TABLE OF CONTENTS:

ALLOIMMUNE HEMOLYTIC ANEMIA................................................................2

INTRODUCTION:........................................................................................................2

CLASSIFICATION OF IMMUNE HEMOLYTIC ANEMIA.................................3

ALLOIMMUNIZATION FROM TRANSFUSION..................................................4

PATHOPHYSIOLOGY:................................................................................................6

CLINICAL PRESENTATION:.....................................................................................8

LABORATORY STUDIES:...........................................................................................9

MANAGEMENT:........................................................................................................10

ALLOIMMUNIZATION DURING PREGNANCY (RhD

ALLOIMMUNIZATION12

BACKGROUND..........................................................................................................13

PATHOPHYSIOLOGY:..............................................................................................13

CAUSES OF MATERNAL ALLOIMMUNIZATION:..............................................14

HEMOLYTIC DISEASE OF INFANTS & NEWBORNS (HDFN)......................16

PATHOPHYSIOLOGY:..............................................................................................17

CLINICAL PRESENTATION:...................................................................................18

LABORATORY STUDIES:.........................................................................................19

MANAGEMENT:........................................................................................................22

DRUG INDUCED ALLOIMMUNIZATION..........................................................29

PATHOGENESIS:......................................................................................................30

SEROLOGICAL DIAGNOSIS:..................................................................................32

REFERENCES:..........................................................................................................33

1

ALLOIMMUNE HEMOLYTIC ANEMIA

INTRODUCTION:

 Alloimmunization is defined as, “an immune response generated in an individual by

an alloantigen from a different individual.”

In alloimmune hemolytic anemia, antibodies are produced against the red blood cells

a person receives in a blood transfusion. If the blood type used for the transfusion is

different than the recipient's blood type, the recipient's immune system can develop

antibodies that attack and destroy the transfused blood cells. 

Alloimmune antibodies also can develop as a result of the mixing of blood between a

pregnant woman and her baby at delivery. If the mother's blood type is Rh-negative

and the baby's is Rh-positive, the mother can produce antibodies against the baby's

blood type. If a mother develops anti-Rh antibodies as a result of one pregnancy, they

can cross the placenta during the next pregnancy and harm the fetus. To prevent this, a

medicine called RhoGam can be given at the time of delivery to block the mother's

body from developing antibodies against the baby's blood type. 

Certain drugs can cause a reaction that develops into hemolytic anemia. These drugs

include high doses of penicillin and related drugs, acetaminophen, quinine and other

drugs to treat malaria, anti-inflammatory drugs, and levodopa. 

Thus Alloimmunization can be broadly divided into three main categories:

1. Alloimmunization after transfusion

2. Maternal Alloimmunization or iso-immunization

2

3. Drug induced alloimmunization

CLASSIFICATION OF IMMUNE HEMOLYTIC

ANEMIA

Antigen Antibody Disease Association

Autoimmune Warm antibody Primary Secondary

Idiopathic Autoimmune disease(SLE)Lymphoproliferative disorders(EBV)Ovarian cystsSome cancersDrugs

Cold antibody Cold haemagglutinin diseaseCold antibody syndromes

Infections, lymphoproliferative disorders

Donath– Landsteiner

Paroxysmal cold hemoglobinuria

Post viral & syphilis

Alloimmune Induced by red cell antigens

Hemolytic transfusion reactionsHDNPost-stem cell allograft

Drug dependent Antibody/macrophage mediatedAntibody/complement mediatedMembrane modification

3

ALLOIMMUNIZATION FROM TRANSFUSION

Allogeneic blood transfusion is a form of temporary transplantation. This procedure

introduces a multitude of foreign antigens and living cells into the recipient that

persist for a variable time. A recipient who is immuno-competent often mounts an

immune response to the donor antigens, resulting in various clinical consequences,

depending on the blood cells and specific antigens involved. The antigens most

commonly involved are classified in the following categories:

Human leukocyte antigens (HLAs)

class I shared by platelets and leukocytes

class II present on some leukocytes

Granulocyte-specific antigens

Platelet-specific antigens (human platelet antigen [HPA])

RBC-specific antigens.

