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Automation to PreciseType and Everything in Between

Jessie Singer MT(ASCP)Transfusion Medicine

Children’s Hospital Los Angeles

Disclosures

• None

Objectives

• Describe the application of molecular testing in the hospital donor center and transfusion service.

• Discuss the impact of molecular testing on serologic antibody identification workups through case studies.

• List the benefits of leveraging software to simultaneously view serology and molecular testing.

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CHLA Overview• 374 Inpatient Beds• Level 1 Trauma Center• CHLA has the largest pediatric

hematology, oncology, and blood and marrow transplant program in the western United States

– Continuously ranked in top 10 in each category

• Rank in top 10 for pediatric cardiology

– Average 10 cardiac surgeries weekly

• Patient breakdown by ethnicity:– African-American— 5 %– Asian— 5 %– Caucasian— 19 %– Latino— 61 %– Native American— 0.08 %– Other— 9 %

CHLA Blood Donor Program

• Average whole blood collections >10,000/year– 50% Mobile and 50% In-House collection sites– Collect ~90% of blood required at CHLA

• All RBC units are serologically typed for C,E,K antigen

Targeted Blood Donation• We are one of the few hospital-based transfusion services

practicing Personalized Transfusion Medicine. By testing our patients and donors at the molecular level, we are able to Target our Collections (Donor Buddy Program) and meet our patients’ antigen specific transfusion needs.– Member of Society for the Advancement of Blood Management

• Targeted Blood Donation program includes molecular testing on:– CHLA Employees – Donors within 5 mile radius of campus– Frequent donors

>2 times a year

– Minorities

• Improve blood collection efficiency by maintaining an optimal inventory of fresh blood products with minimal wastage

• Reduce total purchases of blood products

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Need for Matched RBCs at CHLA• Provide antigen matched blood to Infusion Center for

chronically transfused patients (sickle cell, thalassemia)– CHLA is the largest Sickle Cell center in California– All patients matched for CEK at minimum– Infusion Center requires ~5000 RBC units yearly– Common Antibodies: c, Kpa, Wra, Cw, Jsa, Fyb, Jkb, e

• Growing RBC Exchange program (approximately 12 patients currently)– Increases demand for CEK= and other specially phenotyped RBC

units

• Additional– Oncology patients– Other surgical patients

Transfusion Service Methodologies Utilized

• Solid Phase (primary method)

– Echo (Immucor)

– NEO (Immucor)

• Gel

• LISS

• Reference lab for adsorption studies and

incompatible crossmatches

• PreciseType RBC Phenotype

PreciseType HEA• Multiplexed molecular assay that rapidly predicts genotype of 35 Human

Erythrocyte antigens• 24 polymorphisms associated with 35 RBC antigens

Immucor, 2014

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PreciseType BeadChip Array Overview

Immucor, 2014

PreciseType

Immucor, 2014

PreciseType

Immucor, 2014

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PreciseType

Immucor, 2014

PreciseType

Immucor, 2014

PreciseType

Immucor, 2014

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PreciseType

Immucor, 2014

PreciseType Generated Report

Kpa+ in 2% of

population

Lu(a+b+) 7.5 %

Molecular Phenotyping Patients

• Phenotype may be used to predict antigens most likely to cause alloimmunization

• Assist in antibody ID identification. Molecular phenotype provides whole picture on antibody ID and can be used to support or rule out suspected antibodies

• PreciseType performed on:– Hematology patients (Sickle cell, Thalassemia) – Recently transfused patients– Patients with warm autoantibodies, positive DAT– Patients with complicated antibody ID

• Multiple antibodies• Antibodies to high or low frequency antigens

• Non-specific antibodies

– Bone marrow recipients

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Molecular Phenotyping Donors

• Grow pool of phenotyped donors• Closely match blood required for transfusion• Expand pool of rare donors• Reduce need to purchase special units from

outside facility• Screen for units in which no commercial antisera

is available• Increase recruitment efficiency

• Decrease need to perform sickle screen• Investigate discrepancies in serological testing

(patient phenotype, donor CEK typing)

