still be
detected in the liver and spleen during the first postnatal week (Brugnara C &
Platt OS, 2009). CD34+ cells circulate in fetal blood, but their number has been
reported to decrease rapidly in the first few hours of postnatal life (Li K et al.,
2001). The state of hematopoiesis at the time of birth is reflected in the
composition of CB.
5.2 Normal hemostasis in the neonate
5.2.1 Primary hemostasis
Platelets, derived from megakaryocytes, are a vital component of primary

hemostasis, which provides a rapid response to vascular injury. Megakaryocytic
CFUs have been described to be more abundant in CB, both from full-term and
preterm infants, than in adult peripheral blood (Olson TA et al., 1992). The
concentration of the megakaryocytopoietic cytokine, TPO, is high in the CB of
full-term infants (Sainio S et al., 2000a; Cremer M et al., 2003). Platelets have
been observed in the circulation by 11 weeks of gestation (Bleyer WA et al.
1971), and in the healthy full-term neonate, the platelet count and MPV are
within normal adult ranges (Patrick CH et al., 1987; Sainio S et al., 2000b). In CB
collected for banking, the platelet count has been reported to be at the normal
neonatal level (Aroviita P et al., 2003). Neonatal platelets are ultrastructurally
similar to their adult counterparts (Israels SJ et al., 2003). Platelet function in the
healthy full-term neonate is considered adequate, but preterm infants are prone
to bruising (Manco-Johnson MJ, 2005). Von Willebrand factor is present in higher
levels in the neonate than in the healthy adult (Andrew M et al., 1987). Thus, in
the full-term neonate, primary hemostasis is well developed.
MPV decreases during the platelet life span (McDonald TP et al., 1964; Karpatkin
15
S, 1969). Megakaryocyte ploidy (Bessman JD, 1984) and the extent of the
stimulation of thrombopoiesis (Levin J & Bessman JD, 1983) have been reported
to be associated with MPV in adults. MPV is inversely correlated with platelet
count in adult peripheral blood (O'Brien JR, 1974; Bessman JD et al., 1981; Bain
BJ, 1985). This results in the maintenance of a fairly constant platelet mass
(Thompson CB & Jakubowski JA, 1988), which can be expressed as the
plateletcrit (Giacomini A et al., 2001; Wiwanitkit V, 2004).
Von Willebrand factor, glycoprotein Ia/IIa, and glycoprotein VI bind the platelet
surface to subendothelial matrix proteins, exposed during vascular injury, which
is thought to initiate platelet activation (Crawley JT et al., 2007). Procoagulant
factors and other substances, including PF4, are released from platelet alpha
granules upon activation (Monroe DM et al., 2002). A key element in hemostasis
is the interaction of thrombin with the platelet surface. Both phosphatidylserines
exposed on platelet activation and specific binding proteins, including the
glycoprotein Ib/V/IX complex and the protease-activated receptor PAR1, are
thought to contribute to this process (Dormann D et al., 2000; Heemskerk JW et
al., 2002). In addition to hemostasis, platelets have been reported to play a role
in inflammatory reactions (Boehlen F & Clemetson KJ, 2001).
5.2.2 Secondary hemostasis
Elements of secondary hemostasis, blood clotting and fibrinolytic activity, have

been observed from 10 - 11 weeks of gestation (Bleyer WA et al., 1971). Earlier in
embryogenesis, coagulation proteins are thought to regulate tissue proliferation
and differentiation (Manco-Johnson MJ, 2005).
In the healthy full-term infant, the levels of factor VIII, fibrinogen, and alpha-1-
antitrypsin have been reported to be similar to those in healthy adults, and the
level of alpha-2-macroglobulin is higher. The levels of other pro- and
anticoagulants are at approximately 40 - 70% of normal adult values (Andrew M
et al., 1987). In healthy preterm neonates, the levels are even lower (Andrew M
et al., 1988). Most of the coagulation factors approach their adult levels at six
months of age; protein C is a notable exception. Preterm infants have been
reported to reach the coagulation factor levels of their term peers by the age of
six months (Salonvaara M et al., 2004). Neonatal coagulation factors are
reportedly structurally similar to their adult counterparts, with a few exceptions
(Cantor AB, 2009).
The precursors of factors II, VII, IX, and X, as well as those of proteins C, S, and
Z, undergo vitamin K-dependent carboxylation of their Gla domains (Cantor AB,
16
2009). The concentration of prothrombin, or factor II, in neonates is

approximately half of the 1400 nmol/L observed in the plasma of healthy adults
(Heikinheimo R, 1964; Mann KG, 1999). Whether the low level is due to vitamin K
deficiency and defective carboxylation or reduced production of the precursor
protein is controversial. Undercarboxylated prothrombin has been reported to be
observed in 7% of CB samples from healthy full-term neonates (Bovill EG et al.,
1993). Thus, the low prothrombin level in healthy infants may be partially, but
not entirely, due to vitamin K deficiency.
Several problems hinder the interpretation of coagulation tests in children and

neonates (Monagle P et al., 2010). Conventional coagulation screening tests may
not be suitable for neonates as they fail to consider the postnatal pattern of
coagulation system maturation. In global coagulation assays, which take into
account the entire balance of the pro- and anticoagulant systems, the magnitude
of neonatal thrombin generation has been described to match that of an adult
(Cvirn G et al., 2003; Tripodi A et al., 2008). Neonatal blood has been reported to
clot in just over 5 minutes, about 90 seconds earlier than in healthy adults, after
the addition of a physiological amount of lipidated tissue factor in vitro (Cvirn G et
al., 2003). Thrombotic and hemorrhagic events are rare in the healthy neonate.
Figure 2. Conversion of prothrombin (FII) to thrombin (FIIa)

and the release of prothrombin activation fragment 1+2
(F1+2). FVa = Activated factor V; FXa = Activated factor X.
Tissue factor, exposed due to vascular injury, is thought to initiate coagulation

(Crawley JT et al., 2007). Tissue factor forms a complex with factor VIIa to
activate factors IX and X (Roberts HR et al., 2006). Factors Va and Xa, in turn,
form the prothrombinase complex, which cleaves prothrombin to active
17
thrombin, a key element in coagulation (Heemskerk JW et al., 2002). In this

process, prothrombin activation fragments 1 and 2, which contain the
prothrombin Gla and kringle domains, are released as a F1+2 complex (Figure
2; Aronson DL et al., 1977; Crawley JT et al., 2007). Although thrombin
generation is initiated on tissue factor-bearing cells, such as monocytes and
fibroblasts, the propagation of thrombin generation is thought to occur on the
phospholipid surface of activated platelets (Monroe DM et al., 2002). The Gla and
kringle domains have been reported to localize the prothrombin molecule to
activated platelet surfaces (Crawley JT et al., 2007).
Factors VIIIa and IXa form a complex to enable further factor X activation and,
thus, thrombin generation (Mann KG et al., 2003). Thrombin cleaves fibrinogen
to fibrin, activates platelets, and feeds back on the earlier components of the
thrombin-generation pathway, specifically factors V and VIII, to regulate its
formation. In addition, thrombin activates the anticoagulant pathway in the
thrombin-thrombomodulin complex (Lane DA et al., 2005). Antithrombin, alpha-
2-macroglobulin, and tissue factor pathway inhibitor are the major inhibitors of
thrombin and its generation (Hemker HC & Bguin S, 1995). Alpha-2-
macroglobulin-inactivated thrombin has been observed in higher proportions in
neonates than in adults (Ignjatovic V et al., 2007). In conclusion, the hemostatic
system of the neonate differs from that of an adult in many ways, and these
differences should be considered in CB collection procedures.
5.2.3 Markers of hemostasis activation
Classic coagulation assays, such as activated partial thromboplastin time and

prothrombin time, indicate the time to clot formation (Berntorp E & Salvagno GL,
2008). Coagulation activation can also be assessed with peptides and enzymes
that are released during the activation, as well as with enzyme-inhibitor
complexes. F1+2 is released when prothrombin is cleaved to form thrombin, and
its concentration in plasma is directly proportional to the amount of thrombin
formed. The half-life of F1+2 in circulation is 90 minutes. Fibrinopeptide A
indicates conversion of fibrinogen to fibrin, whereas the inhibition of thrombin by
antithrombin can be detected with the thrombin-antithrombin complex.
Radioimmunoassays and enzyme-linked immunosorbent assays (ELISA) have
been developed for these activation markers (Bauer KA, 1999). F1+2 has been
reported to correlate with fibrinopeptide A in venipuctures in healthy adults
(Miller GJ et al., 1995). These tests indicate ongoing thrombin and fibrin
generation and are sensitive to relatively small amounts of thrombin (Hemker HC
& Bguin S, 1995).
18
More comprehensive clotting assays provide an assessment of the overall

balance of pro- and anticoagulant factors. The test for endogenous thrombin
potential has been developed to assess the potential for thrombin generation in
vivo (Hemker HC & Bguin S, 1995). Some assays allow for the continuous
registration of thrombin generation (Berntorp E & Salvagno GL, 2008).
Diverse substances, including growth factors and chemokines, are released from
platelet alpha granules during platelet activation (Newman PJ & Newman DK,
2009). PF4 and beta-thromboglobulin are examples of activation markers used
in clinical practice (Boehlen F & Clemetson KJ, 2001). Human platelets have been
reported to contain a mean 18 g PF4 / 109 platelets (Weiss HJ et al., 1979). PF4
is also called CXCL4, referring to the CXC subfamily of chemokines, defined by
the amino acid composition of their NH2 terminus. As a chemokine, PF4 is
reported to affect fibroblast chemotaxis and possibly neutrophil and monocyte
recruitment to the inflammatory process (Boehlen F & Clemetson KJ, 2001). The
reference range for PF4 in the plasma of infants aged 1 month to 1 year has
recently been reported to be 132 - 243 IU/mL (Newall F et al., 2009). In addition
to the substances released from platelet granules, substances cleaved from the
platelet surface by thrombin, e.g. soluble glycoprotein V (69 kDa), can be
measured as a sign of hemostasis activation (Ravanat C et al., 2000).
Appropriate sample collection techniques are particularly important in

hemostasis assays, as unsatisfactory venipunctures have been reported to result
in coagulation activation in healthy adult subjects (Miller GJ et al., 1995). In
addition, suitable anticoagulants should be selected to avoid hemostasis
activation in vitro (Bauer KA et al., 1991; Leroy-Matheron C & Goualt-Heilmann
M, 1994). In addition to the potential errors caused by preanalytical factors,
some uncertainty is inherent to all laboratory analyses. This uncertainty can be
reduced e.g. by defining intralaboratory reference values for the assay in
question and by including controls in the analyses.
Coagulation and platelet activation are mutually dependent processes. During

blood clotting, the increase in PF4 parallels that of F1+2 (Shuman MA & Levine
SP, 1980; Rybak ME et al., 1981). However, a small amount of thrombin
(specifically, the amount corresponding to the release of 3 - 5 nmol/L F1+2) has
been reported to induce PF4 secretion (Rybak ME et al., 1981).
Studies on F1+2 levels in newborn infants are scarce; mean levels have ranged
from 0.5 to 3.9 nmol/L (Ballin A et al., 1995; Petaja J et al., 1996; Hyytiainen S et
al., 2006). Some studies have suggested that coagulation may be activated in
healthy neonates due to e.g. stress during delivery (Muntean W et al., 1991;
Franzoi M et al., 2002). Whether platelet activation occurs in healthy infants
remains controversial (Alebouyeh M et al., 1974; Suarez CR et al., 1988;
19
Grosshaupt B et al., 1997).
5.3 Methods for analyzing cord blood cells
5.3.1 Cell counting
Automated hematology analyzers provide information about blood cell

characteristics, including the concentrations of nucleated cells (NC), red blood
cells (RBCs) and platelets, as well as information about cell volume (such as
MPV), hemoglobin content, and the differential count of NCs. For cell counting,
simple analyzers employ the electric impedance method, which is based on the
electrical resistance of blood cells. The hemoglobin concentration can be
determined with the cyanmethemoglobin or the oxyhemoglobin method (Dacie
JV & Lewis SM, 1985). As the analyzers have been developed for adult peripheral
blood, the immature cells present in CB may be erroneously interpreted as e.g.
malignant.
More developed hematology analyzers may employ two or more counting

strategies to allow for more specific differential counting of the cell populations.
In CB, where nucleated RBCs may be included in the total nucleated cell (TNC)
count, a combination of optical and impedance counting has been reported to
result in more accurate enumeration of the white blood cell (WBC) population
(Eichler H et al., 2004). The nucleated RBC count has been reported to correlate
positively with CB CD34+ cells and CFUs (Stevens CE et al., 2002; Eichler H et al.,
2004; Cairo MS et al., 2005) and to predict neutrophil engraftment after CB
transplantation (Stevens CE et al., 2002).
Flow cytometry remains the basic technique for CD34+ cell enumeration. In flow
cytometry, cell populations are identified based on their light scatter properties
and immunofluorescence staining (Sutherland DR et al., 1996). In the CD34+
cell analysis, CD34+ stem cells with weak CD45 expression and low side scatter
are distinguished from mature CD45+CD34- WBCs (Sutherland DR et al., 1994).
In dual-platform assays, the proportion of CD34+ cells in the NC fraction is
determined with flow cytometry, after which the absolute count of CD34+ cells is
calculated based on the NC concentration obtained from a hematology analyzer.
In contrast, in single-platform assays, the absolute number of CD34+ cells can
be directly counted with a known number of fluorescent beads (Gratama JW et
al., 1999).
The International Society of Hematotherapy and Graft Engineering (ISHAGE)
20
guidelines for flow cytometric determination of CD34+ cells are widely used in CB
banking. Guidelines for sample collection and preparation, flow cytometric
analysis, and the gating strategy have been developed (Sutherland DR et al.,
1996). Providing that interlaboratory standardization is achieved, the CD34+ cell
count may offer a more accurate estimate of the number of CB HPCs available for
transplantation than the TNC count.
5.3.2 Hematopoietic cytokines in cell cultures
Before the discovery and purification of specific cytokines, HPC culture systems
employed various conditioned media as a source of cytokine stimulation
(Burgess AW et al., 1977; Fauser AA & Messner HA, 1978; Porter PN et al., 1980).
Purified EPO has been available since the late 1970s (Fauser AA & Messner HA,
1978; Nakahata T & Ogawa M, 1982). The synthetic production of diverse
cytokines in the 1980s and 1990s enabled the development of more
sophisticated culture systems for the detection of different cell types from
various sources. The detection of spontaneous hematopoietic colony formation
without an added cytokine source has been used in the diagnosis of
myeloproliferative disorders (Juvonen E et al., 1987; Dobo I et al., 2001).
TPO is the primary regulator of megakaryocytopoiesis (Kaushansky K, 1995;

Kaushansky K, 2008). However, the proliferative and anti-apoptotic effects of
TPO are not restricted to the megakaryocytic lineage (Yoshida M et al., 1997).
TPO has also been reported to promote erythropoiesis either independently or
due to synergy with EPO (Kaushansky K et al., 1995; Kobayashi M et al., 1995;
Liu W et al., 1999). This may be partially due to the existence of a bipotent
erythroid / megakaryocytic progenitor (Debili N et al., 1996) and the similarity of
TPO and EPO (Kaushansky K, 2008). EPO has been suggested to be required for
the survival and differentiation of RBCs (Krantz SB, 1991; Jung YJ et al., 2007).
However, erythroid cells have also been observed in EPO-free conditions (Sui X et
al., 1996; Corre-Buscail I et al., 2005).
In addition to the so-called lineage-specific cytokines, early-acting cytokines

such as interleukins (IL) 1, 3, 6, and 11, as well as stem cell factor (SCF), may be
required for the expansion of HPCs (Bruno E et al., 1991; Debili N et al., 1993;
Mayani H et al., 1993; Williams JL et al., 1998; Freyssinier JM et al., 1999). A
complex network of multiple cytokines is now recognized to control the
production of each cell type (Metcalf D, 2008).
21
5.3.3 Semisolid progenitor cell cultures
Semisolid culture assays for HPCs from human BM, adult peripheral blood, and
CB were developed in the 1970s (Pike BL & Robinson WA, 1970; Chervenick PA &
Boggs DR, 1971; Kurnick JE & Robinson WA, 1971; Knudtzon S, 1974; Verfaillie
CM et al., 1999). In 1987, the number of CFUs observed in methylcellulose
cultures was reported to correlate with the speed of neutrophil and platelet
engraftment after sibling BM transplantation (Ma DD et al., 1987).
Methylcellulose is the supporting medium currently most frequently employed in

the assessment of hematopoietic colony growth in CB collections.
Methylcellulose is commonly used at a weight-per-volume concentration of 0.8%
(Rubinstein P et al., 1995). In the cell culture, NCs have been incubated at 37C
in a humified atmosphere with 5% CO2 for 14 days (Broxmeyer HE et al., 1989).
Assay systems that allow analysis after seven days' incubation are currently
being developed. The culture system supports the growth of CFU-GM, BFU-E,
and CFU-Mixed colonies (Armitage S et al., 1999a). After incubation, the colonies
are enumerated with a light microscope based on their morphology.
Collagen assays have been suggested to enable sophisticated morphological

analysis of HPCs (Suda T et al., 1984). The collagen gel can be transferred onto a
glass slide for cytochemical or immunocytochemical staining, as well as storage
for reanalysis (Dobo I et al., 1995). Collagen-based assays have been developed
for the detection of megakaryocytic cells (Kanamaru S et al., 2000; Won JH et al.,
2000).
Specific cell types can be recognized with monoclonal antibodies directed against
CD antigens on cell surfaces. Megakaryocytic cells at different stages of
development can be detected with e.g. the CD41, CD42, CD61, and CD62
antigens (Debili N et al., 1992; Guerriero R et al., 1995). Cultured erythroid cells
can be recognized with e.g. glycophorin A (GlyA; CD235a) and CD71 (Loken MR
et al., 1987; Rogers CE et al., 1996). CD71, or the transferrin receptor, is
expressed from the early erythroid colony-forming units to the reticulocyte
stage, while GlyA appears in erythroblasts and is still present in mature RBCs
(Loken MR et al., 1987). The structures containing CD antigens have various
roles in cells. For example, GlyA is partially responsible for maintaining the
surface charge of the RBC and contains the MN blood group antigens (Grace RF &
Lux SE, 2009).
Semisolid CFU assays currently provide the best estimate of the short-term
hematopoietic potential of the CB unit. Although the potential for long-term
hematopoietic recovery is not assessed, the results of CFU assays have been
22
reported to correlate with engraftment after CB transplantation (Migliaccio AR et

al., 2000; Yoo KH et al., 2007). Interlaboratory standardization of CFU assays
has not been achieved. However, these assays are used to evaluate the viability
of the CB unit selected for transplantation (Brand A et al., 2008).
5.3.4 Liquid cultures
In addition to semisolid culture systems for the assessment of CB HPCs,

numerous sophisticated liquid culture assays have been developed. Liquid
culture systems have been designed to assess the expansion potential and
kinetics of HPCs (Migliaccio AR et al., 1994; Traycoff CM et al., 1995; Panzenbock
B et al., 1998; Leberbauer C et al., 2005).
Several studies have aimed at the ex vivo expansion of transplantable materials,

both HPCs (Kogler G et al., 1998; Koller MR et al., 1998; Lam AC et al., 2001;
Shpall EJ et al., 2002; de Lima M et al., 2008) and single cell lineages, such as
erythroid cells (Neildez-Nguyen TM et al., 2002; Giarratana MC et al., 2005;
Miharada K et al., 2006; Baek EJ et al., 2008) and megakaryocytes (Shaw PH et
al., 2003). However, ex vivo expansion may exhaust the potential for long-term
engraftment, as foreseen as early as 1993 (Williams DA, 1993). The cell growth
observed in the cultures may be due to the differentiation of primitive HPCs
rather than the expansion of the early progenitor pool responsible for
engraftment after hematopoietic stem cell transplantation.
5.3.5 Uncertainties of the analyses
The reliability of laboratory analyses depends, e.g., on preanalytical factors, such

as careful sample collection techniques, as well as appropriate instrumentation
and frequent calibration and quality control procedures. Inadequate blood flow
from a venipuncture, insufficient mixing of blood with anticoagulant, and
prolonged tourniquet use may yield erroneous results (Dacie JV & Lewis SM,
1985). The uncertainty of measurement, inherent to all laboratory analyses, can
be evaluated e.g. with the intra-assay and interassay coefficients of variance in a
given analysis.
Efforts have been made to decrease the interlaboratory variability of HPC counts
(Dzik W et al., 1999; Lamana M et al., 1999; Lumley MA et al., 1999; Gratama JW
et al., 2003; Moroff G et al., 2006; Brand A et al., 2008; Flores AI et al., 2009). In
a multilaboratory exercise of the Biomedical Excellence for Safer Transfusion
23
Collaborative, both the intralaboratory and interlaboratory variability of TNC

counts was low, whereas that of CD34+ cell counts was high (Moroff G et al.,
2006). Single-platform flow cytometry assays for CD34 may reduce
interlaboratory variability as compared to dual-platform assays (Brocklebank AM
& Sparrow RL, 2001). In addition, external quality assurance schemes have been
established to improve the reliability of CD34+ cell enumeration (Barnett D et al.,
1998; Gratama JW et al., 2003). To reduce the effect of interlaboratory
variability, a separate, integral sample is cryostored with the CB unit and can be
evaluated at the transplant center (Van Haute I et al., 2004; Netcord-FACT,
2006). The establishment of new techniques and standards may improve CB unit
selection based on the enumeration of HPCs, such as CD34+ cells and CFUs, in
the future.
5.4 Cord blood as a hematopoietic stem cell source
The CFU-GM number observed in semisolid cultures of CB collections has been

reported to be within the range of that required for successful engraftment in BM
transplantation (Broxmeyer HE et al., 1989; Broxmeyer HE et al., 1992). This
observation encouraged the first attempts at CB transplantation, eventually
making CB a feasible source of hematopoietic stem cells.
HPCs have been reported to be present in comparable proportions in CB and BM

