IRL Case Study by Karen Abed, MT (ASCP) SBB

IRL Case Study

Karen Abed, MT(ASCP), SBB

Director, Technical Services

Transfusion Medicine Symposium

August 6, 2016

Disclosures

• I have no financial relationships

related to this presentation.

• I will not be speaking about any

specific commercial product,

device, or medication.

• I will not be speaking of any off

label use of medications or

devices

Objectives

• Identify 2 antibody workup

situations in which red cell

genotyping should be

considered

• Name 2 patient populations that

would benefit from prospective

phenotype/genotype matched

red cells

Case Presentation

• 17 year old African-

American male

• Two day history of:

– Fever

– Shortness of breath

– Productive cough

– Chest pain

• Presumptive diagnosis:

– Acute chest syndrome

Past Medical History

• Sickle cell anemia

– Recurrent pain crises

– Recurrent episodes of acute chest syndrome

– h/o multiple transfusions – last transfusion 4 months ago

• Just moved from mainland

• No red cell phenotype is available

Laboratory Evaluation

• CBC

– WBC = 18.9 x 103/µL

– Hgb = 6.6 g/dL

– Hct = 20%

– Plt = 245,000 x 103/µL

• Other laboratory values

– LDH = 880 U/L

– Bili(T) = 5.9 mg/dL

Transfusion Order

• Request for 3 units red cells ASAP

• Simple transfusion

Blood Type

Forward Type Interp:

Type A

Anti-A 4+

Anti-B 0

Back Type

A cell 0

B-cell 4+

Rh

Interp:

Rh+Anti-D 3+

DU N/A

Antibody Screen (Gel)

• Autoantibodies

• Alloantibodies

• Drug-induced antibodies

• Reagent-related antibodies

Rh-hr KELL DUFFY KIDD LEWIS MNS LUTH TEST RESULTS

Cell

#D C E c e F Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Xga Lea Leb S s M N P1 Lua Lub AHG

1 + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 0 + + 0 + + 0 + 0 + 0 + 3+

2 + 0 + + 0 0 0 0 0 + 0 + 0 + + 0 + 0 + 0 0 0 + + + 0 0 + 3+

3 0 0 0 + + 0 0 0 + + 0 + 0 + 0 + + + + + 0 + + + + +s 0 + 3+

Antibody Identification Panel

Rh-hr KELL DUFFY KIDD LEWIS MNS LUTHTEST

RESULTS

Cell

#D C E c e F Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Xga Lea Leb S s M N P1 Lua Lub AHG

1 + + 0 0 + 0 + 0 0 + 0 + 0 + + + + + + 0 + 0 + 0 + + + + 3+

2 + + 0 0 + 0 0 0 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 + 0 + 3+

3 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 + 0 + + 0 + 0 + 0 + 0 0 + 2+

4 + 0 0 + + + 0 + 0 + 0 + 0 + 0 0 + + + 0 0 + 0 + + +s 0 + 3+

5 0 + 0 + + + 0 0 0 + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 + 3+

6 0 0 + + + + 0 0 0 + 0 + 0 + + 0 0 + + 0 0 0 + + + 0 0 + 3+

7 0 0 0 + + + 0 0 + + 0 + 0 + 0 + 0 + + + 0 + 0 + 0 + 0 + 3+

8 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + 0 + 0 + + + + + 0 0 + 3+

9 0 0 0 + + + 0 0 0 + 0 + 0 + 0 + + + + 0 + + + 0 + + 0 + 2+

10 0 0 0 + + + 0 0 + + 0 + 0 + + + + + + 0 + 0 + + 0 +s 0 + 3+

11 + + 0 0 + 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 + 0 + + 0 + 3+

PC 3+

Attempted Crossmatch

• 3 A-positive red cell units crossmatched

• 3 of 3 incompatible

– AHG

– 1-3+ reactivity

Panel Interpretation

• Panreactive = 2-3+

• Autocontrol: 3+

• Possible interpretations:

– Autoantibodies

– Alloantibodies

» Antibody to a high frequency antigen

» Multiple alloantibodies

» Recently transfused

Direct Antiglobulin Test

• DAT: positive

– Autoantibodies

– Delayed reaction

– Drug-induced antibodies

– Non-specific protein

binding

Forward Type Interp:

