Clinical Significance of Blood Groups

**3**

**Chapter 1**

*Anil Tombak*

**1. Introduction**

Introductory Chapter: Blood

probably, this was the cause of death in some patients.

Groups - From Past to the Future

Because we know there are different blood groups today, doctors can save lives by transferring the right blood to patients. But previously, the blood transfusion was just a dream. This idea was first discussed by the doctors at the time of Renaissance. In later periods, a French doctor transfused calf blood to a patient in the 1600s and the patient died. Of course, blood transfusions which were made unaware of the presence of antigenic differences ended with death. Because of such unsuccessful trials, the blood transfusion gained a bad reputation. In 1817, Dr. James Blundell, an English obstetrician, said that living species had different blood structures, so blood could not be transfused between different species, but only human blood could be given to a human. In the following years, a total of 10 blood transfusions were performed, of which only 4 survived. Dr. Blundell did not know that human blood had different antigens, and people should be transfused with the same blood group antigens. And

Dr. James Blundell Karl Landsteiner identified ABO blood group antigens in 1900. And this was one of the most important steps in safe transfusion. He wondered what would happen

#### **Chapter 1**

## Introductory Chapter: Blood Groups - From Past to the Future

*Anil Tombak*

#### **1. Introduction**

 Because we know there are different blood groups today, doctors can save lives by transferring the right blood to patients. But previously, the blood transfusion was just a dream. This idea was first discussed by the doctors at the time of Renaissance. In later periods, a French doctor transfused calf blood to a patient in the 1600s and the patient died. Of course, blood transfusions which were made unaware of the presence of antigenic differences ended with death. Because of such unsuccessful trials, the blood transfusion gained a bad reputation. In 1817, Dr. James Blundell, an English obstetrician, said that living species had different blood structures, so blood could not be transfused between different species, but only human blood could be given to a human. In the following years, a total of 10 blood transfusions were performed, of which only 4 survived. Dr. Blundell did not know that human blood had different antigens, and people should be transfused with the same blood group antigens. And probably, this was the cause of death in some patients.

Dr. James Blundell

 Karl Landsteiner identified ABO blood group antigens in 1900. And this was one of the most important steps in safe transfusion. He wondered what would happen

when the bloods of healthy people mixed up and sometimes saw clots in healthy blood. When he separated the plasma with the red cells in the blood and mixed the plasma of the different bloods, he realized that clotting was involved in certain mixtures. He gave random names to these plasmas like A, B, and C. Later, C's name was changed to O; after a while, the AB group was found.

Dr. Karl Landsteiner

In the mid-twentieth century, American researcher Philip Levine discovered Rhesus (Rh) factor and classified the blood as Rh (+) and Rh (−).

Dr. Philip Levine

*Introductory Chapter: Blood Groups - From Past to the Future DOI: http://dx.doi.org/10.5772/intechopen.85014* 

 In this still ongoing historical journey, today, blood groups are defined as hereditary characters on the surface of erythrocytes detected by a specific allo-antibody. International Society of Blood Transfusion (ISBT) reported that there are 33 blood group systems and more than 300 blood group antigens for these systems in humans. The structure of blood group antigens may be protein, glycoprotein, and glycolipid. The distribution of these antigens varies between people and societies, and between the human tissues, as well. Some of them are found just at the erythrocytes, at the other blood cells, and at the tissues. Blood groups are of great importance in transplantation, pregnancy, and transfusion. Some functions of blood group antigen are as follows: transport of some biological molecules toward the erythrocyte membrane, cell adhesion, autologous complement regulators, enzymes, receptors for external stimuli, anchors connecting the erythrocyte membrane to the cell skeleton, extracellular carbohydrates that protect the cell from the mechanical and microbial attacks, etc. Blood group antibodies may develop due to various reasons. These may be "natural antibodies" which develop in the first months of life as in ABO system or may be "immune antibodies" which develop due to transfusion, transplantation, or pregnancy. Antibodies against lots of erythrocyte antigens may cause severe transfusion reactions. For this reason, beside the tests where ABO and RhD antigen are evaluated, additional tests are needed to ensure transfusion safety. The goal is to maintain the vitality and the function of erythrocytes in the longest period and prevention of hemolysis after transfusion. For this purpose, to detect and type antibodies that may pose risk, we perform cross-match, antibody screening/identification, direct antiglobulin test and investigate minor blood group antigens.

#### **2. Conclusion**

 Although the recent developments, the biological structure of most blood group antigens and their functions are still unknown, and we still have more ways to walk in this area. This book aims to reveal the latest developments related to "blood groups."

#### **Author details**

Anil Tombak Mersin University, Mersin, Turkey

\*Address all correspondence to: aniltombak@mersin.edu.tr

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**7**

**Chapter 2**

**Abstract**

*John Ayodele Olaniyi*

infusion of blood into the recipients.

**1. Introduction**

Blood Transfusion Reactions

**Keywords:** adverse blood reactions, blood safety, judicious use of blood, clinical features, management, immunological, immediate, infectious

further encourage judicious use of blood and blood components.

0.5–3% of all transfusions result in transfusion reaction.

**2. What is blood transfusion reaction?**

Blood transfusion remains a life-saving therapy and according to World Health Organization (WHO) guidelines, of 10 units per 1000 population, approximately 8 million units of blood are currently needed to meet the transfusion demand for a population of about 800 million [1]. While in the industrialized world, blood provision and blood safety are well established, in Africa, there is limited access to blood, and provision of unsafe blood renders blood safety a major public health concern. Blood transfusion may be needed in circumstances like obstetric hemorrhage, road traffic accidents, armed conflicts, sickle cell disease, anaemias especially in children, malnutrition, HIV, malaria, and parasitic infections. It is therefore important to always highlight the blood transfusion reactions, possible causes, expected symptoms and signs, preventive measures, and appropriate management. This will

Blood transfusion reaction refers to undesirable, unintended, adverse response to the administration of blood, blood components, or derivatives that are well thought-out to be definitely probable or possibly related to this product. About

Blood transfusion reactions can basically be categorized as infectious or noninfectious. The majority of blood transfusion reactions are, nonetheless,

Blood transfusion reaction/adverse transfusion reactions could be fatal/severe or mild, immediate or delayed, immunological or nonimmunological, and infectious or noninfectious, and attention is paid particularly to the incidence, possible causes and pathophysiology, clinical features, and management of each type with the aim of improving awareness and raising consciousness towards improving blood safety and judicious use of blood so as to forestall these blood transfusion reactions as much as possible. This chapter serves as a synopsis to adverse blood reactions, which are very common but apparently more often under-recognized and/or underreported particularly in developing countries. This should sharpen the consciousness of all health practitioners involved in blood transfusion services towards taking measures at preventing transfusion reactions right from donor selection up to the

## **Chapter 2**  Blood Transfusion Reactions

*John Ayodele Olaniyi* 

### **Abstract**

 Blood transfusion reaction/adverse transfusion reactions could be fatal/severe or mild, immediate or delayed, immunological or nonimmunological, and infectious or noninfectious, and attention is paid particularly to the incidence, possible causes and pathophysiology, clinical features, and management of each type with the aim of improving awareness and raising consciousness towards improving blood safety and judicious use of blood so as to forestall these blood transfusion reactions as much as possible. This chapter serves as a synopsis to adverse blood reactions, which are very common but apparently more often under-recognized and/or underreported particularly in developing countries. This should sharpen the consciousness of all health practitioners involved in blood transfusion services towards taking measures at preventing transfusion reactions right from donor selection up to the infusion of blood into the recipients.

**Keywords:** adverse blood reactions, blood safety, judicious use of blood, clinical features, management, immunological, immediate, infectious

#### **1. Introduction**

Blood transfusion remains a life-saving therapy and according to World Health Organization (WHO) guidelines, of 10 units per 1000 population, approximately 8 million units of blood are currently needed to meet the transfusion demand for a population of about 800 million [1]. While in the industrialized world, blood provision and blood safety are well established, in Africa, there is limited access to blood, and provision of unsafe blood renders blood safety a major public health concern. Blood transfusion may be needed in circumstances like obstetric hemorrhage, road traffic accidents, armed conflicts, sickle cell disease, anaemias especially in children, malnutrition, HIV, malaria, and parasitic infections. It is therefore important to always highlight the blood transfusion reactions, possible causes, expected symptoms and signs, preventive measures, and appropriate management. This will further encourage judicious use of blood and blood components.

#### **2. What is blood transfusion reaction?**

Blood transfusion reaction refers to undesirable, unintended, adverse response to the administration of blood, blood components, or derivatives that are well thought-out to be definitely probable or possibly related to this product. About 0.5–3% of all transfusions result in transfusion reaction.

Blood transfusion reactions can basically be categorized as infectious or noninfectious. The majority of blood transfusion reactions are, nonetheless, noninfectious with outcomes ranging from nonsignificant consequences to death [2, 3]. However, the infectious effects are given more prominence than other adverse reactions.

For emphasis, when any unexpected or untoward symptom or sign occurs during or shortly after the transfusion of a blood component, a transfusion reaction must be considered as the precipitating event until confirmed otherwise [4].

### **3. Classification and incidence of adverse events**

Broadly, BTR can be classified as infectious or noninfectious, immunological or nonimmunological, immediate or delayed, and mild or life threatening. The common, well known manifestations to all types of BTR include fever, chills, and urticaria [3, 5, 6] (**Table 1**).

#### **3.1 The acute (life-threatening) BTRs**

	- Allergic reactions
	- Anaphylaxis (IgA-deficient recipient)
	- Lung damage from microaggregates (massive transfusion)
	- Transfusion-associated circulatory overload ("TACO")
	- Bacterial infection (mainly with platelet transfusion)
	- Hypothermia (rapid infusion of refrigerated blood)
	- Citrate toxicity/hypocalcemia (massive transfusion or apheresis)
	- Graft-versus-host disease
	- Air embolism

#### *3.1.2 Classification of transfusion reactions based on immune or nonimmune*

	- Immediate (acute) haemolytic transfusion reaction
	- Febrile nonhemolytic.

*Blood Transfusion Reactions DOI: http://dx.doi.org/10.5772/intechopen.85347* 

	- Delayed haemolytic transfusion reaction.
	- Other delayed reactions.
	- Minor/major allergic.
	- Anaphylaxis.
	- Transfusion transmissible infections (TTIs) (HIV/HBV/HCV).
	- Transfusion-associated circulatory overload (TACO).

**Tables 1, 3** and **5** refer to classification of BTRs.


#### **Table 1.**

*Types of blood transfusion reactions.* 


### **Table 2.**

*Frequency of transfusion reactions.* 

#### **3.2 Frequency of transfusion reactions**

The risk per unit for each adverse event is as stated in **Table 2**.

#### **4. Common signs and symptoms of blood transfusion**

Although the signs and symptoms of BTR will be fully discussed under each type of blood transfusion reaction, it is important that these features be highlighted as it relates to each system.


#### **4.1 Recognition at bedside**

The complex background clinical condition of critically ill patients could mask the symptoms of a serious blood transfusion reaction; therefore, ventilated patients could have increased peak airway pressures, hyperthermia, and changes in urine output or color in the context of a blood transfusion, during a massive transfusion protocol. Therefore, monitoring core temperature, prompt use of measures to avoid hypothermia, using blood warmers, watch for hypocalcaemia, acidosis, and hyperkalemia go a long way in unmasking blood transfusion reactions.

#### **5. Types of transfusion reactions**

#### **5.1 Minor transfusion reaction symptoms**

A BTR is regarded as minor if:


Quick steps to take when temperature increases by >1°C and >38°C (**Table 3**)


#### *Blood Transfusion Reactions DOI: http://dx.doi.org/10.5772/intechopen.85347*

If clerical error is established or additional serious symptoms are identified, do not order for restart of blood transfusion. Instead


#### Suspect


Initiate transfusion reaction if the abovementioned points are excluded in investigation by


The predominant symptom of a fever is most commonly seen in:


#### **5.2 Febrile nonhemolytic transfusion reaction (FNHTR)**

The incidence of FNHTR is 1 in 300 for RBC concentrate transfusion and 1 in 20 for platelet concentrate transfusion.

