Post on 21-Aug-2020
Orthogonal Analysis Workshop
Greg Denomme, Ph.D.
Sequencing at the Tipping Point Meeting
San Diego, December 2010
2 | Life Technologies Proprietary & Confidential | 12/2/2010
•19 Species, >1.2M Assays; custom assays for any species
•135 TaqMan Array Gene Signature Plates
•PreAmp pools for FFPE, laser capture or single/precious cells
•>1,600 predesigned miRNA Assays, 6 species
•~25,000 predesigned long ncRNA
•Custom Assays for any species, any ncRNA
•6 Protein Assays for stem cell differentiation based on proximity ligation assays
•Open Kit for customer-selected Proteins/antibodies
•>4.5M SNP Assays, 2 species, 2,700 Drug Metabolizing (DMEs)
•>1.6M Copy Number (CNV) Assays
•Custom Assays to ANY species
mRNA Gene Expression
nonCoding RNA
Protein
Genetic Variation
TaqMan® Genomic Assays Portfolio• Market leading products for RNA, DNA, & Protein Analysis• Based on Gold Standard TaqMan® Chemistry
3 | Life Technologies Proprietary & Confidential | 12/2/2010
Individual Assays in Tubes
TaqMan Arrays384 well microfludic cards>80 pre-defined pathways/setsCustom Arrays
TaqMan Express Plates96 well optical platesCustom PlatedPredefined gene sets
Assays available in 4 formats
GeneAssist ToolsSearch for assays in agiven pathway or workflow
Software tools simplifyAssay selection & data analysis
UmapItSearch for assays that map to microArray probes
DataAssist, StatMinerData analysis softwareStatistical analysis
TaqMan OpenArraysSNP Browser
TaqMan Genotyper
CopyCaller CNV Analysis
Content, Workflow, Formats…
4 | Life Technologies Proprietary & Confidential | 12/2/2010
LIFE TECHNOLOGIES- ANY PROJECT, ANY SIZE:Complimentary Systems fitting the Customer Need
7900HT or ViiA 7 OpenArray SystemProject Size Minimum 96 or 384 data points
Sample‐Assay combination of 30,000 data points (10 OpenArray plates)
Project Size (best fit)
Assays(10s‐100s); Samples (10s to 1,000s) Assays(10s‐100s); Samples (200s to 100,000s)
Cost per Reaction $0.18‐2.00 $0.07‐.18 GT; $0.20‐.035 GX (see price calcs)Applications Supported
miRNA, mRNA, CNV, SNP, PLA mRNA, SNP, pathogen detection
Development castPCR miRNA, dPCR
5 | Life Technologies Proprietary & Confidential | 12/2/2010
LIFE TECHNOLOGIES- ANY PROJECT, ANY SIZE:Complimentary Systems fitting the Customer Need
Targeted Assay Target Validation
SNaPshot™ kit Sanger Sequencing
Cost per Reaction $2.00‐$6.00 $0.50‐$10
Applications Panel‐based genotyping Full bi‐directional sequence coverage
OpenArray Nanofluidic PCR for High Throughput Red Cell Genotyping
Greg Denomme, PhDDirector of ImmunohematologyBloodCenter of Wisconsin
Objectives
• Review the demand for antigen typed blood• Single nucleotide polymorphism detection
• Identify efficiencies that can be realized from high throughput red cell genotyping (RCG)• Value as a screening tool
• Identify settings where red cell genotyping is applicable• Future applications
Perspective on RBC Antigens
• ~30 - the number of blood group gene loci
• ~300 – number of blood group antigens
• 95% of all blood group antigens are the result of single nucleotide polymorphisms
A PositiveA Positive - represents two antigens
Genotype to Predicted Phenotype
System Antigen Gene SNP rs# NT Change
AA Change VIC FAM
Rh E/e RHCE 609320 C>G P226A G C
Duffy Fya/Fyb FY 12075 A>G G42A A G Fy FY 2814778 A>G promoter G AFyx FY 34599082 G>A R89C G A
Kidd Jka/Jkb JK 1058396 A>G D280N A G
Lutheran Lua/Lub LU 28399653 A>G R77H A G
Kell Jsa/Jsb KEL 8176038 C>T L597P T CK/k KEL 8176058 T>C M193T C T
Dombrock Doa/Dob DO 11276 A>G N265D G AJoa DO 28362798 C>T T117I C T
Transfusion Supportfor the
Alloimmunized Patient
Alloimmunized Transfusion RecipientsBackgroundThere are +15MM transfusions/yr in the US– 1st transfusion/pregnancy ~4% alloimmunization rate– Chronic transfusion? up to 50% transfusion recipients
alloimmunized
What are the trends in Transfusion Medicine?– Physicians recognize chronic transfused people make antibodies– increase demand for antigen-matched blood to avoid Tx
