BLOOD TRANSFUSION

COMPARATIVE STUDY BETWEEN THE USAGE OF WHOLE BLOOD AND WHOLE BLOOD RECONSTITUTED IN NEONATAL

HYPERBILIRUBINEMIA IN EXCHANGE TRANSFUSION

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

In partial fulfilment of the regulations For the award of the degree of

M.D BRANCH - XXI

IMMUNOHAEMATOLOGY &

BLOOD TRANSFUSION

DEPARTMENT OF TRANSFUSION MEDICINE

CHENNAI, INDIA

MAY 2020

ABSTRACT

INTRODUCTION:

Neonatal hyperbilirubinemia is an ongoing problem that requires medical attention and hospital readmission in newborns and approximately 5-10 percent of all newborns require intervention for pathologic jaundice. Exchange Transfusion (ET) is an effective emergency intervention to lower the bilirubin levels in neonates at high risk of bilirubin encephalopathy.

AIM:

To evaluate the efficacy of whole blood and whole blood reconstituted used in Exchange Transfusion (ET) in Neonatal Hyperbilirubinaemia (NNH) due to HDFN.

MATERIAL AND METHODS:

Total 40 neonates with ABO and Rh Hemolytic Disease of Fetus and newborn were included in this study and Exchange Transfusion was carried out in both groups either by Whole Blood or Whole Blood Reconstituted selected by randomization.

RESULTS:

In the present study, the efficacy of exchange transfusion between fresh whole blood and whole blood reconstituted among 20 Neonatal cases of Hyperbilirubinemia each revealed improvement in Haemoglobin and fall in bilirubin level in 7 out of 14 cases of ABO HDFN with WB and in all 8 cases of ABO HDFN with WBR.

We observed improvement in haemoglobin level and fall in serum bilirubin in all cases of Rh HDFN in both groups where “O Rh Negative Whole Blood” and O Rh Negative Red Cells with AB Plasma from male donors with no prior history of blood transfusion was used.(Whole Blood Reconstituted).

CONCLUSION:

In our study, we observed better outcome with whole blood reconstituted than whole blood alone exchange transfusion in bringing back desired level of haemoglobin and serum bilirubin in cases of ABO HDFN. In cases of Rh HDFN there is no difference between WBR and WB exchange transfusion. However, to avoid failure to achieve desired results in cases of ABO HDFN using O Whole blood exchange transfusion, as a preventive measure it is essential to use “O Whole Blood” with titre less than critical level.Further, to avoid unexpected exposure to anti-D in cases of Rh HDFN WBR exchange transfusion, it is necessary to use AB plasma only from male donors without the history of prior transfusion.

KEYWORDS:

Hyperbilirubinemia, Jaundice, Exchange Transfusion

ABBREVIATIONS

Ab - Antibody

AHG - Antihuman globulin

BB - Blood Bank

CNS - Central Nervous System CFR - Case Fatality Rate

CBC - Complete Blood Count

DCT - Direct Coombs Test

ET - Exchange Transfusion

FMH - Feto Maternal Hemorrhage

FFP - Fresh Frozen Plasma

HDFN - Hemolytic Disease of fetus and Newborn ICT - Indirect Coombs Test

LBW - low birth weight

NNF - National Neonatology Forum of India NNH - Neonatal Hyperbilirubinemia

NICU - Neonatal Intensive Care Unit NVD - Normal vaginal delivery

PT - Phototherapy

RBC - Red blood Cell

TSB - Total serum bilirubin

UCB - Unconjugated Bilirubin

UDPGT - Uridine Diphosphoglucuronic acid (UDP)–

glucuronyl transferase VLBW - Very low Birth Weight

WB - Whole Blood

WBR - Whole Blood Reconstituted

TABLE OF CONTENTS

SL.

NO TITLE PAGE

NO

1 INTRODUCTION 1

2 AIM AND OBJECTIVE 4

3 REVIEW OF LITERATURE 5

4 MATERIALS AND METHODS 48

5 RESULTS 65

6 DISCUSSION 86

7 SUMMARY 89

8 CONCLUSION 91

9 REFERENCES 92

10 ANNEXURES

Ethical Committee Clearance Documents Plagiarism Clearance Document

Patient Information Sheet and Consent Form Proforma

Master Chart

Introduction

1

INTRODUCTION

Neonatal hyperbilirubinemia (NNH) is an ongoing problem that requires medical attention and hospital readmission in newborns and approximately 5-10 percent of all newborns require intervention for pathologic jaundice.¹

Although bilirubin may have a physiologic role as an antioxidant, elevations of indirect, unconjugated bilirubin are potentially neurotoxic. Even though the conjugated form is not neurotoxic, direct hyperbilirubinemia indicates a potentially serious hepatic disorders or a systemic illness in neonates.In India the incidence of Neonatal Hyperbilirubinemia varied from 4.3% to 6.5% of all live born babies ^{31, 76}

The serum unconjugated bilirubin level of most neonates raises to >2 mg/dl in the first week of life. This level usually raises in full term neonates to a peak of 6 to 8 mg/dl by 3 to 5 days of age and then falls. A raise to 12 mg/dl is in the physiologic range.³³

In premature neonates, the peak may be 10 to 12 mg/dl on the fifth day of life, possibly rising >15 mg/dl without any specific abnormality of bilirubin metabolism.³. This physiologic jaundice is due to increased bilirubin production, increased enterohepatic circulation, defective conjugation and decreased hepatic excretion of bilirubin.

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Adults appeared jaundice when the serum bilirubin level is >2 mg/dl, and newborns appear jaundiced when it is >7mg/dl.³.The yellow color usually results from the accumulation of unconjugated, nonpolar, lipid-soluble bilirubin pigment in the skin. This unconjugated bilirubin is an end product of heme-protein catabolism from a series of enzymatic reactions by heme-oxygenase and biliverdin reductase. It may also be partly caused by deposition of pigment from conjugated bilirubin, the end product from indirect, unconjugated bilirubin that has undergone conjugation in the liver cell microsome by the enzyme uridine diphosphoglucuronic acid (UDP)–glucuronyl transferase to form the polar, water- soluble glucuronide of bilirubin.³

In some neonates, serum bilirubin level may raise excessively, which can be a cause for concern because unconjugated bilirubin is neurotoxic, can cause death and lifelong neurologic sequelae. Early detection and treatment is important in the prevention of bilirubin induced encephalopathy.⁴

For preventing the Kernicterus and other complications of hyperbilirubinemia, jaundice should be managed by phototherapy or Exchange transfusion (ET). ET is an effective emergency intervention to lower the bilirubin levels in neonates at high risk of bilirubin encephalopathy.⁶. Exchange transfusion (ET) is indicated for avoiding bilirubin neurotoxicity when phototherapy has failed or insufficient to lower total serum bilirubin. Exchange transfusion removes circulating total plasma bilirubin, removes antibody-coated sensitized neonates red blood cells (RBCs) in Hemolytic disease of fetus and newborn (HDFN) and

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mother´s antibodies in plasma, replacing them with RBCs compatible with maternal serum and providing albumin with new bilirubin binding site.⁵

The decision to do exchange transfusion is based on the Total Serum Bilirubin (TSB) value for that postnatal age, level of sickness of the baby, the likely etiology of jaundice, and presence or absence of bilirubin encephalopathy.⁶

A double-volume exchange transfusion (Two 85-mL/kg transfusions for full-term neonates and two 100-mL/kg transfusions for VLBW neonates) removes approximately 70% to 90% of the circulating red cells and approximately 50% of the total bilirubin. ⁷

Exchange Transfusion (ET) can be performed using different blood components, For ‗Rh‘ isoimmunisation, the best choice would be ‗O‘ negative packed cells suspended in AB positive plasma. ‗O‘ negative whole blood or cross- matched baby‘s blood group (Rh negative) may also be used.

