Hematopathology / Umbilical Cord Blood Selection
Evaluation of Volume and Total Nucleated Cell Count
as Cord Blood Selection Parameters
A Receiver Operating Characteristic Curve Modeling Approach
José C. Jaime-Pérez, MD, PhD, Roberto Monreal-Robles, MD, Laura N. Rodríguez-Romo, MD,
Consuelo Mancías-Guerra, MD, José Luís Herrera-Garza, MD, and David Gómez-Almaguer, MD
Key Words: Umbilical cord blood banking; Cord blood selection; CD34+ cord blood cells; Cord blood total nucleated cell content;
Cord blood volume; Receiver operating characteristic analysis; Sensitivity; Specificity
DOI: 10.1309/AJCPFB6EXO7BJVLR
Abstract
The objective of the study was to evaluate the
current standard practice of using volume and total
nucleated cell (TNC) count for the selection of cord
blood (CB) units for cryopreservation and further
transplantation. Data on 794 CB units whose CD34+
cell content was determined by flow cytometry were
analyzed by using a receiver operating characteristic
(ROC) curve model to validate the performance of
volume and TNC count for the selection of CB units
with grafting purposes. The TNC count was the best
parameter to identify CB units having 2 × 106 or more
CD34+ cells, with an area under the ROC curve of
0.828 (95% confidence interval, 0.800-0.856; P < .01)
and an efficiency of 75.4%. Combination of parameters
(TNC/mononuclear cells [MNCs], efficiency 74.7%;
TNC/volume, efficiency 68.9%; and volume/MNCs,
efficiency 68.3%) did not lead to improvement in CB
selection. All CB units having a TNC count of 8 × 108
or more had the required CD34+ cell dose for patients
weighing 10 kg or less.
© American Society for Clinical Pathology
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Umbilical cord blood (UCB) is used as an alternative
source of progenitors for hematopoietic stem cell (HSC)
transplantation. Worldwide, more than 450,000 UCB units
have been stored in CB banks (CBBs), and more than 10,000
UCB transplants have been performed in children and adults
for a variety of disorders,1 including hematologic malignan-
cies, bone marrow failure syndromes, selected hereditary
immunodeficiency states, and metabolic disorders.2-5 The
current status and future trends in this complex and rapidly
evolving field have been recently reviewed.6
HSCs are contained within a population of mononuclear
CD34+ antigen-expressing cells, which typically represent
fewer than 1% of the total leukocytes in CB.7 The CD34+
antigen is the accepted marker for determining the HSC con-
tent in bone marrow, peripheral blood, and UCB.8 Although
no universally accepted guidelines exist, most CBBs use the
combination of product weight (volume) and total nucleated
cell (TNC) count as the main selection factors for cryopreser-
vation, requiring a TNC content from 6 to 10 × 108 for stor-
age9 and a minimal volume between 40 and 60 mL.10-12 The
CD34+ cell content has been shown to influence engraftment
and survival after unrelated UCB transplantation, better pre-
dicting the hematopoietic potential of a CB unit than nucle-
ated cell content.13
For partially mismatched HLA transplants, the number
of CD34+ cells infused is critical, with 1.7 × 105 CD34+ cells
per kilogram of the recipient’s body weight the threshold
dose.13 Taking into account a 10% to 20% cell loss during
freezing-thawing procedures, a dose of 2.0 × 105 CD34+ cells
per kilogram of recipient’s weight before cryopreservation
seems to be more suitable for the selection of CB units for
storage. To know the CD34+ cell number in a CB unit could
Am J Clin Pathol 2011;136:721-726 721
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 Jaime-Pérez et al / Umbilical Cord Blood Selection
be relevant in several aspects, including matching the weight
of the intended recipient with the appropriate unit; also, it
would allow more precise selection of CB units to perform
double-unit transplants for adolescents and adults, as report-
ed.14 However, owing to the lack of standardization of CD34+
cell counting methods, it is currently not possible to compare
CD34+ cell counts among CBBs or transplant centers.
Receiver operating characteristic (ROC) curve analysis is
a graphic representation of the reciprocal relationship between
sensitivity and specificity and provides an objective measure
of the overall diagnostic performance of a test; it has been
frequently used to compare diagnostic tests.15,16 We were able
to locate only 2 published reports on CB selection using ROC