(e.g. 132 vs. 160 myeloid progenitors and 18 vs. 5 multipotent progenitors / 105
NCs; Mayani H & Lansdorp P, 1998). However, it should be noted that the volume
of CB available for collection is limited. Immature lineage-committed CFUs have
been reported to be more abundant in CB than in BM (Hows JM et al., 1992; Kasai
M & Masauzi N, 1998; Mayani H & Lansdorp P, 1998). In addition, the primitive
high proliferative potential - colony-forming cells have been observed in higher
frequencies in CB (Lu L et al., 1993). Long-term culture-initiating cells, which are
considered the most primitive of the HPCs, have been reported to be present in
comparable proportions in CB and BM (about 1 in 13 000 mononuclear cells;
Pettengell R et al., 1994). The high proportion of HPCs in CB may be partially due
to the placental production of colony-stimulating factors (Burgess AW et al.,
1977; Barker JN & Wagner JE, 2003).
5.4.1 Hematopoietic progenitors in cord blood
The TNC count is used to select CB collections for banking, as the heterogeneous
TNC population has been reported to reflect the HPC content of CB (Lim F et al.,
24
1999). NCs consist of mononuclear cells, granulocytes, and nucleated RBCs;

therefore, granulocytosis, for example, leads to an elevated TNC count and may
contribute to an inaccurate estimate of the CB HPC content. The reference range
for WBCs in neonatal blood has been reported to be 9.0 - 30.0 x 109/L (Brugnara
C, 2009). In the CB bank setting, the mean CB WBC concentration has been
reported to be 10.7 x 109/L, with a SD of 3.2 (Aroviita P et al., 2004). Thus, the
theoretical reference range (2 SD) would be 4.3 - 17.1 x 109/L. Although the
WBC concentration is, on average, higher in CB than in adult peripheral blood,
the reference ranges overlap. Nucleated RBCs are present in CB at a variable
frequency of 0 - 35 per 100 WBCs (Perri T et al., 2004; Rolfo A et al., 2007).
The average proportion of CD34+ cells from CB NCs has been reported to be 0.25
- 0.44% (Kanamaru S et al., 2000; Surbek DV et al., 2000; Aroviita P et al.,
2004; Kurtzberg J et al., 2005; Lapierre V et al., 2007). The viability of the
CD34+ cell population has been assessed with dyes such as trypan blue,
ethidium bromide, propidium iodide, SYTO16, and 7-aminoactinomycin D (Kurtz
J et al., 2007; Brand A et al., 2008). However, these analyses may under- or
overestimate HPC viability as compared with CB HPC cultures (Solomon M et al.,
2010). In an interlaboratory exercise with nine participants, high proportions of
viable cells were detected in CB samples with various viability dyes, although no
growth was observed in CFU assays (Brand A et al., 2008).
CFU counts reported from different laboratories may vary due to differences in
assay type, used cytokines, and plated cell type and frequency, among other
things. The mean CFU-TOT concentration of CB has been reported to be 24 -
43/L (Traycoff CM et al., 1994; Migliaccio G et al., 1996; Aroviita P et al, 2003).
Of the different CFU subgroups observed in semisolid cultures, CFU-GM is the
most abundant (11 - 26/L; Abboud M et al., 1992; Traycoff CM et al., 1994;
Migliaccio G et al., 1996; Aroviita P et al., 2003).
The correlation of the TNC count with the total CD34+ cell number in CB
collections has been 0.66 - 0.78 (Lim F et al., 1999; Aroviita P et al., 2003; Adami
V et al., 2005). The TNC count also correlates with the CFU-TOT number observed
in semisolid cultures (0.60 - 0.69); however, the correlation of CD34+ cells and
CFUs has been reported to be higher (0.68 - 0.87; Lim F et al., 1999; Aroviita P et
al., 2003).
In 588 CB collections processed and cryopreserved in 1999 and 2000, the mean
TNC count was reported to be 103 x 107 before processing (Aroviita P et al.,
2003). The mean CD34+ cell count was 2.3 x 106 before processing. The limit for
processing was set at 50 mL without prior cell counting, or 40 mL with at least 80
x 107 TNCs. In an analysis of three CB banks in the Cord Blood Transplantation
Study banking program, the mean TNC count of accepted CB collections was 118
25
x 107 before processing, and the mean CD34+ cell number was 3.4 x 106 in the CB
unit after processing (Kurtzberg J et al., 2005). The limit for processing was set at
60 mL, or 40 mL with at least 60 x 107 TNCs. The mean CFU-TOT number of the CB
unit was reported to be 2.3 x 106 (Cairo MS et al., 2005).
The mean CB unit TNC and HPC counts reported by each bank are dependent on
the acceptance criteria set by individual CB banks. For example, setting a higher
cut-off limit for collected volume leads to higher TNC counts in the accepted CB
collections (Armitage S et al., 1999a). Only 25% of all CB collections have been
reported to be accepted for long-term storage in one CB bank (Lauber S et al.,
2010). The ethnic background of the CB donor infant also affects the TNC count in
the CB unit (Kurtzberg J et al., 2005; Wofford J et al., 2007). Thus, different
acceptance criteria may have to be set for different ethnicities to ensure human
leukocyte antigen (HLA) diversity in the CB bank.
In addition to cell losses during CB processing, the thawing of the CB unit has
been reported to result in a TNC loss of up to 20 percent as compared to the pre-
freeze cell count (Laroche V et al., 2005). Thus, in recent years, there has been a
tendency to set higher limits to collected volume and TNCs for CB collections
accepted for processing in order to ensure the quality of the thawed CB units and
to meet the need for the high HPC doses required for adult patients. The mean
TNC count of collected CB was reported to rise from 136 x 107 in 1998 to 152 x 107
in 2007 in one CB bank (Lecchi L et al., 2009). The rising requirements
emphasize the importance of careful donor selection, as well as HPC preservation
during CB collection, processing, and cryostorage.
5.4.2 Cord blood transplantation
Unrelated donor CB transplantation for severe hematological diseases after

myeloablative conditioning is well established, in particular in pediatric patients.
Examples of recent results are given in Table 1. Successful CB transplantation
after reduced-intensity conditioning has also been reported, both in children and
adolescents (Bradley MB et al., 2007) and in adults (Barker JN et al., 2003; Ballen
KK et al., 2007; Brunstein CG et al., 2007b; Majhail NS et al., 2008; Uchida N et
al., 2008; Bachanova V et al., 2009), including elderly patients. In addition, some
inherited metabolic disorders have been corrected with unrelated donor CB
transplantation (Prasad VK et al., 2008).
Neutrophil and platelet engraftment marks the initial success of the

hematopoietic stem cell transplant. At the time of engraftment, the HPCs of the
graft are capable of producing enough mature blood cells to sustain an adequate
26
peripheral blood cell count. Neutrophil engraftment is defined as the first of three
consecutive days with a neutrophil count over 0.5 x 109/L (Pettengell R, 1998).
Platelet engraftment may be defined as the first of seven consecutive
transfusion-free days with a platelet count over 20 x 109/L or over 50 x 109/L. In
Table 1, to allow for comparison, the time to platelet count over 50 x 109/L is
shown if available.
As the transplant recipient is at a significant risk for morbidity before

engraftment, speedy recovery of neutrophils and platelets is essential.
Engraftment can be evaluated by assessing the degree of chimerism, i.e. the
proportion of donor-derived myeloid and lymphoid cells versus those of patient
origin (McCann SR & Lawler M, 1993). In primary graft failure, engraftment fails
to occur and the graft never produces an adequate number of blood cells. In
secondary graft failure, blood cells are initially produced but the function of the
graft ceases later. RBC recovery may also be delayed after hematopoietic stem
cell transplantation (Solh M et al., 2010).
In graft-versus-host disease (GVHD), T cells in the graft recognize recipient

antigens and elicit an immune response (Haining WN et al., 2009). Acute GVHD is
divided into four grades based on the extent of organ involvement and the
severity of the symptoms (Glucksberg H et al., 1974). Chronic GVHD is
traditionally divided into limited and extensive types (Shulman HM et al., 1980).
HLA matching is essential for the success of hematopoietic stem cell

transplantation, including minimizing the risk of GVHD (Thomas ED, 1999). Six
HLA antigens in the A, B, and DRB1 groups have traditionally been considered in
stem cell transplantation; however, other HLA antigens may also affect
transplantation outcome (Flomenberg N et al., 2004). In addition, the direction
of the HLA mismatch has recently been suggested to affect outcome (Matsuno N
et al., 2009). HLA mismatches seem to be better tolerated in CB than in BM
transplantation (Barker JN et al., 2001). Importantly, however, a better HLA
match has been reported to decrease the TNC dose required to achieve the same
level of transplant-related mortality in CB transplantation (Barker JN et al.,
2010).
Engraftment has been reported to be slower after CB than after BM

transplantation; in contrast, the risk of acute GVHD is reportedly lower (Rocha V
et al., 2001; Rocha V et al., 2004). The risk of chronic GVHD may be similar or
slightly reduced in CB transplantation compared to BM. As the CB unit is available
immediately, without the delays of contacting and testing a potential donor, CB
may be a better alternative in emergency situations. On the other hand,
supporting cells or a new hematopoietic stem cell graft can often be collected
from a BM donor if needed, an alternative not available for CB recipients. In a
27
28
29
follow-up study, one year after CB or BM transplantation in children, from both

related and unrelated donors, a higher frequency of CFUs and long-term culture-
initiating cells was observed in the BM of CB recipients, possibly indicating a
higher potential for long-term hematopoietic reconstitution in CB (Frassoni F et
al., 2003).
Altogether, survival after CB transplantation has been reported to be comparable

to that after BM transplantation (Barker JN et al., 2001; Rocha V et al., 2001;
Laughlin MJ et al., 2004; Rocha V et al., 2004; Takahashi S et al., 2004; Eapen M
et al., 2007; Takahashi S et al., 2007). However, most comparative studies of CB
and BM transplantation have been retrospective, and the patients have not been
matched for diagnosis, disease stage, pre-transplant conditioning, the grade of
HLA matching, or other factors affecting the outcome of hematopoietic stem cell
transplantation. CB transplantation results have been analyzed in
heterogeneous patient groups, and the recipients have often had a poor pre-
transplant prognosis due to e.g. advanced disease. Incomplete HLA matches
have had to be accepted, and the TNC dose/kg patient weight has varied widely
even within the studies. Thus, comparative studies of CB and BM transplantation
are scarce.
In addition to a high degree of HLA matching, an adequate TNC dose is the target
in CB unit selection (Moroff G et al., 2006). In the recent studies summarized in
Table 1, the median TNC dose has been 2.2 - 4.9 x 107 TNCs/kg patient weight. A
high CB unit TNC dose has been associated with higher probability and speed of
neutrophil engraftment (Kurtzberg J et al., 1996; Gluckman E et al., 1997;
Rubinstein P et al., 1998; Locatelli F et al., 1999; Laughlin MJ et al., 2001;
Gluckman E et al., 2004; Gluckman E & Rocha V, 2006). The TNC dose has also
been reported to correlate with platelet engraftment (Rubinstein P et al., 1998;
Gluckman E et al., 2004; Gluckman E & Rocha V, 2006).
In recent studies of one-unit transplantation, the median TNC count of the

transplanted CB units has been approximately 90 - 160 x 107 (see Table 1).
However, the increasing proportion of adult CB recipients has led to rising
requirements for CB unit HPCs and, thus, TNCs. The minimum HPC dose required
for successful transplantation also depends on the degree of HLA mismatching
(Wagner JE et al., 2002). Target TNC doses of 3.0 x 107/kg for HLA-matched units
and 4.0 x 107/kg for HLA-mismatched units have been applied (Verneris MR et al.,
2009). To overcome HLA mismatches at 1 - 2 loci, a cryopreserved TNC dose of at
least 2.5 - 5.0 x 107/kg patient weight has been reported to be required to
minimize CB transplant-related mortality (Barker JN et al., 2010). However, in
Barker and colleagues' study, which included 1061 patients, 56 (5%) of them
HLA-matched to the donor, the lowest transplant-related mortality at three years
was achieved with HLA-matched CB regardless of the TNC dose. Thus, both the
30
available TNC dose and the degree of HLA matching have to be considered when
selecting the best CB unit for each patient.
The transplantation of two CB units to increase the TNC dose has yielded
promising results in adults (Barker JN et al., 2005; Ballen KK et al., 2007;
Brunstein CG et al., 2007b; Verneris MR et al., 2009). However, the probability of
finding two CB units both HLA-matched to the recipient - or even each other - is
low, and, thus, HLA mismatches have had to be accepted. The risk of acute GVHD
may be increased after the transplantation of two CB units (MacMillan ML et al.,
2009). Double-unit transplantation has also been associated with a decreased
risk of relapse, interpreted as a graft-versus-leukemia effect (Verneris MR et al.,
2009).
The CD34+ cell dose (Wagner JE et al., 2002; Terakura S et al., 2007) and the
CFU dose (Migliaccio AR et al., 2000; Yoo KH et al., 2007) have been suggested to
be better determinants of neutrophil and platelet engraftment than the TNC
dose. In some studies, neither the TNC dose nor the CD34+ cell or the CFU-GM
doses have correlated with engraftment (Thomson BG et al., 2000; Iori AP et al.,
2004). The contradictory results may be explained by differences in HPC
enumeration techniques both between and within studies; for example, in some
studies cell counts have been obtained from several CB banks providing the
grafts. Due to problems with the interlaboratory standardization of CD34+ cell
and CFU enumeration, the TNC count is still applied in international registries for
the selection of CB units for transplantation.
5.5 Cord blood collection and banking
The collected CB volume is the first parameter considered in the selection of CB

collections for further evaluation and processing. Mean collected CB volumes of
50 - 103 mL have been reported (Aroviita P et al., 2003; Aufderhaar U et al.,
2003; Zingsem J et al., 2003; Adami V et al., 2005; Askari S et al., 2005;
Kurtzberg J et al., 2005; Meyer TP et al., 2006; Skoric D et al., 2007). Collected
CB volume has been reported to correlate positively with TNCs both before
(Donaldson C et al., 1999; Rogers I et al., 2001 Aufderhaar U et al., 2003;
Nakagawa R et al., 2004; Solves P et al., 2005) and after (George TJ et al., 2006)
processing. Collected volume also correlates positively with the total number of
CD34+ cells before (Rogers I et al., 2001; Aufderhaar U et al., 2003) and after
(Solves P et al., 2005; George TJ et al., 2006) processing, the CFU-TOT number
before (Aufderhaar U et al., 2003) and after (Solves P et al., 2005) processing,
and the CFU-GM number after processing (George TJ et al., 2006). Thus, CB
banks attempt to collect as high a volume as possible without disrupting the
31
delivery or compromising the sterility of the CB collection. Some procedures

proposed to increase the volume of collected CB, such as placental perfusion with
heparinized saline or an anticoagulant, or the use of a pressure device (Khodabux
CM & Brand A, 2009), do not conform to international standards and are thus not
commonly applied in CB collection for banking.
5.5.1 Obstetric and perinatal factors
Obstetric and perinatal data are collected as a part of the routine care of the
neonate, and they are reviewed in order to select only healthy CB donors
(Netcord-FACT, 2006). These data may also be of value in the estimation of CB
HPCs. However, many of the obstetric and perinatal factors are mutually
dependent. The relative importance of each factor can be assessed with multiple
regression analyses, which exclude possible confounding factors. Previous
studies with these analyses are summarized in Table 2. In the published studies,
sample and subgroup sizes as well as definitions of both perinatal factors and
HPCs have varied. Most importantly, the results are dependent on the initial
selection of factors to be analyzed.
Based on the published data, the effect of birth weight and placental weight on
CB HPC counts has been well established. To exclude the possibly confounding
effect of gender and gestational age, birth weight can be expressed relative to
the mean for similar neonates (relative birth weight). To this end, reference data
have been published for several populations (Pihkala J et al., 1989; Kramer MS et
al., 2001; Oken E et al., 2003). Reference data for placental weight, adjusted for
gestational age and gender, have also been published (relative placental weight;
Dombrowski MP et al., 1994; Thompson JM et al., 2007). The birth weight /
placental weight ratio, and, conversely, the placental weight / birth weight ratio,
is also dependent on gestational age (Molteni RA et al., 1978; Dombrowski MP et
al., 1994). Other obstetric factors, such as maternal age, gestational age, and
parity, have also been reported to be associated with CB HPC counts.
CB can be collected after both vaginal delivery and Cesarean section. Vaginal
delivery is associated with gradual physiological changes, while elective
Cesarean section leads to abrupt changes in maternal and fetal physiology.
Notably, when Cesarean sections and vaginal deliveries have been analyzed
separately, birth weight has been reported to correlate with collected CB CFU-
TOT only in Cesarean sections (Aroviita P et al., 2004). Thus, the relative
importance of each perinatal factor for the estimation of CB HPCs may depend on
the mode of delivery. In some studies, assisted and spontaneous delivery
subgroups have been compared. However, as assisted delivery may refer to both
32
33
34
35
Cesarean section and operational vaginal delivery, and as Cesarean sections may
be elective or performed after the commencement of delivery, due to e.g. fetal
distress, this group is heterogeneous with respect to physiological phenomena
during delivery.
Vaginal delivery, as well as Cesarean section performed due to fetal distress, may
be associated with increased stress as opposed to elective Cesarean section.
Umbilical arterial pH has been reported to be lower after vaginal delivery than
after elective Cesarean section (Yoon BH & Kim SW, 1994), and is typically 7.12 -
7.40 after vaginal delivery at term (Lackman F et al., 2001; Kitlinski ML et al.,
2003). The CB EPO level serves as an indirect indicator of intrauterine hypoxia
and has been reported to correlate inversely with umbilical arterial pH (Maier RF
et al., 1993). Accordingly, the EPO level is reportedly higher after vaginal delivery
than after Cesarean section (Widness JA et al., 1984). In vaginal delivery, long
first and second stages have been reported to be associated with low umbilical
arterial pH (Katz M et al., 1987; Yudkin PL et al., 1987; Yoon BH & Kim SW, 1994).
The Apgar scores at one and five minutes of age are frequently used to assess the
extent of fetal distress. In addition, meconium-stained amniotic fluid may be
encountered in conjunction with neonatal distress (Cleary GM & Wiswell TE,
1998).
Endogenous cytokine production due to stress during delivery is hypothesized to

lead to the mobilization of granulocytes, CD34+ cells, and CFUs (Lim FT et al.,
2000; Aufderhaar U et al., 2003), and thus, possibly, to increased HPCs in
collected CB. CB collected after Cesarean section due to fetal distress has been
reported to contain a higher number of NCs, CD34+ cells, and CFUs than that
collected after elective Cesarean section (Manegold G et al., 2008). Healthy
newborn infants rarely suffer extreme stresses during delivery. However,
indicators of stress during delivery have been associated with high CB HPC
counts even in healthy neonates.
The value of obstetric and perinatal factors in the routine assessment of the CB
collection remains undetermined. As CB is collected from healthy, full-term
neonates after uncomplicated gestation and delivery, the associations of
obstetric and perinatal factors and HPCs should only be assessed in a CB bank
setting to avoid drawing conclusions from observations in sick neonates.
5.5.2 Collection methods
A varying proportion of the CB volume remaining in the placenta after clamping

of the umbilical cord can be collected for banking. CB collection methods have
36
been described in several studies (Wagner JE et al., 1992; Rubinstein P et al.,

1993; Harris DT et al., 1994; Bertolini F et al., 1995; Armitage S et al., 1999a;
Elchalal U et al., 2000; M-Reboredo N et al., 2000; Bornstein R et al., 2005).
In a closed CB collection system, after placental separation, the umbilical cord is

disinfected and the blood collected via puncture of the umbilical vessels
(Rubinstein P et al., 1993). Closed collections have been associated with a lower
rate of bacterial contamination than open collections (Bertolini F et al., 1995).
Citrate phosphate dextrose is a commonly used anticoagulant in CB collections,
as it can be utilized through a wide range of anticoagulant-to-blood volume ratios
(Rubinstein P et al., 1993).
CB can be collected from the placenta through the umbilical vessels either before
placental delivery while the placenta is still in utero, or after placental delivery
(ex utero) in a separate collection area (Lasky LC et al., 2002). Studies on the
effect of the collection strategy on CB HPC counts are summarized in Table 3.
Although higher CB volumes may be achieved with the in utero strategy, in most
studies total cell counts have not differed in the two collection methods. In some
studies, an advantage of the in utero method has been observed. However, in
most of the studies, different proportions of vaginal deliveries and Cesarean
sections have been analyzed in the in utero and ex utero groups, which may
confound the possible effect of the collection strategy. In addition, the time to
umbilical cord clamping after delivery, as well as other measures to increase the
volume of collected CB, such as positioning the neonate above the level of the
placenta, may affect total cell counts in the CB collection (Grisaru D et al, 1999;
Khodabux CM & Brand A, 2009).
The selected collection strategy has also been reported to affect bacterial
contamination rates and clotting in the CB collection (M-Reboredo N et al, 2000;
Lasky LC et al, 2002). However, the observed differences may reflect varying CB
bank procedures as much as actual differences between the collection strategies.
Thus, the advantages of each collection strategy remain controversial. The
preferred collection strategy is selected by each CB bank depending, e.g., on the
standard delivery practices of the maternity unit. To ethically justify CB
collection, risks to the donor infant have to be minimized (American Academy of
Pediatrics Committee on Bioethics, 2010). Thus, the normal course of the
delivery must not be disturbed.
Coagulation may be activated upon CB collection. During blood donation in

adults, high fibrinopeptide A levels have been observed at the distal end of the
collecting tube and in the collection bag (Skjonsberg OH et al, 1986). Thus,
standard blood donation equipment causes substantial coagulation activation.
Thrombin generated during blood collection activates platelets (Shuman MA,
37
1986), which, in turn, leads to the release of platelet chemokines from alpha
granules (Boehlen F & Clemetson KJ, 2001). Thrombin has also been reported to
activate megakaryocytes (Cramer EM et al, 1993). Despite differences in the
adult and neonatal hemostatic systems, CB collection procedures are currently
based mainly on adult guidelines. The optimal equipment and procedures to
minimize blood coagulation during CB collection are yet to be determined.
Table 3. Attempts to compare two cord blood collection strategies (in or ex utero) with respect to
high cord blood volume and cell counts.
Reference In Ex Volume TNC Total CD34+ CFU-TOT

utero utero cells number
Surbek DV et 21 19 In utero In utero NG NG

al., 2000 CS CS (p = 0.01) (p = 0.03)
Pafumi C et 21 26 In utero In utero NS NG

al., 2002 CS CS (p < 0.05) (p < 0.05)
Solves P et 264 305 In utero In utero NS In utero*

al., 2003 VD VD (p < 0.05) (p < 0.05) (p < 0.05)
Tamburini A 69 82 NS NS NS NS
et al., 2006 CS CS (CFU-GM)
Wall DA et 293 41 NS NS NG Ex utero*

al., 1997 VD+CS VD+CS (p < 0.001)
Lasky LC et 245 1477 NS NS NS* NS*

al., 2002 NG NG (CFU-GM)
Sparrow RL et 58 99 VD NS NS NS NG
al., 2002 VD 61 CS
Solves P et 98 202 In utero In utero In utero* NG

al., 2005 VD+CS VD+CS (p < 0.001) (p < 0.001) (p < 0.05)
M-Reboredo N 472 104 NS In utero NG NG

et al., 2000 VD CS (p NG)
Yamada T et 126 29 Ex utero NG Ex utero NG

al., 2000 VD CS (p = 0.005) (p = 0.03)
TNC = Total nucleated cells; CFU(-GM) = Colony-forming units (- granulocyte / macrophage);

CFU-TOT = Total CFUs; CS = Cesarean section; VD = Vaginal delivery; NS = No significant
difference; NG = Not given
Cell counts pre-processing unless marked. *Post-processing cell counts analyzed.
38
5.5.3 Cord blood processing
The preservation of as many functional HPCs as possible is a key issue in CB

banking. Long-term cryostorage of CB collections as whole blood requires
substantial storage space. In addition, the thawing of the CB unit after
cryostorage lyses RBCs, as the storage method has been optimized for nucleated
cells (Rubinstein P et al., 1995). The hemoglobin released from these cells is
cytotoxic (Yadav AK et al., 2004). Thus, CB volume reduction procedures have
been described widely (e.g. Harris DT et al., 1994; Bertolini F et al., 1996;
Denning-Kendall P et al., 1996; Dal Cortivo L et al., 2000; Yasutake M et al.,
2001; Eichler H et al., 2003; Adami V et al., 2005).
The volume reduction procedure is based on the different specific gravities of

blood cells. In principle, during centrifugation, RBCs sediment the fastest, while
platelets and plasma are left in the top supernatant, leaving the desired NC layer
in the middle (Loos JA & Wautier JL, 1998). The separation of NCs can be
enhanced with substances such as dextran or hydroxyethyl starch, which
increase the sedimentation rate of RBCs (Strauss RG, 1979). The processing of
the CB collection inevitably leads to cell losses, which can be minimized by
optimizing the volume reduction process.
In 1995, Rubinstein and colleagues described a manual two-phase hydroxyethyl

starch-based CB volume reduction system (Rubinstein P et al., 1995). In the first
step, RBCs are sedimented by centrifugation with hydroxyethyl starch. Then, the
WBC- and platelet-rich supernatant is centrifuged and excess platelet-rich
plasma is removed to obtain the NC fraction. Manual systems based on e.g.
gelatin have also been described (Lazzari L et al., 1996).
As manual methods are labor-intensive, a semi-automated closed system for CB

volume reduction has been developed. In this system, CB platelet-rich plasma
and RBCs are expressed, after centrifugation, into the top and bottom bags of a
triple-bag system, leaving the NC layer in the primary bag (Armitage S et al.,
1999b). To avoid the loss of NCs, protocols with plasma but not RBC reduction
have also been proposed (Chow R et al., 2007).
None of the described methods has been able to fully combat the challenge of the
varying blood volume inherent to CB collection. To this end, a fully automated
closed system with both additive-free and e.g. hydroxyethyl starch-based
protocols has been developed (Zingsem J et al., 2003; Armitage S, 2006;
Lapierre V et al., 2007). The separation chamber of this system allows for the
efficient processing of a large range of CB volumes with an adjustable end volume
(Lapierre V et al., 2007). However, despite the extensive efforts and
39
technological advances, an effective volume reduction process without loss of

HPCs has not been developed.
With the manual and semi-automated methods, mean TNC recoveries of 64 -

91% have been reported after volume reduction (Rubinstein P et al., 1995; M-
Reboredo N et al., 2000; Aroviita P et al., 2003; Takahashi TA et al., 2006; Lecchi
L et al., 2009). The mean CD34+ cell recovery has been 75 - 95% (M-Reboredo N
et al., 2000; Aroviita P et al., 2003; Lecchi L et al., 2009). The automated and
semi-automated systems have been compared in two laboratories (Armitage S
et al., 1999b; Armitage S, 2006; Lapierre V et al., 2007). The TNC recovery was
reportedly better with the automated system than the semi-automated system
(80 - 87% vs. 77 - 83%), while controversial results were reported for HPC
recoveries (CD34+ cells, 86 - 91% vs. 82 - 99%, and CFUs, 91% vs. 103%;
Armitage S et al., 1999b). Recoveries of over 100% are thought to reflect cell
activation during volume reduction. Although no definite improvement of cell
recoveries has been observed with the automated system, this system has
substantially improved the efficiency and speed of CB processing, enabling the
banking of an increasing number of CB units.
5.5.4 Cryopreservation
As the recovery and functionality of CB HPCs after cryostorage and thawing are
essential, several studies concerning cryopreservation methods have been
published (Broxmeyer HE et al., 1989; Harris DT et al., 1994; Rubinstein P et al.,
1995; Donaldson C et al., 1996; Meyer TP et al., 2006; Skoric D et al., 2007).
Controlled-rate freezing to avoid the formation of intracellular ice improves cell
viability and recovery after thawing (Donaldson C et al., 1996; Skoric D et al.,
2007). Dimethyl sulfoxide penetrates living cells and has been used to prevent
freezing damage in them, as originally reported in RBCs (Lovelock JE & Bishop
MW, 1959). The optimal concentration of dimethyl sulfoxide has been reported to
be 5 - 10% (Donaldson C et al., 1996; Meyer TP et al., 2006; Skoric D et al.,
2007). In addition, an extracellular cryoprotectant, such as dextran, has been
used (Rubinstein P et al., 1995).
After thawing of the CB unit at 37C, dimethyl sulfoxide has been removed by
dilution with albumin and dextran, followed by centrifugation. This has been
reported to yield increased cell viability (Rubinstein P et al., 1995). An automated
closed system for washing the CB unit has also been developed (Rodriguez L et
al., 2004). Dilution of the cell suspension in carefully controlled conditions, but
without centrifugation, may reduce cell loss during thawing (Barker JN et al.,
2009a). Thawed unwashed CB units have also been infused without serious
40
toxicity (Hahn T et al., 2003).
The mean recovery of nucleated or mononuclear cells has been reported to be 71

