Clinical and Quality Evaluation of Red Blood
Cell Units Collected Via Apheresis Versus Those
Obtained Manually
Eiman Hussein, MD,1* Azza Enein, MD2
Lab Med Summer 2014;45:238-243
DOI: 10.1309/LMKXJ0Y44GPRSXFG
ABSTRACT
To evaluate the impact of collection procedure on the in vitro quality of
red blood cells (RBC), we studied 30 units of apheresis-prepared RBC
(ARBC) and 30 units of manually collected RBC (MRBC). We performed
assays on day 1 and day 21 of the study, evaluating red cell mass
volume (RCM); rate of hemolysis; pH, and levels of sodium, potassium,
adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG) and
glucose. Eight patients with aplastic anemia received RBC transfusions
of both components and their post-transfusion hematocrit (HCT) levels
were compared. On day 21, we observed a significant drop of sodium
and glucose levels in the ARBC group, compared
New transfusion technology allows selective collection
of different components via apheresis. This process
involves collecting combinations of blood components,
including red blood cells (RBC), from a single donation,
which can reduce donor exposure and transmission
of infectious diseases. Most RBC units used for
transfusion are prepared from anticoagulated whole
blood. Automated collection of RBC via apheresis can
Abbreviations
RBC, red blood cells; Hb, hemoglobin; HCT, hematocrit; FDA, United
States Food and Drug Administration; ARBC, apheresis-prepared RBC;
MRBC, manually collected RBC; CPDA-1, citrate-phosphate-dextrose
adenine; RCM, red cell mass volume; Na, sodium; K, potassium; ATP,
adenosine triphosphate; 2,3-DPG, 2,3-diphosphoglycerate; ACD-A, acid
citrate dextrose; SAGM, saline-adenine-glucose-mannitol; PAGGGM,
phosphate-adenine-guanosine-glucose-gluconate-mannitol
Cairo University Blood Bank, Department of Clinical Pathology, Cairo
University, Egypt; 2Hematology laboratory, Department of Clinical
Pathology, Cairo University, Egypt.
1
*To whom correspondence should be addressed.
E-mail: [email protected]
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Lab Medicine Summer 2014 | Volume 45, Number 3
with the MRBC group (P <.05). ARBC group demonstrated higher
RCM that provided significantly higher HCT values to our group
of anemic patients (P <.05). Hemolysis was significantly lower in
the ARBC group, compared with the MRBC group (P <.05). At day
21, both groups had no detectable 2,3-DPG. Specimens from both
groups retained ATP in sufficiently healthy amounts. The ARBC group
demonstrated higher RCM and lower hemolysis levels compared with
the MRBC group.
Keywords: apheresis-prepared red cells, manually prepared red cells,
hemolysis, in vivo assay, in vitro assay, storage days.
potentially provide a standardized, high-quality product
with consistent RBC content.1
Hemolysis is an important parameter for measuring the
quality of RBC. Hemolysis is caused by disruption of the
RBC membrane and the subsequent release of hemoglobin
(Hb). Hemolysis of RBC can occur during preparation and
during storage. The extent of hemolysis is often defined
as the percentage of free hemoglobin in relation to the
hematocrit (HCT). According to the United States Food and
Drug Administration (FDA) guidelines, hemolysis in stored
RBC units should not exceed 1%. Recently, the FDA has
added that 95% of units should meet quality standards and
that the data for these units must show statistical certainty.
To evaluate the impact of the collection procedure on the
quality of packed RBC, we studied apheresis-prepared
RBC (ARBC) prepared via the Gambro Trima Accel device
(Gambro BCT, Lakewood, Colorado, USA) and manually
collected RBC (MRBC) stored in bags containing citratephosphate-dextrose adenine (CPDA-1) anticoagulant.
Another objective of our study was to investigate the
impact of collection procedure on the viability of RBC after
transfusion by measuring post-transfusion HCT levels.
