winters2010

I M M U N O H E M AT O L O G Y
Polyethylene glycol antiglobulin tube versus gel microcolumn:
influence on the incidence of delayed hemolytic transfusion
reactions and delayed serologic transfusion reactions
_2609
1444..1452
Jeffrey L. Winters, Elie M. Richa, Sandra C. Bryant, Craig D. Tauscher, Brenda J. Bendix,
and James R. Stubbs
BACKGROUND: Our institution has reported on
delayed hemolytic transfusion reaction (DHTR) and
delayed serologic transfusion reaction (DSTR) incidence changes. From January 1993 to June 2003, a
polyethylene glycol (PEG) tube–based technique was
used for red blood cell (RBC) antibody screen. In June
2003, a gel microcolumn technique was implemented.
Impact of this on antibody detection and DHTR and
DSTR incidence was investigated.
STUDY DESIGN AND METHODS: Positive antibody
screen frequency and antibody specificity from January
2002 to March 2003 and July 2003 to September 2004
were compared. Overall incidence of DHTR and DSTR
as well as the number and identity of the RBC antibodies implicated from August 1999 through June 2003
(PEG) and July 2003 through July 2007 (gel) were
compared. The mean length of hospital stay (LOS) and
number of RBC units transfused per patient were
compared.
RESULTS: Equivalent numbers of antibody screens
were performed with equivalent numbers of positive
screens. Significant differences were not seen in the
detection of clinically significant antibodies but significantly fewer clinically insignificant antibodies were
detected with gel. Ninety-six DHTRs and DSTRs were
diagnosed. The LOS and number of transfused RBC
units were not statistically different. A significantly
higher incidence of DHTRs and DSTRs was seen with
PEG compared to the gel.
CONCLUSION: The gel microcolumn method is similar
to the PEG in detecting clinically significant antibodies
but detects fewer clinically insignificant antibodies. The
implementation of gel resulted in a lower incidence of
DHTRs and DSTRs compared to PEG.
1444 TRANSFUSION
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D
elayed complications of transfusion include
delayed hemolytic transfusion reaction
(DHTR) and delayed serologic transfusion
reaction (DSTR). DHTR is the term used to
describe the antibody-mediated destruction of transfused
red blood cells (RBCs) resulting from an anamnestic or
secondary immune response to foreign RBC antigens.1
DSTR is the term used to describe sensitization of transfused RBCs, without hemolysis.2 This may be due to a
primary immune response with the development of a new
antibody or an anamnestic response in an individual
without accompanying evidence of enhanced clearance of
the antibody-coated RBCs.
Previous studies from the Mayo Clinic (Rochester,
MN) demonstrated increasing incidence of DHTRs and
DSTRs from 1978 through 1992.3-6 Pineda and colleagues7
subsequently reported that during the time period 1980 to
1998, a significant increase in the incidence of DSTR and a
trend toward a decrease in the incidence of DHTR was
ABBREVIATIONS: DHTR(s) = delayed hemolytic transfusion
reaction(s); DSTR(s) = delayed serologic transfusion reaction(s);
LOS = length of hospital stay.
From the Department of Laboratory Medicine and Pathology,
Division of Transfusion Medicine, and the Department of
Health Science Research, Division of Biomedical Statistics and
Informatics, Mayo Clinic, Rochester, Minnesota; and the Biological Sciences Division, Department of Pathology, and Blood
Bank/Transfusion Medicine, University of Chicago Medical
Center, Chicago, Illinois.
Address reprint requests to: Jeffrey L. Winters, MD, Department of Laboratory Medicine and Pathology, Division of Transfusion Medicine, Mayo Clinic, 200 First Street SW, Rochester,
MN 55905; e-mail: winters.jeffrey@mayo.edu.
Received for publication November 12, 2009; revision
received December 29, 2009, and accepted January 5, 2010.
doi: 10.1111/j.1537-2995.2010.02609.x
TRANSFUSION 2010;50:1444-1452.
