Download Collection, Processing, and Banking of Umbilical Cord Blood Stem

CE Update
Collection, Processing, and Banking of Umbilical
Cord Blood Stem Cells for Clinical Use in
Transplantation and Regenerative Medicine
David T. Harris, PhD
(Department of Immunobiology, University of Arizona, Tucson, AZ)
DOI: 10.1309/64QG394K1M639L8A
Abstract
Cord blood banking has been the focus of
many medical centers as it can provide a
virtually unlimited source of ethnically-diverse
stem-cell donors. In addition, its recent use
in several regenerative medicine clinical trials
has further spurred the motivation to collect
and bank these stem cells. In the current
study, we review the latest developments in
cord blood banking. We have banked over
195,000 collections at our facility. Collections
were processed by either Ficoll or AXP
methodologies. An average 95% processing
efficiency was obtained. The overall failure
After reading this article, readers should be able to discuss the
collection, processing, and preservation of cord blood samples by the
Cord Blood Registry and explain their current uses and possible uses in
the future.
Bone marrow transplantation (BMT) is an often-used means
of therapy for a variety of diseases, including chemotherapyresistant malignancies and genetic blood disorders (Table 1). For
certain diseases (eg, some leukemias, a variety of aplastic anemias,
and certain immunodeficiencies), it is the only proven treatment
that can achieve long-term patient survival. For other diseases
(eg, autoimmune diseases and some genetic blood disorders), it
could offer the hope of a long-term cure via stem-cell transplantation or gene therapy. If a family member cannot provide a bone
marrow donation, then a search must be performed for a human
leukocyte antigen (HLA)-matched, unrelated volunteer stem-cell
donor. The difficulty of finding suitably HLA-matched donors
for patients needing stem-cell transplant (especially minority patients) has left many patients without treatment.1,2 Furthermore,
graft-versus-host disease (GVHD) occurs more than 50% of the
time in unrelated BMT,3 with approximately half being severe
and life-threatening. Due to the problems of a lack of donors and
Table 1_Current Stem Cell Transplant Uses
Malignancies
Blood disorders
Immunodeficiency
Leukemia
Lymphoma
Multiple myeloma
Hodgkin’s disease
Retinoblastoma
Histiocytosis
Sickle cell anemia
Thalassemia
Aplastic anemia
Thrombocytopenia
Diamond-Blackfan
Amegakaryocytosis
SCID
Ataxia telangiectasia
Wiskott-Aldrich syndrome
DiGeorge syndrome
Kostmann syndrome
Omenn syndrome
Autoimmune diseases
Metabolic disorders
Other inherited disorders
Multiple sclerosis
Leukodystrophy
Osteopetrosis
Systemic lupus
Gaucher disease
Osteogenesis imperfecta
Rheumatoid arthritis
Krabbe disease
Lesch-Nyhan syndrome
Evan syndrome
Hunter syndrome
Tay-Sachs disease
Crohn’s disease Hurler syndrome Niemann-Pick disease
SCID, severe combined immunodeficiency
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rate was less than 4% in terms of samples
containing too few cells to be clinically useful.
Processed samples were frozen in cryovials or
bags and banked in a liquid nitrogen Dewar.
In conclusion, it is possible to simply and
reproducibly harvest, process, and bank cord
blood samples using current technology.
Hematology exam 50801 questions and corresponding answer form
are located after this CE Update article on page 179.
the high incidence of GVHD, researchers have looked to alternate sources of stem cells for transplant.
Work that was begun in the early 1980s revealed that cord
blood (ie, the leftover blood in the umbilical cord and placenta
after the birth of a child) was comparable to bone marrow in
terms of its utility in stem cell transplantation.4-11 Cord blood
offers a number of advantages over bone marrow,11,12 including a lower incidence of GVHD and less strict HLA-matching
requirements, which could increase its availability to transplant
patients. In addition, using related samples instead of unrelated
donor samples can double survival rates for patients.11,13 During the past 10 years, clinical use of cord blood (with more than
8,000 transplants worldwide14) has shown that it is a suitable
alternative to bone marrow.
