Alloantibodies causing Hemolytic Disease of the Newborn and Fetus.

Nancy Benitez, BS, MHS (ASCP)SBB CM Reference Laboratory Director June 6, 2013

Case Saturday Night (SN)

• The referring hospital transfusion service does not have any previous history on this patient as samples were received from the outpatient clinic.

• 32 years old Caucasian Female.

• Second Pregnancy

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SN..cont

…

• They reported that the antibody screen was positive with all cells tested, but due to the limited resources no further testing was performed.

• Samples were sent to our IRL lab for antibody Identification.

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Objectives

  

Review the required serological techniques to determine the antibody specificity and clinical significance of alloantibodies causing Hemolytic disease of the fetus and Newborn. (HDFN) Review the critical role that the Reference laboratory plays in the procurement of uncommon blood to transfuse the newborn. Discuss the importance of the cooperation between patient, attending physician and the laboratory when alloantibodies are detected in the mother.

Routine Protocol First Time Patients

• ABO-Rh • DAT • Screen (Liss and PEG) • Panel • Patient antigen testing.

ABO-Rh determination –O Pos

FORWARD TYPE

ANTI-A ANTI-B ANTI-D 0 0 4

REVERSE TYPE

A1 CELLS B CELLS 4 4

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SN Screen

I II III IV R1R1 R2R2 rr AC D + + 0 C + 0 0 + + c 0 Rh E 0 + 0 e + 0 + Cw 0 0 0 K 0 + 0 Liss Screen k + + + Kell Kp a 0 0 0 Kp b + + + Duffy Fy a + + 0 Fy b + 0 + Jk a + + 0 Kidd Jk b 0 + + M + 0 + MNSs N + + 0 S + 0 0 + + s 0 P P 1 + + 0 Lewis Le a + 0 0 Le b 0 0 + Lu a 0 0 0 Lu Lu b + + + IS 37⁰ C IAT 0 0 0 0 0 1+ 1+ 0 2+ 3+ 3+ 0 I II III R1R1 R2R2 rr D + + 0 C + 0 0 c 0 + + Rh E 0 + 0 e + 0 + Cw 0 0 0 + 0 K 0 k + + + PEG Screen Kell Duffy Kidd MNSs Kp a 0 0 0 Kp b + + + Fy a + + 0 Fy b + 0 + Jk a + + 0 Jk b 0 + + M + 0 + N + + 0 S + 0 0 s 0 + + P P 1 + + 0 Lewis Lu Le a + 0 0 Le b 0 0 + Lu a 0 0 0 Lu b + + + IS 0 0 0 IAT 2+ 3+ 3+

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Patient phenotype

C E c e M N S s K k Fya

+ 0 0 + 0 + + + 0 0

Fyb

+

Jka Jkb

+ 0

Initial Panel - LISS

1 2 3 4 5 6 7 8 9 10 11 rr rr r'r r"r rr R o r R 1 R 1 R 1 R 1 R1R1 w R 2 R 2 R 2 r D 0 0 0 0 0 + + + + + + 0 + C 0 0 + 0 0 + + 0 0 E 0 0 0 + 0 0 0 0 0 + + Rh c + + + + + + 0 0 0 + + e + + + + + + + + + 0 + f + + + + + + NT NT NT NT + Cw 0 0 0 0 0 0 0 0 + 0 0 + + + M + 0 + + + 0 + + + + + MNSs N S 0 + 0 0 + + 0 + + + + 0 0 0 + 0 + 0 + s 0 + + + + + + + + + + P P 1 + + vw + + + vw + + w 0 0 0 + vw Lewis Le a Le b + 0 0 0 0 0 + + + + + + 0 0 0 0 + 0 + 0 0 + + 0 0 + K 0 + 0 0 0 Kell k + + + + + 0 0 + + + + + 0 + + + Duffy Fy a Fy b + + 0 + + + + + 0 0 0 + 0 0 0 + + + + 0 + 0 Jk a Kidd Jk b + 0 0 + + 0 + 0 + + + + + 0 + + + + + 37°C IAT 2+ 2+ 0 1+ 2+ 1+ 3+ 3+ 3+ 3+ 0 2+ 3+ 2+ 0 1+ 2+ 2+ 2+ 2+ 3+ 3+ No.

1 2 3 4 5 6 7 8 9 10 11

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Problems to resolve

• Panel reacting with all cells tested.

• Reactions with different strengths.

• What do you think we should do next.