For ‗ABO‘ HDFN, ‗O‘ group (Rh compatible with baby) packed cells suspended in AB plasma or ‗O‘ group whole blood (Rh compatible with baby) should be used. In other situations baby‘s blood group should be used. All blood must be cross matched against maternal plasma.⁸

Even though NNF guidelines recommend Whole blood reconstituted as the first choice for exchange transfusion in India, many centres practice whole blood only. The study is aimed to establish the role of wholeblood reconstituted for ET in HDFN.

Aim and Objective

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AIM & OBJECTIVE

AIM:

To evaluate the efficacy of whole blood and whole blood reconstituted used in Exchange Transfusion (ET) in Neonatal Hyperbilirubinemia (NNH) due to HDFN.

OBJECTIVE:

To compare bilirubin and hemoglobin values, duration of phototherapy following exchange transfusion between the two groups.

Review of Literature

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REVIEW OF LITERATURE

Hyperbilirubinemia is the most commonly reported cause of readmission during the early neonatal period.^18-20 Jaundice is observed during the first week after birth in approximately 60% of term neonates and 80% of preterm neonates. ²

Kamal A. Patel in their study found that average age of newborn was 3 days (range 0 - 9 days).¹¹⁷ In a study published in lancet reported that, hyperbilirubinemia was a primary diagnosis for severe illness requiring hospital admission in six developing countries and it was the cause for 12–78% of the admissions in the first 6 days of life.¹⁷

Newborn is defined as a child less than 28 days of age.⁹ Depending on the gestational age of mother, the newborn can be classified into term baby and preterm baby. Term birth has been defined as between 37 and 42 weeks of gestation.¹⁰ Preterm birth as any birth before 37 completed weeks of gestation.⁹ According to the Birth weight newborns weighing 2,500 to 4,000 g. was defined as Normal birth weight (NBW)¹² and less than 2,500 g was defined as low birth weight (LBW). ¹¹

INCIDENCE OF NEONATAL JAUNDICE:

From India, Dutta et al reported that severe jaundice represented 15.3% of neonatal admissions, with a Case Fatality Rate (CFR) of 6.7%, leading to 4.4% of the deaths related to jaundice.¹³ An observational study by Bang et al reported that

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severe jaundice had a mortality rate of 7.3/1,000 live births in Indian rural villages.¹⁴

Sgro M et al in their study stated that the incidence of severe hyperbilirubinemia in High Income Countries is currently estimated to be about 31.6/100,000 live births.^21-23

A Kenyan study at a pediatric referral center reported that a total of 306 infants were admitted in the year 2000, with 106 (34.4%) being diagnosed with jaundice; 24 of the jaundiced infants died, giving a Case Fatality Rate of 22.7%.²³

An Egyptian study of 247 babies with TSB ≥ 25 mg/dl admitted in 2008 to the Cairo University Children Hospital, reported that 44 (17.7%) presented with moderate or severe Acute Bilirubin Encephalopathy (ABE) and 26 (10.4%) died, leading to a case fatality rate (CFR) of 56.8% .²³

In a study analyzing NNJ in 3 regions of Indonesia, the prevalence of severe jaundice and acute bilirubin encephalopathy in admitted babies was 6.8 % and 2.2%, respectively.²³

SOURCE OF BILIRUBIN:

Bilirubin is derived from the breakdown of heme-containing proteins in the reticuloendothelial system. The normal newborn produces 6 to 10 mg of bilirubin/kg/day, as opposed to the production of 3 to 4 mg/kg/day in the adult.

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1. Hemoglobin is the major heme-containing protein is red blood cell (RBC).

Hemoglobin released from destruction of senescent RBCs in the reticuloendothelial system is the source of 75% of total bilirubin production.

One gram of hemoglobin produces 34 mg of bilirubin. Accelerated release of hemoglobin from RBCs is the cause of hyperbilirubinemia in isoimmunization, erythrocyte biochemical abnormalities, abnormal erythrocyte morphology, sequestered blood and polycythemia.¹⁶

2. The other 25% of bilirubin is called early-labeled bilirubin. It is derived from hemoglobin released by ineffective erythropoiesis in the bone marrow, from other heme-containing proteins in tissues (e.g., myoglobin, cytochrome, catalase, and peroxidase), and from free heme.¹⁶ (Fig 1)

FIGURE 1

8 BILIRUBIN METABOLISM:

FETAL BILIRUBIN METABOLISM:

Most Unconjugated bilirubin (UCB) formed by the fetus is cleared by the placenta into the maternal circulation. Formation of conjugated bilirubin is limited in the fetus because of decreased fetal hepatic blood flow, decreased hepatic ligandin, and decreased UGT activity. The small amount of Conjugated Biliruin excreted into the fetal gut is usually hydrolyzed by -glucuronidase and reabsorbed. Bilirubin is normally found in amniotic fluid by 12 weeks‘ gestation and is usually gone by 37 weeks‘gestation. ¹⁵

NEONATAL BILIRUBIN METABOLISM:

The heme ring from heme-containing proteins is oxidized in reticuloendothelial cells to biliverdin by the microsomal enzyme heme oxygenase.

This reaction releases carbon monoxide (CO) and iron. Biliverdin is then reduced to bilirubin by the enzyme biliverdin reductase.Catabolism of 1 mol of hemoglobin produces 1 mol each of CO and bilirubin.

1. TRANSPORT:

Bilirubin is nonpolar, insoluble in water, and is transported to liver cells bound to serum albumin. Bilirubin bound to albumin does not usually enter the central nervous system (CNS) and is thought to be nontoxic. Displacement of bilirubin from albumin by drugs, may increase bilirubin toxicity.

9 2. UPTAKE:

Nonpolar, fat-soluble bilirubin (dissociated from albumin) crosses the hepatocyte plasma membrane and is bound mainly to cytoplasmic ligandin (Y protein) for transport to the smooth endoplasmic reticulum. Phenobarbital increases the concentration of ligandin.

3. CONJUGATION:

Unconjugated (indirect) bilirubin (UCB) is converted to water soluble conjugated (direct) bilirubin (CB) in the smooth endoplasmic reticulum by uridine. This enzyme is inducible by phenobarbital and catalyzes the formation of bilirubin monoglucuronide. The monoglucuronide may be further conjugated to bilirubin diglucuronide. Both monoglucuronide and diglucuronide forms of conjugated (direct) bilirubin are able to be excreted into the bile canaliculi against a concentration gradient. At birth, the UDPGT activity level is only 0.1% to 1%

that of the adult.²⁵

4. EXCRETION:

Conjugated (direct) bilirubin in the biliary tree enters the gastrointestinal (GI) tract and is then eliminated in the stool, which contains large amounts of bilirubin. Conjugated (direct) bilirubin is not normally reabsorbed from the bowel unless it is converted back to un conjugated (direct) bilirubin by the intestinal enzyme  glucuronidase. Resorption of bilirubin from the GI tract and delivery back to the liver for reconjugation is called enterohepatic circulation. Intestinal bacteria can prevent enterohepatic circulation of bilirubin by converting

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conjugated (direct) bilirubin to urobilinoids, which are not substrates for - glucuronidase. (Fig 2) Pathologic conditions leading to increased enterohepatic circulation include decreased enteral intake, intestinal atresias, meconium ileus, and Hirschsprung disease.

