Alloimmune hemolytic anemia


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  • Dear Dr. Habib:
    Thank you for posting your report. I pray you are still accessible and my comment reaches you. I have been researching many reports due to my condition and the lack of a diagnosis. I have remained convinced that my condition is the result or a combination of the circumstances of my last third pregnancy, full term with severe maternal hemmoraging and my Rh- factor. The lack of studies and knowledge of medical perssonel provided onestaff to attribute the condition to a self indulgent cause, creating a horrible set of events. I have 3 healthy, academically excelled daughters, 24,18, and 12. About 2 1/2 years ago I becam very ill with signs including but not limited to: extreme fatigue, weakness, shortness of breathe, chest pain, dizziness, memory loss, irritability, high blood pressure, nausea/vomiting, deafening sound in ears of blood rushing, hair loss, sore tongue, etc. After a year of experiencing these symptoms and compromising my business poistion I went to the ER. My hemoglobins were 3.4 and transfusions were immediately starteded. Without the details, I was readmitted 5 months later and again transfused and iron infused. Colonoscopy, Endoscopy, were negative. I had a change in insurance which caused same hospital to administer same treatment, ehree seperate visits, in ER. Not only did insurance change staff approach, but lack of knowledge and interest caused no more testing, etc. I am now ill again with same scenario and attempted to try again with appt to Hemotology Dept. My doctor was young and new and proceeded to convince me that my condition was due to iron deficiancy only, lacking reason to assume or test. He wanted to try double iron infussion only in a week and dismissed the over 12 transfussions as insignificant. I asked about my suspicsions of relatiins to my (O) Rh- and the lab results I read showing anibody-m. First he denied the antibody and then after review stated was unsure but irrelevant. My WC has always been high and all iron is extremely low with no absorption. After changing my insurance to accomodate this facility, I do not trust this facility abd walked out with no treatment. I am looking at possible physicians in Los Angeles. My hemoglobins are extremely low and I am bed ridden. I now have a terrible pain in my back and continue to worsen. I am no longer working and my health has seriously affected my abilities as a single parent, succesful business woman, and everything including ability to walk across small home without severe shortagesof breathe. I am a WF 44yr and have had good health with the exceptions of severe headaches all of my life. I have read your report and still have a strong inclination that my sudden illness is related to the Rh- antibody-m association. I have had anemia all my life. Any suggestion or direction would be greatly appreciated. TN
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Alloimmune hemolytic anemia

  1. 1. TABLE OF CONTENTS:ALLOIMMUNE HEMOLYTIC ANEMIA................................................................2INTRODUCTION:......................................................................................................2CLASSIFICATION OF IMMUNE HEMOLYTIC ANEMIA.................................3ALLOIMMUNIZATION FROM TRANSFUSION..................................................4CONSEQUENCES OF ALLOIMMUNIZATION TO BLOOD:.............................4PATHOPHYSIOLOGY:..............................................................................................6CLINICAL PRESENTATION:...................................................................................8LABORATORY STUDIES:........................................................................................9MANAGEMENT:.......................................................................................................10ALLOIMMUNIZATION DURING PREGNANCY (RhDALLOIMMUNIZATION).........................................................................................12BACKGROUND:.......................................................................................................13EPIDEMIOLOGY:....................................................................................................13PATHOPHYSIOLOGY:............................................................................................14CAUSES OF MATERNAL ALLOIMMUNIZATION:..........................................16HEMOLYTIC DISEASE OF INFANTS & NEWBORNS (HDFN)......................16PATHOPHYSIOLOGY:............................................................................................17CLINICAL PRESENTATION:.................................................................................17CAUSES:.....................................................................................................................18LABORATORY STUDIES:......................................................................................19MANAGEMENT:.......................................................................................................22DRUG INDUCED ALLOIMMUNIZATION..........................................................30PATHOGENESIS:......................................................................................................30SEROLOGICAL DIAGNOSIS:...............................................................................32 1
