PreciseType™ HEA Test

Molecular Technology Personalizes Donor/Patient

Compatibility

The first in the line of Immucor PreciseType personalized-medicine diag-

nostics, the PreciseType HEA (human erythrocyte antigen) test rapidly

and accurately predicts blood compatibility between donors and patients

to help prevent mismatches that can cause a serious immune reaction

(alloimmunization) in patients. Each year, almost five million Americans need

a blood transfusion1. Blood incompatibility remains a significant problem2-5

with lifelong consequences that adds to the burden of healthcare delivery

and may result in life-threatening delays in care. In standard cases, two to

six percent of transfusion patients are alloimmunized. In certain popula-

tions that require multiple transfusions because of blood disorders, such as

sickle cell disease or thalassemia, the alloimmunization rate reaches as high

as 36 percent because the donor blood was not a close enough match to

their own6. Sickle cell disease and thalassemia are inherited blood disor-

ders in which the body makes an abnormal form of hemoglobin, the protein

in red blood cells (RBCs) that carries oxygen.

Knowledge = Power to Improve Health Outcomes

Medical researchers have made great strides over the last two decades in

understanding the genetics behind transfusion reactions and the impor-

tance of a precise phenotype match. Scientists have sequenced the genes

encoding blood-group systems and have identified the genetic mutations

associated with the development of specific antigens. If an antigen-nega-

tive patient receives blood from an antigen-positive donor, it could trigger

an immune reaction, where the blood recipient’s immune system develops

antibodies that can attack and reject the donor RBCs. Immucor’s Pre-

ciseType HEA test leverages the latest advances in medical research and

molecular technology. It is the first FDA-approved test that uncovers the

specific gene variants associated with RBC antigens to provide the clos-

est match now possible between donor and recipient in a single test. The

PreciseType HEA test uses 24 known gene mutations to identify 35 red

blood-cell antigens and three phenotypic variants from 11 blood groups

simultaneously.

Page 2

Personalized Medicine Begins with an Accurate RBC Genotype/ Phenotype Profile

With today’s dual focus on improving health outcomes and lowering health-

care costs, preventing alloimmunization (development of antibodies that

attack antigens seen as “foreign” by the immune system) is the ultimate

goal in transfusion medicine. Accordingly, a best practice for the hospital or

transfusion center is to create a patient phenotype profile with the Precise-

Type test before a patient receives his or her first-ever transfusion. Donor

blood would be similarly profiled and catalogued, with rare types saved for

special cases, to facilitate selection of only those units that are a molecular

match between donor and recipient. If the patient has already received

multiple transfusions, a PreciseType molecular profile remains a critical tool

to identify the best-matched donor blood. A limitation of traditional serol-

ogy testing is that it can be difficult to interpret for some months after a

transfusion when donor red cells remain along with the patient’s own in the

sample. By contrast, the PreciseType assay is a molecular-level test—multi-

ple RBC populations do not compromise interpretability or accuracy.

Sample to Result with the PreciseType HEA Test

The PreciseType process begins with extraction and

isolation of deoxyribonucleic acid (DNA) from a sample

of whole blood.

Sample

Preparation

Manufacturing and Molecular-level PrecisionMolecular-level precision starts with tiny

beads, which are just three microns across—

about one-tenth the diameter of a human hair.

Each bead is stained with a distinct blend of

ultra-violet, blue, and green fluorescent dyes

that gives it a unique color signature. Each

bead also

contains more

than one

million copies

of a particular

“blood-group

specif-

ic probe”

(allele-specific oligonucleotide) that are

bound to its surface. Different colored beads

carry different probes. The probes work like

nanosized docking stations customized to

detect the presence of 24 known gene mu-

tations (antigen-specific polymorphisms) that

determine the RBC antigens. About 4,000

differently colored beads (with about 50 or 60

copies of each color) are affixed randomly to

the surface of a silicon chip to form a single

BeadChip. The fully assembled BeadChips are

then bonded to a glass slide or a plate. The

two available formats are designed to increase

testing efficiency in labs with different through-

puts. The slide format allows eight, and the

plate format allows 96 simultaneous evalua-

tions of patient and/or donor samples. Each

slide and plate is barcoded with a unique ID.

Page 3

A chain reaction makes thousands to millions of

copies of multiple segments of DNA so the mutations

of interest that reside within the amplified segments

can be analyzed.

Following PCR, the excess reactants are deacti-

vated enzymatically. Enzymes then separate the

double-stranded DNA helix into two single strands,

digesting one, and leaving the other intact. The re-

maining single strand of the original DNA segment is

combined with the signal-development reagent and

pipetted onto the surface of each BeadChip, which

are then placed in a hybridizing oven.

STEP ONE:

Multiplex PCR

(polymerase

chain reaction)

STEP TWO:

Post-PCR

Processing

Unlocking Assay ResultsAs part of the manufacturing process, a bead map (i.e. location of the colored beads carrying the blood-group-specific probes) is established for each BeadChip. The bead-map key for all BeadChips manufactured in a lot is stored on a CD, included with each test kit. After running the assay, this unique beadmap key is required to analyze assay results.

What’s in the Kit?The PreciseType HEA Kit includes supplies to

perform 96 tests in two available formats: 12

eight-chip slides or one 96-chip microplate.

The kit includes two boxes.

The first box

contains all the

required PCR,

post-PCR,

and signal-

development

reagents along

with a negative control necessary for the test.