FIGURE 2

PHYSIOLOGIC HYPERBILIRUBINEMIA:

The serum unconjugated bilirubin level of most newborn infants raises to

>2 mg/dL in the first week of life. This level usually raises in fullterm infants to a peak of 6 to 8 mg/dL by 3 to 5 days of age and then falls. A raise to 12 mg/dL is in the physiologic range. In premature infants, the peak may be 10 to 12 mg/dL on the fifth day of life, possibly rising >15 mg/dL without any specific abnormality of bilirubin metabolism.³³ Levels <2 mg/dL may not be seen until one month of age in both full term and premature infants. ³

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This ―physiological jaundice‖ is attributed to the following mechanisms:

A. Increased bilirubin production due to:

1. Increased RBC volume per kilogram and decreased RBC survival (90 days versus 120 days) in infants compared with adults.

2. Increased ineffective erythropoiesis and increased turnover of non hemoglobin heme proteins.

B. Increased enterohepatic circulation caused by high levels of intestinal  glucuronidase, preponderance of bilirubin monoglucuronide rather than diglucuronide, decreased intestinal bacteria, and decreased gut motility with poor evacuation of bilirubin laden meconium.

C. Defective uptake of bilirubin from plasma

NONPHYSIOLOGIC HYPERBILIRUBINEMIA:

The causes of hemolysis in the neonatal period can be broadly grouped into three major categories:

(1) Heritable defects in red cell metabolism, membrane structure, or hemoglobin;

(2) Acquired disorders; and (3) Immune mediated mechanisms

Incidence of non physiologic jaundice:

Anil Narang et al⁷⁶ in their study stated that the incidence of pathological hyperbilirubinemia (TSB >15 mg/dl) was 6.5% and singhal PK study the incidence of pathological hyperbilirubinemia was 5.9% .³¹

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Arif, M. A. et al studied about 414 neonates with jaundice prospectively, of which 306 had nonphysiological jaundice. ¹¹⁹

CAUSES OF INDIRECT HYPERBILIRUBINEMIA A. Increased Hepatic Bilirubin Load

1. Hemolytic disease—immune mediated (positive direct Coombs test) a. Rhesus isoimmunization

b. ABO

c. Minor blood group

2. Hemolytic disease—red blood cell enzyme abnormalities a. Glucose-6-phosphate dehydrogenase deficiency b. Pyruvate kinase deficiency

3. Hemolytic disease—red blood cell membrane defects a. Hereditary spherocytosis

b. Elliptocytosis c. Stomatocytosis d. Pyknocytosis

4. Hemolytic disease—hemoglobinopathies a. Alpha-thalassemia

b. Gamma-thalassemia

5. Extravascular blood (cephalohematoma) 6. Polycythemia

7. Enhanced enterohepatic bilirubin circulation a. Intestinal obstruction, pyloric stenosis

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b. Ileus, meconium plugging, cystic fibrosis c. Breast-milk feeding

B. Decreased Hepatic Bilirubin Clearance 1. Prematurity including latepreterm gestation 2. Hormonal deficiency

a. Hypothyroidism b. Hypopituitarism

3. Impaired hepatic bilirubin uptake a. Patent ductus venosus

b. SLCO1B1 gene polymorphisms

4. Disorders of bilirubin conjugation—UGT1A1 gene variants a. Crigler–Najjar syndrome type I

b. Crigler–Najjar syndrome type II (Arias disease) c. Gilbert disease

5. Enhanced enterohepatic circulation

a. Intestinal obstruction, pyloric stenosis b. Ileus, meconium plugging, cystic fibrosis c. Breast-milk feeding

Evaluation for hyperbilirubinemia should occur before birth and extend through the first few postnatal weeks. Hemolytic anemia caused by isoantibodies in the infant is a major risk factor for severe hyperbilirubinemia and bilirubin neurotoxicity. ⁶⁴

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Sanpavat S in their study found that the commonest cause for hyperbilirubinemia and exchange transfusion was ABO and was observed in 21.3% of their patients.²⁶

Dinesh et al opined that the ABO was the leading cause of neonatal jaundice and HDFN in countries with high human development index.²⁸

Watchko, J. F. et al in their study stated that mother-infant ABO occurs in approximately 15% of all pregnancies, but symptomatic hemolytic disease occurs in only 5% of these infants. Hyperbilirubinemia in infants who have symptomatic ABO hemolytic disease usually is detected within the first 12 to 24 hours after birth. ²⁹ In 15-25% of all maternal/fetal pairs are ABO-incompatible,but ABO HDFN is confined to the roughly 15 of such group O women who have antenatal high titre (512) IgG antibodies.¹¹¹ Kamal A. Patel in their study out of 31 cases of HDFN the most common cause of HDFN was RhD HDFN, which constituted 20 cases (64.51%) while ABO HDFN and other group HDFN (non-ABO, non-RhD) were 08 (25.80%) and 03 (9.6%) respectively. ¹¹⁷

Hemolytic Diseases of Fetus and Newborn

Hemolytic disease of the fetus and newborn (HDFN) is a condition in which the neonatal red cells have a shortened lifespan because antibodies of maternal origin have crossed the placenta where it binds to the corresponding red cell antigen, causing the antibody coated red cells to be destroyed by macrophages in the fetal spleen.²⁷ They are usually IgG alloantibodies but on rare occasions can

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be IgG maternal autoantibodies.The disease begins in intrauterine life and is therefore correctly described as hemolytic disease of the fetus and newborn.²⁷

The fetal hematopoietic tissue initially responds by increasing erythropoiesis and releasing many of the newly produced red cells into the circulation prematurely as nucleated precursors, a condition known as

―erythroblastosis fetalis.‖ Hemolysis causes fetal anemia, which stimulates the production of erythropoietin leading to increased erythropoiesis. Fetal marrow RBC production cannot keep up with the RBC destruction, and extramedullary erythropoiesis (spleen, liver, kidneys, and adrenals) occurs. Hepatosplenomegaly is a hallmark of erythroblastosis fetalis. ³⁵ A resulting decrease in liver production of albumin leads to reduced plasma colloid osmotic pressure, generalized edema, ascites, and effusions known as ―hydrops fetalis.‖ Untreated, hydrops fetalis, with its associated high-output cardiovascular failure, can lead to fetal death. ³⁴

Historical Background of HDFN:

The royal family of England was not spared from the features of HDFN.