INCIDENCE OF DHTRS AND DSTRS, PEG VS. GEL
seen. These findings were felt to be due to multiple factors
including a decrease in the average length of hospital stay
(LOS), decrease in the mean number of RBC units transfused per inpatient, and the implementation of a polyethylene glycol (PEG) antiglobulin RBC antibody detection
technique, which replaced a less sensitive albumin and
papain technique.7
In July 2003, the gel microcolumn technique was
implemented at our institution, replacing the PEG antiglobulin tube technique used previously. This study was
conducted to compare the sensitivity of the gel microcolumn technique and the PEG tube method as well as evaluate the impact of implementation of the gel microcolumn
technique on the incidence of DHTR and DSTR. The frequency of positive antibody screens with each technique,
as well as the specificities detected, was examined. The
incidence of DHTR and DSTR with this system was compared to that seen with the PEG antiglobulin tube technique that had been utilized before July 2003. Potential
factors that could influence the incidence of DHTR and
DSTR, other than the sensitivity of the antibody detection
system, such as LOS and the number of RBC units transfused per patient, were also examined.
MATERIALS AND METHODS
In January 1993, the PEG antiglobulin tube technique was
implemented at our institution as the RBC antibody
screening method. The PEG solution was prepared as
described previously.8 Two drops of serum, one drop of 3%
RBC screening cells, and three drops of PEG solution were
incubated for 15 minutes at 37°C. Testing in anti-human
globulin serum followed this. The 3% RBC screening panel
consisted of three commercially prepared screening cells
from Ortho Clinical Diagnostics. PEG was used as the
enhancement medium for the antibody screening, antibody identification, and crossmatch procedures.
In July 2003, a gel microcolumn technique replaced
the PEG antiglobulin tube technique for pretransfusion
antibody screening. A gel test (ID-MTS, Ortho Clinical
Diagnostics, Raritan, NJ) was used with testing performed
according to manufacturer’s instructions. The gel microcolumn technique utilizes a plastic microcolumn filled
with dextran acrylamide gel containing antiglobulin
reagent. To perform antibody screening, 50 mL of a 0.8%
suspension of reagent RBC screening cells of known phenotype diluted in a low-ionic-strength saline were added
to the labeled microcolumn. Three screening cells were
used for each antibody screen. The cells were commercially prepared and were purchased from Ortho Clinical
Diagnostics specifically for use with the ID-MTS gel test.
Twenty-five microliters of patient plasma was added to the
microcolumns. The microcolumns were then incubated at
37°C for 15 minutes in the MTS incubator. After incubation, the columns were centrifuged in the MTS centrifuge
at 895 rpm for 10 minutes. The front and back of each
microcolumn were read and graded according to manufacturer’s instructions.9 If antibodies in the patient
samples coat the screening RBCs, agglutination by the
antiglobulin reagent in the gel occurs with the gel trapping
the agglutinated RBCs and preventing their migration to
the bottom of the column. In contrast, nonagglutinated
RBCs form a pellet in the bottom of the column. The presence or absence of RBC agglutination is indicated by the
presence of cells trapped at the surface of the gel or at the
bottom of the column, respectively.
DHTRs or DSTRs were suspected when a new RBC
antibody was detected in a recently transfused patient,
within 3 to 4 weeks of transfusion. When this occurred, a
laboratory evaluation was initiated. Direct antiglobulin
testing (DAT) with polyspecific and monospecific
reagents (anti-IgG and anti-C3d) was performed on
patient samples obtained before (when available) and
after transfusion of the implicated RBC units. In the presence of a positive DAT demonstrating IgG, RBC antibodies
were eluted from the patient’s RBCs using an acid-stromal
technique. The eluted antibodies were then identified
using commercially prepared antibody identification
panels. Before August 2003 and after February 2005,
eluates were tested using a PEG tube method and read
with the indirect antiglobulin test. For a 1.5-year period of
the total 4-year period where gel microcolumn was used
as the antibody screen method, from August 2003 to February 2005, the gel microcolumn technique was also used
for antibody identification. For this procedure, the eluate
was added to the gel column along with reagent RBCs of
known phenotype. The columns were treated as described
above.
After the identification of the antibody specificity,
patient cells were typed for the target antigen using commercially prepared or locally prepared typing sera,
depending on the antibody specificity. Standard techniques were used for the DAT, elution, antibody identification, and antigen typing.10 The serologic criteria for
DHTR and DSTR used at our institution have been previously described3-7 and have not changed from these previous reports. All of the following criteria were required for
a diagnosis of DHTR or DSTR to be considered: 1) a new
antibody identified in the patient’s serum, 2) a positive
DAT demonstrating the presence of IgG, 3) an eluate from
the patient’s RBCs demonstrated the presence of the same
antibody that was identified in the serum, and 4) antigen
typing of the patient’s RBCs demonstrated mixed-field
typing for the antigen toward which the antibody in the
patient’s serum and in the eluate was directed. No attempt
was made to determine whether the new antibody represented a primary or a secondary (anamnestic) immune
response.
