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Laboratory Services Guide

Mohan M., heart transplant recipient

2 HEARTCARE LAB SERVICES GUIDE

Introduction to HeartCareHeartCare is a non-invasive blood testing panel comprised of the AlloSure-Heart and AlloMap tests. AlloSure-Heart quantifies donor-derived cell-free DNA (dd-cfDNA) in the plasma of recipients and is a biomarker of allograft cellular injury and potential allograft rejection. AlloSure-Heart utilizes targeted, next generation sequencing (NGS) to measure differences in single-nucleotide polymorphisms (SNPs) between donor and recipient. The AlloMap test is a gene expression profile (GEP) of a recipient’s peripheral blood mononuclear cells utilizing real-time PCR (polymerase chain reaction). The AlloMap score is correlated with the probability that the allograft is quiescent (lack of clinically-significant acute cellular rejection (ACR)) and is associated with the level of a recipient’s immune system activity. These components, AlloSure-Heart dd-cfDNA and AlloMap GEP, provide complementary information about the probability of acute cellular or antibody-mediated rejection and the status of the heart allograft than either component alone.

When used in conjunction with standard clinical assessments, HeartCare aids clinicians in assessing the

probability of rejection in heart transplant recipients. Use of HeartCare in surveillance of heart allograft recipients may help to inform clinical management decisions and may be used along with clinical information when determining the necessity of performing endomyocardial biopsy in patients.

GENE EXPRESSION PROFILING (GEP)CHARACTERIZES RNA EXPRESSION FROM CIRCULATING BLOOD CELLS.

DURING CELLULAR REJECTION, RECIPIENT IMMUNE SYSTEM CHANGES CAN BE MEASURED BY GEP.

CELL-FREE DNA IS DNA IN THE BLOOD THAT ORIGINATES FROM CELLS UNDERGOING CELL INJURY AND DEATH.

WHEN GRAFT INJURY OCCURS, DONOR-DERIVED CELL-FREE DNA (DD-CFDNA) INCREASES IN THE BLOOD.

Immune ActivitymRNA

Graft InjurycfDNA

HEARTCARE® SUMMARYHeartCare testing includes: AlloMap® and AlloSure‐Heart®

HeartCare is for: surveillance of heart transplant recipients for acute rejection in conjunction with standard clinical assessments.

HeartCare testing may begin: for heart transplant recipients who are 2 weeks (≥14 days) post‐transplant (the AlloMap component of HeartCare may begin day 55 post‐transplant).

Limitations:

One month following transfusion: Patients who received transfusions of whole blood or other blood transfusions that contain white blood cell components within one month prior to AlloMap and AlloSure‐Heart testing may have inaccurate test results. Transfusions of washed red blood cells or leukocyte‐depleted, packed red blood cell transfusions are allowed.

Corticosteroid dosage >20 mg/day: Systemic corticosteroid dosage of >20 mg/day of prednisone or equivalent may result in a decreased AlloMap score.

21 days following rejection therapy with steroids: AlloMap performance characteristics have not been established for patients who have received rejection therapy in the 21 days prior to testing.

Heart transplant recipients who are also recipients of other solid organs, who are pregnant, and/or who have received allogeneic blood or bone marrow transplants. In each of these clinical situations, the AlloSure result may be perturbed because there is an additional contribution of cfDNA from non‐recipient genomes that cannot be differentiated from the proportion of cfDNA deriving from the heart allograft.

Multi-organ transplant: The performance characteristics of AlloMap have not been established for recipients of a cardiac allograft and another non‐cardiac organ transplant (e.g. kidney).

Following endomyocardial biopsy: Endomyocardial biopsies performed in the 24 hours prior to AlloSure‐Heart specimen collection may result in increase of dd‐cfDNA.

The clinical validity of AlloMap and AlloSure‐Heart were established in heart transplant recipients who were age 15 or older and at least 55 days post‐transplant.

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Performing Laboratory and Location: CareDx, 3260 Bayshore Blvd., Brisbane, CA 940051‐888‐255‐6627CLIA No: 05D1029609 CAP ID: 7193868

Laboratory Directors: Judith C. Wilber, PhD, D(ABMM) Patrick Joseph, MD

SUPPLIES REQUIRED AND SUPPLIED BY CAREDX HeartCare Specimen Collection Kit – Description of Contents

Specimen collection blood tubes:

• AlloSure: Streck Cell‐Free DNA BCT® (Streck Tube). Cell‐Free DNA BCT contains the anticoagulant K3EDTA and a cell preservative in a liquid medium. Cloudiness or precipitation visible in the tube reagent is an indication of deterioration. Do not use Cell‐Free DNA BCT showing signs of deterioration and please call CareDx Customer Care to request new collection kits.

• AlloMap: BD Vacutainer® CPT™ Cell Preparation Tubes with Sodium Citrate (CPT) (8.0 mL). Protect tubes from direct light.

• Store HeartCare kits between 18°C and 25°C. Check expiration date of each blood tube before use. Do not use expired blood tubes to collect samples. Refer to the AlloMap and AlloSure‐Heart Laboratory Services Guides (LQ‐10004, and LT‐10067, respectively) for additional information about the blood collection tubes, or visit www.bd.com (CPT™ tubes) and streck.com (Cell‐Free DNA BCT® tubes).

HeartCare Tube & Accession Labels card

Packaging and shipping materials:

• Absorbent 2-bay tube pouch

• 95 kPa bag

• Ambient gel pack

• Insulated foil envelope

• List of Contents Card

• FedEx shipping label and Clinical Pak

HeartCare Test Requisition Forms may be ordered separately

To order Supplies

Phone 1-888-255-6627Fax 1-415-287-2456Email [email protected]

ORDERING HEARTCAREHeartCare may be ordered in several ways:

Web Portal: HeartCare may be ordered electronically, via the CareDx Web Portal. One‐time tests or standing orders (up to 12 months) are available through the portal. Please contact Customer Care to sign up for the web portal.

HeartCare Test Requisition Form (TRF): A paper test requisition form is available for ordering HeartCare. Please contact Customer Care for delivery of the hardcopy HeartCare TRFs.

Other Order Form: Some customers prefer to use their own order forms to place laboratory orders. We will accept order forms assuming all Required Information for HeartCare orders (see below) is included.

REQUIRED INFORMATION FOR HEARTCARE ORDERSRegardless of the method used to place HeartCare order, the following information is required and must be included on the test requisition/order received with the specimens. Without complete information on the test requisition/order, test results may be delayed.

Patient and Prescriber Information:

• Patient last name, first name

• Patient medical record number or other unique identifying number

• ICD-10 code

• Patient status

• Prescriber name, NPI, and signature

Phlebotomy and Processing Information:

• Date and time of specimen collection

• Date and time lysate was frozen at -15º C or colder

• Name, address and phone number of the laboratory submitting the specimen

Insurance Information:

• Name of insured

• Insurance provider name

• Insurance Member ID number


The following information must be either on the test order or previously provided to CareDx:

• Date of birth

• Gender

• Transplant date

• Patient address (street, city, state, zip)

• Patient primary phone

SPECIMEN COLLECTION PROCEDUREPreparation

• Identify the patient according to your laboratory’s patient identification policy.

• Prepare the patient for phlebotomy according to your standard procedure.

• Locate your HeartCare TRF, Web Portal or other Order Forms.

• Open the HeartCare Laboratory Specimen Collection Kit and locate the following supplies: One CPT, Two Streck Cell-Free DNA BCT, HeartCare Tube & Accession Labels card.

• Select the tubes and additional supplies needed to perform venipuncture. Check the tube’s expiration date before use.

Draw Order

If collecting additional specimens from the same venipunc-ture, follow the recommendations of your laboratory for the correct order of collection for citrate and EDTA tubes. If your laboratory does not have a recommended draw order, draw tubes in the following order:

AlloMap Specimen Collection Procedure

Step 1: Complete the AlloMap specimen label. Date is MM/DD/YYYY. Write the time to the nearest minute. Do not write on the barcode.

Step 2: Fill one CPT tube to no less than 6 mL. See draw

tube comparison on the back of the Tube and Accession Labels card.

Step 3: Mix filled tube by gentle inversion (10x).

Step 4: Write the collection date, collection time to the nearest minute, and your initials in the red blocks on the label. Print neatly.

Step 5: Affix the labels lengthwise to the tube. Do not cover the tube’s expiration date.

Step 6: Order form must include patient name, DOB, ordering physician, name of lab, draw date and time, and phlebo initials. Affix a barcode label to the upper right corner.

KEEP SPECIMEN AT AMBIENT TEMPERATURE. TAKE CPT TUBE IMMEDIATELY TO THE APPROPRIATE LAB FOR PROCESSING. THE SAMPLE MUST BE FULLY PROCESSED AND PLACED IN THE FREEZER WITHIN THREE 3 HOURS FROM DRAW TIME.

Refer to the AlloMap Laboratory Services Guide, LQ-10004 for detailed instructions how to process, package and ship the AlloMap samples to CareDx.

AlloSure-Heart Specimen Collection Procedure

Step 1: Complete the AlloSure‐Heart specimen labels. Date is MM/DD/YYYY. Do not write on the barcode.

Step 2: Completely fill Streck tubes. If you cannot fill both tubes, collect as much as possible.

Step 3: Mix filled tubes by gentle inversion (10x).

Step 4: Affix the labels lengthwise to the tubes. Do not cover the tube’s expiration date.

Step 5: Order form must include patient name, DOB, ordering physician, name of lab, draw date, and phlebo initials. Affix a barcode label to the upper right corner.

Step 6: For specimen packaging instructions, see the inside of the box lid.

