Immunohematological Disorders

62  Immunohematological Disorders Jay N. Lozier, Pierre Noel

Congenital (primary) or acquired (secondary) immunodeficiencies, medications, and lymphoproliferative and rheumatological disorders are frequently associated with immune-mediated cytopenias. These processes can affect erythrocytes, leukocytes, and platelets (individually or in combination). In this chapter, we address immune-mediated cytopenias of each hematological component, discussing pathophysiology, clinical presentation, differential diagnosis, and treatment options.

IMMUNE-MEDIATED HEMOLYTIC ANEMIA Immune-mediated hemolysis can be autoimmune, alloimmune, idiopathic, or secondary to drugs or other diseases. Regardless of the underlying cause, immunoglobulin G (IgG) or IgM (rarely IgA) antibodies are directed against antigens on the red blood cell (RBC) membrane (Table 62.1).1,2 These disorders can be categorized on the basis of the underlying cause and the type of antierythrocyte antibody that mediates the process.

Autoimmune Hemolysis Mediated by Warm Antibody Although warm-antibody autoimmune hemolytic anemia is rare, it increases in prevalence above the age of 50 years and tends to be more common in women than in men, as is typical of most autoimmune diseases. In this condition, IgG autoantibodies and complement components are present on the RBC surface (see below). Although the autoantibody target is most commonly the Rh antigen, a variety of other targets are known. The specificity of the antibody, however, does not affect the clinical presentation or management of hemolysis.

Drug-Induced Immune Hemolysis Over 125 drugs have been associated with immune hemolysis.3 Cefotetan, ceftriaxone, and piperacillin currently account for over 80% of cases. The prognosis of drug-related immune hemolysis is much better than that of idiopathic hemolysis because the hemolysis stops once the offending drug has been removed. The drugs most commonly associated with fatal hemolytic anemia are cefotetan (8%) and ceftriaxone (6%).3 Fludarabine and cladribine have been associated with immune hemolysis. Following multiple courses of alkylating agents, fludarabine causes autoimmune hemolytic anemia in 20% of patients with chronic lymphocytic leukemia (CLL). The combination of fludarabine and cyclophosphamide with or without rituximab (FC, FCR) may protect against the development of autoimmune hemolytic anemia.3,4 Although the biochemical mechanisms of drug-related immune hemolysis are not completely clear, several hypotheses are generally accepted. Most commonly, complexes of drug and IgG and/or IgM that adsorb to the RBC surface and fix complement. The resultant intravascular hemolysis is acute and often severe enough

to cause renal failure from toxicities of hemoglobin to renal epithelium. A second less common mechanism develops primarily in patients receiving very high doses of penicillin (rarely used) for at least 1 week. High-titer antipenicillin IgG develops and binds to penicillin that is covalently attached to the RBC membranes. The resultant hemolysis is less acute than that caused by immune complexes but can be life-threatening. In a third mechanism, a drug stimulates the production of an antibody that reacts with the patient’s RBCs independently from the drug. Serologically, this antibody is indistinguishable from an idiopathic autoantibody. This has become rare as the use of the primary causal agent, methyldopa (an antihypertensive medication), has declined. Although these autoantibodies commonly cause positive clinical antibody tests (see below), they rarely cause hemolysis in vivo, and when they do, it usually ceases within 2 weeks of discontinuing the drug.

Cold Agglutinin Diseases This type of hemolytic anemia is mediated predominantly by anti-I or anti-i IgM that agglutinates red cells at temperatures well below 37°C.5 These antibodies engage the complement pathway resulting in C3b-mediated RBC phagocytosis mainly by Kupffer cells, whereas membrane-associated complex is a minor mechanism at low IgM titers. The severity of the clinical illness depends on the concentration of the IgM and its “thermal amplitude.” Thermal amplitude describes the temperature range over which it binds to RBCs: for example, antibodies that exclusively bind at 4°C are only active in vitro, whereas those that bind at >30°C can bind to RBCs as they circulate in the periphery and begin the process of complement fixation, which can persist even as the cells return to body core temperatures. The activity of the IgM is also determined by its relative affinity for the I- and i-antigens, which varies from one individual to the next. Two general types of cold agglutinin disease are recognized: a chronic idiopathic disease presenting in patients over age 50 years, caused by monoclonal anti-I IgM, and a transient disease secondary to certain infections (e.g., mycoplasma, Epstein-Barr virus [EBV], cytomegalovirus [CMV]), caused by polyclonal anti-i and anti-I (see Table 62.1). Avoidance of cold environments is important in both categories of cold agglutinin disease. In addition, cold agglutinin disease can be associated with B-cell lymphoproliferative disorders, and this typically is responsive to rituximab and/or rituximab combined with fludarabine.6

Paroxysmal Cold Hemoglobinuria Paroxysmal cold hemoglobinuria (PCH) is caused by anti-P IgG that is very effective in fixing complement and producing intravascular hemolysis.2 Although rare, it is most common in

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TABLE 62.1  Classification of Immune

Hemolytic Disorders Autoimmune

Warm Antibody-Mediated Idiopathic Secondary Drugs, lymphoid malignancies, infections Other autoimmune diseases

Cold Antibody-Mediated Cold agglutinin disease Idiopathic Secondary Infection, lymphoid malignancies

Paroxysmal Cold Hemoglobinuria Idiopathic Secondary to infections

Alloimmune Secondary to red cell transfusions (alloantibodies, isoantibodies) Secondary to fetal-maternal hemorrhage Secondary to transplanted lymphocytes

children following a viral illness and can be managed by avoidance of cold. In the past, it was more commonly associated with syphilis (see Table 62.1). There is also an autoimmune variety of PCH that may require immunosuppression with corticosteroids. Splenectomy is not helpful as a consequence of the fact that the hemolysis is intravascular.

Hemolytic Transfusion Reactions Because individuals with group O RBCs have preformed iso-anti-A and -B, they must only be transfused with group O cells. Similarly, individuals with group A RBCs with preformed anti-B isoantibodies must only receive group A RBCs, and individuals with group B RBCs must only receive group B RBCs. Because of the absence of either group A or B antigens on the surface of group O RBCs, such cells can be used in transfusion of A, B, or AB individuals in emergency situations. Failure to abide by these rules results in acute intravascular hemolysis that can cause renal failure, disseminated intravascular coagulation, and death. Other hemolytic transfusion reactions are caused by alloantibodies, predominantly IgG. Therefore a multiply transfused patient is at risk for hemolysis from alloantibodies if the patient receives incompatible blood. Multiparous women are at similar risk because of exposure to paternal fetal RBC antigens. Fortunately, for unclear reasons, most RBC antigens do not elicit an immune response although the Rh, Kell, Kidd, and Duffy antigens are clearly immunogenic. Exposure particularly to Kidd or Duffy antigens may stimulate alloantibody formation that can rise to sufficient titer to cause hemolysis with an onset typically delayed by approximately 1 week. Such delayed transfusion reactions may be subtle or cause an abrupt drop in hemoglobin with jaundice and hemoglobinuria.

Immune Hemolysis Associated With Transplantation Any transplanted tissue may contain “passenger” lymphocytes from the donor that will survive and proliferate in the recipient if the recipient is sufficiently immunosuppressed.7 When the RBCs of the recipient carry A or B antigen and the donor is ABO incompatible, the transplanted lymphocytes will respond to the recipient RBCs as foreign, and allogeneic anti-A or anti-B

antibodies will be produced and can lead to significant hemolysis. If the transplantation involves hematopoietic stem cells (HSCs), this dilemma will resolve once the donor’s erythropoiesis prevails and the donor lymphocytes are no longer exposed to the recipient RBCs.

