HLA typing using saliva DNA collected with Oragene®
Traditionally, HLA registries and donor recruitment
centers collect DNA samples from volunteers
and potential donors through either a blood sample
or a buccal swab sample. Both of these sample
collection methods pose various challenges thatcan be eliminated using Oragene® to collect DNA
samples from saliva for both low resolution andhigh resolution HLA typing.
Saliva samples collected using Oragene provide
an easier, more reliable and stable method forcollecting DNA for all HLA typing technologies.
Unlike blood samples, Oragene allows volunteers andpotential donors to provide a saliva sample without
assistance, anywhere and at anytime. For example,a volunteer can provide a reliable sample at homeand mail the sample back through the regularpostal system. By eliminating the need for a trained
phlebotomist, the registry improves convenience
for the volunteer and dramatically reduces the costand complexity of the entire process. Saliva collection
simplifies the recruitment process and increases donor
participation, therefore maximizing the likelihoodof finding life-saving donor/recipient matches.
Oragene provides a superior DNA sample compared
to buccal swabs, with a collection protocol that is easyand painless. Oragene stabilizes DNA from saliva forat least 5 years at ambient temperatures allowing
registries a cost-effective banking option that ensures
integrity of the DNA sample without degradation.
Saliva samples collected with Oragene also produce
a high quantity and quality of DNA allowing theHLA typing center to use this sample type for highresolution confirmatory testing.

†In the current study we examined the performanceof Oragene samples using three technologies
commonly used for HLA typing: Sequence-SpecificOligonucleotide Probes (SSOP), Sequence BasedTyping (SBT), and Sequence Specific Primers (SSP).
In addition we investigated the robustness of samples
collected using the Oragene kits. Freshly collected
Oragene/saliva samples were compared to Oragene/saliva samples collected from the same individuals
under the conditions stated below:• 3 freeze/thaw cycles (-20°/+50°C)
or• Ambient temperature storage (~23°C) for 5 years.
The Oragene/saliva samples were purified using theprovided reagents and manual purification protocol
using prepIT®•L2P. The samples were collected,
stored, purified and coded by DNA Genotek Inc.
Purified DNA was shipped to a large US-based CLIAlab† (hereafter referred to as Lab A) for analysis on
the three platforms. The Lab A report describes thesuccessful HLA typing of Oragene/saliva samples
using the three methods described and demonstrates
that long-term storage or extreme temperaturefluctuations produce successful typing results on both
low and high resolution technologies as well as freshlycollected Oragene/saliva samples.

In accordance to confidentiality agreements reference to the third party lab responsible for the report has been removed.

For In Vitro Diagnostic Use
PD-WP-020 Issue 2/2012-05© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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For In Vitro Diagnostic Use

DNA Genotek sample study Oragene HLA validation
With input from DNA Genotek where specified 07/19/10

PurposeLAB A received DNA samples purified fromOragene collected saliva samples (DNA Genotek).
Three different variations of this sample type fromfive different donors were sent. LAB A also included
two in-house control donor samples in the formof buccal swabs. The purpose of this study was todetermine the viability of performing HLA testing onOragene collected saliva samples using the establishedtesting procedures of LAB A’s Testing Division.

Procedure
Samples from five different donors were sent. The fivedonors were labeled HOG1, HOG2, HOG3, HOG4,and HOG5. For each donor, three variations of theOragene collected sample type was received. All
sample type variations arrived as extracted DNA.
To these samples, LAB A added two internal buccal
swab controls; QC22 and QC13. Full sample listingis as follows:
SampleSample typeFirst, the two buccal controls were extracted using
Qiagen spin columns from Qiagen’s blood mini kit.
Once this extraction was complete, the DNA
concentrations of all samples were measured
using a NanoDrop spectrophotometer:

Initial concentrations
Sampleng/µL

260/280

HOG1A

137.30

1.73

HOG1B

171.14

1.85

HOG1C

209.11

1.93

HOG2A

143.80

1.67

HOG2B

52.41

1.81

HOG2C

134.89

1.71

HOG3A

180.38

1.89

HOG3B

110.85

1.85

HOG3C

226.34

1.88

HOG4A

143.661.76

HOG4B

106.90

1.85

HOG4C

207.06

1.83

HOG5A

219.69

1.82

HOG5B

185.04

1.92

HOG’#’A

Oragene sample

HOG’#’B

Oragene sample stored at room
temperature for over 5 years

HOG’#’C

Oragene sample subjected to 3 freeze(-20°C) / thaw (50°C) cycles

QC22

Buccal control

HOG5C

349.53

1.89

QC13

Buccal control

QC22

14.36

1.69

Note: Information in this table provided by DNA Genotek.