Type A

Anti-A 4+

Anti-B 0

Back Type

A cell 0

B-cell 4+

Rh Interp:

Rh+Anti-D 3+

DU N/A

DAT

Poly IgG C3d

3+ 3+ 1+

Evaluation Goals

• Identify clinically significant relevant antibodies

• Select appropriate red cells for transfusion

• Plan for future transfusions

Two questions:

1. What antibodies in plasma?

2. What’s patient’s phenotype?

Evaluation Goals




Two questions:



Adsorptions

• Autoadsorption– Use the patient’s RBCs

– If there is no history of transfusion or pregnancy within the last 3 months

– If there are sufficient patient red cells

• Alloadsorption– Use reagent or donor RBCs of known and complementary

phenotype

– If there is a history of transfusion or pregnancy within the last 3 months

– If there are not enough patient RBCs available for an autoadsorption

Adsorptions

• Top 5 reasons to perform an adsorption

1. Identify underlying clinically significant alloantibodies





Adsorptions

S

k Jka

e

C

Fyb

WAA

WAA

WAAWAA

WAA

WAA

WAAWAA

WAAWAAWAA

WAA

Anti-E

WAA

Anti-E

WAA

WAA

WAA

WAA

WAA

WAA

WAA

WAA

WAA

+

Adsorptions

S

k Jka

e

C

Fyb

Anti-E

Anti-E

WAAWAA

WAA

Alloadsorption

• Warm autoantibody

• Underlying alloantibodies: E, K, Jkb, Fya

Ad

so

rbin

g C

ells

Rh-hr KELL DUFFY KIDD LEWIS MNS LUTH

Cell

#D C E c e F Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Xga Lea Leb S s M N P1 Lua Lub

I + + 0 0 + 0 0 0 0 + 0 + 0 + + 0 0 + + 0 + + 0 + 0 + 0 +

II + 0 + + 0 0 0 0 0 + 0 + 0 + + 0 + 0 + 0 0 0 + + + 0 0 +

III 0 0 0 + + 0 0 0 + + 0 + 0 + 0 + + + + + 0 + + + + +s 0 +

Rh-hr KELL DUFFY KIDD LEWIS MNS LUTH TEST RESULTS

Cell

#D C E c e F Cw V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Xga Lea Leb S s M N P1 Lua Lub I II III

1 + + 0 0 + 0 + 0 0 + 0 + 0 + + + + + + 0 + 0 + 0 + + + + 2+ 0 3+

2 + + 0 0 + 0 0 0 0 + 0 + 0 + 0 + + 0 + + 0 + + + 0 + 0 + 2+ 0 2+

3 + 0 + + 0 0 0 0 0 + 0 + 0 + 0 + 0 + + 0 + 0 + 0 + 0 0 + 2+ 1+ 2+

4 + 0 0 + + + 0 + 0 + 0 + 0 + 0 0 + + + 0 0 + 0 + + +s 0 + 2+ 0 2+

5 0 + + + + + 0 0 + + 0 + 0 + + + + 0 + 0 + 0 + + + + 0 + 3+ 3+ 1+

Evaluation Goals




Two questions:



Phenotype

• Importance

– Shortens list of possible alloantibodies

– Predicts difficulty in finding compatible blood

– Needed if phenotype-matched blood is desired

• What if patient has positive DAT?

– EGA-treatment

• What if patient has been recently transfused?

– Reticulocyte separation

– Hypotonic lysis (sickle cell patients)

– Genotyping

EDTA-Glycine-Acid Treatment

C3d

IgG

EGA

Phenotype RBCs

Incubate

Neutralize

Wash

EGA Treatment

• Cannot be used for phenotyping RBCs from recently transfused (within 2-3 months) patients

• Destroys Kell system (K, k), Bg, and Era antigens– Cannot be used to phenotype for Kell antigens