  2. 2. REFERENCES:..........................................................................................................33 ALLOIMMUNE HEMOLYTIC ANEMIAINTRODUCTION:Alloimmunization is defined as, “an immune response generated in an individual byan alloantigen from a different individual.”In alloimmune hemolytic anemia, antibodies are produced against the red blood cellsa person receives in a blood transfusion. If the blood type used for the transfusion isdifferent than the recipients blood type, the recipients immune system can developantibodies that attack and destroy the transfused blood cells.Alloimmune antibodies also can develop as a result of the mixing of blood between apregnant woman and her baby at delivery. If the mothers blood type is Rh-negativeand the babys is Rh-positive, the mother can produce antibodies against the babysblood type. If a mother develops anti-Rh antibodies as a result of one pregnancy, theycan cross the placenta during the next pregnancy and harm the fetus. To prevent this, amedicine called RhoGam can be given at the time of delivery to block the mothersbody from developing antibodies against the babys blood type.Certain drugs can cause a reaction that develops into hemolytic anemia. These drugsinclude high doses of penicillin and related drugs, acetaminophen, quinine and otherdrugs to treat malaria, anti-inflammatory drugs, and levodopa.Thus Alloimmunization can be broadly divided into three main categories: 1. Alloimmunization after transfusion 2
  3. 3. 2. Maternal Alloimmunization or iso-immunization 3. Drug induced alloimmunization CLASSIFICATION OF IMMUNE HEMOLYTIC ANEMIAAntigen Antibody Disease AssociationAutoimmune Warm antibody Primary Idiopathic Secondary Autoimmune disease(SLE) Lymphoprolifera tive disorders(EBV) Ovarian cysts Some cancers Drugs Cold antibody Cold haemagglutinin disease Cold antibody Infections, syndromes lymphoproliferat ive disorders Donath– Paroxysmal cold Post viral & Landsteiner hemoglobinuria syphilisAlloimmune Induced by red cell Hemolytic transfusion antigens reactions HDN Post-stem cell allograft Drug dependent Antibody/macrophage mediated Antibody/complement mediated Membrane modification 3
  4. 4. ALLOIMMUNIZATION FROM TRANSFUSIONAllogeneic blood transfusion is a form of temporary transplantation. This procedureintroduces a multitude of foreign antigens and living cells into the recipient thatpersist for a variable time. A recipient who is immuno-competent often mounts animmune response to the donor antigens, resulting in various clinical consequences,depending on the blood cells and specific antigens involved. The antigens mostcommonly involved are classified in the following categories: Human leukocyte antigens (HLAs)  class I shared by platelets and leukocytes  class II present on some leukocytes Granulocyte-specific antigens Platelet-specific antigens (human platelet antigen [HPA]) RBC-specific antigens.CONSEQUENCES OF ALLOIMMUNIZATION TO BLOOD:The consequences of alloimmunization to blood include the following clinicalmanifestations:Alloimmunization against RBCs  Acute intravascular hemolytic transfusion reactions (rarely a consequence of alloimmunization and almost always caused by ABO antibodies)  Delayed hemolytic transfusion reactions (DHTRs) (hemolysis caused by RBC all antibodies at least 24 hours post transfusion) 4
  5. 5.  Hemolytic disease in newborns (mothers alloimmunization against fetal antigens, most often resulting from previous pregnancies)Alloimmunization against platelets (platelet-specific or HLA class I antigens)  Refractoriness to platelet transfusion (an increase in the platelet count after platelet transfusion that is significantly lower than expected e.g < 30% of predicted after 10-60 min or < 20% at 18-24h post transfusion)  Post transfusion purpura (thrombocytopenia after transfusion of red cells or other platelet-containing products, associated with presence of platelet allo-antibodies)  Neonatal alloimmune thrombocytopenia (mothers alloimmunization against fetal antigens, most often resulting from previous pregnancies)Alloimmunization against granulocytes  (granulocyte-specific or HLA antigens)Refractoriness to granulocyte transfusion  Febrile non-hemolytic transfusion reactions  Transfusion-related acute lung injury (i-e a transfusion reaction in which donor HLA antibodies react against recipient antigens)Transplant rejection  Alloimmunization against HLA antigens 5
  6. 6.  Alloimmunization against blood cell antigens (in bone marrow transplantation)DHTR and refractoriness to platelet transfusions are most important. Refractoriness togranulocyte transfusions involves either anti-HLA or granulocyte-specific antibodiesand is similar to platelet refractoriness, except that refractoriness to granulocytetransfusions results in the patient failing to respond to the granulocyte transfusions.Granulocyte transfusions are rarely used.PATHOPHYSIOLOGY:The main mechanism for alloimmunization to antigens present in transfused cells mayinvolve presentation of the donor antigens by donor antigen–presenting cells (APCs),i-e monocytes, macrophages, dendritic cells, B cells, to recipient T cells. Recognitionof the MHC class I alloantigens by CD4+ recipient T cells and their subsequentactivation requires a co-stimulatory signal from either the donor or recipient APCs.Alloimmunization by non–leukoreduced platelets involves shared donor HLAantigens (HLA-restricted) and live functional donor APCs. The TH 2 subset of CD4+ Thelper cells secretes interleukin (IL)–4, IL-5, IL-6, and IL-10; activates B cells; andinitiates the antibody response.Refractoriness to platelet transfusions:The presence of HLA antibodies on the platelet surface is the most common cause ofplatelet refractoriness. Other non-HLA antigens present on the platelet surface (eg,platelet-specific antigens, HPA) are also involved in a number of cases. Patients notpreviously sensitized develop antiplatelet antibodies approximately 3-4 weeks afterthe transfusion. Patients previously immunized by transfusion, pregnancy, or organtransplantation develop antiplatelet antibodies as early as 4 days after transfusion. 6
