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1. A Comparative Study of HLA-DRB Typing
by Transcription-Mediated Amplification
with the Hybridization Protection Assay
(TMA/HPA) versus PCR/SSOP
Anajane G. Smith, Koichi Matsubara, Eric Mickelson,
Ahmad Marashi, Lois Regen, Leigh Ann Guthrie,
and John A. Hansen
ABSTRACT: To evaluate alternative human leukocyte
antigen (HLA)-DNA typing methods, we used a system
of transcription-mediated amplification (TMA) with a
probe hybridization protection assay (HPA) in a micro-
titer plate format developed by Chugai Pharmaceutics
Ltd. (Tokyo, Japan) to perform intermediate-level DRB
typing for 502 individual samples. Two hundred fifty-two
samples submitted to our Clinical Immunogenetics Lab-
oratory were prospectively tested concurrently with a lo-
cally developed intermediate-level DRB polymerase chain
reaction/sequence-specific oligonucleotide probe (PCR/
SSOP) assay in a double-blind fashion. In addition, 250
retrospective samples of archived frozen cells or DNA
from clinical and research panels, previously typed by
allele-level DRB1 PCR/SSOP, were chosen to include 66
distinct DRB1 alleles representing Caucasian, American
Black, Asian, and Native American ethnic groups.
Among the prospectively typed samples, except for four
samples with a TMA/HPA microplate handling problem,
a single TMA/HPA allele assignment (1/462 alleles 5
0.2%) was discordant with PCR/SSOP. Among the 250
retrospective samples, a single HPA probe for codon 57
aspartic acid consistently cross-reacted with the codon 57
valine sequence of DRB1*0807. However, TMA/HPA
identified six samples with previous PCR/SSOP typing
errors, all of which involved identification of sequences at
codons 67–71 in samples heterozygous for two DR52-
associated DRB1 alleles. Assay turnaround time from
sample preparation to results was 11 h for 24 samples or
6–7 h for 1–4 samples. In summary, we found the
TMA/HPA DRB typing system to provide rapid, reliable,
and accurate HLA-DRB typing results. The current
TMA/HPA methodology could be improved by use of a
molded plastic cold block to provide more consistent and
secure microtiter plate cooling than the current water/ice
slurry. Nevertheless, this methodology, based on a mi-
crotiter plate format but without the usual plate wash-
ing steps of the traditional ELISA, has superior potential
for microplate handling and reagent distribution with a
robotics system and a work surface incorporating mi-
croplate heating and cooling units. Human Immunology
55, 74–84 (1997). © American Society for Histocom-
patibility and Immunogenetics, 1997. Published by
Elsevier Science Inc.
From the Clinical Immunogenetics Laboratory, Fred Hutchinson Cancer
Research Center, Seattle, Washington, U.S.A. (A.G.S., E.M., A.M., L.R.,
L.A.G., J.A.H.), Diagnostic Technology Labs, Chugai Pharmaceutical
Company, Tokyo, Japan (K.M.), and University of Washington, School of
Medicine, Seattle, Washington, U.S.A. ( J.A.H.).
Address reprint requests to: Dr. Anajane G. Smith, Clinical Immuno-
genetics Laboratory, Fred Hutchinson Cancer Research Center, 1124 Colum-
bia Street, Seattle, WA 98104, U.S.A.
Received February 18, 1997; accepted April 29, 1997
Human Immunology 55, 74–84 (1997)
0198-8859/97/$17.00
© American Society for Histocompatibility and Immunogenetics, 1997.
Published by Elsevier Science Inc. PII S0198-8859(97)00085-2

2. ABBREVIATIONS
EDTA ethylenediamine tetraacetic acid
HLA human leukocyte antigen
HPA hybridization protection assay
IHW International Histocompatibility Workshop
LCL lymphoblastoid cell line
MHC major histocompatibility complex
NMDP National Marrow Donor Program
PBMC peripheral blood mononuclear cells
PCR polymerase chain reaction
RFLP restriction fragment length polymorphism
RLU relative light units
SSOP sequence-specific oligonucleotide probe
TMA transcription-mediated amplification
INTRODUCTION
The cell surface glycoproteins of the human leucocyte
antigen (HLA) system are the products of a complex
array of genes encoded within the human major histo-
compatibility locus (MHC). Analyses of the HLA com-
plex have revealed the importance of typing and match-
ing for these molecules in transplantation [1–5].
Although the traditional serologic and cellular HLA
typing methods can be very accurate, both are signifi-
cantly limited in their ability to recognize allelic variants
and by their dependence on access to viable cells that
normally express HLA molecules. More recently, HLA
typing systems have been developed that directly analyze
the genetic loci that encode the HLA antigens. In studies
for the 10th International Histocompatibility Workshop
(IHW), 1987, a standard method of HLA-DNA typing
using a restriction fragment length polymorphism
(RFLP) Southern blot methodology [6] was evaluated in
80 laboratories throughout the world. Also in 1987, the
WHO Nomenclature Committee for Factors of the HLA
System established a standard nomenclature of HLA
alleles based on DNA sequencing [7]. The 11th IHW,
1991, used the polymerase chain reaction (PCR) for
DNA amplification [8] with a sequence-specific oligo-
nucleotide probe (SSOP) hybridization procedure using a
standardized panel of oligonucleotide primer and probe
reagents to analyze HLA class II genes [9] in 195 labo-
ratories worldwide involved in studies of transplantation,
disease associations, anthropology, and other subjects.
