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From: Methods in Molecular Medicine, vol. 134: Bone Marrow and Stem Cell TransplantationEdited by: M. Beksac © Humana Press Inc., Totowa, NJ

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HLA Typing with Sequence-SpecificOligonucleotide Primed PCR (PCR-SSO)and Use of the Luminex™ Technology

Klara Dalva and Meral Beksac

SummaryThe hybridization products obtained by PCR using sequence-specific oligonucleo-

tides (PCR-SSO) can be traced either by colorimetric- (streptavidin- biotin), X-ray-(digoxigenin-CSPD), or fluorescence- (FITC, PE) based detection systems. To achieve afaster, reliable, automated typing, microbead and fluorescence detection technology havebeen combined and introduced to this field (XMAPTM technology). For each locus, amaximum of 100 microspheres, which are recognizable by their specific color originat-ing from two internal fluorescent dyes, are used. Each microsphere is coupled with asingle probe that is capable of hybridizing with the biotin labeled complementaryamplicon. Once hybridization occurs, it can be quantified via the fluorecence signal origi-nating from fluorescently (Streptavidin-PE) labeled amplicons captured by the beads.Currently, there are two commercially available systems that differ in the scale of probesand the method of amplification or denaturation. One of these will be described in detailin this chapter.

Key Words: PCR-SSO; LuminexTM; XMAPTM technology; HLA typing.

1. IntroductionThe first applications of PCR using sequence-specific oligonucleotides

(PCR-SSO) in the field of human leukocyte antigen (HLA) were started withthe DQA1 locus and followed by class I and other class II specificities (1–8).In order to eliminate ambiguities, protocols that use PCR primers designed toamplify the entire hypervariable region of a particular HLA locus (group-spe-cific primers) were introduced. These PCR products are incubated in the pres-ence of a panel of labeled oligonucleotides designed for the detection ofdifferent polymorphic positions specific for an allele or allelic group (8). The

62 Dalva and Beksac

primers for HLA-A, -B, and -C loci usually give a locus-specific product cov-ering exons 2 and 3. Primer for HLA-DR gives a product from exon 2 (1,7).The introductory chapter on molecular HLA typing authored by Ann-MargaretLittle (Chapter 3) summarizes the main features of PCR-SSO typing and com-pares it with the other methods. Here, we will describe one of the two auto-mated systems using LuminexTM technology, that is capable of typing ofmultiple inviduals simultaneously (9–11).

The xMAPTM technology developed by Luminex (Austin, TX) is a micro-sphere-based, multiplexed, flow cytometric analysis system that makes it pos-sible to combine hybridization with fluorescence detection (12,13). Classi-fication of HLA alleles by Luminex system was first described by Fulton et al.(14). The xMAP technology employs simultaneous applications of up to 100probes, which are already coated on microspheres. To be able to recognizethese probes, 100 shades of two colors that are formed by the ratio of twointernal fluorescent dyes are assigned for each probe. The fluoroanalyzer con-tains a red laser that excites the dyes in the microspheres and categorizes thembased on their dye content. The microspheres are coated with carboxyl groupsin order to achieve a covalent bridge between the oligonucleotides that containterminal amino groups and the beads. Thus, the bound oligonucleotides alsobecome color-coded. In addition to the red laser, the instrument contains agreen laser that is used to quantify fluorescently (Streptavidin-PE) labeledamplicons captured by the beads. Each probe mix contains one ore moreoligonucleotide(s) that react with all alleles within the locus of interest, whichalso serves as an internal control. They are used in the normalization of thevalues during the calculation of reaction patterns. If the minumum fluorescentintensity values defined for this control probes are not achieved, the sampletest must be repeated.

All probe-coated beads can be applied within a well. This unique featureenables one locus typing of 96 samples or more loci on smaller number ofsamples. Currently, there are two commercially available kits. The probe num-ber, specificity, and cutoff levels may vary between lots and commercialsources. A comparison of available probes presented by the two commerciallyavilable sources are presented in Table 1. In this chapter, we will focus on oneof these (Tepnel Life Codes).