Henry VIII‘s first wife, Katherine of Aragon, conceived six times, among which five died in the perinatal period due to features presumed to be of HDFN. ⁹³ The first description of a clinical condition called hydrops fetalis was in 1609, when a French midwife called Louise Bourgeois described the birth of twins: the first was oedematous and died immediately after birth, the second became deeply jaundiced (icterus gravis) and died a few days later.¹⁰⁸ Over the centuries, this clinical picture was recognised and reported as two separate conditions. In 1641,

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Plater contributed a definitive description of hydrops foetalis.The mystery behind many clinically observed scenarios of HDFN involving red cell hemolysis, ranging widely from severely hydropic stillborn fetuses to infants with mild or significant levels of jaundice and kenicterus, were not realized completely until twentieth century.¹⁰⁹

In 1892,Ballantyne described a criteria for diagnosing HDFN, The two conditions were not associated again until 1932, when Diamond et al.

demonstrated that hydrops and kernicterus were two aspects of the same disease in which hemolysis of the red cells of fetuses and neonates resulted in extramedullary erythropoiesis, causing hepatosplenomegaly and an outpouring of erythroblasts into the circulation, a condition they termed erythroblastosis fetalis.¹⁰⁹

In 1938,Dr.Ruth Darrow, a Chicago pathologist who had a baby die of kernicterus, theorized that the cause of the fetal RBC hemolysis was a maternal fetal RBC antibody produced by the transplacental passage of fetal RBCs into the mother‘s circulation and leading to the reverse transplacental passage of the antibody into the fetus with fetal RBC destruction. She postulated that the offending antigen was fetal Hb.¹¹² In 1940, Rh blood group system was described by Landsteiner and Wiener, and in 1941 Levine et al. determined that D antigen in Rh system is the agent for HDFN. ¹¹³ The main cause of sensitization though was stated by Levine in 1940 but was described in detail by Keenan and Pearse in 1963 .¹¹⁴ Until 1960, HDFN due to rhesus blood group system was considered the

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major contributor to the perinatal mortality rates.¹⁵⁵ In 1961, Finn et al. defined the administration of anti-D Ig in the prevention of Rh sensitization and later in 1967 along with two German scientists Schneider and Preisler proved that anti-D is not useful in already sensitized mothers.¹⁵⁷In 1961 Liley, for the first time, described intrauterine transfusion into the abdominal cavity of the fetus as a preventive measure for the disease. ¹¹⁵ Exchange transfusion, introduced by Wallerstein, and induced premature delivery are other treatment options employed for the management of HDFN.¹¹⁶ Kleihauer and Betke developed an extremely sensitive technique which had distinguished fetal erythrocytes from adult erythrocytes on a stained blood smear.¹¹⁸ Bevis introduced the concept of analyzing blood pigments in amniotic fluid for assessing the severity of hemolytic disease.³² Darrow observed that the mother was an unaffected constant factor,first child was seldom affected and the disease resulted from an acquired immune reaction.¹⁵⁸In 1946,Diamond et al developed a method of exchange transfusion for affected infants. ¹²³

Clinical presentation of HDFN :

HDFN should be considered where there is one or more of the following:

• rapidly developing or severe unconjugated hyperbilirubinemia;

• a positive direct antiglobulin test (DAT);

• positive maternal antenatal antibody screening and/or a severely anemic or hydropic fetus;

• haemolysis detected on blood film examination;

• prolonged hyperbilirubinemia.⁴⁰

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Spectrum of hemolytic disease of fetus and newborn 1. ABO hemolytic disease

2. Rh D hemolytic disease, and

3. Hemolytic disease due to alloantibodies other than anti D comprise the complete spectrum of HDFN.

1.ABO HDFN:

ABO incompatibilities can occur in the following situations:

(1) The mother‘s blood type is O, and the neonate‘s blood type is A or B;

(2) The mother‘s blood type is B, and the neonate‘s blood type is A or AB; and (3) The mother‘s blood type is A, and the neonate‘s blood type is B or AB

History of ABO HDFN:

Halbrecht et al described about the first report of a possible association between maternalfetal ABO and neonatal jaundice .⁵³ Biancalna and tenoff in 1930 were the first to prove the antigenicity of the A and B agglutinogens in human species.⁵⁷ Nevanlinna and Stern pointed out that an ABO between mother and fetus could protect the mother from Rh immunization.³⁰

ABO hemolytic disease of the fetus and newborn (ABO HDFN) is the most common maternofetal blood group . Unlike the rhesus disease, it is usually a problem of the neonate rather than the fetus. Anti-A and anti-B occurring in group B and A subjects are predominantly IgM, but in O subjects are at least partly IgG.¹⁷⁵ ABO hemolytic disease occurs almost exclusively in infants of blood group A or B born to O group mothers, because Ig G anti A, anti B, occur more

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commonly in group O than group A or B individuals. High titre of these immune antibodies may not present with adverse effects in utero as A and B antigens are present on cells of all other tissues and body fluid and not only on red cells. The presence of these antigens helps to protect the incompatible fetal red cells by neutralizing the transferred maternal antibody with small amounts of antibody reacting directly with the fetal red cells. ⁵²

Isohemagglutinins are naturally occurring antibodies and are usually IgM, and people who develop IgG isohemagglutinins are typically group O. Since there are fewer A and B antigenic sites on a newborn‘s RBC membrane, and alternative antigenic sites in other tissues to which these antibodies could bind, anti-A or B IgG infrequently causes clinically apparent HDFN.⁵⁴

Incidence:

In Sharad K. Singh et al., in their study stated that, Blood group incompatibilities (ABO and Rh D) are important causes of unconjugated hyperbilirubinemia in neonates requiring treatment. Though less common, non- Rh D can contribute to severe neonatal hyperbilirubinemia, and needs to be investigated. ³⁶

In Najib KS et al study., identified that the cause of severe hyperbilirubinemia was ABO and Rh (5.9%).³⁸ ABO hemolytic disease is estimated to occur in only 3% of pregnancies and requires treatment with ET in

<0.1% of pregnancies.³⁹

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The incidence of ABO HDFN in the United Kingdom is about 2% of all births, but severe hemolytic disease occurs in only 0.03% of births. ⁴² The incidence of ABO HDFN in Blacks.is said to be higher than in Caucasians. ^43-46 This is due to the higher prevalence and titres of immune anti-A and anti-B antibodies in the Black population.^47-51In Roberts et al in their study stated that ,ABO is now the single largest cause of HDFN in the western world. ⁴⁰ Zwiers et al found that ABO reduces the risk not only for D, but also for non-D immunizations. ABO may cause anemia in the first-born child, but Rh rarely does.

The Anti-H, Anti-A, Anti-B that occurs in Bombay Oh mothers can cause HDFN similar to ABO HDFN. ⁵⁶

Frequency in group A and B infants

If group A and B infants were equally liable to the disease, the ratio A/B in white affected infants should be approximately 2.7 : 1. In the series of Fischer (1961) the ratio was 3.7:1, suggesting that, in white people, group B infants are slightly less liable than group A infants to develop hemolytic disease. Peevy et al in their study stated that a positive DAT was found to be relatively commoner in group B than in group A infants, in both white and black people.⁵⁹ B blood group preponderance was found by Kattimani and Ushakiran.⁶⁰

Wang et al stated that ,Anti-B IgG from a group A mother is an infrequent cause of hemolytic disease of the fetus and newborn (HDFN).⁶¹

21 IgG anti-A and anti-B

The simplest and most satisfactory test is to treat the mother‘s serum with a reducing agent to inactivate IgM antibodies and then determine the anti-A or anti- B titre by IAT using an anti-IgG serum .⁶² Using this method, a titre of 512 or more was found to be very suggestive of hemolytic disease. Chen et al. reported that maternal titers of anti-A or B IgG >1:512 correlated with increased risk of HDFN in group O mothers.⁵⁵

Voak and Bowley found that 66% and 90% of sera from mothers delivering babies with HDFN due to anti-A and anti-B, respectively, contain IgG antibodies with an IAT titer greater than 256. ⁶³

Role of IgG subclasses

Janet M. Pollock, John M. Bowman distribution of anti-D subclasses in HDFN was 3% IgG3 alone, 33% IgGl alone, and 64% IgGl and IgG3.⁶⁸ Frankowska and Gorska found that 87.6% pregnant women contained IgG1 Rh antibodies, 23% contained IgG2 antibodies, 56.9% contained IgG3 antibodies, and 7.7% contained IgG4 antibodies. Most commonly, the sera contained IgG1 alone (33.9%) or IgG1 + IgG3 (32.3%); no sera contained IgG2 and/or IgG4 without IgG1 or IgG3. ⁶⁹

2.Rh HDFN

Rh system is more complex than the single antigen system. Five principal Rh antigens D, C, c, E, and e are responsible for the majority of clinically significant antibodies.⁷⁰

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Rh antigens are lipoprotein molecules, which are sparsely located at the erythrocyte surface. About 50 of them can be identified, which indicates the specific complexity of the Rh antigen. D antigen is the most immunogenic and therefore the most important antigen.