KEEP SPECIMEN AT 6°–37°C. SHIP AS SOON AS POS-SIBLE. SPECIMEN MUST ARRIVE AT CAREDX WITHIN 7 DAYS OF DRAWING.

Refer to the AlloSure-Heart Laboratory Services Guide, LT-10067 for detailed instructions how to package and ship the blood specimens to CareDx.

Cell-Free DNA BCT

CPT(Glycolyticinhibitor)

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TEST NAMEAlloSure‐Heart® Donor‐Derived Cell‐Free DNA Test for Heart Transplant Recipients

INTENDED USEThe AlloSure‐Heart test is intended to assess the prob-ability of allograft rejection in heart transplant recipients with clinical suspicion of rejection and to inform clinical decision‐making in such patients, in conjunction with other clinical assessments.

Clinical validity of the AlloSure test was established in heart transplant recipients who were 15 years of age or older and at least two months (55 days) post‐transplant.

PRINCIPLE OF THE TESTThe AlloSure test is a targeted, next-generation sequencing (NGS) assay that measures the amount of donor-derived cfDNA relative to the total amount of cfDNA in a plasma sample. AlloSure accurately quantifies dd-cfDNA in heart transplant recipients without separate genotyping of either the donor or the recipient. The assay can quantify the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs.

Blood is collected from the patient, packaged, and shipped at ambient temperature to CareDx for testing. Donor-derived cell-free DNA is measured via targeted amplification and sequencing of a set of carefully selected and validated single nucleotide polymorphisms (SNPs)

Introduction to AlloSure-HeartAlloSure‐Heart is a non‐invasive blood test that assesses organ health by directly measuring allograft injury. AlloSure‐Heart can accurately determine acute rejection, enabling better management of heart transplant recipients. The AlloSure‐Heart test measures donor‐derived cell‐free DNA (dd‐cfDNA) in heart transplant recipients. Cell‐free DNA is present in plasma as a product of cell injury and death. dd‐cfDNA is fragments of DNA in the recipient’s bloodstream that originate from cells from the donated organ, and therefore are of the donor genotype and indicate specifically the level of graft cell injury and death.

specifically chosen to discriminate among individuals based on genetic sequence (genotype). The AlloSure bioinformatics software calculates the percent dd-cfDNA in the sample tested and applies the QC criteria. Most AlloSure test results are reported to the ordering physician within 2 days from specimen receipt at the CareDx lab.

TEST METHODOLOGY AND VALIDATIONCell-free DNA is purified from plasma collected in Streck cell-free DNA BCT® tubes. Cell-free DNA extracted from plasma is used as the template in a next genera-tion sequencing assay. Based on a proprietary algorithm that uses the known population frequencies of the SNPs sequenced and expected distributions of alleles, the percentage of the cell-free DNA that is derived from the transplanted organ (dd-cfDNA) is computed (Grskovic et al., J Mol Diagn, 2016).

Analytical performance of the assay was characterized and validated using 1,139 samples comprising the NIST-GIAB human reference genome, independently validated reference materials, and clinical samples. The assay can reliably measure dd-cfDNA with a limit of detection (LOD) of 0.15%.

Clinical validation data from the CARGOII study was published in Grskovic et al., J Mol Diagn, 2016. In plasma samples from 53 heart transplant recipients AlloSure‐Heart demonstrated statistically significant increased levels of dd‐cfDNA in samples from patients with biopsy‐confirmed rejection compared to samples from patients negative for rejection as assessed by biopsy (7.4‐fold higher, p=0008).


• Blood cultures – SPS

• Citrate tubes including the CPT tube for AlloMap

• BD Vacutainer SST Gel Separator Tube, Serum Tube (glass or plastic)

• Heparin tube, BD Vacutainer PST Gel Separator Tube with heparin

• EDTA tube

• Streck Cell-Free DNA BCT

• Fluoride (glucose) tube

Step 5: Use a 22 gauge needle or larger bore for phlebotomy. Avoid drawing with a syringe and transferring through the tube’s stopper; hemolysis may result.

Step 6: When using a winged (butterfly) collection set for venipuncture and the Streck Tube is the first tube drawn, draw a partially‐filled non‐additive or EDTA discard tube to eliminate air from the tubing.

Step 7: Since Streck Tubes contain chemical additives, it is important to avoid possible backflow from the tube. To guard against backflow, use the following precautions.

• Keep the patient’s arm in the downward position during the collection procedure.

• Hold the tube with the stopper in the uppermost position so that the tube contents do not touch the stopper or the end of the needle during sample collection.

• Release the tourniquet once blood starts to flow in the tube, or within 2 minutes application.

• Fill both Streck Tubes completely; one tube holds approximately 10 mL.

Step 8: Mix by gentle inversion at least 10 times.

Step 9: On the Tube & Accession Labels card, complete both labels with Patient Last Name, Patient First Name, Date of Birth, Draw Date, and Your Initials. Date format is MM/DD/YYYY. Do not write on the barcode label.

Step 10: Affix the completed labels to the Streck Tubes, vertically aligned. Do not cover the expiration date.

Note: If using a lab-generated label, ensure that Patient Last Name, Patient First Name, Date of Birth, Draw Date, and Phlebotomist’s Initials are included. Affix one of the extra barcode labels from the Tube & Accession Label card provided in the kit to each of the 2 tubes.

Further validation in data from the D‐OAR study published in Khush et al., Am J Transplant 2019, demonstrated that plasma levels of dd‐cfDNA can discriminate acute rejec-tion from no rejection. A total of 2,447 blood specimens were prospectively collected from 740 heart recipients at 26 clinical sites at scheduled intervals, and concomitantly with clinically indicated or surveillance endomyocardial bi-opsies. Venous blood was collected according to AlloSure‐Heart procedures and tested at CareDx. dd‐cfDNA levels were measured in blood plasma using the AlloSure‐Heart test and correlated with allograft rejection status ascer-tained by endomyocardial biopsy. At a 0.15% threshold, AlloSure‐Heart had a 55.9% sensitivity to detect rejection, a 7.8% positive predictive value, and a 97.4% negative predictive value. In a single-center cohort of 33 patients at high risk for antibody-mediated rejection (AMR), also published in Khush et al., Am J Transplant 2019, Allo-Sure-Heart levels were elevated threefold in AMR com-pared to patients without AMR (99 samples, p=0.004).

Patients in the D‐OAR multicenter study who had no clinical signs or symptoms of rejection during the course of the study were considered stable transplant recipients. AlloSure‐Heart tests in this stable population were used to define the baseline AlloSure‐Heart, which has a median of 0.07% dd‐cfDNA. (Khush et al., Am J Transplant 2019).

SPECIMEN COLLECTION AND SHIPPING AlloSure-Heart Specimen Collection Procedure

Step 1: Identify the patient according to your laboratory’s patient identification policy, and prepare the patient for phlebotomy according to your standard procedure.

Step 1: Locate your TRF, Web Portal or other Order Forms.

Step 2: Complete the AlloSure‐Heart specimen labels. Date is MM/DD/YYYY. Do not write on the barcode.

Step 3: Open the Specimen Collection Kit and locate the following supplies:

• Two Streck Cell-Free DNA BCT (check expiration date before use)

• Tube & Accession Labels card

Step 4: If collecting additional specimens from the same venipuncture, follow the recommendations of your laboratory for the correct order of collection for citrate tubes. If your laboratory does not have a recommended draw order, draw tubes in the following order:

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• Mishandled specimens: Specimens that are or have been frozen, or have been shipped on cold packs or ice, or have evidence of mishandling or improper shipping will not be tested.

• Hemolyzed specimens. Specimens showing hemolysis above a trace/slight amount by visual inspection will not be tested.

• Specimens from patients for whom any of the following are true will not be tested:

– Less than 2 weeks (14 days) post-transplant at the time of blood draw

– Known pregnancy at the time of blood draw

– Have multiple transplanted organs

– Have received an allogeneic blood or bone marrow transplant

LIMITATIONS (SPECIAL CONSIDERATIONS) When more than two genomes may be present in the recipient plasma (more than recipient + donor) contribution of cfDNA from each genome is not differentiated by the test. This includes pregnancy, due to the presence of fetal genome DNA in the maternal plasma and multiple‐organ transplant from different donors since both of the grafts introduce a unique genome (e.g. heart and kidney) and each contribute different basal levels of cfDNA confounding interpretation of the results.

A recipient of multiple transplanted organs that all originated from the same donor presents a situation where elevated levels of dd‐cfDNA could have originated from one organ or another or both. If from both, they could be contributing at different baseline levels, confounding interpretation of the results. Therefore, AlloSure‐Heart is not to be used for transplant recipients with multiple transplanted organs from the same donor.

Patients who received transfusions of whole blood or other blood transfusions that contain white blood cell components within one month prior to the AlloSure test may have an inaccurate result. Transfusions of washed red blood cells or leukocyte‐depleted, packed red blood cell transfusions are allowed.

There are some indications that damage to the graft caused by invasive procedures such as endomyocardial biopsy may cause a short‐term elevation of dd‐cfDNA. Until definitive studies are completed, AlloSure‐Heart should not be used on patients wihin 24 hours following an endomyocardial biopsy.

Step 11: You may have an order form from the web portal or an order form from the transplant center. If you do not have an order form, on the blank AlloSure‐Heart Test Requisition Form, please complete:

• Patient Last Name and Patient First Name

• Patient Date of Birth

• Prescriber Name and Location

• Referring Lab Name

Step 12: Affix the first barcode label from the Tube & Accession Labels card to the upper right corner of the order form.