CONSEQUENCES OF ALLOIMMUNIZATION TO BLOOD:

The consequences of alloimmunization to blood include the following clinical

manifestations:

Alloimmunization against RBCs

Acute intravascular hemolytic transfusion reactions (rarely a

consequence of alloimmunization and almost always caused by ABO

antibodies)

Delayed hemolytic transfusion reactions (DHTRs) (hemolysis caused

by RBC all antibodies at least 24 hours post transfusion)

4

Hemolytic disease in newborns (mother's alloimmunization against

fetal antigens, most often resulting from previous pregnancies)

Alloimmunization against platelets (platelet-specific or HLA class I antigens)

Refractoriness to platelet transfusion (an increase in the platelet count

after platelet transfusion that is significantly lower than expected e.g

< 30% of predicted after 10-60 min or < 20% at 18-24h post

transfusion)

Post transfusion purpura (thrombocytopenia after transfusion of red

cells or other platelet-containing products, associated with presence of

platelet allo-antibodies)

Neonatal alloimmune thrombocytopenia (mother's alloimmunization

against fetal antigens, most often resulting from previous pregnancies)

Alloimmunization against granulocytes

(granulocyte-specific or HLA antigens)

Refractoriness to granulocyte transfusion

Febrile non-hemolytic transfusion reactions

Transfusion-related acute lung injury (i-e a transfusion reaction in

which donor HLA antibodies react against recipient antigens)

Transplant rejection

Alloimmunization against HLA antigens

5

Alloimmunization against blood cell antigens (in bone marrow

transplantation)

DHTR and refractoriness to platelet transfusions are most important. Refractoriness to

granulocyte transfusions involves either anti-HLA or granulocyte-specific antibodies

and is similar to platelet refractoriness, except that refractoriness to granulocyte

transfusions results in the patient failing to respond to the granulocyte transfusions.

Granulocyte transfusions are rarely used.

PATHOPHYSIOLOGY:

The main mechanism for alloimmunization to antigens present in transfused cells may

involve presentation of the donor antigens by donor antigen–presenting cells (APCs),

i-e monocytes, macrophages, dendritic cells, B cells, to recipient T cells. Recognition

of the MHC class I alloantigens by CD4+ recipient T cells and their subsequent

activation requires a co-stimulatory signal from either the donor or recipient APCs.

Alloimmunization by non–leukoreduced platelets involves shared donor HLA

antigens (HLA-restricted) and live functional donor APCs. The TH 2 subset of CD4+ T

helper cells secretes interleukin (IL)–4, IL-5, IL-6, and IL-10; activates B cells; and

initiates the antibody response.

Refractoriness to platelet transfusions:

The presence of HLA antibodies on the platelet surface is the most common cause of

platelet refractoriness. Other non-HLA antigens present on the platelet surface (eg,

platelet-specific antigens, HPA) are also involved in a number of cases. Patients not

previously sensitized develop antiplatelet antibodies approximately 3-4 weeks after

the transfusion. Patients previously immunized by transfusion, pregnancy, or organ

6

transplantation develop antiplatelet antibodies as early as 4 days after transfusion.

Macrophages in the liver, spleen, and other tissues of the mononuclear phagocyte

system phagocytize and destroy antibody-coated platelets. Risk factors for developing

antiplatelet antibodies include:

presence > 1 million donor leukocytes in transfused products

transfusing ABO-mismatched platelets

the presence of an intact immune system (i-e, absence of cytotoxic or

immunosuppressive therapy),

Female sex (approximately 75% of cases)

History of multiple transfusions (>20).

Delayed hemolytic transfusion reactions:

DHTRs occur between 24 hours and 3 months (frequently 2 wk) after transfusion and

usually represent a secondary immune response in patients previously immunized by

transfusion or pregnancy. In very rare cases, brisk primary immune response can

result in DHTR after an initial transfusion. Anti-RBC antibody titers frequently (about

50% of the patients with alloimmunization) drop below detectable levels, allowing

incompatible units to be transfused. Transfusion with incompatible RBCs results in

re-stimulation of memory cells and an increase in antibody titer. Antibodies bind to

the surface of RBCs and, depending on the number of antigen-antibody interactions,

activate complement with deposition of C3b usually more than 105 antigenic sites per

cell are required for potent complement activation.

Rarely, binding of immunoglobulin M antibodies to RBCs activates the classic

complement pathway and leads to intravascular hemolysis. RBCs coated with

immunoglobulin G antibodies and/or complement bind to C3b and immunoglobulin

7

Fc receptors present on mononuclear phagocytes and are destroyed by phagocytosis

(i-e extravascular hemolysis). Immunoglobulin G antibodies that efficiently activate

complement (eg, those in Kidd and Duffy systems) tend to cause more intense

extravascular hemolysis compared with antibodies that do not efficiently activate

complement (eg, Rh and Kell).

CLINICAL PRESENTATION:

Delayed hemolytic transfusion reactions:

Hemolysis is usually extravascular, but in some cases, a component of

intravascular hemolysis is present.

Most cases manifest during the second week after transfusion, but the reaction

can occur from 24 hours to 3 months after the transfusion.

Many patients are asymptomatic, and the condition is detected only by

laboratory methods.1

In some patients, fever and/or chills (50%), jaundice (10%), pain (3%), and

dyspnea (1%) can occur.

Rarely, cases may be complicated with renal failure (6%) or disseminated

intravascular coagulation (1%).