Pathophysiological FNHTRs develop in patients that already have anti-leukocyte antibodies. Anti-leukocyte antibodies are raised in multiply transfused patients and multiparous women usually following RBC or platelet transfusions. In addition, donor-derived leukocytes present in platelets and RBC products liberate cytokines in the course of storage of blood and may also mediate NHTRs. Such cytokines include IL1, IL6, IL8, and TNF. Therefore, pre-storage leukoreduction may reduce the accumulation of these biologic mediators and the incidence of febrile, hypotensive, or hypoxic transfusion reactions.

#### **Check for haemolysis**

Perform visual examination of patient's plasma and urine (plasma and urine hemoglobin can be checked but this is not essential).

Blood film may show spherocytosis.

Bilirubin and lactate dehydrogenase (LDH) levels will be raised.

#### **Check for incompatibility**

Check the documentation and the patient's identity. Repeat ABO group of patient pre-transfusion and post-transfusion and of the donor unit(s). Screen the patient for red cell antibodies pre-transfusion and post-transfusion Repeat crossmatch with pre-transfusion and post-transfusion samples. Direct antiglobulin test (DAT) on pre- and post-transfusion samples. Eluate from patient's red cells.

#### **Check for disseminated intravascular coagulation**

Perform blood count and film, coagulation screen, and fibrin degradation products (or D-dimers).

#### **Check for renal dysfunction**

Check blood urea, creatinine, and electrolytes.

#### **Check for bacterial infection**

Take blood cultures from the patient and donor unit including immediate Gram stain.

#### **Immunological investigations**

Check immunoglobulin A (IgA) levels and anti-IgA antibodies.

#### **Table 3.**

*Investigations indicated in transfusion reactions.* 

 Clinical presentation: fever during transfusion or up to 4 hours after. The patient may also experience chills, rigors, nausea and vomiting, and hypotension without fever. FNHTRs typically manifest during or within 4 hours of transfusion with fever (defined as an increase in temperature of 1°C above the patient's baseline temperature, typically to 38°C) with or without chills and/or rigors. Such reactions may also manifest primarily with chills and/or rigors with minimal or absent febrile component particularly in patients receiving antipyretics. Symptoms are self-limited and respond to symptomatic treatment, which includes antipyretics for fever and chills and meperidine for rigors. Close differentials to FNHTRs include acute haemolytic transfusion reaction and septic transfusion reactions and patients' underlying medical condition. Therefore, it is important to do necessary investigations to rule out haemolysis. Leukoreduction has been associated with significant reduction in FNHTRs.

Management: blood transfusion should be stopped immediately and the ordering physician should be informed. Blood transfusion may be restarted cautiously as directed after the thorough investigation (**Table 3** and **Algorithm 1**).

#### **5.3 Acute haemolytic transfusion reaction (AHTR)**

The incidence of AHTR is 1 in 38,000. It is caused by transfusion of incompatible ABO blood group to a patient. It can be fatal with a mortality rate of about 10% and the risk of death is directly proportional to the amount of incompatible blood transfused.

Clinical presentation: fever and chills happen to be the most common feature. Anxiety, pain at the site of infusion, nausea/vomiting, back pain, dyspnea, flushing, wheezing and passage of red color urine, haemoglobinuria, hypotension, renal failure, disseminated intravascular coagulation (DIC), and shock may occur as late/ terminal complications.

#### **Algorithm 1.**

*Algorithm to follow in investigating acute transfusion reaction.* 

Pathophysiology: the ABO isohemagglutinins are compliment fixing and lead to intravascular destruction of transfused red cells which can manifest as hemoglobinemia and haemoglobinuria. Often, fever is the only initial sign. Activation of compliments leads to the release of cytokines like tumor necrosis factor, which is responsible for the fever and chills. The serologic hallmark of acute haemolytic reaction is a positive direct antiglobulin test (DAT), which demonstrates both IgG and compliment on the surface of recipient circulating red cells. Disseminated intravascular coagulation also occurs and bleeding may result.

Possible sources of error/causes include patient misidentification due to clerical error or failure to follow established hospital procedures. Therefore, definitive bedside patient identification, both at the time type and screening specimen, is being obtained, and the time the product is to be administered is very crucial. It has been advocated that the risk of mistransfusion can be greatly reduced by using barcode and radiofrequency chip technologies in order to ensure correct patient identification.

Also, AHTR can occur after platelet transfusions, typically involving a group A patient receiving group O platelets that contain high titer anti-A antibody.

Management: the treatment of AHTR is mainly supportive and it includes taking the following steps:


#### **5.4 Bacterial sepsis or contamination**

The incidence of bacterial contamination for RBC is 1 in 50,000, 1 in 250,000 symptomatic septic reactions, and 1 in 500,000 with fatal bacterial sepsis. The incidence of bacterial contamination for platelet is 1 in 1000 with 1 in 10,000 symptomatic septic reactions and 1 in 60,000 fatal bacterial sepsis. About 10% of transfusion-related deaths are associated with bacterial sepsis.

Clinical presentation: the clinical features are similar to that of AHTRs and comprises of chills, rigors, high grade fever, tachycardia, hypotension, nausea, and vomiting. Disseminated intravascular coagulation (DIC) and shock may occur. Close examination of blood bag may reveal clots and change in color of blood in the bag compared to blood in the segmented tubing. There is no obvious focus of infection in the patient. The reaction typically develops 9–24 hours post transfusion and usually in neutropenic patients.

 Management: such blood transfusion should be discontinued, if suspected and a doctor should be notified immediately who will notify and return the product to the blood bank after careful documentation of events. Necessary investigations should be carried out notably and blood culture samples should be collected. All necessary supportive interventions should be applied as dictated by the patient's clinical condition and the patient should be closely monitored. Also, abnormal bleeding or oozing in a patient during surgery that is equally having blood transfusion may raise suspicion of acute haemolytic transfusion reaction with DIC and appropriate management should be promptly applied (**Algorithm 2**).

#### **5.5 Delayed haemolytic transfusion reaction (DHTRs)**

In DHTRs, the patients develop an alloantibody to an RBC antigen following previous transfusion, pregnancy, or HSCT. Such red blood cell alloantibodies may decrease in titer although remaining clinically important, and hence, the patient has apparently negative antibody screening because the titer of the antibody has fallen below the detectable limit. In the event of a subsequent transfusion, the patient develops an anamnestic immune response to the mismatched antigen leading to delayed antibody-mediated destruction of transfused RBCs.

 Clinical manifestation of AHTRs occurs 5–15 days post transfusion and it comprises haemoglobinuria, jaundice, and pallor as a result of the acute haemolytic process. In the context of a sickle cell disease patient (SCD) that often receives blood transfusion because of hyper-haemolytic crises, these features of haemolytic transfusion reaction are often accompanied by features of vaso-occlusive crisis (VOC), that is, pain, fever, and acute chest syndrome. There is usually worsened anemia and reticulocytopenia. In fact, DHTR is often misdiagnosed as VOC in SCD patient and the patient is unduly further transfused which culminates in multi-organ failure [5–9].

When features of AHTRs manifest, the link to the preceding transfusion is not always obvious. Direct antiglobulin test (DAT) is often positive for IgG, with or without compliment, depending on the antibody if carried out at this point. Also, an eluate may be performed to remove the IgG coating the circulating RBCs in order to identify it because a positive DAT may be unspecific. The antibody screen may also demonstrate the presence of a new antibody, although this may lag behind a positive DAT by a few days. The haemolysis in DHTRs is IgG mediated and thus extravascular; however, it is noteworthy that alloantibodies to Kidd blood group

#### *Blood Transfusion Reactions DOI: http://dx.doi.org/10.5772/intechopen.85347*

#### **Algorithm 2.**

*Necessary steps in the management of blood transfusion reactions.* 

antigens may fix compliment and cause intravascular haemolysis with consequent haemoglobinuria, and occasional instances of severe complications like acute renal failure or disseminated intravascular coagulation have been reported. The antibodies most often implicated in DHTRs are directed against antigens in the Rh (34%), Kidd (30%), Duffy (14%), Kell (13%), and MNSs (4%) [8, 10]

#### *5.5.1 Management of DHTRs*

Ensure leukocyte-poor products as a preventive measure (refer **Algorithm 2**).

#### **5.6 Transfusion-related acute lung injury (TRALI)**

 A consensus definition of TRALI is acute lung injury (ALL) occurring during a transfusion or within 6 hours of completing a transfusion with no other temporarily associated causes of acute lung injury (ALL). ALL is defined as (i) a syndrome of 10 acute onsets, (ii) hypoxemia (PaO2/FiO2 < 300 mm of Hg, O2 saturation < 90% on room air or other clinical evidence), (iii) bilateral pulmonary infiltrates, and (iv) no evidence of circulatory overload [7, 11].

The development of TRALI, which is a potentially life-threatening reaction, is triggered by passive transfusion of donor anti-granulocyte antibodies (anti-HLA or anti HNA antibodies), cytokines, biologically active lipids, or other substances into the recipient. These cause acute lung injury with noncardiogenic pulmonary edema. The signs and symptoms comprise dyspnea, hypoxemia, hypotension, fever, and a chest X-ray showing bilateral lung infiltrates with pulmonary edema (**Figure 1**) [7, 11].

**Figure 1.**  *CXR of a TRALI patient showing pulmonary infiltrates.* 

 Management: aggressive pulmonary support including mechanical ventilation is frequently required. Approximately 80% of patients improve within 48–96 hours and all the patients require oxygen support with approximately 70% needing mechanical ventilation. Infrequently, antibodies in the recipient may react with donor granulocytes that were present in units of RBCs or platelets transfused. Strangely, in some cases of TRALI, neither recipient nor donorderived antibodies can be identified. Other mechanisms have been advanced such as the priming of neutrophils by bioactive lipids that accumulate during blood storage (**Figure 2**) [7, 11].

The United States FDA in 2007 documented that TRALI represented 65% of all transfusion-related fatalities. The widespread implementation of TRALI risk reduction strategies adopted thereafter led to reduction to 37% of transfusion fatalities reported in the 5-year period from 2008 to 2012. TRALI remains the leading cause of death due to transfusion in the US.

The probable incidence rate of TRALI is about 1/5000 transfusions of plasma containing blood product, that is, RBCs, platelets, concentrate, platelet apheresis units, and plasma with a 5–10% fatality rate. TRALI may be difficult to differentiate from manifestations of patients underlying medical problems particularly those of cardiac origin, such as congestive heart failure and fluid overload brought on by transfusion.

Clinical management is supportive with the goal of reversing progressive hypoxemia. There is no universal method to prevent TRALI. Once blood from a particular patient is implicated in a case of TRALI, the donor is excluded from the donor pool. Preventing the first case of TRALI by those donors, however, requires the elimination of all blood donors whose plasma contain anti-HLA or antineutrophil antibodies. For plasma, this is achieved by excluding female donors from the plasma donor pool because multiparous females are most likely among a healthy donor population to have anti-HLA antibodies as a result of sensitization during pregnancy [7, 11]

When this approach was adopted in the UK in late 2003, where 60% of TRALI had been caused by plasma transfusions, no report of TRALI death due to plasma occurred after 2004 (6 deaths occurred in 2005, none from plasma). Major blood suppliers in the US now limit the use of female plasma or screen for HLA or HNA antibodies in multiparous donors. Even with these precautions in place, cases of TRALI in which HLA or any other granulocyte-specific antibodies do not appear to be responsible will not be eliminated.