– Consider K-negative RBCs - if anti-K can’t be ruled-out

Reticulocyte Separation

• Reticulocytes have lower specific gravity than

mature erythrocytes

• Patient’s reticulocytes can be separated from

donor RBCs by centrifugation

– Gradients

– Microhematocrit capillary tubes

• Reticulocytes can then be used for phenotyping

Limitations

• Patient’s reticulocyte count must be adequate

• Large patient blood sample needed

• Separated reticulocytes may be a mixture of

recipient and donor cells

– Wait 1-2 weeks after most recent transfusion so donor

reticulocytes have already matured

– If phenotyping yields mixed field reactions → mixture

of patient and donor reticulocytes

Hypotonic Lysis

• Red cells from sickle cell disease patients more

resistant to lysis in hypotonic NaCl solutions

• Procedure:

– Wash cells in 0.3% NaCl until supernatant no longer

contains grossly visible hemoglobin

– Wash in 0.9% NaCl x 2

– Resuspend and test

Genotyping

• Relies on allele specific single nucleotide polymorphisms

• Uses DNA from recipient WBCs

• Leukopenic patients generally acceptable

• Some assays can use buccal swabs

• Eliminates donor red cell contamination issue

• Eliminates IgG coated red cell interference issue

• Can be done on stored patient DNA

Patient’s Genotype

Genotype

• D+, C-, E-, c+, e+ (R0R0)

• K-

• Jka+, Jkb-

• Fya-, Fyb+**

• M+, N+, S-, s+

• P1+

• Lea-, Leb-

Predicted Phenotype

• D+, C-, E-, c+, e+ (R0R0)

• K-

• Jka+, Jkb-

• Fya-, Fyb-

• M+, N+, S-, s+

• P1+

• Lea-, Leb-

Fyb+**

• Patient has GATA-1 mutation in Fyb gene

• Fyb-negative in red cell phenotype

• Fyb is present on other cells

• Does not need Fyb-negative red cells when transfused.

Transfusion

• 3 units red cells ordered

– Type-compatible (A+)

– E, K, Fya, and Jkb – negative

Antigen Frequencies

Antigen African Americans Asians Caucasians

C 27% 93% 68%

c 96% 80% 47%

E 22% 49% 29%

e 98% 98% 96%

K 2% < 0.01% 9%

Jka 92% 73% 77%

Jkb 49% 76% 74%

Fya 10% 99% 66%

Fyb 23% 8% 83%

Antigen Frequencies


C 27% 93% 68%

c 96% 80% 47%

E 22% 49% 29%

e 98% 98% 96%

K 2% < 0.01% 9%

Jka 92% 73% 77%

Jkb 49% 76% 74%

Fya 10% 99% 66%

Fyb 23% 8% 83%

Antigen Frequencies


C 27% 93% 68%

c 96% 80% 47%

E 22% 49% 29%

e 98% 98% 96%

K 2% < 0.01% 9%

Jka 92% 73% 77%

Jkb 49% 76% 74%

Fya 10% 99% 66%

Fyb 23% 8% 83%

Antigen Frequencies


C 27% 93% 68%

c 96% 80% 47%

E 22% 49% 29%

e 98% 98% 96%

K 2% < 0.01% 9%

Jka 92% 73% 77%

Jkb 49% 76% 74%

Fya 10% 99% 66%

Fyb 23% 8% 83%

Finding Compatible Blood

• Frequency of antigen-negative blood– Type A or O ≈ 77%

– E-negative ≈ 61%

– K-negative ≈ 96%

– Fya–negative ≈ 20%

– Jkb-negative ≈ 25%

• Probability of finding antigen-negative blood– .77 x .61 x .96 x .2 x .25 = 0.023

– 1 in 43 chance of finding one compatible unit

– 1 in 200 if above and C-negative

Finding Matched Blood

Full Phenotype Match

• Type A or O ≈ 77%

• C-negative ≈ 20%

• E-negative ≈ 61%

• K-negative ≈ 96%

• Fya/Fyb–negative ≈ 0.01%

• Jkb-negative ≈ 25%

• S-negative ≈ 70%

• Less than 1 in 10,000

Genotype Match

• Type A or O ≈ 77%

• C-negative ≈ 20%

• E-negative ≈ 61%

• K-negative ≈ 96%

• Fya–negative ≈ 20%

• Jkb-negative ≈ 25%

• S-negative ≈ 70%

• 1 in 333

Case Wrap-Up

• 3 units requested (E-, K-, Fya-, Jkb-)