Step 13: Ship the specimen as soon as possible following the Specimen Shipping Procedure. If you cannot ship the specimens immediately, hold specimens between 6 °C and 37 °C. Do not refrigerate or freeze. Specimens must arrive at CareDx with 7 days of collection.

Specimen Shipping ProcedureTo ship the AlloSure‐Heart specimen:

Step 1: Place the filled Streck tube in the pocket of the absorbent pouch.

Step 2: Place the pouch in the 95 kPa bag and seal.Step 1: Locate your TRF, Web Portal or other Order Forms.

Step 3: Wrap the 95 kPa bag in the gel pack and place inside the foil envelope, and seal.

Step 4: Place the sealed foil envelope in the shipper box.

Step 5: Fold the TRF or order form so that it fits in the shipper box and place it on top of the foil envelope.

Step 6: Place the Tube & Accession Labels card in the box.

Step 7: Place the List of Contents card on the very top; close and seal the box.

Step 8: Label the FedEx Clinical Pak with the shipping label and place the sealed box inside the Clinical Pak. Seal the Pak shut.

Step 9: Ship via FedEx the same day, or as soon as possible.

Specimen Shipping Procedure

• Unlabeled or improperly labeled specimens: Specimens must be labeled with 2 patient identifiers (i.e., First Name & Last Name, and DOB or MRN) and the date the specimen is drawn.


Coefficient of Variation

Thirteen replicate runs were performed on 13 separate days by four operators using two sequencing instruments, and three lots of critical reagents. Mean CV across dd-cfDNA levels = 4.6 % at 3ng input mass.

REFERENCES Blood specimen collection

Streck Cell‐Free DNA BCT® Instructions for Use (visit www.Streck.com for most up‐to‐date version)

College of American Pathologists GEN.40700, GEN.40750, GEN.40490, GEN.40491

PERFORMANCE CHARACTERISTICS Accuracy

Accuracy was assessed across multiple “donor/ recipient” DNA mixtures in three different panels (contrived specimens made from cell lines, range 0.16%-20%, sonicated to 160 bp fragments to mimic cfDNA). Each mixture was run in 6 to 13 replicates at 3ng and 8ng total cfDNA input mass (n=428 results). The slope, intercept, and correlation between the reference results and AlloSure-Heart results were deter-mined for the mixtures (Table 1).

Precision

Thirteen replicate runs of contrived specimens (described above) were performed on 13 separate days by four operators using two sequencing instruments, and three lots of critical reagents. Mean CV across dd-cfDNA levels = 2.7% at 8ng input mass, 4.6 % at 3ng input mass (covers 99% of the population).

Sensitivity

At a cut‐off level of 0.15%, the AlloSure‐Heart test demonstrated 55.9% (95% CI 43.7%‐70.0%) sensitivity to discriminate rejection from no rejection as determined by endomyocardial biopsy (Khush et al., Am J Transplant 2019).

Specificity

At a cut‐off level of 0.15%, the AlloSure‐heart test demonstrated 71.5% specificity (95% CI 69.5%‐74.9%) to discriminate no‐ rejection from rejection (as determined by endomyocardial biopsy). Khush et al., Am J Transplant 2019).

Sensitivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) at three different thresholds are shown in the Table 2 below. Sensitivity, Specificity, PPV and NPV across a broad range of cut-offs are shown in Figure 1

Average 95% CI

3ng

Slope: 0.9552 0.9408, 0.9697

Intercept: -0.00014 -0.00039, 0.00010

R2: 0.9993 - 0.9990, 0.9999

8ng

Slope: 0.9451 0.9317, 0.9586

Intercept: 0.00024 0.00009, 0.00039

R2: 0.9994 0.9992, 1.0000

Threshold Sensitivity Specificity PPV NPV

0.15% 55.9%(43.7%, 70.0%)

71.5%(69.5%, 74.9%)

7.8%(43.7%, 70.0%)

97.4%(96.7%, 98.2%)

0.20% 44.1%(31.6%, 57.9%)

80.4%(78.3%, 82.9%)

8.9%(6.4%, 11.9%)

97.1%(96.4%, 97.8%)

0.25% 32.4%(20.0%, 45.5%)

85.5%(83.3%, 87.7%)

8.8%(5.7%, 12.5%)

96.7%(96.1%, 97.3%)

100%

75%

50%

25%

0%

0.00% 0.25%

0.20%0.25%0.15%

0.50% 0.75%

Threshold

Sensitivity

Specificity

Perc

ent

1.00%

Table 1: Slope, intercept, and correlation between the reference results and AlloSure results

Table 2: Sensitivity, Specificity, PPV and NPV at three different thresholds (95% CI)

Figure 1: Sensitivity, Specificity, PPV and NPV across a broad range of cut-offs. Shaded areas represent the 95% confidence interval for each value.

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JS, Zangwill S, Mitchell ME. (2014). Highly sensitive noninvasive cardiac transplant rejection monitoring using targeted quantification of donor‐specific cell‐free deoxyribonucleic acid. J Am Coll Cardiol 63:1224‐1226.

Lo YM, Tein MS, Pang CC, Yeung CK, Tong KL, Hjelm NM. (1998). Presence of donor‐specific DNA in plasma of kidney and liver‐transplant recipients. Lancet 351:1329‐1330.

Macher HC, Suarez‐Artacho,G, Guerrero JM, Gomez‐Bravo MA, Alvarez‐Gomez S, Bernal‐Bellido C, Domingez‐Pascual I, Rubio A. (2014). Monitoring of Transplanted Liver Health by Quantification of Organ‐Specific Genomic Marker in Circulating DNA from Receptor. PLoS ONE 9:e113987.

Moreira VG GB, Martín JM, Suárez FO, Alvarez FV. (2009) Cell‐free DNA as a noninvasive acute rejection marker in renal transplantation. Clin Chem 11:1958‐1966.

Sigdel T, Vitalone MJ, Tran TQ, Dai H, Hsieh S‐C, Salvatierra O, Sarwal MM. (2013). A Rapid Noninvasive Assay for the

Detection of Renal Transplant Injury. Transplantation 96:97‐101.

Snyder TM, Khush KK, Valantine HA, Quake SR. (2011). Universal noninvasive detection of solid organ transplant rejection. Proc Natl Acad Sci U S A 108:6229‐6234.

Zhang J, Tong K‐L, Li PKT, Chan AWY, Yeung C‐K, Pang CCP, Wong TYH, Lee K‐C, L, YMD. (1999). Presence of Donor‐ and Recipient‐derived DNA in Cell‐free Urine Samples of Renal Transplantation Recipients: Urinary DNA Chimerism. Clin Chem 45:1741‐1746.

CAREDX CLINICAL LABORATORY QUALITY MANAGEMENT Accuracy

Information regarding the CareDx clinical laboratory Quality Management program and current laboratory licensure status will be provided upon request.

Laboratory licenses, permits and accreditations held by the CareDx Laboratory are listed below.

CLIA: 05D1029609 CAP: 7193868 New York: Permit #8193 Pennsylvania: Lab ID #29355A Rhode Island: LCO01129 Maryland: Permit #1254 California: Lab ID: CLF 00332008

AlloSure publications

Grskovic, M., Hiller, D.J., Eubank, L.A., Sninsky, J.J., Christopherson, C., Collins, J.P., Thompson, K., Song, M., Wang, Y.S., Ross, D., et al., (2016). Validation of a Clinical‐Grade Assay to Measure Donor‐Derived Cell‐Free DNA in Solid Organ Transplant Recipients. J Mol Diagn 18, 890‐902.

Khush K, Patel J, Pinney S, Kao A, Alharethi R, DePasquale E, Ewald G, Berman P, Kanwar M, Hiller D, Yee J, Woodward R, Hall S, Kobashigawa J. Non‐invasive Detection of Graft Injury after Heart Transplantation Using Donor‐Derived Cell‐Free DNA: a Prospective Multi‐Center Study. Am J Transplant 2019

Additional relevant publications

Beck J, Bierau S, Balzer S, Andag R, Kanzow P, Schmitz J, Gaedcke J, Moerer O, Slotta JE, Walson P, Kollmar O, Oellerich M, Schutz E. (2013). Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury. Clin Chem 59:1732‐1741.

De Vlaminck I, Valantine HA, Snyder TM, Strehl C, Cohen G, Luikart H, Neff NF, Okamoto J, Bernstein D, Weisshaar D, Quake SR, Khush KK. (2014). Circulating cell‐free DNA enables noninvasive diagnosis of heart transplant rejection. Sci Transl Med 6:241ra277.

De Vlaminck I, Martin L, Kertesz M, Patel K, Kowarsky M, Strehl C, Cohen G, Luikart H, Neff NF, Okamoto J, Nicolls MR, Cornfield D, Weill D, Valantine H, Khush KK, Quake SR. (2015). Noninvasive monitoring of infection and rejection after lung transplantation. Proc Natl Acad Sci U S A 112:13336‐13341.

Gadi VK, Nelson JL, Boespflug, ND, Guthrie, KA, Kuhr, CS. (2006). Soluble Donor DNA Concentrations in Recipient Serum

Correlate with Pancreas‐Kidney Rejection. Clin Chem 52:379‐382.

Gielis EM, Ledeganck KJ, De Winter BY, Del Favero J, Bosmans JL, Claas FH, Abramowicz D, Eikmans M. (2015). Cell‐Free

DNA: An Upcoming Biomarker in Transplantation. Am J Transplant 15:2541‐2551.

