1 Thalassemia, IDA, Megalo, Hemolytic, MPN PDF

disease. However, the complexity and cost of modifying enough stem can be isolated. Powerful transforming gene constructs were placed 747 cells for transplantation is problematic. Expanding a small number of in these cells, and it was found that the cell with the greatest p...

disease. However, the complexity and cost of modifying enough stem can be isolated. Powerful transforming gene constructs were placed 747 cells for transplantation is problematic. Expanding a small number of in these cells, and it was found that the cell with the greatest potential gene-modified stem cells may mitigate that issue. Therefore, under- to produce a malignancy was dependent on the transforming gene. In standing self-renewal offers the potential to facilitate development some cases, it was the stem cell, but in others, the progenitor cell func- of an important new area of stem cell–based medicine. Some limited tioned to initiate and perpetuate the cancer. This shows that cells can understanding of self-renewal exists and, intriguingly, implicates gene acquire stem cell–like properties in malignancy. products that are associated with the chromatin state, a high-order organization of chromosomal DNA that influences transcription. WHAT ELSE CAN HEMATOPOIETIC STEM These include members of the polycomb family, a group of zinc finger– CELLS DO? containing transcriptional regulators that interact with the chromatin Some experimental data have suggested that hematopoietic stem cells structure, contributing to the accessibility of groups of genes for tran- or other bone marrow cells are capable of playing a role in healing scription. One member, Bmi-1, is important in enabling hematopoietic the vascular and tissue damage associated with stroke and myocardial stem cell self-renewal through modification of cell cycle regulators such infarction. These data are controversial, and the applicability of a stem as the cyclin-dependent kinase inhibitors. In the absence of Bmi-1 or cell approach to nonhematopoietic conditions remains experimental. of the transcriptional regulator, Gfi-1, hematopoietic stem cells decline However, reprogramming technology offers the potential for using in number and function. In contrast, dysregulation of Bmi-1 has been readily obtained hematopoietic cells as a source for cells with other associated with leukemia; it may promote leukemic stem cell self- capabilities. Active areas of investigation are to use reprogrammed cells renewal when it is overexpressed. The same is true for the polycomb to generate mature lymphoid cells for immuno-oncology applications gene, Asxl1, that is commonly mutated in myelodysplasia and leukemia. or red cells and platelets to overcome dependency on blood donors. Other transcription regulators have also been associated with self- renewal, particularly homeobox, or “hox,” genes. These transcription STEM CELLS AS TARGETS OF GENE factors are named for their ability to govern large numbers of genes, THERAPY CHAPTER 97 Iron Deficiency and Other Hypoproliferative Anemias including those determining body patterning in invertebrates. HoxB4 Tools to alter gene sequence, expression, and regulation are becoming is capable of inducing extensive self-renewal of stem cells through its increasingly feasible. The hematopoietic stem cell is a target for a wide DNA-binding motif. Other members of the hox family of genes have range of interventions. Lentiviral, retroviral, and adenoviral vectors been noted to affect normal stem cells, but they are also associated with are being used to replace defective genes (e.g., in primary immuno- leukemia. Epigenetic modifiers such as the DNA methyl transferase deficiency diseases). Antisense technology is being applied to block DNMT3a or the dioxygenase involved in DNA demethylation, Tet2, gene expression (e.g., blocking the Bcl11a repression of fetal globin also play a role in stem cell regulation. Like Asxl1, mutations of these expression in sickle cell disease and thalassemia). CRISPR/Cas tech- genes are associated with clonal outgrowth of stem cells bearing the nology is being applied to repair abnormal gene sequences. Precision mutations. These mutations are not sufficient for malignancy, but they genetic manipulations are expanding, and the hematopoietic system is enable clones bearing them to gain dominance and predispose cells to central to it. malignant transformation. They are often referred to as “founder muta- In sum, the stem cell has tremendous healing capacity and is essential tions” because myelodysplastic and leukemic cells appear to evolve from for life. However, if dysregulated, it can threaten the life it maintains. them by DNA sequencing analysis. Understanding how stem cells function, the signals that modify their behavior, and the tissue niches that modulate stem cell responses to CANCER IS SIMILAR TO AN ORGAN WITH injury and disease is critical for more effectively developing stem cell– SELF-RENEWING CAPACITY based medicines. The relationship of stem cells to cancer is an important dimension of adult stem cell biology. Cancer may share principles of organization FURTHER READING with normal tissues. Cancer cells are heterogeneous even within a given Adelman ER, Figueroa ME: Human hematopoiesis: Aging and leu- patient and may have a hierarchical organization of cells with a base of kemogenic risk. Curr Opin Hematol 1:57, 2021. stem-like cells capable of the signature stem cell features: self-renewal Baryawno N et al: A cellular taxonomy of the bone marrow stroma in and differentiation. These stem-like cells might be the basis for perpet- homeostasis and leukemia. Cell 7:1915, 2019. uation of the tumor and represent a slowly dividing, rare population