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Materials and Methods
We designed and performed the study according to the
tenets of the Declaration of Helsinki. A total of 30 ARBC
units versus 30 MRBC units were analyzed. RBC units
were stored for 21 days, under standard blood-banking
conditions at a mean (SD) temperature of 4 (2) Cº. None of
the units were leukoreduced.
before operating the machine. Users load the tubing set
on the top of the device; then, they snap a cassette into
the device to make the machine ready for use.
We performed in vitro studies of RBC on day 1 and day 21,
evaluating absolute red cell mass volume (RCM); pH; rate
of hemolysis; and levels of HCT, sodium (Na), potassium
(K), adenosine triphosphate (ATP), 2,3-diphosphoglycerate
(2,3-DPG), glucose, and plasma Hb.
Whole blood is pumped into the machine and mixed with
acid citrate dextrose (ACD-A) anticoagulant at a controlled
ratio near the access needle. The blood components
separate into layers over the entire circumference of the
centrifuge, according to their density. Each component
leaves the centrifuge via its own channel. RBC collection
is initiated after platelet collection is completed. The
components that are not collected are returned to the
donor via return pump. In this study, we performed
sessions using an initial anticoagulant ratio of 8:1 of whole
blood to ACD-A.
Collection and Preparation of RBC Units
In Vitro RBC Analysis
All RBC units were collected from donors who all met the
American Association of Blood Banks (AABB) eligibility
criteria for donation. We recruited donors from the
apheresis center and the Blood Bank at Cairo University
Hospital, Egypt. Men who weigh at least 155 pounds
qualify for automated donation at our center.
Trima Accel Apheresis Protocol for RBC
Collection
Samples of approximately 5 mL were collected
anaerobically for testing from units immediately after
collection, on day 1 and then subsequently on day
21. We performed in vitro assays of HCT, RCM, Na,
K, ATP, 2,3-DPG, pH, glucose, plasma Hb, and rate
of hemolysis using standard procedures. Specimens
were immediately injected into a blood-gas analyzer
to determine pH at 37 Cº. Na+ and K+ concentrations
were determined via flame photometry. For K+ levels of
greater than 20 mmol/L, we used a urine standard for
calibration. ATP levels were measured enzymatically, as
described by Adams. 2 2,3-DPG levels were measured
as described by Rose and Liebowitz.3 Plasma Hb
was measured using a modification of the Drabkin
cyanmethemoglobin method4 which is read at 540 and
680 nm via spectrophotometer (Beckman Coulter, Inc,
Brea, CA).
We performed apheresis via a single-needle procedure
using a Gambro Trima Accel machine. The target end
points were set to 250 mL of RBC and collected the RBC
along with 6- × 1011 to 9 × 1011 platelets, depending on
donors’ data
We calculated the absolute RBC mass volume using the
following equation:
Absolute RBC mass volume =
total RBC volume × product HCT level
Manual RBC Preparation
We collected a mean (SD) of 450 (20 mL) of whole blood
in triple blood bags, containing 63 mL of (CPD-A). We
separated plasma from the RBC via centrifugation at 4200
g for 10 minutes.
The Trima Accel system is designed to optimize the
efficiency of component collection while maintaining
donor safety; it is an automated continuous-flow
centrifuge that separates whole blood into components
for collection based on donor blood volume and
complete blood count. The system has a single-stage
channel with all blood components flowing in one
direction. The Trima Accel collects platelets, RBC, and
plasma. Users interface with a touch-screen graphical
monitor to manually configure and adjust collections
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Percent hemolysis was determined using the following
equation:
Percentage hemolysis =
(1 - Hct) × plasma Hb (g/L) / Total Hb (g/L)
In Vivo RBC Analysis
We evaluated a total of 60 RBC transfusions in 8
patients, of whom 3 were women and 5 were men; their
mean (SD) age was 29 (6) years. All patients were being
Summer 2014 | Volume 45, Number 3 Lab Medicine
239
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treated for aplastic anemia and had thrombocytopenia.
Their mean (SD) pretransfusion HCT level was 18 (3)%.
None of the patients had bleeding, hemolysis, or sepsis.