reactions+100,000 patients nationwide: SCD, MDS, AA
Increasing demand for antigen typed blood• Increase number of uncommon and rare requests for donors, • increase the number of antigen-typed donors (multiple antigens)
A need for massive screening of blood donors
The list (in order)(1) avoidance of unnecessary transfusions to reduce potentially fatal transfusion
complications;(2) reduction in the risk of transfusion-related acute lung injury; (3) prevention of hemolytic transfusion reactions through patient identification, prevention of
alloantibody formation(4) avoidance of pooled blood products to reduce the risk of transmission of emerging TTIs; (5) WBC reduction of cellular blood components; and(6) pathogen reduction of platelet and plasma components to prevent TTIs
Severe Life-threatening Reactions:FDA Fatality Reports 2005-2008
Antibodies Implicated in Fatal Hemolytic Reactions
http://www.fda.gov/downloads/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/TransfusionDonationFatalities/UCM205620.pdf
Creates Constraints
• Likelihood to fill a request is based on:• Antisera available for testing• Frequency of the absence of an antigen• Total number of donors phenotyped
Large database is better but costly and limited
• Additional problems:• Donor attrition rate : # of donors degrades• Shipping units affects availability• Random donations : feast or famine (frozen blood)
Database size – combined antigen frequencies
• Frequency of a combined antigen negative request:Rh positive, e-neg, S-neg, Fy(a-) Jk(a-)
p = .02 × 0.9 × .45 × .34 × .23 = .00066 [~1:1500]
• Test 10,000 units (Group O, A, and B)
P(4,10000,0.00066) = 10000! × (.00066)4 × (.99934)9996
4!(9996)!
• Testing 10,000 will result in 4 or more units only 90% of the time!
Blood center
HospitalTransfusion Service
Current situation – antigen typing
1
2
Screening
HospitalRequestShip
How the genotyping program evolved
BCW recognized a need to expand the donor antigen-
negative database
• Some reagents are in limited supply, expensive, or not available, e.g. anti-S, anti-Jsa, anti-Doa
Need not being met 100%
• Current antigen typing done manually/semi-automated
• ~15 licensed reagents and 4 or 5 methods
Labor intensive, costly!
• Some transfusion services do their own antigen typing and those results are lost - cannot be linked to the donor’s record
Grossly inefficient
Red Cell Genotyping4 results (A, C, G, T)
• One methodIdentify current
• + + +Identify more
• + … + 32
Taqman ® Chemistry
OpenArray Nanofluidic PCR
33 nL volume
Hydrophilic
Hydrophobic
Technology meets our needs
Throughput capacity:32 SNPs, 91 donors, 4 CTLs ,1 NTC per array12 arrays per day
3072 tests per slide
• Platform capable of 3072 low-volume solution phase assays tested in parallel on a single chip
• Equivalent to • 8 × 384-well • 32 × 96-well plates
OpenArray® Plate
Throughput Capacity
• DNA extraction based on 96 x 4 format
• Set-up formatted for 4, 8 or 12 OpenArrays• 91 + 5 (controls) per array
• Runs performed daily• 25,739 samples tested in 9 weeks (4 days/week)
• 715 samples/day (~8 OpenArrays/day)Yt a/b
Di a/b Hy
Do a/b
Js a/b
Jk a/b
Fy a/b -67
Lu a/b E/e C/c VS 16C
223V
238V
336C
137V Cra Fyx
Lu 8/14 K/k
Kp a/b Joa S/s Cra
Co a/b
Sc 1/2
Jk null
No Call plusOutlier Rate (%)
3.4 0.6 0.3 0.6 1.0 0.6 0.6 0.5 3.9 0.6 0.3 0.5 1.0 0.6 0.7 0.6 0.6 0.7 0.4 5.3 0.9 0.4 0.6 1.6 0.7 0.7 0.3 0.3
Workflow
BT1432394321 BT1432394321 BT1432394321
Insert and seal into cassettes
Cycling and Imaging
Data analysis
OpenArray ® plate loading
Red Cell Genotyping Process
DNA Integrity
Metric DNA]c OD260/280
Imaging(run validation)
Metric FAM/VIC/ROX
Results(assay validation)
Metric DNA Controls
GenotypeRules
Engine
Partner Blood Centers
Run
Acceptance
Data
Reports
DNA Extraction
Results Analysis
Set-upImaging
Red Cell Antigens of Interest
