Value of flowcytometry and PCR-SSP techniques in detection of HLA-B27

Journal of military pharmaco-medicine 7-2013

VALUE OF FLOWCYTOMETRY AND PCR-SSP TECHNIQUES
IN DETECTION OF HLA-B27
Le Xuan Hai*; Nguyen Dang Dung**
Summary
HLA-B27 has been demonstrated to be strongly associated with ankylosing spondylitis (AS), a
chronic inflammatory disease affecting the axial muscoskeletal system. Routine clinical tests detecting
HLA-B27 include cytotoxicity, immunofluorescent, and flowcytometry-based assays, among which the
later showed flowcytometry assays are the best advantages of specificity and rapidity in performance.
In recent years, however, application of PCR-based DNA typing resulted in a more reliable assay for
HLA-B27 detection.
In the present study, we detected HLA-B27 in blood samples of patients suspected to have AS, by
both flowcytometry-based and PCR-SSP-based assays, in order to assess the value of each method
in HLA-B27 detection. The results showed that:
- Flowcytometry-based assay can detect cell surface HLA-B27 expression when positive and
negative events got a clear cut over the cut-off level (the real positive and negative results).
- PCR-SSP-based assay is of much help in detecting HLA-B27 encoding gene when
flowcytometry-based assay was unable to determine the expression of HLA-B27 (the undetermined
results).
* Key words: HLA-B27; Flowcytometry; Polymerase chain reaction (PCR); Ankylosing spodylitis.

INTRODUCTION
HLA-B27 is a protein encoded by an allele of locus HLA-B, one of three loci within the
human class I major histocompatibility complex (MHC). It is well demonstrated that the
expression of HLA-B27 strongly associated with ankylosing spondylitis (AS), a chronic
inflammatory disease affecting the axial muscoskeletal system in human. According to
statistical data, about 1% of people with HLA-B27 positive would develop AS; however, 90 95% of patients with AS had HLA-B27 positive expression, while in patients without the
disease, this incidence was 5 - 8% only. The role of HLA-B27 in pathogenesis of AS has not
been completely understood. A recent study revealed that the HLA-B27 heavy chain had a
tendency to misfold during the assembly of class I complexes in the endoplasmic reticulum

(ER). In experimental HLA-B27 transgenic rats model, HLA-B27 misfolding generates ER
stress and leads to activation of the unfold protein response, which enhances the production
of interleukin (IL)-23 [2]. These findings were consistent with the discovery of IL-23R gene as
an additional susceptibility gene for AS in humans, and have led to new ideas about the role
of HLA-B27 in the AS pathogenesis. Thus, expression of HLA-B27 is considered an
important risk factor for the development of AS in humans. [1, 6, 7].
There are different techniques being adopted in detecting HLA-B27 expression, including
serological testing (based on lymphocytotoxicity test), immunofluorescent technique, or
flowcytometry [5]. Lymphocytotoxicity technique is simple to perform, but has some
limitations itself (low sensitivity and specificity; requirement of 90% or more of viable cells in
blood sample). Immunofluorescent technique improves the sensitivity in HLA-B27 detection,
and allows using sample with low cell viability; yet, the technique is manually performed,
which costs time and labor. Application of flowcytometry (FCM) using fluorescent-labeled
monoclonal antibody specific to HLA-B27 has overcome all disadvantages of techniques
mentioned above.
Recently, the development of DNA typing techniques based on PCR amplification of
target DNA copies allows the detection of HLA-B27 encoding gene with more reliable
results. [3, 8].
In this study, we detected the expression of HLA-B27 in blood samples from patients
suspected to have AS, by both FCM and PCR methods, in order to assess the value of each
method in HLA-B27 detecting assay.


Journal of military pharmaco-medicine 7-2013

MATERIALS AND METHODS
1. Subjects.
123 peripheral blood samples (with EDTA as anticoagulant) sent to Immunology
Laboratory, National Institute of Hematology and Blood Transfusion, for HLA-B27 detection,
during the period of Feb 2013 - April 2013.

2. Materials, reagents.
- Anti-HLA-B27 monoclonal antibody labeled with fluorescein isothiocyanate (anti HLAB27-FITC), and anti-CD3 monoclonal antibody labeled with phycoerythin (anti CD3-PE); red
blood cell lysis buffer (Becton Dickinson, USA)
- EZHighTM DNA Extraction Kit (Texas BioGene Inc, Taiwan).
- MorganTM HLA SSP B27 Typing Kit (Texas BioGene Inc., Taiwan).
3. Methods.
* Flowcytometry technique:
In this assay, we simultaneously utilized anti -HLA-B27- FITC and anti CD3- PE
antibodies for double staining, with steps as followed,
- Mixing 100 µl blood sample with 10 µl anti CD3-PE and 10 µl anti HLA-B27-FITC, then
incubating reaction tube at room temperature (RT) for 20 min, away from direct light.
- Lysing red blood cell (RBC) by adding 2 ml of RBC lysis buffer to the tube, keeping at
RT for 10 min, away from direct light.
- Analyzing the cell suspension on flowcytometer Cytomics FC500 (Beckman Coulter,
USA). Parameters designed for the analysis included: 1. SSC (side scatter) vs. FSC (forward
scatter) windows, for isolation of subpopulations of lymphocyte, neutrophil and monocyte in
the suspension, thus allowing selection of target lymphocyte subpopulation; 2. CD3-PE vs.
HLA-B27-FITC window, allowing analysis of cells double-stained with PE (CD3+ cells) and
FITC (HLA-B27+ cell). Combining parameters from 2 windows will bring in the events with
cell surface co-expression of CD3+ and HLA-B27+ on lymphocytes.
There are 3 possibilities for results of FCM analysis, which depends on density of cell
surface HLA-B27 as well as the fluorescent-labeled anti HLA-B27 antibody itself used in the
assay (see Figure 1)