Each patient received transfusions of both products; all
8 patients received 4 transfusions each of MRBC. Six
patients received 4 ARBC transfusions; the remaining
2 patients received transfusions of 2 units each of
ARBC. All transfused units were stored for less than 7
days. We drew peripheral blood samples from patients
with anemia 24 hours after transfusion of 1 unit and
assessed their HCT levels using a calibrated cell counter
(Cobas Micros, F. Hoffman La Roche, Ltd., Basel,
Switzerland).
Statistical Analysis
We performed standard statistical analysis of the test
results, including calculation of descriptive statistics, mean,
and SD. We used the Student’s t-test for comparative
studies. A P value of less than .05 was considered to be
statistically significant.
Results
In Vitro Evaluation of RBC Count
MRBC had a volume of 240 to 300 mL per unit (mean [SD],
275 [35.5] mL/unit), RCM values of 158.5 to 240.0 mL/unit
(192.5 [3.4] mL/unit), and HCT values of 66% to 80% (70%
[9.5%]). ARBC had a volume of 240 to 250 mL/unit (mean
[SD], 240 [8.5] mL/unit), RCM values of 204 to 237.5 mL/
unit (207.84 [7.7] mL/unit), and HCT values ranging from
85% to 95% (86.6% [9%]).
ARBC showed less variability in HCT level and volume,
compared with MRBC (P <.05). We observed a significant
increase in hemolysis with storage in both products (P
<.05). The platelet count was below the detection limit in
both components. In vitro mean (SD) values for ARBCs
and MRBCs on day 1 and day 21 are compiled in Table 1
and Table 2, respectively.
In Vivo Evaluation of RBC Count
The mean (SD) increase in HCT level after transfusion of
ARBC was 4.5 (1.2%). The mean (SD) increase in HCT level
after transfusion of MRBC was 2.4 (1.3%). The difference
was statistically significant (P <.05).
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Lab Medicine Summer 2014 | Volume 45, Number 3
Discussion
We investigated the impact of collection procedure and
storage on the in vitro characteristics of RBC, evaluating
RCM; pH; rate of hemolysis; and levels of HCT, Na, K,
ATP, 2,3-DPG, pH, glucose, and plasma Hb. We also
prospectively studied the post-transfusion effectiveness
of manually collected versus apheresis-collected RBC in
patients with aplastic anemia.
Our evaluation of the HCT level of ARBC units revealed
that the HCT of these units was not dependent on donor
HCT as it was in whole-blood donation; this was due to
the fact that apheresis machines collect RBC according to
absolute RBC volume. ARBC showed significantly higher
RBC mass that provided higher HCT values to our group
of patients who received transfusions. The RBC mass
difference between the 2 collections can also be explained
by the presence of women in the whole-blood collection
group or possibly a greater number of lower-weight
individuals in that group.
We also observed low variability in the volume of ARBC
in our study. Similar findings have been reported in
previous studies.5,6
We observed a significant drop of Na and glucose on
day 21 in the ARBC group compared with the MRBC
group (P <.05). These data contrast with those reported
by Picker et al,7 which revealed that the amounts of
glucose consumed during 49 days of storage were
higher for the ARBC group compared with the MRBC
group, using saline-adenine-glucose-mannitol (SAGM) as
an RBC-preservative solution.7 However, the lower levels
of glucose in our ARBC group at day 21 may reflect the
amount of glucose in the original anticoagulant rather
than differences in the RBC metabolism between both
products. Storage of both components showed no
relevant drop in pH, and all units had a pH of 6.5 or
greater.
It has been reported8 that damage in ARBC is minimal
compared with that in MRBC. During manual RBC
collection, the whole blood is typically poured onto
the anticoagulant, and some of the RBC are instantly
damaged by the acid, due to change in osmolarity. This
phenomenon has been described as collection injury.8
By contrast, in automated collection, the anticoagulant
is continuously added at a constant small ratio,2 which
minimizes collection injury.