When a DHTR or DSTR was suspected, the clinical
service responsible for the care of the patient was
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WINTERS ET AL.
questioned regarding pertinent signs and symptoms of
hemolysis. Relevant laboratory values (e.g., hemoglobin
[Hb], indirect bilirubin) were recorded for a period of up to
3 weeks after transfusion of the RBC units implicated in
the potential reaction. Each case was reviewed by physicians in the Division of Transfusion Medicine at a daily
conference. The clinical service was contacted as
follow-up continued. Alternative explanations for laboratory abnormalities and/or clinical signs and symptoms
were sought by examining each patient’s primary diagnosis, comorbidities, and medical history.
The presence of an unexpected and otherwise unexplained posttransfusion decline in Hb of at least 1 g/dL
from the immediate posttransfusion level favored a diagnosis of DHTR. However, if alternative explanations for
the decline existed, at least one additional sign or
symptom of hemolysis was required to make the diagnosis, as outlined in Table 1 and used in our previous
studies.3-7 A diagnosis of DHTR or DSTR was made after
sufficient time had elapsed to detect hemolysis and after
an adequate number of laboratory results had been collected and reviewed. This approach to making a diagnosis
of DHTR or DSTR has not changed since originally implemented in 1980 and is identical to that used in our previous studies.3-7 Diagnoses were made while the patient was
still in the hospital or shortly after discharge.
For this study, the results of antibody screens performed from January 2002 to March 2003 (PEG tube
method) and July 2003 to September 2004 (gel microcolumn method) were examined with regard to the total incidence of positive results, the incidence of antibodies
TABLE 1. Signs and symptoms considered
evidence of clinical hemolysis
• Elevation of the serum indirect bilirubin
• Elevation of serum creatinine
• Reduction in serum haptoglobin by at least 50% from the
pretransfusion level
• Hemosiderinuria
• Hemoglobinuria
• Hemoglobinemia
• Unexplained fever
• Decreased urine output
toward antigens within different blood groups, the incidence of clinically significant antibodies, and the incidence of clinically insignificant antibodies that are not
associated with decreased RBC survival. These time
periods were different from those used for the DHTR and
DSTR portion of the study due to limitations in the availability of antibody screening data between August 1999
and January 2002. An F test for the comparison of Poisson
incidence rate was used to compare these incidences for
the two techniques.
DHTR and DSTR data from August 1999 through June
2007 were reviewed. The overall incidence rates of DHTR
and DSTR as well as the incidence rates for RBC antibodies toward antigens within different blood groups were
calculated and compared for the two periods using an F
test. Data on the mean LOS and the number of transfused
RBC units were collected from hospital databases. The
Wilcoxon rank sum test was used to compare these
variables.
RESULTS
From January 2002 to March 2003, a total of 51,814 antibody screens were performed using the PEG tube technique, of which 1044 were positive. From July 2003 to
September 2004, a total of 51,895 antibody screens were
performed using the gel microcolumn method, of which
1005 were positive. The incidence of a positive antibody
screen during the two time periods was not statistically
different (p = 0.19). Table 2 shows the number and incidence of antibodies detected during the two time periods
separated into the blood group containing the antigen
toward which the antibody was detected. Using these
broad antibody categories, no significant differences were
detected between the PEG and gel techniques (all
p ⱖ 0.10).
Table 3 shows the number and percentage of antibodies grouped according to whether the antibodies are
considered to be clinically significant, those which cause
decreased RBC survival or hemolytic disease of the fetus
and newborn (HDFN), or clinically insignificant, not
associated with decreased RBC survival or HDFN. The
gel microcolumn technique detected more clinically
TABLE 2. Incidence of antibodies detected with PEG and gel techniques separated by blood group*
Blood group
Number of antibody screens performed
Kidd
Kell
Duffy
Rh
MNS
* Data are reported as number, IR (95% CI).
IR = incidence rate per 10,000 antibody screens.