Ito Y et al: Turbulence activates platelet biogenesis to enable clinical with distinct regulatory mechanisms, including a relationship with a scale ex vivo production. Cell 3:636, 2018. specialized microenvironment. A subpopulation of self-renewing cells Rodriguez-Fraticelli AE, Camargo F: Systems analysis of hemato- has been defined for some, but not all, cancers. These include myeloid poiesis using single-cell lineage tracing. Curr Opin Hematol 1:18, leukemias where founder mutations appear to enable clones of cells to 2021. expand. With additional mutations, these can serve as the initiating or stem cells of a cancer, and eliminating them may be necessary for curing the patient. Understanding the hierarchical cell organization within cancers and whether eliminating cancer stem cell equivalents can improve cure rates is an area of active investigation. Does the concept of cancer stem cells provide insight into the cellu- lar origin of cancer? The fact that some cells within a cancer have stem cell–like properties does not necessarily mean that the cancer arose in the stem cell itself. Rather, more mature cells could have acquired the self-renewal characteristics of stem cells. Any single genetic event is 97 Iron Deficiency and Other Hypoproliferative Anemias unlikely to be sufficient to enable full transformation of a normal cell to a frankly malignant one. Rather, cancer is a multistep process, and John W. Adamson for the multiple steps to accumulate, the cell of origin must be able to persist for prolonged periods. It must also be able to generate large numbers of daughter cells. The normal stem cell has these properties Anemias associated with normocytic and normochromic red cells and, by virtue of its having intrinsic self-renewal capability, may be and an inappropriately low reticulocyte response (reticulocyte index more readily converted to a malignant phenotype. This hypothesis has 1.5 × 109/L; Cobalamin deficiency is usually due to malabsorption. The only other the platelet count may be moderately reduced, rarely to 350 ng/L), or cobalamin, is the cause of the anemia and to treat only with the have this pattern of raised metabolite levels. These findings bring into appropriate vitamin. In patients who enter the hospital severely ill, question the exact cutoff points for normal MMA and homocysteine however, it may be necessary to treat with both vitamins in large levels. It is also unclear at present whether these mildly raised metabo- doses once blood samples have been taken for cobalamin and folate lite levels have clinical consequences. assays and a bone marrow biopsy has been performed (if deemed Serum homocysteine is raised in both early cobalamin and folate necessary). Transfusion is usually unnecessary and inadvisable. If it deficiency but may be raised in other conditions, for example, chronic is essential, packed red cells should be given slowly, one or two units renal disease, alcoholism, smoking, pyridoxine deficiency, hypothy- only, with the usual treatment for heart failure if present. Potassium roidism, and therapy with steroids, cyclosporine, and other drugs. supplements have been recommended to obviate the danger of the Levels are also higher in serum than in plasma, in men than in pre- hypokalemia but are not necessary. Occasionally, an excessive rise menopausal women, in women taking hormone replacement therapy in platelets occurs after 1–2 weeks of therapy. Antiplatelet therapy, or in oral contraceptive users, and in elderly persons and patients for example, aspirin, should be considered if the platelet count rises with several inborn errors of metabolism affecting enzymes in trans- to >800 × 109/L. sulfuration pathways of homocysteine metabolism. Thus, homo- COBALAMIN DEFICIENCY cysteine levels must be carefully interpreted for diagnosis of cobalamin or folate deficiency. It is usually necessary to treat patients who have developed cobal- amin deficiency with lifelong regular cobalamin injections. In the Tests for the Cause of Cobalamin Deficiency Only vegans, UK, the form used is hydroxocobalamin; in the United States, strict vegetarians, or people living on a totally inadequate diet will cyanocobalamin. In a few instances, the underlying cause of cobal- become vitamin B12 deficient because of inadequate intake. Studies of amin deficiency can be permanently corrected, for example, fish cobalamin absorption once were widely used, but difficulty in obtain- tapeworm, tropical sprue, or an intestinal stagnant loop that is ing radioactive cobalamin and ensuring that IF preparations are free amenable to surgery. The indications for starting cobalamin therapy of viruses has made these tests obsolete. Tests to diagnose PA include are a well-documented megaloblastic anemia or other hematologic serum gastrin, which is raised; serum pepsinogen I, which is low in PA abnormalities and neuropathy due to the deficiency. Patients with (90–92%) but also in other conditions; and gastric endoscopy. Tests for borderline serum cobalamin levels but no hematologic or other IF and parietal cell antibodies are also used, as well as tests for individ- abnormality may be followed to make sure that the cobalamin defi- ual intestinal diseases. ciency does not progress (see below). If malabsorption of cobalamin Patients with atrophic gastritis may also have sufficient occult gas- or rises in serum MMA levels have been demonstrated, however, trointestinal blood loss to have iron deficiency as well as vitamin B12 these patients also should be given regular maintenance cobalamin therapy. Cobalamin should be given routinely to all patients who in the diet after correcting the deficiency with a short course of 775 have had a total gastrectomy or ileal resection. Patients