Transfusion and Apheresis Science xxx (xxxx) xxx–xxx

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Transfusion and Apheresis Science

journal homepage: www.elsevier.com/locate/transci

Red cell storage lesion and the effect of buffy-coat reduction on the
biochemical parameters
⁎
Shamee Shastry , Aaditya Shivhare, Mohandoss Murugesan, Poornima B. Baliga
Department of Immunohematology and Blood Transfusion, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India

A R T I C LE I N FO A B S T R A C T

Keywords: Background: Biochemical and metabolic changes in stored RBC may influence the clinical outcome. We aimed to
Storage lesion study the temporal changes in the biochemical parameters and the effect of buffy-coat reduction on RBC storage
Biochemical changes lesions.
Metabolic changes Materials and methods: A prospective observational study was conducted on fifteen RBC units five each of buffy
Buffy-coat
coat reduced CPD/SAGM (quadruple bags), non-buffycoat reduced CPD/SAGM (triple bags) and non-buffycoat
Leukoreduction
reduced CPDA (double bags). Biochemical parameters such as K+, LDH, pH plasma hemoglobin and percentage
Hemolysis
LDH hemolysis were measured sequentially on day 7,14, 21, 28, 35 and 42. The data was analyzed using SPSS version
Plasma hemoglobin 20.
Shelf life Results: Extracellular K+ and LDH increased rapidly starting from the first week of storage. And the all the
SAGM parameters including percentage hemolysis were significantly higher in RBC stored in CPDA (double bags)
CPDA compared to that stored in SAGM (triple and quadruple). The difference observed in buffy-coat reduced units in
Double bag comparison to the non-leukocyte reduced units were statistically not significant.
Triple bag
Conclusion: The quality of red cells stored in SAGM was superior to that suspended in CPDA measured in terms
Quadruple bag
of percent hemolysis, plasma hemoglobin, potassium and LDH. There was no effect of buffy-coat leukocyte
Red blood cell
Quality of RBC reduction on the red cell storage lesion.

1. Introduction
In vitro storage of RBCs in a liquid medium at lower temperature
slows down their metabolism, however metabolic waste, and cellular
debris accumulate in the suspending fluid, and the RBCs undergo
structural, functional and biochemical alterations. These alterations in
RBCs are termed “storage lesions”. These alterations can be extensive
and are primarily classified into three broad categories as Biochemical,
Biomechanical and Structural changes. These alterations can be ex-
tensive and are primarily classified into three broad categories as bio-
chemical, biomechanical and structural changes. The biochemical
changes within stored RBCs are principally related to alterations in
energy metabolism with depletion of 2.3-DPG and ATP. Biomechanical
changes involves hemolysis, alteration in deformability, lipid perox-
idation, vesiculation, phospholipid conformational changes and al-
teration of Na+ /K+ gradient [1].
As on one hand efforts are being made to discover novel additive
solutions extending the shelf life beyond 42 days, there are researchers
who propose a benefit of transfusing fresher blood due to the possible
deleterious effects of transfusing older units. Several studies are re-
ported in the literature on the effect of stored blood on the clinical
outcome of the patients. However very few researchers have system-
atically analyzed the storage changes in-vitro. In the present study we
assessed the temporal changes in various biochemical parameters in
stored RBC units and compared the results of leukoreduced units with
non-leukoreduced units.
2. Materials and methods
A prospective observational study was conducted to measure the
changes in biochemical parameters during the storage of P Red Blood
Cell units (RBC) in department of immunohematology and blood
transfusion. Institutional ethics committee clearance was taken before
the commencement of the study.
2.1. RBC unit selection and routine quality assessment
Fifteen RBC units five each of buffy coat reduced CPD/SAGM

⁎
Corresponding author.
E-mail addresses: shameeshastry@gmail.com (S. Shastry), aadityashivhare@gmail.com (A. Shivhare), mohandossmurugesan@gmail.com (M. Murugesan),
baliga.poornima@manipal.edu (P.B. Baliga).

https://doi.org/10.1016/j.transci.2019.01.003
Received 7 July 2018; Received in revised form 14 January 2019; Accepted 31 January 2019
1473-0502/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Shamee Shastry, et al., Transfusion and Apheresis Science, https://doi.org/10.1016/j.transci.2019.01.003
S. Shastry, et al. Transfusion and Apheresis Science xxx (xxxx) xxx–xxx

