LABORATORY DIAGNOSIS AND

MANAGEMENT OF HAEMOLYTIC
DISEASE OF NEW BORN
ADEBAYO RAMATALLAH BUKOLA
HAEMATOLOGY AND BLOOD
TRANSFUSION SCIENCE DEPARTMENT,
UNIVERSITY OF ILORIN TEACHING
HOSPITAL.
SUPERVISED BY: MR ALABI
FEBUARY, 2022.
 OUTLINES
• INTRODUCTION
• ETIOLOGY
• TYPES OF HDN
• PATHOGENESIS
• CLINICAL SIGNS AND SYMPTOMS
• LABORATORY DIAGNOSIS
• MANAGEMENT
• PREVENTION
• CONCLUSION
• REFERENCES
 INTRODUCTION
• The term haemolytic disease of the new born and fetus
(HDN) is a destruction of the red blood cells (RBCs) of the
fetus and neonate by antibodies produced by the mother.

• It is a condition in which the life span of the fetal/neonatal

RBC is shortened due to maternal allo-antibodies against red
cell antigens acquired from the father.

(Hendrickson and Delaney, 2016).

 INTRODUCTION CONT
• Increased red cell destruction stimulate increases production
of red cells, many of which enter the circulation prematurely
as nucleated cells hence the term “erythroblastosis foetalis ”.

• Also called Hydrops fetalis as severely affected fetuse may

develop generalized edema called “Hydrops fetalis.”

(Hendrickson and Delaney, 2016).

INTRODUCTION CONT
 ETIOLOGY
• Rhesus incompatibility:
– Hemolytic disease occurs most frequently
– Develops when a Rh-ve mother conceives a fetus which is Rh+ve.
• ABO incompatibility:
– Mother has blood type O and the fetus has blood type A or B or AB.
• Other causes
– Other minor blood group antigens (kell,kid)
– Autoimmune haemolytic anemia

(Kennedy and Krugh, 2005).

 TYPES OF HDN

• ABO hemolytic disease of new born

• Other blood group HDN

– E.g. Rh blood group
– Kell blood group
– Kid blood group etc
(Qureshi et al., 2011).
PATHOGENESIS

(Thompson et al., 2003).

PATHOGENESIS CONT

(Poole and Daniels, 2007).

 CLINICAL SIGN AND SYMPTOMS
• Before birth:
– Anemia
– Heart failure
– Fetal death
• After birth:
– Anemia
– Heart failure
– Hyperbilirubinaemia
– Kernicterus
– Hydrops fetalis
– Severe growth retardation
(Poole and Daniels, 2007).
 LABORATORY DIAGNOSIS
• Blood grouping:
– Mother: rhesus negative
– Father: rhesus positive
– Baby: rhesus positive

• Direct coombs test: positive in infant

• Indirect coombs test: positive in mother
BLOOD TYPE AND RHESUS
INCOMPATIBILITY

(Mancuso, 2017).
 LABORATORY DIAGNOSIS CONT
• CBC
– TLC: normal
– Hb decreased
– MCV, MCH, MCHC: normal or increase
– Platelets: normal to decrease
– Reticulocytosis: increased (6 to 40%)
• Blood smear
– Polychromasia
– Anisocytosis
– Erythroblast (nucleated RBCs)
(Abdellatif, 2020).
COMPLETE BLOOD COUNT (CBC)

(Mancuso, 2017).
 COOMB’S TEST
• DCT PROCEDURE
 Wash patient red cell 3-4 times with normal ionic strength solution (NISS)
 Make a 5% suspension of the washed suspended RBC to a small tube
 Add 1-2 drops of AHG
 Mix well and centrifuge for 1 minutes at 1000rmp
 Re-suspend the cell by gentle agitation and examine macroscopically or
microscopically for agglutination.
 COOMB’S TEST
REPORTS
Agglutination/Haemolysis – Positive
No Agglutination/Haemolysis – Negative
 COOMB’S TEST
• ICT PROCEDURE
 Pool 4-5 Ocells from differebt individual together
 Wash the pooled cells 3-4 times
 Make 3-5% suspension from the wash cells into a new tube

 Centrifuge the patient sample to get the serum

 Add two drops of the suspended washed cells into a new tube
 Add four drops of the patient serum to it
 COOMB’S TEST
 Incubate at 37°c for 45mins
 Wash the incubated cells 3-4 times
 Decant completely after washing
 Add 1-2 drops of AHG
 Light spin at 1000revolution for 10secs
 Resuspend the cells and check for aggluitination macroscopically
and microscopically.
REPORTS
Agglutination/Haemolysis – Positive
No Agglutination/Haemolysis – Negative
 BILIRUBIN ESTIMATION
• Principle: bilirubin reacts with diazo reagent in the presence of
caffeine, benzoate, and acetate as accelerators to form azobilirubin
complex. The color intensity is directly proportional to the amount
of bilirubin in the plasma.
• Procedure:

 Pipette 50µl of sample

 Add 1ml of reagent

(Vasavda et al., 2019).

 BILIRUBIN ESTIMATION
(CONTINUATION)
 20µl of Activator

 Incubate in at room temperature (dark cupboard) for

5minutes.
Reference range:

Conjugated Bilirubin: 0-0.2mg/dl (up to 3.5µmol/L).

