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Hemo ID™ Blood Group Genotyping Panel DNA-based extended antigen typing of erythrocytes, platelets, and neutrophils

Hemo ID Panel at a Glance

The Hemo ID Blood Group Genotyping Panel (Hemo ID Panel) is designed for use with the MassARRAY® System for the identification of key polymorphisms in erythrocyte, platelet, and neutrophil antigens, starting with genomic DNA. With the Hemo ID Panel, you can:

  • Interrogate 167 polymorphisms in 101 antigens (16 blood group systems) and 23 platelet and neutrophil antigens.
  • Perform just the analyses of interest and minimize time and material wastage. 
  • Select any individual Hemo ID Module for focused studies or use the complete Hemo ID Panel for comprehensive analyses. 
    • Obtain predicted phenotypes for up to 3000 samples in 6-8 hours. 
    • Be confident in the accuracy and precision of your results generated with the MassARRAY System2,3 a genetic analysis platform referenced in more than 2,400 scientific publications.
  • Analyze complex Rhesus genotypes and identify variants with assays for RHD zygosity, Del, weak and partial RHD alleles, and RHD-RHCE hybrid alleles.
  • View and print your results with intuitive reports in multiple formats, which allow you to drill down to more detailed data and to select your favorite phenotype designations. (ISBT genotype and phenotype are used as default references when available.)
    • Reports include nine RHD zygosity assay plots to support user assignment of zero, one, or two copies of RHD.
  • Design and run your own additional genotyping assays for blood group variants of interest in your local ethnic population using the on-line Assay Design Suite Software.  
    • Start your assay designs with just the dbSNP rs numbers, or dbSNP data in FASTA format for the SNP positions of interest.
    • The data generated from additional multiplex wells is aggregated and reported with your Hemo ID Panel assay results.

Product Overview 

Hemo ID Blood Group Genotyping Panel
ChemistryiPLEX Pro
Sample TypeGenomic DNA, isolated from whole blood or other tissue
Sample Amount20 ng gDNA/multiplex assay well
Samples per 96W plate1 - 96 samples (depends on the number of multiplex wells in the Module)
Samples per 384W plate1 - 384 samples (depends on the number of multiplex wells in the Module)
No. of wells1-10 wells (depends on Module)
Analytical Sensitivity>95% genotype call rate in internal tests with 20 ng of gDNA per well

 

 

Module Descriptions and Blood Group Systems Assayed

Predicted phenotypes assayed by the Hemo ID Blood Group Genotyping Panel. The Panel contains six Modules, which you can purchase individually or together depending upon your research needs.  Click on the Module name in the first column to view the SNP positions interrogated with each assay. 

Module Name (No. of multiplex reactions)Predicted Phenotypes Assayed
Hemo ID Blood Group Genotyping Panel

Ten multiplex wells
Includes all six Hemo ID Modules for a comprehensive analysis of 16 blood group systems and 23 platelet and neutrophil antigens.
Hemo ID Kell, Kidd, Duffy Module

Single multiplex well
Kell: K/k, Kmod (2 variants), Kpa/Kpb, Jsa/Jsb, KEL11/KEL17, Knull or K0 (7 variants)

Kidd: Jka/Jkb, Jk(a–b–) or Jk0 (3 variants)

Duffy: Fya/Fyb, Fy(a−b−) or Fy0 (GATA), Fy(b+w) or Fyx+
Hemo ID MNS Module

Single multiplex well
M/N, S/s, Mta, Vw, Hut, IVS5 or P2
Hemo ID Rare Blood Groups Module

Two multiplex wells
Kell: K/k, Kpa/Kpb, Jsa/Jsb, KEL11/KEL17

Other rare blood groups: Lua/Lub, Lu8/Lu14, Aua/Aub, Dia/Dib, Wra/Wrb, Yta/Ytb, Coa/Cob, Kna/Knb, McCa/McCb, Sla/Vil, Doa/Dob, Hy, Joa, LWa/LWb, Sc1/Sc2, Cra, Tca/Tcb/Tcc, Ina/Inb, Vel+/Vel–, Vel+weak (See Ref. 1)
Hemo ID RHD-RHCE Broad Module