(quadruple bags), non buffy-coat reduced CPD/SAGM (triple bags) and

non buffy-coat reduced CPDA (double bags) were dedicated to study the
biochemical parameters following informed donor consent. All units
were stored under routine blood bank conditions in a temperature
monitored refrigerator, between 1 °C–6 °C. The additive solution SAGM
contains Sodium chloride (0.877 g), Adenine (0.03 g), Dextrose (0.9 g),
Mannitol (0.525 g) in 100 mL. Mannitol in SAGM functions as a free
radical scavenger and membrane stabilizer [2].
From these units, 10 ml was sampled via sterile connecting device
(Terumo SCD II) to a sample pouch at the day of collection, and sub-
sequently on day 7,14, 21, 28, 35 and 42. The RBC samples were
subjected to the following tests

1 Complete blood count: By an automated hematology analyzer Fig. 1. Changes in the supernatant K+ during red blood cell storage.
Note: In Figs. 1–4, Quadruple (Q), Triple (T) and double (D) refers to the blood
(Sysmex) by Impedance method. The sample was then centrifuged at
bags containing Buffy-coat reduced CPD-SAGM suspended RBCs, Non-leukor-
3300g for 5 min, and the supernatant was separated. The following
educed CPD-SAGM suspended RBCs and Non-leukoreduced CPDA antic-
parameters were measured over the supernatant sample:
oagulated RBCs respectively.
2 Supernatant plasma hemoglobin: Using low plasma hemoglobin
analyzer (Hemocue) by Azide-methemoglobin method and photo-
metry. The supernatant hemoglobin value was used for percent
hemolysis estimation of the bag by the following formula:

(100-Hematocrit of the sample)×Free hemoglobin in supernatant

total hemoglobin of the bag

3 pH of the supernatant using pH meter (Eutech 700) at 22₀C.

4 Supernatant K + and LDH: Using Electrochemiluminescence
method with Cobas 6000-e 601

The lab values were used to analyze the storage changes in the
leukoreduced and non-leukoreduced RBC units.
The lab values were used to analyze the storage changes in the
Fig. 2. Changes in the supernatant LDH during red blood cell storage.
leukoreduced and non-leukoreduced RBC units. This was followed by
the similar one time random sampling of 50 each of CPD-SAGM
(quadruple) and CPDA (double) suspended bags just before issue. The
shelf life of these bags was a continuous variable with different random
days for all bags. They were regrouped as bags with shelf life of: 7( ± 3)
days, 14( ± 3) days, 21( ± 3) days, 28( ± 3) days, 32–35 days.

2.2. Statistical analysis

The data was analyzed using Microsoft excel and SPSS version 20.
Comparison of mean values between two groups was done. The groups
were compared using student t-test Kruskal Wallis test was applied if
the data was non-parametric. 2 × 2 table data analysis was done using
chi-square test to know the p value.

3. Results
Fifteen red blood cell units, 5 each of Buffy-coat reduced CPD-SAGM
suspended (quadruple), Non-leukoreduced CPD-SAGM suspended
(triple) and Non-leukoreduced CPDA anticoagulated (double) were
studied for the following biochemical parameters namely potassium,
lactate dehydrogenase, plasma hemoglobin, and percentage hemolysis.
These parameters were analysed at day zero and then weekly till expiry
date, i.e. Day 0, 7, 14, 21, 28, 35, 42 and compared amongst and within
each other. The effect of buffy-coat reduction was studied by comparing
the quadruple to triple bags.
As shown in Fig. 1, Potassium levels (K+) in RBC from double bags
were significantly higher than the quadruple bags, on all days tested.
When compared to triple bags, the levels though consistently higher
were significant from day 7 onwards. Potassium (K+) levels were
found to be slightly higher in the quadruple bags than triple bags but
none of these differences were statistically significant. The Fig. 2 shows
the LDH levels on different days of storage and a significant difference
Fig. 3. Changes in the supernatant plasma hemoglobin during red blood cell
storage.
was observed when the double bags were compared to quadruple bags
on all days tested. But when compared to triple bags the values were
significantly higher from day 14 onwards. Plasma hemoglobin (Fig. 3)
and percentage hemolysis (Fig. 4) were remarkably low in the SAGM re-
suspended bags (quadruple and triple) up to day 14 following which a
slight increment was observed. There was measurable hemolysis in
CPDA anticoagulated RBC units (double bags) beginning from day 0. It
was significant only after 2 weeks of storage, i.e. on day 21, 28 and 35.
The pH values were more than 6.4 in all the bags at the end of the
storage life.
One hundred and ten bags were further analysed for potassium,
LDH, plasma hemoglobin, and percentage hemolysis. The samples were
collected from these bags just before their issue. Laminar air flow type
2B was used to maintain the sterile conditions and all the samples were