In addition to its use as a substitute for bone marrow, cord
blood has recently been used in a variety of regenerative medicine applications. Work done by McGuckin and colleagues,15,16
Rogers and colleagues,17 Kucia and colleagues,18 and Harris and
colleagues19,20 has shown that cord blood contains a mixture of
pluripotent stem cells capable of giving rise to cells derived from
the endodermal, mesodermal, and ectodermal lineages. Thus,
cord blood appears to be a practical substitute for embryonic
stem cells and readily available for use in tissue engineering and
regenerative medicine. Recently, clinical trials have begun using
cord blood stem cells to treat type 1 diabetes, cerebral palsy,
and peripheral vascular disease among others (Figure 1).21,22
In 1989, this laboratory began a series of studies examining the use of cord blood for transplantation.23-27 These studies
spurred interest in the establishment of a cord blood bank. During this and subsequent work, we established the methodologies that were needed for efficient and reproducible cord blood
collection, processing, and banking for clinical use.28-32 In June
1992, this laboratory established the first cord blood bank in
the world. This cord blood bank later became the Cord Blood
Registry (CBR) in 1996. Currently, the CBR has more than
195,000 cord blood samples banked at our facility, and is the
largest familial (related) cord blood bank in the world. The
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Figure 1_Cord blood stem cells and regenerative medicine. The figure illustrates preclinical and clinical uses of cord blood stem cells in
regenerative medicine.
results presented in this paper will summarize our cord blood
banking efforts, highlighting the success that has been made
with regard to cord blood collection, processing, and storage.
Also, analyses as to collections from surgical and multiple births
will be discussed. Finally, an analysis of the clinical utility of the
collections will be presented.
Collection of Cord Blood
All patients are required to sign informed consent forms
prior to collection of cord blood. All mothers are tested for
infectious diseases as is typically done with blood donors (ie,
reactivity for HIV, hepatitis B and C, etc.). Furthermore, the
collected samples are tested for microbial sterility using an automated system (bioMérieux, Hazelwood, MO).33 Cord blood
(CB) samples are obtained under the auspices of the patient’s
caregiver (ie, physician or midwife). In the majority of cases, the
collections are made after delivery of the infant and ligation of
the cord, prior to expulsion of the placenta. Prior to collection of
CB, the cord is wiped with alcohol or betadine to ensure sterility
of the collection.28
There are a variety of methods used to collect cord blood,
although primarily either large syringes (60 cc) or small bags
(approximately 400 cc) are used. We have found that syringe
collections provide visual feedback to the collector, allowing
them to control not only the rate and volume of the collection, but also to restart collections that have stopped for any
reason. Unattended bag collections, albeit somewhat more facile
to perform, are not able to provide this option. Furthermore,
first-time or inexperienced collectors routinely are able to collect
larger volumes using the syringe method, with greater sterility of
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collection. Routinely, collections are completed within 5 minutes (prior to placental expulsion). Experienced collectors are
able to collect comparable samples using bags or syringes, and
many collectors have expressed the opinion that bag collections
are simpler to perform (noting the above caveats). In our experience, less than 2% of all clients were unable to have CB collected, generally due to rapid birth of the child. Regardless, both
types of collection kits are provided in a sterile condition for use
during surgical deliveries, as well as being pre-anticoagulated and
containing all necessary shipping materials. These kits meet all
regulatory requirements for shipping blood, including double
containment and a crush-resistant container. Furthermore, the
collection kits are insulated and padded for safety during transport, and studies have shown that these kits protect the sample
from temperature extremes during shipment.31 The collected
cord blood is shipped overnight to our laboratory in Tucson,
AZ, to be received within 28 to 34 hours. At the same time, or
prior to the CB collection, blood samples are obtained from the
mother for infectious disease marker (IDM) testing, a regulatory
requirement, using the provided vacutainers.
