420 Perinatal Testing & Hemolytic Disease of Fetus/Newborn Slides

Why do prenatal testing

For early identification of patients at risk for developing HDFN

What is HDFN

Maternal antibodies against antigens on baby's RBCs leading to premature destruction of fetal RBCs. Can lead to death

Which antibodies can cause severe HDFN?

IgG antibodies

anti-D

anti-K

anti-E

anti-c

anti-Fya

anti-Jka

Are ABO antibodies linked to HDFN?

No, they cannot cross the placenta

3 multiple choice options

Reagents that can help determine if IgM or IgG antibodies by destroying IgM reactivity:

Dithiothreital (DTT)

2-Mercaptoethanol (2-ME)

Sulfhydrol reagents that break disulfide bonds in IgM therefore no reactivity

If IgG clinically significant antibodies are found in pregnant women, how would you monitor them during pregnancy?

Antibody titration

Which cells do you use in antibody titration against the patients serum

Homozygous positive RBCs

Titer is tested at which phase

AHG

What titer is clinically significant

A titer of 16 or greater unless it is anti-K, which a titer of 8 is significant

3 multiple choice options

Changes in titer which is clinically significant:

A 2 fold increase in titer

A +10 change in score

Controls for titers

Freeze aliquot of patiet serum/plasma at each appointment to compare at next appointment. Ie: Freeze serum at 26wk appointment to use as the control at their 30wk appointment

4+ reaction score

12

3+ reaction score

10

2+ reaction score

8

1+ reaction score

5

W+ reaction score

2

In order for HDFN to happen, which 3 criteria must be met

1) Mother is Ag negative

2) Father is Ag positive

3) Baby is Ag positive

3 multiple choice options

Which IgG classes are most associated with HDFN

IgG1 and IgG3

3 multiple choice options

Erythroblastosis Fetalis

Seen as a result of antibody mediated destruction of RBCs in HDFN

Rate of RBC destruction in HDFN depends on

Antibody specificity

Antibody strength

# Antigen binding sites

Factors affecting maternal immunization and severity

Antigen exposure

Fetomaternal hemorrhage (>50% at delivery)

Transplacental hemorrhage

Abdominal trauma

Chorionic villus sampling

Amniocentesis

Cordocentesis

Other diagnostic means to monitor RBC destruction of the fetus

Amniocentesis. If bilirubin in amniotic fluid, then likely RBC destruction. Measure change spectrophotometrically at 450nm

Fetal Lung Maturity

No longer recommended

Measured Lecithin to Sphingomyelin

MCA-PSV

Middle cerebral artery peak systolic velocity

How is MCA-PSV used to monitor HDFN

Doppler reading of MCA-PSV compared to median to monitor anemia

Significant anemia MCA-PSV

>=1.5 MoM

3 multiple choice options

If mom is an RhIg candidate, how can you determine how many doses she needs?

Rosette test

Kleihauer-Betke stain

Which test would you do if mom is Rh Negative and baby is Rh Positive

Rosette

When do you do Kleihauer-Betke stain?

If Rosette test is positive

What does KB stain do?

Quantifies how big the fetal-maternal bleed was

Assessing Fetal RBC Destruction - POST DELIVERY

Measure Unconjugated Bilirubin

*High levels are toxic to baby

Lui freeze elution to detect

ABO antibodies

Acid elution to detect

Non-ABO antibodies

Treating fetal rbc destruction post delivery

Ultraviolet phototherapy

Aliquot transfusion

*Hgb and Hct increase, very small amounts from a larger sample, as little as 4cc in a premature baby)

Exchange transfusion

FFP + Less than 7 day old RBCs

3 categories of HDFN

ABO

Rh (D)

Other

ABO Induced HDFN

Mild

Most common cause of HDFN

Typically: O type mom and non-O baby

Usually only requires phototherapy

Can occur in the 1st pregnancy

Rh(D) induced HDFN

Most severe type of HDFN

Usually cannot happen in 1st pregnancy

Increased bilirubin

Should RhIg be given if mom has circulating anti-D?

No

RhIg candidate

Prenatal: Rh neg mom without immunization to D

Postpartum: Rh neg mom without immunization to D with Rh pos baby

One RhIg dose protects against

30mL fetal blood

RhIg given at __ weeks gestation

28

RhIg given following any

1) Invasive procedure

2) Ultrasound scan

3) Blood work

4) UTI

1) Invasive procedure

3 multiple choice options

RhIg should be given within __ hours of birth to Rh positive baby

72

Rosette test cannot be performed if

Baby's Rh type is unknown

Baby is weak D pos

Baby is Rh negative

Rosette test measures

If fetal-maternal hemorrhaging happened during delivery

FetalScreen (Rosette Test) will detect a bleed/exposure of what value?

>10 mL whole blood

3 multiple choice options

Rosette test procedure

Wash maternal RBCs - make a 2-5% susp

Add modified anti-D reagent from kit to 1 drop RBCs

incubate 37C for 15 mins

Wash 4 times, add R2R2 indicator cells from kit

If Rh+ fetal RBCs are in maternal circulation, anti-D attaches, then R2R2 indicator cells attach in a rosette formation

Count the rosettes/field x 5 fields

Usually 3 or 5 rosettes = POS Rosette Test

True or false: A positive rosette test does not need to be quantified

False

Most common method of feto-maternal hemorrhage quantification

Kleihauer-Betke (KB) stain

KB stain principle

Based on the principle that HbF is the major hemoglobin in babies and A1 is the most common in adults. HbF is resistant to acid while HbA1 is NOT. When acid is added, adult red cells will appear as ghost cells while baby RBCs will remain intact. The slide is then stained and baby red cells will be red.

Quantification of KB

% Fetal RBCs are determined out of 2000 cells

If rosette test is negative, administer _ doses of RhIg

1

3 multiple choice options

Rosette test is positive and a KB stain is performed and a total of 45 cells are counted. Calculate how many doses of RhIg mom needs

1) 45 / 2000 * 100 = 2.25% fetal cells

2) 2.25 * 50 = 112.5 mL fetal blood in maternal circulation

3) 112.5 / 30 = 3.75 doses

4) Round up to 4 because decimal is above .5

5) Always add an additional dose because KB isn't precise

6) 5 DOSES OF RHIG

HDFN by other antibodies cannot be prevented with RhIg, how are these patients monitored to try and prevent HDFN?

Closely monitored by titers and/or MCA-PSV

*If indicated, early inducement for birth of the baby may be needed*

O neg mom and A pos baby, what kind of HDFN may we see?

Mostly ABO HDFN because Anti-A and Anti-AB tends to confer protection against D immunization

If a Rh neg mom gives birth to a baby and they are D negative but weak D positive, what do you do next?

Report the type as indeterminate because we dont know if anti-D is coating their cells or if it is truly a weak D expression. Order a type and screen and a KB stain (skip rosette because the test only works reliably if the fetal cells are clearly D-positive...(strong D antigen expression.))

Flow of determining RhIg need

Half life of RhIg is:

25 days