In patients with sickle cell disease, a DHTR can precipitate sickle crisis.

Refractoriness to platelet transfusions:

Frequently, patients with refractoriness to platelet transfusion are

asymptomatic and diagnosed by laboratory methods; however, failure to

achieve haemostatic levels of platelets may preclude these patients from

important procedures, including bone marrow transplantation. 8

Alloimmunization should be avoided at all costs in candidates for bone

marrow transplantation.

Preexisting bleeding resulting from thrombocytopenia may persist after

transfusion of an appropriate therapeutic dose of platelets.2 Rarely,

spontaneous bleeding may occur after prophylactic transfusion of platelets.

LABORATORY STUDIES:

Delayed hemolytic transfusion reactions:

The most reliable laboratory sign is a failure to observe the expected post

transfusion increase in blood hemoglobin levels (approximately 1 g/dl/U) in

the absence of bleeding.

Laboratory signs of hemolysis include elevated lactate dehydrogenase, indirect

bilirubin, and reticulocyte levels and decreased hematocrit and haptoglobin

levels.

Intravascular hemolysis is characterized by the presence of free plasma

hemoglobin and possibly hemosiderinuria.

The results of direct and indirect Coombs test are often positive.

Alloantibodies can be eluted from RBCs, and their specificity can be

determined. Often (about 15-20%), patients with DHTR have multiple

antibodies and some may be detectable only by elution.

Refractoriness to platelet transfusions:

Refractoriness to platelet transfusions is defined as repeated failure to achieve

the expected increment in platelet count after 2 or more platelet transfusions.

9

The expected increment can be calculated based on the number of platelets

transfused and the patient's blood volume 

In general, alloimmunization results in the rapid removal of platelets and in

lower counts at 10 minutes to 1 hour post transfusion, whereas non-immune

causes mostly affect the 4- to 24-hour post transfusion count. Mild

alloimmunization, however, can be present with 1-hour increments within the

reference range.

The percentage of cells to which the patient's serum reacts is referred to as the

panel-reactive antibody (PRA) level. PRA values greater than 20% indicate

significant alloimmunization to HLA antigens and correlate with an increased

risk for platelet refractoriness.

The presence of antiplatelet antibodies can be demonstrated by flow cytometry

or by immunoassays such as the modified antigen capture enzyme-linked

assay, the solid-phase RBC adherence assay, and the monoclonal antibody

immobilization of platelet antigens assay. Most of these assays permit

screening for HLA and HPA antibodies as well as specific identification of the

most commonly involved HPA antigens.

A negative result from platelet antibody screening indicates non-immune

causes of refractoriness.

MANAGEMENT:

Delayed hemolytic transfusion reactions:

Most patients tolerate DHTR well and only require observation and supportive

care.

10

Transfusion support with antigen-negative RBCs. If these RBCs are not

available, weigh the risk of further hemolysis against the indications for

transfusion.

If the load of antigen-positive packed RBCs is large (>5 U), consider

exchange transfusion.

Administer intravenous human immunoglobulin (IVIG) to block further

hemolysis in cases in which antigen-positive blood is transfused.

Refractoriness to platelet transfusions:

Avoiding the use of platelet transfusions as much as possible is important in

allo-immunized patients. Preventive transfusions are not recommended.

Measures to minimize the likelihood and extent of bleeding (eg, rapid

treatment of infection; avoidance of invasive procedures; correction of

coagulation deficiencies, anemia, and renal insufficiency; use of

antifibrinolytic agents) should be used extensively.

After diagnosing alloimmune platelet refractoriness, use the sequence of

measures that follows, initiating each subsequent intervention if the previous

one fails.

o Rule out non-immune, autoimmune, and drug-related causes of platelet

refractoriness, or treat accordingly.

o Consider alternatives to platelet transfusion to control bleeding,

including the use of antifibrinolytic agents such as alpha-aminocaproic

acid, or activated recombinant factor VIIa3

o Transfuse ABO-compatible fresh (aged < 48 h) platelet concentrates.

o Transfuse with platelets from blood relatives.

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o Select HLA-matched platelets. Perform HLA typing of patients who

receive multiple transfusions before they become pancytopenic.

o Select cross-matched platelets.

o The use of HPA1a/5b-negative platelets has been successful in cases of

post-transfusion purpura and neonatal platelet alloimmunization.

o Pre-treat with IVIG before transfusion. IVIG pretreatment can result in

successful recovery after platelet transfusion in patients who are

alloimmunized.

o Use high-dose platelet transfusion. Empirical use of high doses of

random platelet units may result in titration of the antibody,

overwhelming of the mononuclear-phagocyte system, and increased

survival of transfused platelets.

o Attempt large-volume plasmapheresis.

o Consider administering immunosuppressive drugs. While steroids are

not effective the use of vincristine and cyclosporin A has been

successful but requires 2-3 weeks to take effect.