who have folic acid. In any patient receiving long-term folic acid therapy, it undergone gastric reduction for control of obesity or who are is important to measure the serum cobalamin level at regular (e.g., receiving long-term treatment with proton pump inhibitors should once-yearly) intervals to exclude the coincidental development of be screened and, if necessary, given cobalamin replacement. cobalamin deficiency. Replenishment of body stores should be complete with six 1000-μg IM injections of hydroxocobalamin given at 3- to 7-day Folinic Acid (5-Formyl-THF) This is a stable form of fully reduced intervals. More frequent doses are usually used in patients with folate. It is given orally or parenterally to overcome the toxic effects cobalamin neuropathy, but there is no evidence that they produce a of methotrexate or other DHF reductase inhibitors, for example, better response. Allergic reactions are rare and may require desensi- trimethoprim or cotrimoxazole. tization or antihistamine or glucocorticoid cover. For maintenance PROPHYLACTIC FOLIC ACID therapy, 1000 μg hydroxocobalamin IM once every 3 months is Prophylactic folic acid is used in chronic dialysis patients and in satisfactory. Because of the poorer retention of cyanocobalamin, parenteral feeds. Prophylactic folic acid has been used to reduce protocols generally use higher and more frequent doses, for exam- homocysteine levels to prevent cardiovascular disease and for cog- ple, 1000 μg IM, monthly, for maintenance treatment. nitive function in the elderly, but there are no firm data to show Because a small fraction of cobalamin can be absorbed passively any benefit. through mucous membranes even when there is complete failure of physiologic IF-dependent absorption, large daily oral doses Pregnancy In over 70 countries (but none in Europe), food is for- (1000–2000 μg) of cyanocobalamin are used in PA for replacement tified with folic acid (in grain or flour) to reduce the risk of NTDs. (especially in Canada and Sweden) and maintenance of normal Nevertheless, folic acid, 400 μg daily, should be given as a supplement cobalamin status in, for example, food malabsorption of cobalamin. before and throughout pregnancy to prevent megaloblastic anemia Sublingual therapy has also been proposed for those in whom injec- and reduce the incidence of NTDs, even in countries with fortifi- CHAPTER 99 Megaloblastic Anemias tions are difficult because of a bleeding tendency and who may not cation of the diet. The levels of fortification provide up to 400 μg tolerate oral therapy. If oral therapy is used, it is important to mon- daily on average in Chile, but in most countries, it is nearer to itor compliance, particularly with elderly, forgetful patients. This 200 μg, so periconceptual folic acid is still needed. Most if not all author prefers parenteral therapy for initial treatment, particularly the folic acid used in fortification and eaten over three meals a day in severe anemia or if a neuropathy is present, and for maintenance will be converted during absorption to methyltetrahydrofolate. This in PA. Oral B12 therapy even with low doses of 50 μg daily may have compound will not correct the anemia in B12 deficiency. Studies in a larger role in treating food malabsorption of B12. early pregnancy show significant lack of compliance with the folic For treatment of patients with subnormal serum vitamin B12 levels acid supplements, emphasizing the benefit of food fortification. with a normal MCV and no hypersegmentation of neutrophils, a Supplemental folic acid reduces the incidence of birth defects in negative IF antibody test in the absence of tests of B12 absorption is babies born to diabetic mothers. In women who have had a pre- problematic. Some (perhaps 15%) cases may be due to TC I (HC) vious fetus with an NTD, a dose of 5 mg daily is recommended deficiency. Homocysteine and/or MMA measurements may help, but when pregnancy is contemplated and throughout the subsequent in the absence of these tests and with otherwise normal gastroin- pregnancy. testinal function, repeat serum B12 assay after 6–12 months may Infancy and Childhood The incidence of folate deficiency is so help one decide whether to start cobalamin therapy. high in the smallest premature babies during the first 6 weeks of Vitamin B12 injections are used in a wide variety of diseases, often life that folic acid (e.g., 1 mg daily) should be given routinely to neurologic, despite normal serum B12 and folate levels and a nor- those weighing pyruvate kinase > glucose-6- ecules per red cell. As red cells age and become denser, probability is phosphate isomerase > rare deficiencies of other enzymes in the pathway. The increased that a region of the band 3 molecule becomes exposed on the more common enzyme deficiencies are encircled. cell surface and contributes to creating an antigenic site recognizable by low-avidity naturally occurring anti-band 3 IgG antibodies. This pro- cess might be enhanced by the clustering of band 3 molecules favored consequences, except for an increased requirement for erythropoietic by the antibody itself and by the binding of hemichromes arising from factors, particularly folic acid. However, if hemolysis is recurrent or hemoglobin degradation. Senescent red cells thus become opsonized, persistent, the increased bilirubin production favors the formation of and this is the signal for phagocytosis by macrophages in the spleen, gallstones. If a considerable proportion of hemolysis takes place in the in the liver, and elsewhere. This process may become accelerated in spleen, as is often the case, splenomegaly may become increasingly a various ways in HA. feature, and hypersplenism may develop, with consequent neutropenia Another