Simplified Method for Recovery
Cells from Transfused Patients
-
MARION E. REID, M.S., FIMLS AND PEARL T. C. Y.
Separation of autologous and transfused red blood cells from
recently transfused patients is necessary for the proper iden-
tification of any red blood cell alloantibody or autoantibody.
We compared two methods of separation: the standard technic
of microhematocrit centrifugation with phthalate ester solu-
tion, and a simplified method of microhematocrit centrifugation
without the use of esters. Autologous red blood cells were con-
centrated in the top layer of the capillary tube by both methods.
Separation efficacy was comparable, as determined by blood
group antigen reactivity. Good separation was achieved only
in samples drawn three or more days post transfusion. Micro-
hematocrit centrifugation without the use of phthalate esters
is a simple method for the recovery of autologous red blood
cells from recently transfused patients that can be performed
by any standard clinical laboratory. (Key words: Erythrocyte:
autologous; Erythrocyte: separation; Transfusion; Antigen
testing) Am J Clin Pathol 1983; 79: 364-366
SEPARATION of autologous red blood cells from do-
nor red blood cells in transfused patients requires spe-
cialized equipment or reagents or time-consuming ma-
nipulations.2"5 Wallas and associates used phthalate es-
ter solutions with microhematocrit centrifugation to
separate autologous red blood cells on the basis of den-
sity. Since it is time-consuming to prepare and quality
control phthalate ester solutions, this study investigated
whether these solutions were necessary. Recovery of au-
tologous red blood cells from transfused erythrocytes is
valuable in determining the red blood cell blood group
phenotype of a patient who is developing antibodies to
donor red blood cells, and helps determine whether a
positive direct antiglobulin test is because of a delayed
transfusion reaction or warm autoantibodies.
Antigen typing was performed on red blood cells har-
vested after microhematocrit centrifugation and after
phthalate ester density microhematocrit centrifugation5
from in vitro mixtures and from post-transfusion patient
samples. Comparable concentrations of autologous red
blood cells were achieved with or without the use of
pthalate esters.
Materials and Methods
Mixtures of patient and donor red blood cells were
separated using microhematocrit centrifugation, with
Received May 25, 1982; received revised manuscript and accepted
for publication July 26, 1982.
Address reprint requests to Ms. Reid: Blood Bank, San Francisco
General Hospital Medical Center, 1001 Potrero Avenue, San Fran-
cisco, California 94110.
0002-9173/83/0300/0364 $00.95 © /
364
Autologous Red Blood
, M.D.
Blood Bank, Clinical Laboratories, San Francisco General
Hospital Medical Center and Department of Laboratory
Medicine, University of California, San Francisco, California
and without phthalate esters. Whether or not patient red
blood cells were concentrated in the top layer after cen-
trifugation was tested by red blood cell antigen typing.
Preliminary studies were performed on in vitro mixtures
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of fresh and stored erythrocytes; then blood samples
from recently transfused patients were studied.
Test Samples
In vitro red blood cell mixtures. Red blood cell sam-
ples with different ABO groups were washed three times
with 0.9% sodium chloride. Erythrocytes drawn into
EDTA on the day of testing (fresh cells) were mixed with
red blood cells drawn into citrate phosphate dextrose at
least 15 days before (stored cells). The tubes were mixed
for 10 minutes.
Patient samples. Pretransfusion blood samples from
random patients and segments from the donor units
transfused were tested for blood group antigen differ-
ences by standard immunohematology technics.1 The
post-transfusion patient samples were EDTA or clotted
specimens.
Reagents. Commercial blood typing antisera were
used according to the manufacturer's directions. The
phthalate ester solutions were obtained from Dr.
Wallas.*
Red blood cell .separation. Red blood cell samples
were washed three times with saline. When the super-
natant saline was discarded care was taken not to remove
any red blood cells. These washed erythrocyte samples
were mixed thoroughly, and phthalate ester density
curves were established. The solution of ester that best
separated young red blood cells was selected, and four
microhematocrit tubes were prepared.5 An additional
four microhematocrit tubes were loaded with blood but
no phthalate ester. These eight tubes were sealed with
SealEase (Clay Adams, Inc., Parsippany, NJ) and cen-
trifuged together in a microhematocrit centrifuge at
13,500 Xg for 15 minutes. Red blood cells from the
* Vanderbilt University Medical Center, Nashville, Tennessee,
an Society of Clinical Pathologists
Vol. 79 • No. 3 BRIEF SCIENTIFIC REPORT
365
Table 1. Results of In vitro Mixture Red Blood Cell Recovery
Tested with Anti-B Tested with Anti-A
Type or <~eiis
10% Fresh 10% Fresh
Samplef Major Stored Minor Fresh Orig. Estr. Cent.f Orig. Estr. Cent.f