CHLA Patient Population Snapshot(negative antigen expression)

121 Patients Typed• 38 Gata Silencing Mutations• 1 Fyb weak expression• 1 Possible Hybrid C Allele

21%

36%

1%

79%84%83%

97%

0%

100%

0%

92%

0%

36%

30%

19%

36%

17%

36%

60%

4%1%

97%

0%

96%

0% 0%

98%

46%

10%

2% 2%0%

100%

0%

100%

60%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

c C e E V VS K k

Kpa

Kpb Jsa

Jsb

Fya

Fyb

Jka

Jkb M N S s U

Lua

Lub

Dia

Dib

Coa

Cob

Doa

Dob H

y

Joa

LWa

LWb

Sc1

Sc2

HbS

CHLA Donor Population Snapshot (negative antigen expression)

>200 donors phenotyped 10/2016 to 12/2017• 15 Gata Silencing Mutations detected• 2 Possible Hybrid C Allele

19%

41%

1%

78%

96%95%

94%

0%

98%

0%

97%

0%

27%31%

26%27%

23%

35%

52%

6%

0%

99%

0%

95%

0% 0%

95%

40%

17%

0% 0% 0%

100%

0%

100%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

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Tested Populations Compared

19%

41%

1%

78%

96% 95% 94%

0%

98%

0%

97%

0%

27%31%

26%27%

23%

35%

52%

6%0%

99%

0%

95%

0% 0%

95%

40%

17%

0% 0% 0%

100%

0%

100%

0%10%20%30%40%50%60%70%80%90%

100%

c C e E V VS K k

Kpa

Kpb Jsa

Jsb

Fya

Fyb

Jka

Jkb M N S s U

Lua

Lub

Dia

Dib

Coa

Cob

Doa

Dob H

y

Joa

LWa

LWb

Sc1

Sc2

Patients

Donors

21%

36%

1%

79%84%83%

97%

0%

100%

0%

92%

0%

36%30%

19%

36%

17%

36%

60%

4% 1%

97%

0%

96%

0% 0%

98%

46%

10%2% 2% 0%

100%

0%

100%

60%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

c C e E V VS K k

Kpa

Kpb Jsa

Jsb

Fya

Fyb

Jka

Jkb M N S s U

Lua

Lub

Dia

Dib

Coa

Cob

Doa

Dob H

y

Joa

LWa

LWb

Sc1

Sc2

HbS

Patient Population: 19 % Caucasian, 61% Latino, 5% African AmericanDonor Population: 45 % Caucasian, 35% Latino, 3% African American

Example of Impact to Donor Recruitment Program

• 19 year old sickle cell patient added to RBC Exchange program November 2017– B+, E Negative– Anti-Fya (~19% population is type B or O and

negative for Fya)

• Requires 6 units every 4 weeks• Compatible donors

– Historical pool of 70 B and O donors with visits since 2015

– Since live with PreciseType (10/2016), added 55 Fya= B or O donors to recruitment pool

Patient Workups: Case Studies

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Case Study 1 IP

• 10 year old male• Presented to CHLA with scleral icterus• No known transfusion history• Hemoglobin 9.6

Case Study 1 IP (Continued)

• Initial Presentation:– Unable to type ABORH due to

panagglutination• DTT not routinely performed at CHLA

– Solid Phase: All cells 3+– Gel Screen/ Panel: All cells 4+– LISS Screen: All cells 2+ AHG– DAT: Strong Positive IGG, Poly, C3b/C3d

Case Study 1 IP (Continued)

• Sent to Reference Lab– Patient typed as O Positive– Probable Auto anti-E detected via warm and

cold adsorbed serum IAT methodology

• Diagnosed with Warm and Cold Autoimmune Hemolytic Anemia

• Discharged without transfusion

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Case Study 1 IP (Continued)

• 3 months later– Presentation:

• Hemoglobin 7.2• Presents with increased jaundice and back pain

– Workup• Gel screen/panel: All 3+ and 4+• LISS screen: All cells 2+ AHG, autocontrol 3+• DAT: IgG, Poly, C3b/C3d all 3+