Immunopathogenesis The antigens for antierythrocyte IgG are usually proteins, the most clinically important of which are the Rh-associated glycoproteins (RhAG), D, C, c, E, and e.8 In contrast, antierythrocyte IgM is directed at polysaccharides, which include the ABO and I-antigens (I, i) found on the anion and glucose transporter proteins in the RBC membrane.9,10 Antibodies are distinguished by being “warm” and “cold” reactive, respectively, meaning that they bind antigens at core body temperature (warm) or they bind antigens preferentially at lower temperatures (cold) in the peripheral circulation or ex vivo. This distinction results from the different thermodynamics of binding to protein (hydrophobic) and polysaccharide (electrostatic) antigens.10 IgG antibodies typically bind at warm temperatures and IgM antibodies typically bind at cold temperatures, although there can be overlap and exceptions (e.g., the Donath-Landsteiner IgG antibodies that are seen in paroxysmal cold hemoglobinuria). IgG and IgM also differ in their ability to fix complement, and this affects the resulting mechanism of hemolysis. To attach the first component of the classical complement pathway, two IgG molecules must bind in close proximity on the RBC. However, because of its pentameric structure, a single IgM molecule can initiate complement activation. Erythrocyte-bound IgG becomes attached to the Fc receptors of splenic macrophages, which may engulf all or part of the cell or release lysosomal enzymes that digest its membrane (antibodydependent cell-mediated cytotoxicity [ADCC]).11 RBC fragments escaping from this encounter lose more membrane than cytoplasm and become spherical (spherocytes) as a consequence of this change in the surface-to-volume ratio. If IgG has initiated complement activation on the cell surface, binding of C3b to splenic macrophages will augment erythrocyte phagocytosis in the spleen.12 When IgM fixes complement, the process begins in the cooler peripheral circulation, where IgM binds to RBCs. If the amount of IgM bound is relatively high with at least some of it remaining on the cell at 37°C (e.g., anti-A or anti-B isoantibodies), the cascade of complement activation goes to completion. Doughnut-shaped holes are formed in the cell membrane that allow the influx of water and sodium, inducing intravascular osmotic rupture of the cell.11 However, if the IgM elutes from the RBC as it returns to body core temperature, the complement reaction attenuates. In this circumstance, the components remain on the cell but do not cause intravascular hemolysis. Instead, the cells are cleared by hepatic macrophages via complement-binding sites.13 This has important implications for clinical tests for the presence of antibody and complement on the RBC surface during the workup of immune-mediated hemolysis. Antibody-mediated hemolysis causes variable degrees of anemia and reticulocytosis. Intravascular hemolysis releases hemoglobin into the circulation, but this is too small an amount to cause measurable hemoglobinemia, although it will result in consumption of haptoglobin, which is rapidly depleted. In contrast, when hemolysis results from complement-mediated lysis, such as follows an ABO-incompatible blood transfusion, hemoglobinemia becomes massive, overcoming the scavenging capacity of plasma hemoglobin binders

CHAPTER 62  Immunohematological Disorders (haptoglobin, hemopexin, albumin), resulting in hemoglobinuria. Because hemoglobin is toxic to the renal tubular epithelium, renal function may become impaired. RBC membrane fragments released by massive intravascular hemolysis are a rich source of procoagulant phosphatidylserine and can precipitate disseminated intravascular coagulation. In contrast, the consequences of extravascular hemolysis (i.e., via phagocytosis in the reticuloendothelial system of the liver and the spleen) are much less severe. In macrophages, iron is removed from the hemoglobin and recycled to the circulation to support a compensatory reticulocytosis and the heme porphyrin is metabolized to bilirubin. Patients with immune-mediated anemia have an increased incidence of venous thromboembolism, and detection of a lupus anticoagulant in these patients places them at a particularly high risk for this complication.14

Antibody-coated red cells from patient

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Reagent anti-IgG

+

Visible red cell agglutination

Diagnosis With few exceptions, if the mechanism of hemolysis is immune mediated, an anti-RBC antibody can be demonstrated, either on the RBC surface, in serum, or both.1,2 With autoimmune hemolysis, IgG or IgM and/or complement components can be identified by a direct antibody test (DAT), originally known as a direct Coombs test (Fig. 62.1). For this assay, a patient’s RBCs are washed and suspended in buffer. Surface-bound IgG is detected by adding anti-IgG antibody, which, being divalent, can bind to IgG on adjacent RBCs and agglutinate them into visible aggregates. Because of its pentameric structure, IgM on the cells can cause agglutination without the addition of a second antibody. Even when IgM has been previously eluted from the cell surface as a result of warming in the central circulation, its earlier presence in vivo can be detected by telltale remnants of complement that are fixed to the RBC. In this setting, detection requires the addition of anticomplement (e.g., anti-C3dg) antibody. Alloantibodies can also be detected by the DAT if allogeneic RBCs from a previous transfusion are still circulating. If these have been cleared, however, RBC antibodies can be identified in the patient’s serum by adding the serum to a panel of RBCs carrying different antigens. Agglutination is detected as described above; this constitutes the indirect antibody test.

FIG 62.1  The Direct Antibody Test (DAT). The test is positive when immunoglobulin G (IgG: light blue triangles)–coated red blood cells are cross-linked by anti-IgG antibody (dark blue triangles) to form visible cell aggregates. Cell-bound complement and/or IgM can be detected by using anticomplement or anti-IgM reagent antibodies.

CLINICAL PEARLS In the workup of hemolytic anemia, the following clinical laboratory studies provide important clues as to mechanism and may lead to a diagnosis of an immune hemolytic anemia. Direct antibody test (DAT): The presence of antibodies on the surface of red blood cells (RBCs) suggests an immune-mediated hemolysis. The presence of antibody and complement on the surface of the RBC suggests drug-related hemolysis, whereas the presence of complement alone may suggest an immunoglobulin M (IgM) or cold antibody-related hemolysis. Peripheral blood smear: Examination of the peripheral smear and various RBC indices (chiefly the mean corpuscular volume [MCV]) give mechanistic clues to the etiology of the hemolytic process. Immune-mediated hemolysis is characterized by spherocytosis, even microspherocytosis in severe cases, as antibody-coated RBCs traversing the reticuloendothelial system assume a spherical form, rather than that of a normal biconcave disc. The appearance of other pathological forms, such as schistocytes, sickle cells, targets, or tear drop forms (dacrocytes), suggest other causes of hemolysis, such as thrombotic thrombocytopenic purpura (TTP), or mechanical, shear-induced hemolysis (e.g., aortic stenosis), sickle cell disease, thalassemia, or extramedullary hematopoiesis from bone marrow fibrosis, metastasis, or failure. Nucleated RBCs can be seen in any form of hemolytic anemia if it is severe enough.

Reticulocytes: Evaluation of the reticulocyte count indicates whether bone marrow is capable of making new erythrocytes in response to hemolysis. Lactic acid dehydrogenase (LDH): The LDH is typically elevated with ongoing hemolysis, since LDH is an important housekeeping enzyme found in erythrocytes. LDH is found in cells from all tissues, each having a characteristic isoenzyme form of LDH. It is rarely necessary to distinguish LDH from RBCs from other tissue sources, but LDH isoenzyme 1 is the predominant form found in RBCs. Bilirubin: As heme is released from RBCs, it is metabolized to bilirubin, which is glycosylated and then excreted via hepatic metabolism. Initially, as large amounts of heme are released and metabolized, the bilirubin is predominantly indirect bilirubin (unconjugated), and then it is converted to direct (conjugated) bilirubin. This can be altered by cholestasis, either from biliary obstruction or hepatic disease or Gilbert disease, or hepatic immaturity in the premature infant or newborn. Haptoglobin: Haptoglobin is extremely sensitive to even small amounts of hemolysis. Its absence merely confirms that there is a significant hemolysis, but not its extent. The presence of normal haptoglobin effectively rules out significant hemolysis, and the return of measurable amounts of haptoglobin usually signals the end of hemolysis.

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Therapy The first line of therapy is corticosteroids, and 80% of patients achieve a partial or complete response to 1 mg/kg/day of prednisone (orally). Once a response is achieved, the prednisone dose is tapered slowly. Approximately 50% of patients require prednisone at a dose of 15 mg/day or less to maintain the hemoglobin level >10 g/dL, and it may take up to 3 weeks for patients to achieve a response. Patients who do not respond in 3 weeks should be started on second-line therapy. It is estimated that long-term complete responses not requiring prednisone can be achieved in 20% of patients.15

THERAPEUTIC PRINCIPLES Autoimmune Anemia • Hemolytic anemia induced by “warm” antibody • Acute: corticosteroids, transfusion if severe • Chronic: splenectomy, rituximab, immunosuppression • Hemolytic anemia induced by “cold” antibody • Cold agglutinin disease: avoidance of cold; rituximab +/− fludarabine • Paroxysmal cold hemoglobinuria: avoidance of cold; corticosteroids • Hemolytic disease of the newborn • Neonatal exposure to fluorescent light • Intrauterine transfusion or exchange transfusion

Splenectomy and anti-CD20 antibody (rituximab) are considered second-line therapy. Splenectomy is associated with short-term partial or complete responses in two-thirds of patients. The overall response rate to rituximab is approximately 80%, but rituximab is contraindicated in patients with untreated hepatitis B. The rare, but most severe, long-term complication of rituximab therapy is progressive multifocal leukoencephalopathy.15 Danazol, a synthetic anabolic steroid, has been used as a first-line agent in conjunction with prednisone; its effectiveness appears to be less in relapsed or refractory disease. The role of high-dose intravenous immunoglobulin (IVIG) remains controversial; its effectiveness remains to be determined in larger trials. Third-line therapy consists of immunosuppressive agents (e.g., azathioprine, cyclophosphamide, alemtuzumab, mycophenolate mofetil, cyclosporine).1,15

IMMUNE-MEDIATED NEUTROPENIA Immune neutropenia constitutes a heterogeneous group of acquired diseases in which the immune system responds to circulating neutrophils, selectively reducing their level to below 1500 cells/mm3 (Table 62.2).