ALLOIMMUNIZATION DURING PREGNANCY

(RhD ALLOIMMUNIZATION)

Maternal alloimmunization, also known as isoimmunization, occurs when a woman's

immune system is sensitized to foreign erythrocyte surface antigens, stimulating the

production of immunoglobulin G (IgG) antibodies. The most common routes of

maternal sensitization are via blood transfusion or fetomaternal hemorrhage (i-e

12

transplacental passage of fetal erythrocytes) associated with delivery, trauma,

spontaneous or induced abortion, ectopic pregnancy, or invasive obstetric procedures.

These antibodies can cross the placenta during pregnancies in alloimmunized women

and, if the fetus is positive for these specific erythrocyte surface antigens, result in

hemolysis of fetal erythrocytes and anemia. This, in turn, can lead to potentially

disastrous consequences for the fetus, such as hydrops fetalis, a high-output cardiac

failure syndrome.

BACKGROUND:

Among the more than 50 different antigens capable of causing maternal

alloimmunization and fetal hemolytic disease, the Rhesus (Rh) blood group system is

the most common. The Rh blood system is comprised of the c, C, D, e, and E

antigens. The D antigen of the Rh blood group system (Rh D) causes most cases of

severe hemolytic disease. The incidence of fetuses at risk for anemia due to maternal

alloimmunization to red cell antigens has decreased dramatically since the institution

of routine anti-D immune globulin (RhoGAM) prophylaxis for Rh-negative women in

the 1960s. A review of birth certificate data in 2003 reported the incidence of Rh

sensitization to be approximately 6.8 per 1000 live births.4

EPIDEMIOLOGY:

The prevalence of the Rh D–negative blood type is dependent on ethnicity with

whites having the highest prevalence and Asians and American Indians having the

lowest.

Rates of Rh D negativity among ethnic and racial groups are as follows:

13

White - 15-16%

African American - 8%

African - 4%

Basque (region of Spain/France) - 30-35%

Asian - Less than 1%

Asian American - 1%

American Indian/Inuit - 1-2%

Eurasian - 2-4%

PATHOPHYSIOLOGY:

The risk of alloimmunization in a susceptible Rh D–negative woman is significantly

affected by several factors. These factors include the volume of fetomaternal

hemorrhage, the degree of maternal immune response, concurrent ABO

incompatibility, and fetal homozygosity versus heterozygosity for the D antigen.

Fetomaternal hemorrhages have been demonstrated to occur in as many as 75% of

pregnancies, with the frequency increasing as gestation advances and with most cases

occurring during delivery. If transplacental passage of fetal erythrocytes is suspected,

the rosette screening test is used to determine the presence of a fetomaternal

hemorrhage. When a large hemorrhage is suspected, the Kleihauer-Betke test is used

to quantify the volume of hemorrhage so that an appropriate dose of anti-D IgG can

be administered. Hemorrhage volumes sufficient to cause alloimmunization are

produced in 15-50% of births. This volume of fetal blood, which, in more than 50% of

intrapartum cases can be as small as 0.1mL and in rare cases can exceed 30mL, varies

depending on the degree of maternal immune response.

14

ABO blood group status also affects the risk of alloimmunization. With an ABO-

compatible fetus, the overall risk of alloimmunization if not treated with anti-D IgG is

approximately 16%. However, in an ABO-incompatible fetus, the risk is only 1.5-2%.

The protective effect conferred by ABO incompatibility is believed to be due to

maternal destruction and subsequent clearance of the ABO-incompatible fetal

erythrocytes before Rh sensitization can occur.

Approximately 17% of Rh D–negative women who deliver an Rh D–positive fetus

become alloimmunized if anti-D IgG is not administered appropriately. Of note,

because anti-D IgG prophylaxis has reduced the risk of sensitization to less than 1%

of susceptible pregnancies, other alloantibodies have increased in relative importance.

These include antibodies to other antigens of the Rh blood group system (i-e c, C, e,

E) and other atypical antibodies known to cause severe anemia, such as anti-Kell (i-e

K, k), anti-Duffy (i-e Fya), and anti-Kidd (i-e Jka, Jkb).

Despite the dramatic success of anti-D IgG prophylaxis protocols, prevention is not

universal and 0.27% of susceptible women still become Rh D alloimmunized. One

reason for this is failure to follow recommended protocols. Furthermore, a 0.1-0.2%

rate of spontaneous immunization occurs despite prophylaxis. These cases have

been observed in pregnancies in which no prior overt sensitizing events have

occurred. Finally, alloimmunization involving atypical blood groups (eg, Kell and

c blood groups) is not yet preventable. Therefore, understanding and using available

predictive measures and treatment modalities for hemolytic disease of the fetus and

newborn is essential, as is ensuring that the Rh-alloimmunized pregnancy is properly

managed.