consequence of the relative simplicity of red cells is that and/or thrombocytopenia. they have a limited range of ways to manifest distress under hardship; The increased red cell turnover has important consequences. In nor- in essence, any sort of metabolic failure will eventually lead either to mal subjects, the iron from effete red cells is very efficiently recycled by structural damage to the membrane or to failure of the cation pump. the body; however, with chronic intravascular hemolysis, the persistent In either case, the life span of the red cell is reduced, which is the defi- hemoglobinuria will cause considerable iron loss, needing replacement. nition of a hemolytic disorder. If the rate of red cell destruction exceeds With chronic extravascular hemolysis, the opposite problem, iron over- the capacity of the bone marrow to produce more red cells, the hemo- load, is more common, especially if the patient needs frequent blood lytic disorder will manifest as HA. transfusions. Even without blood transfusion, when erythropoiesis is Thus, the essential pathophysiologic process common to all HAs is massively increased, the release of erythroferrone from erythroid cells an increased red cell turnover; in many HAs, this is due at least in part suppresses hepcidin, causing increased iron absorption. In the long to an acceleration of the senescence process described above. The gold run, in the absence of iron-chelation therapy, iron overload will cause standard for proving that the life span of red cells is reduced (compared secondary hemochromatosis; this will cause damage particularly to the to the normal value of about 120 days) is a red cell survival study, which liver, eventually leading to cirrhosis; and to the heart muscle, eventually can be carried out by labeling the red cells with 51Cr and measuring the causing heart failure. fall in radioactivity over several days or weeks (this classic test can now be replaced by a methodology using the nonradioactive isotope 15N). If Compensated Hemolysis versus Hemolytic Anemia Red cell the hemolytic event is transient, it does not usually cause any long-term destruction is a potent stimulus for erythropoiesis, which is mediated  778 by erythropoietin (EPO) produced by the kidney. This mechanism is so proteins also extend to both the outside (extracellular domains) and effective that in many cases the increased output of red cells from the the inside of the cell (cytoplasmic domains). Other proteins are teth- bone marrow can fully balance an increased destruction of red cells. In ered to the membrane through a glycosylphosphatidylinositol (GPI) such cases, we say that hemolysis is compensated. The pathophysiology anchor; these have only an extracellular domain. Membrane proteins of compensated hemolysis is similar to what we have just described, include energy-dependent ion transporters, ion channels, receptors except there is no anemia. This notion is important from the diagnostic for complement components, and receptors for other ligands. The most point of view, because a patient with a hemolytic condition, even an abundant red cell membrane proteins are glycophorins and the so- inherited one, may present without anemia; and it is also important called band 3, an anion transporter that is an integral membrane pro- from the point of view of management because compensated hemo- tein. The extracellular domains of many of these proteins are heavily lysis may become “decompensated,” i.e., anemia may suddenly appear glycosylated, and they carry antigenic determinants that correspond in certain circumstances, for instance in pregnancy, folate deficiency, to blood groups. Underneath the membrane, and tangential to it, is or renal failure interfering with adequate EPO production. Another a network of other proteins that make up the cytoskeleton. The main general feature of chronic HAs is seen when any intercurrent condi- cytoskeletal protein is the spectrin tetramer, consisting of a head- tion, such as an acute infection, depresses erythropoiesis. When this to-head association of two α-spectrin-β-spectrin heterodimers. The happens, in view of the increased rate of red cell turnover, the effect cytoskeleton is linked to the membrane through the ankyrin complex will be predictably much more marked than in a person who does not (that includes also band 4.2) and the junctional complex (that includes have hemolysis. The most dramatic example is infection by parvovirus adducin and band 4.1) (Fig. 100-2). These multiprotein complexes B19, which may cause a rather precipitous fall in hemoglobin—an make membrane and cytoskeleton intimately connected to each other, occurrence sometimes referred to as aplastic crisis. thus supporting membrane stability and at the same time providing the erythrocyte with the important property of deformability. INHERITED HEMOLYTIC ANEMIAS The membrane-cytoskeleton complex has essentially three func- The red cell has three essential components: (1) hemoglobin, (2) the tions: It is an envelope for the red cell cytoplasm; it maintains the membrane-cytoskeleton complex, and (3) the metabolic machinery normal red cell shape; it provides cross-membrane transport of elec- PART 4 necessary to keep hemoglobin and the membrane-cytoskeleton com- trolytes and of metabolites such as glucose and amino acids. In the plex in working order. Diseases caused by inherited abnormalities of membrane-cytoskeleton complex, the individual components are so hemoglobin, or hemoglobinopathies, are covered in Chap. 98. Here we intimately associated with each other that an abnormality of almost will deal with diseases of the other two components. any of them will be disturbing or disruptive, causing mechanical instability of the membrane and/or