- 90% of the pre-freezing counts after CB cryopreservation and thawing (Harris
DT et al., 1994; Laroche V et al., 2005; Meyer TP et al., 2006; Lemarie C et al.,
2007; Skoric D et al., 2007). The proliferative, replating, and engrafting capacity
of CB has been reported to be preserved after cryostorage of at least 15 years
(Broxmeyer HE et al., 2003). Megakaryocyte progenitors have been described to
be more sensitive to the stresses of cryopreservation than other myeloid
progenitors (Xu Y et al., 2004). In compliance with international standards, the
viability of cryopreserved CB units is continuously tested, according to a preset
schedule, in each CB bank. Ultimately, the feasibility of the cryostorage
procedure has been confirmed by successful transplantation of banked CB units.
5.5.5 International standards, accreditation, and registries
Dozens of CB banks and programs have been established to provide a growing

reservoir of CB units for transplantation from unrelated, HLA-matched donors in
e.g. the United States (Rubinstein P et al., 1994; Harris DT, 1996; Alonso JM et
al., 2001; Cairo MS et al., 2005), Europe (Lazzari L et al., 1996; Dal Cortivo et al.,
1998; Jacobs HC & Falkenburg JH, 1998; Querol S et al., 1998; Armitage S et al.,
1999a; Ordemann R et al., 1999; Donaldson C et al., 2000; Rendine S et al.,
2000; Aroviita P et al., 2003), and Asia (Kodera Y, 2008). In the United States, a
national cord blood banking program has been planned (Board on Health
Sciences Policy, 2005). In addition to unrelated CB banking, private banks have
been established to store CB for personal or family use (Kaimal AJ et al., 2009;
Thornley I et al., 2009). Some of these banks may provide CB units for
transplantation to unrelated recipients after storage for a certain time period.
However, the quality of privately banked CB units has recently been reported to
be inferior to that in public CB banks (Sun J et al., 2010). Neither private CB
banking nor CB banking from related donors are discussed further in this thesis.
From the early 1990s, collaboration efforts have been proposed to unify CB bank
procedures (Gluckman E, 1994). Standard operating procedures have been
defined to assure and improve the quality of CB units (Fraser JK et al., 1998).
Eurocord was founded in Europe in 1997 as an effort to create a joint CB unit
registry as well as product and quality control standards (Gluckman E et al.,
1998; Rebulla P et al., 1999). However, quality concerns, such as issues with HPC
viability, remain common in CB units (Scaradavou A et al., 2010; Querol S et al.,
2010).
41
The International Standards for Cord Blood Collection, Banking, and Release for
Administration, developed by Netcord and the Foundation for the Accreditation of
Cellular Therapy (FACT), provide detailed standards for the entire CB banking
process, from donor recruitment to collection, processing, banking,
transportation, and follow-up of transplantation outcome (Netcord-FACT, 2010;
www.factwebsite.org). CB banks may apply for accreditation, awarded by FACT,
to demonstrate adherence to the Netcord-FACT standards (Warkentin PI & FACT,
2003). Other organizations, such as AABB (formerly American Association of
Blood Banks; www.aabb.org), have also developed detailed standards for similar
purposes (Wall DA, 2010). CB banks are now facing licensure by the Food and
Drug Administration in the United States (Wedgeworth S, 2010).
The mother of the CB donor infant must provide written informed consent for the
entire CB banking process (Netcord-FACT, 2010). To ensure suitability of the
newborn infant as a CB donor, maternal and family medical histories are
reviewed with standardized questionnaires. A family history of genetic or
malignant disease, as well as an increased risk for communicable diseases, are
considered grounds for deferral. Details of the pregnancy and delivery are
recorded and reviewed. A maternal blood sample is drawn and tested for
communicable diseases, including human immunodeficiency viruses, hepatitis B
and C, human T cell lymphotropic viruses, cytomegalovirus, and syphilis; the list
is ever-growing as new pathogens are discovered. The collected CB is also tested
for microbial contamination, HLA type, ABO group, and Rh type. The aim is to
provide as safe CB units as possible for banking and transplantation.
The HPC content of the CB collection is estimated with the TNC count, as the
analysis can be standardized (Lim F et al., 1999). In addition, criteria for
collected volume are set to avoid further testing and processing of CB collections
unlikely to fulfill the TNC criterion. Acceptance criteria for collected volume and
the TNC count are defined by individual CB banks. In addition to the TNC count,
the complete blood count with differential, the nucleated RBC count, the total
CD34+ cell count, and the viability of the CB unit are evaluated based on criteria
developed during the validation phase of the CB banking process (Netcord-FACT,
2010).
The assessment of CB unit HPCs, such as total CD34+ cells or CFUs, may provide
a more direct estimate of the hematopoietic potential of the unit than the TNC
count. As interlaboratory standardization of these assays has not been achieved,
the selection of CB units for banking based on the quantitation of hematopoietic
progenitors is currently not feasible. However, CFU assays provide the only
reliable assessment of the viability of the CB unit, despite efforts to develop flow
cytometric methods for viability staining (Brand A et al., 2008).
42
According to current standards, CB units are cryopreserved within 48 hours of

collection (Netcord-FACT, 2010). Integral samples of the CB unit, as well as
samples from other phases of the CB banking process, are cryopreserved for
later analysis. In addition, a defined number of maternal samples is stored for
possible further evaluation. All CB unit data are reviewed before the acceptance
of the unit for long-term cryostorage. Any deviations from standard procedures
during CB collection, transportation, or processing, as well as unexpected
findings in routine tests or in the donor's medical history, are evaluated. If
necessary, the CB unit is discarded or labeled as not suitable for clinical use.
Adequate viability of the CB units in cryostorage is continuously ensured,

according to a predefined schedule, in each CB bank. Current data suggest that
the duration of cryostorage does not affect the engraftment of the CB unit (Jubert
C et al., 2008). When a CB unit is selected as a candidate for transplantation, unit
data are re-reviewed and CB and maternal samples retested. One integral
sample, cryostored with the CB unit, is detached and thawed for confirmatory
HLA typing as well as assays of hematopoietic potential (CFUs) and viability
(Netcord-FACT, 2010). Another integral sample is reserved for testing at the
transplant center. Data on clinical outcomes of CB transplantation are expected
to be reported back to the CB bank to enable further development of banking
processes.
To enable a worldwide search for CB units based on selected unit characteristics,

such as HLA type and the TNC count, CB registries have been established as sub-
organizations of pre-existing BM registries. The Bone Marrow Donors Worldwide
(www.bmdw.org) registry has been established by the European Group for Blood
and Marrow Transplantation. Large registries in the United States, such as the
National Marrow Donor Program CB registry, also participate in the Bone Marrow
Donors Worldwide registry (Confer D & Robinett P, 2008). To this day, 44 CB
banks and registries have contributed a total of 434 744 CB units to this registry
(www.bmdw.org; September 2010). CB banks in Asian cities have united under
the name Asiacord (www.asiacord.umin.jp). The establishment of international
standards and accreditation procedures has enabled the worldwide
implementation of CB banking process and quality control measures.
43
6 AIMS OF THE STUDY
The general aim of this study was to investigate and identify factors related to the
selection and quality of unrelated CB collections for processing and banking in
public CB banks.
More specifically, the aims were
to study CB platelet characteristics, hypothesized to be markers of general

hematopoietic activity, as predictors of HPCs (CD34+ cells and CFUs) in the CB
unit (I)
to evaluate stress-related perinatal data, including umbilical arterial pH, birth

weight, and placental weight, for the estimation of the HPC content of CB units
(II)
to assess activation of the coagulation system and platelets during routine CB

collection (III)
to create a scoring system for the evaluation of the growth of megakaryocytic

cells in processed CB (I), and to describe erythroid growth of thawed
unseparated CB units in liquid culture (IV)
44
7 MATERIALS AND METHODS
7.1 Materials of the original studies
CB collections from the years 1998 - 2009 were analyzed in this study (Figure
3).
Studies I and II: 167 CB collections from a single maternity unit were
processed and cryopreserved at the Finnish CB Bank between September 2004
and August 2005. Six of the processed units did not meet the final selection
criteria for long-term storage due to adverse medical background (four units),
reactivity in communicable disease testing (one unit), or problems during CB
processing (one unit).
In a randomly selected subset of 24 CB units, megakaryocytic colony growth was

analyzed after CB processing.
Study III: Hemostasis activation was assessed in 27 CB collections from two

maternity units collected by the Finnish CB Bank at three time periods (analyses
in the year 1998, n = 11; in the year 2000, n = 10; and in the year 2006, n = 6).
The collections were allocated for use in process control or product development.
Study IV: Seven CB units collected and processed between February 1999 and
January 2007 and thawed for quality control at the Finnish CB Bank between
October 2005 and August 2009 were analyzed. In addition, six CB collections
were used as references.
7.2 Cord blood banking
The Netcord-FACT standards (Netcord-FACT, 2006) were applied in the CB bank,

and the program has been awarded accreditation by FACT (in 2004 and in 2008).
7.2.1 Donor recruitment
Healthy, voluntary pregnant women registered as prospective CB donors at
45
MATERIALS AND METHODS
routine maternity health care or outpatient clinic appointments. The mother's

personal and family medical history was reviewed with standard questionnaires
by the CB bank staff. Relevant perinatal data were obtained from the hospital
records and a maternal blood sample drawn by the CB bank nurse-midwives.
Only CB collections from healthy neonates delivered after uncomplicated
gestation were processed. Umbilical arterial pH was analyzed from a sample
drawn after placental delivery as a part of normal perinatal care at the maternity
unit.
For Study II, relative birth weight, adjusted for gestational age and gender, was
expressed as SDs from the mean (Z score) compared to a Finnish reference
population. (Pihkala J et al., 1989) Relative placental weight, adjusted for
gestational age and gender, was expressed as SDs from the mean (Z score) with
a Norwegian reference population. (Thompson JM et al., 2007) Placental weight
was divided by birth weight to obtain the placental weight / birth weight ratio.
7.2.2 Cord blood collection
A specially trained nurse-midwife collected the CB in a separate laboratory

facility within the maternity unit after vaginal delivery or Cesarean section. All
the collections in this study were performed ex utero. The umbilical cord was
clamped as per the routine procedure of the maternity unit. The placenta was
handed over to the CB bank nurse-midwife and immediately placed in a funnel
designed for CB collection, and the blood was collected with the aid of gravity via
puncture of the umbilical vein (closed system). The collection bag was kept on a
mixer (Biomixer 330; Ljungberg & Kgel Ab, Lule, Sweden, or Optimix V3;
Fenwal Inc, Lake Zurich, IL). Collections with visible clots were discarded.
In Studies I - II, in the last sample series of Study III, and in four of the
collections of Study IV, a CB-Collect double CB bag with 20 mL of citrate
phosphate dextrose was used (T2950, 300/20 mL; Fresenius HemoCare,
Emmer-Compascuum, the Netherlands; 3.27 g citric acid monohydrate, 26.3 g
sodium citrate dihydrate, 2.51 g sodium dihydrogen phosphate dihydrate, and
25.5 g glucose monohydrate per 1000 mL), and an extra 10 mL of anticoagulant
was added to collections 135 mL or over. In the first two sample series of Study
III, and in three of the collections of Study IV, a CB collection bag with 25 mL of
citrate phosphate dextrose was used (nominal volume 150 mL; 791-01U; Pall
Medsep Corporation, Covina, CA; 2.50 - 2.76% sodium citrate dihydrate, 0.21 -
0.23% monobasic sodium phosphate, and 2.42 - 2.68% dextrose
monohydrate). Both collection bags contained two 16-gauge needles.
46
Figure 3. The materials of the original publications.
47
7.2.3 Cord blood processing and cryopreservation
The CB collection bag was individually packed between thermoregulatory

elements (butanediol bags) at the maternity unit and transported to the CB bank.
The temperature was monitored continuously (Tinytalk; Gemini Data Loggers UK
Ltd., Chichester, UK) until processing and a stable temperature was used as an
acceptance criterion for the CB collections.
The CB collections of Studies I - II and IV were processed and cryopreserved at

the CB bank. The volume of the CB collections in Studies I - II was reduced with
an automated cell separation system utilizing the centrifugation principle and
hydroxyethyl starch (UCB-HES; Sepax Biosafe SA, Eysins, Switzerland). In
Study IV, four of the units were processed with the automated system, while the
other three were processed with the manual method described by Rubinstein and
colleagues (Rubinstein P et al., 1995). The CB units in Study IV were
cryopreserved with controlled-rate freezing in 10% dimethyl sulfoxide
(DMSO/Dextran 40; Pall Medsep Corp., Covina, CA) for long-term storage in
liquid nitrogen (-196C; BioArchive; ThermoGenesis Corp., Rancho Cordova,
CA). The units were thawed at the CB bank with the New York thawing protocol
(Rubinstein P et al., 1995), including a washing step to reduce the dimethyl
sulfoxide concentration.
7.3 Laboratory assays
7.3.1 Cell counting with an automated analyzer
CB cells were counted with an automated hematology analyzer (Sysmex K1000

(Studies I - IV) or XT2000 (Study IV); Sysmex Corp., Kobe, Japan, or Coulter JT3
(eight samples in Study III); Coulter Electronics Ltd, Luton, UK).
In Study I, cell concentrations were standardized to exclude the effect of the

varying anticoagulant-to-blood ratio inherent to CB collection techniques and
instead to present the results as if from native CB; these results are referred to as
standardized (Aroviita P et al., 2004).
48
7.3.2 Semisolid progenitor cell assays
HPC assays were performed at the CB bank from processed CB prior to the
addition of cryoprotectant. A standard volume of CB was cultured in duplicate in
methylcellulose at 37C in 5% CO2 for 14 days (MethoCult GF H84434; Stem Cell
Technologies, Vancouver, Canada). After incubation, the colonies were
enumerated with an inverted microscope based on their morphology. The
concentrations of the different CFUs in the CB unit were calculated based on the
results of the microscopy and the plated CB volume. The CFU-TOT number was
defined as the sum of CFU-GM, BFU-E, and CFU-Mixed colonies. The performance
of the analyses was externally evaluated biannually with stem cells from adult
sources (Stem Cell Technologies, Meylan, France).
In Study I, samples from processed CB units (n = 24) were stored refrigerated

(+4 - +8C) for megakaryocytic colony assays. Excess RBCs were removed by
centrifugation, and megakaryocytic colonies were analyzed with a commercial
kit (MegaCult-C; StemCell Technologies, Vancouver, Canada). 100 L of cell
suspension per chamber was plated on double-chambered slides (Nalge Nunc
International, Naperville, IL), four chambers per CB unit, and cultured in a
semisolid collagen assay at 37C in 5% CO2 and 95% humidity for 13 days. The
medium contained 50 ng/mL TPO, 10 ng/mL IL-3, 10 ng/mL IL-6, 1% bovine
serum albumin, 10 g/mL recombinant human insulin, 200 g/mL human
transferrin, 2 mmol/L L-glutamine, and 10-4 mol/L 2-mercaptoethanol in Iscove's
modified Dulbecco's medium (IMDM).
After incubation, the slides were fixed with methanol-acetone (1:3; Merck & Co
Inc, Whitehouse Station, NJ) and immunocytochemically stained with a CD41
(glycoprotein IIb/IIIa) specific monoclonal antibody (Stem Cell Technologies,
Vancouver, Canada). Evans Blue was used to counterstain the nuclei. The slides
were evaluated with a camera-linked inverted brightfield microscope (Olympus
IX 50 microscope and DP12 camera; Olympus, Tokyo, Japan).
Each chamber was assigned a score from 0 (no growth) to 3 (maximum growth)
based on the number, size, and morphology of megakaryocytic colonies (Figure
4). If no megakaryocytic colonies were observed, the chamber was assigned a
score 0; chambers with few small megakaryocytic colonies were scored 1; for a
score 2, scattered large megakaryocytic colonies were observed; and if several
large megakaryocytic colonies were seen, the chamber was assigned a score 3.
Three investigators assessed each chamber independently. The mean of the
scores of four chambers was calculated to yield the final megakaryocytic score
for the CB unit.
49
Figure 4. The scoring of megakaryocytic colonies in semisolid assays of

processed cord blood. Magnification 20 x. (Study I)
7.3.3 Liquid cultures of thawed cord blood units
In Study IV, seven CB units were cultured in a one-step liquid culture immediately
after thawing without further separation. NCs from three fresh CB collections
were separated by density gradient centrifugation (Ficoll-Paque PLUS;
Amersham Biosciences/GE Healthcare, Buckinghamshire, England) and used as
references of the culture conditions.
The cell suspension was plated on 24-well tissue culture plates (Nalge Nunc
International, Naperville, IL) at an NC density of 100 x 103 cells/mL, with the
exception of two cultures with NC densities of 60 and 200 x 103 cells/mL. The final
well volume was 0.93 mL. The culture medium, designed to support
megakaryocytic growth, contained either PF01 (MacoPharma, Tourcoing,
France) or IMDM (Stem Cell Technologies, Vancouver, Canada), serum substitute
BIT9500 (final concentrations 10 mg/mL bovine serum albumin, 10 g/mL
50
recombinant human insulin, and 200 g/mL iron-saturated human transferrin;

Stem Cell Technologies), and the human recombinant cytokines TPO (50 ng/mL),
IL-3 (10 ng/mL), and IL-6 (10 mg/mL), with SCF (10 ng/mL) in three
experiments, all from Stem Cell Technologies. Additionally, in three experiments,
the human-derived serum substitute HIT (albumin, insulin and transferrin; Stem
Cell Technologies) was used to assess cell growth without substances of animal
origin. The cells were incubated at 37C in 5% CO2 for 14 days (Heraeus Heracell
150; Thermo Fisher Scientific Inc, Waltham, MA), with the exception of two
cultures terminated at day 9 for technical reasons. Serial samples were collected
from the cultures for analysis at designated time points.
7.3.4 Flow cytometry
CD34+ cell enumeration was performed at the CB bank with a flow cytometer
(FACSCalibur and CellQuest software; Becton Dickinson, San Jose, CA) with a
dual-platform protocol (ISHAGE; Sutherland DR et al., 1996). The performance
of the analyses was externally evaluated six times a year with stem cells from
adult sources (NEQAS, Sheffield, UK).
In Study IV, erythroid cell surface antigens were analyzed with a flow cytometer
(FACSCalibur and CellQuest software; Becton Dickinson, San Jose, CA). Aliquots
of 100 L were collected from the liquid cultures, stained with FITC- or PE-
conjugated monoclonal antibodies against GlyA (CD235a; Caltag Laboratories,
Burlingame, CA) and CD71 (Becton Dickinson), lysed with FACS Lysing solution
(Becton Dickinson) according to the manufacturer's instructions, and washed
before analysis. Appropriate isotype controls (matched for fluorochrome as well
as immunoglobulin isotype and subclass) were used. Three fresh CB samples
were used as references for flow cytometry.
7.3.5 Cytocentrifugation
In Study IV, 200 L of cell suspension was cytocentrifuged (Cytospin SCA 0030;
Thermo Fisher Scientific Inc, Waltham, MA) onto glass slides (Shandon
Cytoslide; Thermo Fisher Scientific Inc) and fixed with methanol - acetone (1:3;
Merck & Co Inc, Whitehouse Station, NJ). The slides were stained with a GlyA-
specific monoclonal antibody and Evans Blue to counterstain the nuclei (Stem
Cell Technologies, Vancouver, Canada). Additional staining was performed with
benzidine to stain heme-containing proteins (Sigma-Aldrich Corp., St Louis, MO)
with the principle described by McLeod and colleagues (McLeod DL et al., 1974)
51
and with May-Grnwald-Giemsa (Merck & Co Inc). For the benzidine staining, the
slides were incubated in 1% bendizine for two minutes, then placed in 2.5%
hydrogen peroxide for one minute, and finally washed in distilled water for one
minute. For the May-Grnwald-Giemsa staining, the slides were incubated in
May-Grnwald solution for 5 minutes, in Giemsa solution for 15 minutes, and
finally washed in phosphate buffer three times and allowed to air dry. The slides
were evaluated with a camera-linked light microscope (Axioskop 2 Plus
microscope, AxioCam MRc camera, and Axiovision 3.1 software; Carl Zeiss,
Oberkochen, Germany).
7.3.6 ELISA assays for F1+2 and PF4
The samples for hemostasis assays were prepared at the Finnish Red Cross Blood
Service, Department of Hemostasis. Duplicate 10 mL CB samples (F1+2 assay;
Milian Gru-110 tubes, Milian International, Geneva, Switzerland) or 4.5 mL CB
samples (PF4 assay; Diatube H CTAD tubes, Becton Dickinson, Franklin Lakes,
NJ) were centrifuged at 2500 x g at 2 - 8C for 30 minutes. After the
centrifugation, the platelet-free plasma in the middle layer was separated,
mixed, and divided into 1 mL aliquots for storage at -80C until analysis.
The hemostasis assays were performed at the Department of Hemostasis with

commercial kits (Enzygnost F1+2; Dade Behring, Marburg, Germany; and
Asserachrom PF4; Diagnostica Stago S.A.S., Asnires sur Seine, France) utilizing
the ELISA principle. All the assays were performed by the same laboratory
technician, and all the samples of a given series and an internal control were
assayed at the same time.
In the assays, F1+2 or PF4 in the sample plasma was bound to a microtiter plate
coated with anti-human F1+2 or PF4 antibodies; after washing, peroxidase-
conjugated F1+2 or PF4 antibodies were bound to free antigenic determinants on
the plate. The bound enzyme activity was determined with a chromogenic
substrate to the peroxidase by measuring the color intensity, which is
proportional to the concentration of F1+2 or PF4, with a spectrophotometer
(Labsystem Multiskan MCC/340; Thermo Fisher Scientific Inc, Waltham, MA).
The F1+2 assays were performed for all the sample series of Study III; the PF4
assays were only performed for the 1998 and 2000 sample series, and the upper
limit of the measurement was 200 IU/mL in 1998 as the samples were not diluted
for retesting.
The effect of the variable anticoagulant-to-blood ratio inherent to CB collection

was assessed by calculating theoretical values for F1+2 and PF4 in native blood,
52
without the diluting effect of the anticoagulant. The total amount of F1+2 or PF4
in the sample was divided by the volume of native blood (excluding the entire
volume of anticoagulant).
7.4 Ethical considerations
The CB bank program was approved by the ethical committee of the collection
site (Department of Obstetrics and Gynecology, Helsinki University Central
Hospital) according to the provisions of the Declaration of Helsinki and European
and national laws. The Finnish CB Bank was authorized by the National Agency
for Medicines in accordance with tissue safety legislation in 2007. The mothers of
the CB donors provided written informed consent for participation in the program
and permission to use the CB for product development and quality control
purposes.
7.5 Statistics
StatsDirect software (StatsDirect Ltd., Cheshire, UK) was used for statistical
analyses. The normal distribution of the study materials was assessed before
selecting the appropriate statistical methods for each analysis. In Studies I and
II, associations between selected parameters were analyzed with simple and
multivariate linear regression. The concordance of the assessments of different
evaluators was analyzed with the Kendall coefficient of concordance (W). In
Study III, differences within groups were evaluated with the Mann-Whitney-U
test, and associations with Spearman's rank correlation. Two-sided p values of
less than 0.05 were considered statistically significant.
53
8 RESULTS
8.1 Cord blood progenitor cell content (I - II)
The cell characteristics of the 167 CB collections are presented in Table 4. In the
CB units accepted for long-term storage at the time of the study (n = 161),
platelet depletion during processing was 62% (range 40 - 84).
Table 4. Cell characteristics of 167 consecutive processed cord blood

collections analyzed with Sysmex K1000 (Studies I - II).
Median Range
Collected cord blood*
Collected volume, mL 92 42 150
9
Nucleated cell concentration, x 10 /L 15.1 8.5 39.7
7
Total nucleated cells, x 10 139 80.4 425
6
CD34+ cells, x 10 4.09 0.91 31.4
Platelet count, x 109/L 270 161 607
Mean platelet volume, fL 8.7 7.5 11.5
Cord blood unit before

cryopreservation
Volume, mL 20.4 19.4 22.1
Total nucleated cells, x 107 103 59.0 318
+ 6
CD34 cells, x 10 3.66 0.57 31.4
6
CFU-TOT, 10 colonies 2.25 0.53 5.42
9
Platelet count, x 10 /L 448 225 920
* The concentrations are standardized (see Materials and methods for

details).
Without anticoagulant (20-30 mL) and the initial sample for cell
counting.
CFU-TOT = Total colony-forming units
54
RESULTS
Collected CB volume was associated with the NC concentration in collected CB (r

= -0.31, p < 0.001). Collected volume was also associated with the CFU-TOT
number (n = 166, r = 0.48, p < 0.001), CD34+ cell number (r = 0.41, p < 0.001),
and TNCs (r = 0.55, p < 0.001) in the CB unit. In addition, the NC concentration in
collected CB was associated with CB unit CFU-TOT (n = 166, r = 0.23, p < 0.01)
and CD34+ cell (r = 0.36, p < 0.001) numbers.
Table 5. Multiple regression analysis of the effect of platelet

characteristics on cord blood hematopoietic progenitor cell counts
(Study I, unpublished data).
Cord blood unit total nucleated cell number
Predictor variables Unstandardized coefficient b p value

Intercept b0 = -74.34 0.16
Platelet count b1 = -0.024 0.62
Mean platelet volume b2 = 22.16 < 0.0001
Cord blood unit CD34+ cell number