2. Materials

2.1. Specimen

DNA is usually obtained from EDTA or ACD anticoagulated venous bloodsamples. Heparin interferes with Taq polymerase activity and should not beused as an anticoagulant. Final DNA concentration must be 10-200 ng/µL.

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2.2. DNA Extraction

DNA extraction is as described in Chapter 4.

2.3. PCR Amplification

1. Lifecodes HLA-SSO typing kits (HLA-A, -B, -C, -DRB, -DQB ). Each kit con-tains the master mix including 10X PCR buffer, 10X dNTPs, locus-specificbiotin-conjugated primers, and a fluorescently coded probe mix and a dilutionsolution (DS) necessary for the hybridization step.

2. Microcentrifuge.3. Vortex mixer.4. Recombinant Taq polymerase (5 U/µL).5. Nuclease-free water.6. Cooling block or melting ice.7. DNase/RNase-free tubes (0.5 mL).8. DNase/RNase-free thin-wall tubes (0.2 mL).9. PCR thermocycler(s): model with a 96-well format (e.g., Corbett Research CGI-

960, Perkin Elmer 9600 or 9700, MJResearch PTC200 PCH).10. Dispensing equipment: adjustable pipet(s), plastic tips.

2.4. Hybridization

1. Lifecodes HLA-SSO typing kits (HLA-A, -B, -C, DRB, -DQB ).2. Streptavidin-PE (SA-PE) (1 mg/mL, Lifecodes, cat no. 628511).3. Heating block.

Table 1Number of Probes for Human LeukocyteAntigen (HLA) Typing (January 2006)

Locus LabType® SSO Tepnel Life Codes

A 63 64B 100 89C 56 51DRB1 70 68DQA1 11 NADQB1 37 33DPB1 40 59Bw4 supp. 15 NAHLA-DRB(Generic, DR52) NA 82DRB3,4,5 29 NADQA1 11 NA

SSO, sequence-specific oligonucleotides; NA, not assigned.

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4. Ultrasonic bath (Branson).5. Vortex mixer with adjustable speed.6. PCR plates (thin-wall, 96-well, 250 µL/well).7. Polyethylene sealing tape.8. Dispensing equipment: adjustable pipette(s), plastic tips.

2.5. Data Collection

1. Luminex100 Instrument and XY Platform.2. Luminex 100 IS software, Quick Type for Lifematch 2.1 software.3. Sheath fluid.4. Daily maintenance reagents (70% ethanol, double-distilled [dd]H2O).

2.6. Analysis

Quick Type for Lifematch 2.1 software.

3. Method3.1. DNA Extraction

DNA extraction is as described in Chapter 4 (see Notes 1 and 2).

3.2. PCR Amplification

The Lifecodes kits utilize an assymetric PCR that increase the amount ofone primer approx 10 times and allow to generate single-stranded DNA prod-ucts in addition to the double-stranded products (see Note 3).

1. Stand the master mix to reach room temperature.2. Gently vortex for 10 s and centrifuge briefly to ensure salts are in solution (see

Note 4).3. Prepare the amplification mix sufficient for n + 1 samples (Table 2) (see Note 5).4. Pipet 10 µL of genomic DNA isolated by GenoPrepTM (approx 200 ng of DNA)

into the PCR tubes.

Table 2Reaction Components Necessaryfor Amplification (Except DNA)

Amount sufficient for 1 reactionReagent (Prepare for n + 1 reactions)

Master Mix (thawed to room temp.) 15.0 µLddH2O 24.5 µLTaq Polymerase (–20°C) 0.5 µL

Total 40.0 µL

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5. Dispence 40 µL of the amplification mix into the PCR tubes containing the DNAsample.

6. Close the tubes tightly and proceed with amplification.

3.3. PCR Cycling Conditions

Identical PCR cycling conditions are used for all applications (see Notes 6and 7).