analysis; one focused on obstetric factors to establish selec-
tion criteria for CB cryopreservation,17 and the other dealt
with fetal biometry in which prebirth assessment of biparietal
diameter might allow the selection of donors who would yield
CB units of sufficient volume and cell content.18
We decided to use formal statistical analysis and ROC
curve modeling to analyze the standard practice at most CBBs
for using volume and/or the TNC count for the proper selec-
tion of suitable CB units for further transplantation.
Materials and Methods
The characteristics of 794 CB units received between May
2005 and May 2010 at the CBB of the Hematology Depart-
ment, “Dr José E. Gonzalez” University Hospital, School of
Medicine of the Universidad Autónoma de Nuevo León, Mon-
terrey, México, were retrospectively analyzed for this study.
UCB Collection
Informed consent for UCB collection was obtained from
healthy women with uncomplicated pregnancies receiving
care at the Obstetrics Department, University Hospital “Dr.
José Eleuterio González” and other regional hospitals. UCB
was collected by gravity into a 150-mL sterile bag collection
set (Grifols, Barcelona, Spain), containing 25 mL of citrate-
phosphate-dextrose anticoagulant. The CB unit was then
stored on wet ice for transport to the CBB for processing. As
do others,10,11,19 at our CBB, we use volume and the TNC
count as criteria for the selection of CB units for cryopreser-
vation. UCB units were analyzed for nucleated cell counts
and were cryopreserved if their TNC number was at least 8 ×
108 and their volume exceeded 80 mL, including the antico-
agulant. The time from collection to processing was set at 48
hours or less.
Laboratory Processing
After removal of aliquots for routine testing, UCB in
the collection bag was mixed with 6% hydroxyethyl starch
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(HES) in a 1:4 ratio, with 30 mL as the maximum. The buffy
coat was then separated by centrifuging at 45g for 7 minutes
at 10°C according to a method previously described.20 The
UCB/HES mixture and the supernatant plasma were trans-
ferred into a second plasma transfer bag without severing the
connecting tube.
A second centrifugation step was performed at 600g for
15 minutes at 10ºC. The cell suspension was removed into
a processing set (Pall Medical, Covina, CA) and adjusted
to precisely 20 mL. The equipment used to perform the
volume reduction was an automated closed system, Sepax-
100 (Biosafe America, Houston, TX). The cryocyte bag was
placed in the Coolmix-210 (Biosafe America), which kept it
in constant agitation for adding the cryoprotective mixture,
which consists of 2.5 mL of DMSO (dimethyl sulfoxide)
99.9% (Sigma-Aldrich, St Louis, MO) and 2.5 mL of Dextran
40 (Sigma-Aldrich). When the temperature reached 4ºC, the
cryocyte bag was placed in a protective aluminum cassette
(Medical Technology Vertriebs, Altdorf, Germany) and then
deposited horizontally on a level surface inside a controlled-
rate freezer (Cryomed, Thermo Forma, Marietta, OH) until it
reached –90°C. The units were then transferred into the liquid
phase of a nitrogen freezer (Cryoplus, Thermo Forma) for
long-term storage.
Biologic Studies
TNC Counting
TNC counts were determined with an automated hema-
tology analyzer (Sysmex XT 2000, Sysmex, Mundelein, IL)
using a multiangle polarized scatter separation technique,
which provides the primary TNC count. The TNC was calcu-
lated by multiplying the TNC for the total volume of the bag.
The total mononuclear cell (MNC) counts were calculated
by adding the absolute count of lymphocytes and monocytes
reported in the CBC count and then multiplying this value for
the total volume of the bag.
CD34+ Cell Counting
Before cryopreservation, CD34+ cell counts were deter-
mined with the ISHAGE gating strategy21 using monoclonal
antibodies anti-CD45+ fluorescein isothiocyanate (FITC;
Becton Dickinson, San Jose, CA) and monoclonal antihuman
CD34 class III/FITC, clone BIRMA K-3 (DAKO, Glostrup,
Denmark). CD34+ cells were enumerated using a FACSCali-
bur flow cytometer (Becton Dickinson).
Statistical Analysis
Normality of the studied variables was assessed with
the Kolmogorov-Smirnov test. Spearman correlation set at a
significance level of .01 was performed to assess the correla-
tion of volume and TNC and MNC counts with the CD34+
© American Society for Clinical Pathology
 Hematopathology / Original Article