  7. 7. Macrophages in the liver, spleen, and other tissues of the mononuclear phagocytesystem phagocytize and destroy antibody-coated platelets. Risk factors for developingantiplatelet antibodies include:  presence > 1 million donor leukocytes in transfused products  transfusing ABO-mismatched platelets  the presence of an intact immune system (i-e, absence of cytotoxic or immunosuppressive therapy),  Female sex (approximately 75% of cases)  History of multiple transfusions (>20).Delayed hemolytic transfusion reactions:DHTRs occur between 24 hours and 3 months (frequently 2 wk) after transfusion andusually represent a secondary immune response in patients previously immunized bytransfusion or pregnancy. In very rare cases, brisk primary immune response canresult in DHTR after an initial transfusion. Anti-RBC antibody titers frequently (about50% of the patients with alloimmunization) drop below detectable levels, allowingincompatible units to be transfused. Transfusion with incompatible RBCs results inre-stimulation of memory cells and an increase in antibody titer. Antibodies bind tothe surface of RBCs and, depending on the number of antigen-antibody interactions,activate complement with deposition of C3b usually more than 105 antigenic sites percell are required for potent complement activation.Rarely, binding of immunoglobulin M antibodies to RBCs activates the classiccomplement pathway and leads to intravascular hemolysis. RBCs coated withimmunoglobulin G antibodies and/or complement bind to C3b and immunoglobulin 7
  8. 8. Fc receptors present on mononuclear phagocytes and are destroyed by phagocytosis(i-e extravascular hemolysis). Immunoglobulin G antibodies that efficiently activatecomplement (eg, those in Kidd and Duffy systems) tend to cause more intenseextravascular hemolysis compared with antibodies that do not efficiently activatecomplement (eg, Rh and Kell).CLINICAL PRESENTATION:Delayed hemolytic transfusion reactions:  Hemolysis is usually extravascular, but in some cases, a component of intravascular hemolysis is present.  Most cases manifest during the second week after transfusion, but the reaction can occur from 24 hours to 3 months after the transfusion.  Many patients are asymptomatic, and the condition is detected only by laboratory methods.1  In some patients, fever and/or chills (50%), jaundice (10%), pain (3%), and dyspnea (1%) can occur.  Rarely, cases may be complicated with renal failure (6%) or disseminated intravascular coagulation (1%).  In patients with sickle cell disease, a DHTR can precipitate sickle crisis.Refractoriness to platelet transfusions:  Frequently, patients with refractoriness to platelet transfusion are asymptomatic and diagnosed by laboratory methods; however, failure to achieve haemostatic levels of platelets may preclude these patients from important procedures, including bone marrow transplantation. 8
  9. 9. Alloimmunization should be avoided at all costs in candidates for bone marrow transplantation.  Preexisting bleeding resulting from thrombocytopenia may persist after transfusion of an appropriate therapeutic dose of platelets.2 Rarely, spontaneous bleeding may occur after prophylactic transfusion of platelets.LABORATORY STUDIES:Delayed hemolytic transfusion reactions:  The most reliable laboratory sign is a failure to observe the expected post transfusion increase in blood hemoglobin levels (approximately 1 g/dl/U) in the absence of bleeding.  Laboratory signs of hemolysis include elevated lactate dehydrogenase, indirect bilirubin, and reticulocyte levels and decreased hematocrit and haptoglobin levels.  Intravascular hemolysis is characterized by the presence of free plasma hemoglobin and possibly hemosiderinuria.  The results of direct and indirect Coombs test are often positive.  Alloantibodies can be eluted from RBCs, and their specificity can be determined. Often (about 15-20%), patients with DHTR have multiple antibodies and some may be detectable only by elution.Refractoriness to platelet transfusions:  Refractoriness to platelet transfusions is defined as repeated failure to achieve the expected increment in platelet count after 2 or more platelet transfusions. 9
  10. 10. The expected increment can be calculated based on the number of platelets transfused and the patients blood volume  In general, alloimmunization results in the rapid removal of platelets and in lower counts at 10 minutes to 1 hour post transfusion, whereas non-immune causes mostly affect the 4- to 24-hour post transfusion count. Mild alloimmunization, however, can be present with 1-hour increments within the reference range.  The percentage of cells to which the patients serum reacts is referred to as the panel-reactive antibody (PRA) level. PRA values greater than 20% indicate significant alloimmunization to HLA antigens and correlate with an increased risk for platelet refractoriness. • The presence of antiplatelet antibodies can be demonstrated by flow cytometry or by immunoassays such as the modified antigen capture enzyme-linked assay, the solid-phase RBC adherence assay, and the monoclonal antibody immobilization of platelet antigens assay. Most of these assays permit screening for HLA and HPA antibodies as well as specific identification of the most commonly involved HPA antigens. • A negative result from platelet antibody screening indicates non-immune causes of refractoriness.MANAGEMENT:Delayed hemolytic transfusion reactions:  Most patients tolerate DHTR well and only require observation and supportive care. 10
  11. 11.  Transfusion support with antigen-negative RBCs. If these RBCs are not available, weigh the risk of further hemolysis against the indications for transfusion.  If the load of antigen-positive packed RBCs is large (>5 U), consider exchange transfusion.  Administer intravenous human immunoglobulin (IVIG) to block further hemolysis in cases in which antigen-positive blood is transfused.Refractoriness to platelet transfusions:  Avoiding the use of platelet transfusions as much as possible is important in allo-immunized patients. Preventive transfusions are not recommended. Measures to minimize the likelihood and extent of bleeding (eg, rapid treatment of infection; avoidance of invasive procedures; correction of coagulation deficiencies, anemia, and renal insufficiency; use of antifibrinolytic agents) should be used extensively.  After diagnosing alloimmune platelet refractoriness, use the sequence of measures that follows, initiating each subsequent intervention if the previous one fails. o Rule out non-immune, autoimmune, and drug-related causes of platelet refractoriness, or treat accordingly. o Consider alternatives to platelet transfusion to control bleeding, including the use of antifibrinolytic agents such as alpha-aminocaproic acid, or activated recombinant factor VIIa3 o Transfuse ABO-compatible fresh (aged < 48 h) platelet concentrates. o Transfuse with platelets from blood relatives. 11