Several other HLA-DNA typing methods were also de-
scribed at the 11th IHW, including the oligonucleotide
probe hybridization protection assay (HPA) used in this
study [10]. These methods, which differ in the tech-
niques used to detect the many polymorphisms that
characterize HLA alleles, may be more or less useful in a
particular context, depending on the number of samples
to be typed, on the time required for reporting results,
and on the level of resolution needed. Subsequently,
many laboratories have implemented HLA-DNA typing
methods and published reports demonstrating those
methods to be significantly more accurate and reliable
than traditional serologic typing of both HLA class I and
class II genes [11–15]. In particular, the forward dot blot
PCR/SSOP methods developed and implemented by the
contract typing laboratories of the National Marrow Do-
nor Program (NMDP) for DNA-based HLA-DRB typ-
ing of volunteer unrelated donors have been found highly
accurate for medium- and large-scale (50 to over 250
samples per week) testing [16]. The growth of DNA-
based typing has been substantial, and the HLA Nomen-
clature Committee now recognizes at least 83 HLA-A
alleles, 186 HLA-B alleles, 42 HLA-C alleles, 183 DRB1
alleles, and 31 DQB1 alleles [17]. Remarkable as this
development in technology has been, serology is still
widely used for analysis of HLA-DR. It is likely that the
universal application of DNA methods to all HLA typ-
ing, whether in the clinical laboratory or the research
laboratory, will require the availability of commercial
HLA-DNA typing systems that offer standard, specific,
reliable, and affordable reagents. A flexible typing
method should allow for both urgent clinical needs and
high-volume routine or research typing, so that one or
two samples could be typed in less than 8 h or many
samples (.50 per technologist per week) with minimal
requirements for equipment. The massive HLA typing
projects, such as prospective and retrospective DRB typ-
ing of unrelated volunteer stem cell donors throughout
the world [18], continue to have a need for methods
amenable to the automation required for very high-
volume yet accurate typing.
We evaluated a system of transcription-mediated am-
plification (TMA) with probe hybridization protection
assay (HPA) in a microtiter plate format for medium-
resolution DRB typing developed by Chugai Pharma-
ceuticals (Tokyo, Japan). Two hundred fifty-two samples
from patients and family members submitted to the
Clinical Immunogenetics Laboratory for HLA typing
before marrow transplantation were tested prospectively
by TMA/HPA and by locally developed medium-resolu-
tion DRB PCR/SSOP in a double-blind fashion. An
additional 250 samples, previously typed by allele-level
DRB1 PCR/SSOP, were chosen for a retrospective study
by TMA/HPA, including 66 distinct DRB1 alleles rep-
resenting Caucasian, American Black, Asian, and Native
American ethnic groups. We evaluated the sensitivity
and specificity of DRB1 identification at the medium-
resolution level in both the prospective and the retro-
spective studies; in addition, we reviewed the accuracy of
75
HLA-DRB Typing by TMA/HPA vs. PCR/SSOP

3. the HPA probes in identifying known DRB1 allele se-
quences in the retrospective study panel. Our results
demonstrate the excellent sensitivity and specificity of
HLA-DRB typing with the Chugai TMA/HPA system
and the utility of this method for clinical and research
analyses, regardless of whether the situation requires
relatively rapid typing of a few samples or typing in high
volume. Our experience with this assay system suggests
that it might be amenable to automation.
MATERIALS AND METHODS
Study Population
A total of 502 samples representing 500 individuals were
entered into this study. Aliquots from 252 “clinical”
samples obtained from patients and normal family mem-
bers submitted to the Clinical Immunogenetics Labora-
tory, Fred Hutchinson Cancer Research Center over a
3-month period were available for analysis. Among the
first 200 samples, only those with at least 4 million
peripheral blood mononuclear cells above the require-
ments of routine clinical typing were entered sequen-
tially into the study. Among the final 52 prospective
samples, patients and family members were entered se-
quentially without exception. This panel of 252 samples
included 23 patients with a variety of hematologic dis-
orders and 229 normal family members. An additional
250 “retrospective” samples, representing 248 individu-
als, were retrieved from archived frozen cells (n 5 200) or
DNA (n 5 50) that had been analyzed previously by
in-house high-resolution DRB1 PCR/SSOP typing. In-
advertently, two retrospective individuals, one represent-
ing a rare DRB1 allele (DRB1*11082) and the other a
rare allele combination (DRB1*0302, 1001), were tested
in duplicate from independent sample preparations.
Study Design
Prospective clinical samples. Each of the 252 prospective
clinical samples was assigned a sequential code number
upon entry into the study. Among the first 200 samples
entered into the study, peripheral blood mononuclear
cells (PBMC) were isolated by Ficoll–Hypaque density
gradient centrifugation and then aliquoted into duplicate
coded tubes for independent DNA isolation, as described
later. DNA for TMA/HPA testing of the final 52 pro-
spective samples was isolated from 5 ml of whole blood
aliquoted before preparation of the PBMC used for PCR/
SSOP. Subsequently, analysis by the two methods was
carried out independently and blindly by different tech-
nologists in separate laboratories, using the coded sample
number and with no knowledge of any other typing
results or a sample’s relationship to the other study
samples. Within some assay batches, by each method,
samples were identified with ambiguous probe hybrid-
ization patterns so that DRB alleles could not be confi-
dently assigned in the initial typing assays (n 5 23 for
PCR/SSOP and n 5 14 for TMA/HPA). In one TMA/
HPA assay, the luminometer malfunctioned and the
results from one plate, four samples, were lost. These
samples were reassayed before the typing results from the
two methods were compared. The true typing result for
each of the 252 clinical samples was determined by
comparison of the independently derived results of the
two methods and defined as the “consensus” result. If a
sample gave a discordant result in the first comparison,
the typing was repeated by TMA/HPA, PCR/SSOP,
and/or DNA sequencing to obtain a confirmed consen-
sus, which we assumed to be the true typing result.
Retrospective samples. The 250 retrospective samples
were chosen to include a wide variety of DRB1 alleles
representative of Caucasian, American Black, Native
American, and Asian ethnic groups. Because the majority
of the prior typing with this assay technology has been
performed in Japanese laboratories typing samples of
limited ethnic diversity, particular effort was made to
test multiple heterozygous combinations for DRB1 al-
leles rare or absent in the Japanese population. This
retrospective sample panel was analyzed only by TMA/
HPA unless discrepancies were detected between the
TMA/HPA results and the previously known DRB1
PCR/SSOP typing assignments. Discordant samples
were repeated by allele-level PCR/SSOP, TMA/HPA,
and/or DNA sequencing to determine the true typing.
