Hemolytic Disease of the Newborn, Current Methods of Diagnosis and TreatmentTerry Kotrla, MS, MT(ASCP)BB

Objectives

List the classifications of Hemolytic Disease of the Newborn and the most antibody specificities involved.

State the testing to perform on the mother to monitor the severity of HDN.

List the laboratory tests and values which indicate that an infant is affected by HDN both in the fetus and newborn.

State the treatment options for intrauterine treatment of HDN. State the treatment options for HDN in the moderately and

severely affected newborn. State the requirements of blood to be used for transfusion of

the fetus and newborn.

Cause of Hemolytic Disease

Maternal IgG antibodies directed against an antigen of paternal origin present on the fetal red blood cells.

IgG antibodies cross the placenta to coat fetal antigens, cause decreased red blood cell survival which can result in anemia.

Produced in response to previous pregnancy with antigen positive fetus OR exposure to red blood cells, ie transfusion.

Three Classifications of HDN

ABO “Other” – unexpected immune antibodies

other than anti-D – Jk, K, Fy, S, etc. Rh – anti-D alone or may be accompanied

by other Rh antibodies – anti-C, -c, -E or –e.

ABO Hemolytic Disease

Mother group O, baby A or B Group O individuals have anti-A, -B and –A,B in

their plasma, fetal RBCs attacked by 2 antibodies

Occurs in only 3%, is severe in only 1%, and <1:1,000 require exchange transfusion.

The disease is more common and more severe in African-American infants.

“Other” Hemolytic Disease

Uncommon, occurs in ~0.8% of pregnant women.

Immune alloantibodies usually due to anti-E, -c, -Kell, -Kidd or -Duffy.

Anti-K disease ranges from mild to severe over half of the cases are caused by multiple blood

transfusions is the second most common form of severe HDN

Anti-M very rare

Rh Hemolytic Disease

Anti-D is the commonest form of severe HDN. The disease varies from mild to severe.

Anti-E is a mild disease Anti-c can range from a mild to severe disease -

is the third most common form of severe HDN Anti-e - rare Anti-C - rare antibody combinations (ie anti-c and anti-E

antibodies occurring together) - can be severe

HDN

Maternal antibodies destroy fetal red blood cells Results in anemia. Anemia limits the ability of the blood to carry oxygen to the baby's

organs and tissues. Baby's responds to the hemolysis by trying to make more red blood

cells very quickly in the bone marrow and the liver and spleen. Organs enlarge - hepatosplenomegaly. New red blood cells released prematurely from bone marrow and are

unable to do the work of mature red blood cells. As the red blood cells break down, bilirubin is formed.

Babies unable to get rid of the bilirubin. Builds up in the blood (hyperbilirubinemia ) and other tissues and fluids

of the baby's body resulting in jaundice. The placenta helps get rid of some of the bilirubin, but not all.

Complications During Pregnancy

Severe anemia with enlargement of the liver and spleenWhen these organs and the bone marrow cannot compensate for the fast destruction of red blood cells, severe anemia results and other organs are affected.

Hydrops FetalisThis occurs as the baby's organs are unable to handle the anemia. The heart begins to fail and large amounts of fluid build up in the baby's tissues and organs. A fetus with hydrops is at great risk of being stillborn.

Hydrops Fetalis

Clinical Presentation Varies from mild jaundice and anemia to hydrops fetalis (with ascites, pleural and

pericardial effusions) Chief risk to the fetus is anemia. Extramedullary hematopoiesis due to anemia results in hepatosplenomegaly. Risks during labor and delivery include:

asphyxia and splenic rupture. Postnatal problems include:

Asphyxia Pulmonary hypertension Pallor (due to anemia) Edema (hydrops, due to low serum albumin) Respiratory distress Coagulopathies (↓ platelets & clotting factors) Jaundice Kernicterus (from hyperbilirubinemia) Hypoglycemia (due to hyperinsulinemnia from islet cell hyperplasia)

Kernicterus

Kernicterus (bilirubin encephalopathy) results from high levels of indirect bilirubin (>20 mg/dL in a term infant with HDN).

Kernicterus occurs at lower levels of bilirubin in the presence of acidosis, hypoalbuminemia, prematurity and certain drugs (e.g., sulfonamides).

Kernicturus

Affected structures have a bright yellow color. Unbound unconjugated bilirubin crosses the blood-brain barrier and,

because it is lipid soluble, it penetrates neuronal and glial membranes. Bilirubin is thought to be toxic to nerve cells The mechanism of neurotoxicity and the reason for the topography of the

lesions are not known. Patients surviving kernicterus have severe permanent neurologic symptoms

(choreoathetosis, spasticity, muscular rigidity, ataxia, deafness, mental retardation).