Analysis of the last 195,000 CB collections made by more
than 41,000 physicians at 3,128 birthing sites has shown that
cord blood collection is simple and reproducible. Analysis of
the last 100,000 collections made by CBR revealed that more
than 96% of all samples shipped to the cord blood bank arrive
(well) within 36 hours of collection, with more than 75% arriving within 24 hours of collection. The average arrival time of all
samples has been 19.7 hours after collection. Thus, CB collection anywhere in North America (as well as most places worldwide) can be easily accomplished with overnight transport to the
cord blood bank. For all samples, the average range is 70 to 80
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CE Update
cc in size, regardless of the birthing situation (vaginal or surgical
deliveries; Table 2). Cord blood collections from twin and triplet
births are smaller, as expected (as the newborns are generally
smaller), but are routinely large enough for clinical use (Table
3). Further, as more than 99% of all samples are sterile upon
testing, the collection method invariably allows for a CB collection free of microbial and fungal contamination (as assessed
by MacConkey blood agar plate cultures and by an automated
BacT Alert system, bioMérieux). Finally, analysis of these collections for clinical utility revealed that approximately 10% of all
collections did not meet the laboratory’s volume criteria for sample acceptance (ie, greater than 30 cc); however, volume is not
an accurate measure of clinical utility as will be discussed later,
as a high yield of stem cells can be obtained from a relatively
low volume of blood (Table 4). Thus, all collections, regardless
of size, are processed, frozen, and banked as the majority will be
clinically useful for both transplant and regenerative medicine
applications.
12.6 × 106 nucleated cells/cc of cord blood collected and processed. This number of cells is more than 4 times the minimum
number of CB cells that has been successfully used for clinical
transplant. Thus, the failure rate of cord blood collection based
on cell numbers is less than 4% (Table 4). As CD34+ cells constitute approximately 1% of CB mononuclear cells,23 an average
of 6 × 106 CD34+ cells are obtained from a single CB sample
(an average of 5 × 104 CD34+ cells/kg patient body weight is
routinely used for transplant), implying that the average CB
sample contains enough cells to transplant both a child as well as
a full-sized adult recipient.
Samples of all sizes and obtained from all types of birthing/
delivery modes can be easily processed with these methodologies
with high recoveries (Table 1). As expected, samples obtained
from multiple births provide smaller numbers of cells after processing (due to the initial smaller starting volumes) but are well
above minimum thresholds needed for clinical use. Processing
does not introduce a significant risk of contamination when
Processing of Cord Blood
Presently, the vast majority of CB collections are red blood
cell (RBC) reduced prior to cryopreservation. Several methods
are in use to accomplish this goal, including Hespan sedimentation to obtain a modified buffy coat,12 density gradient centrifugation to obtain enriched mononuclear cells (MNC),28 and 2
automated processes (Sepax and automated processing platform
[AXP])34,35 that result in a buffy coat product. The Hespan,
Sepax, and AXP processing methods result in cord blood products composed of all nucleated cell populations found in the
original collection (MNC, neutrophils, some RBCs), while
the Ficoll method enriches for the stem-cell-containing MNC
subpopulation (generally greater than 85% MNC with a few
contaminating neutrophils and nucleated RBC). Cell counts
obtained in the final Ficoll product are generally half the cell
counts found in the other processes for this reason, although the
stem cell recovery may be similar.
Our facility has extensive experience with the use of density
gradient centrifugation and more recently with the automated
AXP process. Both methods reproducibly recover greater than
95% of the cord blood stem cells in a typical collection and result
in a reduced final volume of approximately 20 cc for final storage
(Figure 2). The latter method (AXP) allows for greater throughput
with fixed personnel numbers (increasing the economy of operations) and is an FDA-cleared, functionally closed system (which
is recommended under the current regulatory guidelines36) and is
capable of processing cord blood collections of any volume.
Red blood cell reduction not only facilitates the banking
procedure, but it also helps to eliminate concerns about ABO or
Rh incompatibility between donor and recipient upon clinical
use of the sample. Further, much higher recoveries, as well as
higher cell viabilities, are obtained upon thawing such RBC-depleted products. Also, by performing RBC reduction, the sample
is amenable to immediate use in gene therapy, cell expansion, as
well as storage in multiple aliquots for later multiple uses.