1 cycle 94°C 2 min denaturation 1 cycle 95°C 5 min denaturation 8 cycles 95°C 30 s denaturation

60°C 45 s annealing72°C 45 s extention

32 cycles 95°C 30 s denaturation63°C 45 s annealing72°C 45 s extension

1 cycle 72°C 15 min extension

3.4. Hybridization

This technique excludes the prehybridization denaturation step. Single-stranded DNA is achieved by assymetric PCR and by the addition of a 5-minincubation at 97°C prior to the hybridization (see Notes 8 and 9).

Before starting with the hybridization turn on the Luminex100 instrumentand XY platform to allow warming of the laser at least for 30 min prior to theanalysis.

1. Warm probe mix in 57°C (55–60°C) heat block for 7 (5–10) min to obtain solu-bilization of the components thoroughly. Protect from light to avoid photo-bleaching, do not refreeze bead mixture after thawing, store at 2–8°C (see Note10).

2. In order to suspend the probe carrier beads, sonicate briefly for 15 s and thenvortex for another 15 s (see Note 10).

3. Pipet 5 µL of locus specific PCR product (amplicon) into a thermal cycler 96-well plate (Fig. 1).

4. Aliquot 15 µL of probe mix into each well. When aliquoting for more than 10samples, gently vortex the probe mix for every set of 10 (see Note 11).

5. Seal plate with polyethylene sealing tape (see Note 12).6. Hybridize the samples under the following conditions using a thermocycler:

97°C for 5 min47°C for 30 min56°C for 10 min56°C hold

7. While hybridization is in progress, dilute Streptavidin PE solution within the DS(dilution ratio: 1/200 for one sample 0.85 µL of SA-PE and 170 µL of DS). Pref-

66 Dalva and Beksac

erably prepare the solution for n + 1 samples. Because SA-PE is light-sensitive,keep the DS/SA-PE solution in the dark at room temperature. Do not reuse; dis-card any remaining solution (see Note 13).

8. While the tray is still on the thermocycler at the 56°C “hold” step, serially aliquot170 µL of SA-PE/DS mixture to each well including sample(s). It is critical todilute all of the samples within 5 min following the 10-min incubation at 56°C.

9. Remove the samples from the thermocycler and place in the Luminex100 instru-ment to analyze the samples.

10. Turn off the thermal cycler.

3.5. Analysis

Assay the samples immediatly using the prewarmed Luminex100 instrument.If immediate reading is not possible, protect samples from light. Samples canbe read within 30 min following the SA-PE/DS dilution.

Prior to analysis, set up a “batch run” by which the samples will be ana-lyzed.

3.6. Data Acquisition

The steps described next is a general guide for data acquisition. Daily start-up, calibration, maintenance, and shut-down procedures may be found in theUser’s Manual.

1. Turn on the Luminex100 30 min to 4 h before acquisition of the samples.2. Prior to acquisition, check the level of the sheath fluid, tighten the cap, empty the

waste tank, perform a “prime” and a wash with 70% ethanol, and proceed with awash using the sheath fluid.

Fig. 1. Sample of hybridization data sheet prepared for this application.

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3. Set up a “batch run” by which the samples will be analyzed. For each locus,follow the directions for the analysis.

4. Open Luminex100 IS software from desktop and open “submitted batch.” Followdirections. To analyze multiple loci in one plate, “Create Multi-batch” by addingbatches and click finish after naming the Multi-Batch.

5. Eject the plate holder and place the 96-well plate containing the samples in theXYP heater block present on the plate holder. Click the “retract” icon to start theacquisition.

6. Once the sample tray has been placed into the XY Platform of the Luminex100

instrument, click the “Start Plate” icon to start with acquisition.7. After running of the samples, perform a sanitization wash by rinsing two times

with 70% ethanol and perform “soak” with dH2O.8. Release the pressure on the sheath fluid tank.9. Turn off the instrument. Completed batches are exported automatically as

comma-separated values (csv) and are named as “output.csv” and saved in afolder with batch name defined on the third step. This data are available for theassignments of the results

3.7. Interpretation of Results

Interpretation of the results is performed by using the Quick Type for Life-match 2.1 software with which the opened csv files may be analyzed.