– Reference Lab: • Rouleax and cold autoagglutinins @ RT. • Anti-D detected (presumed autoantibody)• Recommended to transfuse Rh Negative

– No transfusion required

Case Study 1 IP (Continued)3rd presentation:• Autoanti-E and Autoanti-D not demonstrable• Gel Panel: all cells 3+• Liss Screen: all cells 2+ AHG

PreciseType performed:• Decision made to crossmatch AHG-

compatible units negative for K, Jka, Fybuntil they can be ruled out in LISS

• Outcome/ Follow-up: • Patient diagnosed with Evans Syndrome,

controlled predominately by rituximab• Has received 5 RBCs at CHLA• LISS Screen negative 2 years following

initial presentation

Case Study 2 AM

• 16 year old male• History of autoimmune hemolytic anemia

and warm autoantibodies• Presented with fatigue and darkened

urine• No known transfusion history• Hemoglobin 9.0

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Case Study 2 AM (Continued)

• Initial Workup– ABO/RH: O Positive– Solid Phase Screen: All cells 4+– Solid Phase Panel: All cells reactive (varying

reactions)– LISS Screen: All cells 2+ AHG, autocontrol 2+– DAT: Poly 4+, IgG 3+, C3b/C3d 2+

• Patient discharged without transfusion, specimen not sent to reference lab for adsorption study

Case Study 2 AM (Continued)

6 months post initial workup• Presentation:

– Presents with scleral icterus, fatigue, lightheadedness, and dark urine

– Hemoglobin: 4.2

• Workup:– Probable warm autoantibody – Solid phase: all cells reactive– LISS: all cells 2+ AHG– DAT: 4+ Poly, 4+ IgG, 3+ C3b/C3d

• 2 units urgently required• Crossmatched two least incompatible CEK negative RBCs

Case Study 2 AM (Continued)

• Reference Lab:– Acid eluate of RBCs treated with chloroquine

reacted strongly with all RBCs by PEG IAT– Adsorbed Serum contained:

• Anti-E, reactive at RT, 37C, and LISS AGT• Anti-c reactive by LISS AGT• Anti-S reactive by LISS AGT• Probable warm autoantibodies

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Case Study 2 AM (Continued)

• PreciseType Performed– Confirmed negative for E,

c, S

• Conclusion:– Crossmatch least

incompatible E-, c-, S-, K-RBCs

• Follow up: – Patient’s warm Idiopathic

AIHA controlled with steroids

Case Study 3 ES

• ES is a 30 year old Asian male with Thalassemia major– Followed and transfused at CHLA since birth

• Transfused with 2-3 RBCs every 3 weeks• ABO/RH: O Positive• Special Needs: E negative, K negative

RBCs • No known antibodies

Case Study 3 ES (continued)• Workup

– Solid Phase screen: Cells 1 and 2 are 2+ reactive– Gel Screen: all cells negative

– Solid Phase Panel: Jkb suspected (known to react earlier in solid-phase)

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Case Study 3 ES (continued)

• Specimen sent to reference lab

– DAT positive in IGG and complement– Acid elution non reactive– Serum contained no unexpected RBC

antibodies – Anti-Jkb not confirmed, reference did not

test by solid phase at the time– Recommended to conservatively select Jkb

negative units for crossmatch

Case Study 3

• PreciseType predicts sample to be Jkb+

• Rules out suspected anti-Jkb ; variant testing could be considered

• Follow up: Patient has since shown other non-specific reactivity on occasional visits. Warm-autoantibody suspected.