Using either the Ficoll or AXP method, we routinely recover
more than 95% of the original mononuclear cell population in
the sample, as well as more than 95% of the CD34+ cells and
colony-forming units granulocyte-macrophage (CFU-GM) progenitors (both surrogate measures for stem cell recovery). Upon
analysis of the last 15,320 samples processed by the AXP method
(the preferred method for the reasons stated above), we obtained
a final product encompassing 882 × 106 total nucleated cells, or
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Table 2_Comparative Cellular Harvests by Delivery
Route
Type of Collection
N
Volume (cc)
MNC
MNC/cc
Vaginal
C-section
124,057
67,258
71
72
574
528
8.1
7.3
Familial cord blood collections were analyzed according to type of birthing situation: vaginal
deliveries versus surgical deliveries (C-section). Volume is presented as cc of blood minus
anticoagulant. MNC and MNC/cc are presented as x106. No significant differences were
observed between groups. Data is the compilation of products obtained with both Ficoll and
AXP processing methods. MNC, mononuclear cells.
Table 3_Effect of Multiple Births on Cord Blood
Banking Cellular Harvest
Sample
N
Volume (cc)
MNC
Singletons
157,623
71
512
Twins
8,090
53
335
Triplets
507
36
193
Quadruplets
24
37
204
MNC/cc
7.3
6.3
5.4
5.5
Familial cord blood collections were analyzed by number of infants delivered for volume
(minus anticoagulant, presented in cc), total MNC post-processing (x106), and MNC/cc
blood post-processing (x106). Singleton collections were significantly different from all
other collections in terms of volume and total MNC obtained (P<0.05). Twins and triplets
collections were not significantly different from one another. The quadruplet collection was
significantly different from twins and triplets collections (P<0.05) in terms of volume and
total MNC obtained. The MNC/cc was not significantly different between any of the groups.
Data is the compilation of products obtained with both Ficoll and AXP processing methods.
MNC, mononuclear cells.
Table 4_Samples Below Predicted Clinically Usable
Limits
Number of samples below
Number of samples below 30 cc:
19,802 out of 190,000 (10.4%)
1 x 108 TNC: 6,890 out of 190,000 (3.6%)
Familial cord blood collections were analyzed for estimated clinical sufficiency as determined by 2 criteria: absolute volume of blood obtained and total numbers of TNC obtained
post-processing. The numbers of samples below the indicated threshold levels are the percent failure rates for the collections according to each criterion. TNC, total nucleated cells.
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Figure 2_Comparison of processing methods. A comparison of the most commonly-used cord blood processing methods. The percentages are
the MNC/CD34 recovery rates after processing. TNC, total nucleated cells; MNC, mononuclear cells; AXP, automated processing platform.
performed properly, as shown by an overall microbial contamination rate due to processing of <1%.
During the past 14 years we have provided 61 cord blood
samples for use in transplant to 26 different transplant centers
(Figure 3). Although most of our early uses were for typical
transplants (ie, to treat malignant and inherited blood disorders),
recently increased use for regenerative medicine applications has
occurred. Notably, when the samples were thawed at the respective transplant centers, an average post-thaw viability of 90%
was obtained, validating the banking methodology. Significantly,
a sample almost 10 years in storage was thawed with a 99%
recovery, indicating that the samples can probably be stored
indefinitely.
Cryopreservation of Cord Blood
Cord blood samples are cryopreserved using an automated, microprocessor-controlled cell freezer. Briefly, CB cells
are resuspended in ice-cold autologous plasma at densities up
to 300 × 106 cells/mL. An equal volume of cryopreservative
solution containing autologous plasma and the cryoprotectant
dimethylsufoxide (DMSO) is added slowly over the course of
approximately 20 minutes. The cryopreservation protocol uses
a controlled-rate freezing process to slowly reduce the temperature to –180°C. At the end of the freezing procedure the
cells are stored in a specially constructed liquid nitrogen freezer
(MVE, Inc, Laguna Beach, CA) that allows for vapor storage at
liquid nitrogen temperatures.37 Autologous plasma is used for
cryopreservation to avoid exposure to non-self and/or animal
proteins (and the inherent infectious disease risks associated with
such use). A controlled-rate freezer, rather than other methods,
such as methanol immersion, was chosen so that each and
every sample frozen would have a controlled and documented
cryopreservation run, ideal for meeting regulatory guidelines.
Furthermore, this approach produces better results upon thawing as our own experience documents (>90% cell recovery and
viability as determined by independent transplant centers using
the samples for transplant).