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53,
148,
62,
384,
59,
PEG technique
51,814
10.229 (7.662, 13.380)
28.564 (24.147, 33.554)
11.966 (9.174, 15.340)
74.111 (66.883, 81.907)
11.387 (8.668, 14.688)
52,
171,
58,
404,
61,
Gel technique
51,895
10.020 (7.484, 13.140)
32.951 (28.197, 38.277)
11.176 (8.487, 14.448)
77.850 (70.442, 85.824)
11.755 (8.991, 15.099)
p-value
0.46
0.10
0.36
0.25
0.43
INCIDENCE OF DHTRS AND DSTRS, PEG VS. GEL
TABLE 3. Number and percentage of antibodies detected with PEG and gel techniques separated by
clinical significance
Antibody specificity
Clinically significant antibodies
D
C
E
c
e
f
Cw
G
RH 7 (Ce)
K
Kpa
Fya
Fyb
S
s
Jka
Jkb
Jk3
Lua
Dia
Wra
LWa
Coa
Cob
ATLIA
Total clinically significant
Clinically insignificant antibodies
M
N
P1
Lea
Leb
Bg
Cha
Rg
Sda
Ytb
McCa
Sla
HTLA
Passive anti-D
Warm autoantibody
Cold autoantibody
Nonspecific reactivity
Total clinically insignificant
Number
PEG technique
Percentage of positive
antibody screens
Number
Gel technique
Percentage of positive
antibody screens
81
47
192
37
5
0
20
2
0
144
4
59
3
12
4
46
6
1
4
1
0
0
1
2
10
681
7.8
4.5
18.4
3.5
0.5
0
1.9
0.2
0
13.8
0.4
5.7
0.3
1.1
0.4
4.4
0.6
0.1
0.4
0.1
0
0
0.1
0.2
1.0
65.2
99
53
188
44
14
1
4
0
1
169
2
54
4
18
2
39
13
0
5
0
1
1
0
0
16
728
9.9
5.3
18.7
4.4
1.4
0.1
0.4
0
0.1
16.8
0.2
5.4
0.4
1.8
0.2
3.9
1.3
0
0.5
0
0.1
0.1
0
0
1.6
72.4
39
4
35
24
7
3
0
0
3
2
1
1
40
35
72
97
0
363
3.7
0.4
3.4
2.3
0.7
0.3
0
0
0.3
0.2
0.1
0.1
3.8
3.4
6.9
9.3
0
34.8
39
2
2
4
1
12
1
1
1
0
0
0
26
52
75
29
32
277
3.9
0.2
0.2
0.4
0.1
1.2
0.1
0.1
0.1
0
0
0
2.6
5.2
7.5
2.9
3.2
27.6
ATLIA = antibody to low-incidence antigen; HTLA = high-titer low-avidity antibody.
significant antibodies (728 gel vs. 681 PEG), but this was
not significant (p = 0.11). The gel microcolumn technique detected fewer clinically insignificant antibodies
(277 gel vs. 363 PEG, p = 0.0004). In addition, the incidence of selected specificities was compared. Two clinically significant antibodies were significantly different
between PEG and gel techniques. Anti-Cw was detected
significantly more frequently using the PEG technique
(p = 0.01), while anti-e was more frequently detected
with the gel microcolumn technique (p = 0.046). The frequency of detection of seven clinically insignificant antibodies was significantly different between PEG and gel
techniques. Anti-P1 (p = 0.004), anti-Lea (p = 0.005), hightiter low-avidity antibody (p = 0.0495), and cold autoantibodies (p < 0.0001) were detected more frequently with
PEG than with the gel microcolumn technique. Anti-Bg
and passive anti-D were detected significantly more frequently (both p = 0.04) in gel than with the PEG technique. The presence of nonspecific reactivity was not
significantly higher with gel compared to PEG (p = 0.10),
despite the absence of nonspecific reactivity with the
PEG technique.
During the August 1999 to June 2007 time period, 95
subjects (55 females and 40 males) who experienced a
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WINTERS ET AL.
DHTR or DSTR were identified. All of these patients fulfilled the criteria defined above and all were included in
this study. Of note, one patient experienced two reactions
(a DHTR and a separate DSTR identified 2 weeks later),
and as a result, 96 total reactions were identified. The
specificities and frequencies of the antibodies causing
reactions during each time period are listed in Table 4.
Table 5 shows the number and incidence of DHTR
and DSTR for the periods August 1999 to May 2003 and
from July 2003 to June 2007. From August 1999 to May
2003, a total of 32,394 patients were transfused and 27
cases of DHTR and 53 cases of DSTR were detected. From
July 2003 to June 2007, a total of 34,618 patients were
transfused and five cases of DHTR and 11 cases of DSTR
were detected. During the two time periods, there was no
statistical difference in hospital LOS (median, 18 days
[range 2-187] vs. 14 days [range 2-35], p = 0.056) or the
number of transfused RBC units (median 6 units [range
2-81] vs. 4.5 units [range 1-42], p = 0.31). As shown in
Table 5, there was a significantly higher incidence of
DHTR and DSTR in association with the PEG technique
compared to the gel microcolumn technique (p < 0.003).