Hidestrand M, Tomita‐Mitchell A, Hidestrand PM, Oliphant A, Goetsch M, Stamm K, Liang HL, Castleberry C, Benson DW, Stendahl G, Simpson PM, Berger S, Tweddell


Introduction to AlloMap® TestingSince the first successful cardiac transplant in 1967, more than 126,753 such operations have been recorded by the Registry of the International Society for Heart and Lung Transplantation. Improved immunosuppressive agents have resulted in significant reductions in acute rejection rates and improved survival. However, even with modern immunosuppressive therapy, acute cellular rejection can still lead to graft dysfunction, and remains a significant cause of morbidity and mortality.

CareDx is a molecular diagnostics company focused on the discovery, development and commercialization of non-invasive gene expression testing in the areas of transplant medicine and autoimmunity. The company has developed a proprietary method of measuring gene expression that provides additional options to current patient management by physicians specializing in these disease areas.

Figure 1: AlloMap Molecular Expression Testing Process

Principle of the Test

Performed solely in the CareDx CAP-accredited clinical laboratory, the AlloMap test is a service that is intended to aid in the identification of heart transplant recipients with stable allograft function who have a low probability of moderate/severe acute cellular rejection (ACR) at the time of testing, in conjunction with standard clinical assessment.

AlloMap Molecular Expression Testing Process

Figure 1 illustrates the process for AlloMap Molecular Expression Testing. Blood is collected from the patient and subsequently processed, packaged and shipped frozen to the CareDx laboratory. At the CareDx laboratory, gene expression levels are determined via real time PCR for each of the test and control genes and converted to the AlloMap Test Score. The AlloMap Score is then reported to the physician, who determines the next steps for the patient.

CareDx Lab

ClinicLocal Lab

1PATIENT HAS A BLOODSAMPLE DRAWN AT A

LOCAL LAB

3SAMPLE ARRIVES ATCareDx LABORATORY

5GENE EXPRESSION INFORMATION IS TRANSLATED INTO DATA USED BY DOCTORS TO HELP MANAGE PATIENT CARE

6DOCTOR INTERPRETSDATA AND CONTACTSPATIENT

7PATIENT IS NOTIFIEDOF RESULTS

4COMPLEX GENE EXPRESSION

INFORMATION IS EXTRACTED ANDANALYZED FROM BLOOD SAMPLE

2SAMPLE IS PROCESSED,

PACKAGED, AND SHIPPED

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INTENDED USEAlloMap Molecular Expression Testing (the AlloMap Test) is an In Vitro Diagnostic Multivariate Index Assay (IVDMIA) test service, performed in a single laboratory, assessing the gene expression profile of RNA isolated from peripheral blood mononuclear cells (PBMC). AlloMap Testing is intended to aid in the identification of heart transplant recipients with stable allograft function who have a low probability of moderate/severe acute cellular rejection (ACR) at the time of testing in conjunction with standard clinical assessment.

The AlloMap Test is indicated for heart transplant recipients:

• 15 years of age or older

• At least 2 months (≥ 55 days) post-transplant

The performance characteristics of the AlloMap Test were established with samples from heart transplant recipients at least 15 years of age and at least 2 months (≥ 55 days) post-transplant at the time samples were collected.

SUMMARY AND EXPLANATION OF THE TESTThe AlloMap test is based on standard quantitative real-time polymerase chain reaction methodology (qRT-PCR) using RNA isolated from peripheral blood mononuclear cells (PBMC). A whole blood sample is collected in a BD Vacutainer® CPT™ Cell Preparation tube (Becton Dickinson, NJ) with sodium citrate anticoagulant (CPT tube). PBMC are isolated, lysed and the released RNA is stabilized and frozen (PBMC lysate). Following shipment to the CareDx CAP-Accredited laboratory in Brisbane, CA, RNA is then purified from the PBMC lysate. The cDNA is subsequently generated from the RNA for use in the AlloMap test. The AlloMap test is performed using general laboratory equipment and commercially available thermal cycling instruments configured for qRT-PCR testing. The AlloMap test is a panel of 20 gene assays, 11 informative and 9 used for normalization and quality control, which produces gene expression data used in the calculation of an AlloMap Score that ranges from 0-40.

CUSTOMER CARE CONTACT INFORMATION CareDx Customer Care Telephone 1-888-255-6627 Fax 1-415-287-2456Email [email protected]

Ordering Telephone 1-888-255-6627 Fax 1-415-287-2456Email [email protected]

Hours of operation: 6 AM to 5 PM Monday through Friday, 8 AM to 3 PM Saturday, Pacific Time

EQUIPMENT REQUIRED – CENTRIFUGECareDx provides to each laboratory processing AlloMap samples a Drucker 755VES adjustable-speed centrifuge, a Drucker Horizon 24 fixed-speed centrifuge or a Thermo Electron Centra CL2 centrifuge (CL2), and all required accessories.

Note: if, for any reason, an alternate centrifuge must be used to process a sample, call CareDx Customer Care for assistance with centrifuge specifications and RCF settings. Samples that are processed outside of CareDx specifications cannot be tested.

Centrifuge specifications:

• Swing-out bucket rotor; Note: Do not use a fixed-angle rotor

• Tube bucket that accommodates the 16mm x 125mm CPT tube Note: There must be sufficient room between the rotor and the CPT tube when it is inserted into the tube carrier, so that the stoppered end of the tube does not come into contact with the rotor during centrifugation causing the tube to break.

• Relative centrifugal force (RCF): 1796-1970 x g

Initial Set Up

Set up the centrifuge following the manufacturer’s recommended procedures found in the Drucker Manuals or the Centra CL2 Manual that is shipped with the centrifuge unit. Both centrifuges should be set up on a flat and level surface with enough space around the unit to allow for adequate ventilation and airflow (approximately 3 inches on each side and 4 inches at the back of the unit).


• Drucker 755VEW and Horizon 24: To assemble the tube holders, place the flat, black rubber disk in the bottom of the green tube holder first and then insert the black plastic adapter inside tube holder. The black adapter stabilizes the tube during centrifugation, and the black rubber disk will cushion the tube to minimize the risk of tube breakage. During use, press the cone-shaped clear plastic lid down firmly on the tube holder to contain any aerosols that may be created during centrifugation.

• CL2: To assemble the tube buckets, insert one brown tube adapter into each tube holder first, and then drop one black rubber disk down through the adapter to the bottom of the holder-adapter assembly. The brown adapter stabilizes the tube during centrifugation, and the black disk will cushion the tube to minimize the risk of tube breakage. During use, screw the dome shield (lid) onto the tube holder to contain any aerosols that may be created during centrifugation.

Daily Use

Before using the centrifuge, ensure that the speed of the centrifuge is set appropriately (see Section X Specimen Processing Procedure).

Cleaning

Keep your centrifuge, tube holders, adapters and lids clean to ensure good operation and to extend its life.

To clean the sample chamber, use a damp sponge, warm water and a mild liquid detergent suitable for washing dishes by hand. Do not use caustic detergents or detergents that contain chlorine ions, since these attack metals. Remove stubborn stains with a plastic scrub pad. Do not use steel wool, wire brushes, abrasives, or sandpaper, since they create corrosion sites. Never pour water directly into the sample chamber. After cleaning any part of the centrifuge dry it properly, preferably using a clean, absorbent towel.

Broken Tubes/Blood Spill

Follow your laboratory’s safety precautions for handling and disposal of biohazardous contaminated broken glass when carrying out these steps.

Step 1: If a tube breaks during centrifugation, or if blood otherwise becomes aerosolized, do not open the centrifuge for 30 minutes.

Step 2: After opening the centrifuge, disconnect it from its power source.

Step 3: To remove the broken glass and blood, follow your laboratory’s usual safety precautions (such as use of personal protective equipment and engineered safety devices) and carefully remove broken glass from the bowl of the centrifuge.

Step 4: Remove the buckets and dispose of broken glass/blood.

Step 5: Ensure all broken glass is removed, since fragments of broken tubes may lead to additional breakage.

Step 6: Decontaminate bowl, buckets, rotor, lid, and any other grossly contaminated area with 10% sodium hypochlorite. To reduce the chance of corrosion, always decontaminate for the minimum recommended time. After decontaminating, follow the cleaning procedure, above.

Corrosion

Rotors and structural accessories are manufactured and finished to give maximum resistance to corrosion. However, maximum equipment life requires that you continually inspect the rotor cavities for corrosion, especially after using chlorine ion solutions, such as sodium chloride (saline), and sodium hypochlorite (household bleach). These solutions attack most metals. Follow the Cleaning Procedure and clean the rotor, rotor chamber, and accessories (particularly the sample compartments and bucket cups) thoroughly after each use of sodium hypochlorite. Inspect all surfaces under bright light for corrosion; small crevices will grow deeper and cause failure.

If you see signs of corrosion, contact CareDx Customer Care at [email protected].

Power Cord Replacement

Any sign of damage to the power cord should be reported immediately to CareDx Customer Care. Do not use a centrifuge with signs of wear or damage to the power cord.

Decontaminating Equipment before Shipping

Follow the decontamination instructions included in the return shipping box provided by CareDx.

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SUPPLIES REQUIRED AND SUPPLIED BY CAREDX1. BD Vacutainer® CPT™ Cell Preparation Tubes with

Sodium Citrate (CPT). 8.0 mL

• Store tubes at room temperature (18 to 25° C). Protect tubes from direct light.

• Shelf life at 18 to 25° C is printed on each individual tube. Check expiration date of each tube before use.