KEY CONCEPTS Immune Neutropenia • Immune neutropenia in children is caused by antineutrophil antibodies (ANAs). • Immune neutropenia in adults has a more complex etiology. • Immune complex-mediated neutrophil clearance and cell-mediated suppression of myelopoiesis often play a major role.

TABLE 62.2  Causes of Immune

Neutropenia Primary

Isoimmune neonatal neutropenia Autoimmune neutropenia of childhood Adult autoimmune neutropenia

Secondary Systemic autoimmune disease Rheumatoid arthritis (i.e., Felty syndrome) Systemic lupus erythematosus Sjögren syndrome Lymphoproliferative malignancy Large granular lymphocyte (LGL) leukemia Lymphoma

Drug-Induced Antiplatelets—ticlopidine Inflammatory bowel disease drug—sulfasalazine Antipsychotic—clozapine, phenothiazines Antithyroid medications—propylthiouracil, methimazole Retrovirals Antibiotics—beta-lactams, cefepime, trimethoprim-sulfamethoxazole, vancomycin, rifampicin, quinine/quinidine Diuretics—furosemide, spironolactone Antiepileptic—lamotrigine Rituximab, infliximab, etanercept

antibodies usually disappear within 12–15 weeks, but occasionally it can persist as long as 24 weeks after delivery.

Primary Autoimmune Neutropenia Primary autoimmune neutropenia is an antibody-mediated disease that presents commonly in early childhood.17 Patients typically have normal blood counts at birth and develop neutropenia at 3–36 months of age. Children presenting at <2 years of age most commonly recover spontaneously within 2–3 years of diagnosis. Nevertheless, some children, particularly those presenting at older ages or manifesting other autoimmune findings, develop a chronic neutropenia disorder. Primary immune neutropenia (in the absence of other disease manifestations) is less common in adults. Rigorous incidence data are lacking, but a small retrospective study in Sheboygan County, Wisconsin, found an annual incidence of 5–10 cases of primary or secondary neutropenia per 100 000 people.

Neutropenia Associated With Systemic Autoimmune or Lymphoproliferative Diseases Most patients with active systemic lupus erythematosus (SLE) develop neutropenia as part of a more global leukopenia (Chapter 51).18 Separately, a smaller subset develops severe neutropenia, presumably mediated by ANAs. Sjögren syndrome (Chapter 54) and other systemic autoimmune diseases are also sometimes complicated by immune neutropenia. Lymphoproliferative disorders, such as chronic lymphocytic leukemia (Chapter 78), can occasionally be complicated by immune neutropenia as well.

Isoimmune Neonatal Neutropenia

Felty Syndrome

Transient neutropenia analogous to neonatal immune hemolysis or thrombocytopenia develops when IgG antineutrophil antibodies (ANAs) from an allosensitized or autoimmune mother cross the placenta and destroy fetal neutrophils.16 Neutropenia is typically present at birth and resolves spontaneously, as maternal

Immune neutropenia develops in about 1% of patients with rheumatoid arthritis, most commonly (but not exclusively) in association with splenomegaly, a combination designated as Felty syndrome.19 Patients typically express human leukocyte antigen– DR4 (HLA-DR4) and have long-standing seropositive rheumatoid

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arthritis complicated by erosive joint disease, subcutaneous nodules, and/or leg ulcers. The natural history is often marked by repeated infection, with 5-year mortality rates of >30% reported in some studies. The complex pathophysiology of this disorder is discussed elsewhere in detail (Chapter 52).

have been attempts in the past to classify this pattern as pseudo-Felty syndrome, but the clinical findings and course are often indistinguishable from “classic” Felty syndrome. 2. About half the patients with clinically apparent T-LGL leukemia have circulating rheumatoid factor and immune complexes in their blood; about a third, usually patients expressing HLA-DR4, develop clinically significant arthritis, sometimes requiring antiinflammatory agents. Although the reason why clonal T-LGL disorders and autoimmunity often coexist is unclear, the tendency toward overlap is clear. Since the pathophysiology and therapy of both conditions are similar, these problems in classification usually have little impact on the initial management of neutropenia. When a patient with “Felty syndrome” develops aggressive T-LGL leukemia or a patient with T-LGL manifests severe rheumatological symptoms, the clinician must be prepared to alter therapy, as needed, to fit the clinical picture. Although some clinicians have attempted to develop criteria for distinguishing Felty syndrome from T-LGL with pseudo-Felty syndrome, there is now substantial evidence that clonal T-LGL disorders are commonly found in rheumatology patients and that patients with clonal disorders seldom develop a progressive, neoplastic disorder. Conversely, although patients with T-LGL leukemia have a malignancy, it is typically quite indolent and in these cases, the clinical course is often dominated by rheumatological complication and/or neutropenia and not by progressive neoplastic disease.

T-Cell Large Granular Lymphocyte Leukemia

Drug-Induced Immune Neutropenia

Large granular lymphocytes (LGL) are medium to large lymphocytes recognizable on light microscopy by their distinctive azurophilic granules (Fig. 62.2). These cells normally constitute <15% of circulating leukocytes and are composed of two major subsets. One is the natural killer (NK) LGLs that express CD2, CD16, and CD56 and is not linked to neutropenia. The other, T-cell (T) LGLs, expresses CD2, CD3, CD8, and CD57 with or without CD16, a phenotype typical of antigen-stimulated mature CD8 effector T cells. Polyclonal and transient monoclonal expansions of these cells sometimes appear in response to viral infection or other immune stimuli without adverse effect. However, some patients develop an indolent lymphoproliferative disease characterized by the accumulation of an autonomous T-LGL clone in blood and in other lymphoid organs, particularly bone marrow, the liver, and/ or the spleen. Patients with this disease have a remarkably high incidence of immune neutropenia.20,21 Even in the absence of gross marrow involvement, over 80% have a neutrophil count of <2000/ mm3 at presentation, and at some point 30–40% develop severe neutropenia with <500 neutrophils/mm3.20,21 The pathophysiology resembles Felty syndrome in many respects.

A wide variety of medications cause neutropenia.22 In some cases, it may be antibody-mediated (see Table 62.2 for common examples); in others, it may be a direct toxic effect on marrow precursors. Often, the mechanism for neutropenia is unclear. A detailed discussion of this topic is beyond the scope of this chapter, but drug-induced neutropenia must always be considered in the differential diagnosis of acquired neutropenia. Rituximab (anti-CD20) is occasionally associated with late-onset neutropenia (LON).23 LON is typically self-limiting and of no significant clinical consequence; the occurrence and severity of LON may be associated with the total dose of rituximab and the myelotoxicity of the accompanying chemotherapy administered. LON appears to coincide with post-rituximab B-cell recovery. In patients receiving DA-EPOCH (dose-adjusted etoposide/vincristine/doxorubicin/ cyclophosphamide/prednisone regimen) chemotherapy for lymphoma, the incidence of LON was 8%, the median time of onset was 175 days (range of 77–204 days), and the duration was 11–16 days. A list of the most common agents associated with drug-induced neutropenia is included in Table 62.2.

Clinical Overlap Between Felty Syndrome and T-LGL Leukemia

Regulation of Antineutrophil Antibody Production Isoimmune ANA production in pregnant women represents an “appropriate” immune response to foreign antigens. The production of autoimmune ANAs in other settings reflects immune dysregulation. Older studies attributed primary immune neutropenia of childhood to delayed maturation of T cells responsible for regulating B-cell responses. In this view, the spontaneous recovery usually observed reflects the eventual appearance of mature regulatory or inhibitory T cells. However, this hypothesis is not well supported with clinical laboratory data; the cause of antibody production in other autoimmune diseases remains unclear.

FIG 62.2  The Large Granular Lymphocyte (LGL) Is a ModerateSized Lymphocyte Containing Several Distinctive Azurophilic Granules. Additional immunophenotyping is required to distinguish the CD3+, CD8+, CD57+ T-LGL associated with neutropenia from CD16+, CD56+ natural killer (NK)–LGL.

At the extremes these syndromes are easily separable. Patients with classical Felty syndrome have severe rheumatoid arthritis, usually requiring antiinflammatory therapy, incidentally complicated by late neutropenia. This is quite different from the pattern in patients with isolated T-LGL leukemia with neutropenia in the absence of clinical autoimmune disease. Nonetheless, confusing overlap can occur in two settings: 1. More than half the patients with a clinical Felty syndrome may have detectable T-LGL clones in their blood when studied with sensitive flow cytometric or molecular techniques.20 There

Immunopathogenesis

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Part Seven  Organ-Specific Inflammatory Disease this antibody probably is not specific for neutropenia, since it is also present in patients with autoimmune disease who do not have neutropenia.