TMA/HPA Typing
The reagents for genomic DNA extraction, TMA, and
analysis by HPA were included in TMA/HPA DRB
typing kits (Chugai Pharmaceutics, Inc., Tokyo, Japan).
DNA was extracted from 2–4 million PBMC (n 5 200),
from white blood cells recovered from 5 ml of whole
blood after hypotonic lysis of red blood cells (n 5 52), or
from 4–6 million cryopreserved PBMC or B-lympho-
blastoid cell lines (LCL) (n 5 200) after lysis with 4 M
guanidine thiocyanate as previously described [19]. In
addition, 50 of the retrospective study samples consisted
of archived DNA prepared by the salting out method
[9]. TMA [20, 21] of DRB exon 2 was accomplished by
combining 1–3 mg of genomic DNA with generic DRB
primers, dNTPs, rNTPs, and buffer, followed by an
initial thermal cycle (96°C for 6 min, 60°C for 5 min,
and 40°C for 2 min) to denature the DNA and anneal the
primers. Next, 10 ml of reverse transcriptase was added
and a second thermal cycle (5 min at 40°C, 3 min at
96°C, 5 min at 60°C, 1 min at 40°C) was performed to
produce genomic DNA templates. Finally, another
10-ml aliquot containing reverse transcriptase and RNA
polymerase was added and, during a 4-h incubation at
76 A. G. Smith et al.

4. 40°C, millions of copies of RNA amplicons were gener-
ated, which are the targets of the HPA. The HPA was
performed as previously described [17], except that anal-
ysis was carried out in a 96-well microtiter plate con-
taining lyophilized aliquots of 24 acridinium ester-
labeled oligonucleotide probes (Fig. 1), so that up to four
samples were typed in a single plate. The 100-ml TMA
reaction, diluted with 250 ml of Tris/EDTA buffer, was
added in 10-ml aliquots to each of the 24 wells contain-
ing the rehydrated acridinium ester-labeled probes, and
the microtiter plate was incubated at 60°C for 20 min in
a microplate incubator (Tomy Tech, Palo Alto, CA,
USA). Next, 100 ml of hydrolysis reagent was added,
followed by an additional 10-min incubation at 60°C,
after which the microtiter plate was placed in a bed of
shaved ice for at least 5 min. After 10 of 38 samples in
the first two assays performed by one technologist ex-
hibited ambiguous probe reactions, the plate cooling
method was modified so that microtiter plates were
carefully nested in a water/ice slurry for repeat typing of
the 10 ambiguous samples and for all subsequent assays.
The chemiluminescent probe hybridization reactions
were detected in a luminometer (Anthos Biotec, Salz-
berg, Austria) that transmitted the data, in the form of
relative light units (RLU) detected, to a Chugai com-
puter analysis program, which produced a printout of the
probe hybridization values as RLUs and as a percentage
of the control probe RLU value for each sample, along
with the deduced DRB typing. Except for one probe, a
probe hybridization signal of less than 5% of the control
was considered a negative reaction; conversely, a hybrid-
ization signal of greater than 5% was regarded as a
positive signal. A positive reaction for probe S, which
recognizes phenylalanine at codon 47, was set at 1%
because of known weak hybridization signals. Table 1
describes the specific hybridization of the HPA probe
panel with the known DRB alleles. Among the clinical
samples, if a DRB typing assignment was not made by
the computer program, indicating an ambiguous hybrid-
ization pattern, TMA/HPA was repeated before the re-
sults were compared with the concurrent PCR/SSOP
FIGURE 1. TMA/HPA and PCR/SSOP probe locations on
representative DRB alleles. Letters (A, etc.) beside the HPA
and shared probes correspond to the probe names shown in
Tables 1 and 5. M, Probe locations common to HPA and
SSOP; 1, probes unique to HPA; u, probes unique to SSOP.
77
HLA-DRB Typing by TMA/HPA vs. PCR/SSOP

5. typing. Among the retrospective samples, the hybridiza-
tion pattern of each sample was also examined manually
to determine whether the results were consistent with
the known DRB1 alleles present. Discrepant samples
were retested by TMA/HPA, PCR/SSOP, and/or DNA
sequencing.
PCR/SSOP Typing
DNA for PCR/SSOP analysis of the prospectively typed
clinical samples was prepared from 2–3 million white
blood cells with a commercial reagent kit (IsoQuick,
Orca Research, Bothel, WA, USA). Medium-resolution
DRB analysis was performed by a forward dot blot
method with nonradioactive probe labeling as previously
described [13], except with an expanded set of 24 probes
(Fig. 1). Among the prospectively typed clinical sample
set, PCR/SSOP was repeated before comparison with the
TMA/HPA results if the probe hybridization pattern
could not be confidently interpreted by the technologist
performing the assay or by supervisor review.