Intercept b0 = -12.64 0.005
Cord blood unit CFU-TOT number

Intercept b0 = -1.73 0.162
CFU-TOT = Total colony-forming units
8.2 Platelet characteristics (I)
CB platelet count correlated with MPV (r = -0.39, p < 0.001). MPV was associated
with TNCs in the CB unit (r = 0.35, p < 0.001). MPV was also associated with the
total CD34+ cell number (r = 0.42, p < 0.001) and the CFU-TOT number (n =
55
RESULTS
166; r = 0.35, p < 0.001) in the CB unit.
Platelet count was associated with TNCs (r = -0.17, p < 0.05) and the total
CD34+ cell number (r = -0.27, p < 0.001), as well as the CFU-TOT number (n =
166; r = -0.21, p < 0.01) in the CB unit. However, when adjusting for MPV,
platelet count was not statistically significantly independently associated with CB
unit TNCs, CD34+ cells, or the CFU-TOT number (Table 5).
8.3 Perinatal characteristics (II)
Of the 167 neonates, 104 (67%) were born through Cesarean section. This was
due to the high proportion of elective Cesarean sections during the CB bank
operating hours and does not reflect general Cesarean section frequency at the
maternity unit. 52% were male. Other perinatal characteristics are presented in
Table 6.
Umbilical arterial pH correlated with CB unit TNCs (r = -0.29, p < 0.001), total
CD34+ cells (r = -0.31, p < 0.001), and the CFU-TOT number (n = 166, r = -0.32,
p < 0.001).
Both absolute and relative birth weight correlated with CB unit TNCs (r = 0.20; p
= 0.008 for both). Absolute and relative birth weight correlated with total CD34+
cells in the CB unit (r = 0.17, p = 0.03 and r = 0.18, p = 0.02). Relative birth
weight correlated with the CFU-TOT number in the CB unit (n = 166, r = 0.22, p =
0.004). After the exclusion of outliers, the association of absolute birth weight
and CB unit CFU-TOT number did not reach statistical significance (n = 164, r =
0.15, p = 0.05).
Table 6. Perinatal characteristics of 167 consecutive processed cord blood collections

from term infants (Study II).
Umbilical Birth Relative Placental Relative

arterial weight birth weight placental
pH g weight g weight
Median 7.28 3784 +0.5 645 -0.2

Minimum 7.04 2490 -2.4 380 -2.1
Maximum 7.40 4975 +3.4 1000 +2.4
56
RESULTS
Placental weight correlated with TNCs (r = 0.27, p < 0.001), CD34+ cells (r =
0.29, p < 0.001) and the CFU-TOT number (n = 166, r = 0.33, p < 0.001) in the
CB unit. Similarly, relative placental weight correlated with CB unit TNCs (r =
0.24, p = 0.001), CD34+ cells (r = 0.28, p < 0.001), and the CFU-TOT number (n
= 166, r = 0.34, p < 0.001). Using the placental weight / birth weight ratio
(median 17.0%, range 11.8 - 24.5) in the analyses did not improve the
correlations (CB unit TNCs, r = 0.19, p = 0.01; CD34+ cells, r = 0.24, p = 0.002;
and CFU-TOT, n = 166, r = 0.31, p < 0.001).
The mode of delivery affected the correlations of perinatal factors and CB unit
HPCs (Table 7). Birth weight was associated with HPCs only in Cesarean
sections. In contrast, the correlation of umbilical arterial pH with CB unit CD34+
cells and CFUs was higher in vaginal deliveries than in Cesarean sections.
Table 7. Correlations of perinatal factors and cord blood unit cell counts grouped by mode
of delivery (Cesarean section, CS, n = 104, and vaginal delivery, VD, n = 63) (Study II).
Umbilical Birth Relative Placental

arterial pH weight birth weight weight
CS VD CS VD CS VD CS VD
Total nucleated
cells 0.34 0.25* 0.24* NS 0.25* NS 0.23* 0.37
CD34+ cell
number 0.23* 0.35 0.25* NS 0.29 NS 0.33 0.31*
CFU-TOT
number 0.24* 0.45 0.20* NS 0.26 NS 0.36 0.30*
*p < 0.05, p < 0.01. For the other correlations, p < 0.001.
CFU data for one Cesarean section delivery were missing.
CFU-TOT = Total colony-forming units. NS = Not statistically significant.
8.4 Models for the estimation of hematopoietic progenitor cells

(I - II)
The selection of variables for multiple regression analyses was based on data
obtained from univariate analyses, and the aim was to create combined models
57
RESULTS
for the estimation of CB unit HPCs, as well as to confirm the individual

associations of each analyzed variable with CB unit HPC counts.
In multiple regression analyses of platelet characteristics, CB unit HPCs were

best predicted with MPV, collected volume, and the collected CB NC concentration
(total CD34+ cells, R2 = 0.50 and CFU-TOT number, R2 = 0.42).
When perinatal data (umbilical arterial pH, birth weight, and placental weight)
were analyzed, the estimation of CB unit CD34+ cells was most accurate with
2
umbilical arterial pH and collected CB TNCs (R = 0.52). CB unit CFU-TOT were
most accurately estimated with umbilical arterial pH, placental weight, and
collected CB TNCs (R2 = 0.43).
When the models with platelet characteristics and perinatal data were combined,
CB unit CD34+ cells were most accurately estimated with MPV, umbilical arterial
pH, and collected CB TNCs (R2 = 0.55). The CB unit CFU-TOT number was
predicted with MPV, placental weight, umbilical arterial pH, and collected CB
TNCs (R2 = 0.45; all unpublished data).
In addition, models utilizing perinatal data and collected CB volume, instead of

TNCs, were tested. The CB unit CD34+ cell number was most accurately
estimated with umbilical arterial pH and collected CB volume (R2 = 0.29), and the
CFU-TOT number of the CB unit was predicted with umbilical arterial pH,
placental weight, and collected CB volume (R2 = 0.36).
The relative importance of the tested perinatal factors was dependent on the
mode of birth. In vaginal deliveries, both CB unit CFUs and total CD34+ cells were
most accurately predicted with umbilical arterial pH and collected CB TNCs (R2 =
0.53 and R2 = 0.75, respectively). In Cesarean sections, placental weight, birth
weight, and collected CB TNCs predicted CFUs (R2 = 0.37), and CD34+ cells were
most accurately predicted with placental weight and TNCs (R2 = 0.36).
8.5 Hemostasis activation in cord blood collection (III)
The study was undertaken to evaluate the effect of changes in CB bank

procedures on hemostasis activation during CB collection. In 1998, the CB
collection procedure was validated; in 2000, the performance of a new nurse-
midwife after initial training was evaluated; and in 2006, coagulation activation
in a new collection bag was assessed.
Of the 27 CB donor infants, 15 (56%) were born by vaginal delivery, and 18
58
RESULTS
Table 8. Collection characteristics and levels of the hemostasis markers in the three sample
series (Study III).
Collected CPD / Collection Time to F1+2 PF4

volume blood ratio duration processing
mL* 1:x minutes hours nmol/L IU/mL
Year 1998
Median 52 2.1 6.4 1.7 2.8 117
(Range) (8 - 87) (0.3 - 3.5) (3.7 - 9.0) (0.9 - 3.7) (0.8 - 11) (34 - 200)
n = 11 n = 10
Year 2000
Median 42 1.7 2.6 2.1 0.7 104
(Range) (17 - 106) (0.7 - 4.3) (1.5 - 2.8) (1.5 - 3.0) (0.3 - 1.8) (27 - 314)
n = 10
Year 2006
Median 68 3.3 3.1 2.1 0.7 NA
(Range) (58 - 140) (2.7 - 4.7) (2.7 - 4.3) (1.2 - 4.2) (0.3 - 3.8) NA
n=6 n= 5
All samples
Median 57 2.3 3.2 2.0 1.3 113
(Range) (8 - 140) (0.3 - 4.7) (1.5 - 9.0) (0.9 - 4.2) (0.3 - 11) (27 - 314)
n = 27 n = 25 n = 21
*Weight of the collection bag and anticoagulant volume excluded.

Time from cord blood collection to processing of the blood sample.
CPD = Citrate phosphate dextrose. F1+2 = Prothrombin activation fragment 1+2.

PF4 = Platelet factor 4. NA = Not available.
(67%) were male. The median gestational age of the infants was 278 days (range
262 - 289) and the median birth weight 3570 g (n = 21; range 2600 - 5230).
Collection characteristics of the three sample series are presented in Table 8.
The differences in duration of the collection procedure in the three sample series
were statistically significant (1998 and 2000, p < 0.001; 1998 and 2006, p =
0.001; 2000 and 2006, p = 0.002).
The median F1+2 level was 2.8 nmol/L in 1998, 0.7 nmol/L in 2000, and 0.7
nmol/L in 2006 (Table 8). The levels were statistically significantly higher in
1998 than in the later sample series (1998 and 2000, p < 0.001; 1998 and 2006,
p = 0.01). There was no statistically significant difference in F1+2 levels in
vaginal deliveries or Cesarean sections (n = 15; median 0.8 nmol/L, range 0.3 -
5.0, and n = 12; median 1.9 nmol/L, range 0.4 - 11, respectively).
59
RESULTS
The anticoagulant-to-blood ratio was not associated with the F1+2 level. To
further assess the effect of the varying anticoagulant-to-blood ratio, inherent to
CB collection procedures, on the markers of hemostasis activation, standardized
values for the hemostasis markers in native blood were calculated. The
theoretical median level of F1+2 in the entire study was 2.0 nmol/L (range 0.4 -
18).
The median PF4 level was 117 IU/mL in 1998 and 104 IU/mL in 2000 (Table 8).
In 1998, the upper limit of PF4 measurement was 200 IU/mL, and the PF4 level
was at least 200 in three of the samples. The difference in PF4 levels was not
statistically significant. The PF4 level was not statistically significantly different in
vaginal deliveries and Cesarean sections (n = 12; median 93 IU/mL, range 27 -
314, and n = 9; median 117 IU/mL, range 68 - 200, respectively).
The anticoagulant-to-blood ratio was not associated with the PF4 level. The
theoretical median level of PF4 in native blood was 161 IU/mL (n = 21, range 53 -
825). However, this calculation may be confounded by the upper limit of PF4
measurement (200 IU/mL) in the first sample series.
Figure 5. The association of F1+2 (prothrombin activation fragment 1+2) and PF4
(platelet factor 4) (Study III). The 1998 samples are marked with filled squares and the
2000 samples with unfilled squares. The correlation coefficient Rho is shown for the entire
study. The association remained statistically significant when outliers, marked with an
asterisk, were removed (n = 18; Rho = 0.57, p = 0.01).
60
RESULTS
There was an association between F1+2 and PF4 levels in the entire study (n =
21; Spearman's rank correlation coefficient Rho = 0.59, p = 0.006; Figure 5) as
well as in the individual sample series (year 1998, n = 11, Rho = 0.80, p = 0.004;
and year 2000, n = 10, Rho = 0.77, p = 0.01). F1+2 or PF4 levels were not
associated with the NC concentration, hemoglobin level, hematocrit, or platelet
count in the CB collections.
8.6 The hematopoietic potential of cord blood units in vitro
8.6.1 Megakaryocytic cells in a semisolid assay (I)
Three independent investigators analyzed the megakaryocytic colony assays,

and a mean score was calculated for each CB unit. The scores given by the three
investigators were concordant (Kendall coefficient of concordance W = 0.91; p <
0.001), although the distribution of scores varied. Megakaryocytic colonies could
be grown from all 24 samples (mean score 1.92, range 0.67 - 3.00); however, the
growth varied widely among the samples.
Figure 6. The gating strategy in flow

cytometry analyses (Study IV).
Fresh cord blood. Gates R1 and R5
(heterogeneous cell debris) were
excluded from the analyses. Gate
R2: Medium sized events with
variable granularity. Gate R3: Large
events with variable granularity.
Gate R4: Medium sized events with
low granularity.
8.6.2 Erythroid cells in liquid cultures of thawed cord blood (IV)
The storage time of cryopreserved CB units was 1 month - 10 years. The flow
cytometry gating strategy was based on the fresh CB references (Figure 6) and
61
RESULTS
the isotype controls. Based on the results, gates R1 and R5 were excluded from
the analyses of antigen expression and thus, the following results refer to gates
R2 - R4.
At day 0 of the cultures of thawed CB units, 5.9% of the events expressed GlyA.
14% of all events were CD71+, and 4.5% were double-positive for GlyA and
CD71. The majority of both GlyA and CD71 events were medium sized with low
granularity (gate R4).
At day 9, 14% of the events were GlyA+, and 53% expressed CD71. 12% of all
events expressed both GlyA and CD71. The events were medium-sized with
variable granularity (gates R2 and R4).
Figure 7. Cytospin preparations of thawed cord blood cell cultures (Study IV). Staining
with glycophorin A (GlyA; CD235a) and Evans Blue.
62
RESULTS
At day 14, 9.7% of all events expressed GlyA, and 35% were positive for CD71.
5.3% were GlyA+CD71+. The majority of GlyA- and CD71-positive events were
medium sized with variable granularity (gate R2), but events were observed in all
the gates.
On Cytospin slides, GlyA-positive cells of the same size as those in the reference
samples were observed at day 9, and the cells increased by day 14 (Figure 7).
The observed benzidine-positive cells appeared dense and smaller than the
GlyA+ cells.
Figure 8. Cytospin preparations of thawed cord blood cell cultures with added stem cell
factor (SCF) (Study IV). Staining with glycophorin A (GlyA; CD235a) and Evans Blue.
When SCF was included in the cytokine mix with TPO, IL-3, and IL-6, 12% and
14% of the events were GlyA+ at days 9 and 14, respectively. Abundant GlyA-
positive cells were observed on Cytospin slides at day 9 (Figure 8). In the
experiments with human serum albumin instead of bovine serum albumin, no or
scarce GlyA+ cells were observed at days 9 - 14 of the cultures.
63
9 DISCUSSION
The aim of this study was to investigate factors related to the selection and
quality of CB collections for banking. In Studies I and II, novel markers for the
selection of CB collections with high HPC counts were sought. In Study I, high
MPV, thought to indicate general hematopoietic activity, correlated with high CB
unit CD34+ cell and CFU-TOT numbers. In Study II, factors hypothesized to be
associated with perinatal stress, in particular a low-normal umbilical arterial pH,
correlated with high HPC counts in the CB unit. The effect of the perinatal factors
depended on the mode of delivery, with the effect of umbilical arterial pH
pronounced in vaginal deliveries. To enable the use of these novel markers in the
selection of CB collections for processing, theoretical models employing current
CB selection criteria (primarily the TNC count) and the aforementioned markers
were developed.
In Study III, activation of the hemostatic system during CB collection was

observed to decrease when international standards were followed, as well as with
increased experience of the CB bank staff. As hemostasis activation may have
uncontrolled effects on CB cells, assessment of the level of activation could be
implemented as a part of process control in CB banks.
The hematopoietic potential of CB in vitro was assessed in Studies I and IV. In

Study I, a simple scoring system to analyze megakaryocytic growth in processed
CB was developed. Such scoring systems may enable analysis of the growth
potential of a single cell lineage in the CB unit. In Study IV, the erythropoietic
potential of unseparated thawed CB without exogenous erythropoietin was
assessed to characterize cell growth in the heterogeneous cell population of the
infusible CB unit.
9.1 The cell content of cord blood units
The suitability of CB hematopoietic stem cell transplantation for adult patients

has been established in recent years. In fact, more adults than children are
currently transplanted with CB worldwide (Barker JN et al., 2009b). To meet the
need for the high TNC doses required for transplantation into adult patients, the
cell requirements for CB collections have risen over the years. CB unit
processing, cryopreservation, and thawing have been reported to result in
mostly unavoidable cell losses, increasing the need for high collected HPC
64
DISCUSSION
numbers.
The mean TNC count of the CB collections processed during 2004 - 2005 and
analyzed in this study was higher, at 139 x 107, than that reported from the same
bank from the years 1999 - 2000 (103 x 107; Aroviita P et al, 2003). Thus, the
rising HPC requirements are reflected in CB bank practices through, e.g., stricter
criteria for the CB collections accepted for processing, as well as more developed
processing techniques.
Double CB unit transplantation has been used as a strategy to achieve an

adequate cell dose for adult patients (Verneris MR et al., 2009). However, the
dynamics of engraftment in double CB unit transplantation are still under
investigation. On the other hand, the growing number of CB units in worldwide
registries results in higher HLA diversity, which may eventually decrease the TNC
requirement, as HLA matching has been reported to decrease the cell number
required for successful CB transplantation (Barker JN et al., 2010).
HLA-matched CB is still unavailable for a significant proportion of patients. For CB

units mismatched at 1 - 2 HLA loci, a TNC dose of at least 2.5 - 5.0 x 107/kg
patient weight has been reported to be required to achieve minimal transplant-
related mortality (Barker JN et al., 2010). Thus, for an adult patient weighing 70
kg, at least 175 - 350 x 107 TNCs would have to be infused. Only a small
proportion of the CB units currently in cryostorage fulfill this requirement.
Theoretically, to achieve a TNC content of 300 x 107 in the CB collection alone,
150 mL of CB, a volume at the higher end of the normal range, would have to be
9
collected from a neonate with 20 x 10 /L NCs, which is, again, at the upper limit of
the reference range. Clearly, CB banking cannot rely on such random collections.
As the HPC number available for collection in the placenta after birth is restricted,
optimization of donor selection, of collection practices, and of CB banking
procedures is the safest way to increase the HPC number available for
transplantation. Additional factors that predict a high HPC number in the CB unit
have to be sought. In this study, a few such factors were identified.
9.2 Platelet characteristics as determinants of hematopoietic

progenitors
MPV, as an indicator of platelet production (Levin J & Bessman JD, 1983), may
also indicate general hematopoietic activity in the healthy neonate. In the
present study, MPV was associated with the numbers of CD34+ cells and CFUs in
the CB unit; such an association has not been reported before. Other possible
65
DISCUSSION
indicators of hematopoietic activity include the number of reticulated platelets,

representing the immature platelet fraction (Ault KA et al., 1992), and the
platelet - large cell ratio (Kaito K et al., 2005). However, the aforementioned
markers have not been studied in the context of CB banking.
Platelet count also correlated with CB unit HPCs, but the correlation was weaker
than that of MPV. When the effect of MPV was excluded, platelet count was not
independently associated with CB HPCs. Thus, the association observed in
univariate analyses was most likely due to the inverse correlation of MPV and
platelet count. This inverse correlation has been previously reported in adult
peripheral blood (O'Brien JR, 1974; Bessman JD et al., 1981; Bain BJ, 1985).
Measurements of MPV and platelet count are available in automated hematology

analyzers. Thus, they could contribute to the selection of CB collections for
processing. These data can be implemented as a part of a theoretical model for
the estimation of CB unit CD34+ cells and CFUs. Such a model was proposed in
this study and included MPV, as well as collected CB volume and NC
concentration, currently utilized to select CB collections for processing. With
these models, data available before processing could be applied to enable the
processing of CB units with high HPC numbers.
9.3 Perinatal stress factors for the selection of cord blood

collections
Perinatal stress in healthy infants, measured in this study as an umbilical arterial

pH in the lower end of the reference range, was associated with high HPC counts
in the CB unit. Similar observations have been made earlier in collected CB (Lim
FT et al., 2000; Aufderhaar U et al., 2003; Solves P et al., 2005). The effect may
be increased in sick neonates in whom the range of umbilical arterial pH is
greater. However, CB should not be collected in any circumstances associated
with significant fetal distress, in accordance with international standards.
Several other obstetric and perinatal factors have been reported to be associated
with CB HPC counts. The effect of birth weight and placental weight, reported
previously, was confirmed in the present study. The correlation of placental
weight and CB HPCs may stem from the hematopoietic activity previously
observed in the placenta. However, birth weight and placental weight are
currently only applied in the selection of CB collections indirectly, through their
effect on collected CB volume. A theoretical model utilizing perinatal
characteristics and TNCs measured from collected CB was proposed in the
present study. CB unit CD34+ cells could be estimated with umbilical arterial pH
66
DISCUSSION
and collected CB TNCs, and placental weight could be added to estimate the CB
unit CFU-TOT number. The calculated HPC estimates could then be compared
with the acceptance criteria for CB processing set by each bank.
Elective Cesarean sections and vaginal deliveries are physiologically different.

The physiological changes associated with normal vaginal delivery may affect the
cell composition of CB through, e.g., mobilization of HPCs (Lim FT et al., 2000).
However, previous studies comparing factors associated with CB HPCs in the two
modes of delivery are scarce. In one study, the correlation of birth weight and
collected CB CD34+ cell concentration was reported to be higher in Cesarean
sections as compared to vaginal deliveries, and CFU-TOT, in fact, was reported to
correlate with birth weight only in Cesarean sections (Aroviita P et al., 2004).
Parallel observations were made in the present study. Furthermore, in the
present study, the correlation of umbilical arterial pH with CB unit CD34+ cells
and CFUs was stronger in vaginal deliveries. These observations also extended to
the multivariate models created for each mode of delivery so that different
factors were applied to estimate CB unit CD34+ cell and CFU-TOT numbers in
vaginal deliveries and Cesarean sections. Thus, the estimation of CB HPC
numbers may be improved through the adaptation of CB selection criteria
depending on the mode of delivery.
As all the CB collections in this study were performed ex utero, the effect of the
collection strategy on CB unit HPCs was not assessed. Current knowledge of the
differences of in and ex utero collections is inadequate, and the optimal CB
collection circumstances remain undetermined.
9.4 Hemostasis activation during cord blood collection
In addition to optimizing HPC counts, CB banks have focused on providing safer

units through e.g. careful testing for transmissible disease and review of medical
histories. However, in a study of 268 CB units selected for transplantation and
reviewed at the transplant center, over half of the units were reported to have
quality control issues (McCullough J et al., 2005). The absence of quality issues
was recently reported to be associated with membership in CB registries
enforcing strict CB quality standards (Haspel RL & Lamontagne D, 2010).
Hemostasis activation has not been systematically assessed as a part of CB

quality control, although CB collections with visible clots have been discarded or
have undergone special procedures to remove the clot. Due to the many
interconnections of hemostasis with other organ systems, coagulation and
platelet activation could have unwanted effects on the quality of CB units.
67
DISCUSSION
Chemokines, including PF4, released from alpha granules upon platelet

activation are thought to contribute to the side effects of platelet transfusion
(Boehlen F & Clemetson KJ, 2001). In addition, PF4 has been reported to inhibit
megakaryocytopoiesis (Gewirtz AM et al., 1989). Platelet-derived chemokines
have also been reported to promote mesenchymal stromal cell expansion
(Doucet C et al., 2005; Schallmoser K et al., 2007).
In the present study, the levels of the selected hemostasis activation markers
(F1+2 and PF4) were higher in the sample series collected during the set-up of
the CB bank than in those collected after changes in established bank processes.
This may reflect refined CB banking procedures and increased expertise of the
bank staff. In addition, the longer CB collection time in the first series may have
increased the risk of hemostasis activation.
The comparison of the observed F1+2 and PF4 levels to those published earlier is
difficult, as sample selection and collection techniques may be different.
However, in one study of autologous umbilical CB collections, similar mean levels
of F1+2 to those in the later sample series of the present study were reported
(Ballin A et al., 1995). On the other hand, in another study, high F1+2 levels were
observed in the CB of healthy neonates (Hyytiainen S et al., 2006). Previous
studies on CB PF4 levels have employed different analysis techniques than the
present study, and, thus, the results are not comparable; both increased and low
PF4 levels and activities have been reported in neonates (Alebouyeh M et al.,
1974; Suarez CR et al., 1988).
Although F1+2 and PF4 are indicative of mutually dependent processes, namely,
coagulation and platelet activation, their correlation was not perfect, and high
PF4 levels were also observed in samples with low F1+2. As PF4 is abundant in
platelets, detectable levels of it may be released as a response to slight
imperfections in sample collection, processing, and storage. Markers of
coagulation system activation may be more suitable for CB quality control
purposes than those of platelet activation.
As one molecule of F1+2 is released when one molecule of thrombin is formed,

F1+2 is a direct indicator of the presence of thrombin in the sample. Thus, it could
be utilized to assess coagulation activation during CB collection. Optimally,
hemostasis activation should be assessed in CB collections selected for
processing, with predefined acceptance criteria. However, this may be difficult as
any additional sample taken from the CB unit will decrease the HPC number
available for transplantation. Thus, the activation markers could be assessed
periodically from random samples as a part of process control in CB banks.
68
DISCUSSION
9.5 Culture assays of cord blood units
To avoid labor-intensive counting of megakaryocytic colonies, a simple scoring

system for evaluating colony growth in a semisolid assay was developed in this
study. The fixed and stained culture slides were stored and could be re-evaluated
for morphology and the exact number of colonies, if necessary. The
megakaryocytic growth scores given by three independent investigators were
concordant, suggesting that the scoring system could be standardized for use in
routine analyses.
24 random CB collections, 21 of which were accepted for long-term storage for

future clinical use, were analyzed after volume reduction. Although
megakaryocytic colony growth was observed in all the samples, the growth
varied widely. Similar variation has been observed in a study of 134 CB units, in
which the concentration of megakaryocytic CFUs was reported to vary nearly
100-fold (Drygalski A et al., 2000).
Cells of the erythroid lineage were observed in liquid cultures of thawed

unseparated CB units. Such growth without exogenous EPO has not been
reported earlier. Although the differentiation stage of the erythroid cells was not
explored further, the cells did express antigens for both immature and more
developed erythroid precursors (CD71 and GlyA). The proportions of CD71- and
GlyA-positive events were highest at day 9 of the cultures, and, thus, the culture
system may not support long-term erythroid growth. In addition, there was a
tendency for the observed erythroid cells to become more complex (moving from
gate R2 to gate R4) as the culture progressed.
RBCs and megakaryocytes share a common bipotent progenitor (Debili N et al.,