2
S. Shastry, et al.
Fig. 4. Changes in the percentage hemolysis during red blood cell storage.
collected by the principle investigator to minimize any technical var-
iations in sample collection. The data of the bags‟ biochemical para-
meters was clubbed according to their shelf life and bag type, and the
mean values were calculated. The biochemical parameter values thus
obtained were similar to the values of the analytes taken from the
dedicated blood bags.
4. Discussion
Red cell ageing is inevitable and occurs despite ideal refrigerated
storage. Ageing of red cell concentrates occur especially and is 10 times
faster when it is kept at room temperature, i.e. collection, transport,
processing, cross-matching and issue. Multiple theories have been
proposed in the literature to describe the development of storage lesion
in a stored red blood cell unit. A high number of factors affect the
quality of the product. In this study we have assessed the quality of red
cell product at various stages of its storage life.
Loss of RBC integrity during storage results in hemolysis and for-
mation of microparticles. There are studies documenting hemolysis
during storage as a function of time. On reviewing consecutive weekly
data of RBC processed and stored in three different bag types, we ob-
served a substantial amount of hemolysis in the RBC stored in CPDA
from day 0 versus the near absence of hemolysis in the SAGM bags. This
could be attributed to the additional membrane stability provided by
mannitol in the SAGM. Study done by Saini et al supported this finding.
They concluded that the red cell stored in SAGM leads to significantly
lesser hemolysis [3]. The RBC prepared from double bags were cen-
trifuged at 3800 rpm for 7 min with acceleration 6 and deceleration 4,
whereas the SAGM suspended RBCs were centrifuged at 3300 rpm for
9 min with acceleration 8 and deceleration 2 and RBC from triple bag is
prepared by Platelet Rich Plasma (PRP) method, where the initially the
bag will be subjected to soft spin (1500 rpm, for 12 min). Their sub-
jection to higher stress and the absence of mannitol might be the reason
for increased levels of hemolysis from day 0 in the CPDA double bags.
The RBC units prepared by PRP method where the first spin is soft spin
showed significantly lesser supernatant hemoglobin level than that
prepared from double bags. This shows that the initial centrifugal force
induced stress has a significant role in RBC hemolysis. In the study done
by Latham et al, extracellular hemoblobin was around 28 micromol/L
in whole blood suspended in CPDA bags after 35 days of storage and the
corresponding value in the present study in RBC units was much higher
(70 micromol/L or 0.12 g/dL) [4].
In the present study we noted steady increase in the extracellular
potassium level during the cold storage period of RBC. Karon et al
showed that the K+ level on day 42 of storage (Leukodepleted RBC in
additive solution) was 44.2 mmol/L and they noted rapid increase
during the cold storage [5]. Our results were in concordance with their
findings except for RBC units derived from double bags (K+ level;
70.90 mmol/L). Significant increase in plasma K+ and LDH levels were
noted on day 7 compared to day 0. Whereas the significant raise in
3
Transfusion and Apheresis Science xxx (xxxx) xxx–xxx
plasma hemoglobin level and percentage hemolysis were noted from
day 14 onwards. Thus the metabolic changes precedes the hemolysis.
Sawant et al. studied the hemolysis of RBCs during processing and
storage. They studied parameters like potassium LDH plasma he-
moglobin, percent hemolysis, hematocrit, mean corpuscular volume on
day 1, 7, 14, 21 and 28 for CPD-ADSOL, CPD-SAGM and whole blood
units. They observed that rate of change of hemolysis was maximum
during the first week of storage. Though they found significantly higher
levels of potassium and LDH within SAGM, every parameter was within
acceptable range as per guidelines. But they measured these parameters
only till day 28 of storage, as most of the RBC bags are issued by this
time of shelf life [6].
To study the effect of buffy-coat reduction on biochemical para-
meters we compared buffycoat reduced CPD-SAGM bags with non
buffy-coat reduced CPD-SAGM bags. During storage, leukocyte de-
gradation products such as hydrogen peroxide and proteases have been
reported to cause hemolysis on storage [7]. We observed no effect of
buffy-coat reduction over potassium, LDH, plasma hemoglobin and
percent hemolysis. On comparing the values of potassium between
buffy-coat reduced CPD-SAGM, and non buffy-coat reduced CPD-SAGM
bags, we observed that their levels nearly paralleled each other on all
days tested. In the first two weeks of storage, the levels of LDH were
lower in the non buffy-coat reduced CPD- SAGM bags as compared to
the buffy-coat reduced CPD-SAGM bags. While after 2 weeks from day
21 onwards, we noticed a shift. The LDH levels in the non buffy-coat
reduced CPD-SAGM RBCs were higher from this point of time however
the difference was not statistically significant. Plasma hemoglobin and
percent hemolysis of both non buffy-coat reduced CPD-SAGM bags and
buffy-coat reduced CPD-SAGM bags were almost nil in the first week of
storage and the levels almost paralleled each other in the non buffy-coat
reduced and buffy-coat reduced bags, with that in the non buffy-coat
reduced bags being slightly higher than the buffy-coat reduced bags.
The buffy-coat reduced RBCs were subjected to a higher stress during
their component preparation, as compared to the non buffy- coat re-
duced RBCs, which are prepared by the platelet rich plasma(PRP)
method. This could be the confounding factor in our failure to observe
the effect of buffy-coat reduction on the storage biochemical para-
meters indicative of hemolysis. Our findings were similar to those found
by Kamel et al who also observed no effect on plasma hemoglobin and
potassium levels in buffy-coat reduced units [8]. The benefit of leu-
koreduction may not reflect in the parameters that we tested. Re-
searchers have estimated ATP,Annexin V, acetylcholinesterase activity,
lipid peroxides, phosphatidyl serine,CD47 and cellular morphology as
alternative variables which are more closely linked to post transfusion
in vivo recovery [9,10,3]. As we had to reserve the bags till the end of
the shelf life, we considered only fifteen red cell units for the analysis
and this is the limitation of the present study. Unlike the majority of the
published data on this subject we have studied the changes in the
biochemical parameters till the end of the shelf life. We have also done
comparative analysis to observe the effect of buffy-coat reduction on
storage lesion, which is another strength of the study.
To evaluate the effect of stored blood on the clinical outcome,
multicenter, randomized, blinded ABLE trial (Age of Blood Evaluation)
was conducted by Lacroix et al. They used pre storage leukoreduced
RBCs suspended in SAGM and concluded that Transfusion of fresh red
cells, as compared with standard-issue red cells, did not decrease the
90-day mortality among critically ill adults [11]. However there is no
consensus in the published studies regarding the effect stored blood on
clinical outcome.
In the present study we observed that the quality of red cells stored
in SAGM was superior to that suspended in CPDA measured in terms of
percent hemolysis, plasma hemoglobin, potassium and LDH. There was
no effect of buffy-coat leukocyte reduction on the red cell storage le-
sion.
S. Shastry, et al. Transfusion and Apheresis Science xxx (xxxx) xxx–xxx