RESULTS
Prospective Clinical Study
Five of 252 (1.98%) samples prospectively tested in a
double-blind protocol were found to have discrepant
assignments when the results of the TMA/HPA and
PCR/SSOP analyses were compared. Table 2 summarizes
the data with respect to the sensitivity of each method for
detecting a given DRB1 specificity and the specificity of
allele detection by each method. There were no examples
of false positive allele identification by either typing
system; all five discordant typing results involved false
negative allele detection by the TMA/HPA system. Four
of the discordant typing results occurred in the first two
TMA/HPA assays performed by one technologist. In all
four samples, single false negative HPA probe reactions
resulted in lack of detection of DRB1*01 (n 5 3) and
*03 (n 5 1) alleles. Probe A gave hybridization reactions
of 1% of control in three DR1 positive samples, and
probe D had a score of 4% of control in one DR3 positive
TABLE 1 Hybridization of the TMA/HPA probe panel among DRB1, DRB3, DRB4, and DRB5 alleles
ID Codona
DRB allele specificity
A 9–18 DRB1*0101–0104
B 9–16 DRB1*1501–1506, 1601–1608
C 9–16 DRB1*0301–0308, 1101–1104, 1105–1121, 1123–1128, 1301–1316, 1318–1327, 1401–1403, 1405–1409, 1412–1414,
1416–1425
D 74–80 DRB1*0301–0308 (except 03012 and 03022), 0422, 1107, DRB3*0204
E 9–17 DRB1*0401–0423, 1122, 1410
F 55–61 DRB1*1101–1128, 0308, 0415, 1204, 1411
G 66–72 DRB1*0103, 0402, 0414, 1102, 1114, 1116, 1120, 1121, 1301, 1302, 1304, 1308, 1315–1317, 1319, 1322, 1323, 1327,
1416
H 24–31 DRB1*0901
I 72–77 DRB1*0801–0804, 0806–0815, 1604, 0412, 0418, 1123, 1125, 1318, 1403, 1412, 1415, DRB5*0104
J 6–15 DRB1*0701
K 24–30 DRB1*1201–1204
L 26–34 DRB1*1001
M 9–16 DRB1*0801–0813, 0815, 1201–1204, 1105, 1317, 1404, 1411, 1415
N 55–61 DRB1*1401, 1404, 1407, 1410, 1416, 1422, 1425, 0808
O 55–62 DRB1*0405, 0409–0412, 0417, 1303, 1304, 1312, 1321, 1413, 0801, 0803, 0805, 0806, 0810, 0812, 0814
P 34–40 DRB1*0301–0303, 0305–0308, 1109, 1116, 1120, 1301, 1302, 1305, 1306, 1309, 1310, 1315, 1316, 1318, 1320, 1326,
1327, 1402, 1403, 1406, 1409, 1412, 1413, 1417–1419, 1421, 1424
Q 68–73 DRB1*0901, 1113, 1117, 1401, 1404, 1405, 1407, 1408, 1410, 1411, 1414, 1418, 1423, DRB4*0101–0104, 0201N
R 56–62 DRB1*1405, 1418
S 44–50 DRB1*1501–1506, 0301, 0304, 0305, 0307, 0308, 1101–1116, 1118–1128, 1201–1204, 1301, 1302, 1304–1306, 1309–
1311, 1314–1318, 1320–1325, 1327, 1417, 1421
T 63–70 DRB1*0103, 1605, 1607, 0402, 0412, 0414, 0418, 1102, 1114, 1116, 1118–1121, 1201, 1203, 1204, 1301–1304, 1306,
1308, 1310, 1312, 1315–1317, 1319, 1322, 1323, 1327, 1416, 0803, 0810, 0812, 0814, 0815
U 62–70 DRB1*1601, 1603, 1604, 1608, 0415, 1101, 1103–1106, 1109–1112, 1115, 1122–1125, 1127, 1128, 1202, 1305, 1307,
1311, 1314, 1318, 1321, 1324, 1326, 1415, 1422, 1425, 0801, 0802, 0804–0809, 0811, DRB5*0101–0105
V 64–71 DRB1*1602, 1108, 1325, 1403, 1412, 0813
W 55–62 DRB1*0101–0104, 15022, 0301–0307, 0401–0404, 0406–0408, 0413, 0414, 0418–0423, 0802, 0804, 0809, 0813, 1001,
1301, 1302, 1305–1311, 1314–1320, 1322–1325, 1327, 1402, 1403, 1406, 1409, 1412, 1414, 1415, 1417, 1419–1421,
1423, 1424, DRB3*0201–0206
a
Probe locations are illustrated graphically on representative DRB alleles in Fig. 1, including control probe X, which hybridizes to all DRB1, DRB3, DRB4,
and DRB5 alleles.
78 A. G. Smith et al.

6. sample. Because of the particular allele combinations in
these samples, the single probe failures gave hybridiza-
tion patterns consistent with homozygous typing. Re-
peat TMA/HPA typing of those four samples, using the
modified microtiter plate cooling system described in
Materials and Methods, gave scores for all probes of
10–80% of control and typing results concordant with
PCR/SSOP. After this modification of the TMA/HPA
method, two other false negative reactions occurred in a
single sample. This sample typed by PCR/SSOP as
DRB1*03, 1001 but was typed in three separate TMA/
HPA tests as DRB1*1001 and another allele that could
not be determined because of apparent false negative
reactions of HPA probes C (1%) and D (3%) for the
DRB1*03 allele detected in PCR/SSOP. DNA sequence
analysis, after DR52-associated DRB1 specific amplifi-
cation, was consistent with DRB1*0301 at least for
codons 12–87. In subsequent TMA/HPA typing using a
1/3 dilution of the original DNA and an independent
DNA preparation, this sample typed as DRB1*0301,
1001 with no false negative or false positive probe reac-
tions. Table 2 also summarizes the frequency distribu-
tion of the 462 DRB1 specificities assigned among the
252 prospectively typed samples by TMA/HPA and
PCR/SSOP. At least two examples each of the DRB1*01,
02, 03, 04, 06 (13/14), 0701, 08, 0901, 1001, 11, and
12 specificities were detected. The more generic
DRB1*06 designation is used to include both *13 and
*14 because many heterozygous allele combinations pre-
clude assigning a definitive subtype. As expected in this
predominantly Caucasian sample panel, the most fre-
quent specificities were DRB1*06 (16.2%), *02
(15.6%), *04 (13.8%), and *07 (13.8%), and the least
frequent were DR10 (0.4%), DR12 (0.6%), and DR9
(0.8%).