1996). The megakaryocytopoietic cytokine TPO is homologous to EPO
(Kaushansky K et al., 1995) and may be partially responsible for the erythroid
growth observed in the cultures. In addition, the supporting cell types present in
the culture of unseparated CB may secrete endogenous cytokines. Contact with
macrophages has been reported to lead to RBC differentiation (Hanspal M &
Hanspal JS, 1994).
The large-scale production of RBCs for transfusion purposes is a complex process

and requires carefully defined, multi-step culture conditions (Douay L & Andreu
G, 2007; Migliaccio AR et al., 2009). The cell growth observed in the present
study is an indication that cryopreserved and thawed CB units retain the capacity
for erythroid growth in vitro. It also underscores the complexity of the cytokine
network affecting cell growth and differentiation in thawed CB units.
69
DISCUSSION
9.6 Methodological aspects of the study
The CB units analyzed in this study were collected and processed at the Finnish
CB Bank according to international standards. The data obtained from the CB
bank registry were reviewed for completeness and accuracy, and the
characteristics of the study samples were assessed before selecting the
appropriate statistical methods for each analysis. The study was ethically
justified, as judged by an external ethical committee, as knowledge of neonatal
and CB physiology is essential for the development of CB banking.
In Studies I - II, the study sample was collected retrospectively; however, as all
the CB collections accepted for cryostorage during the study period were
included in the analyses, the study design was effectively prospective. However,
the observed associations should be confirmed in another, independent CB bank.
In Study III, due to the variable collected CB volume, the anticoagulant-to-blood

ratio was not constant in the samples. To exclude the effect of the variation,
theoretical levels for F1+2 and PF4 in native blood, without the volume of the
anticoagulant, were calculated, and the levels were parallel to those in the
original measurements. In the first sample series, the upper limit of PF4
measurement was 200 IU/mL, and accurate quantitation of PF4 was thus not
achieved in three samples. To minimize interassay variability, all the samples of a
given series were analyzed simultaneously, and the same laboratory technician
performed all the analyses. However, the results could not be reliably compared
with previously published data due to the lack of international standards for the
assays.
The reliability of the MK scoring procedure developed in Study I was ensured by

assessing the concordance of the scores given by three independent
investigators. In addition, four semisolid assay chambers were analyzed per each
CB unit. The CB units analyzed in Study IV were those cryopreserved by the
Finnish CB Bank for future clinical use and thawed for quality control purposes,
providing an invaluable study material, although the number of CB units
available for analysis may seem small. Although some RBCs are present in
unseparated thawed CB units, the increased proportion of CD71-positive events,
as well as events double-positive for GlyA and CD71, in the cultures at days 9 - 14
led to the conclusion that the observed erythroid cells were formed in the culture.
70
DISCUSSION
9.7 The future of cord blood banking
The number of CB units provided for unrelated transplantation has risen each
year (World Marrow Donor Association, 2009), and the rise is likely to continue in
the future. Several recent studies have reported promising results with reduced-
intensity conditioning regimens before CB transplantation. These new
conditioning regimens may enable CB transplantation in older patients in whom
morbidity related to myeloablative conditioning regimens would have prevented
hematopoietic stem cell transplantation in the past.
In addition to hematopoietic stem and progenitor cells, CB and its surrounding

tissues, such as the placenta and Wharton's jelly of the umbilical cord, have been
reported to contain mesenchymal stromal cells (Lee OK et al., 2004; Baksh D et
al., 2007; Bakhshi T et al., 2008; Brooke G et al., 2009). To optimize the yield of
these non-hematopoietic cells, cord blood banking processes may have to be
developed further. Thus, the entire scale of clinical uses for CB and the structures
surrounding it is just being revealed. In the future, new evidence-based policies
and procedures for CB collection, processing, and storage should be developed
and - through CB banking standards, accreditation, and licensure - implemented
worldwide.
71
10 CONCLUSIONS
Optimal preservation of HPCs has to be ensured at every point of the CB banking

process from collection to processing, cryopreservation, and thawing to provide
CB units for successful transplantation. There have been attempts to increase the
HPC content of CB units through, e.g., improved selection of CB collections for
processing. In the present study, CB platelet characteristics (MPV) and factors
associated with perinatal stress (umbilical arterial pH and placental weight) were
observed to affect the CB unit HPC content. Theoretical models for the estimation
of CB unit HPC counts were developed to enable the use of these factors in the
selection of CB collections. In the information age, such models are easily
adaptable for routine practice.
In addition, as vaginal deliveries and Cesarean sections are physiologically

different, adapting CB selection criteria depending on the mode of delivery may
be required to improve the HPC contents of CB collections.
Coagulation and platelet activation are scarcely evaluated in CB banks. Platelet

activation, in particular, is closely linked to the inflammatory response, and,
further than that, has been reported to play a role in stem cell differentiation,
including inhibition of megakaryocytopoiesis and a suggested role in
mesenchymal stromal cell expansion. For both clinical use and in vitro
applications, the integrity of the cells in collected CB, as well as preservation of
CB HPCs, must be ensured. Thus, care should be taken in CB collection to avoid
unnecessary hemostasis activation. In the present study, coagulation activation
decreased when the CB collection procedure was carefully planned and
international CB banking standards were followed. Assessment of the activation
associated with standard CB collection procedures could be implemented as a
part of process control in CB banks. Novel hemostasis markers may have to be
sought to improve the reliability of the analyses.
HPC culture assays provide a direct estimate of the short-term hematopoietic

potential of the CB unit. To reduce the workload associated with the analysis of
these assays, simple scoring systems for robust analysis of the growth potential
of various cell lineages can be created. The scored assay slides from, e.g.,
semisolid cultures can be stored in CB banks and re-evaluated prior to the
selection of CB units for transplantation. Cryopreserved and thawed,
unseparated CB units retain the capacity for cell growth in vitro, providing a
unique platform for analyses of the complex network of cytokines and cell-to-cell
interactions in the heterogeneous cell population of the CB unit.
72
CONCLUSIONS
In conclusion, CB is a versatile, unique resource for both clinical and research

applications. Vast amounts of data are accumulating in CB banks worldwide;
these data can and should be utilized for further studies of both CB banking
processes and neonatal physiology.
73
11 ACKNOWLEDGEMENTS
This work was carried out at the Finnish Red Cross Blood Service, Helsinki,
Finland, during the years 2004 - 2010. Several people have contributed to this
work; I would like to express my gratitude to all of them, and especially:
The Director of the Institute, Professor Jukka Rautonen, for securing splendid
working facilities. The Director of Advanced Therapies, Research and
Development, Docent Kari Aranko, for providing excellent research facilities, and
for scientific collaboration. The former Director of Clinical Services and Product
Development, Professor Gunnar Myllyl, for supporting the Finnish Cord Blood
Bank project and scientific work at the Blood Service.
My supervisor, Docent Riitta Kekomki, for her determined guidance through

these years, and for sharing with me her vast knowledge of cord blood banking.
The reviewers of this thesis, Docent Pekka Riikonen and Docent Mervi Taskinen,
for insightful comments and inspiring discussions.
My closest collaborators, medical student Mikko Eskola and Sari Mttnen, M.Sc.,
for their advice and friendship through the years. Without them, this work would
surely never have been finished.
My other co-authors: Pekka Aroviita, M.D., Ph.D., for sharing his knowledge of
cord blood banking processes, and for his infectious enthusiasm. Susanna
Sainio, M.D., Ph.D., and Docent Vilho Hiilesmaa, for bringing a valuable clinician's
view into this study. Elina Vahtera, Ph.Lic., for her excellent comments and vast
knowledge of hemostasis.
The staff of the Finnish Cord Blood Bank, for providing me with the materials of
this study and for tirelessly browsing through the archives in search of seemingly
irrelevant data. I would also like to take the opportunity to thank the voluntary
mothers that enable public cord blood banking.
The staff of the Hemostasis Laboratory at the Finnish Red Cross Blood Service, for
performing and interpreting hemostasis assays for the purposes of this study.
Kaija Javela, Ph.D., and Marketta Veihola, Ph.D., for their advice and
encouragement, as well as their continuous interest in my research project.
74
ACKNOWLEDGEMENTS
Seppo Sarna, Professor of Biometrics at the University of Helsinki, for

straightforward statistical consultations. Marja-Leena Hyvnen and Maija
Ekholm, for excellent library services. Juha Eronen and Sanna Kortelainen, for
answering my questions no matter how trivial they may have seemed. Pirjo Nick,
for secretarial assistance. Veikko Rekunen, M.A., for skilful revision of the
language of this thesis.
My colleagues at the department of Research and Development, for their advice

and for the discussions both on and off scientific topics. You are too many to
name.
I would like to express my sincerest thanks to my dear friends, both near and far,
for all the fun times we have shared, for the numerous self-help sessions over tea
or coffee, and for always having someone to knit with.
My parents, Marja and Veikko, are thanked for their love and their support in so
many ways. I would also like to thank my parents-in-law, Arja and Jouko, for
believing in me, and my sister-in-law Sanna and her family, for their friendship.
My sweetest thanks are reserved for my husband Aki, for everything.
Financial support from the Finnish Red Cross Blood Service Research Fund, the
Nona and Kullervo Vre Foundation, the Finnish Association of Hematology, the
Blood Disease Research Foundation, and the Finnish Medical Society Duodecim is
warmly acknowledged.
Helsinki, October 2010
75
12 REFERENCES
Abboud M, Xu F, LaVia M & Laver J. Study of early hematopoietic precursors in human cord
blood. Exp.Hematol. 20:1043-1047, 1992.
Adami V, Malangone W, Falasca E, Marini L, Risso A, Crini S, Toniutti E, Passoni Ferraro E,

Del Frate G, Pittino M, Biffoni F, Rinaldi C & Degrassi A. A closed system for the clinical
banking of umbilical cord blood. Blood Cells Mol.Dis. 35:389-397, 2005.
Alebouyeh M, Unterharnscheidt W & Riegel K. The activity of platelet factor 4 in plasma of

healthy and high risk newborns. Z.Kinderheilkd. 116:137-142, 1974.
Alonso JM, Regan DM, Johnson CE, Oliver DA, Fegan R, Lasky LC & Wall DA. A simple and
reliable procedure for cord blood banking, processing, and freezing: St Louis and Ohio Cord
Blood Bank experiences. Cytotherapy 3:429-433, 2001.
American Academy of Pediatrics Committee on Bioethics. Children as hematopoietic stem

cell donors. Pediatrics 125:392-404, 2010.
Andrew M, Paes B, Milner R, Johnston M, Mitchell L, Tollefsen DM & Powers P. Development

of the human coagulation system in the full-term infant. Blood 70:165-172, 1987.
Andrew M, Paes B, Milner R, Johnston M, Mitchell L, Tollefsen DM, Castle V & Powers P.
Development of the human coagulation system in the healthy premature infant. Blood
72:1651-1657, 1988.
Armitage S, Warwick R, Fehily D, Navarrete C & Contreras M. Cord blood banking in

London: the first 1000 collections. Bone Marrow Transplant. 24:139-145, 1999a.
Armitage S, Fehily D, Dickinson A, Chapman C, Navarrete C & Contreras M. Cord blood

banking: volume reduction of cord blood units using a semi-automated closed system.
Bone Marrow Transplant. 23:505-509, 1999b.
Armitage S. Cord blood processing: volume reduction. Cell Pres.Tech. 4:9-16, 2006.
Aronson DL, Stevan L, Ball AP, Franza BR & Finlayson JS. Generation of the combined
prothrombin activation peptide (F1-2) during the clotting of blood and plasma.
J.Clin.Invest. 60:1410-1418, 1977.
Aroviita P, Teramo K, Westman P, Hiilesmaa V & Kekomaki R. Associations among nucleated

cell, CD34+ cell and colony-forming cell contents in cord blood units obtained through a
standardized banking process. Vox Sang. 84:219-227, 2003.
Aroviita P, Teramo K, Hiilesmaa V, Westman P & Kekomaki R. Birthweight of full-term

infants is associated with cord blood CD34+ cell concentration. Acta Paediatr. 93:1323-
1329, 2004.
Aroviita P, Teramo K, Hiilesmaa V & Kekomaki R. Cord blood hematopoietic progenitor cell
concentration and infant sex. Transfusion 45:613-621, 2005.
Askari S, Miller J, Chrysler G & McCullough J. Impact of donor- and collection-related

variables on product quality in ex utero cord blood banking. Transfusion 45:189-194, 2005.
Atsuta Y, Suzuki R, Nagamura-Inoue T, Taniguchi S, Takahashi S, Kai S, Sakamaki H, Kouzai
76
REFERENCES
Y, Kasai M, Fukuda T, Azuma H, Takanashi M, Okamoto S, Tsuchida M, Kawa K, Morishima Y,

Kodera Y, Kato S & Japan Cord Blood Bank Network. Disease-specific analyses of unrelated
cord blood transplantation compared with unrelated bone marrow transplantation in adult
patients with acute leukemia. Blood 113:1631-1638, 2009.
Aufderhaar U, Holzgreve W, Danzer E, Tichelli A, Troeger C & Surbek DV. The impact of
intrapartum factors on umbilical cord blood stem cell banking. J.Perinat.Med. 31:317-322,
2003.
Ault KA, Rinder HM, Mitchell J, Carmody MB, Vary CP & Hillman RS. The significance of
platelets with increased RNA content (reticulated platelets). A measure of the rate of
thrombopoiesis. Am.J.Clin.Pathol. 98:637-646, 1992.
Bachanova V, Verneris MR, DeFor T, Brunstein CG & Weisdorf DJ. Prolonged survival in
adults with acute lymphoblastic leukemia after reduced-intensity conditioning with cord
blood or sibling donor transplantation. Blood 113:2902-2905, 2009.
Baek EJ, Kim HS, Kim S, Jin H, Choi TY & Kim HO. In vitro clinical-grade generation of red
blood cells from human umbilical cord blood CD34+ cells. Transfusion 48:2235-2245,
2008.
Bain BJ. Platelet count and platelet size in males and females. Scand.J.Haematol. 35:77-
79, 1985.
Bakhshi T, Zabriskie RC, Bodie S, Kidd S, Ramin S, Paganessi LA, Gregory SA, Fung HC &
Christopherson KW. Mesenchymal stem cells from the Wharton's jelly of umbilical cord
segments provide stromal support for the maintenance of cord blood hematopoietic stem
cells during long-term ex vivo culture. Transfusion 48:2638-2644, 2008.
Baksh D, Yao R & Tuan RS. Comparison of proliferative and multilineage differentiation
potential of human mesenchymal stem cells derived from umbilical cord and bone marrow.
Stem Cells 25:1384-1392, 2007.
Ballen KK, Wilson M, Wuu J, Ceredona AM, Hsieh C, Stewart FM, Popovsky MA &
Quesenberry PJ. Bigger is better: maternal and neonatal predictors of hematopoietic
potential of umbilical cord blood units. Bone Marrow Transplant. 27:7-14, 2001.
Ballen KK, Spitzer TR, Yeap BY, McAfee S, Dey BR, Attar E, Haspel R, Kao G, Liney D, Alyea
E, Lee S, Cutler C, Ho V, Soiffer R & Antin JH. Double unrelated reduced-intensity umbilical
cord blood transplantation in adults. Biol.Blood Marrow Transplant. 13:82-89, 2007.
Ballin A, Arbel E, Kenet G, Berar M, Kohelet D, Tanay A, Zakut H & Meytes D. Autologous
umbilical cord blood transfusion. Arch.Dis.Child.Fetal Neonatal Ed. 73:F181-F183, 1995.
Barker JN, Davies SM, DeFor T, Ramsay NK, Weisdorf DJ & Wagner JE. Survival after
transplantation of unrelated donor umbilical cord blood is comparable to that of human
leukocyte antigen-matched unrelated donor bone marrow: results of a matched-pair
analysis. Blood 97:2957-2961, 2001.
Barker JN & Wagner JE. Umbilical-cord blood transplantation for the treatment of cancer.
Nat.Rev.Cancer. 3:526-532, 2003.
Barker JN, Weisdorf DJ, DeFor TE, Blazar BR, Miller JS & Wagner JE. Rapid and complete
donor chimerism in adult recipients of unrelated donor umbilical cord blood transplantation
after reduced-intensity conditioning. Blood 102:1915-1919, 2003.
Barker JN, Weisdorf DJ, DeFor TE, Blazar BR, McGlave PB, Miller JS, Verfaillie CM & Wagner
JE. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance
77
REFERENCES
engraftment in adults with hematologic malignancy. Blood 105:1343-1347, 2005.
Barker JN, Abboud M, Rice RD, Hawke R, Schaible A, Heller G, La Russa V & Scaradavou A. A
"no-wash" albumin-dextran dilution strategy for cord blood unit thaw: high rate of
engraftment and a low incidence of serious infusion reactions. Biol.Blood Marrow
Transplant. 15:1596-1602, 2009a.
Barker JN, Rocha V & Scaradavou A. Optimizing unrelated donor cord blood
transplantation. Biol.Blood Marrow Transplant. 15:154-161, 2009b.
Barker JN, Scaradavou A & Stevens CE. Combined effect of total nucleated cell dose and
HLA match on transplantation outcome in 1061 cord blood recipients with hematologic
malignancies. Blood 115:1843-1849, 2010.
Barnett D, Granger V, Storie I, Peel J, Pollitt R, Smart T & Reilly JT. Quality assessment of
CD34+ stem cell enumeration: experience of the United Kingdom National External Quality
Assessment Scheme (UK NEQAS) using a unique stable whole blood preparation.
Br.J.Haematol. 102:553-565, 1998.
Bauer KA, Barzegar S & Rosenberg RD. Influence of anticoagulants used for blood collection
on plasma prothrombin fragment F1 + 2 measurements. Thromb.Res. 63:617-628, 1991.
Bauer KA. Activation markers of coagulation. Baillieres Best Pract.Res.Clin.Haematol.
12:387-406, 1999.
Benirschke K. Remarkable placenta. Clin.Anat. 11:194-205, 1998.
Berntorp E & Salvagno GL. Standardization and clinical utility of thrombin-generation

assays. Semin.Thromb.Hemost. 34:670-682, 2008.
Bertolini F, Lazzari L, Lauri E, Corsini C, Castelli C, Gorini F & Sirchia G. Comparative study of
different procedures for the collection and banking of umbilical cord blood. J.Hematother.
4:29-36, 1995.
Bertolini F, Battaglia M, Zibera C, Baroni G, Soro V, Perotti C, Salvaneschi L & Robustelli

della Cuna G. A new method for placental/cord blood processing in the collection bag. I.
Analysis of factors involved in red blood cell removal. Bone Marrow Transplant. 18:783-
786, 1996.
Bessman JD, Williams LJ & Gilmer PR. Mean platelet volume. The inverse relation of platelet
size and count in normal subjects, and an artifact of other particles. Am.J.Clin.Pathol.
76:289-293, 1981.
Bessman JD. The relation of megakaryocyte ploidy to platelet volume. Am.J.Hematol.

16:161-170, 1984.
Bleyer WA, Hakami N & Shepard TH. The development of hemostasis in the human fetus
and newborn infant. J.Pediatr. 79:838-853, 1971.
Board on Health Sciences Policy. Cord Blood: Establishing a National Hematopoietic Stem
Cell Bank Program. The National Academies Press, Washington (DC) 2005.
Boehlen F & Clemetson KJ. Platelet chemokines and their receptors: what is their relevance
to platelet storage and transfusion practice? Transfus.Med. 11:403-417, 2001.
Bornstein R, Flores AI, Montalban MA, del Rey MJ, de la Serna J & Gilsanz F. A modified cord
blood collection method achieves sufficient cell levels for transplantation in most adult
patients. Stem Cells 23:324-334, 2005.
78
REFERENCES
Bovill EG, Soll RF, Lynch M, Bhushan F, Landesman M, Freije M, Church W, McAuliffe T,
Davidson K & Sadowski J. Vitamin K1 metabolism and the production of des-carboxy
prothrombin and protein C in the term and premature neonate. Blood 81:77-83, 1993.
Bradley MB, Satwani P, Baldinger L, Morris E, van de Ven C, Del Toro G, Garvin J, George D,
Bhatia M, Roman E, Baxter-Lowe LA, Schwartz J, Qualter E, Hawks R, Wolownik K, Foley S,
Militano O, Leclere J, Cheung YK & Cairo MS. Reduced intensity allogeneic umbilical cord
blood transplantation in children and adolescent recipients with malignant and non-
malignant diseases. Bone Marrow Transplant. 40:621-631, 2007.
Brand A, Eichler H, Szczepiorkowski ZM, Hess JR, Kekomaki R, McKenna DH, Pamphilon D,
Reems J, Sacher RA, Takahashi TA, van de Watering LM & Biomedical Excellence for Safer
Transfusion (BEST) Collaborative. Viability does not necessarily reflect the hematopoietic
progenitor cell potency of a cord blood unit: results of an interlaboratory exercise.
Transfusion 48:546-549, 2008.
Brocklebank AM & Sparrow RL. Enumeration of CD34+ cells in cord blood: a variation on a
single-platform flow cytometric method based on the ISHAGE gating strategy. Cytometry
46:254-261, 2001.
Brooke G, Rossetti T, Pelekanos R, Ilic N, Murray P, Hancock S, Antonenas V, Huang G,

Gottlieb D, Bradstock K & Atkinson K. Manufacturing of human placenta-derived
mesenchymal stem cells for clinical trials. Br.J.Haematol. 144:571-579, 2009.
Broxmeyer HE, Douglas GW, Hangoc G, Cooper S, Bard J, English D, Arny M, Thomas L &
Boyse EA. Human umbilical cord blood as a potential source of transplantable
hematopoietic stem/progenitor cells. Proc.Natl.Acad.Sci.U.S.A. 86:3828-3832, 1989.
Broxmeyer HE, Hangoc G, Cooper S, Ribeiro RC, Graves V, Yoder M, Wagner J, Vadhan-Raj
S, Benninger L & Rubinstein P. Growth characteristics and expansion of human umbilical
cord blood and estimation of its potential for transplantation in adults.
Proc.Natl.Acad.Sci.U.S.A. 89:4109-4113, 1992.
Broxmeyer HE, Srour EF, Hangoc G, Cooper S, Anderson SA & Bodine DM. High-efficiency
recovery of functional hematopoietic progenitor and stem cells from human cord blood
cryopreserved for 15 years. Proc.Natl.Acad.Sci.U.S.A. 100:645-650, 2003.
Brugnara C. Reference values in infancy and childhood. In: Nathan and Oski's Hematology
of Infancy and Childhood (eds. Orkin SH, Nathan DG, Ginsburg D, Look AT, Fisher DE & Lux
SE), 7th edition, Saunders Elsevier, Philadelphia (PA) 2009, pp. 1769-1796.
Brugnara C & Platt OS. The neonatal erythrocyte and its disorders. In: Nathan and Oski's
Hematology of Infancy and Childhood (eds. Orkin SH, Nathan DG, Ginsburg D, Look AT,
Fisher DE & Lux SE), 7th edition, Saunders Elsevier, Philadelphia (PA) 2009, pp. 21-66.
Bruno E, Cooper RJ, Briddell RA & Hoffman R. Further examination of the effects of
recombinant cytokines on the proliferation of human megakaryocyte progenitor cells.
Blood 77:2339-2346, 1991.
Brunstein CG, Setubal DC & Wagner JE. Expanding the role of umbilical cord blood
transplantation. Br.J.Haematol. 137:20-35, 2007a.
Brunstein CG, Barker JN, Weisdorf DJ, DeFor TE, Miller JS, Blazar BR, McGlave PB & Wagner
JE. Umbilical cord blood transplantation after nonmyeloablative conditioning: impact on
transplantation outcomes in 110 adults with hematologic disease. Blood 110:3064-3070,
2007b.
Burgess AW, Wilson EM & Metcalf D. Stimulation by human placental conditioned medium of
79
REFERENCES
hemopoietic colony formation by human marrow cells. Blood 49:573-583, 1977.
Cairo MS, Wagner EL, Fraser J, Cohen G, van de Ven C, Carter SL, Kernan NA & Kurtzberg J.
Characterization of banked umbilical cord blood hematopoietic progenitor cells and
lymphocyte subsets and correlation with ethnicity, birth weight, sex, and type of delivery: a
Cord Blood Transplantation (COBLT) Study report. Transfusion 45:856-866, 2005.
Cantor AB. Developmental hemostasis: relevance to newborns and infants. In: Nathan and
Oski's Hematology of Infancy and Childhood (eds. Orkin SH, Nathan DG, Ginsburg D, Look
AT, Fisher DE & Lux SE), 7th edition, Saunders Elsevier, Philadelphia (PA) 2009, pp. 147-
191.
Charnock-Jones DS & Burton GJ. Placental vascular morphogenesis. Baillieres Best

Pract.Res.Clin.Obstet.Gynaecol. 14:953-968, 2000.
Chervenick PA & Boggs DR. In vitro growth of granulocytic and mononuclear cell colonies
from blood of normal individuals. Blood 37:131-135, 1971.
Chow R, Nademanee A, Rosenthal J, Karanes C, Jaing TH, Graham ML, Tsukahara E, Wang
B, Gjertson D, Tan P, Forman S & Petz LD. Analysis of hematopoietic cell transplants using
plasma-depleted cord blood products that are not red blood cell reduced. Biol.Blood
Marrow Transplant. 13:1346-1357, 2007.
Cleary GM & Wiswell TE. Meconium-stained amniotic fluid and the meconium aspiration
syndrome. An update. Pediatr.Clin.North Am. 45:511-529, 1998.
Confer D & Robinett P. The US National Marrow Donor Program role in unrelated donor
hematopoietic cell transplantation. Bone Marrow Transplant. 42 (Suppl 1):S3-S5, 2008.
Corre-Buscail I, Pineau D, Boissinot M & Hermouet S. Erythropoietin-independent erythroid

colony formation by bone marrow progenitors exposed to interleukin-11 and interleukin-8.
Exp.Hematol. 33:1299-1308, 2005.
Cramer EM, Masse JM, Caen JP, Garcia I, Breton-Gorius J, Debili N & Vainchenker W. Effect
of thrombin on maturing human megakaryocytes. Am.J.Pathol. 143:1498-1508, 1993.
Crawley JT, Zanardelli S, Chion CK & Lane DA. The central role of thrombin in hemostasis.
J.Thromb.Haemost. 5 (Suppl 1):95-101, 2007.
Cremer M, Dame C, Schaeffer HJ, Giers G, Bartmann P & Bald R. Longitudinal

thrombopoietin plasma concentrations in fetuses with alloimmune thrombocytopenia
treated with intrauterine PLT transfusions. Transfusion 43:1216-1222, 2003.
Cross JC, Baczyk D, Dobric N, Hemberger M, Hughes M, Simmons DG, Yamamoto H &
Kingdom JC. Genes, development and evolution of the placenta. Placenta 24:123-130,
2003.
Cvirn G, Gallistl S, Leschnik B & Muntean W. Low tissue factor pathway inhibitor (TFPI)
together with low antithrombin allows sufficient thrombin generation in neonates.
J.Thromb.Haemost. 1:263-268, 2003.
Dacie JV & Lewis SM. Basic haematological techniques. In: Practical Haematology (eds.
Dacie JV & Lewis SM), 6th edition, Churchill Livingstone, New York (NY) 1985, pp. 22-47.
Dal Cortivo L, Marolleau JP, Gluckman E, Chavinie J, Brossard Y & Benbunan M. The Paris
Cord Blood Bank. Bone Marrow Transplant. 22 (Suppl 1):S11, 1998.
Dal Cortivo L, Robert I, Mangin C, Sameshima T, Kora S, Gluckman E, Benbunan M &
80
REFERENCES
Marolleau JP. Cord blood banking: volume reduction using "Procord" Terumo filter.
J.Hematother.Stem Cell Res. 9:885-890, 2000.
de Lima M, McMannis J, Gee A, Komanduri K, Couriel D, Andersson BS, Hosing C, Khouri I,

Jones R, Champlin R, Karandish S, Sadeghi T, Peled T, Grynspan F, Daniely Y, Nagler A &
Shpall EJ. Transplantation of ex vivo expanded cord blood cells using the copper chelator
tetraethylenepentamine: a phase I/II clinical trial. Bone Marrow Transplant. 41:771-778,
2008.
Debili N, Issaad C, Masse JM, Guichard J, Katz A, Breton-Gorius J & Vainchenker W.