cell content of the CB units. ROC curve analysis was applied 1.0
to evaluate the performance of volume, TNC count, and abso-
lute MNC counts as a method for identifying CB units with
a CD34+ cell content of 2 × 106 or more, determining also 0.8

the optimal operating point allowing the best performance

for the CB parameter selected. We arbitrarily added different
0.6

Sensitivity
cutoff points for comparing the performance of the selected
parameter at different values, determining their sensitivity,
specificity, positive and negative predictive values, efficiency, 0.4
and positive and negative likelihood ratios.22-25
The analyzed units were divided in 2 groups depending TNC count
on a CD34+ cell count, 2 × 106 or more or fewer than 2 × 106 0.2 MNC count
Volume
as determined by flow cytometry. Descriptive statistics (per- Reference
centages, mean, SD, median, and range) were used to analyze line
the UCB units group with a count of 2 × 106 or more CD34+ 0.0
0.0 0.2 0.4 0.6 0.8 1.0
cells, presenting the number and total CD34+ cell content of
CB units that would be suitable for transplantation based on 1 – Specificity

the required dose of 2 × 105 CD34+ cells/kg, estimated for
recipients weighing 10 to 40 kg. The statistical package used
was SPSS, version 17.0 (SPSS, Chicago, IL). MedCalc statis-
tical software (MedCalc, Mariakerke, Belgium) was used for
ROC curve analysis.
❚Figure 1❚ Receiver operating characteristic (ROC) curve
analysis comparing the performance of volume (area under
the ROC curve [AUC] = 0.784), total nucleated cell (TNC)
count (AUC = 0.828), and absolute mononuclear cell (MNC)
count (AUC = 0.810) as criteria for selecting cord blood units
suitable for cryopreservation.

Results
We studied 794 CB units received at our CBB during
a 5-year period. By applying the current criteria, we found
that our banking efficiency was 57.5%. The initial analysis
revealed a direct correlation in volume, absolute TNC and
MNC counts, and the total number of CD34+ cells. The TNC
content had the most significant correlation with the number
of CD34+ cells (r = 0.681; P < .01), compared with the MNC
count (r = 0.655; P < .01) and volume (r = 0.581; P < .01).
ROC analysis showed the TNC count to be the parameter
with a superior area under the ROC curve, 0.828 (95% confi-
dence interval [CI], 0.800-0.856), with a significant difference
compared with the MNC count (0.810; 95% CI, 0.780-0.840;
P < .05) and volume (0.784; 95% CI, 0.753-0.816; P < .001)
❚Figure 1❚. There was no significant difference in the area
under the ROC curve between volume and MNC count (P =
.56). A TNC count of 8.32 × 108 was the optimal operating
point chosen by ROC analysis that most accurately allowed
for the selection of CB units meeting the CD34+ cell content
of 2 × 106 (sensitivity, 78.7%; specificity, 70.7%) ❚Table 1❚
and ❚Figure 2❚.
We also evaluated the performance of combining param-
eters (TNC/MNC, efficiency 74.7%; TNC/volume, efficiency
68.9%; and volume/MNC, efficiency 68.3%) in the function
of their best cutoff values derived from the ROC curve with-
out documenting improved performance on CB selection.