reduced red cell deformability, Oncology and Hematology Hemolytic Anemias due to Abnormalities of the Membrane- ultimately causing hemolysis. These abnormalities are almost invari- Cytoskeleton Complex The detailed architecture of the red cell ably inherited mutations; thus diseases of the membrane-cytoskeleton membrane is complex, but its basic design is relatively simple (Fig. 100-2). complex belong to the category of inherited HAs. Before the red cells The lipid bilayer incorporates phospholipids and cholesterol, and lyse, they often exhibit more or less specific changes that alter the it is spanned by a number of proteins that have their hydrophobic normal biconcave disk shape. Thus, the majority of the diseases in this transmembrane domain(s) embedded in the membrane; most of these group have been known for over a century as hereditary spherocy- tosis (HS) and hereditary elliptocytosis (HE). More recently a third morphologic entity, whereby on a blood smear the RhAG round-shaped central pallor of a red cell AChE CD59 is replaced by a linear-shaped central pale area, has earned the name stomatocytosis: ABCB6 because this abnormal shape is related to Rh GLUT1 GPC abnormalities of channel molecules, the Band 3 Band 3 Band 3 underlying disorders are also referred CD47 PIEZO1 KCNN4 to as channelopathies. From an under- standing of the molecular basis of these 4.2 GPA α β p55 Adducin disorders, it has emerged (Table 100-3) that, although these disorders are pre- α-Spectrin Ankyrin β-Spectrin dominantly monogenic, no one-to-one 4.1R Dematin correlation exists between a certain gene and a certain disorder. Rather, what has been regarded as a single disorder (e.g., Tropomyosin Actin protofilament HS) can arise through mutation of one of Self-association site Tropomodulin several genes; conversely, what have been regarded as different disorders can arise through different mutations of the very Ankyrin complex Junctional complex same gene (Fig. 100-3). FIGURE 100-2 The red cell membrane and cytoskeleton. Within the membrane lipid bilayer several integral membrane proteins are shown: band 3 (anion exchanger 1 [AE1]) is the most abundant. PIEZO1 is a mechanoreceptor, KCNN4, a HEREDITARY SPHEROCYTOSIS This Ca2+ activated K+ channel, and ABCB6 is an ion channel: they are important in the regulation of the red cell volume. is most common among this group of Other proteins, e.g., acetylcholinesterase (AChE) and the two complement-regulatory proteins CD59 and CD55, are tethered to the membrane through the glycosylphosphatidylinositol (GPI) anchor: in these cases the entire polypeptide HAs, with an estimated prevalence of chain is extracellular. Many of the membrane proteins bear polypeptide and/or carbohydrate red cell antigens. 1:2000–1:5000 in populations of European Underneath the membrane, the α−β spectrin dimers, that associate head-to-head into tetramers, together with actin ancestry. Its identification is credited to and other proteins, form most of the cytoskeleton. The ankyrin complex, that also involves the band 4.2 protein, and Minkowksy and Chauffard, who, at the the junctional complex, that involves the band 4.1 protein and dematin, connect the membrane to the cytoskeleton. end of the nineteenth century, reported The ankyrin complex provides mainly radial (also called vertical) connections; the junctional complex provides mainly families who had spherocytes in their tangential (also called horizontal) connections: pathogenic changes in the former can cause spherocytosis, whereas pathogenic changes in the latter can cause elliptocytosis; pathogenic changes in spectrin can cause either. Branched peripheral blood (Fig. 100-4A). In vitro lines symbolize carbohydrate moiety of proteins. The various molecules are obviously not drawn to the same scale. studies revealed that the red cells were Additional explanations are found in the text. (Reproduced with permission from N Young et al: Clinical Hematology. abnormally susceptible to lysis in hypo- Philadelphia, Elsevier, 2006.) tonic media; indeed, the presence of  779 TABLE 100-3 Inherited Diseases of the Red Cell Membrane-Cytoskeleton Complex CHROMOSOMAL DISEASE(S) WITH CERTAIN GENE LOCATION PROTEIN PRODUCED MUTATIONS (INHERITANCE) COMMENTS SPTA1 1q22-q23 α-Spectrin HS (recessive) Rare HE (dominant) Mutations of this gene account for about 65% of HE. More severe forms may be due to coexistence of an otherwise silent mutant allele. SPTB 14q23-q24.1 β-Spectrin HS (dominant) Rare HE (dominant) Mutations of this gene account for about 30% of HE, including some severe forms. ANK1 8p11.2 Ankyrin HS (dominant) May account for majority of HS. SLC4A1 17q21 Band 3; also known as AE HS (dominant) Mutations of this gene may account for about 25% of HS. (anion exchanger) or AE1 Southeast Asia ovalocytosis Polymorphic mutation (deletion of nine amino acids); in heterozygotes (dominant) clinically asymptomatic and protective against Plasmodium falciparum. Stomatocytosis Certain specific missense mutations shift protein function from anion (cryohydrocytosis) exchanger to cation conductance. EPB41 1p33-p34.2 Band 4.1 HE (dominant) Mutations of this gene account for about 5% of HE, mostly with prominent morphology but little/no hemolysis in heterozygotes; severe hemolysis in homozygotes. EPB42 15q15-q21 Band 4.2 HS (recessive) Mutations of this gene account for about 3% of HS. RHAG 6p21.1-p11 Rhesus-associated Chronic nonspherocytic Very rare; associated with total loss of all Rh antigens. glycoprotein hemolytic anemia (recessive) CHAPTER 100 Hemolytic Anemias One specific mutation in this gene entails loss of stomatin from the cell membrane, causing overhydrated stomatocytosis. PIEZO1 16q23-q24 PIEZO1 Dehydrated hereditary Also known as