Retrospective Study
The 250 retrospective samples included 66 distinct
DRB1 alleles representing Caucasian, American Black,
Native American, and Asian ethnic groups. Table 3 lists
the two unique DRB1*12 alleles, three alleles each of the
DRB1*01 and *03 families, six alleles of the DRB1*02
family, eight alleles each of the DRB1*08 and *13
families, 11 alleles each of the DRB1*04, *11, and *14
families, and also DRB1*0701, *0901, and *1001 al-
leles. Multiple heterozygous combinations of alleles rep-
resentative of non-Japanese racial groups were success-
fully typed by the TMA/HPA reagents. For example,
DRB1*0302 (n 5 18), found primarily in Black popu-
lations, and DRB1*1305 (n 5 17), common in the
Jewish population, were each detected in 10 different
heterozygous combinations; DRB1*1303 (n 5 25),
found predominantly in Caucasian and Black popula-
tions, was detected in the presence of 13 different DRB1
TABLE 2 Comparison of HLA-DRB1 specificities assigned by PCR/SSOP vs. TMA/HPA among the 252
prospectively typed samples
DRB1 n F (%)
False positive False negative Sensitivity Specificity
HPA SSOP HPA SSOP HPA SSOP HPA SSOP
*01 53 11.5 0 0 3 (0)a
0 0.944 1.00 1.00 1.00
*02 72 15.6 0 0 0 0 1.0 1.00 1.00 1.00
*03 59 12.8 0 0 2 (1)a
0 0.966 1.00 1.00 1.00
*04 64 13.8 0 0 0 0 1.0 1.00 1.00 1.00
*11 43 9.3 0 0 0 0 1.0 1.00 1.00 1.00
*12 3 0.6 0 0 0 0 1.0 1.00 1.00 1.00
*06b
75 16.2 0 0 0 0 1.0 1.00 1.00 1.00
*0701 64 13.8 0 0 0 0 1.00 1.00 1.00 1.00
*08 23 5.0 0 0 0 0 1.00 1.00 1.00 1.00
*0901 4 0.8 0 0 0 0 1.00 1.00 1.00 1.00
*1001 2 0.4 0 0 0 0 1.00 1.00 1.00 1.00
Total 462 0 0 5 (1)a
0 0.992 1.00 1.00 1.00
(0.998)a
n is the number of each DRB1 specificity identified as the true typing result. F (%) gives the frequency of each DRB1 specificity identified among the prospectively
typed sample panel expressed as n/462 3 100.
a
Four of the discordant typing results occurred in the first two TMA/HPA assays done by one technologist, with false negative identification of DRB1*01(n 5
3) and DRB1*03 (n 5 1). After modifying the microtiter plate cooling technique, as described in Materials and Methods, only one additional sample was found
to be discordant when TMA/HPA and PCR/SSOP results for the 252 clinical samples were compared. In the latter sample, PCR/SSOP identified DRB1*1001,
03, whereas TMA/HPA detected DRB1*1001 and another allele that could not be defined in three separate tests. Excluding the four discordant typings that were
resolved with the methodology modification, only a single DR3 allele (0.2%) was discordant among the 462 DRB1 alleles detected. Ultimately, this sample was
typed as concordant with PCR/SSOP using both the original DNA sample diluted 1:3 and an independent DNA preparation.
b
DRB1*13 and *14 alleles are reported as the combined designation *06 because many DRB1 allele combinations preclude definitive subtyping assignments with
the medium-resolution reagents used in these studies.
79
HLA-DRB Typing by TMA/HPA vs. PCR/SSOP

7. alleles; and DRB1*0411 (n 5 7), characteristic of Native
Americans, was identified in seven different combina-
tions. In the TMA/HPA typing of the retrospective
samples, all 483 DRB1 specificities previously assigned
by PCR/SSOP were detected, with two exceptions.
DRB1*1402 and *1406 alleles that occurred in the
presence of DR3 (n 5 2) were not detectable by the HPA
probe panel. These latter two samples were assigned by
TMA/HPA as DRB1*03 homozygous or as DRB1*03
and a possible *06. Evaluated at the medium-resolution
level, there were no false positive allele assignments in
the TMA/HPA typing of this panel of samples. In addi-
tion, to evaluate the accuracy of the HPA probes in
predicting the DRB1 sequences present in a sample, we
also examined the hybridization of 12 HPA probes to
polymorphisms at codons 37, 47, 57, 60, and 67–71. In
this analysis, TMA/HPA results were discordant with
previous PCR/SSOP typing in seven samples (Table 4).
Repeat DR52-associated DRB1 allele-level PCR/SSOP
determined that TMA/HPA analysis was correct in six of
these samples, all of which involved identification of
sequences at codons 67–71 in samples heterozygous for
two DR52-associated DRB1 alleles. Five of these in-
volved identification of single nucleotide differences in
the sequences that encode phenylalanine vs. isoleucine at
codon 67, including three samples with DRB1*1102 vs.
*1103 in the presence of DRB1*1301. However, one
sample, typed by PCR/SSOP as DRB1*0405, 0807,
showed a consistent cross-reaction with the HPA probe
for codon 57 aspartic acid in three separate tests. Repeat
PCR/SSOP and DNA sequence analysis confirmed the
presence of the codon 57 serine (DRB1*0405) and valine
(DRB1*0807) sequences previously identified by PCR/
SSOP.
To evaluate objectively the specificity and sensitivity
of the 23 HPA probes that hybridize variably among the
DRB alleles, we also examined the relative positive and
negative signals of these probe reactions among the ret-
rospective sample set. A control probe hybridizing to all
DRB alleles was used to monitor the level of amplifica-
tion. Control probe values ranged from 40,882 to
1,115,861 relative light units (RLU), with 19 samples
having less than 100,000 RLU, 154 samples having
100,000–500,000 RLU, and 77 samples having greater
than 500,000 RLU. The HPA probe hybridization values
summarized in Table 5 were derived by dividing the
RLU detected by the luminometer in each probe well by
the RLU detected in the control probe well for that
sample. The average positive probe reaction among all 23
probes was 45%, with a range of 9% for probe S, which
detected the codon 47 phenylalanine (47F) sequence, to
95% for probe W, which identified the codon 57 aspartic
acid sequence (57D). An expected positive probe signal
less than 5% was considered to be a false negative reac-
tion except for probe S, which consistently gave low
(1–30%) positive signals. Excluding the latter probe,
TABLE 3 DRB1 alleles (n 5 483) identified among the 250 retrospective samples
DR1 n DR2 n DR3 n DR4 n DR11 n DR12 n DR13 n DR14 n DR8 n
0101 15 1501 7 0301 18 0401 15 1101 30 1201 37 1301 14 1401 7 0801 8
0102 10 15021 4 0302 18 0402 6 1102 28 1202 8 1302 21 1402 4 0802 3
0103 15 15022 1 0304 1 0403 1 1103 21 1303 25 1403 1 0803 4
1503 4 0404 6 1104 9 1304 7 1404 8 0804 3
1601 2 0405 6 1105 1 1305 17 1405 1 0806 1
1602 2 0407 4 1106 1 1306 1 1406 6 0807 3
0409 3 11081 1 1310 3 1407 2 0811 1
0410 2 11082 3 1312 2 1408 1 0814 2
0411 7 1110 1 1409 1
0412 1 1113 1 1417 1
0419 1 1119 1 1418 1
In addition, DRB1*0701 (n 5 22), DRB1*0901 (n 5 3), and DR10 (n 5 19) were included in the retrospective samples.