Vapor phase storage prevents cross-sample contamination.
Certain viruses, such as hepatitis and papilloma viruses, are
known to survive exposure to liquid nitrogen and have been
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shown to cross-contaminate bags of bone marrow during liquid
nitrogen storage.38 If samples are to be banked for indefinite
periods of time then this aspect becomes a major concern, and
efforts must be made to minimize if not eliminate this potential
problem.
We have chosen to cryopreserve our CB samples in multiple aliquots for several reasons. Multiple aliquots allow for
future use of the stem cells in cell expansion, in gene therapy, or
for regenerative medicine uses that may only require a fraction
of the frozen sample. Thus, it is not necessary to thaw the entire
sample unless absolutely needed, avoiding the damaging effects
of repeated episodes of freezing/thawing. Multiple aliquots also
allow for repeated testing of the sample if needed to resolve any
issues of misidentification or sample potency.
Banking of Cord Blood
Our CB samples are stored in the vapor phase of the largest liquid nitrogen Dewars commercially available (MVE, Inc,
Figure 3_Categories of cord blood stem cell use. An illustration of
the types of applications for cord blood samples banked by the Cord
Blood Registry. Data is shown as the percentage of uses for each
application along with the absolute numbers of samples.
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CE Update
model 1830). Even in the absence of continuous liquid nitrogen
availability, these devices will maintain their –196°C temperatures for more than 1 week. Other facilities may use liquid phase
liquid nitrogen storage, but then cross-contamination becomes
an issue unless other measures are implemented (eg, storage
container overwraps composed of impermeable materials). As a
matter of precaution (and to meet regulatory requirements), the
Dewars are hard-piped to a 9,000-gallon liquid nitrogen container on-site at the facility, with the liquid nitrogen container
connected via telemetry directly to the liquid nitrogen supplier,
eliminating the concern of running out of liquid nitrogen. Each
Dewar is monitored continuously for liquid nitrogen levels and
Dewar temperature. There are multiple backup and fail-safe
systems in place, including the immediate availability of spare
Dewars. The entire facility is alarmed and monitored, and it
requires 2 individuals with separate access codes to access the
Dewars. Overall, the bank is extremely secure against any unforeseen events.
Conclusions
As described above, the collection, processing, and banking
of CB for immediate or future clinical use can be reproducibly
performed with the proper methodology. It is important to note
that all procedures used in the cord blood banking endeavor described herein have met and passed regulatory scrutiny (including AABB accreditation). Such regulatory compliance comes at a
price but is essential in providing the assurance to clients and the
transplant physician that each sample is banked under optimal
conditions and will continue to be in optimal condition years
later, if needed. Although many individuals elect to bank cord
blood for its potential use in the treatment of cancer and genetic
blood disorders, more and more clients are banking upon uses
that are only now coming to light. Such uses include autologous
stem-cell transplants for the treatment of autoimmune diseases,
stem-cell applications in tissue engineering and regenerative
medicine, and stem-cell gene therapy (Tables 2 and 3). Already,
stem cells have been shown to be able to repair damaged heart
and nervous tissues, to treat type 1 diabetes, to give rise to liver
and corneas, to treat children born with neurological defects,
and to cure severe combined immunodeficiency disease via
gene therapy. In the next decade, there will undoubtedly be additional uses that are not yet anticipated. If so, then the odds of
use for a family-banked CB sample will increase from 3 in 1,000
for the treatment of cancer, to upwards of 1 in 5 for use in regenerative medicine (Figure 4).39 As cord blood stem cells come
Figure 5_The Cord Blood Registry Facility. Top panel: the CBR facility
from the outside. Bottom panel: the cord blood storage facility.
to be realized as the next-best alternative to embryonic stem
cells, then the routine banking of CB stem cells for use by the
family or newborn should become a routine and standard part
of the birthing process. LM
Acknowledgments: I would like to acknowledge the invaluable technical assistance of the Cord Blood Bank personnel that
have made this study possible, particularly Heather Brown, Jody
Gorton, and Aaron McGaffey. I would also like to acknowledge
the numerous physicians, midwives, and nurses that have participated in the collection of the cord blood samples.
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