Rh D negative people do not have the D antigen in the Caucasian population, 85% of people are Rh positive and 15% Rh negative .^{71, 73} The frequency of Rh negative women than is more common for Caucasian women (15%) than African American (5%) and is less common in Asian women .^71-75

The first antibody to be described as a cause of HDFN was anti-D. Until the use of Rh immunoprophylaxis in 1968, anti-D was, by far, the most common antibody to cause HDFN, being responsible for about 98% of all cases.^{84, 85}

In Giancarlo Maria Liumbruno et al study stated that before 1945, about 50% of all fetuses with HDFN died of kernicterus or hydrops foetalis.

Subsequently, progress in treatment, in industrialised countries the mortality decreased to 2-3%; this mortality rate was then very considerably further reduced with the introduction of anti-D immunoprophylaxis to prevent maternal-foetal antiRh(D) alloimmunisation.³⁷

Before the widespread adoption of FMH screening and immunoprophylaxis with anti-D immunoglobulin, the incidence of infant mortality from HDFN in England and Wales in 1970 was 1.2 per 1,000 births. By 1989, this figure dropped to 0.02 per 1,000 births. ⁸⁶

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DCe is the most common haplotype in Caucasians (42%), Native Americans (44%), and Asians (70%).¹⁰⁴ With widespread use of RhD immunoglobulin, the focus has shifted to the non- RhD antibodies causing isoimmunization. Other Rh antigens include C, c, E, and e antigens

Rh antibodies implicated in HDFN are:

• Severe HDFN—anti-D and anti-c ^77,78

• Mild disease—anti-C, anti-E, and anti-e. ^79-83

Factors Influence the Immunogenicity ²⁴ 1. Class and subclass of antibody 2. Strength/quantity of antibody

3. Presence, or strength, of antigen on fetal RBCs 4. Efficiency of placental transfer

5. Efficiency of fetal reticuloendothelial system

6. Competition effect of antigen present in fetal body fluids or fetal tissue 7. Maternal blocking antibodies (capable of blocking fetal macrophage

receptors)

Maternal Alloimmunization

Sensitization to an antigen occurs when the immune system encounters an antigen for the first time and mounts an immune response. In the case of HDFN caused by Rh , an Rh D negative mother may first encounter the D antigen while

24

being pregnant with an Rh D positive child, or by receiving a blood transfusion of Rh D positive blood.

Urbaniak, S. J. at el in their study described that at the first exposure to Rh D positive blood, Rh D negative individuals who will seroconvert produce small amounts of IgM. With a second exposure the reaction is rapid with the production of IgG and as little as 0.03ml of Rh D positive red blood cells (RBC) can elicit a secondary response. ⁸⁸

The risk of Rh D alloimmunization after first birth is 16 % if the fetus is ABO compatible with the mother, 2 % if it is not compatible and 2 % after termination of pregnancy. Rh sensitization refers to the level fetomaternal hemorrhage (3 % < 0.1 ml and 22% > 0.1 ml).^{87- 90}

Fetomaternal hemorrhage (FMH) occurs spontaneously during pregnancy and its likelihood increases with gestational age (from 3% in I trimester to 12% in II trimester and 45% in III trimester).^{91, 92}

Detection of feto-maternal hemorrhage

Qualitative Screens for Detection of Fetal RBCs in Maternal Circulation Rosette screen

The rosette screen is a highly sensitive method to qualitatively detect 10 ml or more of fetal whole blood, or 0.2% fetal cells in the maternal circulation.If the rosette test is negative, one vial (300 lg) of RhIg is sufficient to prevent immunization in 99% of patients. ⁹⁴

25

Quantitative Tests for Measuring Fetal RBCs in Maternal Circulation Kleihauer-Betke acid-elution test

The Kleihauer-Betke acid-elution test, originally described by Kleihauer, Braun, and Betke in 1957.⁹⁵ relies on the principle that fetal RBCs containing mostly fetal hemoglobin (HbF) are resistant to acid elution whereas adult hemoglobin is acid-sensitive.

A modified version of the Kleihauer-Betke test was proposed by Clayton et al., who found that preparing the citric acid-phosphate buffer at pH 3.2 resulted in optimal detection of FMH as small as 0.5 mL .^96-98Both over and underestimation of Fetomaternal Haemorrhage have been reported but most of the studies report the tendency of the Kleihauer-Betke test to overestimate FMH. 99,100-103

Flow cytometry

Flow cytometry using monoclonal antibodies directed against HbF has some important advantages over the Kleihauer- Betke test in the quantitation of FMH.

Prevention of Rh Isoimmunization Primary Prevention

Antenatal Prophylaxis

It can be achieved by giving prophylactic dose of anti-D immunoglobulins. If no prophylaxis is given, it is estimated that 1 % of Rh-D negative women would develop antibodies by the end of first Rh-D positive

26

pregnancy. Around 7–9 % of additional women would be sensitized at the time of delivery. Another 7–9 % would develop antibodies during 6 months following delivery. Therefore around 17 % women would become sensitized by the second pregnancy. ^41,111

There are various thoughts regarding the one dose of 300 µg at 28 week versus two doses of 100–120 µg each at 28 and 34 week. Crowther CA et al in their study stated that the risk of RhD alloimmunisation during or immediately after a first pregnancy is about 1.5%. Administration of 100ug (500IU) anti-D at 28 weeks and 34 weeks gestation to women in their first pregnancy can reduce this risk to about 0.2% and the adoption of such a policy will need to consider the costs of prophylaxis. ¹⁶¹

Postpartum Prophylaxis

The FMH which occurs at the time of delivery is covered by prophylactic anti-D within 72 h of birth. A dose of 300 µg of anti-D is given when the baby‘s blood group is Rh-D positive.(Fig 3) If anti-D dose is missed within 72 h, it can be given up to 28 days of delivery with some benefit.¹⁶²

27 FIGURE 3 Secondary prevention

Before 1995, isoimmunized pregnancies were monitored by serial amniocentesis to detect bilirubin levels in amniotic fluid with the help of spectrophotometry. The Liley‘s and Freda‘s graphs are available to manage these pregnancies on the basis of amniotic fluid optical density at 450 nm. The value of OD 450 indicates degree of hemolysis and the fetal outcome . Bullock R et al in their study concluded that management of isoimmunized pregnancies with serial

28

amniocentesis has been now given up in favour of measurement of MCA-PSV because it requires invasive testing with a potential to cause FMH and increase in antibody titers.¹⁶³

Maciuleviciene R et al in their study concluded that the sensitivity of the MCA-PSV was 94.4% in the case of the subgroup of fetuses with severe anemia and the sensitivity of the MCA-PSV test decreased in less anemic fetuses and was 77.3% in the subgroup with moderate anemia and 32% in the subgroup with mild anemia. ¹⁶⁴

Intrauterine Transfusion (IUT)

IUT is considered to be most effective in management of isoimmunized pregnancy where fetus is anemic and not mature enough to be delivered.