– 3 units identified

– Transfused without transfusion reaction

• If full prospective matching required

– 1 frozen unit available

– 2 imported from mainland

Conclusion

• Multiply transfused patients can be challenging– Alloantibodies

– Autoantibodies

• Evaluations can be time-consuming

• Phenotypes can be helpful but often not possible in these patients

• Genotyping is now readily available– Multiply-transfused patients

– Sickle-cell disease

– Thalassemia

– daratumumab

Sickle Cell Disease

• Group of inherited red cell diseases

– Homozygous – sickle cell anemia

– Heterozygous – sickle cell trait

• Abnormal hemoglobin – hemoglobin S

– Can form stiff rods within red cell – crescent or sickle shape

– Not flexible and can stick to vessel walls, causing a blockage

that slows or stops the flow of blood

» Symptoms – e.g. pain crises

» Organ damage

– hemolysis

SCD Prognosis

• Life-long disease

• Severity varies widely

• Life expectancy

– 1974 – 14 years

– Today – 40-60 years

• Cure – hematopoietic stem cell transplant (HSCT)

Who’s At Risk

• African American

– 1 in 13 - sickle cell trait

– 1 in 365 - sickle cell disease

• Hispanic – 1 in 16,300

• Southern European

• Middle Eastern

• Asian Indian

Transfusion

• Acute vs chronic

• Simple vs exchange

Transfusion in SCD

• Acute Complications

– Simple transfusion

» Symptomatic ACS + hgb ≥ 1g/dL

» Acute splenic sequestration + anemia

» Aplastic crisis

» Symptomatic anemia

– Exchange transfusion

» Symptomatic, severe ACS

– Simple or exchange

» Stroke

» Hepatic sequestration

» Intrahepatic cholestasis

» Multisystem organ failure

• Chronic complications

– Simple or exchange

» Previous stroke

» Increased transcranial Doppler

reading (> 200 cm/sec)

• Transfusion not indicated

– Acute complications» Uncomplicated pain crisis

» Priapism

» Asymptomatic anemia

» Acute kidney injury

– Chronic complications» Recurrent splenic sequestration

NHLBI, Evidence-based management of sickle cell disease: expert panel report, 2014

Complications of Transfusion

• All complications possible

• Specifically:

– Alloimmunization

– DHTR

– Hyperhemolysis

– Iron overload

Alloimmunization

• Rates– Alloimmunization ≈ 20-35% (6-85%)

– Autoantibody formation ≈ 4-10%

– DHTR ≈ 5-20%

• Antibodies– Rh antibodies ≈ 50-66%

– K and Jk ≈ 20%

• Phenotype-matching– 0-7% alloimmunization rate

Recommendations for Transfusion

• Leukoreduced

• Hemoglobin S–negative

• Phenotype-matching– Rh and Kell antigens

– Consider Kidd and Duffy

– Avoid known significant alloantibodies

• Chronic transfusion patients– Maintain HgbS level < 30%

– Immediately prior to next transfusion

• Acute transfusion– Not getting chronic transfusion therapy

– Keep target Hgb < 10 g/dL - hyperviscosity

Fya- / Fyb-

• 67% African Americans

– Mutation in Fyb allele (in GATA-1 region)

» Blocks expression of Fyb on red cells

» Fyb present on other cells

» Don’t make anti-Fyb

• Malaria resistance

– Fy antigen is receptor for:

» Plasmodium vivax

» Plasmodium knowlesiGATA-1

Blood Bank of Hawaii

• Universal leukoreduction

• HgbS-negative

– Low rate in Hawaii donors

– Testing is available

• Phenotype-matched blood

– Prevention

– Alloimmunized

• Chronic therapy – similar to thalassemia patients

Antigen Typed Red Cells

• Increased typing of red cells – Rh and K– Donor testing laboratory

– Licensed reagents

– No need to serologically confirm

• Future– Additional screening for Jk, Fy, Ss

– Non-licensed reagents

• Genotyping– Patients who may require frequent transfusion

» Sickle cell disease

» Thalassemia

» Warm autoimmune hemolytic anemia

– Recently transfused

IRL Case Study by Karen Abed, MT (ASCP) SBB

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