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Let’s review the patient phenotype

Jka Jkb C E c e M N S s K k Fya

+ 0 0 + 0 + + + 0 0

Fyb

+ + 0

FICIN Treated Panel

1 2 3 4 5 6 7 8 9 10 11 rr rr r'r r"r rr R o r R 1 R 1 R 1 R 1 R1R1 w R 2 R 2 R 2 r D 0 0 0 0 0 + + + + + + C 0 0 + 0 0 0 0 0 + + + + + E 0 0 0 + 0 0 0 0 0 + + + + Rh c + + + + 0 0 0 0 + e + + + + + + + + + f + + + + + + NT NT NT NT + 0 0 0 0 + Cw 0 0 0 0 0 0 + + + + + M + 0 + + + 0 + + 0 + + + 0 0 + MNSs N 0 S + 0 + + + + 0 0 + 0 + 0 + + s 0 + + + + + + + + P P 1 + + vw + + + vw + + w 0 0 0 + vw + 0 0 Lewis Le a Le b + 0 0 0 0 0 + + + + + 0 + 0 0 + 0 0 + 0 + 0 + 0 + 0 0 K 0 0 0 Kell k + + + + + + + 0 + + + + + + Duffy Fy a Fy b + + + 0 + + + + 0 0 0 0 + 0 0 + + + + + 0 + Jk a + Kidd Jk b 0 0 + + 0 + 0 + + + + 0 + + + 0 + + IAT 3+ 4+ 3+ 4+ 3+ 3+ 0 0 0 4+ 3+ No.

1 2 3 4 5 8 9 6 7 10 11

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Selected Cell Panel: FICIN Treated Cells

Rh Donor/ Vial # D C E c e f C w MNSs P Lewis Lu V M N S s P 1 Le a Le b Lu a Lu b K k Kell Kp a Kp b Js a Duffy Kidd X Js b Fy a Fy b Jk a Jk b Xg a B6917 9 550063 + + 0 0 + 0 0 + 0 + + + 0 + 0 + + 0 0 + 0 + + + 0 + + + + 0 0 + 0 + 0 0 + + 0 + 0 + + + 0 + 0 + 0 + + 0 0 9 M2975HM 7 + + 0 0 + 0 0 + 0 0 + w + 0 + + 0 + 0 0 + 0 0 + + Additional Antigens 06/21/13 Do(a+) 06/11/13 Co(a+) Co(b-) Di (a+) 5/25/2013 PEG IAT 0 0 0

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0.01 DTT TREATED PLASMA

Donor/ Vial # Rh D C E c e f C w MNSs P Lewis Lu V M N S s P 1 Le a Le b Lu a Lu b K k Kell Kp a Kp b Js a Js b Duffy Kidd X Fy a Fy b Jk a Jk b Xg a B8392 3 B8390 20 B1051 2 + + 0 0 + + + 0 0 + + + 0 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + 0 + 0 + + + + + + 0 0 + + 0 + 0 0 + + + 0 + 0 + 0 + + + + + + + 0 + 0 + + + + 0 0 + 0 + + + 0 + + 0 0 + + Additional Antigens Co(b+) 06/28/13 6/28/2013 Co(b+) Bg(a+) 06/21/13 Do(a+) PEG PEG IAT 0 0 0 IAT 2+ 1+ 2+ 0.01 DTT Treated plasma Control

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Titration Studies

Plasma TEST Cell 3144 Testing phase IAT Neat 3+

TITER - ANTI-E

(1:2) 3+ (1:4) 3+ (1:8) 2+ (1:16) 2+ (1:32) 1+ (1:64) (1:128) (1:256) 0 0 Plasma TEST Cell 3342 Testing phase IAT Neat 3+

TITER - ANTI- c

(1:2) 3+ (1:4) 3+ (1:8) 2+ (1:16) 1+ (1:32) 0 (1:64) (1:128) (1:256) 0 0

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Summary of mom’s results

Antibody

Anti-c Anti-E Anti-M

Titration

1:16 1:32 N/A

Immunoglobulin

IgG IgG IgM

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Why Patient follow up……

 Maternal IgG antibodies (anti-c, E) could be directed against the antigen(s) present on the fetal red blood cells.

 IgG antibodies cross the placenta to coat fetal antigens, cause decreased red blood cell survival and produce anemia and HDNF.

 Prevention for future pregnancies.

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SECTION TWO

Case Saturday Night Baby (SNB)

• One of our local transfusion facilities requested testing for a baby sample with a possible Hemolytic Disease of the Newborn and Fetus .

• Mother’s records were immediately retrieve from the computer system

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Case Saturday Night Baby (SNB)

 The hospital also confirmed that the mother has a history of anti-c, -E.

 RBCs fresh as possible, preferably <5 days old.