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Table 1. In Vitro Characteristics of ARBC and MRBC on Study Day 1
Mean (SD)
In Vitro Variable (Units of Measure)
ARBC
MRBC
P Value
Hematocrit %
Absolute RBC mass volume (ml/U)
Plasma Hg (mg/dL)
K (mmol/L)
Hemolysis corrected for hematocrit %
Na (mmol/L)
Glucose (mg/dL)
ATP (mmol/L)
2,3-DPG (mmol/L)
pH
86.6 (9)
207.8 (7.7)
37 (14)
3.29 (0.13)
0.09 (0.03)
130 (5)
440 (13)
3.4 (0.03)
2.25 (0.081)
7.2 (0.08)
70 (9.5)
192.5 (3.4)
25 (17)
1.29 (0.18)
0.19 (0.05)
133 (5)
541 (20)
3.1 (0.1)
2.3 (0.09)
7 (0.06)
<.05
<.05
<.05
<.05
<.05
.19
>.05
.27
.21
.18
ARBC, apheresis-collected red blood cells; MRBC, manually collected red blood cells; RBC, red blood cell; Hg, hemoglobin; K, potassium; Na, sodium; ATP, adenosine
triphosphate; 2,3-DPG, 2,3-diphosphoglycerate
Table 2. In Vitro Characteristics of ARBC and MRBC on Study Day 21
Mean (SD)
In Vitro Variable (Units of Measure)
ARBC
MRBC
P Value
Hematocrit %
89 (7) 73 (9)
<.05
Absolute RBC mass volume (ml/U)
213.6 (0.6)
200.7 (3.2)
<.05
Plasma Hg (mg/dL)
170 (15.5)
80 (9)
<.05
K (mmol/L)
55 (7)
40 (1.8)
<.05
Hemolysis corrected for hematocrit %
0.41 (0.22)
0.7 (0.18)
<.05
Na (mmol/L)
67 (4)
118 (10)
<.05
Glucose (mg/dL)
180 (35)
270 (20)
<.05
ATP (mmol/L)
3 (0.04)
3 (0.3)
.2
2,3-DPG (mmol/L)
00NA
pH
6.7 (0.04)
6.7 (0.04)
.21
ARBC, apheresis-collected red blood cells; MRBC, manually collected red blood cells; RBC, red blood cell; Hg, hemoglobin; K, potassium; Na, sodium; ATP, adenosine
triphosphate; 2,3-DPG, 2,3-diphosphoglycerate
Holme et al9 reported a slightly lower rate of hemolysis
(mean [SD], 0.44 [0.26] vs 0.61% [0.50%]), and lower
supernatant K+ levels (50 [3] vs 53 [3] mEq/L) for ARBC
prepared via the Haemonetics apheresis machine
(Haemonetics Corporation, Braintree, MA) compared with
manual units.9 These findings are in accordance to those
of Moog and colleagues,5 who compared filtered SAGM
ARBC units collected by Haemonetics machine with those
collected manually. Our results demonstrated significantly
higher levels of K+ and plasma Hb for the ARBC group on
day 1 and day 21, when compared to the MRBC group.
The plasma Hb values that we report for the ARBC group
on day 1 (mean [SD], 37 [17] mg/dL) contrast with values
reported in previous studies that used Trima10 and Amicus5
apheresis machines (Fenwal, Inc., Lake Zurich, IL).
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Because of the different HCT values demonstrated in our
study, it was essential to correct hemolysis for the HCT,
to avoid overestimating the hemolysis percentage. We
observed significantly lower rates of hemolysis in ARBC
compared with MRBC.
Picker et al7 reported different results, in which
MRBC had an advantage for hemolysis at the end
of specimen storage and were superior in energy
maintenance. This was indicated by less ATP
degradation and K+ leakage, which could be a
possible consequence of less citrate. The authors also
demonstrated higher methemoglobin formation with
ARBC, compared with MRBC, due to differences in
glucose metabolism.7
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Mean percentages of hemolysis for stored ARBC, as
reported in the literature, ranged from .05% to .8%.1,7,10-12
This variation may likely be related to the different
apheresis machines and to the different additive solutions
that various study groups used.