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[3] Saini N, Basu S, Kaur R, Kaur J. Assessment of changes in plasma hemoglobin and
None. potassium levels in red cell units during processing and storage. Transfus Apher Sci
2015;52(3):319–25. Elsevier Ltd.
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[5] Karon BS, Van Buskirk CM, Jaben EA, Hoyer JD, Thomas DD. Temporal sequence of
Dr. Shamee Shastry, conceptualized, planned the study and drafted major biochemical events during Blood Bank storage of packed red blood cells.
the manuscript. Blood Transfus 2012;10(4):453–61.
[6] Sawant R, Jathar S, Rajadhyaksha S, Kadam P. Red cell hemolysis during processing
Dr. Aaditya Shivahare: Performed the tests, collected the data and and storage. Asian J Transfus Sci 2007;1(2):47.
analyzed the results. [7] Heaton WAL, Holme S, Smith K, Brecher ME, Pineda A, AuBuchon JP, et al. Effects
Dr. Mohandoss and Dr. Poornima Baliga: Assisted in analyzing the of 3-5 log10pre-storage leucocyte depletion on red cell storage and metabolism. Br J
Haematol 1994;87(2):363–8.
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burden of packed and buffy-coat depleted red blood cell units on red cell storage
Disclosure of conflicts of interest lesion. Blood Transfus 2010;8(4):260–6.
[9] Prowse CV, de Korte D, Hess JR, van der Meer PF. Commercially available blood
storage containers. Vox Sang 2014;106(1):1–13.
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tassium, haemoglobin and Annexin V in supernatants. Transfus Apher Sci
Acknowledgement 2002;26(2):139–43.
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