Laboratory Findings

Vary with severity of HDN and include: Anemia Hyperbilirubinemia Reticulocytosis (6 to 40%) ↑ nucleated RBC count (>10/100 WBCs) Thrombocytopenia Leukopenia Positive Direct Antiglobulin Test Hypoalbuminemia Rh negative blood type or ABO incompatibility Smear: polychromasia, anisocytosis, no spherocytes

Blood Smear

Polychromasia Anisocytosis Increase NRBCs no spherocytes

Blood Bank Testing

Bilirubin Nomogram

Total Serum Bilirubin (TSB) monitored to determine risk of kernicterus.

Measure bilirubin in cord blood and at least every 4 hours for the first 12 to 24 hours. Plot bilirubin concentrations over time.

Transcutaneous Monitoring

Transcutaneous bilirubinometry can be adopted as the first-line screening tool for jaundice in well, full-term babies.

This leads to about 50% decrease in blood testing. http://tinyurl.com/36jazx

Intrauterine Transfusion (IUT)

Given to the fetus to prevent hydrops fetalis and fetal death. Can be done as early as 17 weeks, although preferable to wait until

20 weeks Severely affected fetus, transfusions done every 1 to 4 weeks until

the fetus is mature enough to be delivered safely. Amniocentesis may be done to determine the maturity of the fetus's lungs before delivery is scheduled.

After multiple IUTs, most of the baby’s blood will be D negative donor blood, therefore, the Direct Antiglobulin test will be negative, but the Indirect Antiglobulin Test will be positive.

After IUTs, the cord bilirubin is not an accurate indicator of rate of hemolysis or of the likelihood of the need for post-natal exchange transfusion.

Intrauterine Transfusion

An intrauterine fetal blood transfusion is done in the hospital. The mother may have to stay overnight after the procedure.

The mother is sedated, and an ultrasound image is obtained to determine the position of the fetus and placenta.

After the mother's abdomen is cleaned with an antiseptic solution, she is given a local anesthetic injection to numb the abdominal area where the transfusion needle will be inserted.

Medication may be given to the fetus to temporarily stop fetal movement. Ultrasound is used to guide the needle through the mother's abdomen into

the fetus's abdomen or an umbilical cord vein. A compatible blood type (usually type O, Rh-negative) is delivered into the

fetus's abdominal cavity or into an umbilical cord blood vessel. The mother is usually given antibiotics to prevent infection. She may also be

given tocolytic medication to prevent labor from beginning, though this is unusual.

Intrauterine Transfusion Increasingly common and relatively safe procedure since the development of high

resolution ultrasound particularly with colour Doppler capability. MCA Doppler velocity as a reliable non-invasive screening tool to detect fetal anemia.

The vessel can be easily visualized with color flow Doppler as early as 18 weeks’ gestation. In cases of fetal anemia, an increase in the fetal cardiac output and a decrease in blood

viscosity contribute to an increased blood flow velocity

Intrauterine Transfusion

The risk of these procedures is now largely dependent on the prior condition of the fetus and the gestational age at which transfusion is commenced.

Intrauterine Transfusion

Titer greater than 32 for anti-D and 8 for anti-K OR four fold increase in titer indicates need for analysis of amniotic fluid.

Amniocentesis Perform at 28 wks if HDN in previous child Perform at 22 wks if previous child severely affected Perform if maternal antibody increases before 34th wk.

High values of bilirubin in amniotic fluid analyses by the Liley method or a hemoglobin concentration of cord blood below 10.0 g/mL.

Type fetus -recent development in fetal RhD typing involves the isolation of free fetal DNA in maternal serum. In the United Kingdom, this technique has virtually replaced amniocentesis for fetal RhD determination in the case of a heterozygous paternal phenotype

Maternal plasma exchange may be instituted if the fetus is too young for intrauterine transfusion.

Liley Graph

Selection of Blood

CPD, as fresh as possible, preferably <5 days old. A hematocrit of 80% or greater is desirable to minimize

the chance of volume overload in the fetus. The volume transfused ranges from 75-175 mL

depending on the fetal size and age. CMV negative Hemoglobin S negative IRRADIATED O negative, lack all antigens to which mom has

antibodies and Coomb’s compatible.

Treatment of Mild HDN

Phototherapy is the treatment of choice. Phototherapy process slowly

decomposes/converts bilirubin into a nontoxic isomer, photobilirubin, which is transported in the plasma to the liver.

HDN is judged to be clinically significant (phototherapy treatment) if the peak bilirubin level reaches 12 mg/dL or more.

Bilirubin Degradation by Phototherapy

Phototherapy

The therapy uses a blue light (420-470 nm) that converts bilirubin so that it can be excreted in the urine and feces.