15

CAUSES OF MATERNAL ALLOIMMUNIZATION:

Blood transfusion

Fetomaternal hemorrhage

Antepartum

Intrapartum

Abortion

Therapeutic

Spontaneous

Molar pregnancy

Ectopic pregnancy

Placental abruption

Abdominal trauma

Obstetric procedures

Amniocentesis

Chorionic villus sampling (CVS)

Percutaneous umbilical blood sampling

External cephalic version

Manual removal of the placenta

HEMOLYTIC DISEASE OF INFANTS &

NEWBORNS (HDFN)

 The perinatal effects of maternal Rh alloimmunization are now referred to as

hemolytic disease of the fetus and newborn and fetal manifestations of the disease are

16

more appreciated with newer technologies such as cordocentesis and fetal

ultrasonography.

PATHOPHYSIOLOGY:

After sensitization, maternal anti-D antibodies cross the placenta into fetal circulation

and attach to Rh antigen on fetal RBCs, which form rosettes on macrophages in the

reticuloendothelial system, especially in the spleen. These antibody-coated RBCs are

lysed by lysosomal enzymes released by macrophages and natural killer lymphocytes

and are independent of the activation of the complement system.

Reticulocytosis is noted when fetal Hb deficit exceeds 2 gm/dl compared with

gestational age norms. Tissue hypoxia develops as fetal anemia becomes severe.

When the hemoglobin (Hb) level drops below 8 g/dl, a rise in umbilical arterial lactate

occurs. When the Hb level drops below 4g/dl, increased venous lactate is noted.

Hydrops fetalis occurs when fetal Hb deficit exceeds 7 g/dl, and starts as fetal ascites

and evolves into pleural effusions and generalized edema. The various mechanisms

responsible for hydrops are hypoalbuminemia secondary to depressed liver function,

increased capillary permeability, iron overload secondary to hemolysis, and increased

venous pressures due to poor cardiac function.5

Hemolysis associated with ABO incompatibility exclusively occurs in type-O mothers

with fetuses who have type A or type B blood. Hemolysis due to anti-A is more

common than hemolysis due to anti-B.

CLINICAL PRESENTATION:

17

An infant born to an alloimmunized mother shows clinical signs based on the severity

of the disease. The typical diagnostic findings are jaundice, pallor,

hepatosplenomegaly, and fetal hydrops in severe cases. The jaundice typically

manifests at birth or in the first 24 hours after birth with rapidly rising unconjugated

bilirubin level. Occasionally, conjugated hyperbilirubinemia is present because of

placental or hepatic dysfunction in those infants with severe hemolytic disease.

Anemia is most often due to destruction of antibody-coated RBCs by the

reticuloendothelial system, and, in some infants, anemia is due to intravascular

destruction. The suppression of erythropoiesis by intravascular transfusion (IVT) of

adult Hb to an anemic fetus can also cause anemia. Extramedullary hematopoiesis can

lead to hepatosplenomegaly, portal hypertension, and ascites.

Anemia is not the only cause of hydrops. Excessive hepatic extramedullary

hematopoiesis causes portal and umbilical venous obstruction and diminished

placental perfusion because of edema. Increased placental weight and edema of

chorionic villi interfere with placental transport. Fetal hydrops results from fetal

hypoxia, anemia, congestive cardiac failure, and hypoproteinemia secondary to

hepatic dysfunction. Commonly, hydrops is not observed until the Hb level drops

below approximately 4 g/dl (Hct < 15%)5 . Clinically significant jaundice occurs in as

many as 20% of ABO-incompatible infants.

CAUSES:

Common causes of hemolytic disease of the newborn

Rh system antibodies

18

ABO system antibodies

Uncommon causes - Kell system antibodies

Rare causes

Duffy system antibodies

MNS and s system antibodies

No occurrence in hemolytic disease of the newborn

Lewis system antibodies

P system antibodies

LABORATORY STUDIES:

CBC COUNT:

Anemia: Measurements are more accurate using central venous or arterial

samples rather than capillary blood.

Increased nucleated RBCs, reticulocytosis, polychromasia, anisocytosis,

spherocytes, and cell fragmentation

The reticulocyte count can be as high as 40% in patients without

intrauterine intervention.

The nucleated RBC count is elevated and falsely elevates the leukocyte

count, reflecting a state of erythropoiesis.

Spherocytes (< 40%) are more commonly observed in cases of ABO

incompatibility. Glucose does not correct the autohemolysis in ABO

incompatibility unlike hereditary spherocytosis.

In severe hemolytic disease, schistocytes and burr cells may be

observed, reflecting ongoing disseminated intravascular coagulation.

19

A low reticulocyte count is observed in fetuses provided with

intravascular transfusion in utero and with Kell alloimmunization.