Expression of CD34 and platelet glycoproteins during human megakaryocytic
differentiation. Blood 80:3022-3035, 1992.
Debili N, Masse JM, Katz A, Guichard J, Breton-Gorius J & Vainchenker W. Effects of the
recombinant hematopoietic growth factors interleukin-3, interleukin-6, stem cell factor,
and leukemia inhibitory factor on the megakaryocytic differentiation of CD34+ cells. Blood
82:84-95, 1993.
Debili N, Coulombel L, Croisille L, Katz A, Guichard J, Breton-Gorius J & Vainchenker W.

Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow.
Blood 88:1284-1296, 1996.
Denning-Kendall P, Donaldson C, Nicol A, Bradley B & Hows J. Optimal processing of human

umbilical cord blood for clinical banking. Exp.Hematol. 24:1394-1401, 1996.
Dobo I, Allegraud A, Navenot JM, Boasson M, Bidet JM & Praloran V. Collagen matrix: an
attractive alternative to agar and methylcellulose for the culture of hematopoietic
progenitors in autologous transplantation products. J.Hematother. 4:281-287, 1995.
Dobo I, Mossuz P, Campos L, Girodon F, Allegraud A, Latger-Cannard V, Boiret N, Pineau D,

Wunder E, Zandecki M, Praloran V & Hermouet S. Comparison of four serum-free, cytokine-
free media for analysis of endogenous erythroid colony growth in polycythemia vera and
essential thrombocythemia. Hematol.J. 2:396-403, 2001.
Dombrowski MP, Berry SM, Johnson MP, Saleh AA & Sokol RJ. Birth weight-length ratios,
ponderal indexes, placental weights, and birth weight-placenta ratios in a large population.
Arch.Pediatr.Adolesc.Med. 148:508-512, 1994.
Donaldson C, Armitage WJ, Denning-Kendall PA, Nicol AJ, Bradley BA & Hows JM. Optimal
cryopreservation of human umbilical cord blood. Bone Marrow Transplant. 18:725-731,
1996.
Donaldson C, Armitage WJ, Laundy V, Barron C, Buchanan R, Webster J, Bradley B & Hows
J. Impact of obstetric factors on cord blood donation for transplantation. Br.J.Haematol.
106:128-132, 1999.
Donaldson C, Buchanan R, Webster J, Laundy V, Horsley H, Barron C, Anderson N, Bradley B

& Hows J. Development of a district Cord Blood Bank: a model for cord blood banking in the
National Health Service. Bone Marrow Transplant. 25:899-905, 2000.
Dormann D, Clemetson KJ & Kehrel BE. The GPIb thrombin-binding site is essential for
thrombin-induced platelet procoagulant activity. Blood 96:2469-2478, 2000.
Douay L & Andreu G. Ex vivo production of human red blood cells from hematopoietic stem
cells: what is the future in transfusion? Transfus.Med.Rev. 21:91-100, 2007.
Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy X & Lataillade JJ. Platelet lysates
promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-
81
REFERENCES
based therapy applications. J.Cell.Physiol. 205:228-236, 2005.
Drygalski A, Xu G, Constantinescu D, Kashiwakura I, Farley T, Dobrila L, Rubinstein P &

Adamson JW. The frequency and proliferative potential of megakaryocytic colony-forming
cells (Meg-CFC) in cord blood, cytokine-mobilized peripheral blood and bone marrow, and
their correlation with total CFC numbers: implications for the quantitation of Meg-CFC to
predict platelet engraftment following cord blood transplantation. Bone Marrow Transplant.
25:1029-1034, 2000.
Dzik W, Sniecinski I & Fischer J. Toward standardization of CD34+ cell enumeration: an

international study. Biomedical Excellence for Safer Transfusion Working Party. Transfusion
39:856-863, 1999.
Eapen M, Rubinstein P, Zhang MJ, Stevens C, Kurtzberg J, Scaradavou A, Loberiza FR,

Champlin RE, Klein JP, Horowitz MM & Wagner JE. Outcomes of transplantation of unrelated
donor umbilical cord blood and bone marrow in children with acute leukaemia: a
comparison study. Lancet 369:1947-1954, 2007.
Eichler H, Kern S, Beck C, Zieger W & Kluter H. Engraftment capacity of umbilical cord blood
cells processed by either whole blood preparation or filtration. Stem Cells 21:208-216,
2003.
Eichler H, Seetharaman S, Latta M, Kurtz J & Moroff G. Comparison of total nucleated cell
measurements of UC blood samples using two hematology analyzers. Cytotherapy 6:457-
464, 2004.
Elchalal U, Fasouliotis SJ, Shtockheim D, Brautbar C, Schenker JG, Weinstein D & Nagler A.
Postpartum umbilical cord blood collection for transplantation: a comparison of three
methods. Am.J.Obstet.Gynecol. 182:227-232, 2000.
Ende M & Ende N. Hematopoietic transplantation by means of fetal (cord) blood. A new
method. Va.Med.Mon. 99:276-280, 1972.
Fauser AA & Messner HA. Granuloerythropoietic colonies in human bone marrow,

peripheral blood, and cord blood. Blood 52:1243-1248, 1978.
Flomenberg N, Baxter-Lowe LA, Confer D, Fernandez-Vina M, Filipovich A, Horowitz M,

Hurley C, Kollman C, Anasetti C, Noreen H, Begovich A, Hildebrand W, Petersdorf E,
Schmeckpeper B, Setterholm M, Trachtenberg E, Williams T, Yunis E & Weisdorf D. Impact
of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone
marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on
transplantation outcome. Blood 104:1923-1930, 2004.
Flores AI, McKenna DH, Montalban MA, De la Cruz J, Wagner JE & Bornstein R. Consistency
of the initial cell acquisition procedure is critical to the standardization of CD34+ cell
enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units
and cryopreserved aliquots. Transfusion 49:636-647, 2009.
Franzoi M, Simioni P, Luni S, Zerbinati P, Girolami A & Zanardo V. Effect of delivery

modalities on the physiologic inhibition system of coagulation of the neonate. Thromb.Res.
105:15-18, 2002.
Fraser JK, Cairo MS, Wagner EL, McCurdy PR, Baxter-Lowe LA, Carter SL, Kernan NA, Lill
MC, Slone V, Wagner JE, Wallas CH & Kurtzberg J. Cord Blood Transplantation Study
(COBLT): cord blood bank standard operating procedures. J.Hematother. 7:521-561,
1998.
Frassoni F, Podesta M, Maccario R, Giorgiani G, Rossi G, Zecca M, Bacigalupo A, Piaggio G &
82
REFERENCES
Locatelli F. Cord blood transplantation provides better reconstitution of hematopoietic

reservoir compared with bone marrow transplantation. Blood 102:1138-1141, 2003.
Freyssinier JM, Lecoq-Lafon C, Amsellem S, Picard F, Ducrocq R, Mayeux P, Lacombe C &

Fichelson S. Purification, amplification and characterization of a population of human
erythroid progenitors. Br.J.Haematol. 106:912-922, 1999.
Galloway JL & Zon LI. Ontogeny of hematopoiesis: examining the emergence of

hematopoietic cells in the vertebrate embryo. Curr.Top.Dev.Biol. 53:139-158, 2003.
Gangenahalli GU, Singh VK, Verma YK, Gupta P, Sharma RK, Chandra R & Luthra PM.
Hematopoietic stem cell antigen CD34: role in adhesion or homing. Stem Cells Dev.
15:305-313, 2006.
Gatti RA, Meuwissen HJ, Allen HD, Hong R & Good RA. Immunological reconstitution of sex-
linked lymphopenic immunological deficiency. Lancet 2:1366-1369, 1968.
Gentry T, Deibert E, Foster SJ, Haley R, Kurtzberg J & Balber AE. Isolation of early
hematopoietic cells, including megakaryocyte progenitors, in the ALDH-bright cell
population of cryopreserved, banked UC blood. Cytotherapy 9:569-576, 2007.
George TJ, Sugrue MW, George SN & Wingard JR. Factors associated with parameters of
engraftment potential of umbilical cord blood. Transfusion 46:1803-1812, 2006.
Gewirtz AM, Calabretta B, Rucinski B, Niewiarowski S & Xu WY. Inhibition of human

megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4
peptide. J.Clin.Invest. 83:1477-1486, 1989.
Giacomini A, Legovini P, Gessoni G, Antico F, Valverde S, Salvadego MM & Manoni F. Platelet

count and parameters determined by the Bayer ADVIA 120 in reference subjects and
patients. Clin.Lab.Haematol. 23:181-186, 2001.
Giarratana MC, Kobari L, Lapillonne H, Chalmers D, Kiger L, Cynober T, Marden MC,

Wajcman H & Douay L. Ex vivo generation of fully mature human red blood cells from
hematopoietic stem cells. Nat.Biotechnol. 23:69-74, 2005.
Gluckman E, Broxmeyer HA, Auerbach AD, Friedman HS, Douglas GW, Devergie A, Esperou
H, Thierry D, Socie G & Lehn P. Hematopoietic reconstitution in a patient with Fanconi's
anemia by means of umbilical-cord blood from an HLA-identical sibling. N.Engl.J.Med.
321:1174-1178, 1989.
Gluckman E. European organization for cord blood banking. Blood Cells 20:601-608, 1994.
Gluckman E, Rocha V, Boyer-Chammard A, Locatelli F, Arcese W, Pasquini R, Ortega J,

Souillet G, Ferreira E, Laporte JP, Fernandez M & Chastang C. Outcome of cord-blood
transplantation from related and unrelated donors. Eurocord Transplant Group and the
European Blood and Marrow Transplantation Group. N.Engl.J.Med. 337:373-381, 1997.
Gluckman E, Rocha V & Chastang C. Cord blood banking and transplant in Europe.
Eurocord. Bone Marrow Transplant. 22 (Suppl 1):S68-S74, 1998.
Gluckman E, Rocha V, Arcese W, Michel G, Sanz G, Chan KW, Takahashi TA, Ortega J,
Filipovich A, Locatelli F, Asano S, Fagioli F, Vowels M, Sirvent A, Laporte JP, Tiedemann K,
Amadori S, Abecassis M, Bordigoni P, Diez B, Shaw PJ, Vora A, Caniglia M, Garnier F, Ionescu
I, Garcia J, Koegler G, Rebulla P, Chevret S & Eurocord Group. Factors associated with
outcomes of unrelated cord blood transplant: guidelines for donor choice. Exp.Hematol.
32:397-407, 2004.
83
REFERENCES
Gluckman E & Rocha V. Donor selection for unrelated cord blood transplants.
Curr.Opin.Immunol. 18:565-570, 2006.
Gluckman E, Rocha V, Ionescu I, Bierings M, Harris RE, Wagner J, Kurtzberg J, Champagne

MA, Bonfim C, Bittencourt M, Darbyshire P, Fernandez MN, Locatelli F, Pasquini R &
Eurocord-Netcord and EBMT. Results of unrelated cord blood transplant in Fanconi anemia
patients: risk factor analysis for engraftment and survival. Biol.Blood Marrow Transplant.
13:1073-1082, 2007.
Glucksberg H, Storb R, Fefer A, Buckner CD, Neiman PE, Clift RA, Lerner KG & Thomas ED.
Clinical manifestations of graft-versus-host disease in human recipients of marrow from
HL-A-matched sibling donors. Transplantation 18:295-304, 1974.
Grace RF & Lux SE. Disorders of the red cell membrane. In: Nathan and Oski's Hematology
Gratama JW, Braakman E, Kraan J, Lankheet P, Levering WH, Van Den Beemd MW, Van Der
Schoot CE, Wijermans P & Preijers F. Comparison of single and dual-platform assay formats
for CD34+ haematopoietic progenitor cell enumeration. Clin.Lab.Haematol. 21:337-346,
1999.
Gratama JW, Kraan J, Keeney M, Sutherland DR, Granger V & Barnett D. Validation of the
single-platform ISHAGE method for CD34(+) hematopoietic stem and progenitor cell
enumeration in an international multicenter study. Cytotherapy 5:55-65, 2003.
Grisaru D, Deutsch V, Pick M, Fait G, Lessing JB, Dollberg S & Eldor A. Placing the newborn
on the maternal abdomen after delivery increases the volume and CD34 cell content in the
umbilical cord blood collected: an old maneuver with new applications.
Am.J.Obstet.Gynecol. 180:1240-1243, 1999.
Grosshaupt B, Muntean W & Sedlmayr P. Hyporeactivity of neonatal platelets is not caused

by preactivation during birth. Eur.J.Pediatr. 156:944-948, 1997.
Guerriero R, Testa U, Gabbianelli M, Mattia G, Montesoro E, Macioce G, Pace A, Ziegler B,

Hassan HJ & Peschle C. Unilineage megakaryocytic proliferation and differentiation of
purified hematopoietic progenitors in serum-free liquid culture. Blood 86:3725-3736,
1995.
Gutman JA, Leisenring W, Appelbaum FR, Woolfrey AE & Delaney C. Low relapse without
excessive transplant-related mortality following myeloablative cord blood transplantation
for acute leukemia in complete remission: a matched cohort analysis. Biol.Blood Marrow
Transplant. 15:1122-1129, 2009.
Hahn T, Bunworasate U, George MC, Bir AS, Chinratanalab W, Alam AR, Bambach B, Baer
MR, Slack JL, Wetzler M, Becker JL & McCarthy PL. Use of nonvolume-reduced
(unmanipulated after thawing) umbilical cord blood stem cells for allogeneic
transplantation results in safe engraftment. Bone Marrow Transplant. 32:145-150, 2003.
Haining WN, Duncan C & Lehmann LE. Principles of bone marrow and stem cell
transplantation. In: Nathan and Oski's Hematology of Infancy and Childhood (eds. Orkin
SH, Nathan DG, Ginsburg D, Look AT, Fisher DE & Lux SE), 7th edition, Saunders Elsevier,
Philadelphia (PA) 2009, pp. 397-452.
Hanspal M & Hanspal JS. The association of erythroblasts with macrophages promotes
erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this
contact. Blood 84:3494-3504, 1994.
84
REFERENCES
Harris DT, Schumacher MJ, Rychlik S, Booth A, Acevedo A, Rubinstein P, Bard J & Boyse EA.
Collection, separation and cryopreservation of umbilical cord blood for use in
transplantation. Bone Marrow Transplant. 13:135-143, 1994.
Harris DT. Experience in autologous and allogeneic cord blood banking. J.Hematother.
5:123-128, 1996.
Haspel RL & Lamontagne D. Prethaw predictors of cord blood unit quality. Transfusion
50:265, 2010.
Heemskerk JW, Bevers EM & Lindhout T. Platelet activation and blood coagulation.
Thromb.Haemost. 88:186-193, 2002.
Heikinheimo R. Coagulation studies with fetal blood. Biol.Neonat. 7:319-327, 1964.
Hemker HC & Beguin S. Thrombin generation in plasma: its assessment via the
endogenous thrombin potential. Thromb.Haemost. 74:134-138, 1995.
Hogan CJ, Shpall EJ & Keller G. Differential long-term and multilineage engraftment
potential from subfractions of human CD34+ cord blood cells transplanted into NOD/SCID
mice. Proc.Natl.Acad.Sci.U.S.A. 99:413-418, 2002.
Hows JM, Bradley BA, Marsh JC, Luft T, Coutinho L, Testa NG & Dexter TM. Growth of human
umbilical-cord blood in longterm haemopoietic cultures. Lancet 340:73-76, 1992.
Hyytiainen S, Wartiovaara-Kautto U, Ulander VM, Kaaja R, Heikinheimo M & Petaja J. The

procoagulant effects of factor V Leiden may be balanced against decreased levels of factor
V and do not reflect in vivo thrombin formation in newborns. Thromb.Haemost. 95:434-
440, 2006.
Ignjatovic V, Greenway A, Summerhayes R & Monagle P. Thrombin generation: the

functional role of alpha-2-macroglobulin and influence of developmental haemostasis.
Br.J.Haematol. 138:366-368, 2007.
Iori AP, Cerretti R, De Felice L, Screnci M, Mengarelli A, Romano A, Caniglia M, Cerilli L,

Gentile G, Moleti ML, Giona F, Agostini F, Pasqua I, Perrone MP, Pinto MR, Grapulin L, Testi
AM, Martino P, De Rossi G, Mandelli F & Arcese W. Pre-transplant prognostic factors for
patients with high-risk leukemia undergoing an unrelated cord blood transplantation. Bone
Israels SJ, Rand ML & Michelson AD. Neonatal platelet function. Semin.Thromb.Hemost.
29:363-372, 2003.
Jacobs HC & Falkenburg JH. Umbilical cord blood banking in The Netherlands. Bone Marrow
Transplant. 22 Suppl 1:S8-10, 1998.
Jan RH, Wen SH, Shyr MH & Chiang BL. Impact of maternal and neonatal factors on CD34+
cell count, total nucleated cells, and volume of cord blood. Pediatr.Transplant. 12:868-873,
2008.
Jones J, Stevens CE, Rubinstein P, Robertazzi RR, Kerr A & Cabbad MF. Obstetric predictors
of placental/umbilical cord blood volume for transplantation. Am.J.Obstet.Gynecol.
188:503-509, 2003.
Jubert C, Wagner E, Bizier S, Vachon MF, Duval M & Champagne MA. Length of cord blood
unit cryopreservation does not impact hematopoietic engraftment. Transfusion 48:2028-
2030, 2008.
85
REFERENCES
Jung YJ, Cha JE, Kim HJ, Ju SY, Cho SJ, Cho KA, Kim LS, Woo SY, Park JW, Seoh JY & Ryu KH.
Erythropoietin-independent and -dependent stages during in vitro erythropoiesis. Acta
Haematol. 118:222-225, 2007.
Juvonen E, Partanen S & Ruutu T. Colony formation by megakaryocytic progenitors in

essential thrombocythaemia. Br.J.Haematol. 66:161-164, 1987.
Kaimal AJ, Smith CC, Laros RK, Caughey AB & Cheng YW. Cost-effectiveness of private
umbilical cord blood banking. Obstet.Gynecol. 114:848-855, 2009.
Kaito K, Otsubo H, Usui N, Yoshida M, Tanno J, Kurihara E, Matsumoto K, Hirata R, Domitsu

K & Kobayashi M. Platelet size deviation width, platelet large cell ratio, and mean platelet
volume have sufficient sensitivity and specificity in the diagnosis of immune
thrombocytopenia. Br.J.Haematol. 128:698-702, 2005.
Kanamaru S, Kawano Y, Watanabe T, Nakagawa R, Suzuya H, Onishi T, Yamazaki J,

Nakayama T, Kuroda Y & Takaue Y. Low numbers of megakaryocyte progenitors in grafts of
cord blood cells may result in delayed platelet recovery after cord blood cell transplant.
Stem Cells 18:190-195, 2000.
Karpatkin S. Heterogeneity of human platelets. I. Metabolic and kinetic evidence

suggestive of young and old platelets. J.Clin.Invest. 48:1073-1082, 1969.
Kasai M & Masauzi N. The characteristics of umbilical cord blood (UCB) and UCB
transplantation. Semin.Thromb.Hemost. 24:491-495, 1998.
Katz M, Lunenfeld E, Meizner I, Bashan N & Gross J. The effect of the duration of the second
stage of labour on the acid-base state of the fetus. Br.J.Obstet.Gynaecol. 94:425-430,
1987.
Kaushansky K. Thrombopoietin: the primary regulator of platelet production. Blood

86:419-431, 1995.
Kaushansky K, Broudy VC, Grossmann A, Humes J, Lin N, Ren HP, Bailey MC,
Papayannopoulou T, Forstrom JW & Sprugel KH. Thrombopoietin expands erythroid
progenitors, increases red cell production, and enhances erythroid recovery after
myelosuppressive therapy. J.Clin.Invest. 96:1683-1687, 1995.
Kaushansky K. Historical review: megakaryopoiesis and thrombopoiesis. Blood 111:981-

986, 2008.
Khodabux CM & Brand A. The use of cord blood for transfusion purposes: current status.
Vox Sang. 97:281-293, 2009.
Kirkland MA. A phase space model of hemopoiesis and the concept of stem cell renewal.
Exp.Hematol. 32:511-519, 2004.
Kitlinski ML, Kallen K, Marsal K & Olofsson P. Gestational age-dependent reference values
for pH in umbilical cord arterial blood at term. Obstet.Gynecol. 102:338-345, 2003.
Knudtzon S. In vitro growth of granulocytic colonies from circulating cells in human cord
blood. Blood 43:357-361, 1974.
Kobayashi M, Laver JH, Kato T, Miyazaki H & Ogawa M. Recombinant human thrombopoietin
(Mpl ligand) enhances proliferation of erythroid progenitors. Blood 86:2494-2499, 1995.
Kodera Y. The Japan Marrow Donor Program, the Japan Cord Blood Bank Network and the
Asia Blood and Marrow Transplant Registry. Bone Marrow Transplant. 42 (Suppl 1):S6,
86
REFERENCES
2008.
Kogler G, Callejas J, Sorg RV, Fischer J, Migliaccio AR & Wernet P. The effect of different
thawing methods, growth factor combinations and media on the ex vivo expansion of
umbilical cord blood primitive and committed progenitors. Bone Marrow Transplant.
21:233-241, 1998.
Koller MR, Manchel I, Maher RJ, Goltry KL, Armstrong RD & Smith AK. Clinical-scale human
umbilical cord blood cell expansion in a novel automated perfusion culture system. Bone
Kramer MS, Platt RW, Wen SW, Joseph KS, Allen A, Abrahamowicz M, Blondel B, Breart G &
Fetal/Infant Health Study Group of the Canadian Perinatal Surveillance System. A new and
improved population-based Canadian reference for birth weight for gestational age.
Pediatrics 108:E35, 2001.
Krantz SB. Erythropoietin. Blood 77:419-434, 1991.
Krause DS, Fackler MJ, Civin CI & May WS. CD34: structure, biology, and clinical utility.
Blood 87:1-13, 1996.
Kurnick JE & Robison WA. Colony growth of human peripheral white blood cells in vitro.
Blood 37:136-141, 1971.
Kurtz J, Seetharaman S, Greco N & Moroff G. Assessment of cord blood hematopoietic cell
parameters before and after cryopreservation. Transfusion 47:1578-1587, 2007.
Kurtzberg J, Laughlin M, Graham ML, Smith C, Olson JF, Halperin EC, Ciocci G, Carrier C,
Stevens CE & Rubinstein P. Placental blood as a source of hematopoietic stem cells for
transplantation into unrelated recipients. N.Engl.J.Med. 335:157-166, 1996.
Kurtzberg J, Cairo MS, Fraser JK, Baxter-Lowe L, Cohen G, Carter SL & Kernan NA. Results
of the cord blood transplantation (COBLT) study unrelated donor banking program.
Transfusion 45:842-855, 2005.
Kurtzberg J, Prasad VK, Carter SL, Wagner JE, Baxter-Lowe LA, Wall D, Kapoor N, Guinan
EC, Feig SA, Wagner EL, Kernan NA & COBLT Steering Committee. Results of the Cord Blood
Transplantation Study (COBLT): clinical outcomes of unrelated donor umbilical cord blood
transplantation in pediatric patients with hematologic malignancies. Blood 112:4318-
4327, 2008.
Kurtzberg J. Update on umbilical cord blood transplantation. Curr.Opin.Pediatr. 21:22-29,