Table 6 shows the number and incidence of DHTR
and DSTR during the two time periods separated into the
blood group containing the antigen toward which the
antibody causing the reaction was directed. Looking at
these broad categories of major blood groups, a significantly greater number of DHTRs and DSTRs were seen
TABLE 4. Distribution of antibodies involved in DSTRs and DHTRs
Antibody involved in reaction
C
E
c
e
RH 7 (Ce)
Fya
Fyb
Jka
Jkb
K
M
S
C, Jka
E, Jka, K
E, K
E, S
E, c
e, C
Frequency*
0
19
7
2
0
16
2
19
5
1
2
2
1
0
1
1
1
1
PEG technique
Percentage of reactions
0
23.75
8.75
2.5
0
20
2.5
23.75
6.25
1.25
2.5
2.5
1.25
0
1.25
1.25
1.25
1.25
Frequency
1
5
1
1
1
3
0
2
0
1
0
0
0
1
0
0
0
0
Gel technique
Percentage of reactions
6.25
31.25
6.25
6.25
6.25
18.75
0
12.5
0
6.25
0
0
0
6.25
0
0
0
0
* One patient experienced both DHTR and DSTR, so there are 96 total reactions in 95 patients.
TABLE 5. Incidence of DHTRs and DSTRs with PEG and gel techniques*
Number of people transfused
DHTRs detected number/incidence
DSTRs detected number/incidence
PEG technique
(August 1999–May 2003)
32,394
27, 8.335 (5.493-12.127)
53, 16.361 (12.256-21.401)
Gel technique
(July 2003–July 2007)
34,618
5, 1.444 (0.469-3.371)
11, 3.178 (1.586-5.685)
p-value
0.002
<0.0001
* Data are reported as number, IR (95% CI).
IR = incidence rate per 10,000 patients transfused.
TABLE 6. Incidence of DHTRs and DSTRs by blood group antibody in PEG and gel techniques*
Blood group containing antigen toward
which the antibody was directed
Kidd
Kell
Duffy
Rh
MNS
PEG technique
25, 7.717 (4.994, 11.393)
2, 0.617 (0.075, 2.230)
18, 5.557 (3.293, 8.782)
33, 10.187 (7.012, 14.306)
5, 1.543 (0.501, 3.602)
* Data are reported as number, IR (95% CI).
IR = incidence rate per 10,000 people with transfusions.
1448 TRANSFUSION
Volume 50, July 2010
3,
2,
3,
10,
Gel technique
0.867 (0.179, 2.533)
0.578 (0.070, 2.087)
0.867 (0.179, 2.533)
2.889 (1.385, 5.312)
0, 0 (0, 1.066)
p-value
0.005
0.47
0.01
0.001
0.02
INCIDENCE OF DHTRS AND DSTRS, PEG VS. GEL
TABLE 7. Antibody specificities identified in
more than five instances of DHTRs or DSTRs*
PEG
Antibody
E
C
Fya
Jka
Jkb
DHTR
4 (15)
0 (0)
6 (22)
9 (33)
2 (7)
Gel
DSTR
15 (28)
7 (13)
10 (19)
10 (19)
3 (6)
DHTR
2 (40)
0 (0)
1 (20)
1 (20)
0 (0)
DSTR
3 (27)
1 (9)
2 (18)
1 (9)
0 (0)
* Data are reported as number (%).
during the time period when the PEG technique was used
for all major blood groups, except Kell.
Finally, Table 7 shows alloantibody specificities seen
in five or more instances of DHTR and DSTR in the two
time periods. Because of the small number of DHTRs and
DSTRs caused by individual specificities, statistical analysis comparing the incidence of individual specificities
between the two periods would not be valid and was not
performed.
DISCUSSION
Factors that influence the incidence of DHTRs and DSTRs
include LOS, mean number of RBC units transfused per
patient, and the sensitivity of the antibody detection
system used in pretransfusion screening.7 As the mean
LOS decreases, there is a reduced opportunity to detect
delayed transfusion complications. This could result in a
failure to detect the appearance of the antibody resulting
in an apparent decrease in the incidence of DHTRs and
DSTRs. Also, decreased LOS could result in the potential to
misclassify a DHTR as a DSTR due to a failure to detect
clinical hemolysis. Such misclassifications may result in
the erroneous conclusion of a decreased incidence of
DHTR accompanied by a concurrent increase in the incidence of DSTRs. In this study, LOS was not significantly
different between the two time periods, excluding this as
an explanation for the difference in incidence of DHTR
and DSTR.