#### *Blood Transfusion Reactions DOI: http://dx.doi.org/10.5772/intechopen.85347*

#### **Figure 2.**

*Pathophysiologic mechanisms in TRALI. This figure illustrates inflammatory processes in the lung both during the "first hit" and during the "second hit" (acute phase) where the inflammatory processes are heightened than the "first hit". Mediators of inflammation in the infused blood products (FFP > platelets > RBCs) containing donor anti-granulocyte antibodies (anti-HLA and anti-HNA antibodies) along with cytokines and biologically active lipids activate inflammatory cascade through polymorph-nuclear cells (PMNs) with resultant capillary injury. As shown in the "second hit" section of the lung, the capillaries are congested, endothelial cells are swollen and inflamed, and there is increased platelet deposition and aggregation. The interstitial becomes more enlarged. There is increased adherence and migration of neutrophils of activated adhesion molecules (ICAM-1, P-Selectin, L-Selectin). Also, alveolar macrophages liberate inflammatory cytokines (IL-1b, IL-6, IL-8) and activated neutrophils elaborate PAF, NET compliments, and oxidant proteins. All these culminated in lung injury with noncardiogenic pulmonary edema causing hypoxemia, hypotension, pulmonary infiltrates, and fever.* 

Therefore, strict transfusion criteria for plasma-rich blood products, early recognition, and prompt clinical management are the keys to dealing with these potentially fatal transfusion reactions.

Reporting suspected cases of TRALI to the blood bank is also important in limiting potential risk to other patients by quarantine of any co-components from the same donation and evaluating the donor with possible exclusion from future donation if TRALI is confirmed [7, 11].

#### **5.7 Transfusion-associated circulatory overload (TACO)**

 The incidence of TACO is 1/100 and the risk factors in TACO include patients with limited cardiopulmonary reserve, that is, the very young and the very old, high volume transfusion, background renal, or cardiac disease.

The onset is usually 1–2 hours post transfusion. TACO manifests as shortness of breath, cough, chest tightness, cyanosis, rales, orthopnea tachycardia, distended jugular veins, S3 gallop, and pulmonary edema, which are consistent with cardiac decompensation following volume overload [7, 12].

It is important that the vital signs of a patient under general anesthesia and on blood transfusion be continually monitored in order to be able to detect these features early and to be able to prevent TACO.

#### Managing


However, it is important to bear in mind the differences between TACO and TRALI (**Table 4**).

#### **5.8 Allergic transfusion reactions**

Allergic reactions following blood transfusions can be mild and frequently manifested by urticarial rash. Many urticarial reactions are donor-specific and thus do not occur with subsequent transfusion.

#### *5.8.1 Management*

If a recipient experiences multiple urticarial reactions, premedication with antihistamines should be considered.

 Washed products re-suspended in albumin or saline may be considered in severe cases. While removing plasma through washing mitigates allergic reactions, washing platelets impair platelet functions and lead to accelerated clearance after transfusion.

Antihistamines generally alleviate symptoms of allergic reactions but have not been proven to prevent them [7, 13].

#### *5.8.2 Anaphylactic reaction*

The incidence of anaphylactic reaction is put at 1 in 40,000 and the clinical presentation is characterized by widespread rash, shortness of breath cough, tachycardia, flushing, and anxiety.


#### **Table 4.**

*Differences between TRALI and TACO.* 

#### *Blood Transfusion Reactions DOI: http://dx.doi.org/10.5772/intechopen.85347*

Severe IgA-deficient patients may make anti IgA antibody that can cause anaphylactic reaction, but this is a rare occurrence. Considering that approximately 1 in 1200 people is IgA deficient with anti-IgA antibodies and that passively transfused anti IgA antibodies do not cause allergic reactions, the pathophysiology of recurrent and severe allergic transfusion reactions in IgA deficiency is incompletely understood. Washed RBCs, washed platelets, and/or platelet and plasma products from IgA-deficient donors should be transfused only when a patient has severe IgA deficiency and a concern for anaphylactic reactions. Most IgA-deficient patients, even those with anti IgA, have no adverse reactions to transfusion. There are also reports of patients with deficiency of haptoglobin and various complement components such as C4a (Rogers antigen) or C4b (Chido antigen) developing anaphylactic reactions to platelets [7, 13].

#### *Management*: as illustrated in **Table 5** and **Algorithm 1**.

If hives/rash covers <25% of body stop transfusion; do the following: clerical check, notify physician, and notify blood bank.

If clerical error is identified or there are serious symptoms do not restart transfusion, the following should be ensured:



#### **Table 5.**

*The protocol to follow in the case of emergence of fever during blood transfusion.* 

### **6. Infectious complications**

#### **6.1 Approximate risk per transfused unit of various infectious agents**

The risk per transfused units for each infectious agent is as shown in **Table 6** [7].


**Table 6.** 

*Infectious complications of transfusion.* 

#### **6.2 Bacterial and parasitic transmissions by transfusion**

 In the United States, bacterial contamination of platelet products has been recognized as the most common cause of transfusion-associated morbidity and mortality owing to an infectious source. It exceeds hepatitis, HIV, and other viral sources put together. It was noted that the frequency of bacterial contamination is as high as 1 in 1000 to 1 in 2000 platelet units. It results in clinical sepsis after 1 in 4000 platelet transfusions before preventive measures were put in place. As an example, the introduction of bacterial screening has reduced the risk of septic transfusion reactions for apheresis platelets, and it has declined to approximately 1 in 75,000 with the risk of a fatal septic reaction declining to approximately 1 in 500,000 [7, 14, 15].

Efforts to detect the presence of bacteria in platelet units before dispensing to a patient include incubating an aliquot of the unit in a culture system and using a rapid strip immunoassay for bacterial antigens. Other less sensitive methods for detection using a surrogate marker for evidence of bacterial metabolism, such as a low pH, in an aliquot of the platelet suspension have been discontinued. While platelet products are typically contaminated by Gram-positive cocci, such as coagulase-negative Staphylococci, sepsis associated with transfusion of RBC units is most often due to Gram-negative organisms, particularly Yersinia enterocolitica.

 Red blood cell contamination with Yersinia enterocolitica had resulted in bacteremia and septic shock which is often catastrophic. This Gram-negative organism can survive during refrigerated storage and lead to bacteremia or septic shock in the transfused recipient. Malarial transmission by transfusion is very common in Africa where malaria is known to be endemic but uncommon in Europe and America but cases are occasionally reported [7, 15].

#### **6.3 Hepatitis**

The estimated risk of post-transfusion hepatitis C is 1 per 1.1 million units transfused with current use of anti-hepatitis C virus antibody tests and nucleic acid testing.

Post transfusion hepatitis occasionally still develops despite the exclusive use of volunteer blood donors and screening of donor blood for hepatitis B and hepatitis C viruses. Transfusion-related hepatitis C virus infection is usually subclinical and anicteric in most cases but frequently becomes chronic and often results in clinically significant liver dysfunction [7, 15].

 The risk of HBV transmission by transfusion decreased from 1:220,000 to approximately 1:750,000 after implementation of HBV DNA testing. Photochemical pathogen inactivation strategies appear both efficacious and relatively sparing in terms of qualitative platelet function, although decreases in quantitative platelet recovery have been observed in some studies [7, 15].

#### **6.4 HIV and human T-cell lymphotropic viruses**

The risk of acquiring HIV-1 or HIV-2 infection as a result of transfusion currently is estimated to be 1 in 1.5 million. Nucleic acid amplification testing for HIV has reduced the window of serologic conversion from 16 days to about 9 days. The use of heat-treated concentrates, solvent detergent-treated products, and recombinant factor concentrates has essentially eliminated HIV as a therapy-risk for hemophiliacs [7, 15].

#### **6.5 Human T-cell lymphotropic virus 1 (HTLV-1)**

This is a retrovirus associated with adult T-cell leukemia or lymphoma and tropical spastic paraparesis. Screening for HTLV-1 in blood donors is currently performed in the United States because asymptomatic blood donors can transmit this virus. Several cases of neuropathy had been reported in transfused recipients before the availability of testing.

HTLV-2, a related virus with antigenic cross-reactivity to HTLV-1, is endemic in certain Native American populations and also has been found in a high proportion of intravenous drug users. The risk of HTLV transmission by transfusion using current test methods is approximately 1 in 2.7 million [7, 15].

#### **6.6 West Nile virus (WNV)**

WNV became known to the US during the 2002 (WNV) epidemic in the United States wherein 23 individuals acquired WNV after blood transfusion. The characteristic clinical features manifested include fever, confusion, and encephalitis which developed within days to weeks of transfusion. As a result, blood centers implemented nucleic acid-based testing to screen all donations for WNV.

In a survey of 2.5 million donations in 2003, 601 donations (0.02%) were found to contain WNV. A subsequent follow-up study detected no cases of transfusiontransmitted WNV infection among recipients of tested blood; however, rare breakthrough transmissions have been reported [7, 15].

#### **6.7 Parvovirus B19**

 Rare transmissions of parvovirus B19 by transfusion have been recognized. A recent study documented persistence of low levels of parvovirus B19 DNA in a high percentage of multi-transfused patients. The long-term clinical implications of this finding currently are unknown. Parvovirus (and other viruses without a lipid envelope such as hepatitis A virus) is not eliminated by solvent detergent treatment.

 Acute parvovirus B19 infection can result in impaired erythropoiesis and can cause an aplastic crisis in patients with sickle cell disease and other hemolytic diseases. Infection with this virus can also result in significant fetal harm when a pregnant woman is infected during weeks 9–20 of pregnancy. There is no currently available blood donor screening assay for this virus [7, 15].

#### **6.8 Cytomegalovirus (CMV)**

CMV resides in leukocytes, and leukocytes inevitably contaminate RBC and platelet concentrate products. Hence, they are capable of transmitting CMV infection. Transfusion-transmitted CMV infection is an important issue in transfusion of cellular blood products to neonates, particularly low-birth-weight infants born to seronegative mothers, HSCT recipients, and other highly immunosuppressed patients [7, 15].

The risk of acquiring CMV from transfusions is particularly high when pretransplantation serologic testing reveals that neither the HPSC donor nor the recipient has been previously exposed to CMV. In addition, transplantation recipients are at increased risk for transplantation-associated CMV reactivation when either the donor or the recipient is seropositive for CMV before transplantation. The latter consideration often affects the choice of HPSC donors. For these reasons, some institutions use blood products obtained exclusively from CMV-seronegative donors when providing blood products to neonatal recipients or recipients of HPSC transplantations.

However, as noted earlier, a landmark randomized comparison of leukoreduced versus CMV-seronegative blood components in CMV-seronegative HSCT recipients (with seronegative donors) found no significant difference in the incidence of CMV infection, and CMV disease as a composite outcome and most transplantation centers [7, 15].

In practice, prestorage leukoreduced blood components will be used for CMV prevention. Other institutions simply use leukoreduced blood products in all recipients, regardless of CMV status. The latter strategy has the additional advantage of reducing the risk of alloimmunization to HLA antigens and thus of developing refractoriness to platelet transfusions.

#### **6.9 Parasites**

**Malaria**: malarial transmission by transfusion is common in malarial endemic regions of Africa. In nonmalarial endemic areas, donors with a history of residence in a malaria-endemic area or travel associated with a risk of malarial exposure are deferred for up to 3 years, depending on the exposure.

**Chagas disease**: Trypanosoma cruzi parasites can survive several weeks of storage in blood, and contamination of blood products with this organism is already a significant problem in parts of South America. Therefore, the immigration of individuals from South America to the United States raises concerns that Chagas disease may emerge as a common transfusion-transmitted infection [7, 15].

An FDA-approved blood donor-screening test for antibodies to *T. cruzi* is available. Blood donors only need to be tested at their first donation.