 CMV and Hemoglobin S negative  Irradiated  Requested O Neg, c, E negative (extremely difficult combination)

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Laboratory Hospital Findings- Baby

      Positive Direct Antiglobulin Test ABO-Rh: O Neg Anemia Hyperbilirubinemia Reticulocytosis (6 to 40%) Abnormal Smear

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Baby ABO-Rh & DAT Results

Forward Type

Anti-A Anti-B 0 0 Anti-D 0 Rh Control 0

Interpretation

O NEG Poly 2+

Direct Antiglobulin Test (DAT)

IgG C3 Interpretation 2+ 0 Positive

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SN Baby Elution

Rh Kell Duffy Kidd MNSs P Lewis Lu I R1R1 II R2R2 III rr D + + 0 C + 0 0 c 0 + + E 0 + 0 e Cw K k Kp a Kp b Fy a Fy b Jk a Jk b M N + 0 + 0 0 0 0 + 0 + + + 0 0 0 + + + + + 0 + 0 + + + 0 0 + + 0 0 + + + 0 S + 0 0 s 0 + + P 1 Le a Le b Lu a Lu b + + 0 0 + + 0 0 0 0 + 0 0 + + Elution IAT 0 3+ 3+

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SN Baby Elution

1 2 3 4 5 6 7 8 9 10 11 rr rr r'r r"r rr R o r R 1 R 1 R 1 R 1 R1R1 w R 2 R 2 R 2 r + +

D

0 0 0 0 0 + + + + 0 0 0 + 0 0

C

0 0 + + + + +

E

0 0 0 + 0 0 0 0 0

Rh c

+ + + + + + + + 0 0 0 0 +

e

+ + + + + + + + +

f

+ + + + + + NT NT NT NT +

Cw

0 0 0 0 0 0 0 0 0 0 + + + + + +

M

+ 0 + + + 0 + + 0 + +

MNSs N

0 + 0 0 +

S

+ 0 + + + + 0 0 + 0 + 0 + +

s

0 + + + + + + + +

P P 1

+ + vw + + + vw + + w 0 0 0 + vw + 0 0

Lewis Le a Le b

+ 0 0 + 0 0 0 + + + + 0 + 0 0 + 0 0 + 0 + 0 + 0

K

0 + 0 0 0 0

Kell k

+ + + + + + + 0 + + + + + +

Duffy Fy a Fy b

+ + + + 0 + + + 0 0 0 0 + 0 0 + + + + + 0 + + + 0

Jk a

+

Kidd Jk b

0 0 + 0 + + + + 0 + + + 0 + +

IAT

3+ 4+ 3+ 4+ 3+ 3+ 0 0 0 4+ 3+ No.

1 2 3 4 5 8 9 6 7 10 11

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Newborn- D testing

EGA treated cell

Immediate Spin AHG Rh Control

Anti-D (1) Anti-D (2) Anti-D (3) Interpretation 0 0 0 0 0 0 D Negative 0 0 0 Negative

Where could we find blood

• Where and how will we find a unit of blood that is Rh(D) negative and also c negative, as most of the units are dce/dce.

• Blood of this type was not available in the lab

The Rh complex

• The D antigen always travel with the C, c and E or e

• Antigens cannot be separated

Prevalence of Rh Haplotypes

Fisher-Race Haplotype Modified Wiener Haplotype Prevalence (%) White

Rh Positive

Dce DcE Dce DCE R 1 R 2 R 0 R z 42 14 4 <0.01

Rh Negative

Ce Ce cE CE r r' r'' r y 37 2 1 <0.01

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Baby’s Rh Haplotype

R 1 Mother r' F a t h e r R 1 r R 1 R 1 R 1 R 1 r' rr'

• Baby is dce / dCe Heterozygous for the Cc Antigen

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Prevalence Rh Haplotypes

GENOTYPE

dce/dce rr dce/dCe rr' dCe/dCe r'r'

DONOR FREQUENCY (%)

15.1020

0.7644

0.0097

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Baby SN….Final outcome…..

   Baby’s total serum bilirubin level was at 16 mg/dL and did not increase after phototherapy.

The Newborn was almost a Full-term an exchange transfusion was not necessary.

Baby was not severely affected because he was heterozygous for the c antigen.

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Be ready……

• Think ahead, plan ahead, prepare ahead and work ahead. • Communication among the medical team and blood provider is crucial to prevent additional complications.

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References

1. Issitt PD, Anstee DJ

. Applied Blood Group Serology. Montgomery Scientific Publications; Durham, NC; 1998.

2. AABB Technical Manual, 17 th edition.

3.Mollison, PL; Engelfriet CP and Contreras M (1997).

Blood Transfusion in Clinical Medicine

(10th ed.). Oxford, UK: Blackwell Science.

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Questions and comments.

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