A reduction in RBC antigen exposure due to a
decrease in RBC transfusion could also influence the incidence of DHTRs and DSTRs. The transfusion of fewer RBC
units per patient, due to changes in medical practice,
would expose patients to fewer foreign RBC antigens. This
would result in a decrease in the opportunity for a patient
to develop an immune response to an RBC antigen that
would, in turn, decrease DHTR and DSTR incidence. The
mean number of units of RBCs transfused per patient
during the two time periods was not significantly different. Differences in RBC unit transfusion practices do not
explain the difference in the incidence of DHTRs and
DSTRs seen.
Finally, the sensitivity of the antibody detection
system used in pretransfusion screening can influence
DHTR and DSTR incidence. RBC antibodies present at the
time of pretransfusion testing, but with reactivity below
the threshold of detection of an antibody detection
system, could result in a DHTR or a DSTR. If an antibody is
not detected, RBC units expressing the antigenic target of
the antibody could be transfused. This could result in an
anamnestic response by the patient’s immune system
with the development of what appears to be a “new” antibody as the titer of the antibody increases. This would
result in a DHTR or DSTR, depending on the patient’s
ability to clear the antibody-coated RBCs. The incidence
of DHTRs and DSTRs should be lower when more sensitive RBC antibody detection systems are employed since
RBC alloantibodies will be more readily detected during
pretransfusion testing. The result will be the provision of
antigen-negative RBC units with the avoidance of antigenic stimulation.
The gel microcolumn technique was first described
by Lapierre and colleagues in 1990.11 It offers several
potential advantages over tube techniques in that it can be
automated, the test endpoints lack the variability resulting
from different operators performing the test as seen in
tube-based methods, and the test endpoints are stable for
up to 24 hours such that results can be reviewed at a later
time if necessary.12 Another potential advantage over tube
techniques would be increased sensitivity due to the fact
that the gel microcolumn method does not require a wash
step, which could potentially remove weakly bound
antibodies.12
The clinical performance of RBC antibody detection
systems varies and can be evaluated by comparing the
ability of the systems to detect clinically significant antibodies (“wanted antibodies” such as antibodies to Rh,
Kell, and Kidd system antigens) and those that are not
clinically significant (“unwanted antibodies” such as coldreacting alloantibodies, cold-reacting autoantibodies, and
high-titer low-avidity antibodies).13 Determining the clinical performance of new systems usually involves retrospective studies where the frequency of antibodies
identified during two time periods are compared or
studies, either prospective or retrospective, using stored
samples, that test samples in parallel with both methods
being evaluated.14 Another way to evaluate the clinical
performance of two antibody detection systems is by
determining the incidence of hemolytic transfusion reactions during the time periods when the systems were in
use.14 Limited reports have been published on the use of
these methods to compare the gel microcolumn and PEG
tube techniques.15,16
Novaretti and colleagues15 prospectively performed
antibody screening of 10,123 random samples submitted
to their reference laboratory with both the PEG tube technique and the gel microcolumn method. Of the samples
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WINTERS ET AL.
tested, 628 (6.2%) contained RBC alloantibodies. The gel
microcolumn technique identified 196 reactive samples
that were negative with the PEG tube technique. These
discordant samples included 152 antibodies directed
toward antigens in the Rh blood group system, 18 anti-K
antibodies, and seven anti-Jka. Two samples reacted only
using the PEG tube technique but not with the gel technique (one anti-K and one anti-Dia) while 430 samples
were reactive using both methods.15 The difference in the
number of antibodies detected by each system was significant (p < 0.01) favoring the gel system. This prospective
study using fresh samples demonstrated superior sensitivity of the gel technique compared to the PEG tube
technique.15
Combs and Bredehoeft16 compared the sensitivity of
the PEG tube technique to the gel microcolumn method
by comparing both the frequency of clinically significant
and clinically insignificant antibodies and the incidence
of DHTRs and DSTRs in two consecutive 1-year periods
where the different techniques were used for pretransfusion testing. During the period that the PEG tube technique was used, a total of 3,085 antibodies were detected.