 **Babesiosis**: this has been identified in receiving platelets, refrigerated RBCs, and even frozen-thawed RBCs. Cases have been reported in New England and the upper Midwest. Various tests are being evaluated for donor screening in areas endemic for Babesia [7, 15].

#### **7. Conclusions**

 This chapter serves as a synopsis to adverse blood reactions which are very common but apparently more often under-recognized and/or under-reported particularly in developing countries. This should sharpen the consciousness of all health practitioners involved in blood transfusion services towards taking measures at preventing transfusion reactions right from donor selection up to the infusion of blood into the recipients.

#### **Conflict of interest**

No conflict of interest.

### **Notes/Thanks/Other declarations**

Chapter 12; Transfusion medicine: American Society of Hematology Self-Assessment Program served as a good template on which this chapter is built.

### **Author details**

John Ayodele Olaniyi College of Medicine, University College Hospital, University of Ibadan, Ibadan, Nigeria

\*Address all correspondence to: ayodeleolaniyi8@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Tapko J, Mainuka P, Diarra-Nama AJ. Status of Blood Safety in the WHO African Region: Report of 2006 Survey. Brazaville, Republic of Congo: WHO Regional Office for Africa; 2006. Available from: http//wwwáfro.who.int/ en/divisions-a-programmes/dsd/healthtechnologies-a-laboratories.html

[2] Suddock JT, Crookston K. Transfusion Reactions. StartPearls Publishing; 2018

[3] Land KJ, Townsend M, Goldman M, Whitaker BI, Perez GE, Wiersum-Osselton JC. International validation of harmonized definitions for complications of blood donations. Transfusion. 2018;**58**(11):2589-2595

[4] Webert K. Transfusion Reactions: Are Those Symptoms Normal? [PowerPoint slides]. 2015. Retrieved from http://www.transfusion.ca/

[5] Fung MK, editor. Non-infectious complications of blood transfusion. Chapter 27. In: AABB Technical Manual. 18th ed. Bethesda: AABB; 2014

[6] Popovsky M, editor. Transfusion Reactions. 3rd ed. Bethesda: AABB Press; 2007

 [7] Savage W, Bakdash S. Transfusion medicine, Chapter 12. In: American Society of Haematology Self-Assessment Program Textbook. 6th ed. 2016. Available from: www.ash.sap.org

[8] Jasinski S, Glasser CL. Catastrophic delayed hemolytic transfusion reaction in a patient with sickle cell disease without alloantibodies: Case report and review of literature. Journal of Pediatric Hematology/Oncology. 2018. DOI 10.1097/MPH00000000001307. PMID: 30179992

[9] Fasano RM, Meyer EK, Branscomb J, White MS, Gibson RW, Eckman

JR. Impact of red blood cell antigen matching on alloimmunization and transfusion complications in patients with sickle cell disease: A systematic review. Transfusion Medicine Reviews. 2018;**26**

[10] Siddon AJ, Kenney BC, Hendrickson JE, Tormey CA. Delayed haemolytic and serologic transfusion reactions: Pathophysiology, treatment and prevention. Current Opinion in Hematology. 2018;**25**(6):459-467

[11] Tariket S, Sut C, Hamzeh-Cognasse H, Laradi S, Garraud O, Cognasse F. Platelet and TRALI: From blood component to organism. Transfusion Clinique et Biologique. 2018;**25**(3):204-209

[12] Garraud O, Cognasse F, Laradi S, Hamzeh-Cognasse H, Peyrard T, Tissot JD, et al. How to mitigate the risk of inducing transfusion associated adverse reactions. Transfusion Clinique et Biologique. 2018;**25**(4):262-268

[13] Frazier SK, Higgins J, Bugajski A, Jones AR, Brown MR. Adverse reactions to transfusion of blood products and best practices for prevention. Critical Care Nursing Clinics of North America. 2017;**29**(3):271-290

[14] Erony SM, Marshall CE, Gehrie EA, Boyd JS, Ness PM, Tobian AAR, et al. The epidemiology of bacterial culture-positive and septic transfusion reactions at a large tertiary academic center: 2009 to 2016. Transfusion. 2018;**58**(8):1933-1939

[15] Block EM, Vermeuilen M, Murphy E. Blood transfusion safety in Africa: A literature of infectious diseases and organizational challenges. Transfusion Medicine Reviews. 2012;**26**(2):164-180. NIH Public Access authored Manuscript. DOI: 10.1016/j. tmrv.2011.07.006

#### Chapter 3

## Hemolytic Disease of the Fetus and Newborn

Soumya Das

#### Abstract

Hemolytic disease of the fetus and newborn (HDFN) also called as "erythroblastosis fetalis" is characterized by the increased rate of red blood cells (RBCs) destruction. Hemolysis should always be investigated even if the anemia is mild and apparently trivial. The principle clues which suggest hemolytic anemia includes: increased number of reticulocytes and/or circulating nucleated RBCs, unconjugated hyperbilirubinemia, a positive direct antiglobulin test and characteristic changes in red cells in the blood films. Based on etiology, hemolysis in newborn can be immune or non-immune mediated. The immune-mediated hemolysis due to blood group incompatibility between the mother and the fetus is the main cause of HDFN.

Keywords: hemolysis, blood groups

#### 1. Introduction

Hemolytic disease of the fetus and newborn (HDFN) also called as "erythroblastosis fetalis" is characterized by the increased rate of red blood cell (RBC) destruction. Hemolysis should always be investigated even if anemia is mild and apparently trivial. The principle clues which suggest hemolytic anemia include increased number of reticulocytes and/or circulating nucleated RBCs, unconjugated hyperbilirubinemia, a positive direct antiglobulin test, and characteristic changes in red cells in the blood films [1, 2]. Based on etiology, hemolysis in newborn can be immune or nonimmune mediated. The nonimmune causes include α-thalassemia, RBC membrane, or enzyme defects [1]. The immune-mediated hemolysis due to blood group incompatibility between the mother and the fetus is the main cause of HDFN. Immune-mediated hemolysis of fetal red cells, due to blood group incompatibility, occurs when there is transplacental passage of maternal antibody active against paternal red cell antigen of the infant [2–4]. Both naturally occurring and immune antibodies are implicated leading to a spectrum of clinical sequela, ranging from anemia and hyperbilirubinemia to fetal hydrops, kernicterus, and death [3, 4]. Although more than 60 different RBC antigens are capable of eliciting an antibody response, significant morbidity is associated primarily with D antigen of Rh group [2]. The prevalence of red cell antibodies other than anti-D with the potency to induce HDFN is about 1 in 500 pregnancies [5–7].

#### 2. History

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 [8]. The welldocumented description of HDFN was made in 1609 by the midwife in the French literature. The case was a twin gestation in which the first fetus was stillborn and the second twin developed jaundice and succumbed soon after birth [9]. 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 [10, 11]. The main cause of sensitization though was stated by Levine in 1940 but was described in detail by Keenan and Pearse in 1963 [12]. In 1961 Liley, for the first time, described intrauterine transfusion into the abdominal cavity of the fetus as a preventive measure for the disease [13]. Exchange transfusion, introduced by Wallerstein, and induced premature delivery are other treatment options employed for the management of HDFN [14, 15]. Until 1960, HDFN due to rhesus blood group system was considered the major contributor to the perinatal mortality rates. 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 [16, 17]. This was a major breakthrough in prevention of sensitization with administration of 400 μg of human anti-D globulin within 72 h after delivery. Since 1971, the WHO recommends empirical use of anti-D Ig following any sensitizing event including after delivery of Rh-positive newborn and for abortions.

#### 3. Blood group antigens and antibodies

An antigen is any substance which, when introduced into the body of an immunocompetent individual, stimulates the immune system by production of an antibody by interacting with the immunoglobulin receptor of B cell. Each red cell membrane is a bi-phospholipid layer containing millions of antigen on its surface [18]. The blood group antigens are either protein or carbohydrate structures present on the red cell membrane. An individual's blood group is determined by the antigen expressed on the surface of red cell membrane. The carbohydrate antigens are expressed as it is, while the protein antigens stretch in the bi-phospholipid layer by transmembrane proteins [19].

Antibodies are recognition proteins found in the serum and other body fluids of vertebrates that react specifically with the antigens that induce their formation. Antibodies belong to a family of globular proteins called immunoglobulins. The terms antibody and immunoglobulins are used synonymously. They are produced by the lymphocyte-plasma cell system. Antibodies bind antigen, fix complement, facilitate phagocytosis, and neutralize toxic substances in the circulation [18]. IgG, IgM, and IgA are the most significant from the point of view of transfusion medicine. Most clinically significant antibodies are IgG type, reacting at body temperature (37°C) with the antigens, and cause significant in transfusion reactions as they are a class of antibodies produced in response to nonself-antigens on the blood products. IgM antibodies are mostly naturally occurring antibodies [19].

#### 4. Alloimmunization during pregnancy

Alloimmunization can be caused due to pregnancy, blood transfusion, or tissue/ organ transplantation or grafting, due to genetic difference between the individuals [20]. On exposure to a foreign red cell antigen, the immune system is activated which is mediated by lymphocytes. The first step involved is recognition of the antigen by T cell. The recipient's helper T cell interacts with the MHC class II


Table 1.

Sensitizing events for feto-maternal haemorrhage [24].

molecule expressed on the donor red cells. Following the initial interaction, T cells trigger a second signal for the B lymphocytes, in order to stimulate humoral immune response [20, 21]. Initially, IgM class of antibody is produced in primary response to an antigen and is formed as early as 4 weeks to 3 months period. However, there is a switch of IgM to IgG class during the secondary response [20]. The secondary response is more rapid, potent, and specific than the primary.

Placenta is a natural barrier present between mother and fetus. Only IgG antibodies can cross the placenta. The transfer is mediated by the neonatal Fc receptors (FcRn) [6]. The immunoglobulin is bound and transported by FcRn of syncytiotrophoblast which also protects IgG molecule from normal serum protein catabolism. In the first trimester, there is relatively less transfer; however, it subsequently increases exponentially in the second and third trimester. Mean concentration in the fetus at the 24th week of pregnancy is 1.8 g/dL. The IgG antibody levels are higher in the fetus than in the mother toward the term [22]. Of the four subclasses of IgG antibody, IgG3 and IgG1 are more efficient in RBC hemolysis than IgG2 and IgG4, though all the four classes are efficiently transferred across the placenta [23].

#### 4.1 Sensitizing events

Transplacental feto-maternal hemorrhage occurs in over 75% of pregnancies. The average volume of fetal blood in the maternal circulation following delivery is less than 1 mL in 96% of pregnancies [24]. As the pregnancy progresses, the possibility of feto-maternal hemorrhage increases, 3% in first trimester, 12% in the second, 45% in the third, and 64% at the time of delivery as shown by Bowman [22]. It has been reported that as little as 0.1 mL of antigen-positive blood is sufficient to cause sensitization in an antigen-negative mother [23]. Feto-maternal hemorrhage can occur due to various antenatal and postnatal events in Table 1.

#### 5. Pathophysiology for HDFN

HDFN is the destruction of fetal and newborn red cells by maternal alloantibodies specific for the inherited paternal red cell alloantigens. While IgM is usually detected in the maternal circulation during primary response, IgG is found during secondary response, which appears about 5–15 weeks after feto-maternal hemorrhage. Because exposure to fetal red cells and resulting maternal alloimmunization typically occurs late during pregnancy and at delivery, and IgM does not cross the placenta, the fetus and newborn of the first pregnancy are rarely affected. Reexposure to red cell antigen during subsequent pregnancies produces IgG in sufficient concentration [4].