The steps described next are only a general guide to data analysis.1. Select “Final Typing Assignments.”2. Select file(s) to be analyzed.3. Verify the number of counted events to be greater than 60 for each probe includ-

ing the controls in each sample (this information presents in “data type” and countsection of csv files ).

4. The values for the concensus probes for each sample must exceed the minumummedian fluorescent intensity (MFI) (see Notes 4 and 7). These values can befound on the threshold tables supplied with each kit or can be loaded to the ana-lyzer by the technical services during updating process. The minumum thresh-olds are lot-specific.

5. For each probe, compare the normalized value with the threshold values suppliedwith the kits. The software makes this assignment automatically. However, thecurrent result must be validated before continuing to the next step.

Normalized values are calculated as:

MFI (probe) – MFI (control blank or probe)

MFFI (concensus) – MFI (control blank or conccensus)

6. If the measured value for a particular probe falls above the maximum thresholdof a negative assignment and below the minumum value of a positive assign-ment, the software evaluates this value as “undetermined” (see Note 14).

7. Save and print the results.

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4. Notes

1. Poor-quality, degraded, or insufficient amount of DNA may lead to reaction fail-ures. Check DNA quality and purity. Reisolate DNA.

2. A sample contamination may lead to failure to yield an HLA typing result.3. The differences between Lifecodes and LabType are: the amplification policy

(asymetric PCR vs regular PCR) and the availability of ready to use mixtures andthe amplification conditions; the number of probes are also currently different,and are summarized in Table 1.

4. Pipetting errors owing to poor homogenization of reagents (Master Mix) mayresult in reaction failures (low MFI for the control probes). This can be avoidedby prewarming the Master Mix at 37°C for 5 min.

5. Poor-quality Taq polymerase may result in reaction failures. Use only recombi-nant Taq.

6. Before proceeding to hybridization, check sample amplification by gel electro-phoresis.

7. PCR program errors, temperature failures, and poor contact with the PCR blockmay lead to multiple SSO failures. Perform gel electrophoresis to be sure ofsample amplification.

8. For LabType SSO, there is a separate 10-min denaturation/neutralization stepprior to hybridization that is applied by the addition of denaturation buffer andneutralization buffers, respectively.

9. For LabType SSO, the hybridization conditions are different. There are threewash steps prior to labeling with SA-PE and one wash step after labeling in orderto eliminate unbound probes.

10. A probe mix that is not suspended properly may lead to low bead counts and, asa result, interpretation failures.

11. Poor-quality tubes/pipet tips may lead to the adhesion of the beads to their sur-face and may cause too-low bead counts.

12. Evaporation during hybridization may lead to random failures in varioussamples within a batch or batches. In situations in which partial use of platesare planned, keep one row empty on each side of the samples to allow space fortight sealing.

13. Warm DS at 45°C for 5 min upon arrival. Before the labeling, warm the DS toroom temperature and vortex, store at room temperature until pipetting. DS/SA-PE mixture must be protected from light.

14. If more than two probes are indeterminate, repeat the assay. If less than two probesare indeterminate, analyze the sample with the probe as negative and repeat withthe probe as positive.

a. One of the choices provide a match. continue to the next step.b. Both choices provide matches, reassay the sample.c. Neither choices produce a match, check for the other possible incorrectly

assigned probes, check for an amplification failure, and repeat the assay.

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9. LifeMATCH Website (http://www.lifematchhla.com). Accessed June 2006.10. http://www.tepnel.com/life_codes/hla_testing_services.asp/11. One Lambda Inc. (http://www.onelambda.com). Accesed June 2006.12. Wu, Y. Y. and Csako, G. (2006) Rapid and/or high throughput genotyping for

human red blood cell, platelet,and leukocyte antigens, and forensic applications.Clin. Chimica Acta 363, 165–176.

13. Dunbar, A. S. (2006) Applications of Luminex® MAP technology for rapid, highthroughput multiplexed nucleic acid detection. Clinica Chimica Acta 363, 71–82.

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