The second

box includes

the barcoded

BeadChip

slides or plate,

along with

a disk that

holds the chip-specific bead-map key, neces-

sary to analyze results after running the assay.

Page 4

In the warm, humid environment of the oven, DNA

sequences hybridize to the blood-group-specific

probes on the BeadChip. If the DNA sequence in the

sample matches perfectly with the probe sequence on the BeadChip, the

probe will be extended through the incorporation of fluorescently tagged

nucleotides.

If there is not a precise match, elongation does not occur.

STEP THREE:

Hybridization

Limitations of SerologyAt the March 2014 meeting of the FDA

Blood Products Advisory Committee,

Orieji Illoh, MD,OBRR, deputy director in

the FDA’s Office of Blood Research and

Review, Division of Blood Applications,

outlined some of the limitations of serology

in her introductory statement. Dr. Illoh said

that in many cases, there are no licensed

antisera to most rare antigens and when

licensed antisera do exist, availability can

sometimes be limited. She added that rou-

tine screening of large numbers of donor

units for all potential antigens is hampered

because serological processes are time

consuming and resource intensive. Dr. Illoh

also highlighted the difficulty in determining

antigen specificity in a person who has

had recent transfusions, given the multiple

populations of red cells that remain in the

blood sample. In addition, she noted that

the presence of an auto-antibody (a

positive DAT) can also affect serology

results, as could differences in reagent

reactivity.7

Page 5

Next, the Beadchips are imaged with Immucor’s AIS

(Array Imaging System), a specially equipped fluores-

cence microscope-based reader. The reader detects

where there is a fluorescent signal in the assay image,

and then determines which particular blood-group-spe-

cific probe was associated with the signal. Immucor’s proprietary BASIS™

software suite then uses proprietary algorithms to interpret the results, and

generates a report detailing the blood group genotype and phenotype.

Hands-on time from multiplex PCR through results

reporting is about one-and-a-half hours

Not including sample preparation, running the PreciseType HEA assay from

multiplex PCR through results reporting takes approximately six hours for

up to 96 samples. This allows for completion of the assay within a single

work shift.

STEP FOUR:

Analysis &

Results

Becoming the New Standard of

Care in Molecular Red Blood

Cell Matching

The use of serology to determine red

blood cell antigen phenotypes has

been very successful in providing safer

transfusions. However, there are limita-

tions (see sidebar on page four), which

are addressed by the use of molecular

technology, like the Immucor Precise-

Type assay. The company introduced a

research-use-only version of its molec-

ular HEA assay in 20058. Since then,

more than 80 sites have conducted

approximately 700,000 tests, including

many of the largest donor centers

and most-prominent medical centers

around the globe. The test received

a CE IVD mark in May of 2010, and

currently 35 centers are using the tech-

nology outside of the U.S., primarily

in Europe. With its approval of the

PreciseType HEA test, the FDA has

ushered in a new era—where molecular

blood donor/recipient matching can

finally become the standard of care.

References

1. NIH National Heart, Lung, and Blood Institute. Who Needs a Blood Transfu-sion? Available at: http://www.nhlbi.nih.gov/health/health-topics/topics/bt/whoneeds.html Accessed May 22, 2014.

2. US Food and Drug Administration. Annual Summary for Fiscal Year 2010. 2010. Fatalities Reported to FDA Following Blood Collection and Transfu-sion. Available at: http://www.fda.gov/biologicsbloodvaccines/safetyavail-ability/reportaproblem/transfusiondonationfatalities/ucm254802.htm. Accessed May 10, 2014.

3. Immunohematology. 2012; 28:1–30. Available at: http://www.redcross.org/images/MEDIA_CustomProductCatalog/m7240089_28_1_12.pdf. Accessed May 10, 2014

4. Kochman SA. Role of the Food and Drug Administration in the use of molecular techniques in immunohematology. Transfusion. 2007;47(1 Sup- pl):3S–9S. Available at: http://www.med.upenn.edu/cstr/documents/clinical-problem.pdf. Accessed May 10, 2014

5. Mazonson P, Efrusy M, Santas C et al. The HI-STAR study: resource uti-lization and costs associated with serologic testing for antibody-positive patients at four United States medical centers. Transfusion. 2014;54:271–277. Abstract available online at: http://onlinelibrary.wiley.com/doi/10.1111/trf.12176/abstract. Accessed May 10, 2014.

6. Transfusion Medicine Reviews. Vol 22, No 2 (April), 2008: pp 117-132. Available at: http://imunohematologia.com.br/ello_plugins/content/images/file/51_artigo_transfusion_medicine_reviews.pdf

7. Transcript FDA Center for Biologics Evaluation and Research 109th Meet-ing of the Blood Products Advisory Committee. March 18, 2014. Available at: http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeet-ing-Materials/BloodVaccinesandOtherBiologics/BloodProductsAdvisory-Committee/UCM392257.pdf. Accessed May 10, 2014

8. Hashmi G, Shariff T, Seul M, Vissavajjhala P, HueRoye K. A flexible ar-ray format for large-scale, rapid blood group DNA typing. Transfusion. 2005;45:680–688. Abstract available at: http://onlinelibrary.wiley.com/doi/10.1111/j.1537-2995.2005.04362.x/abstract. Accessed May 10, 2014

©2014 Immucor, Inc.⎥ rev. 01-2014 ⎥ 530-00002

MEDIA CONTACT

Caroline Grossman

781.771.5579

caroline.grossman@gmail.com

To learn more, call 855.IMMUCOR (855.466.8267), or visit www.immucor.com.