❚Table 1❚
Performance Evaluation for Different ROC-Derived TNC Cutoff Values for Cord Blood Selection

TNC Cutoff Value (× 108)

Performance Measure 8.0 8.32* 9.0 10.0 11.0 12.0 13.0

Sensitivity (%) 80.9 78.7 69.7 59.6 49.5 40.3 33.2

Specificity (%) 65.6 70.7 76.8 84.1 90.8 93.9 96.6
Negative predictive value (%) 70.7 70.1 64.1 59.4 55.9 52.5 50.4
Positive predictive value (%) 76.9 79.2 81.0 84.0 88.5 90.3 93.3
Efficiency (%) 74.5 75.4 72.6 69.7 66.6 62.4 59.4
Positive likelihood ratio 2.4 2.7 3.0 3.7 5.4 6.6 9.8
Negative likelihood ratio 0.3 0.3 0.4 0.5 0.6 0.6 0.7

ROC, receiver operating characteristic; TNC, total nucleated cell.

* Optimal operating point chosen by ROC analysis.

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 Jaime-Pérez et al / Umbilical Cord Blood Selection

1.0 It is interesting that when applying the ROC-selected

TNC cutoff as selection criterion to the 466 CB units having
a CD34+ cell content of 2 × 106 or more, the CD34+ cell
0.8
median content increased by 11%, from 3.4 × 106 to 3.8 ×
106, although at the expense of a 21% (n = 99) decrease in
0.6
the number of CB units available for transplantation. This
Sensitivity

approach would lead to an increase in the percentage of units

stored that could be safely grafted, particularly for patients
0.4 weighing 15 to 25 kg ❚Table 2❚ and ❚Figure 3❚.

0.2
0.0
0.0 0.2 0.4 0.6
1 – Specificity
❚Figure 2❚ Receiver operating characteristic (ROC) curve
analysis of absolute total nucleated cell count with an area
under the ROC curve of 0.828 (95% confidence interval,
0.800-0.856; P < .01). The optimal ROC-derived cutoff (square)
shown has a sensitivity of 78.7% and a specificity of 70.7%.
The characteristics of the CB units meeting a CD34+
cell content of 2 × 106 or more (n = 466 [58.7%]) were fur-
ther analyzed. The median volume obtained was 106.3 mL
(range, 48.0-213.2 mL) with a median TNC content of 11.0
× 108 (range, 2.5 × 108-36.6 × 108) and a median CD34+ cell
count of 3.4 × 106 (range, 2.0 × 106-19.4 × 106) after volume
reduction and before cryopreservation. Based on the require-
ment of 2 × 105 CD34+ cells per kilogram of recipient weight
before freezing the CB, this approach would make it possible
to use 100%, 37.3%, 15.2%, and 7.5% of CB units for patients
weighing 10, 20, 30, or 40 kg, respectively (data not shown).
Discussion
Although different studies have been performed regarding
selection, collection, and processing of CB,17,26-30 improve-
0.8 1.0
ment in methods is needed to maximize resource utilization
without compromising quality for clinical use, to increase
efficacy, and to reduce the cost of CB grafting.
The TNC count had the largest area under the ROC
curve, confirming that it is the primary driver of the CD34+
cell content in CB. Combining 2 parameters to select CB
did not lead to an improvement in the relationship between
sensitivity and specificity; therefore, the TNC count as a
single criterion seems to be sufficient for selecting CB units
for cryopreservation. Most of the CB units not meeting the
CD34+ cell content of 2 × 106 or more had a TNC count
below the ROC-selected value. These units would not have
been processed for CD34+ cell content had this cutoff point
been applied. It is worth mentioning that of all CB units with a
TNC count more than the ROC-selected value, only 20% had
a CD34+ cell count of less than 2 × 106. These units, however,
could potentially be used for double partially HLA-matched
UCB transplantation in adults who otherwise are not eligible
for allogeneic grafting owing to the lack of a suitable single-
UCB unit, as previously reported.14