Of these, 71% were considered clinically significant while
29% were considered “unwanted” or clinically insignificant. During the year in which the gel technique was
used, 2715 antibodies were identified of which 80% were
considered “wanted” and 20% were considered clinically
insignificant. Combs and Bredehoeft considered these
findings to indicate equivalent sensitivity, equivalent
ability to detect clinically significant antibodies, and
superior specificity in that fewer clinically insignificant
antibodies were detected by the gel microcolumn technique.16 During the period when the PEG tube technique
was used, one DHTR occurred due to anti-S. In addition,
there were 21 missed alloantibodies (two anti-D, four
anti-E, three anti-c, three anti-Jka, two anti-Jkb, three
anti-Fya, one combination of anti-c and anti-E, and one
with a combination of anti-E and anti-Jka), which
resulted in DSTR in 19 patients. With the gel microcolumn technique, no DHTRs were seen and there were 11
missed alloantibodies (one anti-D, two anti-c, three
anti-K, one anti-Jka, and two with the combination of
anti-E and anti-Fya), which caused DSTRs in nine
patients.16 The authors reported that overall, the gel technique detected slightly more Rh and Kell antibodies but
fewer anti-Jka antibodies.16
Our study, like that performed by Combs and Bredehoeft, determined sensitivity by examining the frequency
with which antibodies were detected in two time periods
using two different methods. Our finding was that the gel
microcolumn method of detecting antibodies showed
similar sensitivity to that of the PEG tube method. These
findings are similar to those reported by Combs and Bredehoeft. Like Combs and Bredehoeft, we did detect more
antibodies to Rh and Kell blood group system antigens but
1450 TRANSFUSION
Volume 50, July 2010
these differences were not significant (Rh p = 0.25, Kell
p = 0.10). Similarly, fewer examples of anti-Jka were seen
with gel but again, the difference was not significant.
Enhanced sensitivity of the gel microcolumn method was
not demonstrated across multiple blood group systems in
our study, with an insignificant increase in the incidence
in antibodies detected (Table 2).
In examining the frequency of clinically significant
and clinically insignificant antibodies, we saw similar frequency to that reported by Combs and Bredehoeft, 66%
significant and 34% insignificant versus 71 and 29%16 for
PEG tube method and 72% significant and 28% insignificant versus 80 and 20%16 for gel. Overall, the detection of
clinically significant antibodies was not statistically different between the two methods in our study. Two clinically
significant antibodies did demonstrate significant differences between PEG tube method and gel. Anti-Cw was
more frequently detected with PEG tube and anti-e was
more frequently detected with gel. A possible explanation
for the former could be differences in the antigen profiles
of the screening cells used in the two methods. The frequency with which the Cw antigen is present on screening
cells varies between manufacturers. During the PEG
period, commercially prepared 3% screening cells from
Ortho Clinical Diagnostics were used. During the period
when the gel microcolumn technique was used, 0.8%
screening cells from Ortho Clinical Diagnostics, specifically for use with the ID-MTS system, were used. It is possible that the presence of the Cw antigen was less frequent
in these screening cells. This would explain the lower incidence of anti-Cw detection with the gel method. This
explanation is not valid when speaking about the e
antigen, however, as most if not all screening cells will
have this antigen present.
Overall, the gel microcolumn technique did demonstrate a significantly lower frequency of detecting clinically insignificant antibodies. Exceptions were the
detection of anti-Bg and passive anti-D, which were more
frequently seen with the gel microcolumn technique.
Our study demonstrated a significant decrease in the
incidence of DHTR or DSTR for all blood groups when the
gel microcolumn technique was used, with the exception
of the Kell blood group system. For the Kell blood group
system, a significant difference in the incidence of DHTRs
or DSTRs was not seen between the time periods (Table 6).
With regard to the sensitivity of the two systems when
used for screening, in our hands, both were equivalent
with regard to the detection of antibodies to all blood
group system antigens, including Kell system antigens
(Table 2). Combs and Bredehoeft did report three anti-K
antibodies causing DSTR during the gel technique period
but felt that overall, based on the frequency of clinically
significant antibodies identified, the gel microcolumn
technique was more sensitive to Kell system antibodies
than PEG tube technique.16 Similarly, Novaretti and
INCIDENCE OF DHTRS AND DSTRS, PEG VS. GEL
coworkers15 reported that one anti-K was detectable only
by the PEG tube method and 18 instances of anti-K that
were detected only by the gel microcolumn technique. In
our study, only two cases of DHTR or DSTR due to antibodies to Kell system antigens were seen in each time
period. In all four cases, the antibody causing the reaction
was anti-K. The small number of cases means that this
finding should be interpreted with caution considering
the previously reported findings by Combs and Bredehoeft and Novaretti and colleagues. These findings point
out that not all antibodies to a given antigen will be
detected by a given test system.
