Discrimination of HLA-B27 alleles by group-specific amplification followed by solid-phase sequencing

Blasczyk, Rainer; Weber, M.; Lessen, Antje van; Schwella, N.; Huhn, D.; Salama, Abdulgabar

doi:10.1016/0198-8859(95)00147-6

Cited by 14 publications

(15 citation statements)

References 18 publications

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“…Sequence‐based typing methods have been described for HLA‐B in general and for B*27 in particular (19, 21, 40–44) . Although in most approaches primers are used that amplify all HLA‐B alleles, in some of them the sequence differences found in the three new HLA‐B*27 alleles could interfere with correct amplification.…”

Section: Discussionmentioning

confidence: 99%

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B*27 in molecular diagnostics: Impact of new alleles and polymorphism outside exons 2 and 3

2002

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HLA-B*27 is known to be associated with ankylosing spondylitis and several methods have been applied to determine its presence or absence. In this report two molecular methods were used for detection of B*27. The polymerase chain reaction sequence-specific primer (PCR-SSP) method was performed to detect the presence or absence of B*27, whereas the sequence-based typing method (SBT) was used to identify the B*27 subtype. The PCR-SSP method used to detect B*27 was updated to enable the detection of all B*27 alleles. The typing results obtained by this method were compared with the serological typings of 262 individuals. Fifty of them were found to be B*27 positive by PCR-SSP and 46 also showed positive serological reactions with B27-specific sera. The four discrepancies were the result of the presence of B*2712 in three individuals and B*2715 in one individual; both alleles showed no serological reactions with B27-specific antisera. With SBT the sequences of exons 1 through 4 were determined to unequivocally assign the B*27 alleles. Eleven different subtypes were detected in 78 individuals, including three new B*27 alleles: B*27054, B*2715 and B*2717. The allele B*27054 showed an allelic drop out when exon 3 was amplified. Three differences with B*27052 were demonstrated; one in exon 1, one in intron 1 and one in intron 2, the latter being responsible for the allelic drop out. The B*2715 allele was serologically not detectable with several B27-specific sera, but showed Bw4-positive reactions. The sequence of B*2715 showed two mismatches with B*2704. The sequence of B*2717 showed one mismatch with B*27052 at position 248 (A-->T), which was considered to be a conserved position in all B alleles.

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Section: Discussionmentioning

confidence: 99%

“…Separate amplification reduces the chance of a complete allelic drop out in both amplifications. In the B*27 sequencing approach, used to discriminate the different B*27 alleles (19), primer MW09 is used as the 3′primer for group‐specific amplification. This primer is located at positions 559–576 in exon 3.…”

Section: Discussionmentioning

confidence: 99%

B*27 in molecular diagnostics: Impact of new alleles and polymorphism outside exons 2 and 3

2002

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“…The typing of patients with low B27 antigen expression may become problematic if the cutoff between B27 negative and B27 positive samples is high. Hence, a major advantage of the method of CD3 gating and MESF quantitation is that this gating method may minimize the number of indeterminate cases, thus saving the cost and delay in turnaround time with the requisite confirmation testing by HLA-ABC family studies, nucleotide sequencing of B27 (24), and PCR-SO typing (25).…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric HLA-B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation

et al. 1996

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The determination of HLA‐B27 (B27) status is important in the diagnosis of ankylosing spondylitis, Reiter's disease, and other arthropathies. Flow cytometric (FCM) typing of B27 is a relatively new method which allows for rapid turnaround time and low cost. However, different leukocyte populations may manifest significant variation in staining intensity with B27 antibodies. Therefore, gating utilizing only light scatter properties of cells may lead to high readings in some B27‐negative samples. Fluorescence gating on CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore, quantitative FCM measurements, as afforded through use of molecules of equivalent soluble fluorochrome (MESF) units, can minimize day‐to‐day and instrument‐to‐instrument variabilities in fluorescence measurements. We compared CD3 gating to light scatter gating and MESF analysis on 123 specimens in a 4‐month period and found: (1) CD3 gating gave the lowest nonspecific B27 antibody binding among B27‐negative subjects; (2) there was no significant difference in MESF values between CD3 gating and light scatter gating of B27‐positive samples; (3) there was a wide range of B27 antibody binding fluorescence intensities among B27‐positive subjects; (4) identification of patients with low B27 expression may require the use of CD3 gating in order to avoid costly confirmation testing; (5) fluorescent standard beads are stable for routine use in a clinical flow cytometry laboratory. © 1996 Wiley‐Liss, Inc.

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“…For automated Sanger-sequencing there are different methods available, which in turn vary significantly with respect to the effort required to carry them out and the accuracy of the results obtained. When using a one-lane sequencer (PE Applied Biosystems) the work load can be reduced further by using terminators with different fluorescent labels (dye terminator technique) [163,167,170,177]. With the new enzymes AmpliTaq FS (PE Applied Biosystems) or Thermo Sequenase (Amersham Pharmacia Biotech), almost identical results are obtained but is less time consuming (cycle sequencing) [175].…”

Section: Sequencing-based Typing (Pcr-sbt)mentioning

confidence: 99%

New Diagnostic Methods in Oncology and Hematology

Huhn¹

1998

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Discrimination of HLA-B27 alleles by group-specific amplification followed by solid-phase sequencing

Cited by 14 publications

References 18 publications

B*27 in molecular diagnostics: Impact of new alleles and polymorphism outside exons 2 and 3

B*27 in molecular diagnostics: Impact of new alleles and polymorphism outside exons 2 and 3

Flow cytometric HLA-B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation

New Diagnostic Methods in Oncology and Hematology

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