2. CareDx Frozen Shipper Pack

• Materials required to properly ship the specimen to CareDx for testing

3. AlloMap Sample Processing Reagents Kit

Consult Instructions for Use (REF LQ-10016)

Contents of the Kit

PBS 10 centrifuge tubes containing 5.0 mL phosphate buffered saline

LDX 10 tubes containing 1.8 mL of LyseDx™ lysing reagent:

• 1% Beta-Mercaptoethanol in buffer RLT (Contains guanidinium thiocya-nate)

PIP 10 sterile disposable transfer pipettes

PI 1 Package insert with instructions for use

QRC 1 Quick reference card

Reagent Storage and Stability

• Sample Processing Reagents: Store sample processing reagents at room temperature (18 to 25° C). Check expiration date printed on each reagent tube before use.

• Processing reagents are single use only. Keep reagent tubes closed until use. Once reagent tubes are opened, use immediately. Do not recap and store for later use.

Ordering Supplies

To order AlloMap testing supplies, contact CareDx Customer Care at [email protected]. Please allow at least a two week lead time for replenishing supplies.

WARNINGS AND PRECAUTIONS• When drawing blood for use in the AlloMap test, follow

your laboratory’s universal precautions for bloodborne pathogens.

• Follow your laboratory’s policy for personal protective equipment (PPE) during sample preparation.

• AlloMap Sample Processing reagents are single use only.

• LyseDx™ lysing reagent contains beta-mercaptoethanol and guanidinium thiocyanate which can cause skin and eye irritation and is considered toxic. Avoid contact with skin, mouth, eyes and clothing.

• Do not mix LyseDx lysing reagent with sodium hypochlorite (chlorine bleach). When cleaning LyseDx reagent spills, the area must first be wiped down with soapy water, after which 10% sodium hypochlorite may be used, if necessary.

• Do not mix reagents between lots.

IMPORTANT: Gene expression continues to change in live cells, therefore the time from phlebotomy to freezing the cell lysate must be no longer than 3 hours.

References http://allomap.com/healthcare-providers/allomap-sample-preparation-training

Safety Data Sheet, Phosphate Buffered Saline

Safety Data Sheet, Lyse Dx

Safety Data Sheet, Dry Ice (CO2)

ALLOMAP TEST REQUISTION FORMS (TRF) AND TEST ORDERSAlloMap Test Requisition Forms (TRF) are pre-printed with your lab information and can be ordered at [email protected]. If you receive a TRF used to order an AlloMap test that has a different lab name on it, please cross out the lab name on the form and add your lab name.

AlloMap tests may be ordered in a number of ways including (examples below):

• AlloMap TRF, the two-page carbon form which contains tube labels for labeling the AlloMap specimens or the single page form without labels (use tube labels from Labels for Lab Use card to label tubes).


• Order sheet, or lab script. Ensure all required specimen and patient information is provided. Attached additional sheets or a TRF as needed.

• Web portal order

• EMR (e.g. EPIC) or LIS order. Ensure all required specimen and patient information is provided. Attached additional sheets or a TRF as needed.

AlloMap TRF completed by transplant center: If the patient presents with a completed AlloMap test requisition form or the transplant center has provided it to your site, use that form to identify the patient, record specimen collection and processing information and label the specimen. Send all paperwork you receive from the transplant center with the processed specimen to CareDx.

Test is ordered using an alternate method (not on an AlloMap TRF): If the patient does not arrive with the AlloMap TRF and the transplant center has not provided it, (i.e., phone orders, faxed orders, lab script or test order form), use a blank TRF and write the patient’s first and last name, date of birth, and medical record number on the AlloMap test requisition. Use this TRF to identify the patient, record specimen collection and processing information. Place an accession label (bar code) on the specimen, the original order and the TRF. Send all paperwork you receive from the transplant center with the processed specimen to CareDx.

Specimen Collection and Processing Information: Specimen collection date, specimen collection time, and specimen in freezer time MUST BE FILLED OUT FOR EVERY TEST.

Comments: Enter any comments relevant to the specimen collection, processing or patient status.

The TRF must accompany the patient specimen into your processing area to record the “In-freezer time” and so that both the tube and the TRF can be labeled with a barcode sticker. Extra barcode labels for labeling paperwork are provided on the TRF (2-page carbon form) and on the “Labels for Lab Use” labels card for use with the one-page TRF. The barcode labels uniquely identify a patient’s specimen. DO NOT use the same barcode label for more than one patient’s specimen. Improperly labeled and unlabeled specimens will not be tested.

Requisitions must accompany patient specimens when they are sent to CareDx. Keep a copy of the TRF for your records, and send a copy to CareDx with the processed patient specimen.

SPECIMEN COLLECTION PROCEDUREAll processing must be completed at room temperature and the lysate from processed specimens must be frozen within 3 hours of blood collection. Freeze the lysate at -15° C or colder, or on dry ice, and ship frozen to CareDx for testing. Specimens should be shipped the day the specimen was collected. If it is not possible to ship the same day as collection (e.g. specimen drawn after FedEx pick-up or on a weekend) ship the specimen to CareDx the next business day.

Collection Steps

Step 1: Identify the patient according to your laboratory’s patient identification policy.

Step 2: Prepare the patient for phlebotomy according to your standard procedure.

Step 3: Select a CPT tube and additional supplies needed to perform venipuncture. Check the tube’s expiration date before use.

Step 4: If collecting additional specimens from the same venipuncture, follow the recommendations of your laboratory for the correct order of collection for citrate tubes. If your laboratory does not have a recommended draw order, draw blood tubes in the order shown below.

Cell-Free DNA BCT

CPT(Glycolyticinhibitor)

Step 5: Collect 8 mL of blood into the CPT tube. Allow the tube to fill completely - flow may slow at the end of the draw.

Note: Minimum blood volume for the 8.0 mL CPT tube is 6 mL. CPT tubes with less than 6 mL of blood are not acceptable for AlloMap testing. Do not process CPT tubes containing less than 6 mL of blood; collect a new specimen for use.

Step 6: Gently invert the tube at least 10 times to fully mix the blood with anticoagulant.

Step 7: Label the tubes according to your laboratory’s procedure for specimen identification.

15

Step 8: Place a bar-coded label from the AlloMap Test Requisition Form (TRF) or “Labels for Lab Use” label card on the CPT tube.

Step 9: On the TRF, fill in the specimen collection date and time.

Step 10: Immediately send the specimen and the TRF to the processing area.

Step 11: Do not refrigerate the specimen.

SPECIMEN PROCESSING PROCEDURE Use only AlloMap Specimen Processing Reagents; do not substitute other general-purpose reagents or supplies. Do not mix reagents between lots. Check the expiration date on each reagent tube before use. The centrifuge speed settings listed in these instructions are for the Drucker 755VES and the Thermo CL2 centrifuges only. The Drucker Horizon 24 centrifuge is a fixed-speed centrifuge. Both centrifugation steps using the Drucker Horizon 24 are performed “as-set”; no user adjustment needs to be made.

If, for any reason, an alternate centrifuge must be used to process a specimen, refer to section V. EQUIPMENT REQUIRED – CENTRIFUGE for centrifuge specifications and call CareDx Customer Care for assistance to ensure that centrifuge specifications and RCF settings are met. Specimens that are processed outside of CareDx specifications cannot be tested.

Step 1: Following specimen collection, make sure the CPT tube is mixed well by inverting it at least 10 times prior to centrifugation. Process specimens at room temperature (18 to 25° C). Centrifuge the CPT tube for 15 minutes at 3400 rpm in the Drucker 755VES or CL2 centrifuge and at the “as-set” speed in the Drucker Horizon 24 centrifuge. Be sure the centrifuge is balanced by centrifuging an even number of specimens or by using a balance tube.

Step 2: From the AlloMap Sample Processing Reagents Box, obtain the centrifuge tube containing phosphate buffered saline ( PBS ).

Step 3: Label the centrifuge tube with a barcode label from the patient’s AlloMap TRF or Labels for Lab Use Card. Place the barcode label in a vertical orientation in the designated space on the PBS reagent label, indicated by: #

Note: Perform the next two steps immediately following centrifugation. Delay may result in the time from specimen collection to freezing to exceed three hours.

Step 4: After centrifugation, invert the CPT tube at least 10 times to re-suspend the separated mononuclear cells into the plasma.

Step 5: Pour the plasma/mononuclear cell mixture (the layer above the gel) from the CPT tube into the labeled centrifuge tube that contains PBS. Cap and invert at least 10 times to mix.

Step 6: Centrifuge for 5 minutes at 3400 rpm in the Drucker 755VES or CL2 centrifuge and “as-set” in the Drucker Horizon 24 centrifuge. Be sure the centrifuge is balanced by centrifuging an even number of specimens or by using a balance tube.

Step 7: The mononuclear cells will form a pellet in the bottom of the tube. Open the tube and place the cap open end up on a clean surface. Pour off and discard the supernatant. Remove as much supernatant as possible by touching the rim of the tube to a clean paper towel or absorbent pad to get the last drop. The cell pellet is sticky and will not easily fall out of the tube.

Step 8: Pipette all of the contents from the LyseDx lysing reagent tube ( LDX ) onto the cell pellet, using the transfer pipette supplied ( PIP ). Vigorously pipette the cell pellet and the LyseDx lysing reagent up and down until the cells have completely disappeared and the lysate is clear. If you are doing this step properly, you will have a foamy mix of cells and reagent, which becomes clear when all cells are lysed. If the pellet is drawn into the bulb of the pipette, draw the LyseDx into the bulb to rinse the pellet down into the tube. Continue mixing until the pellet has been completely lysed and no visible cellular material sticks to the pipette.

Note: Do not vortex.

Step 9: When lysis is complete, replace and tighten the cap on the centrifuge tube, freeze upright at -15° C or colder until shipped to CareDx for AlloMap testing.