  12. 12. o Select HLA-matched platelets. Perform HLA typing of patients who receive multiple transfusions before they become pancytopenic. o Select cross-matched platelets. o The use of HPA1a/5b-negative platelets has been successful in cases of post-transfusion purpura and neonatal platelet alloimmunization. o Pre-treat with IVIG before transfusion. IVIG pretreatment can result in successful recovery after platelet transfusion in patients who are alloimmunized. o Use high-dose platelet transfusion. Empirical use of high doses of random platelet units may result in titration of the antibody, overwhelming of the mononuclear-phagocyte system, and increased survival of transfused platelets. o Attempt large-volume plasmapheresis. o Consider administering immunosuppressive drugs. While steroids are not effective the use of vincristine and cyclosporin A has been successful but requires 2-3 weeks to take effect. ALLOIMMUNIZATION DURING PREGNANCY (RhD ALLOIMMUNIZATION)Maternal alloimmunization, also known as isoimmunization, occurs when a womansimmune system is sensitized to foreign erythrocyte surface antigens, stimulating theproduction of immunoglobulin G (IgG) antibodies. The most common routes ofmaternal sensitization are via blood transfusion or fetomaternal hemorrhage (i-etransplacental passage of fetal erythrocytes) associated with delivery, trauma, 12
  13. 13. spontaneous or induced abortion, ectopic pregnancy, or invasive obstetric procedures.These antibodies can cross the placenta during pregnancies in alloimmunized womenand, if the fetus is positive for these specific erythrocyte surface antigens, result inhemolysis of fetal erythrocytes and anemia. This, in turn, can lead to potentiallydisastrous consequences for the fetus, such as hydrops fetalis, a high-output cardiacfailure syndrome.BACKGROUND:Among the more than 50 different antigens capable of causing maternalalloimmunization and fetal hemolytic disease, the Rhesus (Rh) blood group system isthe most common. The Rh blood system is comprised of the c, C, D, e, and Eantigens. The D antigen of the Rh blood group system (Rh D) causes most cases ofsevere hemolytic disease. The incidence of fetuses at risk for anemia due to maternalalloimmunization to red cell antigens has decreased dramatically since the institutionof routine anti-D immune globulin (RhoGAM) prophylaxis for Rh-negative women inthe 1960s. A review of birth certificate data in 2003 reported the incidence of Rhsensitization to be approximately 6.8 per 1000 live births.4EPIDEMIOLOGY:The prevalence of the Rh D–negative blood type is dependent on ethnicity withwhites having the highest prevalence and Asians and American Indians having thelowest.Rates of Rh D negativity among ethnic and racial groups are as follows:  White - 15-16% 13
  14. 14.  African American - 8%  African - 4%  Basque (region of Spain/France) - 30-35%  Asian - Less than 1%  Asian American - 1%  American Indian/Inuit - 1-2%  Eurasian - 2-4%PATHOPHYSIOLOGY:The risk of alloimmunization in a susceptible Rh D–negative woman is significantlyaffected by several factors. These factors include the volume of fetomaternalhemorrhage, the degree of maternal immune response, concurrent ABOincompatibility, and fetal homozygosity versus heterozygosity for the D antigen.Fetomaternal hemorrhages have been demonstrated to occur in as many as 75% ofpregnancies, with the frequency increasing as gestation advances and with most casesoccurring during delivery. If transplacental passage of fetal erythrocytes is suspected,the rosette screening test is used to determine the presence of a fetomaternalhemorrhage. When a large hemorrhage is suspected, the Kleihauer-Betke test is usedto quantify the volume of hemorrhage so that an appropriate dose of anti-D IgG canbe administered. Hemorrhage volumes sufficient to cause alloimmunization areproduced in 15-50% of births. This volume of fetal blood, which, in more than 50% ofintrapartum cases can be as small as 0.1mL and in rare cases can exceed 30mL, variesdepending on the degree of maternal immune response. 14
  15. 15. ABO blood group status also affects the risk of alloimmunization. With an ABO-compatible fetus, the overall risk of alloimmunization if not treated with anti-D IgG isapproximately 16%. However, in an ABO-incompatible fetus, the risk is only 1.5-2%.The protective effect conferred by ABO incompatibility is believed to be due tomaternal destruction and subsequent clearance of the ABO-incompatible fetalerythrocytes before Rh sensitization can occur.Approximately 17% of Rh D–negative women who deliver an Rh D–positive fetusbecome alloimmunized if anti-D IgG is not administered appropriately. Of note,because anti-D IgG prophylaxis has reduced the risk of sensitization to less than 1%of susceptible pregnancies, other alloantibodies have increased in relative importance.These include antibodies to other antigens of the Rh blood group system (i-e c, C, e,E) and other atypical antibodies known to cause severe anemia, such as anti-Kell (i-eK, k), anti-Duffy (i-e Fya), and anti-Kidd (i-e Jka, Jkb).Despite the dramatic success of anti-D IgG prophylaxis protocols, prevention is notuniversal and 0.27% of susceptible women still become Rh D alloimmunized. Onereason for this is failure to follow recommended protocols. Furthermore, a 0.1-0.2%rate of spontaneous immunization occurs despite prophylaxis. These cases havebeen observed in pregnancies in which no prior overt sensitizing events haveoccurred. Finally, alloimmunization involving atypical blood groups (eg, Kell and cblood groups) is not yet preventable. Therefore, understanding and using availablepredictive measures and treatment modalities for hemolytic disease of the fetus andnewborn is essential, as is ensuring that the Rh-alloimmunized pregnancy is properlymanaged. 15