TABLE 4 Seven retrospective samples with
discordant TMA/HPA and PCR/SSOP
DRB1 typing
Sample ID PCR/SSOP TMA/HPA Correct method
RN097 *0803, 1201 *0803, 1202 TMA/HPA
RN146 *1103, 1301 *1102, 1301 TMA/HPA
RN053 *1102, 1301 *1103, 1301 TMA/HPA
RN161 *1202, 1202 *1201/1203, 1202 TMA/HPA
RN187 *1102, 1301 *1103, 1301 TMA/HPA
RN197 *1101, 1103 *1101, 1104 TMA/HPA
RDNA23a
*0405, 0807 Codon 57 S and D PCR/SSOP
TMA/HPA probe hybridization suggested errors in the PCR/SSOP DRB1
high-resolution typing of six samples. Review of previous SSOP results found
that the DRB1 alleles in these heterozygous samples had not been separately
amplified and probed. Repeat SSOP typing with allele-specific primers for
DR8, DR11, and/or DR12 [23] found that the pattern of probe hybridization
in the TMA/HPA tests was correct.
a
TMA/HPA probe W (Table 5), specific for the codon 57 aspartic acid (D)
sequence, gave positive reactions (25–27%) in three separate TMA/HPA tests
for RDNA23, which was identified as DRB1*0405, 0807 by PCR/SSOP and
DNA sequencing.
80 A. G. Smith et al.

8. only three false negative HPA probe reactions, 0.06% of
all probe reactions, occurred among retrospective sam-
ples; repeat testing of the three samples gave probe
values greater than 5%. Although for any particular
probe there was a wide range of positive hybridization
signals, the vast majority of probe reactions were greater
than 10 times the average negative signal for that probe.
Only 15 positive probe reactions (0.3% of all probe
reactions) fell below that level, with 11 of those found
among the reactions of probes T (FLED) and U (ILED),
codons 67–70, with DR12 sequences. To determine
whether there was a systematic problem associated with
DR12 alleles, we examined the hybridization scores for
probes T and U among 29 DR12 positive samples in
which the codon 67–70 probe signals could be evaluated
independent of the other DRB allele present. In those
samples, probe T gave an average signal of 8% (3–16%)
and probe U gave 13% (5–27%), compared with a mean
30% signal for U and T among the entire sample set.
Because this substantial decrease in signals suggested a
possible amplification problem among DR12 alleles, we
compared the mean hybridization values for probe M
(codons 9–16) among 43 DR12 positive samples
(mean 5 54%) versus 30 DRB1*08 or *1404 positive
samples (mean 5 63%). Although the average probe M
signal was slightly decreased (14%) among DR12 sam-
ples, it appears unlikely that reduced DR12 amplifica-
tion caused the 73% decrease in probe T and 57%
decrease in probe U signals among DR12 positive sam-
ples. The average negative HPA probe reaction among
the 23 variable probes was less than 1%, with the ex-
ception of probe R for the codon 57 aspartic acid nucle-
TABLE 5 Analysis of the specificity and sensitivity of HPA probe hybridization among the retrospective
sample set
Probe
No.
positive
Ave. %b
(positive)
Range
(positive) No. , 5%c
No. 5–10%d
Ave. %b
(negative)
Range
(negative) No. . 5%e
DR specificitya
Codon
A 1 9–18 40 63 16–213 0 0 ,1 0–4 0
B 2: DRB1 9–16 20 48 20–89 0 0 ,1 0–1 0
C 3, 6, 11 9–16 188 89 24–216 0 0 ,1 0–2 0
D 3 (4, 11, B3) 74–80 37 23 13–42 0 0 ,1 0–11 1
E 4 (11, 14) 9–17 55 31 10–80 0 0 ,1 0–1 0
F 11 (4, 12) 55–61 84 75 30–130 0 0 ,1 0–1 0
G 1, 4, 6, 11 66–72 90 65 34–153 0 0 ,1 0–5 0
H 9 24–31 3 86 46–144 0 0 ,1 0–3 0
I 8, 6 (2, 4, 11) 72–77 29 33 13–49 0 0 ,1 0–1 0
J 7 6–15 26 41 20–78 0 0 ,1 0–5 0
K 12 24–30 47 34 17–78 0 0 ,1 0–1 0
L 10 26–34 19 57 16–76 0 0 ,1 0–1 0
M 8, 12 (11, 6) 9–16 78 60 17–118 0 0 ,1 0–1 0
N 14 (8) 55–61 18 31 15–65 0 0 ,1 0–2 0
O 4, 6, 8 55–62 68 43 17–143 0 0 ,1 0–23 1
P 3, 6, (2, 11) 34–40 94 33 10–83 0 0 ,1 0–6 1
Q 9, 14, B4 (11) 68–73 97 30 4–80 1 2 ,1 0–6 1
R 1405, 1418 56–62 2 15 10–20 0 0 1.2 0–21 5
S 2, 3, 6, 11, 12 44–50 178 9 1–30 55 67 ,1 0–1 0
T 2, 4, 6, 8, 11, 12 63–70 121 30 3–67 2f
4f
,1 0–3 0
U 1, 2, 4, 6, 8, 11, 12 62–70 150 30 5–66 0 5f
,1 0–7 1
V 2, 6 (8, 11) 64–71 7 16 6–21 0 1 ,1 0–6 1
W 1, 3, 4, 6, 8, 10, B3 55–62 223 95 17–214 0 0 ,1 0–27 1
a
DRB specificities in which the probe sequences are found. Specificities in parentheses indicate an uncommon occurrence of a probe sequence within a DRB
specificity. The letters (A, B, C, etc.) in the first column refer to the HPA probe locations in Fig. 1. For the specific DRB allele hybridization patterns of each
HPA probe, see Table 1. DR6 includes both DRB1*13 and 14 alleles.