In the current study, we observed significant increases
in hemolysis with storage in specimens from the ARBC
and MRBC groups: from .09 (.03%) and .19 (.05%) to 0.41
(.22%) and .7 (.22%), respectively. All units met the United
States standards of less than 1% hemolysis.
None of our RBC units were leukoreduced. It has been
demonstrated13-15 that leukocytes can significantly
contribute to RBC hemolysis during storage, due to
the release of enzymes, especially proteases. Other
studies16,17 showed an increase in hemolysis even with
leukoreduction.
Many factors can also affect RBC quality, including the
duration and force of centrifugation. The temperature of
blood components during processing and storage can
also contribute to hemolysis. The significant increase in
hemolysis with MRBC may indicate that processing and/or
storage of MRBC were not optimized.
Hemolysis in our MRBC group can also be related to the
low Hb content of the specimens from those RBC units.
A previous study18 demonstrated higher rates of in vitro
hemolysis in RBC with low Hb content.
Several biochemical changes occur during storage that
can affect recovery and survival of RBC in the recipient.
The most dramatic changes are rapid decrease of ATP
and 2,3-DPG. 2,3-DPG is important in the regulation of
oxygen delivery by RBC.19 After transfusion, low levels of
2,3-DPG will increase in vivo. It takes at least 24 hours
before normal levels are reached in healthy volunteers,
although slower recoveries have been described in
some patients. 20 ATP is a critical energy source for the
overall functioning of RBC. Heaton21 demonstrated that
ATP levels should be higher than 2.7 µmol per g of Hb,
to yield more than 90% chance for a 24-hour recovery
of 75% or higher. 21 Our ATP values of both components
were well preserved during storage. Our data on ATP
levels are consistent with those obtained in previous
studies.5,22,23
The initial mean (SD) concentrations of 2,3-DPG for
specimens from our MRBC and ARBC groups were
2.25 (0.08) and 2.3 (0.09) mmol/L, respectively. On day
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Lab Medicine Summer 2014 | Volume 45, Number 3
21, both components had no detectable 2,3-DPG. We
suggest that the high citric acid content and associated
acidity in ACD-A explains the poor maintenance of 2,3DPG levels in both groups. Knutson and colleagues23
suggested that 2,3-DPG levels can be maintained by
changing the anticoagulant used or by rapid cooling of
the RBC unit immediately after preparation. Recently,
it has been demonstrated19 that resuspension of RBC
in phosphate-adenine-guanosine-glucose-gluconatemannitol (PAGGGM) can provide a high-quality unit
throughout 35 days of storage, with 2,3-DPG levels higher
than 10 µmol per g of Hb, ATP levels higher than 5 µmol
per g of Hb, and hemolysis levels of less than 0.2%.
Although the storage lesion is well documented, whether
the age of RBC can adversely affect the outcome of
transfusion remains in dispute.24-27 A 2006 study28 reported
that transfusion of stored or recently donated blood to
hypoxic patients has the same ability to restore evoked
brain-stem potentials. In a previous study,29 however, it
had been reported that transfusion of older stored RBCs
produces extravascular hemolysis and circulating non–
transferrin-bound iron, which may enhance complications
such as infection. Four large multicenter trials are currently
studying the impact of storage days on clinical outcomes.
Strategies regarding storage age of RBC should await the
results of such ongoing trials.30
One limitation of the current study is that although
measurement of HCT levels 24 hours after transfusion
can reflect short-term viability, tagging studies may be
required to draw definitive conclusions regarding RBC
viability.
Conclusion
Compared with the MRBC group, the ARBC group
demonstrated less variability in volume, higher RCM, and
lower rates of hemolysis. Transfusion of ARBCs that had
been stored for less than 1 week provided significantly
higher HCT levels than transfusion of MRBCs for our
cohort of patients with anemia. LM
Acknowledgments
We thank the staff members of the Department of Internal
Medicine, Hematology division, Cairo University, Egypt, for
their cooperation. We also thank the staff members of the
Cairo University Apheresis Center and the Blood Bank for
their technical support.
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