  16. 16. CAUSES OF MATERNAL ALLOIMMUNIZATION:  Blood transfusion  Fetomaternal hemorrhage  Antepartum  Intrapartum  Abortion  Therapeutic  Spontaneous  Molar pregnancy  Ectopic pregnancy  Placental abruption  Abdominal trauma  Obstetric procedures  Amniocentesis  Chorionic villus sampling (CVS)  Percutaneous umbilical blood sampling  External cephalic version  Manual removal of the placenta HEMOLYTIC DISEASE OF INFANTS & NEWBORNS (HDFN)The perinatal effects of maternal Rh alloimmunization are now referred to ashemolytic disease of the fetus and newborn and fetal manifestations of the disease are 16
  17. 17. more appreciated with newer technologies such as cordocentesis and fetalultrasonography.PATHOPHYSIOLOGY:After sensitization, maternal anti-D antibodies cross the placenta into fetal circulationand attach to Rh antigen on fetal RBCs, which form rosettes on macrophages in thereticuloendothelial system, especially in the spleen. These antibody-coated RBCs arelysed by lysosomal enzymes released by macrophages and natural killer lymphocytesand are independent of the activation of the complement system.Reticulocytosis is noted when fetal Hb deficit exceeds 2 gm/dl compared withgestational age norms. Tissue hypoxia develops as fetal anemia becomes severe.When the hemoglobin (Hb) level drops below 8 g/dl, a rise in umbilical arterial lactateoccurs. When the Hb level drops below 4g/dl, increased venous lactate is noted.Hydrops fetalis occurs when fetal Hb deficit exceeds 7 g/dl, and starts as fetal ascitesand evolves into pleural effusions and generalized edema. The various mechanismsresponsible for hydrops are hypoalbuminemia secondary to depressed liver function,increased capillary permeability, iron overload secondary to hemolysis, and increasedvenous pressures due to poor cardiac function.5Hemolysis associated with ABO incompatibility exclusively occurs in type-O motherswith fetuses who have type A or type B blood. Hemolysis due to anti-A is morecommon than hemolysis due to anti-B.CLINICAL PRESENTATION: 17
  18. 18. An infant born to an alloimmunized mother shows clinical signs based on the severityof the disease. The typical diagnostic findings are jaundice, pallor,hepatosplenomegaly, and fetal hydrops in severe cases. The jaundice typicallymanifests at birth or in the first 24 hours after birth with rapidly rising unconjugatedbilirubin level. Occasionally, conjugated hyperbilirubinemia is present because ofplacental or hepatic dysfunction in those infants with severe hemolytic disease.Anemia is most often due to destruction of antibody-coated RBCs by thereticuloendothelial system, and, in some infants, anemia is due to intravasculardestruction. The suppression of erythropoiesis by intravascular transfusion (IVT) ofadult Hb to an anemic fetus can also cause anemia. Extramedullary hematopoiesis canlead to hepatosplenomegaly, portal hypertension, and ascites.Anemia is not the only cause of hydrops. Excessive hepatic extramedullaryhematopoiesis causes portal and umbilical venous obstruction and diminishedplacental perfusion because of edema. Increased placental weight and edema ofchorionic villi interfere with placental transport. Fetal hydrops results from fetalhypoxia, anemia, congestive cardiac failure, and hypoproteinemia secondary tohepatic dysfunction. Commonly, hydrops is not observed until the Hb level dropsbelow approximately 4 g/dl (Hct < 15%)5 . Clinically significant jaundice occurs in asmany as 20% of ABO-incompatible infants.CAUSES:  Common causes of hemolytic disease of the newborn  Rh system antibodies  ABO system antibodies 18
  19. 19.  Uncommon causes - Kell system antibodies  Rare causes  Duffy system antibodies  MNS and s system antibodies  No occurrence in hemolytic disease of the newborn  Lewis system antibodies  P system antibodiesLABORATORY STUDIES:CBC COUNT:  Anemia: Measurements are more accurate using central venous or arterial samples rather than capillary blood.  Increased nucleated RBCs, reticulocytosis, polychromasia, anisocytosis, spherocytes, and cell fragmentation  The reticulocyte count can be as high as 40% in patients without intrauterine intervention.  The nucleated RBC count is elevated and falsely elevates the leukocyte count, reflecting a state of erythropoiesis.  Spherocytes (< 40%) are more commonly observed in cases of ABO incompatibility. Glucose does not correct the autohemolysis in ABO incompatibility unlike hereditary spherocytosis.  In severe hemolytic disease, schistocytes and burr cells may be observed, reflecting ongoing disseminated intravascular coagulation. 19
  20. 20.  A low reticulocyte count is observed in fetuses provided with intravascular transfusion in utero and with Kell alloimmunization.  Neutropenia: This condition seems to be secondary to stimulation of erythropoiesis in favor of myelopoiesis. However, neutrophilia can be observed after intrauterine transfusion because of an increase in circulating cytokines (granulocyte-macrophage colony-stimulating factor).  Thrombocytopenia: This condition is common, especially after intrauterine or exchange transfusions because of platelet-poor blood product and suppression of platelet production in favor of erythropoiesis.HYPOGLYCEMIA:Hypoglycemia is common and is due to islet cell hyperplasia and hyperinsulinism6The abnormality is thought to be secondary to release of metabolic byproducts such asglutathione from lysed RBCs. Hypokalemia, hyperkalemia, and hypocalcemia arecommonly observed during and after exchange transfusion.SEROLOGIC TEST FINDINGS:  Indirect Coombs test and direct antibody test results are positive in the mother and affected newborn. Unlike Rh alloimmunization, direct antibody test results are positive in only 20-40% of infants with ABO incompatibility.7 In a recent study,8 positive direct antibody test findings have a positive predictive value of only 23% and a sensitivity of only 86% in predicting significant hemolysis and need for phototherapy, unless the findings are strongly positive (4+). This is because fetal RBCs have less surface expression of type-specific antigen compared with adult cells. A prospective study has shown that the 20