Three multiplex wells
Rhesus status: DD vs. Dd vs. dd , CC vs. Cc vs. cc, EE vs. Ee vs. ee, RHCE vs. RHD exon screening [promoter, intron 1, exon 3, 4, 5, 6, 7 (2x), intron 7, exon 9 (2x), exon 10], RHc at RHCE*201A, RHCw

RHD weak type: Type 41, Type 66

RHD category & D– C+: cat DIII 4, cat DIVa, cat DIVa type, cat III 4-8 or DCes type 1, cat IIIsub or DCes type 1, Dces type 1 & 2 @ RHD-CE*1006T

D–: RHD(W16X), RHD(Y401X), RHD psi, RHD psi IVS3-19 dupl 37, RHD(IVS3+2T>A) null

RHDel: RHD(delA147), RHD(IVS3+1G>A)el, RHD(X418L)el D– (CE): RHD-CE(2-9)-D+203C, RHD-CE(2-9)-D-2+268A

Other Hybrids: RHD-CE(1-9)-D, RHD-CE(2-9)-D or RHD-CE(3-9)-D, RHD-CE(2-7)-D or RHD-CE(3-7)-D, RHD-CE(4-7)-D, RHCE-Ce-D(6-10); RHD-D-CE(3)-D, RHD-D-CE(3-5)-D, RHD-D-CE(3-6)-D, RHD-D-CE(4)-D, RHD-D-CE(4-5)-D, RHD-D-CE(4-6)-D, RHD-D-CE(5)-D, RHD-D-CE(5-9)-D, RHD-D-CE(6-9)-D, RHD-D-CE(7)-D, RHD-D-CE(7-9)-D, RHD-D-CE(1048C, 8-9)-D; RHCE-D(1-3)-CE, RHCE-ce-D(4-9)-ce, RHCE-Ce-D(4)-ce, RHCE-Ce-D(4)-ce + 455A, RHCE-CE-D(5)-CE, RHCE-ce-D(5)-ce
Hemo ID RHD Variant Module

Two multiplex wells
RHD weak type: Type 1, Type 1.1, Type 2, Type 3, Type 4.0, Type 4.1, Type 4.2 or DAR1, Type 4.3, Type 5, Type 11, Type 15, Type 17, Type 34, Type 47

RHD category: cat DIII 6, cat DIII 7, cat DV 5, cat DVI1-DVI4, cat DVII

Other partial RHD: DAU1-DAU7, DFL, DFR1-DFR4, DHK, DNB, DOL1 or DAK, DOL3, DVL-2

RHDel: RHD(K409K)el

Normal RHD: DAU0

RHCE variants: RHCE ce(G385A), RHCE CeMA
Hemo ID HPA/HNA Module 

 Single multiplex well
HNA: HNA-1a/b/ab/ac/bc /c, HNA-3a/b, HNA-4a/bw, HNA-5a/bw

HPA ( See Ref. 4 ): HPA-1a/b, HPA-2a/b, HPA-3a/b, HPA-4a/b, HPA-5a/b, HPA-6a/bw, HPA-15a/b

 

 

Running the Hemo ID Panel on the MassARRAY System

 The process time from sample to results starting with purified DNA is 8 hours (depending upon the speed of thermo cycling). Hands-on time is approximately 60 minutes without liquid handling system.

Step 1: Order Hemo ID Panel or Hemo ID Modules

The Hemo ID Blood Group Genotyping Panel and the individual Hemo ID Modules contain all the required components for the analysis of either 192 or 768 DNA samples. The contents include multiplex PCR Primer Mixes, Extension Primer Mixes, PCR Accessory Set, PCR Enzyme, iPLEX Pro Reagent Set and SpectroCHIP II and Resin Kit. See ordering information below.

Step 2: Prepare & Run Sample

Start with 10 ng of DNA per multiplexed assay well, purified using any standard method. (See ordering information for the number of wells used by each Hemo ID Module.) Following locus-specific multiplexed PCR to amplify targeted regions of genomic DNA, shrimp alkaline phosphatase (SAP) is added to dephosphorylate any remaining nucleotides. Next, oligonucleotide primers, designed to anneal immediately upstream of each polymorphic site of interest, are added together with a polymerase and terminating nucleotides. The polymerase extends each oligonucleotide primer into the polymorphic site by adding a single complementary base. 