Platelets with bound IgG

Macrophage with FcRγIIA receptor

FIG 62.3  Antibody-Bound Platelets Are Cleared From the Circulation by Binding of the Fc Domain of Immunoglobulin G (IgG) to FcγIIIA Receptors on Macrophages and Other Cells. The cross-linking of the macrophage receptors sets off a cascade of internal signaling that leads to increased expression of the inhibitory FcγIIB receptors (not shown).

Antibody Specificity The most important target for antibody responses is the Fcγ receptor IIIb (FcγRIIIb), a low-avidity granulocyte-specific Fcγ receptor that binds IgG immune complexes (Fig. 62.3). This cell surface protein, a glycosyl phosphatidylinositol-linked variant of CD16 selectively expressed on neutrophils, contains several highly immunogenic polymorphisms.24 Human neutrophil antigen 1a (HNA-1a) and HNA-1b (previously designated NA1 and NA2) are two related polymorphic forms of FcγRIIIb. Antigenic differences are attributed to a cluster of five base substitutions, all found within exon 3. The two products can be distinguished because two of the base substitutions in HNA-1a disrupt sites of N-linked glycosylation, thereby lowering the size of the protein by ~30 000 Daltons (Da). The frequency of the gene for HNA-1a (FCGR3B) varies from 0.3 to 0.55 in different ethnic groups. HNA-1c (previously designated SH) is another immunogenic polymorphism related to FcγRIIIb. This antigen, present in 5% of northern Europeans, is attributable to an amino acid polymorphism at position 266 in a (reduplicated) copy of the HNA-1b allele of FcγRIIIb. FcγRIIIb-null phenotype, present in 0.1% of the northern European population, occurs in individuals homozygous for an extensive gene deletion involving all of FcγRIIIb. Because affected individuals have never been exposed to endogenous FcγRIIIb, they often develop a broad antibody response after neutrophil exposure through transfusion or pregnancy. Other HNAs have also been identified.25 Perhaps most clinically relevant is HNA-2a (previously NB1), a neutrophil glycoprotein designated CD177. Antibodies against this target have been reported in isoimmune and autoimmune neutropenia. HNA-3a (previously 5b) and HNA-4a (previously MART), an epitope on CD11b, are also immunogenic, but antibody formation against these is seen primarily in heavily transfused individuals and not in autoimmune neutropenia. As described below, individuals with chronic immune neutropenia may develop antibodies against other surface antigens, including CD11b/CDI8 and CD35.26 Using a sophisticated antigen discovery strategy, patients with Felty syndrome were found to have a high incidence of antibodies against an intracellular antigen eukaryotic elongation factor 1A-1 (eEF1A-1). Although the finding has been confirmed,

Patterns of Autoantibody Specificity Isoimmune neonatal neutropenia is associated with maternal IgG iso- or autoantibodies that can be generated in response to each of the polymorphic alloantigens noted above, particularly polymorphisms affecting FcγRIIIb. Immune neutropenia in childhood is most commonly associated with IgG directed against the autoantigens HNA-1a and/or HNA-1b.26 Sera from affected patients often also bind (albeit more weakly) to neutrophils expressing the alternative allele, and in some series, more than half the patients with this entity were shown to produce antibodies capable of binding to nonpolymorphic elements within FcγRIIIb. Using monoclonal antibody (mAb)–specific immobilization of granulocyte antigens (MAIGA) to search for antibodies directed against other neutrophil surface molecules, more complex patterns can be recognized. In one study, autoantibodies against FcγIIIb were seen in 27%, against CD11b/CDI8 (CR3) in 21%, against CD35 (CR1) in 14%, and FcγRII in 2% of patients’ sera.27 Prognostically, patients with antibody responses confined to HNA-1a and/or HNA-1b are more likely to have uncomplicated, self-limiting disease; patients with specificity for nonpolymorphic determinants on the FcγRIIIb molecule and other antigens more often have a generalized immune disorder and more persistent neutropenia.26 Although antineutrophil assays are not highly quantitative, a recent retrospective study has suggested that patients with low levels of ANAs may have a more favorable long-term prognosis.28 In adults, it is often difficult clinically to distinguish immune neutropenia from nonimmune idiopathic neutropenia. Consequently, the sensitivity and specificity of antibody assays in this setting are uncertain. In general, antibodies against HNA-1a or HNA-1b are less common, and antibodies against surface receptors, such as CD11b/CD18 (CR3), are more common in older children and adults than in young children. Sera from patients with Felty syndrome20 and T-LGL leukemia21 are often positive in ANA assays. Interpretation of these results is complicated by the high incidence of nonspecific immune complexes in these populations, which may bind nonspecifically through Fc and complement receptors to the neutrophil surface. Indeed, because it is difficult to distinguish these two types of binding, the incidence of “true” ANAs in these syndromes remains uncertain. Detectable antineutrophil antibodies are low in titer or absent in most patients carefully studied with either diagnosis.20,21 Impact of Antibodies and Immune Complexes on Neutrophil Survival There is good experimental evidence that both ANAs and immune complexes can induce neutropenia in vivo. The relative importance of reversible sequestration and neutrophil destruction in inducing neutropenia varies with the experimental model, the character of the antibody/immune complex, spleen size, and presumably other factors as well. The detection of antineutrophil antibodies in serum, however, does not automatically predict accelerated immune clearance of neutrophils in vivo. Some antibodies bind well to neutrophils under in vitro assay conditions, without provoking neutrophil destruction in vivo.29 In part, this reflects the inability of these crude assays to distinguish effective from ineffective binding of immunoproteins.

CHAPTER 62  Immunohematological Disorders Myelopoiesis in Immune Neutropenia In primary immune neutropenia, bone marrow is typically normocellular or mildly hypercellular with an increased proportion of early myeloid forms (particularly myelocytes and promyelocytes) and decreased mature forms (neutrophils, bands, and metamyelocytes), a pattern designated maturation arrest. Although maturation arrest can also be seen in a number of other diseases, in this setting, it suggests an expansion in immature precursors with early release of mature components into blood. Rigorous kinetic studies in children with primary neutropenia are not available, but the available data suggest myelopoiesis in this setting is increased. The findings are more complex in Felty syndrome and T-LGL leukemia. In vivo neutrophil kinetic studies and in vitro assays of marrow function often document reduced myelopoiesis in these settings.20,21 This has been attributed to T-cell–mediated and cytokine-mediated suppression. T-LGL leukemia cells constitutively express Fas ligand on their surface and also release significant quantities of soluble Fas ligand into plasma in vivo. Reduced myelopoiesis in T-LGL leukemia and some patients with Felty syndrome may be linked to apoptosis instigated by the binding of Fas ligand expressed on the abnormal cells to Fas expressed on the surface of myeloid precursors.21 Whatever the precise mechanism, reductions in myelopoiesis appear to be a common element in patients with these forms of secondary immune neutropenia.

Diagnosis

Clinical Presentation Isoimmune neutropenia presents at birth and may persist for up to 6 months. Self-limiting primary autoimmune neutropenia typically presents in early childhood. In older children and adults, neutropenia is more commonly associated with other systemic autoimmune disease, especially rheumatoid arthritis and SLE or T-LGL leukemia. Drug-induced neutropenia must always be considered in patients taking medications. Laboratory Findings Blood counts typically demonstrate isolated neutropenia, sometimes with monocytosis. More generalized leukopenia, anemia, and/or thrombocytopenia suggest concurrent SLE or a primary bone marrow disorder, especially aplastic anemia or myelodysplasia. Examination of the blood film for evidence of abnormalities in other cell lines or increased numbers of LGL is essential. The persistent presence of >2000 LGL/mm3 for 6 months in itself is diagnostic of T-LGL leukemia; however, normal LGL counts in blood do not preclude this diagnosis. Perhaps a quarter of patients with T-LGL leukemia and immune neutropenia have fewer than 500 monoclonal LGL/mm3 in blood.21 The evaluation of patients with small T-LGL clones detected in blood on flow cytometric or molecular testing without clear tissue infiltration remains problematic. At least some of these patients probably have self-limiting “T-cell gammopathies of unknown origin” (Chapter 80) unassociated with overt lymphoproliferation or autoimmunity. Bone marrow findings in immune neutropenias (as briefly reviewed above) can vary substantially. Perhaps the most important function of the bone marrow examination is to rule out hypoplasia/aplasia, myelokathexis, marked megaloblastic dysplastic changes, or abnormal infiltration with nonhematopoietic cells,

