Rapid LIFECODES SSO typing kits –

White Paper
Shalaka Patel, PhD
IMMUCOR
Stamford CT

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Background
Human Leukocyte Antigen (HLA) Class I and II genes are the most polymorphic genes in the human
genome. Sequence‐specific oligonucleotide (SSO) based HLA typing, has been used routinely in several
tissue typing laboratories for greater than 20 years3,4. The LIFECODES HLA‐SSO typing kits are used to
type for HLA Class I and Class II alleles based on the Luminex xMAP technology. SSO probes are attached
to Luminex Microspheres designed for use with the Luminex Instrument. Each of the different SSO
probes may be homologous to a sequence within the amplified DNA that is unique to an allele or group
of alleles. The hybridization of the amplified genomic DNA with these unique SSO probes yields
information needed to determine the HLA genotype of the sample.

The total time taken by the current LIFECODES amplification and hybridization protocols is 3 hours and
15 minutes. We have developed the rapid protocol which has cut this time to 1 hour and 32 minutes,
while still maintaining the performance characteristics of the current LIFECODES SSO products. In
addition, we have reduced the quantity of template DNA required for testing.

Rapid Assay Protocol

Need for faster protocol:
1. Deceased donor testing: Shorter time to result is required for HLA testing on a deceased donor.
2. High volume labs: Faster protocol required for labs with high volume samples.
3. Saves HLA lab personnel time: Decrease in protocol time increases productivity.

Development of Rapid Protocol:

The aim was to develop a faster protocol that uses less quantity of reagents and DNA amounts while
maintaining the performance LIFCODES HLA SSO kits. For feasibility testing, a combination of 12
amplification protocols and 7 hybridization protocols were tested on 12 LIFECODES HLA SSO typing kits –
A, A eREs, B, B eRES, C eRES, DRB1, DRB1 eRES, DRB345, DQA, DQB, DPB and Null kits using well
characterized samples. A final amplification and hybridization protocol described below was chosen
based on performance equivalency with the current protocol. All testing was done on the 96‐Well
GeneAmp® PCR sSystem 9700 (MAX mode) and the Veriti™ 96‐Well Thermal Cycler (9700 MAX mode).

Rapid Protocol
The current LIFECODES HLA‐SSO typing kits require 2 hours and 30 minutes for amplification and 45
minutes for the hybridization step. The total assay time, excluding Luminex analysis and tech
preparation time is approximated 3 hours and 15 minutes. The new protocol reduces the amplification
time to 1 hour and 12 minutes and the hybridization time to 20 minutes. To achieve better heat
conductivity, the PCR volume was reduced to 20 µL. In addition, the quantity of genomic DNA used in
the PCR reaction was reduced to 40‐120ng while maintaining the analytical DNA sensitivity of 2‐6 ng/µL
of PCR.

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Table 1 highlights the changes between the current and the rapid protocol:

Table 1
Current LIFECODES HLA SSO Kits Rapid LIFECODES HLA SSO Kits
Amplification (AMP) Protocol 2 hours and 30 minutes 1 hour and 12 minutes
Hybridization (HYB) Protocol 45 minutes 20 minutes
Total AMP+HYB time 3 hours and 15 minutes 1 hours and 32 minutes
PCR Reaction Volume 50 µL 20 µL
DNA Quantity 100‐300ng 40‐120ng
(2‐6 ng/µL of PCR) (2‐6 ng/µL of PCR)

Table 2 describes the changes between the current and rapid amplification protocols

Table 2
Current LIFECODES HLA SSO Kits Rapid LIFECODES HLA SSO Kits
95°C 5 min 1 cycle 95°C 3 min 1 cycle

95°C 30 sec 95°C 15 sec

60°C 45 sec 8 cycles 60°C 30 sec 12 cycles
72°C 45 sec 72°C 30 sec
Amplification Protocol
95°C 30 sec 95°C 10 sec
63°C 45 sec 32 cycles 63°C 30 sec 28 cycles
72°C 45 sec 72°C 30 sec

72°C 15 min 1 cycle 72°C 2 min 1 cycle

Table 3 describes the changes between the current and rapid amplification protocols

Table 3
Current LIFECODES HLA SSO Kits Rapid LIFECODES HLA SSO Kits
97 °C 5 min 97 °C 2 min
Hybridization Protocol 47°C 30 min 47°C 10 min
56 °C 10 min 56 °C 8 min
56 °C hold 56 °C hold

Validation Studies with Rapid Protocol

The purpose of the following studies was to show equivalency of the Rapid LIFECODES SSO protocol to
the current LIFECODES protocol. All testing was done on the 96‐Well GeneAmp® PCR sSystem 9700
(MAX mode) and the Veriti™ 96‐Well Thermal Cycler (9700 MAX mode).