2009.
Lackman F, Capewell V, Gagnon R & Richardson B. Fetal umbilical cord oxygen values and
birth to placental weight ratio in relation to size at birth. Am.J.Obstet.Gynecol. 185:674-
682, 2001.
Lam AC, Li K, Zhang XB, Li CK, Fok TF, Chang AM, James AE, Tsang KS & Yuen PM. Preclinical
ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of
culture; the media, serum supplements, and growth factors used; and engraftment in
NOD/SCID mice. Transfusion 41:1567-1576, 2001.
Lamana M, Albella B, Rodriguez F, Regidor C & Bueren JA. Conclusions of a national

multicenter intercomparative study of in vitro cultures of human hematopoietic
progenitors. Bone Marrow Transplant. 23:373-380, 1999.
Lane DA, Philippou H & Huntington JA. Directing thrombin. Blood 106:2605-2612, 2005.
87
REFERENCES
Lapierre V, Pellegrini N, Bardey I, Malugani C, Saas P, Garnache F, Racadot E, Schillinger F &

Maddens S. Cord blood volume reduction using an automated system (Sepax) vs. a semi-
automated system (Optipress II) and a manual method (hydroxyethyl starch
sedimentation) for routine cord blood banking: a comparative study. Cytotherapy 9:165-
169, 2007.
Laroche V, McKenna DH, Moroff G, Schierman T, Kadidlo D & McCullough J. Cell loss and
recovery in umbilical cord blood processing: a comparison of postthaw and postwash
samples. Transfusion 45:1909-1916, 2005.
Lasky LC, Lane TA, Miller JP, Lindgren B, Patterson HA, Haley NR & Ballen K. In utero or ex
utero cord blood collection: which is better? Transfusion 42:1261-1267, 2002.
Lauber S, Latta M, Kluter H & Muller-Steinhardt M. The Mannheim Cord Blood Bank:
experiences and perspectives for the future. Transfus.Med.Hemother 37:90-97, 2010.
Laughlin MJ, Barker J, Bambach B, Koc ON, Rizzieri DA, Wagner JE, Gerson SL, Lazarus HM,
Cairo M, Stevens CE, Rubinstein P & Kurtzberg J. Hematopoietic engraftment and survival
in adult recipients of umbilical-cord blood from unrelated donors. N.Engl.J.Med. 344:1815-
1822, 2001.
Laughlin MJ, Eapen M, Rubinstein P, Wagner JE, Zhang MJ, Champlin RE, Stevens C, Barker
JN, Gale RP, Lazarus HM, Marks DI, van Rood JJ, Scaradavou A & Horowitz MM. Outcomes
after transplantation of cord blood or bone marrow from unrelated donors in adults with
leukemia. N.Engl.J.Med. 351:2265-2275, 2004.
Lazzari L, Corsini C, Curioni C, Lecchi L, Scalamogna M, Rebulla P & Sirchia G. The Milan
Cord Blood Bank and the Italian Cord Blood Network. J.Hematother. 5:117-122, 1996.
Leberbauer C, Boulme F, Unfried G, Huber J, Beug H & Mullner EW. Different steroids co-
regulate long-term expansion versus terminal differentiation in primary human erythroid
progenitors. Blood 105:85-94, 2005.
Lecchi L, Perego L, Garcea F, Ratti I, Brasca M, Dotti D, Cimoni S, Pezzali I, Celeste T,

Giovanelli S, Butti B, De Fazio N, Lopa R & Rebulla P. Ten-year quality control of a
semiautomated procedure of cord blood unit volume reduction. Transfusion 49:563-569,
2009.
Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL & Chen TH. Isolation of multipotent
mesenchymal stem cells from umbilical cord blood. Blood 103:1669-1675, 2004.
Lemarie C, Esterni B, Calmels B, Dazey B, Lapierre V, Lecchi L, Meyer A, Rea D, Thuret I,

Chambost H, Curtillet C, Chabannon C & Michel G. CD34+ progenitors are reproducibly
recovered in thawed umbilical grafts, and positively influence haematopoietic
reconstitution after transplantation. Bone Marrow Transplant. 39:453-460, 2007.
Leroy-Matheron C & Gouault-Heilmann M. Influence of conditions of blood sampling on

coagulation activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin
complexes and D-dimers) measurements. Thromb.Res. 74:399-407, 1994.
Levin J & Bessman JD. The inverse relation between platelet volume and platelet number.
Abnormalities in hematologic disease and evidence that platelet size does not correlate
with platelet age. J.Lab.Clin.Med. 101:295-307, 1983.
Li K, Yau FW, Fok TF, So KW, Li CK & Yuen PM. Haematopoietic stem and progenitor cells in
human term and preterm neonatal blood. Vox Sang. 80:162-169, 2001.
Lim F, Beckhoven J, Brand A, Kluin-Nelemans J, Hermans J, Willemze R, Kanhai H &
88
REFERENCES
Falkenburg J. The number of nucleated cells reflects the hematopoietic content of umbilical
cord blood for transplantation. Bone Marrow Transplant. 24:965-970, 1999.
Lim FT, Scherjon SA, van Beckhoven JM, Brand A, Kanhai HH, Hermans JM & Falkenburg JH.
Association of stress during delivery with increased numbers of nucleated cells and
hematopoietic progenitor cells in umbilical cord blood. Am.J.Obstet.Gynecol. 183:1144-
1152, 2000.
Liu W, Wang M, Tang DC, Ding I & Rodgers GP. Thrombopoietin has a differentiative effect
on late-stage human erythropoiesis. Br.J.Haematol. 105:459-469, 1999.
Locatelli F, Rocha V, Chastang C, Arcese W, Michel G, Abecasis M, Messina C, Ortega J,

Badell-Serra I, Plouvier E, Souillet G, Jouet JP, Pasquini R, Ferreira E, Garnier F & Gluckman
E. Factors associated with outcome after cord blood transplantation in children with acute
leukemia. Eurocord-Cord Blood Transplant Group. Blood 93:3662-3671, 1999.
Loken MR, Shah VO, Dattilio KL & Civin CI. Flow cytometric analysis of human bone
marrow: I. Normal erythroid development. Blood 69:255-263, 1987.
Loos JA & Wautier JL. Leukocyte depletion: a biotechnical transfusion story.

Transfus.Clin.Biol. 5:64-79, 1998.
Lovelock JE & Bishop MW. Prevention of freezing damage to living cells by dimethyl
sulphoxide. Nature 183:1394-1395, 1959.
Lu L, Xiao M, Shen RN, Grigsby S & Broxmeyer HE. Enrichment, characterization, and
responsiveness of single primitive CD34 human umbilical cord blood hematopoietic
progenitors with high proliferative and replating potential. Blood 81:41-48, 1993.
Lumley MA, Burton A, Billingham LJ, McDonald DF, Czarnecka HM & Milligan DW. Quality
assurance of CFU-GM assays: inter-laboratory variation despite standard reagents.
Eur.J.Haematol. 62:32-37, 1999.
Ma DD, Varga DE & Biggs JC. Donor marrow progenitors (CFU-Mix, BFU-E and CFU-GM) and
haemopoietic engraftment following HLA matched sibling bone marrow transplantation.
Leuk.Res. 11:141-147, 1987.
MacMillan ML, Weisdorf DJ, Brunstein CG, Cao Q, DeFor TE, Verneris MR, Blazar BR &
Wagner JE. Acute graft-versus-host disease after unrelated donor umbilical cord blood
transplantation: analysis of risk factors. Blood 113:2410-2415, 2009.
Maier RF, Bohme K, Dudenhausen JW & Obladen M. Cord blood erythropoietin in relation to
different markers of fetal hypoxia. Obstet.Gynecol. 81:575-580, 1993.
Majhail NS, Brunstein CG, Tomblyn M, Thomas AJ, Miller JS, Arora M, Kaufman DS, Burns
LJ, Slungaard A, McGlave PB, Wagner JE & Weisdorf DJ. Reduced-intensity allogeneic
transplant in patients older than 55 years: unrelated umbilical cord blood is safe and
effective for patients without a matched related donor. Biol.Blood Marrow Transplant.
14:282-289, 2008.
Mancinelli F, Tamburini A, Spagnoli A, Malerba C, Suppo G, Lasorella R, de Fabritiis P &

Calugi A. Optimizing umbilical cord blood collection: impact of obstetric factors versus
quality of cord blood units. Transplant.Proc. 38:1174-1176, 2006.
Manco-Johnson MJ. Development of hemostasis in the fetus. Thromb.Res. 115 (Suppl

1):55-63, 2005.
Manegold G, Meyer-Monard S, Tichelli A, Pauli D, Holzgreve W & Troeger C. Cesarean
89
REFERENCES
section due to fetal distress increases the number of stem cells in umbilical cord blood.
Transfusion 48:871-876, 2008.
Mann KG. Biochemistry and physiology of blood coagulation. Thromb.Haemost. 82:165-

174, 1999.
Mann KG, Butenas S & Brummel K. The dynamics of thrombin formation.

Arterioscler.Thromb.Vasc.Biol. 23:17-25, 2003.
Matsuno N, Wake A, Uchida N, Ishiwata K, Araoka H, Takagi S, Tsuji M, Yamamoto H, Kato

D, Matsuhashi Y, Seo S, Masuoka K, Miyakoshi S, Makino S, Yoneyama A, Kanda Y &
Taniguchi S. Impact of HLA disparity in the graft-versus-host direction on engraftment in
adult patients receiving reduced-intensity cord blood transplantation. Blood 114:1689-
1695, 2009.
Mayani H, Dragowska W & Lansdorp PM. Cytokine-induced selective expansion and

maturation of erythroid versus myeloid progenitors from purified cord blood precursor
cells. Blood 81:3252-3258, 1993.
Mayani H & Lansdorp PM. Biology of human umbilical cord blood-derived hematopoietic
stem/progenitor cells. Stem Cells 16:153-165, 1998.
McCann SR & Lawler M. Mixed chimaerism; detection and significance following BMT. Bone
McCullough J, McKenna D, Kadidlo D, Schierman T & Wagner J. Issues in the quality of

umbilical cord blood stem cells for transplantation. Transfusion 45:832-841, 2005.
McDonald TP, Odell TT & Gosslee DG. Platelet size in relation to platelet age.
Proc.Soc.Exp.Biol.Med. 115:684-689, 1964.
McLeod DL, Shreeve MM & Axelrad AA. Improved plasma culture system for production of
erythrocytic colonies in vitro: quantitative assay method for CFU-E. Blood 44:517-534,
1974.
Metcalf D. Hematopoietic cytokines. Blood 111:485-491, 2008.
Meyer TP, Hofmann B, Zaisserer J, Jacobs VR, Fuchs B, Rapp S, Weinauer F & Burkhart J.
Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical
cord blood. Cytotherapy 8:265-276, 2006.
Migliaccio AR, Migliaccio G & Adamson JW. Expansion of human neonatal progenitor cells in
vitro under serum-deprived conditions. Blood Cells 20:424-428, 1994.
Migliaccio AR, Adamson JW, Stevens CE, Dobrila NL, Carrier CM & Rubinstein P. Cell dose
and speed of engraftment in placental/umbilical cord blood transplantation: graft
progenitor cell content is a better predictor than nucleated cell quantity. Blood 96:2717-
2722, 2000.
Migliaccio AR, Whitsett C & Migliaccio G. Erythroid cells in vitro: from developmental
biology to blood transfusion products. Curr.Opin.Hematol. 16:259-268, 2009.
Migliaccio G, Migliaccio AR, Petti S, Mavilio F, Russo G, Lazzaro D, Testa U, Marinucci M &
Peschle C. Human embryonic hemopoiesis. Kinetics of progenitors and precursors
underlying the yolk sac - liver transition. J.Clin.Invest. 78:51-60, 1986.
Migliaccio G, Baiocchi M, Hamel N, Eddleman K & Migliaccio AR. Circulating progenitor cells
in human ontogenesis: response to growth factors and replating potential. J.Hematother.
90
REFERENCES
5:161-170, 1996.
Miharada K, Hiroyama T, Sudo K, Nagasawa T & Nakamura Y. Efficient enucleation of

erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells.
Nat.Biotechnol. 24:1255-1256, 2006.
Mikkola HK, Gekas C, Orkin SH & Dieterlen-Lievre F. Placenta as a site for hematopoietic
stem cell development. Exp.Hematol. 33:1048-1054, 2005.
Miller GJ, Bauer KA, Barzegar S, Foley AJ, Mitchell JP, Cooper JA & Rosenberg RD. The
effects of quality and timing of venepuncture on markers of blood coagulation in healthy
middle-aged men. Thromb.Haemost. 73:82-86, 1995.
Mohyeddin Bonab MA, Alimoghaddam KA, Goliaei ZA & Ghavamzadeh AR. Which factors
can affect cord blood variables? Transfusion 44:690-693, 2004.
Molteni RA, Stys SJ & Battaglia FC. Relationship of fetal and placental weight in human
beings: fetal/placental weight ratios at various gestational ages and birth weight
distributions. J.Reprod.Med. 21:327-334, 1978.
Monagle P, Ignjatovic V & Savoia H. Hemostasis in neonates and children: pitfalls and
dilemmas. Blood Rev. 24:63-68, 2010.
Monroe DM, Hoffman M & Roberts HR. Platelets and thrombin generation.
Arterioscler.Thromb.Vasc.Biol. 22:1381-1389, 2002.
Moroff G, Eichler H, Brand A, Kekomaki R, Kurtz J, Letowska M, Pamphilon D, Read EJ,

Porretti L, Lecchi L, Reems JA, Sacher R, Seetharaman S, Takahashi TA & Biomedical
Excellence for Safer Transfusion (BEST) Collaborative. Multiple-laboratory comparison of in
vitro assays utilized to characterize hematopoietic cells in cord blood. Transfusion 46:507-
515, 2006.
M-Reboredo N, Diaz A, Castro A & Villaescusa RG. Collection, processing and

cryopreservation of umbilical cord blood for unrelated transplantation. Bone Marrow
Transplant. 26:1263-1270, 2000.
Muntean W, Danda M & Rosegger H. Thrombin-antithrombin III complex and D-dimer in

neonates: signs of thrombin generation during birth. In: Perinatal Thrombosis and
Hemostasis (eds. Suzuki S, Hathaway WE, Bonnar J & Sutor AH), Springer-Verlag, Tokyo
1991, pp. 57-64.
Nakagawa R, Watanabe T, Kawano Y, Kanai S, Suzuya H, Kaneko M, Watanabe H, Okamoto

Y, Kuroda Y, Nakayama T & Chugoku-Shikoku Cord Blood Bank. Analysis of maternal and
neonatal factors that influence the nucleated and CD34+ cell yield for cord blood banking.
Transfusion 44:262-267, 2004.
Nakahata T & Ogawa M. Hemopoietic colony-forming cells in umbilical cord blood with
extensive capability to generate mono- and multipotential hemopoietic progenitors.
J.Clin.Invest. 70:1324-1328, 1982.
Nakahata T, Gross AJ & Ogawa M. A stochastic model of self-renewal and commitment to

differentiation of the primitive hemopoietic stem cells in culture. J.Cell.Physiol. 113:455-
458, 1982.
Neildez-Nguyen TM, Wajcman H, Marden MC, Bensidhoum M, Moncollin V, Giarratana MC,

Kobari L, Thierry D & Douay L. Human erythroid cells produced ex vivo at large scale
differentiate into red blood cells in vivo. Nat.Biotechnol. 20:467-472, 2002.
91
REFERENCES
Netcord-FACT. International Standards for CB Collection, Banking, and Release for

Administration, 3rd edition, FACT Accreditation Office, Omaha (NE) 2006.
Netcord-FACT. International Standards for Cord Blood Collection, Banking, and Release for
Administration, 4th edition, FACT Accreditation Office, Omaha (NE) 2010.
Newall F, Johnston L, Ignjatovic V, Summerhayes R & Monagle P. Age-related plasma

reference ranges for two heparin-binding proteins - vitronectin and platelet factor 4.
Int.J.Lab.Hematol. 31:683-687, 2009.
Newman PJ & Newman DK. Platelets and the vessel wall. In: Nathan and Oski's Hematology
Nicola NA & Johnson GR. The production of committed hemopoietic colony-forming cells
from multipotential precursor cells in vitro. Blood 60:1019-1029, 1982.
Novak JP & Stewart CC. Stochastic versus deterministic in haemopoiesis: what is what?
Br.J.Haematol. 78:149-154, 1991.
O'Brien JR. A relationship between platelet volume and platelet number.

Thromb.Diath.Haemorrh. 31:363-365, 1974.
Ogawa M, Porter PN & Nakahata T. Renewal and commitment to differentiation of

hemopoietic stem cells (an interpretive review). Blood 61:823-829, 1983.
Oken E, Kleinman KP, Rich-Edwards J & Gillman MW. A nearly continuous measure of birth
weight for gestational age using a United States national reference. BMC Pediatr. 3:6,
2003.
Olson TA, Levine RF, Mazur EM, Wright DG & Salvado AJ. Megakaryocytes and
megakaryocyte progenitors in human cord blood. Am.J.Pediatr.Hematol.Oncol. 14:241-
247, 1992.
Omori A, Manabe M, Kudo K, Tanaka K, Takahashi K & Kashiwakura I. Influence of obstetric

factors on the yield of mononuclear cells, CD34(+) cell count and volume of
placental/umbilical cord blood. J.Obstet.Gynaecol.Res. 36:52-57, 2010.
Ordemann R, Petzold K, Holig K, Schaffer B, Mauersberger S & Ehninger G. Experiences of

the Dresdner Cord Blood Bank, supported by the Deutsche Knochenmarkspenderdatei.
Semin.Thromb.Hemost. 25:575-578, 1999.
Orkin SH & Zon LI. Hematopoiesis and stem cells: plasticity versus developmental
heterogeneity. Nat.Immunol. 3:323-328, 2002.
Pafumi C, Farina M, Bandiera S, Cavallaro A, Pernicone G, Russo A, Iemmola A, Chiarenza

M, Leonardi I, Calogero AE, Calcagno A & Cianci A. Differences in umbilical cord blood units
collected during cesarean section, before or after the delivery of the placenta.
Gynecol.Obstet.Invest. 54:73-77, 2002.
Panzenbock B, Bartunek P, Mapara MY & Zenke M. Growth and differentiation of human

stem cell factor/erythropoietin-dependent erythroid progenitor cells in vitro. Blood
92:3658-3668, 1998.
Parikh SH, Mendizabal A, Martin PL, Prasad VK, Szabolcs P, Driscoll TA & Kurtzberg J.
Unrelated donor umbilical cord blood transplantation in pediatric myelodysplastic
syndrome: a single-center experience. Biol.Blood Marrow Transplant. 15:948-955, 2009.
92
REFERENCES
Pasca AM & Penn AA. The placenta: the lost neuroendocrine organ. NeoReviews 11:e64-
e77, 2010.
Patrick CH, Lazarchick J, Stubbs T & Pittard WB. Mean platelet volume and platelet
distribution width in the neonate. Am.J.Pediatr.Hematol.Oncol. 9:130-132, 1987.
Perri T, Ferber A, Digli A, Rabizadeh E, Weissmann-Brenner A & Divon MY. Nucleated red
blood cells in uncomplicated prolonged pregnancy. Obstet.Gynecol. 104:372-376, 2004.
Petaja J, Peltola K, Sairanen H, Leijala M, Kekomaki R, Vahtera E & Siimes MA. Fibrinolysis,
antithrombin III, and protein C in neonates during cardiac operations.
J.Thorac.Cardiovasc.Surg. 112:665-671, 1996.
Pettengell R, Luft T, Henschler R, Hows JM, Dexter TM, Ryder D & Testa NG. Direct
comparison by limiting dilution analysis of long-term culture-initiating cells in human bone
marrow, umbilical cord blood, and blood stem cells. Blood 84:3653-3659, 1994.
Pettengell R. Haematopoietic recovery following transplantation. In: Blood Stem Cell

Transplantation (eds. Reiffers J, Goldman JM & Armitage JO), 1st edition, Martin Dunitz,
London 1998, pp. 157-170.
Petterson TE, Gabriel M, Tiedemann K, Teague L, Shaw PJ, Baker D, Bolton-Jones R, Tapp H,
Oswald C, Vowels MR & O'Brien TA. Outcome following unrelated cord blood transplant in
136 patients with malignant and non-malignant diseases: a report from the Australian and
New Zealand children's haematology and oncology group. Bone Marrow Transplant.
43:207-215, 2009.
Pihkala J, Hakala T, Voutilainen P & Raivio K. Characteristic of recent fetal growth curves in
Finland. Duodecim 105:1540-1546, 1989.
Pike BL & Robinson WA. Human bone marrow colony growth in agar-gel. J.Cell.Physiol.
76:77-84, 1970.
Porter PN, Ogawa M & Leary AG. Enhancement of the growth of human early erythroid
progenitors by bone marrow conditioned media. Exp.Hematol. 8:83-88, 1980.
Prasad VK, Mendizabal A, Parikh SH, Szabolcs P, Driscoll TA, Page K, Lakshminarayanan S,
Allison J, Wood S, Semmel D, Escolar ML, Martin PL, Carter S & Kurtzberg J. Unrelated donor
umbilical cord blood transplantation for inherited metabolic disorders in 159 pediatric
patients from a single center: influence of cellular composition of the graft on
transplantation outcomes. Blood 112:2979-2989, 2008.
Querol S, Gabarro M, Amat L, Gonzalez S, Gomez MD, de la Calle O, Madoz P, Badell I &
Garcia J. The placental blood program of the Barcelona Cord Blood Bank. Bone Marrow
Transplant. 22 (Suppl 1):S3-S5, 1998.
Querol S, Gomez SG, Pagliuca A, Torrabadella M & Madrigal JA. Quality rather than
quantity: the cord blood bank dilemma. Bone Marrow Transplant. 45:970-978, 2010.
Quesenberry PJ, Colvin GA & Lambert JF. The chiaroscuro stem cell: a unified stem cell
theory. Blood 100:4266-4271, 2002.
Ravanat C, Freund M, Mangin P, Azorsa DO, Schwartz C, Moog S, Schuhler S, Dambach J,

Cazenave JP & Lanza F. GPV is a marker of in vivo platelet activation - study in a rat
thrombosis model. Thromb.Haemost. 83:327-333, 2000.
Rebulla P, Lecchi L, Porretti L, Poli F, Ratti I, Mozzi F & Sirchia G. Practical placental blood
banking. Transfus.Med.Rev. 13:205-226, 1999.
93
REFERENCES
Rendine S, Curtoni ES, di Celle PF, Berrino M, Bertola L, Barbanti M, Saracco P, Fazio L, Gay E
& Dall'Omo AM. Analysis of the Turin umbilical cord blood bank registry. Transfusion
40:813-816, 2000.
Roberts HR, Hoffman M & Monroe DM. A cell-based model of thrombin generation.
Semin.Thromb.Hemost. 32 (Suppl 1):32-38, 2006.
Robin C, Bollerot K, Mendes S, Haak E, Crisan M, Cerisoli F, Lauw I, Kaimakis P, Jorna R,

Vermeulen M, Kayser M, van der Linden R, Imanirad P, Verstegen M, Nawaz-Yousaf H,
Papazian N, Steegers E, Cupedo T & Dzierzak E. Human placenta is a potent hematopoietic
niche containing hematopoietic stem and progenitor cells throughout development.
Cell.Stem Cell. 5:385-395, 2009.
Rocha V, Chastang C, Souillet G, Pasquini R, Plouvier E, Nagler A, Locatelli F, Saarinen U,

Cornu G, Bernaudin F & Gluckman E. Related cord blood transplants: the Eurocord
experience from 78 transplants. Eurocord Transplant group. Bone Marrow Transplant. 21
(Suppl 3):S59-S62, 1998.
Rocha V, Cornish J, Sievers EL, Filipovich A, Locatelli F, Peters C, Remberger M, Michel G,

Arcese W, Dallorso S, Tiedemann K, Busca A, Chan KW, Kato S, Ortega J, Vowels M, Zander
A, Souillet G, Oakill A, Woolfrey A, Pay AL, Green A, Garnier F, Ionescu I, Wernet P, Sirchia
G, Rubinstein P, Chevret S & Gluckman E. Comparison of outcomes of unrelated bone
marrow and umbilical cord blood transplants in children with acute leukemia. Blood
97:2962-2971, 2001.
Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A, Jacobsen N, Ruutu T, de

Lima M, Finke J, Frassoni F, Gluckman E, Acute Leukemia Working Party of European Blood
and Marrow Transplant Group & Eurocord-Netcord Registry. Transplants of umbilical-cord
blood or bone marrow from unrelated donors in adults with acute leukemia. N.Engl.J.Med.
351:2276-2285, 2004.
Rodriguez L, Azqueta C, Azzalin S, Garcia J & Querol S. Washing of cord blood grafts after
thawing: high cell recovery using an automated and closed system. Vox Sang. 87:165-172,
2004.
Rogers CE, Bradley MS, Palsson BO & Koller MR. Flow cytometric analysis of human bone
marrow perfusion cultures: erythroid development and relationship with burst-forming
units-erythroid. Exp.Hematol. 24:597-604, 1996.
Rogers I, Sutherland, Holt D, Macpate F, Lains A, Hollowell S, Cruickshank B & Casper RF.
Human UC-blood banking: impact of blood volume, cell separation and cryopreservation on
leukocyte and CD34(+) cell recovery. Cytotherapy 3:269-276, 2001.
Rolfo A, Maconi M, Cardaropoli S, Biolcati M, Danise P & Todros T. Nucleated red blood cells
in term fetuses: reference values using an automated analyzer. Neonatology 92:205-208,
2007.
Rubinstein P, Rosenfield RE, Adamson JW & Stevens CE. Stored placental blood for
unrelated bone marrow reconstitution. Blood 81:1679-1690, 1993.
Rubinstein P, Taylor PE, Scaradavou A, Adamson JW, Migliaccio G, Emanuel D, Berkowitz RL,
Alvarez E & Stevens CE. Unrelated placental blood for bone marrow reconstitution:
organization of the placental blood program. Blood Cells 20:587-596, 1994.
Rubinstein P, Dobrila L, Rosenfield RE, Adamson JW, Migliaccio G, Migliaccio AR, Taylor PE &
Stevens CE. Processing and cryopreservation of placental/umbilical cord blood for
unrelated bone marrow reconstitution. Proc.Natl.Acad.Sci.U.S.A. 92:10119-10122, 1995.
94
REFERENCES
Rubinstein P, Carrier C, Scaradavou A, Kurtzberg J, Adamson J, Migliaccio AR, Berkowitz RL,

Cabbad M, Dobrila NL, Taylor PE, Rosenfield RE & Stevens CE. Outcomes among 562
recipients of placental-blood transplants from unrelated donors. N.Engl.J.Med. 339:1565-
1577, 1998.
Russell N, Gratwohl A & Schmitz N. The place of blood stem cells in allogeneic
transplantation. Br.J.Haematol. 93:747-753, 1996.
Rybak ME, Lau HK, Tomkins B, Rosenberg RD & Handin RI. Relationship between platelet
secretion and prothrombin cleavage in native whole blood. J.Clin.Invest. 68:405-412,
1981.
Sainio S, Javela K, Kekomaki R & Teramo K. Thrombopoietin levels in cord blood plasma and
amniotic fluid in fetuses with alloimmune thrombocytopenia and healthy controls.
Br.J.Haematol. 109:330-335, 2000a.
Sainio S, Jarvenpaa AL, Renlund M, Riikonen S, Teramo K & Kekomaki R. Thrombocytopenia

in term infants: a population-based study. Obstet.Gynecol. 95:441-446, 2000b.
Salonvaara M, Riikonen P, Vahtera E, Mahlamaki E, Heinonen K & Kekomaki R.