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which might suggest an alternative diagnosis. The marrow examination may also be helpful in confirming T-LGL leukemia.30 Detection of antineutrophil antibodies.  Antibodies are assayed clinically using indirect assays (i.e., by measuring the binding of antibodies from patient sera to fixed granulocytes from unrelated individuals). The granulocyte immunofluorescence test (GIFT), which exploits flow cytometry for detection, is most commonly used because of its high sensitivity. The granulocyte agglutination test (GAT) is less sensitive but is particularly valuable used in conjunction with GIFT to detect antibodies against HNA-3a or HNA-1b. Once the presence of an antibody has been confirmed, MAIGA is a valuable technique in identifying the target molecule recognized by the antibody, information that may be very helpful in identifying antibody specificity and in distinguishing granulocyte-specific antibodies from alloantibodies directed against HLA determinants. More precise epitope typing still requires a panel of granulocytes of varied phenotype. Unfortunately, to date, granulocyte panels are both difficult to prepare and impossible to store. Consequently, antibody typing remains a laborious, difficult task. At the second international granulocyte serology workshop, 12 centers independently tested a series of unknown sera. Many laboratories could detect strong HNA-1a antibodies, but the success rate was much lower in defining HNA-1b or HNA-2a antibodies, and individual laboratories varied greatly in their proficiency.25 Clinical use of antineutrophil antibody studies. In young children with neutropenia, a positive result is very helpful in distinguishing between immune-mediated and congenital causes. Isoimmune and congenital disease may be apparent from birth, and the former usually resolves within 2–6 months. Using GIFT or GAT assays, ANAs can be detected in more than 70% of children with primary immune neutropenia. When both are used in tandem the yield increases further. A strong positive result strongly supports the diagnosis of immune neutropenia. However, a negative result does not exclude the diagnosis.28 Primary autoimmune neutropenia in adults is difficult to distinguish from the ill-defined entity chronic idiopathic neutropenia.1 Because there is no “gold standard” for distinguishing immune disease from nonimmune disease in this setting, the diagnostic sensitivity and specificity of the ANA assays are unclear. Assays are positive in perhaps a third of adults referred with chronic neutropenia, and a positive result in the absence of other systemic autoimmune disease certainly supports a diagnosis of immune neutropenia. Again, a negative result does not preclude an immune etiology, but it is more consistent with chronic idiopathic neutropenia. In patients with systemic autoimmune disease or T-LGL leukemia, hyperglobulinemia and circulating immune complexes greatly complicate laboratory evaluation. ANA assays are frequently positive even in the absence of neutropenia. Since the specificity of a positive result is low, its diagnostic value is very limited, and the clinician must be vigilant for other possible causes, especially drug-induced neutropenia.

Therapy

Overview All patients with neutrophil counts below 1000/mm3 have some increased risk of infection, but some remain asymptomatic even with absolute neutrophil counts of 500/mm3 or less. Growth factors can usually improve neutropenia and reduce the risk of infection, but given their expense, inconvenience, and possible side effects, they should be reserved for use in patients with a

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very low count, or a previous pattern of frequent infection. The indications for immunosuppressives, steroids, and splenectomy are more complex.

IMMUNE-MEDIATED THROMBOCYTPENIAS Immune Thrombocytopenia

THERAPEUTIC PRINCIPLES Immune Neutropenia • Palliative treatment of neutropenia is reserved for patients with a neutrophil count below 500/mm3 or recurrent infection. • Recombinant granulocyte–colony-stimulating factor (G-CSF) is the most effective single agent for palliating neutropenia. • Immunosuppressive agents, steroids, and splenectomy are reserved for patients with persistent or refractory neutropenia or with other detrimental manifestations of systemic autoimmunity.

Colony-Stimulating Factors Controlled trials are lacking in this disease setting, but granulocyte–colony-stimulating factor (G-CSF) or granulocyte macrophage–colony-stimulating factor (GM-CSF) usually enhance neutrophil counts in each of the clinical groups discussed. Because of their safety, speed, and efficacy, they have replaced steroids and splenectomy as first-line symptomatic therapy. They should be used at the lowest effective dose, with particular caution in patients with systemic autoimmune disease, as they are prone to leukocytoclastic vasculitis as a complication of therapy.18 Immunosuppressive Agents Because disease is usually self-limiting and responsive to G-CSF, immunosuppressive agents are seldom used in children with primary immune neutropenia. Chronic low-dose methotrexate is considered first-line therapy for patients with Felty or LGL leukemia–associated neutropenia. Cyclophosphamide and cyclosporine are considered second-line therapy for LGL leukemia, and purine analogues, splenectomy, and alemtuzumab are considered third-line therapy for LGL leukemia. Other Therapy Each of the following treatments can be effective in reversing neutropenia, but their use has diminished considerably in recent years. IVIG can temporarily reverse neutropenia, particularly in children, probably by blocking Fc receptors responsible for triggering neutrophil destruction. However, G-CSF, which is more convenient to administer and at least as effective, has largely replaced use of IVIG. Splenectomy and steroids can each reduce immune destruction by suppressing the body’s capacity to clear IgG- and complement-coated cells. Over a longer time frame, these treatments can also suppress antibody production, in the first case by removing a major site of production and in the second by reducing antineutrophil antibody production and blocking T-cell–mediated myelosuppression. They can reverse neutropenia in many patients, but their long-term impact on outcome remains unclear. Given their risk and side effects, both modalities are generally reserved for patients resistant to CSFs and low-dose immunosuppressives. Prophylactic Antibiotics Where recurrent infection is a problem, oral trimethoprim– sulfamethoxazole (TMP-SMX) is commonly used for prophylaxis, particularly in children. This approach is very reasonable, given its success in other immunocompromised groups, but it has not been tested in a controlled trial. Immunization with pneumococcal vaccine is also recommended in situations where therapeutic splenectomy has been used or is being planned.

KEY CONCEPTS Immune Thrombocytopenic Purpura • • • •

Antibody-mediated destruction or decreased production of platelets Both B and T cells important in etiology Clearance of platelets mediated by Fcγ receptors Diagnosis depends on: • Clinical presentation of low platelets • Exclusion of other causes of thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune syndrome involving antibody and cell-mediated destruction of platelets.31 ITP can occur in the absence of an identified predisposing factor (primary ITP) or in association to drug exposure or other identified cause (secondary ITP). Recent recommendations from an international working group recommend that a platelet count of <100 × 109/L be required for diagnosis.32 ITP During Pregnancy and the Neonatal Period Gestational thrombocytopenia is the most common cause of thrombocytopenia in pregnancy, affecting 5% of all pregnancies and accounting for 75% of cases of pregnancy-associated thrombocytopenia. Gestational thrombocytopenia develops in the late second or third trimester and is not associated with an increased incidence of pregnancy-related complications or the delivery of thrombocytopenic offspring. Preeclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome are also present in the third trimester and are associated with thrombocytopenia. Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome need to be considered in the differential diagnosis of thrombocytopenia of pregnancy. ITP accounts for 5% of pregnancy-associated thrombocytopenia.33 Lack of hypertension, fever, hemolytic anemia, uremia, and liver function abnormalities serve to distinguish ITP from these other conditions. Neonatal Alloimmune Thrombocytopenia and Posttransfusion Purpura Neonatal alloimmune thrombocytopenia (NAT) caused by antihuman platelet antigen 1a (HPA-1a) antibodies occurs in 1 : 1250 pregnancies in the Caucasian population. Severe hemorrhage occurs in 1 : 12 500–1 : 25 000 pregnancies. NAT is caused by maternal antibodies against paternally derived antigens on fetal platelets, most commonly HPA-1a. These antibodies cross the placenta and sensitize fetal HPA-1a–positive platelets, which are then removed in the spleen. Two percent of Caucasian women carry the less frequent HPA-1b and can be immunized against HPA-1a during pregnancy (25%) or at the time of delivery (75%). The HPA-1a antigen is most efficiently presented by HLA-DRB3*0101; 35% of the women at risk (with HPA-1b and HLA- DRB3*01:01) are immunized.34 In most circumstances, first-time cases of NAT are identified following the birth of a markedly thrombocytopenic neonate; antenatal management is, thus, only possible in subsequent pregnancies. Screening all pregnancies for NAT is under evaluation in several countries. Treatment of NAT consists of maternal administration of high doses of IVIG and corticosteroids.35 Platelet transfusions can induce antibodies to either HPA or antigens encoded by the major histocompatibility complex

CHAPTER 62  Immunohematological Disorders (MHC). Eight percent of transfused patients mount detectable antibodies to HPA antigens and 45% to HLA antigens. Platelet antibodies cause rapid clearance of transfused platelets and lead patients to become refractory to platelet transfusions.36 Posttransfusion purpura (PTP) can follow transfusion with platelets or RBCs (in which platelets are found in numbers sufficient to sensitize the recipient).