Step 10: On the TRF, fill in the time the processed specimen was put in the freezer, or into the dry ice if specimen is to be packaged immediately for shipping.


SPECIMEN ACCEPTANCE CRITERIASpecimens

To be acceptable for AlloMap molecular expression testing, a lysate specimen must meet the following acceptance criteria:

1. Minimum labeling requirements: The centrifuge tube containing the lysate must be labeled with:

• The CareDx accession number that corresponds to the accompanying TRF, or

• Patient name, or a unique patient identifier, such as the medical record number, and the date and time the specimen was drawn. Inadequately labeled specimens will not be tested and will be discarded.

2. Specimen age and condition: The specimen must be:

• Prepared from at least 6 mL blood drawn in BD Vacutainer® CPT™ Cell Preparation Tubes with Sodium Citrate. Refer to section titled “Specimen Collection Procedure”.

• Collected from a patient who is 2 months (at least 55 days) post-transplant.

• Processed with in-date AlloMap Sample Processing Reagents as indicated by the expiration date printed on the individual reagent tubes.

• Processed according to Section X. Specimen Processing Procedure and frozen at -15° C or colder within three hours of collection.

• Shipped to and received at CareDx frozen on dry ice.

Note: Specimens that do not meet the conditions above will not be tested and will be discarded.

Test Order / Test Requisition Form (TRF)

1. No testing will be performed without an authorized test order. A completed TRF/order must be sent to CareDx with the processed specimen.

2. All test orders must include:

• Patient first and last name, or other patient identifier

• A second identifier such as medical record number, or other unique identifying number

• Name and address of the authorized person (physician or transplant coordinator) requesting the test or his/her authorized designee

• Name, address and phone number of the laboratory submitting the specimen

• Date and time of specimen collection

• Date and time lysate was frozen at -15° C or colder

• Diagnosis information (ICD-9 code)

• Any additional information relevant and necessary for accurate reporting of the AlloMap test result

3. The following information must be either on the test order or previously provided to CareDx:

• Date of birth

• Gender

• Transplant date

4. Customer Care will contact the laboratory or transplant center to obtain any required information that is missing from the TRF. All communication(s) required to obtain the missing information will be documented by the CareDx representative.

Unacceptable Specimen

• CareDx Customer Care or designee will contact the requesting facility and referring laboratory before discarding a specimen that is unacceptable for testing.

• If the specimen is to be discarded and not used for testing, a new specimen will be requested.

• A written report stating the reason for rejection and discard will be sent to the transplant center.

SPECIMEN SHIPPING PROCEDURE Important Safety Notes

The frozen lysate is derived from blood. Follow your laboratory’s universal precautions for bloodborne pathogens.

When handling dry ice, follow your laboratory’s safety procedures.

• Never handle dry ice with your bare hands. Dry ice has a temperature of -109° F (-78° C), cold enough to freeze skin cells and cause an injury similar to a burn. When handling dry ice, always use insulated gloves.

• Use dry ice in a ventilated area.

17

• Store dry ice in an appropriate dry ice storage chest. DO NOT USE AIRTIGHT CONTAINERS as these may provide an explosive hazard.

• Dry ice is a skin and eye irritant. Avoid contact with skin, mouth, eyes, and clothing.

• Use personal protective equipment – gloves, eye protection, and lab coat or apron.

Equipment, Materials, and Supplies

CareDx supplies the Frozen Shipper Pack which includes the following:

• Aqui-Pak 6-Bay absorbent pouch

• 95 kPa Specimen Transport Bag

• Dry ice label filled out with 2.3 kg in all sections and affixed to outer shipping box

• List of Contents card

• Cooler

• Outer shipping box

• Monday through Thursday FedEx Shipping Packet containing:

– Priority Alert Airbill, pre-printed for Priority or First Overnight service, depending upon service level required

• Friday FedEx Shipping Packet containing:

– Priority Alert Airbill, pre-printed for Saturday HOLD service

– Saturday Delivery stickers

– HOLD Sticker addressed to 900 Gateway Blvd., South San Francisco, CA 94080

• 9 x 12 plastic zip bag

• Instructions for Shipping Card

Your laboratory will provide the following:

• Dry ice – pellet form is recommended, approximately 1.5 cm x 4 cm

• Packing tape for outer box

Packaging and Shipping

IMPORTANT: Once you begin this process, work quickly. Do NOT let the specimens thaw. Do not expect dry ice to

last more than 24 hours. Ship specimens the same day collected to ensure the quickest turnaround time for results.

Step 1: Prepare the cooler by removing the contents (absorbent pouch, bags, labels, etc.).

Step 2: Have the dry ice ready; keep the specimen tube(s) in the freezer as long as possible.

Step 3: Insert the frozen specimen tube(s) into the Aqui-Pak 6-Bay absorbent pouch. The pouch can hold a maximum of 6 specimen tubes.

Step 4: Roll the pouch with the tube(s) and put it into the 95 kPa Specimen Transport Bag. Seal this bag closed. Each bag holds one absorbent pouch with up to 6 tubes.

Step 5: The cooler can hold up to 6 specimens. If you have more than 6 specimens, use a second Frozen Shipper Pack.

Step 6: Place the Transport Bag with the specimen(s) in the cooler and pack with pellets or small chunks of dry ice. Snow-like dry ice will not last 24 hours. Fill the cooler with as much dry ice as possible; generally, a cooler with up to 6 specimens holds approximately 5 pounds (2.3 kgs) of dry ice. If there are more than 6 tubes to ship, use a second Frozen Shipper Pack.

Step 7: Place the lid on the cooler. DO NOT tape the lid shut.

Step 8: Fold the test requisition forms in half, place them in the 9 x 12 plastic zip bag and place it on top of the lid.

Step 9: Place the List of Contents card on top of the plastic zip bag.

Step 10: Close the outer box and seal with packing tape.

Step 11: The Dry Ice Shipping label has been filled in with your laboratory name and the maximum amount of dry ice in kilograms that will fit in the box. This label has already been affixed to the shipping box.

Step 12: To expedite specimens, CareDx uses the FedEx Priority Alert service. With this service, FedEx tracks each shipment and ensures that it arrives at the destination on time. Be sure to use only the special, Priority Alert Airbills that come with the CareDx Frozen Shippers. Priority Alert cannot be used with FedEx Online, so please follow these instructions. Based on the day of the week that you are shipping, use the appropriate FedEx Shipping Packet.


For FedEx shipments sent Monday through Thursday:

Select the Packet marked “USE OVERNIGHT DELIVERY PACKETS ONLY ON MONDAY—THURSDAY SHIPMENTS TO CareDx”.

Ensure that the Priority Alert FedEx airbill, a multi-part paper form, is marked in Section 4a for FedEx Priority or First Overnight service. Follow the instructions below for proper completion of the airbill.

For FedEx shipments sent Friday:

Select the Packet marked “USE SATURDAY DELIVERY PACKETS ONLY ON FRIDAY SHIPMENTS TO CareDx”.

Step 1: Place the yellow and white Saturday Delivery stickers on the outside of the box. Do not cover any of the markings on the box.

Step 2: Place the white and orange HOLD sticker on top of the shipper box next to the airbill. Ensure that the address on the sticker is 900 Gateway Blvd., South San Francisco, CA 94080.

Step 3: Ensure that the Priority Alert FedEx airbill, a multi-part paper form, is marked in Section 3 for HOLD Saturday at FedEx Location. Follow the instructions below for proper completion of the airbill.

Completion of the Airbill for all shipments using the multi-part paper Priority Alert FedEx airbill:

Step 1: Enter the shipment date and enter or check that the correct sender information is entered in Section 1.

Step 2: Enter or check that the correct recipient information is entered in Section 3.

Step 3: Enter or check that the Dry Ice Section is marked and it includes the weight of the dry ice in kilograms (2.3 kg for 5 lbs. of dry ice) in Section 6. The weight entered must match the weight on the Dry Ice Shipping Label.

Step 4: Enter or check that the number of packages and total weight (3 kgs) is entered in Section 7 (2.3 kgs of dry ice + 0.7 kg for the shipper, tubes and paperwork).

Step 5: When finished, remove and keep the top copy of the airbill for your records.

Step 6: Affix the airbill to the top of the shipper box.

Step 7: Arrange for FedEx Express shipping pickup.

References

DOT Hazardous Materials Regulations for Diagnostic Specimens, sec. 173.199: diagnostic specimens and used health care products.

IATA Package Instruction 650

IATA Packing Instruction 904 – Dry Ice

INTERPRETATION OF RESULTS An AlloMap Score between 0 and 40 is calculated based on the gene expression levels of the test and control genes. The score and additional data are provided to the clinician on a Test Report. The range of AlloMap scores observed in 1002 CARGO samples tested was 2 to 39 (see Performance Characteristics section below).

The performance characteristics of the AlloMap Test were established with samples from heart transplant recipients at least 15 years of age and at least 2 months (≥ 55 days) post-transplant at the time samples were collected.

The use of AlloMap scores in the management of heart transplant patients is defined for each patient by the physician based on their clinical experience and knowledge of their patient populations. Expected Negative Predictive Values (NPV) will be dependent upon the threshold selected by the physician.

PERFORMANCE CHARACTERISTICS The CARGO Study2

Polymerase Chain Reaction (PCR) technology provides a sensitive, specific, and reproducible measurement of gene expression. The emergence of this technology along with the development of statistical and database tools capable of storing and analyzing gene expression data made it possible for CareDx to develop the AlloMap test for use in monitoring cardiac allograft rejection. This technology was used in the CARGO (Cardiac Allograft Rejection Gene Observational) study, a multi-center collaboration that provided the basis for the development of the AlloMap test. CARGO was a prospective, observational, multi-center protocol active from 2001 to 2005 whose prime objectives were to develop:

• A sample archive of RNA samples prepared from peripheral blood obtained concurrently during endomyocardial biopsy (EMB) procedures

19

• A database of clinical information collected at the time of EMB. The study enrolled 737 subjects at 9 heart transplant centers in the United States and yielded an archive of data and RNA samples for 5834 clinical visits. Clinical data was entered into the study database.