Neutropenia: This condition seems to be secondary to stimulation of

erythropoiesis in favor of myelopoiesis. However, neutrophilia can be

observed after intrauterine transfusion because of an increase in circulating

cytokines (granulocyte-macrophage colony-stimulating factor).

Thrombocytopenia: This condition is common, especially after intrauterine or

exchange transfusions because of platelet-poor blood product and suppression

of platelet production in favor of erythropoiesis.

HYPOGLYCEMIA:

Hypoglycemia is common and is due to islet cell hyperplasia and hyperinsulinism6

The abnormality is thought to be secondary to release of metabolic byproducts such as

glutathione from lysed RBCs. Hypokalemia, hyperkalemia, and hypocalcemia are

commonly observed during and after exchange transfusion.

SEROLOGIC TEST FINDINGS:

Indirect Coombs test and direct antibody test results are positive in the mother

and affected newborn. Unlike Rh alloimmunization, direct antibody test

results are positive in only 20-40% of infants with ABO incompatibility.7 In a

recent study,8 positive direct antibody test findings have a positive predictive

value of only 23% and a sensitivity of only 86% in predicting significant

hemolysis and need for phototherapy, unless the findings are strongly positive

(4+). This is because fetal RBCs have less surface expression of type-specific

20

antigen compared with adult cells. A prospective study has shown that the

titers of maternal immunoglobulin G (IgG) anti-A or anti-B may be more

helpful in predicting severe hemolysis and hyperbilirubinemia.

Although the indirect Coombs test result (neonate's serum with adult A or B

RBCs) is more commonly positive in neonates with ABO incompatibility, it

also has poor predictive value for hemolysis. This is because of the differences

in binding of IgG subtypes to the Fc receptor of phagocytic cells and, in turn,

in their ability to cause hemolysis.

IgG2 is more commonly found in maternal serum but has weak lytic activity,

which leads to the observation of little or no hemolysis with a positive direct

antibody test result. On the other hand, significant hemolysis is associated

with a negative direct antibody test result when IgG1 and IgG3 are

predominant antibodies, which are in low concentration but have strong lytic

activity, crossing to neonatal circulation.

In newborns with hemolytic disease due to anti-c or anti-C antibodies, direct

antibody test results may be negative, and the diagnosis is established after

indirect Coombs testing.

IMAGING STUDIES:

High-resolution ultrasonography has been a major advance in detection of early

hydrops and has also reduced the fetal trauma and morbidity rate to less than 2%

during percutaneous umbilical blood sampling (PUBS) and placental trauma during

amniocentesis. High-resolution ultrasonography has been extremely helpful in

directing the needle with intraperitoneal transfusion (IPT) and intravascular

transfusion (IVT) in fetal location.21

Table 2: Comparison oh Rh and ABO incompatibility:

CHARACTERISTICS Rh ABO

Clinical Aspects First born 5% 50%

Later pregnancies More severe No increased

severity

Still born/hydrops Frequent Rare

Severe anemia Frequent Rare

Jaundice Moderate to

severe/frequent

Mild

Late anemia Frequent Rare

Laboratory

Findings

Direct antibody test Positive Weakly positive

Indirect Coombs

test

Positive Usually positive

Spherocytosis Rare Frequent

MANAGEMENT:

Management of maternal alloimmunization

As a rule, serial maternal antibody titers are monitored until a critical titer of 1:32,

which indicates that a high risk of fetal hydrops has been reached. At this point, the

fetus requires very intense monitoring for signs of anemia and fetal hydrops. In Kell

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alloimmunization, hydrops can occur at low maternal titers because of suppressed

erythropoiesis, and, thus, a titer of 1:8 has been suggested as critical.

Maternal titers are not useful in predicting the onset of fetal anemia after the first

affected gestation. Large differences in titer can be seen in the same patient between

different laboratories, and a newer gel technique produces higher titer results than the

older tube method. Therefore, standard tube methodology should be used to determine

critical titer, and a change of more than 1 dilution represents a true increase in

maternal antibody titer. For all the antibodies responsible for hemolytic disease of the

newborn (HDN), a 4-fold increase in any antibody titer is typically considered a

significant change that requires fetal evaluation9.

When indicated, amniocentesis can be performed as early as 15 weeks' gestation

(rarely needed in first affected pregnancy before 24 weeks' gestation) to determine

fetal genotype and to assess the severity. Maternal and paternal blood samples should

be sent to the reference laboratory with amniotic fluid sample to eliminate false-

positive results (from maternal pseudogene or Ccde gene) and false-negative results

(from a rearrangement at the RHD gene locus in the father).