Total Bilirubin: 0.2-1.0mg/dl (up to 17µmol/L).

(Vasavda et al., 2019).
 MANAGEMENT
• If there is evidence of erythroblastosis notify pediatrics
team for possibility delivery of a compromised new born.
• Before birth ( Antenatal):
– Intrauterine transfusion
– Early induction of labor when
• Pulmonary maturity has been attained
• Fetal distress is present or
• 35 to 37 weeks of gestation have passed
– The mother may also undergo plasma exchange to reduce
circulating levels of antibody as much as 75%.
(Stockman and Alacron, 2011).
INTRAUTERINE TRANSFUSION

(Stockman and Alacron, 2011).

 MANAGEMENT CONT
• Treatment depends on the severity of condition:
– Phototherapy
– Transfusion with compatible RCC
– Exchange transfusion with a blood type compatible with
both the infant and the mother
– Supportive care
• Temperature stabilization
• Monitoring
• Sodium bicarbonate (correction of acidosis)
• Oxygen ( assisted ventilation)
(Stockman and Alacron, 2011).
 PHOTOTHERAPY

(Stockman and Alacron, 2011).

EXCHANGE TRANSFUSION

(Stockman and Alacron, 2011).

TRANSCUTAENOUS MONITORING

(Stockman and Alacron, 2011).

 MANAGEMENT CONT
• For mother (ANTENATAL): Rh-ve mothers (pregnant with a Rh+ve
infant are given)
– Rh immune globulin (RhIG) to prevent sensitization to D antigen
RhoGAM protects against early effects of transplancenta haemorrhage
(as recommended by the American College of Gynecologist).
• At 28 weeks during pregnancy
• At 36weeks after pregnancy
– Plasma exchange to reduce circulating levels of antibody by as much
as 75%.
– Close monitoring of fetal well-being, as reflected by Rh titers,
amniocentesis results and sonography.
(Wang et al., 2005).
 MANAGEMENT CONT
• For mother (post natal)
– Rh-ve mother with Rh+ve infant
– Injection of RhoGAM must be given within 72hours of
delivery of the newborn.

(Wang et al., 2005).

 PREVENTION
• Determine Rh status of the mother
• If the mother is not sensitized, reduced the risk of future
sensitization
• If the mother is sensitized, determine whether the fetus is at
risk and monitor accordingly
• To prevent isoimmunization of yet unimmunizeds mother give
Anti Rh D IgG (RhoGAM) IntraMuscular at 28 weeks of
gestation.
(Onesimo et al., 2010).
 CONCLUSION

• Haemolytic disease of newborn occurs when IgG antibodies

produced by the mother against the corresponding antigen
which is absent in her, crosses the placenta and destroy the
red blood cells of the fetus.
• Proper early management of Rh-HDN saves lives of a child and
future pregnancies.
• ABO- HDN is usually mild
• Other blood group antigens can also cause HDN.
 REFERENCES
• Abdellatif, M. (2020). "Massage therapy for the treatment of
neonatal jaundice: A systematic review and network
meta analysis". Journal of Neonatal Nursing, 26 (1): 17-
24. 
• Hendrickson JE, Delaney M (2016) Hemolytic Disease of the
Fetus and Newborn: Modern Practice and Future
Investigations. Transfus Med Rev.
• Kennedy MS, Krugh D (2005) Hemolytic disease of the
newborn and fetus. In: Harmening DM (ed.). Modern Blood
Banking and Transfusion practices, (5th edn), FA Davis,
Philadelphia, USA.
 REFERENCES
• Mancuso, C. (2017). "Bilirubin and brain: A pharmacological
approach". Neuropharmacology, 118: 113-123.
• Moise KJ (2008) Fetal anemia due to non-Rhesus-D red-cell
alloimmunization. Semin Fetal Neonatal Med 13: 207-
214.
• Mollison PL, Engelfret CP, Contreras M, editors (2005) Blood
transfusion in clinical medicine, (11th edn), Oxford
Blackwell Science, New Jersey. Pg no:508.
• Onesimo R, Rizzo D, Ruggiero A, Valentini P (2010)
Intravenous Immunoglobulin therapy for anti-E hemolytic
disease in the newborn. J Matern Fetal Neonatal Med 23:
1059-1061.
 REFERENCES
• Poole J, Daniels G (2007) Blood group antibodies and their significance in
transfusion medicine. Transfus Med Rev 21: 58-71.
• Qureshi H, Massey E, Kirwan D, Davies T, Robson S, et al. (2014) BCSH
guideline for the use of anti-D immunoglobulin for the prevention of
haemolytic disease of the fetus and newborn. Transfus Med 24: 8-20.
• Stockman JA, de Alarcon PA (2001) Overview of the state of the art of Rh
disease: history, current clinical management, and recent progress. J
Pediatr Hematol Oncol. 23: 385-393.
• Thompson S, Eggington J, Dodd A (2003) Late developing red cell
antibodies in pregnancy. Transfusion Medicine 13: 8-9.
• Wang M, Hays T, Ambruso DR, Silliman CC, Dickey WC (2005)
Hemolytic disease of the newborn caused by a high titer anti-group B
IgG from a group A mother. Pediatr Blood Cancer 45: 861-862.
THANKS
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