Drug-Induced Thrombocytopenia Drug-induced thrombocytopenia (DITP) is an idiosyncratic immune-mediated reaction. The drug-dependent antibodies bind to specific epitopes on platelet surface glycoproteins only in the presence of the sensitizing drug. The drugs bind noncovalently and reversibly to platelets, commonly to GPIIb-IIIa and GPIb-V-IX, and also to the antibody. The Fab domains of the antibodies bind to the drug-platelet epitope. Drug-dependent antibodies inducing thrombocytopenia typically develop 1–2 weeks after exposure to a drug; exceptions to this rule include eptifibatide, tirofiban, and abciximab, as naturally occurring antibodies to these drugs can cause thrombocytopenia within a few hours of the first exposure. Thrombocytopenia with platelet counts frequently below 20 × 109/L develops acutely, recovery occurs 1–2 days after discontinuation of the drug and is usually complete after 1 week, but rarely thrombocytopenia persists for several weeks. Quinidine, quinine, rifampin, tegretol, TMP-SMX, vancomycin, danazol, acetaminophen, abciximab, eptifibatide, tirofiban, and gold salts are the most common culprits. Treatment consists of discontinuing the offending drug; platelet transfusions are sometimes necessary.37,38 Heparin-induced thrombocytopenia (HIT) is a special case that is caused by antibodies to platelet factor 4 (PF4)–heparin complexes. It can be associated with life-threatening thrombosis. The antibody–PF4–heparin complex activates platelets, resulting in a high risk of both arterial and venous thrombotic events.39 Thrombocytopenia occurs as a result of clearance of platelet aggregates induced by the antibody and usually appears 5–7 days after treatment with heparin (or low-molecular-weight heparin) unless a patient has been previously exposed to heparin. In the event of prior exposure, especially within the last 100 days, thrombocytopenia can occur within 1 day of heparin administration. Even small doses of heparin given as “flushes” to maintain intravenous catheter patency can be sufficient to cause HIT with thrombosis. In cases of suspected HIT, all heparins should be stopped, and an alternative anticoagulant agent, such as the direct thrombin inhibitors argatroban and lepirudin, should be used. Fondaparinux is an anticoagulant synthesized from the pentasaccharide core of the heparin molecule and may be another option for the treatment of HIT, since it binds to HIT antibodies but does not activate platelets and cause thrombosis. In cases of confirmed or strongly suspected HIT, anticoagulation should be continued for 3 months because the risk of thrombosis persists in this group of patients. Coumadin is generally used for long-term anticoagulation; it should be started concomitant with a heparin alternative because of the increased risk of thrombosis during the initial depletion of anticoagulant factors (proteins C and S) by warfarin (Coumadin). The role of direct oral anticoagulants that target thrombin or activated factor X remains to be defined.

Pathogenesis In the majority of patients the underlying defects leading to autoantibody production remain unclear. In some patients, ITP follows exposure to viral or bacterial antigens. Molecular mimicry appears to play a role in the development of self-reactive platelet

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antibodies following infections. Human immunodeficiency virus (HIV), hepatitis C virus (HCV), and Helicobacter pylori infections have been associated with ITP. H. pylori CagA antigen appears to cross-react with platelet antigens,40 which may explain the association with ITP and H. pylori infection. Interestingly, empiric treatment of Helicobacter infection with amoxicillin, clarithromycin, and proton pump inhibitors in patients suspected of infection leads to 53% remission of ITP whether or not Helicobacter is eradicated, suggesting immunomodulatory effects of treatment that do not involve elimination of the bacterial antigen with cross-reactivity to platelet antigens.41 Patients who are not infected with H. pylori but are treated empirically with antibiotics do not have a significant platelet response rate (6.5%), which suggests that empiric therapy is not useful in the absence of a documented infection.41 NAT is caused by maternal antibodies against the HPAs that the fetus carries but which the mother lacks (most commonly HPA-1a). It is more likely to cause intracerebral hemorrhage (10–20% of cases) than is maternal ITP, and it is very likely to recur if it has occurred in a previous pregnancy.42 IVIG and platelet transfusions using maternal platelets (to ensure that the offending antigen is not present in the transfused product) are often necessary. Antibodies against human platelet antigens are also responsible for PTP, in which the recipient has an acquired antibody directed against a platelet antigen on the donor platelets. It is not understood why the antibodies also destroy the patient’s own “antigennegative” platelets. Treatment consists of IVIG and corticosteroids, and sometimes plasma exchange.34 Transplantation-mediated alloimmune thrombocytopenia (TMAT) may occur as the result of donor-origin antibodies produced by passenger B cells directed against the recipient platelet alloantigen (HPA-1a).43 The most common epitopes for platelet antibodies in ITP are the platelet GPIIb/IIa and GPIb-IX receptors.44 The autoantibodies serve as opsonins resulting in the clearance of platelets by FcγR-bearing cells in the reticuloendothelial system (see Fig. 62.3). There is upregulation of genes involved in cell-mediated cytotoxicity via CD3+CD8+ T lymphocytes,45 with T-helper 1 (Th1)–associated cytokines predominating.46 Regulatory T cells (Tregs) are decreased,47 and B-cell activation is increased.48 There is evidence of suppression of megakaryopoiesis by both T lymphocytes49 and ITP plasma/IgG.50 In addition, megakaryocyte and platelet production are dependent on thrombopoietin signaling through binding to the Mpl receptor, and patients with ITP have reduced thrombopoietin levels despite the presence of low platelet counts.51

Laboratory Diagnosis52 Peripheral blood smear examination is important to evaluate for the presence of schistocytes, leukocyte adhesion bodies in MYH-9-related disease, and giant or large platelets in inherited thrombocytopenias and to exclude ethylenediaminetetraacetic acid (EDTA)–dependent platelet agglutination. Bone marrow aspirate, biopsy, flow cytometry, and cytogenetics should be considered in patients older than 60 years of age and in patients with systemic symptoms. The detection of H. pylori with the urea breath test is recommended in adults, and testing in children is recommended in high prevalence areas. Routine serological evaluation for HIV and HCV is recommended in adults. Baseline Igs (IgG, IgA, IgM) should be measured in both adults and children to diagnose such conditions as common variable immunodeficiency and selective IgA deficiency (Chapter 34), in which ITP is a common complication.

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Antiplatelet antibody testing to specific platelet glycoproteins is not routinely recommended; the test is neither sensitive nor specific for ITP. Routine testing for anticardiolipin antibodies is not recommended in the absence of symptoms of antiphospholipid syndrome. DAT should be obtained if hemolytic anemia is suspected and in patients in whom anti-D antiglobulin treatment is considered. Blood group Rh(D) typing should be obtained if anti-D antiglobulin treatment is considered. Where a microangiopathic process is evident, especially with concomitant renal failure, fever, or cognitive impairment, measurement of ADAMTS13 (ADintegrin-like And Metalloprotease with ThromboSpondin type 13 motifs) is warranted to rule out TTP.

Therapy of ITP53 THERAPEUTIC PRINCIPLES Immune Thrombocytopenic Purpura (ITP) • Primary goal—prevent bleeding • Treatment not usually necessary if platelets >30 000/µL • Prednisone and/or intravenous immunoglobulin (IVIG) usual first medications • Anti-D may avert/delay splenectomy • Treat refractory ITP with immunosuppressives • Mycophenolate mofetil, cyclosporine, alemtuzumab, cyclophosphamide, rituximab (not approved by the US Food and Drug Administration [FDA] for this use) • Splenectomy for persistent disease (immunize patient with pneumococcal vaccine before surgery)

In most circumstances, the goals of therapy are to keep the platelet count above 30 × 109/L and to minimize toxicity. In the absence of hemostatic comorbidity, trauma, or surgery, intracranial hemorrhage is rare in patients with a platelet count above 20 × 109/L. Treatment of ITP should be individualized, but first-line therapy usually consists of corticosteroids supplemented with either IVIG or anti-Rh(D) in Rh(D)-positive, nonsplenectomized patients. Intravascular hemolysis, disseminated intravascular coagulation, and renal failure have been reported with the use of anti-Rh(D). Anti-Rh(D) should be avoided in patients with underlying hemolysis or a positive result of the DAT that is not the result of prior therapy. Several uncontrolled studies suggest that bolus oral therapy with oral dexamethasone for 1–4 cycles increases response rates and prolongs remission duration. Second-line therapy should be initiated if there is absence of a robust response at 1 month or once significant steroid related toxicity supervenes. Two-thirds of patients obtain a durable long-term remission following splenectomy, and these patients should receive immunizations with the pneumococcal, Haemophilus influenzae type b, and the quadrivalent meningococcal vaccines before splenectomy. A single course of rituximab (anti-CD20) (375 mg/m2 weekly for 4 weeks) is associated with 40% complete remission at 1 year and 15–20% complete remission at 5 years. Patients who relapse after an initial response usually respond to a second course. Rituximab is contraindicated in patients with active hepatitis B. Cases of multifocal leukoencephalopathy have been reported in HIV-negative patients treated with rituximab. The thrombopoietin receptor agonists (TRAs) romiplostim and eltrombopag have shown significant sustained activity in patients with ITP. Romiplostim is administered subcutaneously and eltrombopag orally. Increased bone marrow reticulin fibrosis has been described in some patients receiving