  21. 21. titers of maternal immunoglobulin G (IgG) anti-A or anti-B may be more helpful in predicting severe hemolysis and hyperbilirubinemia.  Although the indirect Coombs test result (neonates serum with adult A or B RBCs) is more commonly positive in neonates with ABO incompatibility, it also has poor predictive value for hemolysis. This is because of the differences in binding of IgG subtypes to the Fc receptor of phagocytic cells and, in turn, in their ability to cause hemolysis.  IgG2 is more commonly found in maternal serum but has weak lytic activity, which leads to the observation of little or no hemolysis with a positive direct antibody test result. On the other hand, significant hemolysis is associated with a negative direct antibody test result when IgG1 and IgG3 are predominant antibodies, which are in low concentration but have strong lytic activity, crossing to neonatal circulation.  In newborns with hemolytic disease due to anti-c or anti-C antibodies, direct antibody test results may be negative, and the diagnosis is established after indirect Coombs testing.IMAGING STUDIES:High-resolution ultrasonography has been a major advance in detection of earlyhydrops and has also reduced the fetal trauma and morbidity rate to less than 2%during percutaneous umbilical blood sampling (PUBS) and placental trauma duringamniocentesis. High-resolution ultrasonography has been extremely helpful indirecting the needle with intraperitoneal transfusion (IPT) and intravasculartransfusion (IVT) in fetal location. 21
  22. 22. Table 2: Comparison oh Rh and ABO incompatibility: CHARACTERISTICS Rh ABOClinical Aspects First born 5% 50% Later pregnancies More severe No increased severity Still born/hydrops Frequent Rare Severe anemia Frequent Rare Jaundice Moderate to severe/ Mild frequent Late anemia Frequent RareLaboratory Direct antibody test Positive Weakly positive Indirect Coombs Positive Usually positiveFindings test Spherocytosis Rare FrequentMANAGEMENT:Management of maternal alloimmunizationAs a rule, serial maternal antibody titers are monitored until a critical titer of 1:32,which indicates that a high risk of fetal hydrops has been reached. At this point, thefetus requires very intense monitoring for signs of anemia and fetal hydrops. In Kellalloimmunization, hydrops can occur at low maternal titers because of suppressederythropoiesis, and, thus, a titer of 1:8 has been suggested as critical.Maternal titers are not useful in predicting the onset of fetal anemia after the firstaffected gestation. Large differences in titer can be seen in the same patient betweendifferent laboratories, and a newer gel technique produces higher titer results than theolder tube method. Therefore, standard tube methodology should be used to determine 22
  23. 23. critical titer, and a change of more than 1 dilution represents a true increase inmaternal antibody titer. For all the antibodies responsible for hemolytic disease of thenewborn (HDN), a 4-fold increase in any antibody titer is typically considered asignificant change that requires fetal evaluation9.When indicated, amniocentesis can be performed as early as 15 weeks gestation(rarely needed in first affected pregnancy before 24 weeks gestation) to determinefetal genotype and to assess the severity. Maternal and paternal blood samples shouldbe sent to the reference laboratory with amniotic fluid sample to eliminate false-positive results (from maternal pseudogene or Ccde gene) and false-negative results(from a rearrangement at the RHD gene locus in the father).Fetal Rh-genotype determination in maternal plasma has become routine in othercountries and will soon be offered in the United States. Fetal cell free DNA accountsfor 3% of the total circulating maternal plasma DNA. It is subjected to real-time PCRfor the presence of RHD gene-specific sequences and has been found to be accurate in99.5% of cases. The SRY gene (in the male fetus) and DNA polymorphisms in thegeneral population (in the female fetus) are used as internal controls to confirm thefetal origin of the cell-free DNA.10Serial amniocentesis is begun at 10-14 day intervals to monitor the severity of thedisease in the fetus. All attempts should be made to avoid transplacental passage ofneedle which can lead to fetomaternal hemorrhage (FMH) and a further rise inantibody titer. Serial delta-OD 450 values are plotted on the Queenan chart or theextended Liley chart to evaluate the risk of fetal hydrops. 23
  24. 24. Traditional management of alloimmunized patients with serial amniocenteses (asdepicted below) was based on which zone the delta OD450 measurement falls into onthe Liley or Queenan curves. Evidence from several studies, including Lileys originalwork, indicates that mild or no hemolytic disease occurs in zone 1; intermediatedisease occurs in zone 2 (transitional between mild and severe hemolysis); and severedisease, including the development of hydrops within the week, occurs in zone 3.Based on this evidence, once serial measurements are started, if a zone 1 reading isobtained, monitoring the delta OD450 approximately every 3 weeks is reasonable.However, with a trend into zone 2, the frequency of testing should increase to every1-2 weeks depending on the steepness of the slope of the curve and the closeness ofthe measurement to zone 3. 24