❚Table 2❚
Descriptive Statistics for Cord Blood Units With a CD34+ Cell Count of 2 × 106 or More According to TNC Cutoff Selection
Criterion and the Requirement of 2 × 105 CD34+ Cells per Kilogram of Patient Weight, Estimated for 10 to 40 kg (n = 466)*

CD34+ MNC (× 106) Patient Weight (kg)†

TNC Cutoff (× 108) Mean ± SD Median 10 20 30 40

8.0 4.4 ± 2.5 3.7 377 164 (43.5) 70 (18.6) 35 (9.3)

8.32‡ 4.5 ± 2.6 3.8 367 163 (44.4) 70 (19.1) 35 (9.5)
9.0 4.6 ± 2.6 3.9 325 156 (48.0) 67 (20.6) 35 (10.8)
10.0 4.9 ± 2.7 4.2 278 148 (53.2) 67 (24.1) 35 (12.5)
11.0 5.2 ± 2.9 4.6 231 136 (58.9) 64 (27.7) 35 (15.2)
12.0 5.3 ± 3.0 4.6 188 114 (60.6) 54 (28.7) 29 (15.4)
13.0 5.7 ± 3.0 4.9 155 106 (68.4) 51 (32.9) 27 (17.4)

MNC, mononuclear cells; TNC, total nucleated cell.

* All units met the required dose for patients weighing 10 kg; 89 units containing ≥2 × 106 CD34+ did not meet the 8 × 108 TNC content.
† For 20-40 kg, data are given as number (percentage) in relation to the value in the 10-kg column.
‡ Optimal operating point chosen by receiver operating characteristic analysis.

724 Am J Clin Pathol 2011;136:721-726 © American Society for Clinical Pathology

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40
38
36
34
Patient’s Weight (kg)
32
30
28
26
24
22
20
18
16
14
12
10
0.0 20.0
Percentage of Acceptable Cord Blood Units
❚Figure 3❚ Percentage of cord blood units that would be available for transplantation as a function of patient weight
using units meeting a CD34+ cell count ≥2 × 106 determined by flow cytometry before cord blood cryopreservation,
compared with the application of the parameter total nucleated cell (TNC) count ≥8.32 × 108 selected by receiver operating
characteristic curve analysis.
Because CD34+ cell determination demands significant
time and resources, we used our data for applying ROC curve
analysis to validate current CB parameters as surrogates for
CD34+ cell counting and ability to provide reliable, effective,
and cost-effective criteria for the appropriate identification
of CB units suitable for transplantation, avoiding the cost
of processing units with a high probability of not having the
required CD34+ cell content for cryopreservation and grafting
purposes. We confirmed that the TNC count is the parameter
that better correlates with the CD34+ cell content in UCB, in
agreement with previous reports.26,31,32
ROC curve analysis validated the current standard of TNC
content of 8 × 108 or more as an appropriate cutoff parameter
for the selection of CB units containing an adequate number
of CD34+ MNCs for cryopreservation for grafting purposes.
From the Hematology Department, Internal Medicine Division,
“Dr. José Eleuterio González” University Hospital of the School
of Medicine of the Autonomous University of Nuevo León,
Monterrey, México.
Address reprint requests to Dr Jaime-Pérez: Servicio de
Hematología, Hospital Universitario “Dr. José E. González,”
Edificio “Dr. Rodrigo Barragán,” 2° piso, Avenida Madero y
Gonzalitos S/N, Colonia Mitras Centro, Monterrey, Nuevo León,
México, CP 64460.
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