Lotus study analyzed outcome after a total of 1284 IUTs performed in 451 fetuses in a 20-year period and found that the alloimmunization was due to RhD in 80%, Kell in 12% and Rh c in 5% of the cases. Twenty-six percent of the fetuses were hydropic at the first transfusion and the mean gestational age at first transfusion was 26 weeks; the mean number of transfusion was 3. They concluded that the vast majority (>95%) of children had a normal neurodevelopmental outcome.¹⁶⁶

Intravenous immunoglobulins (IVIg)

High doses of IVIg to mother have been tried in the management.

However, the results are not promising and the fetus still requires transfusion.

29

Deka et al. studied the effect of high dose of IVIg in iso-immunized mother as primary therapy in six patients and found it to be beneficial. Deka et al. had also studied the effect of direct fetal intravenous transfusion of immunoglobulins and reported that these fetuses required less number of transfusions and the rate of fall in fetal hemoglobin was slower in the intervention group.¹⁶⁵

3.Hemolytic disease due to other antibodies

Historically, the next most common antibody after anti-D was anti-E, followed by anti-c, -Jka, and -K, followed by anti-C, -s, -e, -cE, -Fya. A 12-year study conducted in Sweden comprising of 765 pregnant women found out 836 alloantibodies and its percentages were: D-9.0%, E-6.1%, C-4.3%, Cw-1.2%, c- 4.5%, e-0.1%, Kell-5.7%, Duffy-3.1%, MNSs-4.2%, Kidd-1.2%, Lutheran-1.6%, P1-5.7%, Lewis-28.8%, Others-14.4%.¹⁰⁵ A prospective South Indian study carried out in 624 antenatal women gave a positive antibody screen in 9 cases (1.44%); anti-D-6 (0.96%), anti-D with anti-c -2(0.32%) and anti-M-1 (0.16%).¹⁰⁶ A study conducted in same setting among 2469 D negative females showed a D- alloimmunisation of 3.12% and frequency of Rh-D HDFN was 2.28%.^107.

PREVENTION

Evaluation and Treatment of Neonatal Hyperbilirubinemia Clinical Manifestations

Acute Bilirubin Encephalopathy And Kernicterus:

American Academy of Pediatrics Clinical Practice Guideline describes about the phases of acute Bilirubin Encephalopathy ⁶⁴

30

Early phase is severely jaundiced infants become lethargic and hypotonic and suck poorly.

Intermediate phase is characterized by moderate stupor, irritability, and hypertonia. The infant may develop a fever and high-pitched cry, which may alternate with drowsiness and hypotonia. The hypertonia is manifested by backward arching of the neck (retrocollis) and trunk (opisthotonos).

Advanced phase, in which central nervous system damage is probably irreversible, is characterized by pronounced retrocollis-opisthotonos, shrill cry, no feeding, apnea, fever, deep stupor to coma, sometimes seizures, and death.

Chronic form of bilirubin encephalopathy:

Surviving infants may develop a severe form of athetoid cerebral palsy, auditory dysfunction, dentalenamel dysplasia, paralysis of upward gaze, and, less often, intellectual and other handicaps. ¹²¹

The period of prominent brain pigmentation lasts for only approximately 7 to 10 days, and this phase is accompanied by the commencement of the neuronal changes that result in chronic (post kernicteric) bilirubin encephalopathy.¹²²

Clinical evaluation of jaundice:

Jaundice is detected by blanching the skin with finger pressure to observe the color of the skin and subcutaneous tissues. Jaundice progresses in a cephalocaudal direction. The highest bilirubin levels are typically associated with jaundice below the knees and in the hands, although there is substantial overlap of

31

serum bilirubin levels associated with jaundice progression. Visual inspection is not a reliable indicator of serum bilirubin levels.

Kramer et al¹²⁰ in their study, stated that in newborn infants, progressive hyperbilirubinemia is accompanied by a caudal advancement of dermal icterus which begins at the face and proceeds to the trunk, the extremities, and finally to the palms and soles. All infants were examined at least once every 24 hours, and those infants with rapidly advancing icterus, two to four times daily.

The appearance of dermal icterus was confined to the face and neck (zone 1) when the serum bilirubin concentration was between 4 and 8 mg/100 ml. The dermal icterus progressed to the trunk as far as the umbilicus (zone 2) at levels between 5 and 12 mg/100 ml. As dermal icterus advanced to the groin and upper thighs (zone 3), the serum bilirubin levels were between 8 and 16 mg/100 ml. The knees and elbows to the ankles and wrists (zone 4) became icteric between levels of 11 and 18 mg/100 ml. The feet and hands, including the palms and soles (zone 5), became icteric at serum bilirubin levels of 15 mg/100 ml or higher. ¹²⁰(Fig 4)

FIGURE 4

32 Clinical tests

Visual inspection is not an accurate method to determine bilirubin levels and often missed severe hyperbilirubinemia.¹²⁴ All infants who appear jaundiced should be evaluated TSB measurement.

The American Academy of Pediatrics recommends the following laboratory tests for all infants with jaundice: neonatal blood type, direct Coombs test, complete blood count and smear, and serum bilirubin level, antibody titer.

It is possible to measure bilirubin concentration using capillary or venous blood samples or transcutaneously. In Leslie GI et al in their study there is no systematic difference between the results of capillary or venous samples. ^125,126

A study were conducted in Spain ¹²⁷, India ^128, Turkey ¹²⁹ and Israel ¹³⁰. The study population in three studies included healthy term babies (≥ 37 weeks) and serum bilirubin was measured within 24 hours of birth.The Indian study ¹²⁸ included healthy babies with gestational age > 35 weeks and serum bilirubin (TSB) level of < or = 6 mg/dl was measured at 24 ± 6 hours of age . In their studies , hyperbilirubinemia was defined as serum bilirubin levels ≥ 16.95mg/dl and its prevalence ranged from 2.9% to 12.0%.^127-130

After birth, the infant should be assessed for jaundice at a minimum of every 8 to 12 hours by physical examination and TSB should be assessed.The result should be interpreted based on the nomogram. Reassessment should be based on the zone in which the bilirubin falls on the nomogram. (Fig 5)

33

The bilirubin level should be interpreted according to the infants age in hours. When evaluating need for phototherapy or exchange transfusion,total bilirubin level should be used. Direct bilirubin is not subtracted from the total, except possibly if it constitutes >50% of total bilirubin.

FIGURE 5

34 Diagnosis of HDFN

The direct antiglobulin test

Identification of antibody on infant‘s RBCs (if result of direct Coombs test is positive).

The DAT (Coombs test) is one of the cornerstones of diagnosis of HDFN.