• Rh antigens• C/c (intron 2 and 103), E/e (226), VS (245), V(336)
• Other clinically significant antigens• M/N, S/s, K/k, Kp(a/b), Js(a/b), Fy(a/b), Jk(a/b), Do(a/b), Jo(a), Hy,
Lu(a/b), Di(a/b), Co(a/b)
• Rare antigens• Lu(8/14), In(a/b), Cr(a-), Sc(1/2), Tc(a/b), Yt(a/b)
• Variants• DIIIa/DIV(455), E/e variants, hrB, hrS, Fy(a-b-), Fy(x), JknullFinn
• Quality improvements• D (in Rh negative donors, RoHAR and Crawford(233E)
Blood Product Delivery
HospitalTransfusion Service
Current situation – antigen typing
1
2
BCW
Screening
HospitalRequestShip
Looking ahead: hospital portal
Blood center
30,000 donors43 antigens
= 1.3 x 106
1:50001:100
Red CellGenotyping
(Screened genotypes)
Rare Blood Types
Screening
DNA ��
HospitalRequestShip
BCW
HospitalPortal
HospitalTransfusion Service
Phenotypes confirmed prior to transfusion
Transfusion needs met
IRL / Donor Services
Genotyping
Patient Donors
Inventory
RFID
Ags
Blood Center
Exchan
geAgs
Ags Ags
Hospital Service
RCG Database
Phenotype requests – 6 months
Special Phenotype Requests (N = 580 Requests)
Rh41%
Kell20%
Kidd13%
Duffy15%
MNS10%
Misc1%
1
10
100
1000
10000
100000
1000000
1 16 31 46 61 76 91 106 121 136 151 166 181 196 211 226 241 256 271 286 301 316 331 346 361 376 391 406 421 436 451 466 481 496 511 526 541 556 571
Database size
Donor Red Cell Genotyping - Applications• Identify antigen negative genotypes
• Limits amount of regulatory phenotyping to those ‘in demand’units (10%)
• Reduces need to hold large numbers of screened units• Provides new support to hospital blood banks
• Rare and uncommon frequency units• Supports ARDP• Screening assay Test-of-record
• QA tool• phenotype ≠ genotype (flag)• Impact of weak antigens: Fyx, D (Weak D Types, Del)
• Donor recruitment/donation value:• Information used as a recruitment tool?
Discussion
Thank you!
Genotype Frequencies Repeat (65%)Cauc Ethnic0.585 0.065
# of Donors 30,000 17,550 1,950Ag neg
Selected Inventory All Donors S/K/Fya/Jka1RoRo/Ror 606 202Fy(a-b-) 1,943 643rr Fy(a-b-) 623 214Doa- 3,169 1055R1R1 5,134 1696R2R2 479 16
6,219 394Rare Types All Donors
hrB- 6k- 88kpb- 3Jsb- 39Jknull 6Lub- 16Lu14- 2Dib- 3Coa- 10Inb- 29Hy- 15Joa- 16Cra- 29Tca- 4Yta- 15Sc1- 15
294 689
Can a blood center work smarter?• Likelihood to fill a request is based on:
Donor/recipient allelic differencesWhere the unit is > 5 days (St. Elsewhere) Shipping unit(s) affects availability (difficult to model)
RFID – location, location, location Center � Hospital communication
Genotype donors does cost = efficiencies
TATs, efficiencies, # genotyped, portal, location
Tx Populations – Today and Tomorrow• Sickle Cell disease and other chronic Tx’n
• practice is gaining momentum for at risk patients
• Women of childbearing age (extension of K Ag match)• Prevent alloimmunization (Rh, Kell, MNS, Fy, Jk)
• NICU• Should we invest heavily in this very young population?
• History of transfusion/pregnancy• Avoid post-transfusion immune-mediated hemolysis
The Future
• Recognize that patient become alloimmunized• Antigen detection Antibody identification
• Identify clinically relevant genotypes• Regardless of recent transfusion
• Scan patients for at-risk alleles• Lack of a high prevalence and other risk alleles
• Identify the appropriate genotyped unit• Ensure compatibility prior to transfusion
Genotype matching a reality
How the genotyping program evolved
• Additional need to find rare antigen types and multiple antigen-negative blood types• Transfusion of patients with antibodies• Preventing alloimmunization - antigen-matched requests
• Identification of rare types requires large scale screening• Phenotyping by hemagglutination (?)
• Multiple antigen-negative types requires an efficient multiple analytical testing approach
Red Cell Genotyping solves these problems
BCW’s early high volume experience
• 427 blood donors genotyped• 2,037 phenotype:genotype comparisons• 4 of Phenotype errors• 2 Genotype variants