QC13

13.03

1.81

PD-WP-020 Issue 2/2012-05© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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2For In Vitro Diagnostic Use

HLA testing was performed on all samples using
three different methods. For all methods, the HLA-A,
HLA-B, and HLA-DRB1 regions were typed. First,
SSOP testing was performed using LAB A’s in-housemethodology and established procedures. Next, SBT
was performed. Sequencing was done for all threeloci using kits manufactured by Celera Diagnostics,
and distributed by Abbott Molecular. EPG’s weregenerated using ABI genetic analyzers and readusing Assign 3.5+ software from Conexio Genomics.
Finally, SSP testing was performed on all samples
for a single HLA-B allele using Invitrogen SSP kits.
All results were based on the IMGT 2.24 database.

Results from all testing methods were compiled
and checked for concordance. Finally, raw data wasanalyzed for quality differences between the threevariations of the Oragene collected saliva samples.

Results
After SSOP testing was completed, all typings wererecorded and checked for concordance between
the sample types of the different donors. The SSOP
typings are as follows:

SSOP resultsSample

HLA-A

HLA-A

HLA-B

HLA-B

HLA-DRB1

HLA-DRB1

HOG1A

02:XX

23:XX

39:XX

49:0101

11:01

11:04

HOG1B

02:XX

23:XX

39:XX

49:0101

11:01

11:04

HOG1C

02:XX

23:XX

39:XX

49:0101

11:01

11:04

HOG2A

24:XX

68:XX

07:XX

27:XX

03:01

15:01

HOG2B

24:XX

68:XX

07:XX

27:XX

03:01

15:01

HOG2C

24:XX

68:XX

07:XX

27:XX

03:01

15:01

HOG3A

01:XX

02:XX

08:XX

39:XX

01:01

03:01

HOG3B

01:XX

02:XX

08:XX

39:XX

01:01

03:01

HOG3C

01:XX

02:XX

08:XX

39:XX

01:01

03:01

HOG4A

02:XX

–15:XX

44:XX

04:01

12:DUKV

HOG4B02:XX

–

15:XX

44:XX

04:01

12:DUKV

HOG4C

02:XX

–

15:XX

44:XX

04:01

12:DUKV

HOG5A

03:XX

33:XX

07:XX

14:XX

01:02

15:01

HOG5B

03:XX

33:XX

07:XX

14:XX

01:02

15:01

HOG5C

03:XX

33:XX

07:XX

14:XX

01:02

15:01

QC22

02:XX

–

44:XX

57:XX

07:01

11:01

QC13

02:XX

03:XX

07:XX

27:XX

08:01

15:01

PD-WP-020 Issue 2/2012-05© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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3For In Vitro Diagnostic Use

Data quality for all samples was very good. The dot
intensity from positive reactions was clear for all
Oragene collected saliva samples. Typings wereobtained for all samples.

Once SBT testing was completed, all typings wererecorded and checked for concordance between thesample types of the different donors. The SBT typings
are as follows:

SBT results
Sample

HLA-A

HLA-A

HLA-B

HLA-B

HLA-DRB1

HLA-DRB1

HOG1A

02:CEZE

23:BRXU

39:DAUV

49:BAXH

11:BCP

11:04

HOG1B

02:CEZE

23:BRXU

39:DAUV

49:BAXH

11:BCP

11:04

HOG1C

02:CEZE

23:BRXU

39:DAUV

49:BAXH

11:BCP

11:04

HOG2A

24:BSMU

68:BDDJ

07:ANVB

27:EKN

03:01

15:01

HOG2B

24:BSMU

68:BDDJ

07:ANVB

27:EKN

03:01

15:01

HOG2C

24:BSMU

68:BDDJ

07:ANVB

27:EKN

03:01

15:01

39:BZDK†

01:AH

03:01

HOG3A

01:CENZ

02:CCCZ

08:TM†

HOG3B

01:CENZ

02:CCCZ

08:01

39:BMFM

01:AH

03:01

HOG3C

01:CENZ

02:CCCZ

No typeNo type

01:AH

03:01

HOG4A

02:ANGA–

15:CTFG

44:CTFF

04:01

12:DUKV

HOG4B

02:ANGA–

15:CTFG

44:CTFF

04:01

12:DUKV

HOG4C

02:ANGA–

15:CTFG

44:CTFF

04:01

12:DUKV

HOG5A

03:XKS

33:01

07:ANVB

14:02

01:02

15:01

HOG5B

03:XKS

33:01

07:ANVB

14:02

01:02

15:01

HOG5C

03:XKS

33:01

07:ANVB

14:02

01:02

15:01

QC22

02:ANGA–

44:AMUT

57:01

07:01

11:BCP

QC13

02:ANGA

03:XKS

07:DEZW

27:EHNR

08:01

15:01

† HARP failed.