Fetal Rh-genotype determination in maternal plasma has become routine in other

countries and will soon be offered in the United States. Fetal cell free DNA accounts

for 3% of the total circulating maternal plasma DNA. It is subjected to real-time PCR

for the presence of RHD gene-specific sequences and has been found to be accurate in

99.5% of cases. The SRY gene (in the male fetus) and DNA polymorphisms in the

general population (in the female fetus) are used as internal controls to confirm the

fetal origin of the cell-free DNA.10

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Serial amniocentesis is begun at 10-14 day intervals to monitor the severity of the

disease in the fetus. All attempts should be made to avoid transplacental passage of

needle which can lead to fetomaternal hemorrhage (FMH) and a further rise in

antibody titer. Serial delta-OD 450 values are plotted on the Queenan chart or the

extended Liley chart to evaluate the risk of fetal hydrops.

Traditional management of alloimmunized patients with serial amniocenteses (as

depicted below) was based on which zone the delta OD450 measurement falls into on

the Liley or Queenan curves. Evidence from several studies, including Liley's original

work, indicates that mild or no hemolytic disease occurs in zone 1; intermediate

disease occurs in zone 2 (transitional between mild and severe hemolysis); and severe

disease, including the development of hydrops within the week, occurs in zone 3.

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Based on this evidence, once serial measurements are started, if a zone 1 reading is

obtained, monitoring the delta OD450 approximately every 3 weeks is reasonable.

However, with a trend into zone 2, the frequency of testing should increase to every 1-

2 weeks depending on the steepness of the slope of the curve and the closeness of the

measurement to zone 3.

Early ultrasonography is performed to establish correct gestational age. Frequent

ultrasonographic monitoring is also performed to assess fetal well-being and to detect

moderate anemia and early signs of hydrops.

The peak systolic middle cerebral artery (MCA) Doppler velocity has proved to be a

reliable screening tool to detect fetal anemia. The MCA is easily visualized with

color-flow Doppler; pulsed Doppler is then used to measure the peak systolic velocity

just distal to its bifurcation from the internal carotid artery. Because the MCA velocity

increases with advancing gestational age, the result is reported in multiples of median

(MOMs). In recent studies, the sensitivity for detection of moderate and severe fetal

anemia has been proven to be 100%, with a false-positive rate of 10% at 1.5

MOM.11 It has been shown to reduce the need for invasive diagnostic procedures such

as amniocentesis and cordocentesis by more than 70%.12

MCA Doppler studies can be started as early as 18 weeks' gestation but are not

reliable after 35 weeks' gestation13 . It has also been used to time the subsequent fetal

transfusion and to diagnose anemia from multiple causes, such as in twin-twin

transfusion. The MCA slope from 3-weekly readings is now used to predict fetal risk

for severe anemia14

25

26

Management of the sensitized neonate

Mild hemolytic disease accounts for 50% of newborns with positive direct antibody

test results. Most of these newborns are not anemic (cord hemoglobin [Hb] >14 g/dL)

and have minimal hemolysis (cord bilirubin < 4 mg/dL). Apart from early

phototherapy, they require no transfusions. However, these newborns are at risk of

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developing severe late anemia by 3-6 weeks of life. Therefore, monitoring their Hb

levels after hospital discharge is important.

Moderate hemolytic disease accounts for approximately 25% of affected neonates.

Moderate hemolytic disease of newborn is characterized by moderate anemia and

increased cord bilirubin levels. These infants are not clinically jaundiced at birth but

rapidly develop unconjugated hyperbilirubinemia in the first 24 hours of life.

Peripheral smear shows numerous nucleated RBCs, decreased platelets, and,

occasionally, a large number of immature granulocytes. These newborns often have

hepatosplenomegaly and are at risk of developing bilirubin encephalopathy without

adequate treatment. Early exchange transfusion with type-O Rh-negative fresh RBCs

with intensive phototherapy is usually required. Use of IVIG in doses of 0.5-1 g/kg in

a single or multiple dose regimen have been able to effectively reduce need for

exchange transfusion.15

A prospective randomized controlled study has shown early high-dose IVIG 1 g/kg at

12 hours of age to reduce duration of phototherapy and hospital stay and to prevent

exchange transfusion in neonates with moderate-to-severe Rh

isoimmunization.16 These newborns are also at risk of developing late

hyporegenerative anemia of infancy at 4-6 weeks of life. However, one randomized

double-blind placebo-controlled trial failed to show the benefit of prophylactic IVIG

therapy 0.75 g/kg within 4 hours of age in severely affected neonates who were

treated with intrauterine transfusion for Rh isoimmunization.17

Severe hemolytic disease accounts for the remaining 25% of the alloimmunized

newborns who are either stillborn or hydropic at birth. The fetal hydrops is

predominantly caused by a capillary leak syndrome due to tissue hypoxia,

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hypoalbuminemia secondary to hepatic dysfunction, and high-output cardiac failure

from anemia. About half of these fetuses become hydropic before 34 weeks' gestation

and need intensive monitoring and management of alloimmunized gestation as

described earlier. Mild hydrops involving ascites reverses with IVTs in only 88% of

cases with improved survival but severe hydrops causing scalp edema and severe

ascites and pleural effusions reverse in 39% of cases and are associated with poor

survival.