1 O A (Not tested) 3 10 8
2 O B 5 8 8 (Not tested)
3 A B 5 11 11 10 3 3
4 A B 5 8 8 12 2 3
5 A B 5 9 9 (Not tested)

* All reactions were mixed field. ester solution; Cent. = tests on top layer after separation with microhematocrit centrifugation
t Orig. = Original sample before separation; Estr. = Tests on top layer after separation with alone.

tubes containing phthalate ester solutions were collected
as described by Wallas.5 A 5-mm column of red blood
cells was collected from the top of the other four tubes
by cutting the tubes. Both preparations of red blood cells
were washed twice and resuspended to 4% with 0.9%
ples 2 to 5. At the same time, stored cells decreased in
the top layer, as test scores with anti-A decreased in
samples 3 and 4. Separation was comparable with and
without ester solutions.
The results of red blood cell separation in patient sam-

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sodium chloride. Separated red blood cell suspensions
from the patient samples were coded by an independent
worker, and then tested without knowing the content
of the tubes. Results were read microscopically.
Agglutination scores' of blood group antigen testing
on red blood cells obtained after separation by micro-
hematocrit centrifugation alone were compared to the
scores obtained on erythrocytes separated using the
phthalate solutions.
Results
The results of red blood cell separation of in vitro
mixtures of stored and fresh cells are shown in Table
1. Fresh cells were concentrated in the top layer after
centrifugation, as test scores with anti-A increased with
sample 1, and test scores with anti-B increased with sam-
ples are shown in Table 2. In cases 1 to 6, antigen neg-
ative patients were transfused with antigen positive
blood; in cases 4 and 6, patients required massive trans-
fusion when D negative blood was unavailable. In cases
7 to 10, antigen positive patients received antigen neg-
ative blood. If the patient red blood cells lacked the
antigen being tested (cases 1 to 6), a decrease in test
score after separation would be expected if autologous
cells were successfully concentrated. In contrast, if the
patient red blood cells possessed the antigen being tested
(cases 7 to 10), then a stronger positive test would be
expected. Separation was not always complete, i.e.,
mixed field agglutination present, but concentration of
the autologous red blood cell population was obtained
by both methods. Separation by microhematocrit cen-
trifugation alone was as good as that with ester solution.

Table 2. Results of Testing Red Blood Cells Recovered from Transfused Patient Samples
Number of
Units
Transfused $ Number of Days Agglutination Scoresf
Patient Antigen from Transfusion Tested with
Case Type ag+ ag- to Sample Anti- Orig. Estr. Cent.

1 c- 3 0 2 C 5 4 4
2 C- 1 1 4 C 3 3 0
3 1 1 5 3 0 0
4
c-
D- 6 8 3
c
D 8 3 4
5 E- 1 1 4 E 3 0 0
6 D- 6 26 1 D 8 7 4
6 3 D 5 0 0
7 E+ 8 3 1 E 7 8 8
6 E 8 10» 10*
8 D+ 0 1 1 D 10 10 10
11 D 10 11* 11*
9 D+ 1 1 1 D 10 10 10
10 E+ 1 1 1 E 8 9 9

* Not mixed field reactions. alone.

