Orthogonal Analysis Workshop -...

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


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…


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


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


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


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

Orthogonal Analysis Workshop -...

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