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Limit of Detection studies
One of the changes in the rapid protocol was to decrease the amount of DNA added per PCR reaction.
The range in the rapid protocol is 40‐120ng per PCR reaction. Testing was performed on a range of DNA
inputs – 20ng, 30ng, 40ng, 80ng, 120ng and 180ng. Ten well‐characterized samples were tested in
duplicate on all 12 of the LIFECODES SSO typing kits – A, A eRES, B, B eRES, C eRES, DRB1, DRB1 eRES,
DRB345, DQA, DQB, DPB and Null. The % concordances and % failures are listed in Table 4 below.

Table 4
Kit 20 ng 30 ng 40 ng 80 ng 120 ng 180 ng
A %C %F %C %F %C %F %C %F %C %F %C %F
A eRES 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
B 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
B eRES 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
C eRES 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DRB1 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DRB1eRES 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DRB345 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DQA 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DQB 100% 0% 95% 0% 95% 0% 95% 0% 95% 0% 100% 0%
DPB 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
Null 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
%C: % Concordance %F: % Failure

Typing with Well Characterized samples

Approximately 130 Well Characterized samples were tested on all 12 of the LIFECODES SSO typing kits –
A, A eRES, B, B eRES, C eRES, DRB1, DRB1 eRES, DRB345, DQA, DQB, DPB and Null. The % concordances
and % failures are listed in Table 5.

Table 5
% Failures (# of % Concordant (# of Lower Boundary, One‐Sided
HLA Locus
samples) samples) Exact Method (95% CI) 5
HLA‐A 0% (0/131) 100% (131/131) 97.74%
HLA‐A eRES 0% (0/130) 100% (130/130) 97.72%
HLA‐B 0% (0/130) 100% (130/130) 97.72%
HLA‐B eRES 0% (0/131) 100% (131/131) 97.76%
HLA‐C eRES 0% (0/130) 100% (130/130) 97.72%
HLA‐DPB 0% (0/130) 100% (130/130) 97.72%
HLA‐DQA 0% (0/130) 100% (130/130) 97.72%
HLA‐DQB 0% (0/130) 100% (130/130) 97.72%
HLA‐DRB1 0% (0/131) 100% (131/131) 97.74%
HLA‐DRB1 eRES 0% (0/130) 100% (130/130) 97.72%
HLA‐DRB 3/4/5 0% (0/130) 100% (130/130) 97.72%
Null Allele, Class I 0% (0/145) 100% (145/145) 97.95%
Null Allele, Class II 0% (0/133) 100% (133/133) 97.77%

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Sample Type and DNA Extraction method
The quality of DNA samples is an important variable that can affect accurate typing with HLA SSO kits.
Most DNA extraction kits are designed to eliminate possible PCR inhibitors. The most common PCR
inhibitor in blood is “heme”2. Commonly used methods for DNA extraction from blood are either
magnetic or column based. Therefore, we verified that the LIFECODES Rapid SSO protocol worked with
the two commonly used anti‐coagulants EDTA and ACD on whole blood and buffy coat samples
extracted with the 3 commonly used DNA extraction methods, 2 of which were column based and 1 was
magnetic (Table 6). Testing was done on all 12 of the LIFECODES SSO typing kits – A, A eRES, B, B eRES, C
eRES, DRB1, DRB1 eRES, DRB345, DQA, DQB, DPB and Null The % concordances and % failures are listed
in Table 7.