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4For In Vitro Diagnostic Use

For the HLA-A and HLA-B loci, exons 2, 3, and 4
were sequenced in the forward and reverse direction.
For the DRB1 region, exon 2 was sequenced in theforward and reverse direction. For some donors,Heterozygous Ambiguity Resolution Primers(HARPs) were used to sequence a single allele insamples with cis/trans ambiguities. The data quality
for all reactions could best be expressed throughassigned base call scores (BCS) for each sample.
The Assign software gives each sample and HARPa BCS based on the average signal intensity, signal
to noise ratio, alignment, and peak characteristics.
These scores are between 0 and 100 with 100 being
the best quality. The BCS of each sample andcorresponding HARPs are as follows:

SBT BCSSample

HLA-A

HARP

HLA-B

HOG1A

87.413625

43.176211

85.261557

85.590717

42.53158

HOG1B

86.540146

42.709251

80.976886

81.223629

42.52105

HOG1C

86.951338

43.797357

84.380779

85.945148

42.04211

HOG2A

87.420925

85.97201943.165217

79.662447

84.763713

HOG2B

87.087591

86.006083

43.130435

79.126582

80.64557

HOG2C

87.519465

85.07056

42.843478

86.059072

86.092827

HOG3A

86.051095

83.690998

081.485232

85.227848

HOG3B

86.332117

84.913625

29.725664

82.160338

85.118143

HOG3C

86.53163

16.305147

084.616034

87.172996

HOG4A

89.190998

68.391727

30.360947

87.56962

81.860759

HOG4B

89.473236

86.104623

42.805882

81.320675

86.008439

HOG4C

89.20073

86.187348

42.882353

80.400844

85.303797

HOG5A

86.541363

84.585158

42.327511

86.151899

82.835443

HOG5B

87.153285

83.718978

41.148472

79.64135

84.493671

HOG5C

86.80292

81.363747

37.495652

85.021097

85.755274

QC2288.875912

80.998783

85.409283

83.417722

QC1385.675182

85.762774

71.85654

85.561181

43.806167

PD-WP-020 Issue 2/2012-05© 2012 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

HARP

HLA-DRB1

Group 2

HARP

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5For In Vitro Diagnostic Use

BCS for most samples are well within the normalrange. Please note however, that HARP scores werealways lower due to being sequenced in only a single
direction. The HARP for HOG3A and HOG3C failed.
Also, the general sequence for HOG3C failed.
A single HLA-B SSP kit was run to type a single
HLA-B allele for each sample. Once testing wascompleted, all typings were recorded and checked forconcordance between the sample types of the different
donors and controls. The SSP typings are as follows:

SSP results
Sample

HLA-B

HOG1A

39:01

HOG1B

39:01

HOG1C

39:01

HOG2A

07:02

HOG2B

07:02/07:44†

HOG2C

07:02

HOG3A

39:01

HOG3B39:01

HOG3C

39:01

HOG4A

44:02

HOG4B

44:02

HOG4C

44:02

HOG5A

07:02

HOG5B

07:02

HOG5C

07:02

QC22

No type

QC13

07:02

† Single lane failure, unrelated to sample quality.

Data quality was very good for nearly all samples.
This assessment was based on the presence andintensity of all positives and all positive control
bands. Also taken into account was the absenceof bands from any non-specific priming. Eight lanesor positive control bands failed for QC22 and 1 failedfor HOG2B. Aside from these failures, band intensityand presence for all positives and positive control
bands was excellent for all samples. For every sample,
there was little or no evidence of bands showing
non-specific priming.

Conclusions
After compiling all data, result concordance waschecked. Results for each donor with all sample
type variations were concordant across all three tests.
Both LAB A internal QC samples matched expectedtypings for all tests.
The SSOP data quality was very good for all
variations of the Oragene collected saliva samples.
The SBT data quality was very good for all sample
variations of HOG1, 2, 4, and 5. For HOG3A andHOG3C, the HARP reaction failed. Furthermore,HOG3C failed completely while HOG3B worked
well for the general sequence as well as the HARP.
The reason for these failures could not be easily
determined. More testing would be needed todetermine the root cause. For the purpose ofthis study, it is important to note that the data
quality matched between all variations of donors
HOG1, 2, 4, and 5.
The SSP data quality was excellent for all sample
variations of the Oragene collected saliva samples.
One lane failed for variation B for donor HOG2
causing the inclusion of one additional allele
possibility, however, this is a very common
occurrence with SSP testing. Data quality between
variations matched one another, and was equal toor better than that of the LAB A controls. This isbest exhibited by the fact that LAB A QC22 failedfor this test.
Based on these results, the extracted DNA ofthe three tested variations of Oragene collectedsaliva samples, Oragene/saliva is viable for HLA
testing using the procedures established at LAB A’s
Testing Division.

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®Oragene and prepIT are registered trademarks of DNA Genotek Inc. All other brands and names contained herein are the property of their respective owners.
All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

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