CareDx sequenced 25000 clones from leukocyte cDNA libraries and incorporated more than 8000 probe sequences representing these clones into custom microarrays and used them to examine gene expression. Patient blood samples were obtained at the time of biopsy and the expression levels of these 8000 genes were determined and compared to the biopsy result. A subset of more than 250 candidate genes showed promise as markers to discriminate rejection from no rejection.

The next phase of the CARGO study used RT-PCR to measure gene expression levels of the candidate genes. These studies provided highly quantitative and reproducible measures of expression levels for each gene and were correlated with the biopsy readings and overall clinical results.

Clinical Validation

Clinical validation was performed to evaluate the performance characteristics of the AlloMap test and statistical informativeness of the AlloMap test in cardiac transplant patients. Samples used for the study were derived from the CARGO study. The objective of the clinical validation was to estimate predictive values for the AlloMap test for ACR in the intended clinical population.

Diagnostic Classifications

This study used the following definition for rejection: a local biopsy grade ≥ 3A that was also assigned grade ≥ 3A by at least one of the three panel pathologists (“confirmed rejection”). All local ≥ 3A biopsies were graded by the central pathologists. The “no rejection” class included all samples that did not qualify as rejection.

Samples and Patients

A total of 300 samples, from 154 patients enrolled in CARGO were assayed with the AlloMap test but were not used to develop the AlloMap test algorithm. These samples conformed to the following inclusion criteria:

• At least 55 days since transplantation, and

• More than 30 days after administration of immunosuppressive therapy for treating rejection.

Statistical Analysis

The specific objectives of the clinical validation were:

• To test the informativeness of the test as measured by Receiver Operator Characteristic (ROC) curve analysis, specifically Area Under the Curve (AUC)

• To estimate the clinical performance of the test for all integer AlloMap test scores (0-40) and indicated patients in two time periods post-transplant: 55-182 days (“2-6 months”) and ≥ 183 days (“> 6 months”).

Clinical Performance Metrics Estimated:

• Percentage of AlloMap test scores below a specific AlloMap test score

• Negative Predictive Value (NPV) for ACR for scores below a specific AlloMap test score

• Positive Predictive Value (PPV) for ACR for scores at or above a specific AlloMap test score

Results and Conclusions

The AUC calculated from the 300 samples from 154 patients was 0.67 with 95% confidence interval from 0.56 to 0.78 calculated by bootstrap. The AUC for the 55-182 days post-transplant period was 0.71 with 95% confidence interval from 0.56 to 0.84. The AUC for the ≥ 183 days post-transplant period was 0.67 with 95% confidence interval from 0.50 to 0.88.

Clinical Performance Characteristics

Table 1, on page 12, provides the clinical performance characteristics for two to six months post-transplant and greater than 6 months post-transplant.

Reproducibility/Precision3

Reproducibility for the AlloMap test process was established for the following variables:

• Run to Run

• Operator to Operator

• Lot-to-lot, plate-to-plate and plate section-to-section

Reproducibility of AlloMap scores was analyzed in healthy donor and patient samples from the Specimen Handling and Testing Reproducibility study. Run-to-run and operator-to-operator variability was analyzed in Arm A data. Lot-to-lot, plate-to-plate and section-to-section variability was analyzed in Arm C data. The AlloMap test process


demonstrated acceptable precision, summarized below in Tables 2-16, with respect to coefficients of variation (CV’s). Summary of maximum CV’s are:

• Overall CV’s (Tables 2, 3, 4, and 5):

– 4 Donor Samples (Arm A): ≤ 6.3%

– 6 Patient Samples (Arm A): ≤ 10.4%

– Donor Sample (Arm C): 3.8%

– Pooled Patient Sample (Arm C): 4.4%

• Run-to-run CV’s (Tables 4, 5, 6)

– Donor Samples: ≤ 9.2%

– Patient Samples: ≤ 11.9%

• Operator-to-operator CV’s (Tables 7, 8)



• Lot-to-lot CV’s (Tables 11, 12)



• Plate-to-plate CV’s (Tables 13, 14)



• Section-to-section CV’s (Tables 15, 16)



Coefficients of variation are acceptable and comparable between donors and patients.

AlloMapScore

> 2 – 6 Months (n=166 samples) > 6 Months (n=134 samples)AlloMapScore%- Pts

Belowppv

≥ 3a(2r)PPV

Std. Err.NPV

< 3A(2R)NPV

Std. Err.%- Pts Below

PPV ≥ 3A(2R)

PPVStd. Err.

NPV < 3A(2R)

NPV Std. Err.

≤ 19 < 22.4 ≤ 2.7 % ≤ 0.1 % 100.0 % 0.0 % ≤ 5.4 ≤ 1.8 % 0.0 % 100.0 % 0.0 % ≤ 19

20 24.3 % 2.8 % 0.2 % 100.0 % 0.0 % 8.1 % 1.8 % 0.1 % 100.0 % 0.0 % 20

21 33.6 % 2.5 % 0.4 % 98.8 % 0.8 % 9.8 % 1.9 % 0.1 % 100.0 % 0.0 % 21

22 38.8 % 2.7 % 0.5 % 98.9 % 0.7 % 11.0 % 1.9 % 0.1 % 100.0 % 0.0 % 22

23 41.8 % 2.9 % 0.5 % 99.0 % 0.6 % 14.1 % 2.0 % 0.1 % 100.0 % 0.0 % 23

24 47.5 % 3.2 % 0.6 % 99.1 % 0.6 % 18.4 % 2.1 % 0.1 % 100.0 % 0.0 % 24

25 56.0 % 3.8 % 0.7 % 99.3 % 0.5 % 22.1 % 2.2 % 0.1 % 100.0 % 0.0 % 25

26 61.4 % 3.8 % 0.9 % 99.0 % 0.5 % 26.8 % 2.3 % 0.1 % 100.0 % 0.0 % 26

27 63.6 % 3.4 % 1.0 % 98.7 % 0.5 % 31.6 % 1.9 % 0.4 % 98.7 % 0.9 % 27

28 68.3 % 3.3 % 1.1 % 98.5 % 0.5 % 39.1 % 2.1 % 0.5 % 98.9 % 0.7 % 28

29 73.7 % 4.0 % 1.3 % 98.6 % 0.4 % 40.8 % 2.1 % 0.5 % 99.0 % 0.7 % 29

30 77.2 % 4.6 % 1.6 % 98.6 % 0.4 % 50.6 % 2.1 % 0.6 % 98.7 % 0.6 % 30

31 81.0 % 3.3 % 1.6 % 98.2 % 0.4 % 54.1 % 2.3 % 0.7 % 98.8 % 0.6 % 31

32 85.6 % 2.9 % 2.0 % 98.0 % 0.3 % 63.1 % 2.9 % 0.9 % 99.0 % 0.5 % 32

33 89.4 % 4.0 % 2.7 % 98.1 % 0.3 % 72.4 % 3.8 % 1.3 % 99.1 % 0.4 % 33

34 91.7 % 5.0 % 3.5 % 98.2 % 0.3 % 79.1 % 4.1 % 1.7 % 98.9 % 0.4 % 34

35 94.5 % 5.7 % 4.8 % 98.1 % 0.2 % 84.1 % 4.0 % 2.2 % 98.7 % 0.4 % 35

36 97.3 % 7.6 % 13.8 % 98.1 % 0.2 % 90.2 % 5.4 % 3.2 % 98.7 % 0.3 % 36

37 97.8 % 9.5 % 21.1 % 98.1 % 0.2 % 91.7 % – – 98.4 % 0.2 % 37

38 100.0 % – – 97.9 % 0.0 % 96.5 % – – 98.2 % 0.0 % 38

39 100.0 % – – 97.9 % 0.0 % 97.7 % – – 98.3 % 0.0 % 39

Table 1: Clinical Performance Characteristics of the AlloMap Test

21

Donor n Mean SD CV (%)

D1 28 30.047 1.654 5.5%

D2 28 30.678 1.105 3.6%

D3 30 28.365 1.771 6.2%

D4 30 30.139 1.897 6.3%

Patient n Mean SD CV (%)

Stanford1 8 27.456 1.642 6.0%

Stanford2 8 34.481 0.598 1.7%

Stanford3 8 31.656 2.014 6.4%

UCLA1 6 33.720 0.295 0.9%

UCLA2 4 34.858 1.262 3.6%

UCLA3 6 24.263 2.534 10.4%

Run: 1 2

Donor n Mean SD CV (%) n Mean SD CV (%)

D2 28 30.678 1.105 3.6% 3.6% 3.6% 3.6% 3.6%

D3 30 28.365 1.771 6.2% 6.2% 6.2% 6.2% 6.2%

D4 30 30.139 1.897 6.3% 6.3% 6.3% 6.3% 6.3%

D4 30 30.139 1.897 6.3% 6.3% 6.3% 6.3% 6.3%

Table 2: Overall statistics for 4 donor samples (D1-D4), 4 runs per sample, 4 operators (Op1-Op4) per run and 2 reagent plates per operator

Table 3: Overall statistics for 6 patient samples (Stanford1-Stanford3 and UCLA1-UCLA3), 2 runs per sample, 2 operators (Op1 and Op2) per run and 2 reagent plates per operator. NOTE: no run 2/ operator 2 data obtained for UCLA samples

Table 4: Descriptive statistics for run to run assessment for donor samples

Table 6: Descriptive statistics for run to run assessment for patient samples

Table 5: Descriptive statistics for run to run assessment for donor samples (cont.)