:
t Orig. = Original sample before separation; Estr. = Tests on top layer after separation with $ ag+ antigen-positive; a g - = antigen-negative.
ester solution; Cent. = tests on top layer after separation with microhematocrit centrifugation
366 REID AND TOY
Separation of patient cells was better when the post-
transfusion sample was drawn at least three days after
transfusion. In patient samples tested three to eleven
days after transfusion, recovery of autologous cells was
good in six of six, as shown by score changes of 2 to 5.
However, in patient samples tested only one to two days
post-transfusion, autologous cells were concentrated sig-
nificantly in only one of the six cases, and scores changes
were 0-1. In cases 6,7, and 8, separation improved upon
retesting several days later.
Discussion
Transfused donor red blood cells were partially sep-
arated from autologous red blood cells by microhemato-
crit centrifugation without phthalate ester solutions. The
separation of red blood cells by this simple procedure
was comparable to the method using phthalate esters.5
Separation of autologous red blood cells from trans-
fused donor red blood cells is valuable whenever antigen
phenotyping of patient red blood cells is necessary for
a complete serologic workup and is particularly valuable
if the transfused patient develops alloantibodies to the
donor red blood cells. Separation of patient red blood
cells also may be useful for direct antiglobulin testing
(DAT) in differentiating delayed transfusion reaction
from warm autoantibodies.3,6 Centrifugal technics for
separating patient from donor red blood cells have been
available for some time.2"4 However, these technics re-
quire relatively large volumes of blood and repeated cen-
trifugations,2 a specialized centrifuge,4 long centrifuga-
tion time,3 or specialized reagents.5 Since the need for
separation of autologous red blood cells is not common,
a simplified procedure that does not require special tech-
nical proficiency, equipment, or reagents would be of
value.
A.J.C.P. • March 1983
By comparing reaction strengths of red blood cell an-
tigen testing, this study shows that autologous red blood
cells can be separated from transfused donor red blood
cells without phthalate ester solutions.
The likelihood is greater that the donor red blood cells
will have increased in density in vitro in proportion to
the length of time they have remained in the patient's
circulation. This longer time period also increases the
likelihood that the patient will have produced reticu-
locytes. Thus, the separation should be better.
The method described does not require special anti-
coagulants, handling, equipment, reagents, practice, or
experience. In addition, this procedure is quick, eco-
nomical, and practical for any laboratory where stan-
dard microhematocrit centrifugation is available.
Acknowledgment. The authors would like to thank Ms. S. Ellisor
and Dr. T. Drake for their helpful comments during the preparation
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of this manuscript, Ms. J. Hendrickson for editorial assistance, and
Ms. E. Jacobs for secretarial assistance.
References
1. AABB Technical Manual, 8th ed., Washington, DC, American
Association of Blood Banks, 1981
2. Constandoulakis M, Kay HEM: Observations on the centrifugal
segregation of young erythrocytes. A possible method of geno-
typing the transfused patient. J. Clin Pathol 1959; i 2:312-318
3. Croucher BEE: Differential diagnosis of delayed transfusion re-
action. A Seminar on Laboratory Management of Hemolysis.
Edited by CA Bell. Washington, DC, American Association of
Blood Banks, 1979, p 157
4. Renton PH, Hancock JA: A simple method of separating eryth-
rocytes of different ages. Vox Sang 1964; 9:183-186
5. Wallas CH, Tanley PC, Gorrell LP: Recovery of autologous eryth-
rocytes in transfused patients. Transfusion 1980; 20:332-336
6. Wallas CH, Tanley PC, Gorrell LP: Utilization of a cell separation
technique to evaluate patients with a positive direct antiglob-
ulin test. Transfusion 1982; 22:26-30