Table 6

Categories Total samples analyzed:

EDTA 120
Anti‐coagulants
ACD 120
QIAamp DNA Mini kit – Column based 80
Extraction Method Maxwell DNA kit ‐ Magnetic 80
Puregene Blood DNA kit – Column based 80
Whole Blood 120
Sample types
Buffy Coats 120

Table 7
Anti‐coagulants Sample Type Extraction methods
ACD EDTA Whole Buffy Coat QiaAMP Maxwell Puregene
Kit
Blood
%C %F %C %F %C %F %C %F %C %F %C %F %C %F
A 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
A eRES 100% 0.8% 100% 0% 100% 0.8% 100% 0% 100% 0% 100% 0% 100% 1.3%
B 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
B eRES 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
C eRES 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DRB1 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DRB1eRES 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DRB345 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DQA 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
DQB 100% 0% 98.3% 0% 100% 0% 98.3% 0% 97.5% 0% 100% 0% 100% 0%
DPB 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
Null 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0% 100% 0%
%C: % Concordance
%F: % Failure

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Some HLA testing laboratories also used buccal swabs as source of DNA. Therefore, we verified that the
LIFECODES Rapid SSO protocol also worked with buccal swabs extracted from 3 commonly used DNA
extraction kits – QiaAMP DNA Mini kit, Reliaprep gDNA tissue miniprep system and Gentra Puregene
buccal cell kit. The % concordances and % failures are listed in Table 8.

Table 8
Buccal Swab:
Extraction methods
Kit
QiaAMP ReliaPrep Puregene
%C %F %C %F %C %F
A 100% 0% 100% 0% 100% 0%
A eRES 100% 0% 100% 0% 100% 0%
B 100% 0% 100% 0% 100% 0%
B eRES 100% 0% 100% 0% 100% 0%
C eRES 100% 0% 100% 0% 100% 0%
DRB1 95% 0% 100% 0% 100% 0%
DRB1eRES 100% 0% 100% 0% 100% 0%
DRB345 100% 0% 100% 0% 100% 0%
DQA 100% 0% 100% 0% 100% 0%
DQB 100% 0% 100% 0% 100% 0%
DPB 100% 0% 100% 0% 100% 0%
Null 100% 0% 100% 0% 100% 0%
%C: % Concordance
%F: % Failure

Reproducibility of the assay

To determine reproducibility, it is important to determine the variable involved with the manufacturing
and testing of the product. For the LIFECODES SSO assay, the variables involved are different
manufactured lots and testing operators. The following study was performed on HLA‐B (as a
representative for HLA Class I) and HLA‐DRB1 (as a representative for HLA Class II):
The study was carried out by 3 different operators using 3 different lots of HLA‐B and HLA‐DRB1 using 16
well characterized samples tested in duplicate. The test results are shown in Table 9.

Table 9
HLA-B HLA-DRB1
Test criteria
Concordance Failure Concordance Failure
Operator 1 (3 lots) 99% 0% 100% 1%
Operator 2 (3 lots) 100% 0% 100% 0%
Operator 3 (3 lots) 100% 0% 100% 0%
Lot 1 (3 operators) 99% 0% 100% 0%
Lot 2 (3 operators) 100% 0% 100% 1%
Lot 3 (3 operators) 100% 0% 100% 0%

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In addition to the above studies ‐ 80 LIFECODES QC samples were tested four times and a panel of 32
LIFECODES QC samples were tested two times. Testing was done on all 12 of the LIFECODES SSO typing
kits – A, A eRES, B, B eRES, C eRES, DRB1, DRB1 eRES, DRB345, DQA, DQB, DPB and Null. All kits yielded
comparable results to the studies described earlier.

External studies were performed at 4 sites with ~120 samples tested on A, B and DRB1 kits. All sites
yielded comparable results to the results from internal studies described earlier.

Conclusions
Rapid protocol for LIFECODES SSO kits was developed that maintained the current performance
characteristics, but had the following advantages:
1. Shorter time to result
2. Use of lesser DNA template
3. Equivalent kit performance with Rapid protocol

References
1. Abu Al‐Soud, W and Peter Rådström (1998) Capacity of nine thermostable DNA polymerases to
mediate DNA amplification in the presence of PCR‐inhibiting samples. Appl Environ Microbiol,
64: 3748‐3753
2. Akane, A. et al. (1994) Identification of the heme compound copurified with deoxyribonucleic
acid (DNA) from bloodstains,a major inhibitor of polymerase chain reaction (PCR) amplification.
J. Forensic Sci. 39, 362–72.
3. Dunckley H (2012) HLA typing by SSO and SSP methods. Methods Mol Biol. 882:9‐25.
4. Erlich H.(2012) HLA DNA typing: past, present, and future. Tissue Antigens. 80(1):1‐11.
5. CJ Clopper and ES Pearson (1934) The use of confidence or fiducial limits illustrated in the case
of the binomial. Biometrika 26:404‐413

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