xerocytosis with pseudohyperkalemia. Patients may (mechanosensitive ion stomatocytosis (dominant) present with perinatal edema. channel component 1 channel) KCNN4 19q13.31 KCNN4 Dehydrated hereditary Clinical presentation similar to that of PIEZO1 mutants. Intermediate stomatocytosis (dominant) conductance calcium- activated potassium channel protein 4 (Gardos channel) ABCB6 2q35-q36 ATP-binding cassette Familial pseudohyperkalemia Increased potassium leakage upon storage in blood bank condition: this subfamily B member 6 (dominant) can cause hyperkalemia in the recipient. ABCB6 mutation is present in 0.3% of blood donors. SLC2A1 1p34.2 GLUT1 glucose Overhydrated hereditary Associated with serious neurological manifestations. transporter stomatocytosis Note: PIEZO1, KCNN4, ABCB6, and GLUT1 are channel molecules; conditions associated with mutations in the respective genes are appropriately named channelopathies. Abbreviations: HE, hereditary elliptocytosis; HS, hereditary spherocytosis. osmotic fragility became the main diagnostic test for HS. Today we through a dual mechanism. On one hand, like in many other HAs, the know that HS, thus defined, is genetically heterogeneous; i.e., it can spleen itself is a major site of destruction; on the other hand, because arise from a variety of mutations in one of several genes (Table 100-3). the red cells in HS are less deformable, transit through the splenic It has been also recognized that the inheritance of HS is not always circulation makes them more prone to vesiculate, thus accelerating autosomal dominant (with the patient being heterozygous); indeed, their demise. some of the most severe forms are instead autosomal recessive (with When there is a family history, it is usually easy to make a diagnosis the patient being homozygous). based on features of HA and typical red cell morphology. However, Clinical Presentation and Diagnosis The spectrum of clinical severity of family history may be negative for at least two reasons. First, the HS is broad. Severe cases may present in infancy with severe anemia, patient may have a de novo mutation, i.e., a mutation that has taken whereas mild cases may present in young adults or even later in life. place in a germ cell of one of the patient’s parents or early after zygote The main clinical findings are jaundice, an enlarged spleen, and often formation. Second, the patient may have a recessive form of HS gallstones; indeed, it may be the finding of gallstones in a young person (Table 100-3). In such cases, more extensive laboratory investigations that triggers diagnostic investigations. are required, including osmotic fragility, the acid glycerol lysis test, the The variability in clinical manifestations that is observed among eosin-5’-maleimide (EMA)–binding test, and SDS-gel electrophoresis patients with HS is largely due to the different underlying molecular of membrane proteins; these tests are usually carried out in laboratories lesions (Table 100-3). Not only are mutations of several genes involved, with special expertise in this area. Sometimes a definitive diagnosis can even different mutations of the same gene can give very different clin- be obtained only by molecular studies demonstrating a mutation in one ical manifestations. In milder cases, hemolysis is often compensated of the genes underlying HS (Table 100-3). (see above), but changes in clinical expression may be seen even in the same patient because intercurrent conditions (e.g., pregnancy, infec- tion) may cause decompensation. The anemia is usually normocytic TREATMENT with the characteristic morphology that gives the disease its name. An Hereditary Spherocytosis increased mean corpuscular hemoglobin concentration (MCHC >34) and increased red cell distribution width (RDW >14%) associated with We do not have a causal treatment for HS; i.e., no way has yet been normal or slightly decreased MCV on an ordinary blood count report found to correct the basic defect in the membrane-cytoskeleton should raise the suspicion of HS. The spleen plays a key role in HS structure. Given the special role of the spleen in HS (see above),  780 ANK1 EPB42 HS SPTB SPTA1 EPB41 SLC4A1 HE SLC2A1 RHAG PIEZ01 HSt KCNN4 ABCB6 FIGURE 100-3 Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary stomatocytosis (HSt) are three morphologically distinct forms of congenital hemolytic anemia. It has emerged that each one can arise from mutation of one of several genes and that different mutations of the same gene can give one or another form. (See also Table 100-3.) Genes encoding membrane proteins are in PART 4 black; genes encoding cytoskeleton proteins are in green; genes encoding proteins in the junctional and ankyrin complexes are in purple. splenectomy is often beneficial. Current recommendations are to Oncology and Hematology proceed with splenectomy at the age of 4–6 years in severe cases, to delay splenectomy until puberty in moderate cases, and to avoid splenectomy in mild cases. Partial splenectomy can be considered in certain cases; and it is helpful to know about the outcome of splenectomy in the patient’s affected relatives. Before splenectomy, vaccination against encapsulated bacteria (Neisseria meningitidis and Streptococcus pneumonia) is imperative; penicillin prophylaxis after splenectomy is controversial. Along with splenectomy, chole- cystectomy should not be carried out automatically; but it should be carried out, usually by the laparoscopic approach, whenever it is clinically indicated. HEREDITARY ELLIPTOCYTOSIS HE is at least as heterogeneous as HS, both from the genetic point of view (Table 100-3, Fig. 100-3) and from the clinical point of view. The global incidence of HE is 1:2000–4000 subjects. Again, it is the shape of