Run: 1 2


D2 8 29.175 1.752 6.0% 8 29.702 2.284 7.7%

D3 8 32.306 0.648 2.2% 8 30.314 0.887 2.9%

D4 8 28.071 2.594 9.2% 8 28.740 1.418 4.0%

D4 8 29.105 2.607 9.0% 8 31.185 1.986 6.4%

Run: 1 2

Patient n Mean SD CV (%) n Mean SD CV (%)

Stanford1 4 27.911 1.878 6.7% 4 27.001 1.487 5.5%

Stanford2 4 34.748 0.442 1.3% 4 34.214 0.670 2.0%

Stanford3 4 32.868 1.410 4.3% 4 30.444 1.887 6.2%

UCLA1 4 33.730 0.354 1.0% 2 33.700 0.241 0.7%

UCLA2 2 34.320 0.319 0.9% 2 35.396 1.875 5.3%

UCLA3 4 23.556 2.804 11.9% 2 25.679 1.585 6.2%


Operator: Op3 Op4


D1 8 30.258 0.829 2.7% 8 28.723 1.748 6.1%

D2 8 30.932 1.507 4.9% 8 30.574 0.931 3.0%

D3 8 28.759 1.437 5.0% 8 28.567 2.082 7.3%

D4 8 31.342 0.838 2.7% 8 29.453 1.240 4.2%

Table 7: Descriptive statistics for operator to operator assessment for donor samples

Table 8: Descriptive statistics for operator to operator assessment for patient samples

Table 9: Overall statistics for 1 donor sample using 3 reagent lots (made from 3 distinct lots of raw materials), 4 plates per lot and 4 sections per plate

Table 12: Descriptive statistics for reagent lot assessment for patient samples

Table 10: Overall statistics for 1 pooled patient samples using 3 reagent lots (made from 3 distinct lots of raw materials), 4 plates per lot and 4 sections per plate

Table 11: Descriptive statistics for reagent lot assessment for donor samples

Operator: Op1 Op2


D1 6 30.631 1.896 6.2% 6 30.947 1.294 4.2%

D2 6 29.957 0.971 3.2% 6 31.198 0.437 1.4%

D3 6 28.649 1.241 4.3% 8 27.557 2.110 7.7%

D4 6 30.072 2.499 8.3% 8 29.671 2.399 8.1%

Operator: Op1 Op2

Patient n Mean SD CV (%) n Mean SD CV (%)

Stanford1 4 26.843 1.552 5.8% 4 28.069 1.697 6.0%

Stanford2 4 34.459 0.851 2.5% 4 34.502 0.328 1.0%

Stanford3 4 31.722 1.396 4.4% 4 31.590 2.740 8.7%

UCLA1 4 33.663 0.162 0.5% 2 33.835 0.562 1.7%

UCLA2 4 34.858 1.262 3.6% 0 — — —

UCLA3 4 23.644 2.771 11.7% 2 25.503 2.114 8.3%

N Mean SD CV(%)

48 31.630 1.214 3.8%

N Mean SD CV(%)

48 28.875 1.273 4.4%

Lot n Mean SD CV(%)

1 16 31.279 1.470 4.7%

2 16 31.258 0.920 2.9%

3 16 32.354 0.878 2.7%

Lot n Mean SD CV(%)

1 16 28.611 1.299 4.5%

2 16 28.582 1.284 4.5%

3 16 29.431 1.114 3.8%

23

LIMITATIONS OF PROCEDUREPatient population

The AlloMap test has not been validated for use in patient populations other than heart transplant recipients who are at least 15 years old and at least 55 days post-transplant. Corticosteroid dosage

Systemic corticosteroid dosage of > 20 mg/day of prednisone or equivalent may artificially decrease the AlloMap score.4

Following rejection therapy

The CARGO Clinical Validation study excluded all samples from patients who had received rejection therapy within the past 21 days. The performance characteristics of AlloMap testing in such samples, therefore, have not been established.2

Following transfusion

The CARGO Clinical Validation study excluded all samples from patients who had received a transfusion within the past 30 days. The performance characteristics of AlloMap testing in such samples, therefore, have not been estab-lished.2

Dual Organ Transplant Populations

The performance characteristics of AlloMap testing in a recipient of a cardiac allograft and another non-cardiac organ transplant (e.g. kidney) have not been established.

INTERFERING SUBSTANCESAssessment of Endogenous and Exogenous Interferents

Table 17 (on back page) outlines endogenous and exogenous substances and concentrations tested. No interference was observed at the concentrations tested for any of the substances evaluated.

Genomic DNA Contamination3

Genomic DNA (gDNA) was evaluated for potential inter-ference on AlloMap test Scores. Results demonstrate that samples may contain up to 25% gDNA without interfering with the AlloMap test score and that CareDx sample pro-cessing and QC procedures provide adequate control over potential gDNA interference with the AlloMap test results.

Cytomegalovirus Infection3

To determine if immune system activation associated with cytomegalovirus (CMV) viremia affected the performance of the AlloMap test, AlloMap test scores were compared between CMV viremic patients and CMV non-viremic patients. Results indicated that the immune system activation associated with CMV infection present in peripheral blood samples at the time of sample collection did not interfere with the performance of the AlloMap test.

Table 13: Descriptive statistics for plate to plate assessment for donor samples

Table 15: Descriptive statistics for plate section to section assessment for donor samples (Note: UL=upper left quadrant of plate; LL = lower left quadrant of plate; UR=upper right quadrant of plate; LR=lower right quadrant of plate.

Table 16: Descriptive statistics for plate section to section assessment for patient samples (Note: UL=upper left quadrant of plate; LL = lower left quadrant of plate; UR=upper right quadrant of plate; LR=lower right quadrant of plate.

Table 14: Descriptive statistics for plate to plate assessment for patient samples

Plate n Mean SD CV(%)

1 12 31.428 1.594 5.1%

2 12 31.864 0.960 3.0%

3 12 31.467 1.010 3.2%

4 12 31.762 1.289 4.1%

Plate n Mean SD CV(%)

1 12 28.834 1.116 3.9%

2 12 29.359 1.323 4.5%

3 12 28.958 1.304 4.5%

4 12 28.349 1.287 4.5%

Sections n Mean SD CV(%)

UL 12 31.771 1.035 3.3%

LL 12 31.072 1.764 5.7%

UR 12 31.735 1.026 3.2%

LR 12 31.942 0.759 2.4%

Sections n Mean SD CV(%)

UL 12 29.244 0.997 3.4%

LL 12 28.558 1.645 5.8%

UR 12 28.896 1.036 3.6%

LR 12 28.801 1.373 4.8%

CAREDX CUSTOMER CAREContact: Phone 1-888-255-6627 Fax 1-415-287-2456 Email [email protected]

Hours (PST): Monday–Friday, 6:00 am – 5:00 pm, Saturday, 8:00 am – 3:00 pm, closed Sunday

Order Supplies: Email [email protected]

© 2019 CareDx, Inc. All service marks or trademarks are owned or licensed by CareDx, Inc. or its affiliates. All rights reserved.

LT-10067 Revision 2 Effective 2019-10

Assessment of Immunosuppression Therapy3

To determine if immunosuppression therapy affected the performance of the AlloMap test, serum drug levels for cyclosporine A, tacrolimus and sirolimus were evaluated from 700 clinical samples. The level of concordance between AlloMap test scores and biopsy grades were compared in samples with therapeutic drug levels versus those samples with drug levels greater than the therapeutic range. Results show there was no detectable effect of drug level on AlloMap test scores.

CAREDX CLINICAL LABORATORY QUALITY MANAGEMENT Information regarding the CareDx clinical laboratory Quality Management program and current laboratory licensure status will be provided upon request.

Laboratory licenses, permits and accreditations held by the CareDx Laboratory are listed below.

CLIA: 05D1029609 CAP: 7193868 New York: Permit #8193 Pennsylvania: Lab ID #29355A Rhode Island: LCO01129 Maryland: Permit #1254 California: Lab ID: CLF 00332008

REFERENCES1. Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R.

Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY) 1992:10, 413-417.

2. Deng M. et al. Noninvasive Discrimination of Rejection in Cardiac Allograft Recipients using Gene Expression Profiling. Am J Transplant 2006. 6:150-160.

3. Data on file at CareDx.

4. Starling RC, Deng MC, Kobashigawa JA, Walther D, Wohlgemuth JG, Rosenberg S, Mehra MR. The influence of corticosteroids on the alloimmune molecular signature for cardiac allograft rejection. J Heart Lung Transplant. 2006;24 (suppl):S65.

Material Concentrations Tested Recommended CLSI Test Concen-trations*

Endogenous

Hemoglobin (HgB) 2.0 mg/mL 2.0 mg/mL

Bilirubin 20 mg/dL 342 µmol/L (20 mg/dL)

Triglyceride 37 mmol/L 37 mmol/L

Exogenous

Heparin 13.3 U/mL 0.35 – 1.0 U/mL

Acetylsalicylic Ac-id 3.62 mmol/L 3.62 mmol/L

Acetaminophen 1324 µmol/L 1324 µmol/L

Table 17: Interference testing of endogenous and exogenous substances3

* Clinical and Laboratory Standards Institute, CLSI EP7-A2 (Vol.25, No 27, 2005) recommended test concentrations.

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