b
HPA probe hybridization values were derived by dividing the relative light units (RLU) detected in the well of a particular probe by the RLU detected for the
control probe (X) of that sample, to give the percentage of control probe for each of the 23 specific probe reactions for each sample. Among the retrospective sample
set, the range of control probe (X) values was 40,882–1,115,861 RLU, with 19 samples having X less than 100,000, 154 samples with X between 100,000 and
500,000, and 77 samples with X greater than 500,000.
c
An expected positive probe hybridization value of less than 5% was considered to be a false negative reaction except for probe S (47F), which consistently gave
low (1–30%) positive signals. Repeat testing of the three samples with false negative HPA reactions gave probe values greater than 5% of control.
d
Expected positive probe values of 5–10% were considered weak reactions but were not repeated.
e
Expected negative probe values greater than 5% of control were considered to be false positive reactions and HPA testing was repeated. Repeat analysis of the
10 samples with 12 false positive HPA reactions all gave probe values less than 5% of control except for the consistent false positive reaction of probe W with
a sample having DRB1*0807, 0405.
f
All 11 weak or false negative HPA reactions for probes T and U (FLED and ILED, respectively) were found among DR12 positive samples.
81
HLA-DRB Typing by TMA/HPA vs. PCR/SSOP

9. otide sequence unique to DRB1*1405 and *1418, with
an average negative probe reaction of 1.2%. An expected
negative HPA probe reaction with a signal above 5% was
defined as a false positive and the test was repeated.
Twelve false positive reactions occurred among eight
different HPA probes, with five of the false positive
reactions associated with probe R. All of the false posi-
tive reactions were resolved with repeat TMA/HPA anal-
ysis except for the consistently positive reaction of probe
W (57D) with the sequence present on DRB1*0807,
previously mentioned above as the only discrepant TMA/
HPA typing in the retrospective sample set. In three
separate tests, probe W gave scores of 25–27% with the
DRB1*0807, 0405 sample.
DISCUSSION
The large-scale DNA typing of HLA class II alleles in
over 60,000 unrelated marrow donors and quality-
control samples by multiple laboratories for the National
Marrow Donor Program have demonstrated the PCR/
SSOP method to be highly accurate and reliable, with an
average of just 1% discrepant DRB1 assignments among
the 4636 quality-control samples tested [16]. These fa-
cilities have demonstrated the feasibility of DRB typing
over 250 samples per week using, predominately, in-
house developed PCR/SSOP systems that may include
automation of some assay processes. However, despite
this remarkable level of typing, there are still millions of
volunteer donors already recruited in registries world-
wide that do not have DRB typing [18] and more donors
continue to volunteer. Current constraints on high-vol-
ume DNA typing relate primarily to physical limitations
of the assay systems and to their costs. In comparison
with the HPA process—which consists of a 20-min
incubation, a reagent addition, two short incubations (10
and 5 min), and a 5-min automated luminescence read-
ing and analysis program—the forward dot blot SSOP
typing systems employed by the majority of the NMDP
contract typing laboratories and even the various reverse
probe format assays remain somewhat cumbersome in
their hybridization and washing processes. Projects such
as HLA-DNA typing of volunteer stem cell donors il-
lustrate the need for accurate commercial HLA typing
systems flexible enough to be used in both clinical lab-
oratories and in high-volume situations. We have carried
out a comprehensive evaluation of the medium-resolu-
tion HLA-DRB DNA typing system developed by Chu-
gai Pharmaceutics (Tokyo, Japan), which employs a tran-
scription-mediated DNA amplification method with an
oligonucleotide probe hybridization protection assay
(TMA/HPA). In one phase of this study, 252 samples
were prospectively typed in a double-blind protocol con-
currently by TMA/HPA and by a locally developed me-
dium-resolution DRB PCR/SSOP assay. The results of
this comparative study demonstrated a high degree of
concordance between the two methods in the assignment
of DR1 through DR12 specificities. In one sample typed
by TMA/HPA, a DRB1*03 allele was detected but not
clearly identified in the presence of a DRB1*1001 allele.
Because this sample was correctly typed after preparation
of a new DNA sample, this initial discrepancy was
apparently due to poor quality of DNA in the initial
sample. The typing problems of the four other discrepant
samples were apparently caused by an inadequate plate
cooling process, which was identified and corrected after
the first two problematic assays by one technologist
(described in Materials and Methods). In these 4 samples
as well as the 10 other samples in those two assays with
ambiguous HPA probe patterns, single HPA probes gave
false negative signals, presumably when the microtiter
plate cooling method failed to stop the probe hydrolysis
reaction. This experience suggests that the Chugai typ-
ing system might be improved by using a molded freez-
able form fitting closely to the bottom of the microtiter
plate instead of the current water/ice slurry process.
In the other phase of this study, 250 samples, previ-
ously typed to the allele level by local PCR/SSOP, were
analyzed by TMA/HPA. Evaluated at the medium-reso-
lution typing level, TMA/HPA testing detected all of
the 483 DRB1 alleles present except in two samples in
which DRB1*1402 or *1406 alleles were not detected in
the presence of DRB1*0301 because of the absence of an
HPA probe for the LLEQR sequence at codons 67–71.