The sensitized fetal red cells by maternal IgG antibody are unable to continue in the circulation, and these red cells are destroyed by the fetal spleen resulting in anemia. Compensatory erythropoiesis is induced by fetal anemia initially [25]. The exception to this rule is antibodies Kell blood group system and MNS system which cause destruction of erythroid progenitor cells, causing early anemia without erythroblastosis [26, 27]. Hyperdynamic circulation tries to compensate anemia in the fetus, which subsequently leads to cardiomegaly, and finally fetal hydrops develops [25]. Due to hemolysis of the fetal cell, there is rise in the bilirubin level in the fetus. In utero, the bilirubin is excreted by the mother, when it is transported across the placenta, so the severity of hyperbilirubinemia is not observed. After delivery, the hemolysis continues, but the comparatively immature liver of the neonate is unable to sufficiently conjugate the excess of bilirubin. This subsequently leads to severe hyperbilirubinemia and, when left untreated, could result in "kernicterus" [4, 25, 28].

### 6. Clinical relevance of different red cell alloantibody specificities

The risk of developing severe HDFN depends on several factors, including Ig class, specificity of the red cell alloantibodies, and level of expression of the involved blood group antigen on the fetal red cells and other tissues as shown in Table 2 [25].

#### 6.1 Rh blood group system

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, but over 50 different Rh antigens have been described [29].


#### Table 2.

Red cell antibody specificities in reference to induce HDFN [25].

#### Hemolytic Disease of the Fetus and Newborn DOI: http://dx.doi.org/10.5772/intechopen.85316

D is by far the most immunogenic of all the Rh antigens. Hence, it is common in clinical practice to equate D with Rh and to use the terms Rh-positive and Rhnegative to describe "D-positive" and "D-negative" [30].

Rh antibodies implicated in HDFN are:


#### 6.1.1 D antigen and antibodies

The D antigen carried by the RhD proteins is the most immunogenic and most important blood group antigen leading to HDFN. There is no antithetical antigen to D [4]. The first blood group antigen to be associated with HDFN was described by Levine et al. in 1945 [9]. About 15% of Western world and 8% of blacks are Dnegative [38, 39]. If a unit of D-positive blood is transfused to a D-negative recipient, the recipient will form anti-D in around 90% of cases, and subsequently Dpositive red cells cannot be given safely to these patients [29].

Sensitization to D antigen can occur in reaction to less than 0.1 mL of fetal blood, resulting in formation of anti-D in the maternal circulation [22]. Before 1945, more than 50% of all fetus with HDFN died of kernicterus or hydrops fetalis, and anti-D was the most common associated [9, 40]. With improvement of treatment, in industrialized countries the mortality reduced to 2–3%. But anti-D is still among the most frequently detected antibodies in sensitized pregnancies.

Very early, it was understood that, if the D-negative mother was carrying an ABO-compatible D-positive fetus, her risk of Rh immunization was 16%. If the Dpositive fetus was ABO-incompatible, the risk was only 2%. So, the overall risk of Rh immunization is 13.2% [9].

Not only in Rh-negative pregnancies does anti-D causes HDFN, but also case reports have been reported for HDFN due to anti-D in Rh-positive pregnancies [41– 43]. These are mostly due to the Rh variants: weak D, Du, and partial D described by molecular analysis [30].

#### 6.1.1.1 Blocked D phenomenon

The blocking of D antigen sites by IgG anti-D in severe cases of HDFN is a rare phenomenon explained by Wiener in 1944 [44]. Only a handful of case reports have been described in the literature [45–48]. The coating of maternal anti-D IgG on the D-positive red blood cells (RBCs) of the newborn gives false-negative D typing, when IgM typing reagent anti-D is used. This phenomenon is not limited to anti-D, but is seen with other blood groups [49]. BCSH describes guideline for resolving such cases [50].

#### 6.1.1.2 Rh immunoglobulin prophylaxis

Rh immunoglobulin (RhIG) prophylaxis for D-negative pregnant women is now the international cornerstone for prevention of maternal alloimmunization to the D antigen and subsequent HDFN [50, 51].

During the mid-1960s, experiments were carried out in various parts of the world for preventing HDFN due to anti-D. Clinical trials showed that, when unimmunized mothers who have delivered D-positive infants, were given RhIG prevented the development of anti-D in the mother. RhIG is obtained from the

human plasma. RhIG has to be given within 72 h after delivery of a D-positive infant. Since 1968, RhIG is licensed for prevention of HDFN [9, 21, 22].

The effectiveness of RhIG in order to prevent isoimmunization is determined by adequate dosage and should be administered before initiation of Rh isoimmunization [50].

There are various mechanisms describing the role of RhIG in preventing HDFN. Though antigenic epitopes are not fully masked by anti-D, they are still available for immune system recognition. But anti-D is be able to destroy RBCs without triggering the adaptive immune response, by inhibition of FcgammaRIIB signaling in B cells which is called as antibody-mediated immune suppression (AMIS) [21, 52, 53]. The T-cell response and memory may still be intact.

Various studies were carried out in the 1970s after systemic implementation of systemic anti-D prophylaxis, which showed reduction in HDFN from 16 to 0.3% [54].

The standard guidelines recommend to administer RhIG as soon after delivery as the infant is determined to be D-positive or latest within 72 hours after delivery or after any antenatal procedure, where the risk of feto-maternal hemorrhage is high as shown in Table 3 [50]. It has been shown experimentally that at least partial protection is afforded by giving RhIG up to 13 days after exposure to D-positive RBCs. Rh prophylaxis therefore is recommended up to 28 days after delivery, with the understanding, however, that the longer the prophylaxis after delivery is delayed, the less likely it is to be effective [28, 55].

In 2014, Cohen et al. described a case report on severe HDFN caused due to passive transfer of anti-D from maternal RhIG [56].

#### 6.1.2 Other Rh system antibodies other than anti-D

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. DCe is the most common haplotype in Caucasians (42%), Native Americans (44%), and Asians (70%) [57].

#### 6.1.3 Anti-c

Anti-c is usually described as the next most common cause of severe HDFN after anti-D. Various case reports have been reported, stating that anti-c isoimmunization can cause HDFN from mild to severe degree [31, 32, 58, 59]. A titer of more than 1:32 is associated with hydrops fetalis as described by David et al. [58]. BCSH guidelines state that women with anti-c should be retested following the same protocol as for anti-D [50]. Quantification of the antibodies is expressed in terms of IU/mL. Mothers with antibody concentration of less than 7.5 IU/mL are advised to continue the pregnancy, while 7.5–20 IU/mL are at a risk of moderate HDFN and more than 20 IU/mL, severe HDFN. It should be kept in mind that anti-c causes delayed anemia in neonate [50].

#### 6.1.4 Anti-D + anti-C or anti-G

D-positive or C-positive RBCs have G antigen which was first described by Allen and Tippett in 1958 [60]. The G antigen is co-distributed either with C or D antigen which causes anti-G to appear serologically as anti-C plus anti-D [60]. During pregnancy, it is apparently important to distinguish between anti-D, anti-G, and anti-D + C. As the pregnancies without anti-D are candidates for the administration

#### Hemolytic Disease of the Fetus and Newborn DOI: http://dx.doi.org/10.5772/intechopen.85316


#### Table 3.

Anti-D prophylaxis and quality of evidence available [50, 54, 55].

of RhIG. The administration of RhIG can be avoided if anti-D has already developed. D-negative mothers with anti-G are potential candidates to receive RhIG in order to prevent formation of anti-D. It also avoids the associated social or medicolegal complications [61]. The clinical significance of anti-G alone in causing mild to severe HDFN still remains controversial [62]. The isolation of anti-G by double adsorption and elution is a tedious and relatively complex procedure [63]. The technique to distinguish anti-D + C from anti-G is recently described by Fatima et al. [64].

#### 6.1.5 Anti-C, anti-E, anti-e, and others

Anti-RhC, anti-RhE, and anti-Rhe antibodies are of Rhesus family and usually occur in low titer in conjunction with anti-RhD antibody. Their presence can be additive to the hemolytic effect of the anti-RhD on the fetus [65, 66]. Various reports have been published on pregnancies alloimmunized only to RhE [36, 37].

Hardy and Napier in their review of red blood cell antibodies among Rh-positive women in South and Mid Wales over a 30-year period (1948–1978) described two infants with hemolytic disease caused by anti-C [67].

Anti-e is usually a very rare cause of HDN; the disease is usually mild [68].

In addition to the above antibodies, there are many other antibodies belonging to Rh family which are associated with HDFN [35, 69, 70].

#### 6.2 Kell blood group system

A mnemonic goes "Duffy dies, Kell kills, and Lewy lives" [71]. Kell blood group system is clinically significant in terms of transfusion medicine and perinatology. It relates to the polymorphic nature of the Kell protein. It is also associated with the Kx and Gerbich blood group systems. K is formed in fetuses of 10–11 weeks and k at 6–7 weeks of gestation [72, 73].

Alloimmunization to Kell blood group antigens is due to previous blood transfusion or feto-maternal hemorrhage induced during pregnancy [73]. Kell alloimmunization is the second major cause for fetal hemolytic anemia, with a reported and still increasing incidence in a large US series of 3.2 in 1000 and affecting 1 in 10,000 neonates [74].

#### 6.2.1 Anti-K

Anti-K antibodies differ from the other blood group system antibodies that cause HDFN in suppressing fetal non-hemoglobinized erythropoiesis, causing severe anemia and often death of the fetus. The high bilirubin level is not a characteristic feature as the precursor cells are destroyed. Amniocentesis therefore does not give an indication of the severity of the disease. Successful management of RBCalloimmunized pregnancies depends on early detection of fetal anemia and timely intervention by intrauterine blood transfusions [75–77].

Perinatal survival in severe Kell alloimmunization was only 58% as recorded after implementation of routine screening nationwide in the Netherlands from 1988 to 2005 [75].

In some countries it is usually practiced to give K-negative red cells for girls and women of childbearing age group [78].

#### 6.3 Detection of feto-maternal hemorrhage (FMH)

#### 6.3.1 KB test

As mentioned earlier, feto-maternal hemorrhage increases as the pregnancy progresses: 3% in the first trimester, 12% in the second, 45% in the third, and 64% at the time of delivery [22]. These fetal cells which have crossed the placenta can be detected by acid elution method of differential staining described by Kleinhauer and Betke in 1960 [79].

#### 6.3.2 α-Fetoprotein (α-FP)

α-FP is an analogue of albumin [6]. Seppala and Ruoslahti in 1972 and Caballero et al. in 1977 used α-FP as an index of transplacental hemorrhage (TPH) [80, 81].

#### 6.3.3 Others

Apart from quantification, the serological methods for detecting transplacental hemorrhage are also available. This includes rosetting test and flow cytometry.

Rosetting test is not sensitive, as it needs at least 15 mL or more of fetal cells to be present in the maternal circulation to give a positive result [19].

Flow cytometry is the most sensitive test technique in detecting the amount of TPH [19, 82].

The other surrogate markers of FMH include enzyme-linked antiglobulin test (ELAT), placental alkaline phosphatase (PLAP), polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH).

#### 6.4 Antibody screening

It had been a common practice to screen the sera of all Rh(D)-negative pregnant women for Rh antibodies. Later, when it was found that Rh(D)-positive women could also have babies with HDFN due to Rh antibodies (other than anti-D) and non-Rh antibodies, it was suggested that sera from all pregnant women should be screened for antibodies [6, 25, 83].

#### 6.4.1 Screening methods

The indirect antiglobulin test (IAT) using reagent red cells suspended in low ionic strength saline (LISS) is the most suitable method for detection of clinically significant red cell antibodies [84]. Column agglutination methods, liquid-phase tube tests, and solid-phase methods have also been found to be suitable [50].

#### 6.4.2 Guidelines for antenatal antibody screening

Various countries have developed guidelines for screening of antenatal cases. Scientific Section Coordinating Committee of the American Association of Blood Banks (AABB) has issued guidelines (not AABB standards) for serological testing of pregnant women [85]. Table 4 shows the recommendations for prenatal testing.