Negative

Undetermined

Positive

Figure 1: Flowcytometry analysis of cell surface HLA-B27 on TCD3+ lymphocytes.
Polymerase chain reaction (PCR): utilizing PCR-SSP (sequence specific primer)
technique to detect HLA-B27 encoding gene, with steps as followed:
- Extracting DNA by conventional method, using EZHighTM DNA Extraction Kit (BioGene
Inc., Taiwan); total time of DNA extraction (for one sample) is about 30 min.


Journal of military pharmaco-medicine 7-2013
- Preparing for PCR-SSP: performed as guided by the provider of the reagents, MorganTM
HLA SSP B27 Typing Kit (total time for preparation is about 10 min).
- Running PCR cycles (including DNA denaturation, annealing and extension steps) with
temperature and time parameters as showed in Table 1.
Table 1: Time and temperature in PCR cycles.
STEPS

0

N
CYCLES

1

OF

TEMPERATU

TIME

960C

2,5 min

RE

1

0

2

10

96 C

15 sec

650C

60 sec

0

15 sec

0

62 C

50 sec

720C

30 sec

95 C
3
4

22

0

1

4C

Until finished

- PCR product electrophoresis: to detect copies of HLA-B27 encoding gene (previously
amplified by PCR), the product after PCR is electrophoresed on 2% agarose gel in 0.5X TBE
buffer (89 mM Tris Base; 89 nM boric acid; 2 mM EDTA, pH 8).
+ Melting agarose/TBE buffer by microwave (about 1 min), then leaving the gel to cool
down to 60 - 700 C (about 5 min). Adding ethidium bromide (0,5 µg/ml) to agarose gel, and
then casting the gel with 4 mm thickness, making the wells in gel with 3 mm width. Leaving
gel to harden for 40 min. Put the gel in electrophoresis tank, then pouring in the tank with
0.5X TBE buffer to 5mm depth (for the buffer to fully cover the gel surface).
+ Transferring PCR product into appropriate wells in the gel. Running electrophoresis at
10 V/cm for about 12 min. Visualizing the electrophoresis bands with a UV lamp, and
photographing the gel with visualized bands.
+ The results of PCR-SSP analysis can be expressed as either of the two forms, as

followed: 1. positive: a band showed in position specific for HLA-B27 allele; and, 2. negative:
no specific band showed.
RESULTS AND DISCUSSION
Results of FCM analysis to detect cell surface HLA-B27 on 123 blood samples were
shown in table 2.
Table 2: Results of FCM analysis to detect cell surface HLA-B27.
0

RESULT

N OF SAMPLES

INCIDENCE (%)

Negative

27

22.0

Undetermined

34

27.6

Positive

62

50.4

Total

123

100

In this FCM analysis to detect HLA-B27, we have set a cut-off value for the expression of
HLA-B27 on cell surface, as followed: samples with more than 85% of CD3+ cells locating in
HLA-B27+ area will be defined as HLA-B27 positive; samples with less than 15% of CD3+
cells in HLA-B27+ area will be defined as HLA-B27 negative; finally, samples with 15 - 85%
of CD3+ cells in HLA-B27+ area will be defined as undetermined (see Figure 1). [5]
Before the application of FCM technique, the detection of HLA-B27 was based on
cytotoxicity technique, using antibody specific to HLA-B27 in combination with rabbit
complement: cells with HLA-B27 on their surface will be bound by the specific antibody, and
then complement fixation will attack and get the cells die, making them stained with trypan


Journal of military pharmaco-medicine 7-2013
blue or eosin red – the dead cell staining reagents). The principle of the technique makes it
dependent on percentage of viable cells in the sample before testing: there should be at
least 90% of viable cell in the sample for the assay to be validated. Compared to this
technique, the immunofluorescent technique in general, and FCM technique in particular
(with the use of fluorescent-labeled anti HLA-B27 antibody), showed many advantages.
Especially, the result of FCM technique in detecting HLA-B27 is somewhat quantitative, and
independent from cell’s viability in the sample. Due to this advantage, FCM technique helps
reduce false positive (which is sometimes relatively high in cytotoxicity testing). However,
data in Table 2 showed that 34/123 tested sample (27.6%) were undetermined in terms of
HLA-B27 expression. The reason for this indetermination might be due to cross reactivity of