The overall decline in the incidence of DHTR or DSTR
with the use of the gel microcolumn technique is difficult
to explain. Such a decline could occur due to changes in
risks for DHTR or DSTR such as decreased LOS or
decreased RBC exposure or if the sensitivity of the two
detection systems were different. We failed to demonstrate
any significant differences in LOS and RBC exposure
between the time periods when the PEG tube and gel
microcolumn techniques were used. We also could not
demonstrate differences in sensitivity between the two
methods with regard to clinically significant antibodies.
Another consideration would be the fact that for a 1.5-year
period during the time when the gel microcolumn technique was used for antibody screening, it was also used for
antibody identification of the eluates. During the period
that the gel technique was used for eluate antibody ID, a
total of two DHTRs (1.3/year) and four DSTRs (2.7/year)
were identified (data not shown). During the period when
PEG tube method was used for eluate antibody ID, three
DHTRs (1.2/year) and seven DSTRs (2.8/year) were identified (data not shown). These numbers are similar. As
already stated, the PEG method and the gel microcolumn
technique demonstrated similar sensitivities and therefore this should not have influenced the incidence of
DHTRs and DSTRs. The possible exceptions would be for
the two clinically significant antibodies where differences
in sensitivity were identified, anti-Cw and anti-e. During
both periods, there were no instances where anti-Cw was
implicated in DHTRs or DSTRs. With regard to anti-e, the
number of cases of DHTRs and DSTRs due to anti-e were
similar in the two periods, two (2.5%) with the PEG tube
method and one (6.25%) with the gel microcolumn technique. It therefore appears that the use of the gel method
for antibody identification of the eluates does not explain
the decline in the incidence in DHTRs and DSTRs
observed. The explanation for the lower incidence of
DHTRs or DSTRs with the gel technique is not readily
apparent.
While our study found findings similar to those seen
by Combs and Bredehoeft, similar sensitivity between
the two methods for clinically significant antibodies,
fewer clinically insignificant antibodies detected by gel,
and a decreased frequency of both DHTRs and DSTRs
with the gel microcolumn technique, our study has
several advantages over that of Combs and Bredehoeft.
First, we were able to perform a statistical analysis of the
incidence of antibodies detected with the two methods
when used for antibody screening, not simply a listing of
antibody frequencies. This showed that while differences
in detection seen in our study are comparable to those
reported by Combs and Bredehoeft, not all of these were
significant. Another advantage was that we were able to
exclude additional factors that could explain differences
in the incidence of DHTRs and DSTRs independent of
the test system used to perform antibody screening.
Unlike the study by Combs and Bredehoeft, we excluded
changes in LOS and mean number of RBC units transfused per patient as possible explanations for the
observed decline in the incidence of these reactions. We
also examined the incidence of reactions over longer
periods, 4 years before and after implementation of the
gel microcolumn technique, and examined more cases of
DHTRs and DSTRs.
A weakness of our study is the inability to identify the
incidence of DHTRs and DSTRs with the two techniques
with regard to individual antibody specificities. This
resulted from the small number of DHTRs and DSTRs due
to single antibody specificities that occurred during the
two time periods. These small numbers precluded a
meaningful statistical analysis. As a result, the incidence
of DHTRs and DSTRs due to antibodies to antigens within
specific blood groups was compared. To legitimately
examine individual specificities, a larger sample size of
DHTRs and DSTRs would be required. To increase automation in our laboratory, the gel microcolumn technique
was discontinued in July 2007 and replaced with a solidphase RBC adherence technique precluding further identification of DHTRs and DSTRs in patients screened with
the gel microcolumn technique.
In summary, the gel microcolumn method detected
similar numbers of antibodies compared to the PEG tube
method. We could not demonstrate enhanced sensitivity
for clinically significant antibodies. The gel microcolumn
technique did, however, detect fewer clinically insignificant antibodies. This would be beneficial in routine daily
laboratory work because it would minimize necessary
evaluation of these antibodies. The incidence of DHTRs
and DSTRs demonstrated a significant decrease after
implementation of the gel microcolumn technique, suggesting enhanced safety of the gel technique compared to
the PEG tube method.
CONFLICT OF INTEREST
The authors certify that they have no affiliation with or financial
involvement in any organization or entity with a direct financial
interest in the subject matter or materials discussed in this
manuscript.
Volume 50, July 2010
TRANSFUSION 1451
WINTERS ET AL.
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