Case Study 4 TA

• 12 year old female• History of ITP and family history of

thalassemia• No transfusion history • Presented at outside hospital with

anemia, lethargy, jaundice, and dark urine

• Hgb 4.1

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Case Study 4 TA (Continued)

• Initial workup– ABO/ Rh: A Positive– Gel Screen/Panel: All Cells 3+– Solid Phase Screen (Echo): All cells 4+– LISS Screen: All Cells 3+– Eluate: All cells 3+ in gel – DAT: Poly 3+, IgG +, c3b/c3d negative

• Reference Lab– Serum contained warm autoantibodies

reactive by LISS, PEG, and ficin AGT

Case Study 4 TA (continued)

• PreciseType– Patient is negative for K, Fya, Jkb

• Decision made to conservatively crossmatch units that are K, Fya, and Jkbnegative and least incompatible with patient’s unadsorbedserum at AHG

• Received 11 RBCs during stay in 2015– Discharged with hemoglobin 7.3

Case Study 4 TA (continued)

• Patient returns 2018 (almost 3 years post- initial presentation)• Hgb 6.5 • Solid Phase Screen (NEO) :

• DAT: Poly 3+, IgG 3+, C3b/C3d negative

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Case Study 4 TA (continued)

• NEO

• Autocontrol 3+• E, c not ruled out

– Anti-S ruled out on additional panel

Case Study 4 TA (continued)• Eluate: All cells 3+ or 4+

– Strong warm autoantibody

• Only antigens not ruled out in multiple panels: E, c– Patient phenotype E+ c+ – Add more panels/ liss/gel

• Conclusion: autoanti-E and autoanti-c

• Transfusion requirements:– AHG compatible E-neg, c-neg (while Auto-E and Auto-c

presenting) and K-neg RBCs

• Follow Up:

Case Study 5 JM

• 7 month old male• History of biliary atresia admitted to

CHLA with increased abdominal growth• Lab results consistent with liver failure

and added to liver transplant list• Hgb: 7.0• Patient had received one aliquot of RBCs

at outside hospital 3 weeks prior

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Case Study 5 JM (continued)

• Workup– ABO/RH: O Positive– Solid Phase Screen (NEO): cell 3 is 1+

• need to rule out K, Jkb, Leb

– Gel Screen: All cells negative

Case Study 5 JM (continued)• Autocontrol and DAT Negative• Solid phase panel: All antigens ruled out, anti-Jkb still suspected. • Added special instruction to crossmatch Jkb negative RBCs (1 RBC transfused)

Case Study 5 JM (Continued)

• Anti-Jkbprediction supported with PreciseType

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Case Study 5 JM (Continued)

• Follow up visit• Solid Phase

panel: “?” on almost all homozygous positive Jkbcells

Solid Phase Panel

Case Study 5 JM (continued)

• Gel Screen: w+ in 3 cell• Gel Panel: w+ in all homozygous positive Jkb cells• Autocontrol: 2+, no antibody eluted

Case Study 5 JM (continued)

• Conclusion: Anti-Jkb predicted earlier through solid phase and supported by PreciseType

• Follow Up:– Patient received liver transplant – 2 Jkb negative units transfused in OR, 3 aliquots

post-OP– Transfusion required 4 months after transplant,

anti-Jkb not demonstrable

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Leveraging software to simultaneously view serology and

molecular testing

Blood Bank LIS

• Our blood bank LIS, SafeTrace TX displays antibodies and antigens collectively in the patient profile.

• PreciseType/BASIS does not interface directly with our Blood Bank LIS and Donor Services LIS

• Molecular phenotype antigens must be manually resulted as a test batch for 35 antigens in both LIS software.

• Manual entry eliminated with leveraging software, ImmuLINK

Benefits of a Leveraging Software

• Addition of ImmuLINK to workflow– Transmit automated CEK and molecular antigen testing results via

automation to donor and transfusion service LIS• Reduce transcription error from manual entry• Reduce tech time of antigen result entry and review

– Easily view all serology and molecular testing results for patients to provide full picture of patient workup and history

– Print reports with panel images and reactions for all ordered tests on a specimen (including Antibody ID and Molecular testing)

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Report Example #1

Report Example #2

Molecular and Serology displayed on one report

• Future plan:– Continue to grow inventory of donors with

historical molecular phenotype• Grow targeted blood donor program• Build dashboards for snapshot of screened units in

inventory

– Further match patient transfusion needs to proactively prevent alloimmunization in hypertransfused patients

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Questions?