The BCSH Task Force has also laid guidelines in 2007 for blood grouping and antibody testing in pregnancy as follows [50]:


#### Table 4.

AABB recommendations for prenatal testing [85].


Australian and New Zealand guidelines have been adapted from AABB recommendations for pregnant women [85, 86]. However, repeat testing of RhD-negative women only at 28 weeks, prior to administering RhIG, is becoming the accepted protocol in most Australian centers, eliminating the norm of antibody screening at 34–36 weeks. In New Zealand, in the absence of routine antenatal prophylaxis, the normal practice is to test RhD-negative women at 28 and 36 weeks of gestation [86].

The latest guidelines for alloimmunized pregnancy framed and followed in Japan since 2014 are as follows [87]:


#### 6.5 Methods of red cell antibody detection and identification

Antibody detection plays a critical role in detection and monitoring of antenatal cases who are at risk of delivering neonates with HFDN [88].

Most of the clinically significant antibodies are IgG in nature, which are nonagglutinating or incomplete antibodies, so they can only sensitize red cells but cannot produce agglutination. Coombs et al. in 1945 described "antiglobulin test" for detection of these non-agglutinating antibodies [89].

The presence of red cell antibodies in patient's serum or plasma and an in vitro reaction between red cell detection are demonstrated by indirect antiglobulin test [89]. Indirect antiglobulin test is considered to be the most effective and reliable method for detection of clinically significant antibodies [84]. Several studies have shown that the column agglutination test is better than tube and solid-phase tests.

The gel technique has shown sensitivity as compared with conventional test tube (CTT) methods (93.5–100% for CAT vs. 50% for CTT) [90–93]. The

sensitivity of SPRCA has been found to be superior to CTT and comparable with that of CAT [94].

#### 6.6 Quantification of antibody in the serum

Titration is a semiquantitative method to estimate the strength and concentration of antibodies present in a serum sample [95]. Titers give only rough estimates of the amount of antibody bound to the target RBCs and do not measure the amount of antibody remaining free in solution at the endpoint of agglutination [96]:


There are various methods of performing the titration, conventional tube test (CTT), or by gel microcolumn assay (GMA) [95, 97]. A study by Thakur et al. showed that gel technique is more sensitive for antibody detection. It does not show a linear correlation with tube titers in predicting the outcome in RhD-sensitized women, while Rachel et al. suggested that GMA gives comparable results to the CTT in titrating alloantibodies to Rh and Kell antigens [95, 97].

#### 6.7 Amniotic fluid analysis

Amniocentesis is helpful in determining the overall condition of the fetus and is mostly indicated when the clinically significant maternal antibody titer is 1:32 or greater in the fourth or fifth month of pregnancy [4, 19, 98]. If there is a history of a previous pregnancy complicated by HDN, amniocentesis is indicated regardless of the present maternal serum antibody titer.

In 1961, Liley developed a chart depicting change in amniotic fluid bilirubin levels (delta OD450) with period of gestation, with three zones delineating the severity of rhesus disease [99]. The chart is useful only after the 27th week of gestation. Currently, cordocentesis is the only reliable means of assessing the fetal condition accurately prior to 27 weeks. In 1993, Queenan proposed a chart showing delta OD450 from 14 to 40 weeks, with four zones to guide management [100].

#### 6.8 Other methods for assessing the severity of HDFN

#### 6.8.1 Ultrasound

Some of the pathophysiological changes in the fetus due to anemia could be shown using ultrasound [6]. Real-time sonography accurately predicted the clinical course in 86% of the cases, with no false-positive predictions [101].

With recent advances, Doppler ultrasonography, which measures fetal hemodynamics, has been used, and it gives better results in predicting fetal anemia as early as the 18th week. The Doppler assessment of peak systolic velocity in the middle cerebral artery (MCA-PSV) is done [102]. It is hypothesized that faster rate of blood flow indicates a more severely anemic fetus, with severe anemia being an indicator of fetal hydrops.

#### 6.8.2 Fetoscopy, percutaneous umbilical blood sampling (cordocentesis), and chorionic villus biopsy

Fetoscopy is a technique in which the second-trimester fetus can be visualized directly and fetal blood (or other tissues) can be sampled through an endoscope introduced transabdominally into the amniotic cavity. The technique is only reliably successful at 16 weeks of gestation and later. Fetoscopy carries a mortality rate of 5% as compared to a 1–2% mortality rate after midtrimester amniocentesis. The fetal blood can be tested for blood type, DAT, hemoglobin, and hematocrit [96]. MacKenzie and coworkers suggested that the technique would be of benefit in cases where the father of the baby is known to be heterozygous for the offending blood group antigen in the following situations:


#### 6.8.3 Prenatal determination of fetal blood groups

The father's probable genotype is predicted. If the father is thought to be homozygous, the baby is assumed to possess the putative antigen. If the father is heterozygous, there is a 50% chance that the baby is antigen-positive [6, 96]. Antenatal genotyping of the fetus is now in widespread use as an aid to the clinical management in cases where there is a possibility of occurancy of hemolytic disease of the newborn [121]. Molecular genotyping is a major clinical application which has led to the determination basis of blood group antigens expressed, most of which have been defined at the level of the gene. All assays used are dependent on the polymerase chain reaction amplification of fetal DNA derived from.

#### 6.8.4 In vitro predictive tests utilizing functional cellular assays

They are based on the in vivo mechanisms of RBC immune destruction. The interactions with the monocytes are measured by recording RBC adherence/ phagocytosis by a monocyte monolayer assay (MMA), antibody-dependent cellular cytotoxicity (ADCC) using 51Cr-labeled RBCs, or a chemiluminescence test (CL) using luminol. These tests are not very accurate in predicting the severity of HDFN [6, 96].

#### 6.9 Antenatal treatment of hemolytic disease

### 6.9.1 Plasma exchange in mother

A known therapeutic approach for red cell alloimmunization is plasmapheresis in the mother in order to reduce maternal antibody titer [103]. In the current era, plasma exchange treatment appears to be useful in cases of HDFN developed early in pregnancy (before 20 weeks). The American Society for Apheresis in 2013 proposed that plasmapheresis should be considered early in pregnancy (from the 7th to the 20th week) and continued until IUT can be safely administered (approx. 20 weeks of gestation) [104].

#### 6.9.2 Absorption of alloantibodies onto red cells

Plasma containing the antibodies is drawn from the patient, the antibodies are absorbed using appropriate cells, and then plasma is returned to the patient [19]. During the 1980s, this procedure was attempted by Robinson and Yoshida et al. for an Rh-immunized woman [105, 106].

#### 6.9.3 Intravenous immunoglobulin given to the mother

The use of intravenous immunoglobulin (IVIG) to the mother is one of the alternative strategies developed in past 20 years for the management of severely alloimmunized pregnancies [107]. The mechanism by which IVIG might act is saturation of FcRn, thereby inhibiting placental transfer of anti-D to the fetus as shown by Morgan et al. in 1991 [108]. A single course of 2 g/kg over 5 days or repeated weekly injections of 1 g/kg have been tried in conjunction with plasma exchange or with intravascular transfusion of the fetus [109]. There is no other description of the dosing during the antenatal period.

#### 6.9.4 IVIG given to the fetus

IVIG given to the fetus did not show any beneficial effect [19].

#### 6.9.5 Intrauterine transfusion

In 1970, Pontuch stated five strategies as a preventive measure for HDFN, which are valid to this day [110]:


One of the oldest methods, introduced by Sir William Liley, is intraperitoneal fetal blood transfusion into the abdominal cavity of the fetus under X-ray guidance [111]. The donor cells were absorbed into the fetal circulation via the

subdiaphragmatic lymphatics and thoracic duct, which, in conjunction with the use of amniotic fluid analysis for bilirubin levels, markedly improved the management of Rh-sensitized pregnancies.

From the time of its initiation, IUT has come a long way. Initially, it was intraperitoneal, but in 1981, Rodeck et al. described intravascular transfusion by using the umbilical cord and fetoscope [112].

The formula for calculation of blood volume for IUT is shown below [113]:


Red cell prerequisites for IUT as described by BCSH guidelines are as follows [113]:


The target of a single IUT is to reach Hct 48–55% in non-hydropic fetuses [103].

#### 6.9.6 Induction of GvHD

Immunomodulatory effect due to transfusion to the fetus by IUT due to the HLA group of the donor causes GvHD [19].

#### 6.9.7 Premature delivery

Premature induction of labor may be considered, as after the birth of the infant, placental transfer of antibody ceases [6, 28].

#### 6.10 Postnatal intervention for hemolytic disease

#### 6.10.1 Exchange transfusion

The main purposes of exchange transfusion in HDFN are as follows [28, 114]:

1. With sedimented red cells, it can raise the hematocrit without increasing the blood volume of a severely affected erythroblastotic newborn infant in the first minutes of life.


The method was introduced by Diamond (1947). Blood was withdrawn and injected, intermittently, through a plastic catheter passed up the umbilical vein [110].

For HDFN double-volume exchange is mostly preferred, where approximately 85% of the blood volume is replaced and will cause an approximate reduction of 50% in pre-exchange bilirubin level [114–116]. The American Academy of Pediatrics (AAP) has published guidelines, recommending not to initiate early ET, such as within the first 12 h of birth. The bilirubin threshold for the start of ET depends on the gestational age at which the baby is born [117].

Components used in exchange transfusion.

Red cells for exchange transfusion should meet the following BCSH criteria [113, 118]:


Fresh frozen plasma (FFP)—The RBCs are suspended in AB plasma in order to provide plasma proteins, coagulation factors, and albumin [113]. Reconstituted whole blood is prepared by adding appropriate amount of FFP into the RBC unit which is preservative-free. The hematocrit of the unit should be 40–45% [119]. Volume of blood for exchange is calculated using an estimate of the neonate's circulating blood volume [113]:


Volume to be exchange ¼ 2 � circulating blood volume

The volume to be given is calculated below [119]:

Total volume <sup>ð</sup>in mLÞ ¼ infant'<sup>s</sup> weight in kg � <sup>85</sup> <sup>∗</sup> mL=kg � <sup>2</sup> Absolute volume of RBCs required ðin mLÞ ¼ total volume � 0:45 ðdesired hematocritÞ Absolute volume Actual volume of RBCs required <sup>ð</sup>in mLÞ ¼ Hematocrit of unit after any manipulation Necessary volume of FFP ¼ total volume required � actual volume of RBCs required �ð85–100 mL=kg; depending on estimated blood volumeÞ

#### 6.10.2 Phototherapy

Phototherapy has been proven to be effective in treatment of hyperbilirubinemia by denaturing the bilirubin at appropriate wavelength [28]. AAP and NICE have laid down guidelines for initiation of treatment, considering gestational age, birth weight, and cause of hyperbilirubinemia [117, 120].

#### 6.10.3 Intravenous immunoglobulin

IVIG blocks the Fc receptor sites on the cells of the reticuloendothelial system, thus preventing the hemolysis of sensitized cells. It is mostly used for ABO HDFN and is not very effective in anti-D-mediated HDFN [25, 28].