  25. 25. Early ultrasonography is performed to establish correct gestational age. Frequentultrasonographic monitoring is also performed to assess fetal well-being and to detectmoderate anemia and early signs of hydrops.The peak systolic middle cerebral artery (MCA) Doppler velocity has proved to be areliable screening tool to detect fetal anemia. The MCA is easily visualized withcolor-flow Doppler; pulsed Doppler is then used to measure the peak systolic velocityjust distal to its bifurcation from the internal carotid artery. Because the MCA velocityincreases with advancing gestational age, the result is reported in multiples of median(MOMs). In recent studies, the sensitivity for detection of moderate and severe fetalanemia has been proven to be 100%, with a false-positive rate of 10% at 1.5MOM.11 It has been shown to reduce the need for invasive diagnostic procedures suchas amniocentesis and cordocentesis by more than 70%.12MCA Doppler studies can be started as early as 18 weeks gestation but are notreliable after 35 weeks gestation13 . It has also been used to time the subsequent fetaltransfusion and to diagnose anemia from multiple causes, such as in twin-twintransfusion. The MCA slope from 3-weekly readings is now used to predict fetal riskfor severe anemia14 25
  26. 26. 26
  27. 27. Management of the sensitized neonateMild hemolytic disease accounts for 50% of newborns with positive direct antibodytest results. Most of these newborns are not anemic (cord hemoglobin [Hb] >14 g/dL)and have minimal hemolysis (cord bilirubin < 4 mg/dL). Apart from earlyphototherapy, they require no transfusions. However, these newborns are at risk of 27
  28. 28. developing severe late anemia by 3-6 weeks of life. Therefore, monitoring their Hblevels after hospital discharge is important.Moderate hemolytic disease accounts for approximately 25% of affected neonates.Moderate hemolytic disease of newborn is characterized by moderate anemia andincreased cord bilirubin levels. These infants are not clinically jaundiced at birth butrapidly develop unconjugated hyperbilirubinemia in the first 24 hours of life.Peripheral smear shows numerous nucleated RBCs, decreased platelets, and,occasionally, a large number of immature granulocytes. These newborns often havehepatosplenomegaly and are at risk of developing bilirubin encephalopathy withoutadequate treatment. Early exchange transfusion with type-O Rh-negative fresh RBCswith intensive phototherapy is usually required. Use of IVIG in doses of 0.5-1 g/kg ina single or multiple dose regimen have been able to effectively reduce need forexchange transfusion.15A prospective randomized controlled study has shown early high-dose IVIG 1 g/kg at12 hours of age to reduce duration of phototherapy and hospital stay and to preventexchange transfusion in neonates with moderate-to-severe Rhisoimmunization.16 These newborns are also at risk of developing latehyporegenerative anemia of infancy at 4-6 weeks of life. However, one randomizeddouble-blind placebo-controlled trial failed to show the benefit of prophylactic IVIGtherapy 0.75 g/kg within 4 hours of age in severely affected neonates who weretreated with intrauterine transfusion for Rh isoimmunization.17Severe hemolytic disease accounts for the remaining 25% of the alloimmunizednewborns who are either stillborn or hydropic at birth. The fetal hydrops ispredominantly caused by a capillary leak syndrome due to tissue hypoxia, 28
  29. 29. hypoalbuminemia secondary to hepatic dysfunction, and high-output cardiac failurefrom anemia. About half of these fetuses become hydropic before 34 weeks gestationand need intensive monitoring and management of alloimmunized gestation asdescribed earlier. Mild hydrops involving ascites reverses with IVTs in only 88% ofcases with improved survival but severe hydrops causing scalp edema and severeascites and pleural effusions reverse in 39% of cases and are associated with poorsurvival.Management of ABO incompatibilityManagement of hyperbilirubinemia is a major concern in newborns with ABOincompatibility. The criteria for exchange transfusion and phototherapy are similar tothose used in Rh alloimmunization. IVIG has also been very effective whenadministered early in the course. Tin(Sn) porphyrin a potent inhibitor of hemeoxygenase, the enzyme that catalyzes the rate-limiting step in the production ofbilirubin from heme, has been shown to reduce the production of bilirubin and reducethe need for exchange transfusion and the duration of phototherapy in neonates withABO incompatibility.Tin or zinc protoporphyrin or mesoporphyrins have been studied in newborns. Theymust be administered intramuscularly in a dose based on body weight, and theireffectiveness appears to be dose related in all gestations.18 Their possible toxic effectsinclude skin photosensitization, iron deficiency, and possible inhibition of carbonmonoxide production. Their use in Rh hemolytic disease of newborn has not beenreported. Their routine use cannot be recommended yet because of lack of long-termsafety data. 29
  30. 30. DRUG INDUCED ALLOIMMUNIZATIONIt is rare but in some cases may be acute, severe and even life threatening. Four mainmechanisms have been proposed for antibody dependent drug-induced hemolyticanemia: drug adsorption, immune complex and membrane modifications lead toantibody reacting with novel epitopes and the true auto-antibody induced hemolyticanemia. The same dose at different doses and repeated usage may activate differentmechanisms but the main underlying mechanism is membrane modification.Diagnosis of drug-induced hemolytic anemia is made in three stages (1) diagnosis ofDAT positive hemolytic anemia (2) careful drug history (3) serological examinationof specific antibodies.PATHOGENESIS:Drug adsorption mechanism: IgG antibodies and extravascular hemolysisdrugs in this group readily form hapten-carrier complexes with plasma proteins,which enhance drug specific antibody production. Prototype drug is penicillinalthough cephalosporins and other penicillin derivatives have also been implicated.90% individuals receiving penicillin produce clinically insignificant IgM anti-penicillin antibodies. When high dose I/V penicillin is given, drug is adsorbed ontothe red cell surface and become non-specifically attached to red cell surface proteins.A minority of patients on high dose I/V penicillin therapy ( >1million units daily)develops high titer IgG antibodies which attach to the drug bound to the red cellsurface and result in extravascular hemolysis. If unrecognized and large dosescontinued then complement fixation and acute I/V hemolysis may occur. 30