TRAs. Initial dosing of eltrombopag should be reduced by 50% in patients of Southeast Asian origin. Danazol is an attenuated androgen administered orally and has been used, with some success, as a steroid-sparing agent. Third-line therapy is reserved for patients who are unresponsive to or ineligible for first- and second-line therapies. These agents include cyclophosphamide, azathioprine, mycophenolate mofetil, cyclosporine, alemtuzumab, and vinca alkaloids. One novel approach to ITP consists of blockade of the FcγIIIA receptors on macrophages in the spleen that mediate uptake of antibody-bound platelets. Prior attempts at FcγIIIA receptor blockade to ameliorate ITP using mAbs to FcγIIIA have improved platelet counts in half the patients refractory to other treatments but were abandoned because of nausea, vomiting, fever, and rarely anaphylaxis that were presumed to be caused by immune stimulation from cross-linking of FcγIIIA receptors by bivalent IgG. Preclinical studies suggest monovalent anti-FcγIIIA fused to albumin (to prolong the half-life) may block the FcγIIIA receptor without stimulating the immune system, and this approach may be tested clinically, again.54

Therapy of ITP During Pregnancy and the Neonatal Period

Patients with ITP with platelet counts higher than 20–30 × 109/L usually do not require treatment during the first and second trimesters of pregnancy; platelet counts should be monitored more closely as delivery approaches. Obstetrical anesthesiologists recommend a minimum platelet count of 75 × 109/L to allow administration of spinal or epidural anesthesia. A platelet count of at least 50 × 109/L is generally considered adequate to allow a Cesarean section.52 First-line therapy consists of prednisone and IVIG. Prednisone is initiated at a low dose (10–20 mg) and then adjusted to the minimum dose that produces a hemostatically effective platelet count. Corticosteroids may exacerbate hypertension, diabetes, and osteoporosis during pregnancy and are associated with weight gain and psychosis. Corticosteroid use in the first trimester has been associated with congenital anomalies, such as orofacial clefts, and low-dose corticosteroids have no impact on the fetal platelet count. IVIG is an effective means of increasing the platelet count rapidly and is preferred over prednisone in this setting. The American Society of Hematology (ASH) guidelines consider IVIG to be an appropriate first-line agent for severe thrombocytopenia or for thrombocytopenic bleeding in the third trimester. Anti-Rh(D) is safe and effective for the mother and fetus in nonsplenectomized Rh(D)-positive patients.33,52 Second-level therapy before delivery may require utilizing higher doses of corticosteroids combined with IVIG to achieve an adequate platelet count. Splenectomy is an option for patients who fail corticosteroids and IVIG. Remission of ITP is achieved in 75% of patients. Splenectomy is optimally performed during the second trimester. The risk of inducing premature labor is high during the first trimester, and the enlarged uterus renders the procedure difficult if not impossible during the third trimester. Vinca alkaloids, rituximab, danazol, thrombopoietin receptor agonists, and immunosuppressive drugs (other than azathioprine) should be avoided during pregnancy.33,52 Management of parturition is based on the finding that there is no correlation between platelet counts or ITP status of the mothers and the development of neonatal thrombocytopenia. The most reliable predictor of neonatal thrombocytopenia is a history of thrombocytopenia at delivery in a prior sibling.33 In a large meta-analysis, fetal platelet counts below 50 × 109/L were

CHAPTER 62  Immunohematological Disorders observed in 10.1%, and fetal platelet counts below 20 × 109/L were observed in 4.2%.42 The risk of intracranial hemorrhage is below 1%, and the risk of fetal platelet count determination by either fetal scalp sampling or umbilical blood sampling likely exceeds the risk of fetal intracranial hemorrhage, negating its clinical utility. The mode of delivery has no impact on the incidence of intracranial hemorrhage. Platelet counts should be monitored for 1 week following delivery. In a study of 61 patients, 66% of neonates had a further drop in platelets following delivery; the nadir occurred at day 2, and the counts stabilized or began to rise at day 7.34 ASH guidelines recommend that children with a platelet count below 20 × 109/L or those with hemorrhage be treated with IVIG. The concurrent use of corticosteroids is controversial because of the risk of neonatal sepsis. Neonates with platelet counts below 50 × 109/L should have brain imaging performed to detect occult intracranial hemorrhage.

ON THE HORIZON • For many years immunosuppressive therapy for immune thrombocytopenic purpura (ITP) has consisted of corticosteroids and infusions of intravenous immunoglobulins (IVIGs), and more recently anti-Rh antibodies (in suitable candidates), and/or rituximab in patients whose disease is refractory to first-line therapy. Splenectomy may be indicated in those who fail medical therapy altogether. • All ITP seems to be mediated by interaction of antibody-coated platelets with Fc receptors in the reticuloendothelial system of the spleen (and the liver). Many investigators have sought to interrupt the interaction between immunoglobulin (Ig)–coated platelets and Fcγ receptors by use of monoclonal antibodies to the Fcγ receptor itself. However, the use of traditional murine monoclonal antibodies (mAbs) to Fcγ has been associated with adverse events, perhaps as a result of cross-linking of Fcγ receptors on the surface of macrophages, resulting in undesirable immune stimulation and even anaphylaxis. One strategy to circumvent these problems would be to use monovalent Fab fragments that would bind but not cross-link the Fcγ receptors. Yu et al.54 have reported on the design of a monovalent fusion protein composed of the single chain variable region (scFv) of the anti-FcγRIIIA antibody, fused to human serum albumin so as to increase its size and prolong its half-life. These investigators have shown in a mouse model of ITP that the novel construct can prevent IgG-platelet interaction with FcγRIIIA receptors without harmful immune stimulation.54 If this can be replicated in larger animal models, it may be eventually tested for use in humans with ITP. Further, in theory this approach could be applied to other diseases, such as autoimmune hemolytic anemia.

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REFERENCES 1. Barros MM, Blajchman MA, Bordin JO. Warm autoimmune hemolytic anemia: recent progress in understanding the immunobiology and treatment. Transfus Med Rev 2010;24:195–210. 2. Petz LD, Garratty G. Classification and clinical characteristics of autoimmune hemolytic anemias. In: Petz LD, Garratty G, editors. Immune hemolytic anemias. 2nd ed. Philadelphia, PA: Churchill Livingstone; 2004. p. 61. 3. Garratty G. Immune hemolytic anemia associated with drug therapy. Blood Rev 2010;24:143–50. 4. Hoffman PC. Immune hemolytic anemia—selected topics. Hematology Am Soc Hematol Educ Program 2009;80–6. 5. McNicholl FP. Clinical syndromes associated with cold agglutinins. Transfus Sci 2000;22:125–33.