the red cells (Fig. 100-4B) that gives the name to the condition, but there is no direct correlation between the elliptocytic morphology and clinical severity. In fact, some mild or even asymptomatic cases may have nearly 100% elliptocytes (or oval- ocytes). Indeed, the diagnosis of HE is generally incidental, because hemolysis may be compensated and there may be no anemia, although this may become evident in the course of infection. One particular in-frame deletion of nine amino acids in the SLC4A1 gene encoding band 3 underlies the so-called Southeast Asia ovalocytosis (SAO): it is not a disease, but rather a polymorphism with a frequency of up to 5–7% in certain populations (e.g., Papua New Guinea, Indonesia, Malaysia, Philippines), presumably as a result of malaria selection; it is asymptomatic in heterozygotes and probably lethal in homozygotes. The cases of HE with the most severe HA are those with biallelic mutations of one of the genes involved (see Fig. 100-3), and these are said to have pyropoikilocytosis (HPP): here the instability of the cytoskeleton protein network may result from decreased tetrameriza- tion of spectrin dimers. The red cell volume is decreased (MCV: 50–60 fL), and all kinds of bizarre poikilocytes are seen on the blood smear FIGURE 100-4 Peripheral blood smear from patients with membrane-cytoskeleton (Fig. 100-4C). HPP patients have splenomegaly and often benefit from abnormalities. A. Hereditary spherocytosis. B. Hereditary elliptocytosis, heterozygote. splenectomy. C. Pyropoikilocytosis, with both alleles of the α-spectrin gene mutated. Channelopathies These rare conditions (see Fig. 100-3) are char- acterized by abnormalities in red cell ion content and alteration of ery- dehydrated stomatocytosis (DHS; also referred to as xerocytosis) is a throcyte volume. Cation leak can cause hyperkalemia; in some cases, (usually compensated) macrocytic hemolytic disorder, with increased this leak is accelerated in the cold (the resulting spuriously high serum MCHC (generally higher than 36 g/dL) associated with mild jaundice. K+ is then referred to as pseudo-hyperkalemia). The less rare form, Mutations in either PIEZO1, encoding an ion channel activated by pressure (mechanoreceptor), or in KCCN4, encoding the Ca2+ activated functions: (1) to provide energy in the form of ATP, and (2) to prevent 781 K+ channel (Gardos channel) have been recognized to cause DHS (see oxidative damage to hemoglobin and to other proteins by providing Table 100-3). sufficient reductive potential; the key molecule for this is NADPH. Another form is overhydrated stomatocytosis (OHS): this too is ABNORMALITIES OF THE GLYCOLYTIC PATHWAY Because red cells, macrocytic (MCV >110 fL), but the MCHC is low (3 g/d) ally with a history of NNJ, who may present with anemia, unexplained Phenazopyridine Acetaminophen jaundice, or gallstones later in life. The spleen may be enlarged. The Phenacetin severity of anemia ranges in different patients from borderline to Other Rasburicase Vitamin K analogues Doxorubicin transfusion dependent. The anemia is usually normo-macrocytic, with reticulocytosis. Bilirubin and LDH are increased. Although hemolysis Naphthalene is, by definition, chronic in these patients, they are also vulnerable to Methylene blue Ascorbic acid (>1 g) Probenecid acute oxidative damage, and therefore the same agents that can cause a Marketed as Lapdap from 2003 to 2008. AHA in people with the ordinary type of G6PD deficiency will cause severe exacerbations in people with CNSHA associated with G6PD but a highly distinctive feature of this condition is a morphologic 785 deficiency. In some cases of CNSHA, the deficiency of G6PD is so abnormality of the red cells known as basophilic stippling. The con- severe in granulocytes that it limits their capacity to produce an oxida- dition is rare, but it probably ranks third in frequency among red cell tive burst, with consequent increased susceptibility to some bacterial enzyme defects (after G6PD deficiency and PK deficiency). The ane- infections. mia is lifelong, of variable severity, and may benefit from splenectomy. Laboratory Diagnosis The suspicion of G6PD deficiency can be con- Familial (Atypical) Hemolytic-Uremic Syndrome (aHUS) firmed by semiquantitative methods often referred to as screening This term is used to designate a group of rare disorders, mostly affect- tests, which are suitable for population studies and can correctly clas- ing children, characterized by microangiopathic HA with presence of sify male subjects, in the steady state, as G6PD normal or G6PD defi- fragmented erythrocytes in the peripheral blood smear, thrombocy- cient. However, in clinical practice, a diagnostic test is usually needed topenia (usually mild), and acute renal failure. (The word atypical in when the patient has had a hemolytic attack: whereby the oldest, most this phrase should be consigned to history: it was introduced originally G6PD-deficient red cells have been selectively destroyed, and young to distinguish this condition from the hemolytic-uremic syndrome red cells, having higher G6PD activity, are being released into the [HUS] caused by infection with Escherichia coli producing the Shiga circulation. Under these conditions, only a quantitative test can give toxin, regarded as typical.) The genetic basis of atypical HUS (aHUS) a definitive result. In males, this test will identify normal hemizygotes has been elucidated. Studies of >100 families have revealed that those and G6PD-deficient hemizygotes; among females, some heterozygotes family members who developed HUS had mutations in any one of will be missed, but those who are at most risk of hemolysis will be iden- several genes encoding complement regulatory proteins: complement