Step 3: Transfer Analyte to SpectroCHIP Array

The Extension products (analyte) are desalted and approximately 10 nL transferred using the MassARRAY Nanodispenser to SpectroCHIP® (silicon supports pre-spotted with matrix crystals that facilitate mass spectrometry). 

Step 4: Acquire Data

SpectroCHIP Arrays loaded with analyte are inserted into the MassARRAY Analyzer 4 MALDI-TOF mass spectrometer. The Extension analyte/matrix co-crystals are irradiated by a laser, inducing desorption and ionization. The positively charged molecules accelerate into a flight tube towards a detector. The time of arrival at the detector is proportional to the mass of the individual molecules. After data processing, a spectrum is produced with relative intensity on the y-axis and mass on the x-axis.

Step 5: View Genotypes and Predicted Phenotypes

After spectra are obtained with the MassARRAY System, the Typer Software generates customizable, interactive Hemo ID Reports that can be viewed in a web browser and easily printed. The data sets are also available as digital files that can be transferred to LIMS systems using standard procedures. 

 

Results Reporting

The Hemo ID Panel reporting software provides a Summary Report (Figure 1), listing the plates analyzed and the total number of samples in the batch. Next the software identifies and lists disqualified samples, discordant samples, and samples in wells generating low quality data (poor signal-to-noise ratio or poor extension rate). Users can dynamically view or hide different parts of the report and can drill down to individual Module Reports and Sample Reports. 

Navigate through a full example Hemo ID Panel Report produced from the analysis of a batch of 47 commercially available HapMap gDNA samples with the Hemo ID Panel.

Figure 1. Hemo ID Panel Summary Report

 

Figure 2. Hemo ID Panel Module Report for the Hemo ID Kell/Kidd/Duffy Module

Data for the report below was generated from the analysis of 47 HapMap samples. The three tables at the top show predicted phenotypes and frequencies for each blood group system in the sample batch, and the lower table shows the individual predicted phenotypes for each sample within the sample batch.

Navigate through a full example Hemo ID Panel Report 

 

RHD Zygosity Analysis

 The Hemo ID RHD-RHCE Broad Module contains nine RHD copy number assays, which provide users with information for RHD zygosity determination. When a large population of samples is analyzed with one of the assays, as shown in  Figure 3 below, three distributions are evident, corresponding to samples predicted to have zero, one, or two copies of RHD

 Figure 3. RHD copy number analysis of 760 samples assayed using assay RHDCE Exon 4*514

The orange line shows the position of a single sample that appears to have one copy of RHD, and thus a predicted phenotype of RhDd.

 Figure 4. RHD copy number analysis of a single sample with all 9 zygosity analysis assays.

The robustness of the RHD zygosity analysis in the Hemo ID RHD-RHCE Broad Module is enhanced with the use of nine different assays that probe single nucleotide differences between the RHD and RHCE genes. Each histogram below displays the data generated from a specific assay for all 47 samples in one experiment. The orange bar in each plot indicates the position of the sample of interest in the background of all the samples analyzed with that assay. The analysis appears to indicate that the sample most likely contains 1 copy of the RHD gene. 

 

 

Designing and Running Custom Assays for other Alleles or Blood Group Systems

You can design additional genotyping assays for blood group variants prevalent in your research population using the on-line Assay Design Suite software (or contract the work to the Assays by Agena Bioscience team for custom designs.) Start your designs with just the dbSNP rs numbers, or dbSNP data in FASTA format for the SNP positions of interest. Order the suggested PCR and extension primers from your favorite vendor and perform the additional SNP assays using iPLEX Pro reagent sets from Agena Bioscience. The data generated from the additional custom assay designs is easily aggregated to any Hemo ID Panel assays performed at the same time.