855

6. Berentsen S, Randen U, Vagan AM, et al. High response rate and durable remissions following fludarabine and rituximab combination therapy for chronic cold agglutinin disease. Blood 2010;116:3180–4. 7. Petz LD. Immune hemolysis associated with transplantation. Semin Hematol 2005;42:145–55. 8. Avent ND, Reid ME. The Rh blood system: a review. Blood 2000;95:375–87. 9. Mollison PL, Engelfriet CP, Contreras M. Immunology of red cells. In: Mollison PL, Engelfriet CP, Contreras M, editors. Blood transfusion in clinical medicine. 10th ed. London: Blackwell Science; 1997. p. 60. 10. Hughes-Jones NC. Red-cell antigens, antibodies and their interaction. Clin Haematol 1975;4:29–43. 11. Petz LD, Garratty G. Mechanisms of immune hemolysis. In: Petz LD, Garratty G, editors. Immune hemolytic anemias. 2nd ed. Philadelphia, PA: Churchill Livingstone; 2004. p. 133. 12. Kurlander RJ, Rosse WF, Logue GL. Quantitative influence of antibody and complement coating of red cells on monocyte-mediated cell lysis. J Clin Invest 1978;61:1309–19. 13. Frank MM, Schreiber AD, Atkinson JP, et al. Pathophysiology of immune hemolytic anemia. Ann Intern Med 1977;87:210–22. 14. Moise KJ. Red blood cell alloimmunization in pregnancy. Semin Hematol 2005;42:169–78. 15. Lechner K, Jager U. How I treat autoimmune hemolytic anemias in adults. Blood 2010;116:1831–8. 16. Maheshwari A, Christensen RD, Calhoun DA. Immune-mediated neutropenia in the neonate. Acta Paediatr Suppl 2002;91:98–103. 17. Capsoni F, Sarzi-Puttini P, Zanella A. Primary and secondary autoimmune neutropenia. Arthritis Res Ther 2005;7:208–14. 18. Starkebaum G. Chronic neutropenia associated with autoimmune disease. Semin Hematol 2002;39:121–7. 19. Balint GP, Balint PV. Felty’s syndrome. Best Pract Res Clin Rheumatol 2004;18:631–45. 20. Mohan SJ, Maciejewski JP. Diagnosis and therapy of neutropenia in large granular lymphocyte leukemia. Curr Opin Hematol 2009;16:27–34. 21. Lamy T, Loughran TP. How I treat LGL leukemia. Blood 2011;117:2764–74. 22. Tesfa D, Keisu M, Palmblad J. Idiosyncratic drug-induced agranulocytosis: Possible mechanisms and management. Am J Hematol 2009;84:428–34. 23. Grant C, Wilson WH, Dunleavy K. Neutropenia associated with rituximab therapy. Curr Opin Hematol 2010;18:49–54. 24. Bux J. Molecular nature of granulocyte antigens. Transfus Clin Biol 2001;8:242–7. 25. Bux J, Chapman J. Report on the second international granulocyte serology workshop. Transfusion 1997;37:977–83. 26. Bruin MC, von dem Borne AE, Tamminga RY, et al. Neutrophil antibody specificity in different types of childhood autoimmune neutropenia. Blood 1999;94:1797–802. 27. Bux J, Behrens G, Jaeger G, et al. Diagnosis and clinical course of autoimmune neutropenia in infancy: analysis of 240 cases. Blood 1998;91:181–6. 28. Kobayashi M, Nakamura K, Kawaguchi H, et al. Significance of the detection of anti-neutrophil antibodies in children with chronic neutropenia. Blood 2002;99:3468–71. 29. McCullough J, Weibler BJ, Clay M, et al. Effect of leukocyte antibodies on the fate in vivo of indium-111-labelled granulocytes. Blood 1981;58:164–70. 30. Morice WG, Kurtin PJ, Tefferi A, et al. Distinct bone marrow findings in T-cell granular lymphocytic leukemia revealed by paraffin section immunoperoxidase stains for CD8. TIA-1, and granzyme B. Blood 2002;99:268–74. 31. Cines DB, Bussel JB, Liebman HA, et al. The ITP syndrome: pathogenic and clinical diversity. Blood 2009;113:6511–21. 32. Rodeghiero F, Stasi R, Gernsheimer T, et al. Standardization of terminology, definitions of outcome criteria in immune thrombocytopenic purport of adults and children: report from an international working group. Blood 2009;113:2386–93. 33. Stavrou E, McCrea KR. Immune thrombocytopenia in pregnancy. Hematol Oncol Clin North Am 2009;23:1299–316.

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Part Seven  Organ-Specific Inflammatory Disease

34. Husebekk A, Killie MK, Kjeldsen-Kragh J, et al. Is it time to implement HPA-1 screening in pregnancy? Curr Opin Hematol 2009;16:497–502. 35. Vinograd CA, Bussel JB. Antenatal treatment of fetal alloimmune thrombocytopenia: a current perspective. Haematologica 2010;95:1807–11. 36. Kiefel V, Konig C, Kroll H, et al. Platelet antibodies in transfused patients. Transfusion 2001;41:766–70. 37. George JN, Aster RH. Drug-induced thrombocytopenia: pathogenesis, evaluation, and management. Hematology Am Soc Hematol Educ Program 2009;153–8. 38. Aster RH, Curtis BR, McFarland JG, et al. Drug-induced immune thrombocytopenia: pathogenesis, diagnosis, and management. J Thromb Haemost 2009;7:911–18. 39. Otis SA, Zehnder JL. Heparin-induced thrombocytopenia: current status and diagnostic challenges. Am J Hematol 2010;85:700–6. 40. Takahashi T, Yujiri T, Shinohara K, et al. Molecular mimicry by Helicobacter pylori CagA protein may be involved in the pathogenesis of H. pylori-associated chronic idiopathic thrombocytopenic purpura. Br J Haematol 2004;124:91–6. 41. Arnold DM, Bernotas A, Nazi I, et al. Platelet count response to H. pylori treatment in patients with immune thrombocytopenic purpura with and without H. pylori infection: a systematic review. Haematologica 2009;94:850–6. 42. Burrows RF, Kelton JG. Pregnancy in patients with idiopathic thrombocytopenic purpura: assessing the risks for the infant at delivery. Obstet Gynecol Surv 1993;48:781–8. 43. West KA, Anderson DR, McAlister VC, et al. Alloimmune thrombocytopenia after organ transplantation. N Engl J Med 1999;341:1504–7. 44. McMillan R. Antiplatelet antibodies in chronic adult immune thrombocytopenic purpura: assays and epitopes. J Pediatr Hematol Oncol 2003;25:S57–61.

45. Olson B, Anderson PO, Jernas M, et al. T-cell mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura. Nat Med 2003;9:1123–4. 46. Panitsas FP, Theodoropoulou M, Kouraklis A, et al. Adult chronic idiopathic thrombocytopenic purpura (ITP) is the manifestation of a type-1 polarized immune response. Blood 2004;103:2645–7. 47. Yu J, Heck S, Patel V, et al. Defective circulating CD25 regulatory T cells in patients with chronic immune thrombocytopenic purpura. Blood 2008;112:1325–8. 48. Emmerich F, Bal G, Barakat A, et al. High-level serum B-cell activating factor and promoter polymorphisms in patients with idiopathic thrombocytopenic purport. Br J Haematol 2007;136:309–14. 49. Olson B, Ridell B, Carlsson L, et al. Recruitment of T cells in the bone marrow of ITP patients possibly due to elevated expression of VLA-4 and CX3CR1. Blood 2008;112:1078–84. 50. McMillan R, Wang L, Tomer A, et al. Suppression of in vitro megakaryocytic production by antiplatelet autoantibodies from adult patients with chronic ITP. Blood 2004;103:1364–9. 51. Aledort LM, Hayward CP, Chen MG, et al. Prospective screening of 205 patients with ITP, including diagnosis, serological markers, and the relationship between platelet counts, endogenous thrombopoietin, and circulating anti-thrombopoietin antibodies. Am J Hematol 2004;76:205–13. 52. Provan D, Stasi R, Newland AC, et al. International consensus report on the investigation and management of primary immune thrombocytopenia. Blood 2010;115:168–86. 53. Vesely SK, Perdue JJ, Rizvi MA, et al. Management of adult patients with persistent idiopathic thrombocytopenic purpura following splenectomy: a systematic review. Ann Intern Med 2004;140:112–20. 54. Yu X, Menard L, Prechl J, et al. Monovalent Fc receptor blockade by an anti–Fcγ receptor/albumin fusion protein ameliorates murine ITP with abrogated toxicity. Blood 2016;127:132–8.

CHAPTER 62  Immunohematological Disorders

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MULTIPLE-CHOICE QUESTIONS 1. Which of the following treatments is LEAST likely to improve platelet counts in an patient with type A- blood and immune thrombocytopenic purpura (ITP)? a. Rituximab 375 mg/m2 IV b. Prednisone 1 mg/kg PO c. WinRho 250 IU/kg (50 micrograms/kg) IV d. IVIG, 2 g/kg continuous IV infusion over 48 hours 2. Which is true of hemolysis caused by a “cold” antibody elicited by exposure to drugs? a. Usually caused by immunoglobulin G (IgG) antibody against Rh antigens b. Usually accompanied by a direct antibody test (DAT) (Coombs test) that is positive for IgM and IgG and negative for complement c. Usually accompanied by a DAT (Coombs test) that is negative for IgM and IgG and positive for complement d. Usually caused by a Donath-Landsteiner IgG against Rh antigen on the surface of the red blood cell (RBC)

3. Hemolytic anemia of the newborn: a. Usually occurs when the mother of the infant is Rh+ and the father is Rh− b. Is more likely in a first pregnancy than subsequent pregnancies between the same two parents c. May be prevented by administration of RhoGam at week 28 of pregnancy when the parents are Rh incompatible d. Is more common if the parents are ABO incompatible 4. Neutropenia associated with administration of rituximab: a. Is usually self-limiting b. Precludes future use of rituximab c. Typically occurs 7–10 days after administration of rituximab d. Does not respond to administration of granulocyte–colonystimulating factor (G-CSF)