It is a screening test for nonagglutinating antibodies present on an individual's RBCs. If maternal serum contains an IgG class, immunoglobulin directed against a fetal RBC antigen transplacental passage of this antibody will result in RBC antibody coating and a positive neonatal DAT. Blood group with a positive DAT is listed by the Subcommittee on Hyperbilirubinemia of the American Academy of Pediatrics (AAP) as a major risk factor for the development of severe hyperbilirubinemia and also as a risk factor for neurotoxicity.^64,65

In Rh D hemolytic disease, the DAT may be strongly positive without clinical signs of disease; whereas in ABO hemolytic disease, clinical features may exist with only a weak / negative DAT. Elution will also be necessary when the diagnosis of HDFN is in doubt, as in rare cases of ABO with a negative DAT.

Eluates from Infant’s RBCs.

Elute anti-A or anti-B from group A or B infants born to a group O mother, even if the DAT is negative. Van Rossum HH et al in their study stated that in cases of a strong clinical suspicion for HDFN together with a DAT, a second, more sensitive test such as eluate screening should be performed in order to rule out or confirm the diagnosis.⁶⁷

35

MANAGEMENT OF UNCONJUGATED HYPERBILIRUBINEMIA:

In July 2004, the Subcommittee on Hyperbilirubinemia of the American Academy of pediatrics (AAP) published its clinical practice guideline on the management of hyperbilirubinemia in the newborn infant >35 weeks of gestation,describes 2 categories of risk factors.⁶⁴

1. Laboratory and clinical Factors that help to assess the risk of subsequent severe hyperbilirubinemia.

Important Risk Factors for Severe Hyperbilirubinemia

 Predischarge TSB measurement in high-risk or high-intermediate zone

 Lower gestational age

 Exclusive breastfeeding, especially if it is not going well and infant has excessive weight loss

 Jaundice in the first 24 hours of age

 Isoimmune or other hemolytic disease

 Previous sibling with jaundice

 Cephalhematoma or significant bruising

 East Asian race

2. Laboratory and Clinical Factors that might increase the risk of brain damage in an infant who has hyperbilirubinemia

36

Risk Factors for Hyperbilirubinemia Neurotoxicity

 Isoimmune hemolytic disease

 G6PD deficiency

 Asphyxia

 Sepsis

 Acidosis

 Albumin less than 3.0 mg/dL

Phototherapy for jaundice

Figure 6 –Guidelines for phototherapy

The goal of therapy is to lower the concentration of circulating bilirubin or keep it from increasing.Phototherapy achieves this by using light energy to change

37

the shape and structure of bilirubin,converting it to molecules that can be excreted even when normal conjugation is deficient. (Fig 7)

―Intensive phototherapy‖ implies irradiance in the blue-green spectrum (wavelengths of approximately 430–490 nm) of at least 30 μW/cm² per nm (measured at the infant‘s skin directly below the center of the phototherapy unit) and delivered to as much of the infant‘s surface area as possible.

FIGURE 7

A decrease in bilirubin levels of 6% to 20% of initial levels can be expected in the first 24 hours of standard phototherapy.¹⁴² The American Academy of Pediatrics (AAP) found that one needs to treat 6 to 10 otherwise

38

healthy jaundiced neonates with TSB ≥15 mg/dl by phototherapy in order to prevent the TSB in one infant from rising above 20 mg/dl.⁶⁴ Newman TB et al in their study showed that the number needed to treat varies widely and was dependent on gestational age, sex, and age in hours.¹³¹When using intensive phototherapy, a decrease of 0.5 mg/dL (8.6 µmol/L) per hour can be expected in the first 4 to 8 hours. When phototherapy is discontinued, one can expect a rebound increase in TSB levels of 1 to 2 mg/dL.¹⁴³

Dennery PA et al in their study described that, Discontinuation of phototherapy is not standardized.Therefore, clinical judgment is recommended .¹³² Exchange transfusion and phototherapy have traditionally been the primary modes of treatment for affected newborn infants to reduce both mortality and the risk of kernicterus..¹⁵⁶

39 EXCHANGE TRANSFUSION

The method was introduced by Diamond (1947).¹³⁴ Exchange transfusion is the treatment of choice for hyperbilirubinemia when the most aggressive intervention is necessary.

The most common indication for neonatal exchange transfusion currently is hemolytic disease of the fetus and newborn due to maternal isoimmunization to blood groups other than Rh D.¹³² Exchange transfusion should be performed in infants with TSB levels in the range indicated by the nomogram, (Fig 7) with TSB levels of 25 mg per dL or greater, and with jaundice and signs of acute bilirubin encephalopathy.⁶⁴ Exchange transfusion was generally performed using the umbilical vein and citrate phosphate dextrose adenine (CPDA) blood.Aliquots of 5–10 ml of blood are removed from the infant over 15–20 seconds while the same volume of donor blood is administered over 60–90 seconds, dependent upon infant stability.

Infant size and blood volume:

Volume of blood for exchange is calculated using an estimate of the neonate‘s circulating blood volume:

 Term infants: 80–160 mL/kg

 Preterm infants: 100–200 mL/kg.¹³⁵

40 Double volume exchange transfusion

 2 x circulating blood volume

 Replaces approximately 85% of the blood volume and will cause an approximate reduction of 50% in pre-exchange bilirubin level.

Single volume exchange transfusion

 1 x circulating blood volume

 Replaces approximately 60% of the blood volume

The volume to be given is calculated below: ¹³⁶

Total Volume of PC= Total Volume of WBR Desired ×HCT of WBR HCT of PC

Total volume of FFP = Total Volume of WBR −Volume of PC

This RCT, carried out in Switzerland, compared DVET with single-volume exchange transfusion (SVET) in the management of ABO hemolytic disease.

Twenty babies were included, of whom 15 (75%) were male. The mean gestational age of the sample was 39.5 ± 1.0 weeks, the mean birth weight was 3305 ± 392 g, the mean age at entry to study was 17.9 ± 6.1 hours, and the mean serum bilirubin was 207 ± 45 micromol/litre. There was no statistically significant difference between SVET and DVET in mean reduction of serum bilirubin, mean duration of adjunctive phototherapy and level of rebound hyperbilirubinemia.¹³⁷ Amato et al in their small study comparing SVET with DVET in babies with

41

hemolytic jaundice due to ABO . The study noted no significant difference in the short term outcomes.¹³⁸ Lathe et al in their Studies have shown that the double volume exchange removes 80 - 85 % of RBC and single volume exchange removes about 65% of the circulating RBCs.¹³⁹

Mechanisms.

Indications for exchange transfusion

1. When phototherapy fails to prevent a raise in bilirubin to toxic levels.

2. To correct anemia and improve heart failure in hydropic infants with haemolytic disease.

3. To stop hemolysis and bilirubin production by removing antibody and sensitized RBCs.

Indications for immediate exchange transfusion in hemolytic disease:

a. The cord bilirubin level is >4.5 mg/dL and the cord hemoglobin level is under 11 g/dL.

b. The bilirubin level is rising >1 mg/dL/hour despite phototherapy.

c. The hemoglobin level is between 11 and 13 g/dL, and the bilirubin level is rising >0.5 mg/dL/hour despite phototherapy.

d. The bilirubin level is 20 mg/dL, or it appears that it will reach 20 mg/dL at the rate it is rising.

e. There is progression of anemia in the face of adequate control of bilirubin by other methods.