 

Avoiding Sample Mix-Ups

 It is estimated that ~2% of samples stored in biobanks and other repositories are incorrectly annotated. Use Agena Bioscience’s iPLEX Pro Sample ID Panel to test DNA extracted from blood, cell lines, and/or tissues in order to track samples, identify sample mismatches and/or sample duplication, and quantify input DNA prior to further genotyping assays.

 The Sample ID Panel is an ideal research tool for qualifying samples prior to PCR-based assay methods on the MassARRAY® System or next generation sequencing platforms, and provides:

  • A high degree of discriminatory power – greater than 1.0 x 1018 with comprehensive SNP coverage
  • Estimated number of amplifiable DNA copies  
  • An effective single-well assay requiring ≤ 10 ng gDNA per sample.

For more information visit the iPLEX® Pro Sample ID Panel product page.

 

Ordering Information

 Each Hemo ID Blood Group Genotyping Panel Kit includes: 

Reagent sets are available in 96-well and 384-well formats, and are designed to be used with the MassARRAY® System with Typer Application Software v4.0.119 or higher.

Ordering Information: 

Product NameNumber of Multiplex Wells RequiredCatalog Number
(Sufficient for 192 Samples)
Catalog Number
(Sufficient for 768 Samples)
Hemo ID Blood Group Genotyping Panel10196002196000
Hemo ID Module – Kell/Kidd/Duffy1196012196010
Hemo ID Module – MNS1196022196020
Hemo ID Module – Rare Blood Groups2196032196030
Hemo ID Module – RHD-RHCE Broad3196042196040
Hemo ID Module – RHD Variant2196052196050
Hemo ID Module – HPA & HNA1196062196060
Hemo ID Bundle – KKD/MNS/Rare BG4196072196070
Hemo ID Bundle – RHD-RHCE Broad & Variant5196082196080

 

 

 

 

 

Custom Services

 The Assays by Agena Bioscience Custom Services team provides expert services that can extend your laboratory’s capabilities by designing additional blood group genotyping assays to meet your specific needs. Contact Assays by Agena Bioscience for more information.

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References

1. Storry, JR, Jöud, M, Christophersen, MK, Thuresson, B., et al. Homozygosity for a null allele of SMIM1 defines the Vel-negative blood group phenotype. Nat Genet. 2013; 45(5):537-541.

2. Gassner, C., Meyer, S., Frey, BM., Vollmert, C. Matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry-based blood group genotyping--the alternative approach. Transfus Med Rev. 2013;27(1):2-9.

3. Meyer, S., Vollmert, C., Trost, N., Brönnimann, C., Gottschalk, J., Andreas, B.,. Frey, BM., and Gassner, C. High-throughput Kell, Kidd, and Duffy matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry–based blood group genotyping of 4000 donors shows close to full concordance with serotyping and detects new alleles. Transfusion, 2014 (on-line).

4. Technology for platelet polymorphism typing is made available under a license from BloodCenter Research Foundation and BloodCenter of Wisconsin. US Patent Nos. 5,652,357; 5,972,601; 5,780,229; 5,670,337.

Note: The MassARRAY System, MassARRAY Analyzer 4, Hemo ID Blood Group Genotyping Panel, iPLEX Gold, iSEQ, QGE, EpiTYPER, TYPER software, MelaCarta Panel, OncoCarta Panel, LungCarta Panel, iPLEX ADME PGx Pro Panel, Assays by Agena Bioscience Oligo Sets, UltraSEEK Oncogene Panel, OncoFOCUS Panel, SpectroCHIP Array, iPLEX Pro Sample ID Panel, Assay Design Suite Software and Assay Explorer are for Research Use Only. Not for use in diagnostic procedures.

Note:  The MassARRAY System, MassARRAY Analyzer 4, Hemo ID Blood Group Genotyping Panel, iPLEX Gold, iSEQ, QGE, EpiTYPER, TYPER software, MelaCarta Panel, OncoCarta Panel, LungCarta Panel, iPLEX ADME PGx Pro Panel, Assays by Agena Bioscience Oligo Sets, UltraSEEK Oncogene Panel, OncoFOCUS Panel, SpectroCHIP Array, iPLEX Pro Sample ID Panel, Assay Design Suite Software and Assay Explorer are for Research Use Only. Not for use in diagnostic procedures.