  31. 31. Immune complex mechanism: complement activated acute intravascularhemolysisMost common drugs include rifampicin, phenacetin, quinine, quinidine,hydrochlorothiazide and chlorpropomide. More recently iv cephalosporins anddiclofenac are indicated. Hapten carrier complexes are formed between these drugsand plasma proteins, leading to the production of drug specific antibodies. Once drugantibodies are formed reintroduction results in immune complexes to form, which areadsorbed onto the red cell surface and complement is activated. Classically hemolysisoccurs on the second or subsequent exposure exposure to the drugand may developwithin minutes or hours of drug ingestion.Membrane modification mechanism:Cephalosporin in addition to the drug adsorption mechanism, can cause a positiveDAT by modifying red cell membrane components. cisplatin and carboplatin has alsobeen reported to cause immune hemolytic anemia by this method. As a result, avariety of plasma proteins including immunoglobulin and complement, may attach viaa non-immune mechanism to the red cell membrane. This may result in finding of apositive DAT but rarely causes immune hemolytic anemia.Autoimmune mechanism:In this case the antibodies show no Rh specificity when tested against Rhnull cells.some drugs may produce hemolysis by both the immune mechanism and autoimmunemechanism depending on the circumstances 31
  32. 32. SEROLOGICAL DIAGNOSIS:Drug adsorption and membrane modification mechanism:  The DAT is usually positive with IgG1 or IgG and C3 on red cell surface.  The red cell elutae or serum donot react against normal or enzyme modified red cells  Warm reacting drug specific antibody in the eluate or serum is only detected after preincubation of the test red cells with the appropriate drug.Immune complex mechanism:  DAT is usually positive but may be negative if performed immediately after brisk episode of hemolysis.  Red cell eluate is not reactive even in the presence of drug  Drug specific antibody is best detected by preincubating the patient’s serum with the drug in solution to allow immune complexes to form.  Drug metabolite may be detected by preincubating drug metabolite obtained from the serum or urine of a volunteer with patient’s serum. 32
  33. 33. REFERENCES: 1. Heddle NM, Soutar RL, OHoski PL, Singer J, McBride JA, Ali MA. A prospective study to determine the frequency and clinical significance of alloimmunization post-transfusion. Br J Haematol. Dec 1995;91(4):1000-5 2. Kerkhoffs JL, Eikenboom JC, van de Watering LM, van Wordragen- Vlaswinkel RJ, Wijermans PW, Brand A. The clinical impact of platelet refractoriness: correlation with bleeding and survival. Transfusion. Sep 2008;48(9):1959-65 3. Poon MC. The evidence for the use of recombinant human activated factor VII in the treatment of bleeding patients with quantitative and qualitative platelet disorders. Transfus Med Rev. Jul 2007;21(3):223-36. 4. Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Munson ML. Births: final data for 2002. Natl Vital Stat Rep. Dec 17 2003;52(10):1-113. 5. Moise KJ. Hemolytic disease of the fetus and newborn. In: Creasy RK, Resnik R. Maternal-fetal Medicine: Principles and Practice. 6th edition. Philadelphia: WB Saunders; 2008:477-503 6. Vidnes J, Finne H. Immunoreactive insulin in amniotic fluid from Rh- immunized women. Biol Neonate. 1977;31(1-2):1-6 7. Romano EL, Hughes-Jones NC, Mollison PL. Direct antiglobulin reaction in ABO-haemolytic disease of the newborn. Br Med J. Mar 3 1973;1(852):524-6. 8. Murray NA, Roberts IA. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed. Mar 2007;92(2):F83-8 9. Bowman J. The management of hemolytic disease in the fetus and newborn. Semin Perinatol. Feb 1997;21(1):39-44 33
  34. 34. 10. Moise KJ Jr. Red blood cell alloimmunization in Pregnancy. Seminars in Hematology. 2005;42:169-17811. Segata M, Mari G. Fetal anemia: new technologies. Curr Opin Obstet Gynecol. Apr 2004;16(2):153-812. Zimmerman R, Carpenter RJ, Durig P, et al. Longitudinal measurement of peak systolic velocity in the fetal middle cerebral artery for monitoring pregnancies complicated by red cell alloimmunisation: a prospective multicentre trial with intention-to-treat. BJOG. Jul 2002;109(7):746-5213. ACOG Practice Bulletin No. 75: management of alloimmunization. Obstet Gynecol. Aug 2006;108(2):457-64.14. Opekes D, seward G, Vandenbussche F, et al. Minimally invasive management of rh alloimmunization: Can amniotic fluid delta OD 450 be replaced by Doppler studies? A prospective study multicenter trial. Am J Obstet Gynecol. 2004;191:S315. Gottstein R, Cooke RW. Systematic review of intravenous immunoglobulin in haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed. 2003 Jan;88(1):F6-10. 88(1);2003:F6-10.16. Elalfy MS, Elbarbary NS, Abaza HW. Early intravenous immunoglobin (two- dose regimen) in the management of severe Rh hemolytic disease of newborn- a prospective randomized controlled trial. Eur J Pediatr. Apr 2011;170(4):461-717. Smits-Wintjens VE, Walther FJ, Rath ME, Lindenburg IT, Te Pas AB, Kramer CM, et al. Intravenous immunoglobulin in neonates with rhesus hemolytic disease: a randomized controlled trial. Pediatrics. Apr 2011;127(4):680-6. 34
  35. 35. 18. Kappas A. A method for interdicting the development of severe jaundice in newborns by inhibiting the production of bilirubin. Pediatrics. Jan 2004;113(1 Pt 1):119-23 35