The highly polymorphic nature of the DR13 and DR14
alleles [22] makes it very difficult to detect all DR6
alleles in the presence of DRB1*03 or *11 alleles with a
limited probe panel. Using the panels of 24 probes
described in Table 1 for HPA or SSOP typing, only two
DRB1*13 alleles (*1309 and *1320) are not detected in
the presence of DRB1*03 by either method. Similarly,
three DRB1*13 alleles (*1307, *1311, and *1314) are
not detected by either method in the presence of the
most common DRB1*11 alleles, *1101 and *1104.
However, seven DRB1*14 alleles (*1402, *1406,
*1409, *1417, *1419, *1420, and *1421) are not de-
tected in the presence of DRB1*03, and one
(DRB1*1420) is not detected in the presence of
DRB1*11 by the TMA/HPA probes, whereas only
DRB1*1419 and *1421 are not detected in the presence
of DRB1*03 by the PCR/SSOP panel. The addition of
the single probe for the LLEQR sequence (codons 67–71)
to the HPA panel would allow detection of all DRB1*14
alleles except *1419 and *1421 in the presence of either
DRB1*03 or *11. Any sample identified as homozygous
for DRB1*03,*11, *13, or *14 by intermediate-level
DRB typing should be analyzed by high-resolution anal-
ysis to confirm homozygosity.
82 A. G. Smith et al.

10. Examined at the DRB1 allele level, TMA/HPA typ-
ing detected errors in previous PCR/SSOP typing of six
samples with two DR52-associated DRB1 alleles, all
involving sequences at codons 67–71. Repeat PCR/SSOP
typing of these samples using the current panel of allele-
specific primers and probes [23] for the high-resolution
analysis of DR52-associated DRB1 alleles determined
that the TMA/HPA result was correct. Only one addi-
tional retrospective sample remained discordant, with
the single consistent cross-reaction of the HPA probe W
for codon 57 aspartic acid with the single base mismatch
in the codon 57 valine sequence present in DRB1*0807.
In summary, we found the TMA/HPA DRB typing
system to provide reliable and accurate typing results
using DNA freshly prepared from 2 million PBMC, 5 ml
whole blood after hypotonic red cell lysis, or 2–4 million
cryopreserved B-LCL, or using archived DNA stored up
to 4 years at 220°C. The transcription-mediated ampli-
fication system appears to be robust and reliable, because
only 2.4% among all 502 study samples were repeated
because of insufficient amplification. Although the TMA
protocol used in this assay system required 5–6 h, the
two initial thermal cycles followed by a 4-h isothermal
incubation at 40°C minimizes the requirement for ex-
pensive thermal cycling equipment, especially in high-
volume situations. Except for the two early HPA assays
with the plate cooling methodology problem, the num-
ber of prospectively typed clinical samples that required
repeat analysis (because of an ambiguous probe reaction
pattern or insufficient amplification) before comparison
of typing results was nearly equivalent between TMA/
HPA (7.8%) and PCR/SSOP (9.2%), and only a single
DR3 allele of a single TMA/HPA sample typing was
discordant when results were compared. Among the 250
retrospective samples, a single HPA probe consistently
cross-reacted with a single DRB1 allele sequence; how-
ever, HPA probes were more specific than previous PCR/
SSOP in the detection of sequences for codons 67–71
among samples heterozygous for two alleles of
DRB1*08, 11, 12, or 13. Although consistently low
positive HPA signals for two probes (T and U) at codons
67–70 were detected among DR12 alleles, these low
hybridization values were, nevertheless, highly specific
and did not result in any discrepant typings. Similar low
signals for the two equivalent probes used in local PCR/
SSOP assays have been observed among DR12 positive
samples. Because these weak reactions do not appear to
be the result of reduced DR12 amplification, we specu-
late that DR12 alleles may develop secondary structure
in their TMA or PCR amplified fragments, which results
in decreased probe binding at codons 67–71.
In this study, most TMA/HPA assays were done in
batches of 20 or 24 samples, including a negative (no
DNA) control sample to monitor the amplification re-
agents for potential contamination. At this level, turn-
around time from DNA isolation through data analysis
was 10–11 h, with 6–7 h of actual hands-on laboratory
work. In a high-throughput situation, two technologists
could easily isolate, amplify, and analyze over 144 sam-
ples in 48 h using the same set of amplification temper-
ature cycler, microplate incubator, and luminometer. For
the typing of one to four samples, which was done in this
study for two repeat typings, turnaround time would be
6–7 h. Although the 4.5-h isothermal TMA process is
about 2 h longer than standard PCR profiles, the 50–
60-min HPA assay and analysis time gives a total typing
time (,6 h) for a single sample that is comparable to
current commercially available reverse probe assays based
on membrane or microtiter plate formats. Thus, this
flexible method could be used for both high- and low-
volume typing situations, including solid organ trans-
plantation. In addition, the Chugai TMA/HPA method-
ology, based in a microtiter plate format that requires
only sequential sample and reagent additions with short
incubations and eliminates the usual plate washing steps
of the traditional ELISA format, appears to have superior
potential for automation. Although this application has
not yet been developed, robotic systems for microtiter
plate manipulations are widely available.
The exquisite sensitivity and specificity demonstrated
by the HPA probes in this study suggests that this
methodology might be successfully miniaturized. The
application of 10 times as many probes (960) to a single
plate would allow the simultaneous DRB typing of 40
samples, potentially increasing typing volumes by a fac-
tor of 10. Such technology might also be applied to HLA
class I typing, which requires the use of probes to over
100 polymorphisms. Very likely it will only be through
the commercial competition of multiple HLA-DNA typ-
ing technologies that the cost-effective very high-
throughput typing systems will be developed that will
allow complete HLA-DNA typing for such massive
projects as the worldwide unrelated marrow donor reg-
istries.
ACKNOWLEDGMENTS
This work was supported by grants from the National Insti-
tutes of Health, numbers AI33484 and CA18029, from the
Allison Atlas Foundation, and from Chugai Pharmaceutics,
Ltd., Tokyo, Japan.
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