### Author details

Soumya Das

Department of Transfusion Medicine, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India

\*Address all correspondence to: sdas317@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapteris distributed underthe terms oftheCreative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hemolytic Disease of the Fetus and Newborn DOI: http://dx.doi.org/10.5772/intechopen.85316

#### References

Haematology. In: Rennie JM, editor. TR, Greene MF, editors. Creasy and Rennie & Robertson's Textbook of Resnik's Maternal-Fetal Medicine: Neonatology. 5th ed. China: Elsevier Ltd; 2012. pp. 755-790

[2] Maheshwari A, Carlo WA. Hemolytic disease of the Newborn (erythroblastosis fetalis). In: Kliegman RM, Stanton BF, Schor NF, St Geme JW III, Behrman RE, editors. Nelson Textbook of Pediatrics. 19th ed. New Delhi: Thomas Press India Ltd; 2012. pp. 615-619

[3] Maitra A. Disease of infancy and childhood. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cortan Pathologic Basis of Disease. 8th ed. New Delhi: Elsevier Inc.; 2010. pp. 447-486

[4] Kennedy MS. Perinatal issues in transfusion practices. In: Roback JD, Grossman BJ, Harris T, Hillyer CD, editors. Technical Manual. 17th ed. Maryland, United States: AABB; 2011. pp. 631-645

[5] Poole J, Daniels G. Blood group antibodies and their significance in transfusion medicine. Transfusion Medicine Reviews. 2007;21(1):58-71

[6] Klein HG, Anstee DJ, editors. Hemolytic disease of the fetus and the newborn. In: Mollison Transfusion in Clinical Medicine. 12th ed. Blackwell Scientific; 2014. pp. 499-549

[7] Koelewijn JM, Van Der Schoot CE, Bonsel GJ, De Haas M. Effect of screening for red cell antibodies, other than anti-D, to detect hemolytic disease of fetus and newborn: A population study in the Netherlands. Transfusion. 2008;48:941-952

[8] Moise KJJ. Hemolytic disease of the fetus and newborn. In: Creasy RK,

[1] Murray NA, Roberts IAG. Resnik R, Iams DJ, Lockwood CJ, Moore Principles and Practice. 2014

> [9] Bowman J. Thirty-five years of Rh prophylaxis. Transfusion. 2003;43: 1661-1666

[10] Landsteiner K, Weiner A. An agglutinable factor in human blood recognized by immune sera for rhesus blood. Proceedings of the Society for Experimental Biology and Medicine. 1940;43:223-229

[11] Levine P, Katzin E, Burham L. Isoimmunization in pregnancy: Its possible bearing on the etiology of erythroblastosis fetalis. Journal of the American Medical Association. 1941; 116:825-827

[12] Keenan H, Pearse W. Transplacental transmission of fetal erythrocytes. American Journal of Obstetrics & Gynecology. 1963;86:1096

[13] Liley A. Intrauterine transfusion of fetus in haemolytic disease. British Medical Journal. 1963;2:1107

[14] Wallerstein H. Treatment of severe erythroblastosis by simultaneous removal and replacement of blood of the newborn infant. Science. 1946;103: 's Blood 583-584

> [15] Chown B. The place of early induction in the management of erythroblastosis fetalis. Canadian Medical Association. 1958;78:252-256

[16] Finn R, Sheppard P, Lehane D, Kulke W. Experimental studies on the prevention of Rh hemolytic disease. British Medical Journal. 1961;1486:490

[17] Schneider JPO. Die profylaxe der thesis-sensibilisierung mit

Immunoglobulin anti-D. Ärztliche Forschung. 1967;21:11

[18] Kindt TJ, Goldsby RA, Osborne BA, editors. Antigens and Antibodies. In: Kuby Immunology. 6th ed. New York: W. H Freeman and Company; 2007. pp. 76-106

[19] Klein HG, Anstee DJ. Immunology of red cells. In: Klein HG, Anstee DJ, editors. Mollison's Blood Transfusion in Clinical Medicine. 12th ed. Wiley Blackwell; 2014. pp. 53-118

[20] Urbaniak J. Alloimmunity to human red blood cell antigens. Vox Sanguinis. 2002;83(Suppl. 1):293-297

[21] Brinc D, Lazarus AH. Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn. Hematology American Society of Hematology. 2009:185-191

[22] Bowman J. RhD hemolytic disease of the newborn. The New England Journal of Medicine. 1998;339(24):1775-1777

[23] Kennedy MS. Hemolytic disease of the fetus and newborn (HDFN). In: Harmening D, editor. Modern Blood Banking & Transfusion Practices. 6th ed. New Delhi: Jaypee; 2013. pp. 427-438

[24] Egbor M, Knott P, Bhide A. Red-cell and platelet alloimmunisation in pregnancy. Best Practice & Research. Clinical Obstetrics & Gynaecology. 2012;26:112-132

[25] de Haas M, Thurik FF, Koelewijn JM, van der Schoot CE. Haemolytic disease of the fetus and the newborn. Vox Sanguinis. 2015;109:99-113

[26] Vaughan J, Manning M, Warwick RM, et al. Inhibition of erythroid progenitor cells by anti-Kell antibodies in fetal alloimmune anemia. The New England Journal of Medicine. 1998;338: 798-803

[27] Heathcote D, Carroll T, Flower R. Sixty years of antibodies to MNS system hybrid glycophorins: What have we learned? Transfusion Medicine Reviews. 2011;25:111-124

[28] Armstrong B, Smart E. Haemolytic diseases. International Society of Blood Transfusion Science Series. 2008;3: 93-109

[29] Klein HG, Anstee DJ, editors. The Rh blood group system (including LW and RHAG). In: Mollison's Blood Transfusion in Clinical Medicine. 12th ed. Oxford, UK: Wiley Blackwell; 2014. pp. 167-213

[30] Daniels G, editor. Rh and RHAG blood group system. In: Human Blood Groups. 3rd ed. Oxford, UK: Blackwell Scientific; 2013. pp. 182-258

[31] Sheeladevi CS, Suchitha S, Manjunath GV, Murthy S. Hemolytic disease of the newborn due to anti-c isoimmunization: A case report. Indian Journal of Hematology and Blood Transfusion. 2013;29(3):155-157

[32] Shastry S, Bhat S. Severe hemolytic disease of newborn in a Rh D-positive mother: Time to mandate the antenatal antibody screening. The Journal of Obstetrics and Gynecology of India. 2012:1-2

[33] Negi G, Singh GD. Anti Rh hemolytic disease due to anti C antibody: Is testing for anti D antibodies enough? Indian Journal of Hematology and Blood Transfusion. 2012;28(2): 121-122

[34] Chao A-S, Chao A, Ho S-Y, Chang Y-L, Lien R. Anti-E alloimmunization: A rare cause of severe fetal hemolytic disease resulting in pregnancy loss. Case Reports in Medicine. 2009;2009:1-2

[35] Ranasinghe E, Goodyear E, Burgess G. Anti-Ce complicating two consecutive pregnancies with increasing Hemolytic Disease of the Fetus and Newborn DOI: http://dx.doi.org/10.5772/intechopen.85316

severity of haemolytic disease of the newborn. Transfusion Medicine. 2003; 13:53-56

[36] Moran P, Robson S, Reid M. Anti-E in pregnancy. British Journal of Obstetrics and Gynaecology;107: 1436-1438

[37] Joy SD, Rossi KQ, Krugh D, O'Shaughnessy RW. Management of pregnancies complicated by anti-E alloimmunization. The American College of Obstetricians and Gynecologists. 2005;105(1):24-28

[38] Chou ST, Westhoff CM. The Rh system. In: Roback JD, Grossman BJ, Harris T, Hillyer CD, editors. Technical Manual. 17th ed. Maryland, United States: AABB; 2011. pp. 389-410

[39] Whittle MJ. Rhesus haemolytic disease. Archives of Disease in Childhood. 1992;67:65-68

[40] Liumbruno GM, D'Alessandro A, Rea F, Piccinini V, Catalano L, Calizzani G, et al. The role of antenatal immunoprophylaxis in the prevention of maternal-foetal anti-Rh(D) alloimmunisation. Blood Transfusion. 2010;8:8-16

[41] Filbey D, Berseus O, Carlberg M. Occurrence of anti-D in RhD-positive mothers and the outcome of the newborns. Acta Obstetricia et Gynecologica Scandinavica. 1996;75: 585-587

[42] Prasad MR, Krugh D, Rossi KQ, O'Shaughnessy RW. Anti-D in Rh positive pregnancies. American Journal of Obstetrics and Gynecology. 2006; 195(4):1158-1162

[43] Lacey PA, Caskey CR, Werner DJ, Moulds JJ. Fatal hemolytic disease of a newborn due to anti-D in an Rh-positive Du variant mother. Transfusion. 1983; 23(2):91-94

[44] Wiener AS. A new test (blocking test) for Rh sensitization. Proceedings of the Society for Experimental Biology and Medicine. 1944;56:173-176

[45] Lee E. Blocked D phenomenon. Blood Transfusion. 2013;11:10-11

[46] Moiz B, Salman M, Kamran N, Shamsuddin N. Blocked D phenomenon. Transfusion. 2008;48(8):1545-1546

[47] Sulochana PV, Rajesh A, Mathai J, Sathyabhama S. Blocked D phenomenon, a rare condition with Rh D haemolytic disease of newborn-A case report. International Journal of Laboratory Hematology. 2008;30(3): 244-247

[48] Verma A, Sachan D, Bajpayee A, Elhence P, Dubey A, Pradhan M. RhD blocking phenomenon implicated in an immunohaematological diagnostic dilemma in a case of RhD-haemolytic disease of the foetus. Blood Transfusion. 2013;11(1):140-142

[49] Lee E, Redman M, Owen I. Blocking of fetal K antigens on cord red blood cells by maternal anti-K. Transfusion Medicine. 2009;19:139-140

[50] Gooch A, Parker J, British Committee for Standards in Haematology Blood Transfusion Task Force. Guideline for Blood Grouping and Antibody Testing in Pregnancy. 2007. pp. 252-262

[51] American College of Obstetricians and Gynecologists. ACOG practice bulletin No. 75: Management of alloimmunization during pregnancy. Obstetrics and Gynecology;108(2): 457-464

[52] Uhr J, Moller G. Regulatory effect of antibody on the immune response. Advances in Immunology. 1968;8:81-127

[53] Kumpel B, Elson C. Mechanism of anti-D-mediated immune suppression—A paradox awaiting resolution? Trends in Immunology. 2001;22:26-31

[54] de Haas M, Finning K, Massey E, Roberts DJ. Anti-D prophylaxis: past, present and future. Transfusion Medicine. 2014;24(1):1-7

[55] Fung KFK, Eason E. Prevention of Rh Alloimmunization. SOGC Clin Pract Guidel. No. 133 2003. pp. 1-9

[56] Cohen DN, Johnson MS, Liang WH, McDaniel HL, Young PP. Clinically significant hemolytic disease of the newborn secondary to passive transfer of anti-D from maternal RhIG. Transfusion. 2014;54(11): 2863-2866

[57] Dean L. The Rh blood group. In: Blood Groups and Red Cell Antigens. Bethesda: National library of medicine (US), NCBI; 2006. pp. 1-6

[58] Hackney DN, Knudtson EJ, Rossi KQ, Krugh D, O'Shaughnessy RW. Management of pregnancies complicated by anti-c isoimmunization. The American College of Obstetricians and Gynecologists. 2004;103(1):24-30

[59] Rath MEA, Smits-Wintjens VEHJ, Walther FJ, Lopriore E. Hematological morbidity and management in neonates with hemolytic disease due to red cell alloimmunization. Early Human Development. 2011;87:583-588

[60] Allen F, Tippett P. A new Rh blood type which reveals the Rh antigen G. Vox Sanguinis. 1958;3:321-330

[61] Palfi M, Gunnarsson C. The frequency of anti-C + anti-G in the absence of anti-D in alloimmunized pregnancies. Transfusion Medicine. 2001;11:207-210

[62] Hadley A, Poole G, Poole J, et al. Haemolytic disease of the newborn due to anti-G. Vox Sanguinis. 1996;71: 108-112

[63] Vos G. The evaluation of specific anti-G (CD) eluate obtained by a double absorption and elution procedure. Vox Sanguinis. 1960;5:472-478

[64] Baía F, Muñiz-Diaz E, Boto N, Salgado M, Montero R, Ventura T, et al. A simple approach to confirm the presence of anti-D in sera with presumed anti-D+C specificity. Blood Transfusion. 2013;11(3):449-451