Development of selected coagulation factors and anticoagulants in preterm infants by the
age of six months. Thromb.Haemost. 92:688-696, 2004.
Sanz J, Sanz MA, Saavedra S, Lorenzo I, Montesinos P, Senent L, Planelles D, Larrea L,

Martin G, Palau J, Jarque I, Martinez J, de la Rubia J, Moscardo F, Romero M, Luna I,
Montava A, Canabate S & Sanz GF. Cord blood transplantation from unrelated donors in
adults with high-risk acute myeloid leukemia. Biol.Blood Marrow Transplant. 16:86-94,
2010.
Scaradavou A, Smith KM, Hawke R, Schaible A, Abboud M, Kernan NA, Young JW & Barker
JN. Cord blood units with low CD34+ cell viability have a low probability of engraftment
after double unit transplantation. Biol.Blood Marrow Transplant. 16:500-508, 2010.
Schallmoser K, Bartmann C, Rohde E, Reinisch A, Kashofer K, Stadelmeyer E, Drexler C,

Lanzer G, Linkesch W & Strunk D. Human platelet lysate can replace fetal bovine serum for
clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 47:1436-
1446, 2007.
Shaw PH, Gilligan D, Wang XM, Thall PF & Corey SJ. Ex vivo expansion of megakaryocyte
precursors from umbilical cord blood CD34 cells in a closed liquid culture system. Biol.Blood
Shpall EJ, Quinones R, Giller R, Zeng C, Baron AE, Jones RB, Bearman SI, Nieto Y, Freed B,
Madinger N, Hogan CJ, Slat-Vasquez V, Russell P, Blunk B, Schissel D, Hild E, Malcolm J,
Ward W & McNiece IK. Transplantation of ex vivo expanded cord blood. Biol.Blood Marrow
Transplant. 8:368-376, 2002.
Shulman HM, Sullivan KM, Weiden PL, McDonald GB, Striker GE, Sale GE, Hackman R, Tsoi
MS, Storb R & Thomas ED. Chronic graft-versus-host syndrome in man. A long-term
clinicopathologic study of 20 Seattle patients. Am.J.Med. 69:204-217, 1980.
Shuman MA & Levine SP. Relationship between secretion of platelet factor 4 and thrombin
generation during in vitro blood clotting. J.Clin.Invest. 65:307-313, 1980.
Shuman MA. Thrombin-cellular interactions. Ann.N.Y.Acad.Sci. 485:228-239, 1986.
Skjonsberg OH, Kierulf P, Fagerhol MK & Godal HC. Thrombin generation during collection
and storage of blood. Vox Sang. 50:33-37, 1986.
95
REFERENCES
Skoric D, Balint B, Petakov M, Sindjic M & Rodic P. Collection strategies and

cryopreservation of umbilical cord blood. Transfus.Med. 17:107-113, 2007.
Solh M, Brunstein C, Morgan S & Weisdorf D. Platelet and red blood cell utilization and
transfusion independence in umbilical cord blood and allogeneic peripheral blood
hematopoietic cell transplants. Biol.Blood Marrow Transplant. 2010.
Solomon M, Wofford J, Johnson C, Regan D & Creer MH. Factors influencing cord blood
viability assessment before cryopreservation. Transfusion 50:820-830, 2010.
Solves P, Moraga R, Saucedo E, Perales A, Soler MA, Larrea L, Mirabet V, Planelles D,

Carbonell-Uberos F, Monleon J, Planells T, Guillen M, Andres A & Franco E. Comparison
between two strategies for umbilical cord blood collection. Bone Marrow Transplant.
31:269-273, 2003.
Solves P, Perales A, Moraga R, Saucedo E, Soler MA & Monleon J. Maternal, neonatal and
collection factors influencing the haematopoietic content of cord blood units. Acta
Haematol. 113:241-246, 2005.
Sparrow RL, Cauchi JA, Ramadi LT, Waugh CM & Kirkland MA. Influence of mode of birth and
collection on WBC yields of umbilical cord blood units. Transfusion 42:210-215, 2002.
Stevens CE, Gladstone J, Taylor PE, Scaradavou A, Migliaccio AR, Visser J, Dobrila NL,
Carrier C, Cabbad M, Wernet P, Kurtzberg J & Rubinstein P. Placental/umbilical cord blood
for unrelated-donor bone marrow reconstitution: relevance of nucleated red blood cells.
Blood 100:2662-2664, 2002.
Storms RW, Trujillo AP, Springer JB, Shah L, Colvin OM, Ludeman SM & Smith C. Isolation of
primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase
activity. Proc.Natl.Acad.Sci.U.S.A. 96:9118-9123, 1999.
Strauss RG. In vitro comparison of the erythrocyte sedimenting properties of dextran,

hydroxyethyl starch and a new low-molecular-weight hydroxyethyl starch. Vox Sang.
37:268-271, 1979.
Suarez CR, Gonzalez J, Menendez C, Fareed J, Fresco R & Walenga J. Neonatal and maternal
platelets: activation at time of birth. Am.J.Hematol. 29:18-21, 1988.
Suda T, Nemoto K, Izumi T, Miyawaki S & Maekawa T. Colony assay using collagen gel: a
better technique for studying the cell morphology of colonies. Exp.Hematol. 12:245-249,
1984.
Sui X, Tsuji K, Tajima S, Tanaka R, Muraoka K, Ebihara Y, Ikebuchi K, Yasukawa K, Taga T,

Kishimoto T & Nakahata T. Erythropoietin-independent erythrocyte production: signals
through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.
J.Exp.Med. 183:837-845, 1996.
Sun J, Allison J, McLaughlin C, Sledge L, Waters-Pick B, Wease S & Kurtzberg J. Differences

in quality between privately and publicly banked umbilical cord blood units: a pilot study of
autologous cord blood infusion in children with acquired neurologic disorders. Transfusion
2010.
Surbek DV, Visca E, Steinmann C, Tichelli A, Schatt S, Hahn S, Gratwohl A & Holzgreve W.
Umbilical cord blood collection before placental delivery during Cesarean delivery increases
cord blood volume and nucleated cell number available for transplantation.
Am.J.Obstet.Gynecol. 183:218-221, 2000.
Sutherland DR & Keating A. The CD34 antigen: structure, biology, and potential clinical
96
REFERENCES
applications. J.Hematother. 1:115-129, 1992.
Sutherland DR, Keating A, Nayar R, Anania S & Stewart AK. Sensitive detection and
enumeration of CD34+ cells in peripheral and cord blood by flow cytometry. Exp.Hematol.
22:1003-1010, 1994.
Sutherland DR, Anderson L, Keeney M, Nayar R & Chin-Yee I. The ISHAGE guidelines for
CD34+ cell determination by flow cytometry. International Society of Hematotherapy and
Graft Engineering. J.Hematother. 5:213-226, 1996.
Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W & Lansdorp PM. Characterization and
partial purification of human marrow cells capable of initiating long-term hematopoiesis in
vitro. Blood 74:1563-1570, 1989.
Takahashi S, Iseki T, Ooi J, Tomonari A, Takasugi K, Shimohakamada Y, Yamada T, Uchimaru

K, Tojo A, Shirafuji N, Kodo H, Tani K, Takahashi T, Yamaguchi T & Asano S. Single-institute
comparative analysis of unrelated bone marrow transplantation and cord blood
transplantation for adult patients with hematologic malignancies. Blood 104:3813-3820,
2004.
Takahashi S, Ooi J, Tomonari A, Konuma T, Tsukada N, Oiwa-Monna M, Fukuno K, Uchiyama

M, Takasugi K, Iseki T, Tojo A, Yamaguchi T & Asano S. Comparative single-institute
analysis of cord blood transplantation from unrelated donors with bone marrow or
peripheral blood stem-cell transplants from related donors in adult patients with
hematologic malignancies after myeloablative conditioning regimen. Blood 109:1322-
1330, 2007.
Takahashi TA, Rebulla P, Armitage S, van Beckhoven J, Eichler H, Kekomaki R, Letowska M,

Wahab F & Moroff G. Multi-laboratory evaluation of procedures for reducing the volume of
cord blood: influence on cell recoveries. Cytotherapy 8:254-264, 2006.
Tamburini A, Malerba C, Mancinelli F, Spagnoli A, Ballatore G, Bruno A, Crescenzi F, de

Fabritiis P & Calugi A. Evaluation of biological features of cord blood units collected with
different methods after Cesarean section. Transplant.Proc. 38:1171-1173, 2006.
Terakura S, Azuma E, Murata M, Kumamoto T, Hirayama M, Atsuta Y, Kodera Y, Yazaki M,

Naoe T & Kato K. Hematopoietic engraftment in recipients of unrelated donor umbilical cord
blood is affected by the CD34+ and CD8+ cell doses. Biol.Blood Marrow Transplant.
13:822-830, 2007.
Terstappen LW, Huang S, Safford M, Lansdorp PM & Loken MR. Sequential generations of
hematopoietic colonies derived from single nonlineage-committed CD34+CD38-
progenitor cells. Blood 77:1218-1227, 1991.
Thomas E, Storb R, Clift RA, Fefer A, Johnson FL, Neiman PE, Lerner KG, Glucksberg H &
Buckner CD. Bone-marrow transplantation (first of two parts). N.Engl.J.Med. 292:832-
843, 1975.
Thomas ED. Bone marrow transplantation: a review. Semin.Hematol. 36:95-103, 1999.
Thompson CB & Jakubowski JA. The pathophysiology and clinical relevance of platelet
heterogeneity. Blood 72:1-8, 1988.
Thompson JM, Irgens LM, Skjaerven R & Rasmussen S. Placenta weight percentile curves
for singleton deliveries. BJOG 114:715-720, 2007.
Thomson BG, Robertson KA, Gowan D, Heilman D, Broxmeyer HE, Emanuel D, Kotylo P,
Brahmi Z & Smith FO. Analysis of engraftment, graft-versus-host disease, and immune
97
REFERENCES
recovery following unrelated donor cord blood transplantation. Blood 96:2703-2711, 2000.
Thornley I, Eapen M, Sung L, Lee SJ, Davies SM & Joffe S. Private cord blood banking:
experiences and views of pediatric hematopoietic cell transplantation physicians. Pediatrics
123:1011-1017, 2009.
Till JE, McCulloch EA & Siminovitch L. A stochastic model of stem cell proliferation, based on
the growth of spleen colony-forming cells. Proc.Natl.Acad.Sci.U.S.A. 51:29-36, 1964.
Traycoff CM, Abboud MR, Laver J, Brandt JE, Hoffman R, Law P, Ishizawa L & Srour EF.
Evaluation of the in vitro behavior of phenotypically defined populations of umbilical cord
blood hematopoietic progenitor cells. Exp.Hematol. 22:215-222, 1994.
Traycoff CM, Kosak ST, Grigsby S & Srour EF. Evaluation of ex vivo expansion potential of
cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting
dilution analysis. Blood 85:2059-2068, 1995.
Tripodi A, Ramenghi LA, Chantarangkul V, De Carli A, Clerici M, Groppo M, Mosca F &

Mannucci PM. Normal thrombin generation in neonates in spite of prolonged conventional
coagulation tests. Haematologica 93:1256-1259, 2008.
Uchida N, Wake A, Takagi S, Yamamoto H, Kato D, Matsuhashi Y, Matsumura T, Seo S,

Matsuno N, Masuoka K, Kusumi E, Yuji K, Miyakoshi S, Matsuzaki M, Yoneyama A &
Taniguchi S. Umbilical cord blood transplantation after reduced-intensity conditioning for
elderly patients with hematologic diseases. Biol.Blood Marrow Transplant. 14:583-590,
2008.
Van Haute I, Lootens N, De Smet S, De Buck C, Verdegem L, Vanheusden K, Pinxteren J &

Vandekerckhove B. Viable CD34+ stem cell content of a cord blood graft: which
measurement performed before transplantation is most representative? Transfusion
44:547-554, 2004.
Verfaillie CM, Ploemacher R, Di Persio J, Sutherland R, Serke S, Johnsen H, Noga S, Negrin

R & Stem Cell Evaluation and Manipulation Committee. Assays to determine hematopoietic
stem cell content in blood or marrow grafts. Cytotherapy 1:41-49, 1999.
Verneris MR, Brunstein CG, Barker J, MacMillan ML, DeFor T, McKenna DH, Burke MJ, Blazar
BR, Miller JS, McGlave PB, Weisdorf DJ & Wagner JE. Relapse risk after umbilical cord blood
transplantation: enhanced graft-versus-leukemia effect in recipients of 2 units. Blood
114:4293-4299, 2009.
Wagner JE, Broxmeyer HE & Cooper S. Umbilical cord and placental blood hematopoietic
stem cells: collection, cryopreservation, and storage. J.Hematother. 1:167-173, 1992.
Wagner JE, Kernan NA, Steinbuch M, Broxmeyer HE & Gluckman E. Allogeneic sibling
umbilical-cord-blood transplantation in children with malignant and non-malignant
disease. Lancet 346:214-219, 1995.
Wagner JE, Rosenthal J, Sweetman R, Shu XO, Davies SM, Ramsay NK, McGlave PB, Sender
L & Cairo MS. Successful transplantation of HLA-matched and HLA-mismatched umbilical
cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host
disease. Blood 88:795-802, 1996.
Wagner JE, Barker JN, DeFor TE, Baker KS, Blazar BR, Eide C, Goldman A, Kersey J, Krivit W,
MacMillan ML, Orchard PJ, Peters C, Weisdorf DJ, Ramsay NK & Davies SM. Transplantation
of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant
diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and
survival. Blood 100:1611-1618, 2002.
98
REFERENCES
Wall DA, Noffsinger JM, Mueckl KA, Alonso JM, Regan DM, Johnson CE, Weinstein DL,
Duarte LM & Winn HN. Feasibility of an obstetrician-based cord blood collection network for
unrelated donor umbilical cord blood banking. J.Matern.Fetal.Med. 6:320-323, 1997.
Wall DA. Regulatory issues in cord blood banking and transplantation. Best
Pract.Res.Clin.Haematol. 23:171-177, 2010.
Wardrop CA & Holland BM. The roles and vital importance of placental blood to the newborn
infant. J.Perinat.Med. 23:139-143, 1995.
Warkentin PI & FACT. Voluntary accreditation of cellular therapies: Foundation for the
Accreditation of Cellular Therapy (FACT). Cytotherapy 5:299-305, 2003.
Wedgeworth S. Cord blood workshop addresses licensure questions. AABB News p. 6-7,
April 2010.
Weiss HJ, Witte LD, Kaplan KL, Lages BA, Chernoff A, Nossel HL, Goodman DS &
Baumgartner HR. Heterogeneity in storage pool deficiency: studies on granule-bound
substances in 18 patients including variants deficient in alpha-granules, platelet factor 4,
beta-thromboglobulin, and platelet-derived growth factor. Blood 54:1296-1319, 1979.
Widness JA, Clemons GK, Garcia JF, Oh W & Schwartz R. Increased immunoreactive
erythropoietin in cord serum after labor. Am.J.Obstet.Gynecol. 148:194-197, 1984.
Williams DA. Ex vivo expansion of hematopoietic stem and progenitor cells - robbing Peter
to pay Paul? Blood 81:3169-3172, 1993.
Williams JL, Pipia GG, Datta NS & Long MW. Thrombopoietin requires additional
megakaryocyte-active cytokines for optimal ex vivo expansion of megakaryocyte
precursor cells. Blood 91:4118-4126, 1998.
Wiwanitkit V. Plateletcrit, mean platelet volume, platelet distribution width: its expected
values and correlation with parallel red blood cell parameters. Clin.Appl.Thromb.Hemost.
10:175-178, 2004.
Wofford J, Kemp J, Regan D & Creer M. Ethnically mismatched cord blood transplants in
African Americans: the Saint Louis Cord Blood Bank experience. Cytotherapy 9:660-666,
2007.
Won JH, Cho SD, Park SK, Lee GT, Baick SH, Suh WS, Hong DS & Park HS. Thrombopoietin is
synergistic with other cytokines for expansion of cord blood progenitor cells.
J.Hematother.Stem Cell Res. 9:465-473, 2000.
World Marrow Donor Association. Unrelated Cord Blood Banks / Registries. Annual Report
2009. 11th edition, 2009.
Xu Y, Kashiwakura I & Takahashi TA. High sensitivity of megakaryocytic progenitor cells

contained in placental/umbilical cord blood to the stresses during cryopreservation. Bone
Yadav AK, Paul BN, Naik S, Saxena AK & Patel DK. Human hemoglobin shares bioactivities
ascribed to human tumor necrosis factor-alpha. Immunopharmacol.Immunotoxicol.
26:559-572, 2004.
Yamada T, Okamoto Y, Kasamatsu H, Horie Y, Yamashita N & Matsumoto K. Factors affecting

the volume of umbilical cord blood collections. Acta Obstet.Gynecol.Scand. 79:830-833,
2000.
99
REFERENCES
Yao AC, Moinian M & Lind J. Distribution of blood between infant and placenta after birth.
Lancet 2:871-873, 1969.
Yasutake M, Sumita M, Terashima S, Tokushima Y, Nitadori Y & Takahashi TA. Stem cell
collection filter system for human placental/umbilical cord blood processing. Vox Sang.
80:101-105, 2001.
Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M, Leary AG, Olweus J, Kearney J
& Buck DW. AC133, a novel marker for human hematopoietic stem and progenitor cells.
Blood 90:5002-5012, 1997.
Yoo KH, Lee SH, Kim HJ, Sung KW, Jung HL, Cho EJ, Park HK, Kim HA & Koo HH. The impact
of post-thaw colony-forming units-granulocyte/macrophage on engraftment following
unrelated cord blood transplantation in pediatric recipients. Bone Marrow Transplant.
39:515-521, 2007.
Yoon BH & Kim SW. The effect of labor on the normal values of umbilical blood acid-base
status. Acta Obstet.Gynecol.Scand. 73:555-561, 1994.
Yoshida M, Tsuji K, Ebihara Y, Muraoka K, Tanaka R, Miyazaki H & Nakahata T.

Thrombopoietin alone stimulates the early proliferation and survival of human erythroid,
myeloid and multipotential progenitors in serum-free culture. Br.J.Haematol. 98:254-264,
1997.
Yudkin PL, Johnson P & Redman CW. Obstetric factors associated with cord blood gas values
at birth. Eur.J.Obstet.Gynecol.Reprod.Biol. 24:167-176, 1987.
Zingsem J, Strasser E, Weisbach V, Zimmermann R, Ringwald J, Goecke T, Beckmann MW &

Eckstein R. Cord blood processing with an automated and functionally closed system.
Transfusion 43:806-813, 2003.
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NEW APPROACHES TO IMPROVE THE CORD BLOOD UNIT

HEMATOPOIETIC PROGENITOR CELL CONTENT

Finnish Red Cross Blood Service

Department of Obstetrics and Gynecology

To be publicly discussed, with the permission of the Faculty of Medicine,

Riitta Kekomki, M.D., Ph.D.

Pekka Riikonen, M.D., Ph.D.

Mervi Taskinen, M.D., Ph.D.

Freja Ebeling, M.D., Ph.D.

ISBN 978-952-5457-23-0 (print)

In 167 CB units collected and processed at a national CB bank, mean platelet

Stress during delivery is hypothesized to lead to mobilization of HPCs through

Theoretical models were created to enable the use of platelet characteristics

Hemostasis activation during CB collection was assessed with prothrombin

Culture assays provide information about the hematopoietic potential of the CB

erythroid cells were observed in liquid cultures of cryostored and thawed,

3 List of original publications .................................................................. 9

5 Review of the literature ..................................................................... 11

6 Aims of the study ............................................................................. 44

7 Materials and methods ..................................................................... 45

7.2.1 Donor recruitment ........................................................... 45

BFU-E Burst-forming unit - erythroid

I Eskola M*, Rekunen S*, Aroviita P, Mttnen S, Hiilesmaa V, Sainio S &

II Juutistenaho S, Eskola M, Sainio S, Aranko K & Kekomki R. Association of

III Juutistenaho S, Vahtera E, Aranko K & Kekomki R. Prothrombin activation

IV Juutistenaho S*, Mttnen S*, Eskola M, Aranko K & Kekomki R. Erythroid

*These authors contributed equally to the study.

In addition, some unpublished data, in particular on multiple regression models

The first successful bone marrow (BM) transplantations to reconstitute the

CB banks have been established to enable unrelated CB transplantation

5.1.1 Theories of hematopoiesis

Whether the differentiation of stem cells to more committed progenitors is a

The hierarchical model of hematopoiesis has been challenged with modern

5.1.2 Hematopoietic progenitors

In CB banking, HPCs are recognized based on their surface markers (cluster of

CD34+ cells constitute a heterogeneous population, and more specific markers

In 1974, Knudtzon reported the growth of granulocytic progenitor cell colonies in

assays nor the immunophenotyping of CB HPCs have been standardized.

CB is also reported to contain primitive HPCs with high replating efficiency

Figure 1. The fetomaternal interface of the placenta.

5.1.3 The human placenta

The placenta has recently been suggested to serve as a hematopoietic organ

The blood volume in the fetoplacental circulation has been reported to be

5.1.4 Hematopoiesis in the fetus

gestation (Migliaccio G et al., 1986). Whether definitive, intraembryonic

5.2 Normal hemostasis in the neonate

5.2.1 Primary hemostasis

Platelets, derived from megakaryocytes, are a vital component of primary

5.2.2 Secondary hemostasis

Elements of secondary hemostasis, blood clotting and fibrinolytic activity, have

2009). The concentration of prothrombin, or factor II, in neonates is

Several problems hinder the interpretation of coagulation tests in children and

Figure 2. Conversion of prothrombin (FII) to thrombin (FIIa)

Tissue factor, exposed due to vascular injury, is thought to initiate coagulation

thrombin, a key element in coagulation (Heemskerk JW et al., 2002). In this

5.2.3 Markers of hemostasis activation

Classic coagulation assays, such as activated partial thromboplastin time and

More comprehensive clotting assays provide an assessment of the overall

Appropriate sample collection techniques are particularly important in

Coagulation and platelet activation are mutually dependent processes. During

Grosshaupt B et al., 1997).

5.3 Methods for analyzing cord blood cells

5.3.1 Cell counting

Automated hematology analyzers provide information about blood cell

I Eskola M, Rekunen S, Aroviita P, Mttnen S, Hiilesmaa V, Sainio S &

IV Juutistenaho S, Mttnen S, Eskola M, Aranko K & Kekomki R. Erythroid