Soft eye shields are placed on the baby to protect their eyes from damage that may lead to retinopathy due to the bili lights.

Phototherapy

Lightweight, fiberoptic pad delivers up to 45 microwatts of therapeutic light for the treatment of jaundice while allowing the infant to be swaddled, held and cared for by parents and hospital staff.

Compact unit is ideal for hospital and homecare.

Exchange Transfusion

Exchange Transfusion

Full-term infants rarely require an exchange transfusion if intense phototherapy is initiated in a timely manner.

It should be considered if the total serum bilirubin level is approaching 20 mg/dL and continues to rise despite intense in-hospital phototherapy.

The procedure carries a mortality rate of approximately 1% and there may be substantial morbidity

Goals of Exchange Transfusion

Remove sensitized cells. Reduce level of maternal antibody. Removes about 60 percent of bilirubin from the plasma,

resulting in a clearance of about 30 percent to 40 percent of the total bilirubin.

Correct anemia by providing blood that will have normal survival.

Replacement with donor plasma restores albumin and any needed coagulation factors.

Rebound – usually a 2 volume exchange is needed as bilirubin in tissues will return to blood stream.

Testing Baby

Antibody elution testing from cord red blood cells. ABO/D typing

If baby received intrauterine transfusions will type as O negative If baby’s Direct Antiglobulin Test is strongly positive due to anti-

D may get FALSE NEGATIVE immediate spin reaction with reagent anti-D (blocking phenomenon), weak D (Du) test will be STRONGLY positive

Antibody screen Coomb’s crossmatch antigen negative donor.

Testing Mom

Type and screen on mom. Identification of unexpected antibodies. More than 40 antigens have been identified as

causing HDN. Select blood that lacks antigens to which mom

has antibodies. Perform coomb’s crossmatch with Mom and

baby’s blood.

Selection of Donor Blood

CPD, as fresh as possible, preferably <5 days old.

CMV negative Hemoglobin S negative Irradiated if possible

Preparation of Donor Unit

Physician will specify a hematocrit. Reconstitute donor unit with plasma. Most facilities prefer to use group O red

cells and AB plasma. Reference for procedure at end of this

presentation.

Summary

Three types of HDN vary in severity. Laboratory testing key to diagnosing and

monitoring- great care to be taken when interpreting ABO/D typing on affected infants.

Therapy dependent on severity: phototherapy alone or with transfusion.

References Urgent Clinical Need for Accurate and Precise Bilirubin Measurements in the United States to Prevent

Kernicterus, 2004 http://www.clinchem.org/cgi/content/full/50/3/477 Identifying newborns at risk of significant hyperbilirubinaemia: a comparison of two recommended

approaches, http://adc.bmj.com/cgi/content/full/90/4/415 Predictive Ability of a Predischarge Hour-specific Serum Bilirubin for Subsequent Significant

Hyperbilirubinemia in Healthy Term and Near-term Newborns, 1999, http://pediatrics.aappublications.org/cgi/content/full/103/1/6

Neonatal Jaundice, 2005, http://int-pediatrics.org/PDF/volume_20/20_1/47_54_ip20_1.pdf HDN, Answers.com, http://www.answers.com/topic/hemolytic-disease-of-the-newborn Hemolytic Disease of the Newborn, UCSF Children’s Hospital,

http://www.ucsfhealth.org/childrens/health_professionals/manuals/42_Hemol.pdf Lucile Packard Children’s Hospital at Stanford,

http://www.lpch.org/DiseaseHealthInfo/HealthLibrary/hrnewborn/hdn.html Intrauterine Transfusion, http://www.sufw.com.au/default.asp?cid=1%7C6%7C1 The Procedure for Preparation of Whole Blood for Neonatal Exchange Transfusion,

http://www.cbbsweb.org/enf/attachments/sop_reconstitute_blood_mass.pdf California Blood Bank Society discussion on neonatal transfusions

http://www.cbbsweb.org/enf/2002/txneonatalpract.html Irradiation of Blood Products, recommendations, http://www.scbcinfo.org/publications/bulletin_v2_n1.htm Intrauterine Tranfusion: IOI Consecutive Cases treated at Queen Charlotte's Maternity Hospital,

http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1587950&blobtype=pdf Management of Rhesus Alloimmunization in Pregnancy, 2005,

http://www.touchbriefings.com/pdf/1421/ACFA2C2.pdf Molecular Diagnosis in Prenatal Medicine, http://pages.unibas.ch/diss/2004/DabsB_7129.pdf Rh phenotype prediction by DNA typing and its application to practice,

http://www.uni-ulm.de/~wflegel/RH/TME/trans173.pdf Many references to molecular blood group testing http://www.ihop-net.org/UniPub/iHOP/bng/91718.html Created April 2007