[65] Howard H, Martlew V, Mcfadyen I, Clarke C, Duguid J, Bromilow I, et al. Consequences for fetus and neonate of maternal red cell Allo-immunisation. Archives of Disease in Childhood Fetal and Neonatal Edition. 1998;78:62-66

[66] Nordvall M, Dziegiel M, Hegaard HK, Bidstrup M, Jonsbo F. Red blood cell antibodies in pregnancy and their clinical consequences : Synergistic effects of multiple specificities. Transfusion. 2009;49:2070-2075

[67] Kornstad L. New cases of irregular blood group antibodies other than anti-D in pregnancy. Frequency and clinical significance. Acta Obstetricia et Gynecologica Scandinavica. 1983;62(5): 431-436

[68] Chapman J, Waters A. Haemolytic disease of the newborn due to rhesus anti-e antibody. Vox Sanguinis. 1981; 41(1):45-47

[69] Kollamparambil TG, Jani BR, Aldouri M, Soe A, Ducker DA. Anti-Cw alloimmunization presenting as hydrops fetalis. Acta Paediatrica. 2005;94:499-501

[70] Dajak S, Stefanović V, Capkun V. Severe hemolytic disease of fetus and newborn caused by red blood cell antibodies undetected at first-trimester screening. Transfusion. 2011;51(7): 1380-1388

[71] Fortner KB. The Johns Hopkins Manual of Gynecology and Obstetrics. 2007. p. 238

Hemolytic Disease of the Fetus and Newborn DOI: http://dx.doi.org/10.5772/intechopen.85316

[72] Leger RM. Blood group terminology and the other blood groups. In: Harmening D, editor. Modern Blood Banking & Transfusion Practices. 6th ed. New Delhi: Jaypee; 2013. pp. 172-215

[73] Klein HG, Anstee DJ, editors. Other red cell antigens. In: Mollison's Blood Transfusion in Clinical Medicine. 12th ed. Oxford, UK: Wiley Blackwell; 2014. pp. 214-258

[74] Tovey L. Haemolytic disease of the newborn—The changing scene. British Journal of Obstetrics and Gynaecology. 1986;93:960-966

[75] Kamphuis MM, Lindenburg I, van Kamp IL, Meerman RH, Kanhai HHH, Oepkes D. Implementation of routine screening for Kell antibodies: Does it improve perinatal survival? Transfusion. 2008;48(5):953-957

[76] Grant S, Kilby M, Meer L, Weaver J, Gabra G, Whittle M. The outcome of pregnancy in Kell alloimmunisation. British Journal of Obstetrics and Gynaecology. 2000;107:481-485

[77] van Wamelen DJ, Klumper FJ, de Haas M, Meerman RH, van Kamp IL, Oepkes D. Obstetric history and antibody titer in estimating severity of Kell alloimmunization in pregnancy. Obstetrics and Gynecology. 2007; 109(5):1093-1098

[78] Daniels G. Other blood groups. In: Roback JD, Grossman BJ, Harris T, Hillyer CD, editors. Technical Manual. 17th ed. Maryland, United States: AABB; 2011. pp. 411-436

[79] Kleinhauer K, Braun E, Betke K. Demonstration von fetalen haemoglobin in den erytrozyten eines blutausstrichs. Klin Wochenschr. 1957;35:637-640

[80] Caballero C, Vekemans M, Lopez del Campo J. Serum alpha-fetoprotein in adults, in women during pregnancy, in children at birth, and during the first

week of life: A sex difference. American Journal of Obstetrics and Gynecology. 1977;127:384

[81] Seppala M, Ruoslahti E. Alpha fetoprotein in amniotic fluid: An index of gestational age. American Journal of Obstetrics and Gynecology. 1972;114: 595-598

[82] Fong EA, Davies JI, Grey DE, Reid PJ, Erber WN. Detection of massive transplacental haemorrhage by flow cytometry. Clinical and Laboratory Haematology. 2000;22(6):325-327

[83] de Haas M, Van der Schoot E. Prenatal screening. International Society of Blood Transfusion Science Series. 2013;8:6-10

[84] Bromilow IM, Adams KE, Hope J, Eggington JA, Duguid JK. Evaluation of the ID-gel test for antibody screening and identification. Transfusion Medicine. 1991;1(3):159-161

[85] Judd WJ. Practice guidelines for prenatal and perinatal immunohematology, revisited. Transfusion. 2001;41:1445-1452

[86] The Royal Australian and New Zealand College of Obstetricians and Gynaecologists. Guidelines for Blood Grouping and Antibody Screening in the Antenatal and Perinatal Setting. Aust New Zeal Soc Blood Transfus Ltd; 2007. pp. 1-24

[87] Minakami H, Maeda T, Fujii T, Hamada H, Iitsuka Y, Itakura A, et al. Guidelines for obstetrical practice in Japan: Japan Society of Obstetrics and Gynecology (JSOG) and Japan Association of Obstetricians and Gynecologists (JAOG) 2014 edition. The Journal of Obstetrics and Gynaecology Research. 2014;40(6):1469-1499

[88] Trudell KS. Detection and identification of antibodies. In: Harmening DM, editor. Modern Blood Banking & Transfusion Practices. 6th ed. New Delhi: Jaypee; 2013. pp. 216-240

[89] Green RE, Klostermann DA. The Antiglobulin test. In: Harmening D, editor. Modern Blood Banking & Transfusion Practices. 6th ed. New Delhi: Jaypee; 2013. pp. 101-118

[90] Das S, Chaudhary R, Khetan D. A comparison of conventional tube test and gel technique in evaluation of direct antiglobulin test. Hematology. 2007;12: 175-178

[91] Nathalang O, Chuansumrit A, Prayoonwiwat W, Siripoonya P, Sriphaisal T. Comparison between the conventional tube technique and the gel technique in direct antiglobulin tests. Vox Sanguinis. 1997;72:169-171

[92] Novaretti M, Jens E, Pagliarini T, Bonifacio S, Dorlhiac-Llacer P, Chamone D. Comparison of conventional tube test technique and gel microcolumn assay for direct antiglobulin test: A large study. Journal of Clinical Laboratory Analysis. 2004;18:255-258

[93] Bajpai M, Kaur R, Gupta E. Automation in immunohematology. Asian Journal of Transfusion Science. 2012;6(2):140-144

[94] Weisbach V, Kohnhäuser T, Zimmermann R, Ringwald J, Strasser E, Zingsem J, et al. Comparison of the performance of microtube column systems and solid-phase systems and the tube low-ionic-strength solution additive indirect antiglobulin test in the detection of red cell alloantibodies. Transfusion Medicine. 2006;16:276-284

[95] Finck R, Lui-Deguzman C, Teng S-M, Davis R, Yuan S. Comparison of a gel microcolumn assay with the conventional tube test for red blood cell alloantibody titration. Transfusion. 2013;53(4):811-815

[96] Petz LD, Garratty G, editors. Hemolytic disease of Fetus and

Newborn. In: Immune Hemolytic Anemia. United States of America: Elsevier; 1980. pp. 517-572

[97] Thakur MK, Marwaha N, Kumar P, Saha S, Thakral B, Sharma R, et al. Comparison of gel test and conventional tube test for antibody detection and titration in D-negative pregnant women: Study from a tertiary-care hospital in North India. Immunohematology. 2010; 26(4):174-177

[98] Kurtz EM, Pappas AA, Cannon A. Laboratory identification of erythroblastosis fetalis. Annals of Clinical and Laboratory Science. 1982; 12(5):388-397

[99] Liley A. Liquor amnii analysis in management of pregnancy complicated by rhesus immunization. American Journal of Obstetrics and Gynecology. 1961;82:1359

[100] Scott F, Chan FY. Assessment of the clinical usefulness of the "Queenan" chart versus the "Liley" chart in predicting severity of rhesus isoimmunization. Prenatal Diagnosis. 1998; 18(11):1143-1148

[101] Divakaran TG, Waugh J, Clark TJ, Khan KS, Whittle MJ, Kilby MD. Noninvasive techniques to detect fetal anemia due to red blood cell alloimmunization: A systematic review. Obstetrics and Gynecology. 2001;98: 509-517

[102] Mari G. Middle cerebral artery peak systolic velocity: Is it the standard of care for the diagnosis of fetal anemia? Journal of Ultrasound in Medicine. 2005;24(5):697-702

[103] Papantoniou N, Sifakis S, Antsaklis A. Therapeutic management of fetal anemia : Review of standard practice and alternative treatment options. Journal of Perinatal Medicine. 2013;41: 71-82

[104] Schwartz J, Winters JL, Padmanabhan A, Balogun RA, Delaney Hemolytic Disease of the Fetus and Newborn DOI: http://dx.doi.org/10.5772/intechopen.85316

M, Linenberger ML, et al. Guidelines on the use of therapeutic apheresis in clinical practice—Evidence-based approach from the writing Committee of the American Society for apheresis: The sixth special issue. Journal of Clinical Apheresis. 2013;28:145-284

[105] Robinson A. Unsuccessful use of absorbed autologous plasma in Rhincompatible pregnancy (letter). The New England Journal of Medicine. 1981; 305:1346

[106] Yoshida Y, Yoshida H, Tatsumi K, et al. Successful antibody elimination in severe M-incompatible pregnancy. The New England Journal of Medicine. 1981; 305:460-461

[107] Gottstein R, Cooke RWI. Systematic review of intravenous immunoglobulin in haemolytic disease of the newborn. 2003;6–11

[108] Morgan C, Cannell G, Addison R, et al. The effect of intravenous immunoglobulin on placental transfer of a platelet- specific antibody: Anti-PlA1. Transfusion Medicine. 1991;1:209-216

[109] Chitkara U, Bussel J, Alvarez M, Lynch L, Meisel R, Berkowitz R. Highdose intravenous gamma globulin: Does it have a role in the treatment of severe erythroblastosis fetalis? Obstetrics and Gynecology. 1990;76(4):703-708

[110] Santavy J. Hemolytic disease in the Newborn-history and prevention in the world and the Czech Republic. Biomedical Papers of the Medical Faculty of the University Palacky, Olomouc, Czech Republic. 2010;154(2): 147-151

[111] Oepkes D. The modern management of red cell alloimmunisation. Royal College of Obstetricians and Gynaecologists. 2003; 5:15-20

[112] Rodeck C, Kemp J, Holman C, Whitmore D, Karnicki J, Austin M. Direct intravascular fetal blood transfusion by fetoscopy in severe rhesus isoimmunisation. Lancet. 1981;1: 625-627

[113] Boulton F. Transfusion guidelines for neonates and older children. British Journal of Haematology. 2004;124(4): 433-453

[114] Phibbs RH, Francisco S. Advances in the theory and practice of exchange transfusions. California Medicine. 1966; 105(6):442-453

[115] Li B, Jiang Y, Yuan F, Ye H. Exchange transfusion of least incompatible blood for severe hemolytic disease of the newborn due to anti-Rh17. Transfusion Medicine. 2010;20(1): 66-69

[116] Rath MEA, Lindenburg ITM, Brand A, Van Kamp IL, Oepkes D, Walther FJ. Exchange transfusions and top-up transfusions in neonates with Kell haemolytic disease compared to Rh D haemolytic disease. Vox Sanguinis. 2011;100:312-316

[117] Paediatrics AA. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics. 2004;114: 297-316

[118] Green-top Guideline: The Management of Women with Red Cell Antibodies during Pregnancy. Royal College of Obstetricians and Gynaecologists; 2014. p 65

[119] Goodstein M. Neonatal red cell transfusion. In: Herman J, Manno C, editors. Pediatric Transfusion Therapy. Bethesda: AABB; 2002. p. 65

[120] Neonatal Jaundice. NICE Clin Guidel; 2010. p 98

[121] Avent ND. Antenatal genotyping of the blood groups of the Fetus. Vox Sanguinis. 1998;74:365-374

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Section 2

Future Perspectives

Section 2