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Bilirubin levels in normal term infants increase from birth, reaching peak levels of 5 to 7 mg/dL around days 3 to 5 of life and then decline by days 7 to 10.^144-146 Asian, Native American, and other populations of primarily breastfed infants have a much different pattern, with their peak levels being higher, reached later, and lasting longer.¹⁴⁴ Breastfeeding newborns have been shown to have mean peak TSB levels of 8 to 9 mg/dL.^147-149

In a review of 2840 infants, Bhutani and colleagues found that the 95th percentile was a level of 17.5 mg/dL.¹⁵⁰ Newman and colleagues also found a TSB level of 17.5 mg/dL to be the 95th percentile in a review of 51,387 infants.¹⁵¹ Maisels and colleagues reported the 95th percentile to be 15.5 mg/dL in a study of infants in the United States, Hong Kong, Japan, and Israel. ¹⁵

Blood for exchange transfusion

ET can be performed using many different combinations of blood components, including fresh whole blood (< 5 days) and packed RBCs reconstituted with fresh frozen plasma (FFP).⁸

Whole Blood

Whole Blood in CPDA-1 is 35 days and has an expiration date of 21 days when stored at 1 to 6˚C. The minimum hematocrit of Whole Blood units is usually approximately 33%.¹⁷⁶

43 RBC

RBCs in anticoagulant-preservative in CPDA-1 have a shelf life of 35 days at 1 to 6˚ C with a hematocrit of 80%.¹⁷⁶

FFP

FFP is plasma collected either from a single unit WB collection or by apheresis. FFP must be placed in the freezer within 8 hours of collection; FFP has a shelf life of 12 months when stored at –18˚ C or colder. FFP, once thawed, has a shelf life of 24 hours at 1 to 6˚ C.¹⁷⁶

Fresh Blood

Whole blood or red blood cells concentrates less than 5 days old ¹⁷⁷ from the time of collection are considered fresh. Fresh blood provides the greatest oxygen-carrying capacity because it has the maximum level of 2, 3 - diphosphoglycerate (2, 3-DPG) and minimum amount of potassium as compared with older blood. 2, 3-DPG and potassium may be important in the premature neonates. ¹⁷⁶ Newborns sometimes need fresher blood because they have high percentage of fetal hemoglobin, which does not release oxygen to the tissues as well as the adult hemoglobin. ¹¹⁰

Whole blood reconstituted

It consists of RBCs combined with ABO-compatible FFP. Following recombination, the product can be stored at 1 to 6˚C for up to 24 hours.

44 Guidelines for exchange transfusion

BCSH guidelines NNF guidelines AABB guidelines

 Group O, compatible with any maternal antibody.

 irradiated

 ‗fresh‘ less than 5 days old,

 Hct 0.5-0.6.

 negative for high-titre anti-A and anti-B antibodies

‘Rh’ isoimmunization,

 O negative packed cells suspended in AB positive plasma.

 O negative whole blood or cross-matched baby‘s blood group (Rh negative) may also be used.

‘ABO’ isoimmunization,

 O group (Rh

compatible) packed cells suspended in AB plasma

 O group whole blood (Rh compatible with baby) should be used.

Other situations

 baby‘s blood group should be used. All blood must be cross matched against maternal plasma.

 fresh (<7 days old),

 irradiated, and

 whole blood

reconstituted

 hematocrit 45–50%

made from PRBCs and FFP collected in CPD.

Rh

 Rh-negative, cross- matched against the mother.

ABO

 Type O Rh-negative or Rh compatible with the mother and infant, be cross-matched against the mother and infant, and have a low titer of naturally occurring anti- A or anti-B abs.

S. Yigit et al studied 381 infants, 300 were transfused with whole blood, whereas 81 infants were transfused with O red cells suspended in A or B plasma.

The Reexchange rate was higher in the whole blood group, compared with the erythrocyte and plasma group.

Use of erythrocyte and plasma provided 30% reduction in the number of ETs per patient. Eight hours after the first ET, mean bilirubin levels were 84% of the pre-exchange values in the whole blood group and 73% of the pre-exchange values in the erythrocyte and plasma group and they concluded that the use of O

45

red cells re-suspended in AB plasma for the ET in cases of ABO hemolytic disease.¹⁴⁰

Kamal A. Patel in his study was to review and establish the practice of exchange transfusion with reconstituted blood in neonates and to observe fall of bilirubin and also raise in hemoglobin. Out of the 31 cases, 20 were of Rhesus (Rh) hemolytic disease of fetus and newborn, while ABO and other blood groups constituted 08 and 03 hemolytic disease of fetus and newborn cases respectively.

They found that the average post-exchange fall in serum indirect bilirubin was (53.47%) and average raise in hemoglobin level was 3.06 gm/dl in all 31 cases and concluded that the reconstituted blood is immunologically much safer and better than whole blood for purpose of exchange transfusion in hemolytic disease of fetus and newborn because of its superiority in minimizing transfusion reactions.¹⁴¹

Dharmesh Chandra Sharma et al¹⁷⁹ in their study of Out of 110 cases, 61 (55.5%) were of RhD HDFN whereas ABO and other group HDFN cases were 30 (27.3%) and 19 (17.3%) respectively. An average post-ET fall in indirect serum bilirubin by 54.6% and correction of anemia by 3.7 gm/dl were reported in the study. They concluded that an average post-ET fall in indirect serum bilirubin and correction of anemia was found to be more significant when compared to other studies.

46 Post exchange bilirubin:

Hemoglobin conversion to bilirubin is rapid and that erythrocyte destruction in the post exchange period could affect the bilirubin level during this time. The erythrocytes which would be considered as a source of hemolysis in the postexchange period would be nonexchanged infant cells and new erythrocytes released from the bone marrow of the infant after the procedure. The volume of infant blood remaining after exchange transfusion can be calculated for each procedure but usually is 10 to 15 per cent of the initial cell population. ¹⁶⁷ The susceptibility of these erythrocytes to rapid destruction is unchanged by the exchange transfusion or possibly enhanced. ¹⁶⁸

Giblett found the half-life of CrS tagged Rh-negative donor cells in a postexehange Rh-incompatible erythroblastic infant to be 16 days rather than the normal 26 days.¹⁶⁹ Hemolysis of the donor cell population in this instance as well as in other situations may contribute to the postexchange bilirubin raise. Allen and Diamond and Dillon and Krivit et al in their study stated that active production of erythrocytes by the bone marrow of the infant after exchange transfusion has been reported. ^170,171 These authors also found that the rate of erythrocyte production varied inversely with the hemoglobin concentration. Sisson and associates studied the effect of using sedimented donor blood to achieve a higher postexchange hemoglobin mass and found a decrease in the degree of bilirubin rebound in infants receiving sedimented cells.¹⁷²

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Potential complications of exchange transfusion

Thrombocytopenia , hypocalcemia, hypomagnesemia, hypoglycemia, arrhythmia, cardiac arrest, respiratory or metabolic acidosis, rebound metabolic alkalosis, and complications associated with blood transfusion and umbilical vessel catheterization.

Steiner and colleagues performed a retrospective review of 107 patients who received 141 exchange transfusions between 1986 and 1996.¹⁵² They did not find a significant difference in transfusion-related complications. Transient asymptomatic complications in the form of thrombocytopenia and hypocalcaemia were observed in 11% of neonates following exchange transfusion. Badice Z has reported this in 6% of his patients. ¹⁵³ While Patra et al showed such complications in 24% of their Patients. ¹⁵⁴

Materials and Methods