anti HLA-B27 antibody with other HLA-B antigens, such as HLA-B7 [4]. Other groups have
published similar findings about a certain rate of samples giving indetermination in HLA-B27
expression when analyzed by FCM [1, 4].
PCR-SSP allows detecting the expression of HLA-B27 at the level of encoding gene, and
thus, may help avoiding cross reactivity seen in cytotoxicity-based assays, or in assays
using labeled antibodies. In this study, in order to assess the ability in detecting HLA-B27
expression of FCM and PCR methods, we intentionally selected 30 samples out of 123
samples tested for HLA-B27 expression by FCM, among which 10 samples had positive
results, 10 had negative results, and 10 had undetermined results with cell surface HLA-B27
expression. Then we performed PCR-SSP using the primers pair specific for the HLA-B27
allele, to detect the HLA-B27 encoding gene in 30 selected samples. The results were
presented in Table 3 and 4:
Table 3: Results in detecting expression of HLA-B27 by PCR-SSP.
FCM

NEGATIVE

UNDETERMINED

POSITIVE

(< 15%)

(15 - 85%)

(> 85%)

Positive

0

4

10

14

Negative

10

6

0

16

Total

10

10

10

30

PCR-SSP

TOTAL

Table 4: Comparisons of the 2 techniques in HLA-B27 detection.
FCM

PCR-SSP

RESULTS

Positive (> 85%)

+

10/10

100

-

0/10

0

+

0/10

0

-

10/10

100

+

4/10

40

-

6/10

60

Negative (< 15%)
Undetermined (15 - 85%)

INCIDENCE %

Data in table 4 showed that 10/10 (100%) samples with HLA-B27 positive detected by
FCM also gave positive results for HLA-B27 allele when tested by PCR-SSP technique;
similarly, 10/10 (100%) samples with HLA-B27 negative by FCM had HLA-B27 allele
negative. However, PCR-SSP technique had detected HLA-B27 allele in 4/10 (40%)
samples undetermined of HLA-B27 expression by FCM; in addition, PCR-SSP technique
gave negative result for HLA-B27 allele in 6/10 remaining samples undetermined of HLA-


Journal of military pharmaco-medicine 7-2013

B27 by FCM technique (Figure 2).

Figure 2: Results of PCR-SSP product electrophoresis.
H2O: distilled water; neg: negative control (a sample without HLA-B27 by PCR-based HLA
typing); pos: positive control (a sample without HLA-B27 by PCR-based HLA typing); M:
DNA ladder markers. Samples giving HLA-B27 positive include: pos, 810, 870, 871, 902,
923, 934; samples with negative results: neg, 812, 924, 927.
It was revealed by these data that in cases of HLA-B27 indetermination by FCM
technique, the use of PCR-SSP technique proved to be a good option, which helped giving
clear results. In fact, PCR-based HLA typing assays (known as DNA typing) have been
replacing conventional HLA typing assays (the serological assays, using either HLA-specific
monoclonal or polyclonal antibodies), both nationally and internationally. Within the
framework of this study, the results once again highlighted the value of PCR-SSP technique
in HLA typing, this time with a specific allele, namely HLA-B27, making the assay reliable,
and thus, helpful in diagnosis of AS.
Table 5: Comparisons between FCM and PCR-SSP in their performance.
TECHNIQUE

FCM

Sample preparation
Analysis
analyzer

on

Ability to run single
test
Cost
Requirement

equipment

of

20 - 30 min (including RBC
lysis)
~2 min

PCR-SSP
2 hours (including DNA extraction, PCR
running time)
30 min (electrophoresis, interpretation of
results)

Easy, flexible

Inappropriate; batch assay preferable

~ 300.000 VND/test

~ 350.000 VND/test

Flowcytometry and popular lab
equipment

PCR
equipment
and
(electrophoresis system, UV lamp)

others

Table 5 showed several comparisons between the FCM and PCR-SSP methods in
detecting HLA-B27, in terms of assay performance. In general, FCM method is simple to
perform. FCM method is more flexible than PCR method, and FCM can be performed on
single sample without inconvenience. With less total testing time, FCM method gives faster
assay result than PCR method does. Taking all advantages of FCM method together, this
method can be used for HLA-B27 screening assay. Meanwhile, PCR-SSP technique, though
taking longer time for assay compared to that in FCM, can help avoiding indetermination in
HLA-B27 expression. Thus, PCR-based assay for HLA-B27 detection should only be used
when FCM-based assay gave undetermined result in HLA-B27 expression.


Journal of military pharmaco-medicine 7-2013

CONCLUSIONS
In clinical HLA-B27 detection assays,
- Flowcytometry-based assay can detect cell surface HLA-B27 expression when positive
and negative events got a clear cut over the cut-off level (the real positive and negative
results).
- Polymerase chain reaction-based assay is of help in detecting HLA-B27 encoding gene
when flowcytometry-based assay was unable to determine the expression of HLA-B27 (the
undetermined results).
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Levering WH, Wind H, Sintnicolaas K et al. Flow cytometric HLA-B27 screening: crossreactivity patterns of commercially available anti-HLA-B27 monoclonal antibodies with other
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