Management of ABO incompatibility

Management of hyperbilirubinemia is a major concern in newborns with ABO

incompatibility. The criteria for exchange transfusion and phototherapy are similar to

those used in Rh alloimmunization. IVIG has also been very effective when

administered early in the course. Tin(Sn) porphyrin a potent inhibitor of heme

oxygenase, the enzyme that catalyzes the rate-limiting step in the production of

bilirubin from heme, has been shown to reduce the production of bilirubin and reduce

the need for exchange transfusion and the duration of phototherapy in neonates with

ABO incompatibility.

Tin or zinc protoporphyrin or mesoporphyrins have been studied in newborns. They

must be administered intramuscularly in a dose based on body weight, and their

effectiveness appears to be dose related in all gestations.18 Their possible toxic effects

include skin photosensitization, iron deficiency, and possible inhibition of carbon

monoxide production. Their use in Rh hemolytic disease of newborn has not been

reported. Their routine use cannot be recommended yet because of lack of long-term

safety data.

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DRUG INDUCED ALLOIMMUNIZATION

It is rare but in some cases may be acute, severe and even life threatening. Four main

mechanisms have been proposed for antibody dependent drug-induced hemolytic

anemia: drug adsorption, immune complex and membrane modifications lead to

antibody reacting with novel epitopes and the true auto-antibody induced hemolytic

anemia. The same dose at different doses and repeated usage may activate different

mechanisms but the main underlying mechanism is membrane modification.

Diagnosis of drug-induced hemolytic anemia is made in three stages (1) diagnosis of

DAT positive hemolytic anemia (2) careful drug history (3) serological examination

of specific antibodies.

PATHOGENESIS:

Drug adsorption mechanism: IgG antibodies and extravascular hemolysis

drugs in this group readily form hapten-carrier complexes with plasma proteins,

which enhance drug specific antibody production. Prototype drug is penicillin

although cephalosporins and other penicillin derivatives have also been implicated.

90% individuals receiving penicillin produce clinically insignificant IgM anti-

penicillin antibodies. When high dose I/V penicillin is given, drug is adsorbed onto

the red cell surface and become non-specifically attached to red cell surface proteins.

A minority of patients on high dose I/V penicillin therapy ( >1million units daily)

develops high titer IgG antibodies which attach to the drug bound to the red cell

surface and result in extravascular hemolysis. If unrecognized and large doses

continued then complement fixation and acute I/V hemolysis may occur.

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Immune complex mechanism: complement activated acute intravascular

hemolysis

Most common drugs include rifampicin, phenacetin, quinine, quinidine,

hydrochlorothiazide and chlorpropomide. More recently iv cephalosporins and

diclofenac are indicated. Hapten carrier complexes are formed between these drugs

and plasma proteins, leading to the production of drug specific antibodies. Once drug

antibodies are formed reintroduction results in immune complexes to form, which are

adsorbed onto the red cell surface and complement is activated. Classically hemolysis

occurs on the second or subsequent exposure exposure to the drugand may develop

within minutes or hours of drug ingestion.

Membrane modification mechanism:

Cephalosporin in addition to the drug adsorption mechanism, can cause a positive

DAT by modifying red cell membrane components. cisplatin and carboplatin has also

been reported to cause immune hemolytic anemia by this method. As a result, a

variety of plasma proteins including immunoglobulin and complement, may attach via

a non-immune mechanism to the red cell membrane. This may result in finding of a

positive DAT but rarely causes immune hemolytic anemia.

Autoimmune mechanism:

In this case the antibodies show no Rh specificity when tested against Rhnull cells.

some drugs may produce hemolysis by both the immune mechanism and autoimmune

mechanism depending on the circumstances

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SEROLOGICAL DIAGNOSIS:

Drug adsorption and membrane modification mechanism:

The DAT is usually positive with IgG1 or IgG and C3 on red cell surface.

The red cell elutae or serum donot react against normal or enzyme modified

red cells

Warm reacting drug specific antibody in the eluate or serum is only detected

after preincubation of the test red cells with the appropriate drug.

Immune complex mechanism:

DAT is usually positive but may be negative if performed immediately after

brisk episode of hemolysis.

Red cell eluate is not reactive even in the presence of drug

Drug specific antibody is best detected by preincubating the patient’s serum

with the drug in solution to allow immune complexes to form.

Drug metabolite may be detected by preincubating drug metabolite obtained

from the serum or urine of a volunteer with patient’s serum.

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