tified. Of course, G6PD deficiency also can be diagnosed by DNA test- factor H (CFH), CD46 or membrane cofactor protein (MCP), comple- ing. Currently easy-to-use “point of care” tests for G6PD deficiency are ment factor I (CFI), complement component C3, complement factor B becoming available, geared especially to the prospect of mass adminis- (CFB), thrombomodulin, and others. Thus, whereas all other inherited tration of PQ or of the newly introduced derivative tafenoquine. HAs are due to intrinsic red cell abnormalities, this group is unique CHAPTER 100 Hemolytic Anemias in that hemolysis results from an inherited defect external to red cells TREATMENT (Table 100-1). Because the regulation of the complement cascade has considerable redundancy, in the steady state any of the above abnor- G6PD Deficiency malities can be tolerated. However, when an intercurrent infection or The AHA of G6PD deficiency is largely preventable by avoiding some other trigger briskly activates complement the deficiency of one exposure to triggering factors of previously screened subjects. Of of the complement regulators becomes critical. Endothelial cells get course, the practicability and cost-effectiveness of screening depend damaged, especially in the kidney; at the same time, and partly as a on the prevalence of G6PD deficiency in each individual commu- result of this, there will be brisk hemolysis (thus, the more common nity. Favism is entirely preventable in G6PD-deficient subjects by Shiga toxin–related HUS (Chap. 166) can be regarded as a pheno- not eating fava beans. Drug-induced hemolysis can be prevented by copy of aHUS). aHUS is a severe disease, with up to 15% mortality testing for G6PD deficiency before prescribing; in many cases one in the acute phase and up to 50% of cases progressing to end-stage can use alternative drugs. When AHA develops and once its cause is renal disease (ESRD). Not infrequently, aHUS undergoes spontaneous recognized, no specific treatment is needed in most cases. However, remission. Because it is an inherited abnormality, it is not surprising if the anemia is severe, it may be a medical emergency, especially in that, given renewed exposure to a trigger, the syndrome will tend to children, requiring immediate action, including blood transfusion. recur; when it does, the prognosis is always serious. The traditional This has been the case with an antimalarial drug combination treatment has been plasma exchange, which will supply the deficient containing dapsone (called Lapdap, introduced in 2003) that has complement regulator. This has changed since the introduction of the caused severe acute hemolytic episodes in children with malaria anti-C5 complement inhibitor eculizumab (see “Paroxysmal Nocturnal in several African countries; after a few years, the drug was taken Hemoglobinuria”) was found to greatly ameliorate the microangio- off the market. If there is acute renal failure, hemodialysis may be pathic picture, with improvement in platelet counts and in renal func- necessary, but if there is no previous kidney disease, recovery is the tion, thus abrogating the need for plasma exchange, which is not always rule. The management of NNJ associated with G6PD deficiency is effective and not free of complications. Because the basis of aHUS is no different from that of NNJ due to other causes. genetic, and relapses are always possible even after complete remission, In cases with CNSHA, if the anemia is not severe, regular folic there is a rationale for continuing eculizumab indefinitely, especially in acid supplements and regular hematologic surveillance will suffice. order to prevent ESRD. Patients who relapsed after discontinuing ecu- It will be important to avoid exposure to potentially hemolytic lizumab have responded again. Discontinuation of eculizumab might drugs, and blood transfusion may be indicated when exacerbations be reasonable especially in patients heterozygous for a MCP mutation. occur, mostly in concomitance with intercurrent infection. In rare However, there is no evidence base at the moment for balancing the patients, regular blood transfusions may be required, in which case pros and cons of lifetime eculizumab (a very expensive drug). appropriate iron chelation should be instituted. Unlike in HS, there ACQUIRED HEMOLYTIC ANEMIA is no evidence of selective red cell destruction in the spleen; how- ever, in practice, splenectomy has proven beneficial in severe cases. Mechanical Destruction of Red Cells Although red cells are characterized by the remarkable deformability that enables them to squeeze through capillaries narrower than themselves for thousands of Other Abnormalities of the Redox System As mentioned previously, GSH times in their lifetime, there are at least two situations in which they is a key player in the defense against oxidative stress. Inherited defects succumb to shear, if not to wear and tear; the result is intravascular of GSH metabolism are exceedingly rare, but each one can give rise hemolysis, resulting in hemoglobinuria (Table 100-6). One situation to chronic HA (Table 100-4). A rare, peculiar, and severe but usually is acute and self-inflicted, march hemoglobinuria. Why sometimes a self-limited HA occurring in the first month of life, called infantile marathon runner may develop this complication, whereas on another poikilocytosis, may be associated with deficiency of glutathione perox- occasion, this does not happen, we do not know (perhaps her or his idase (GSHPX) due not to an inherited abnormality, but to transient footwear needs attention). A similar syndrome may develop after pro- nutritional deficiency of selenium, an element essential for the activity longed barefoot ritual dancing or intense playing of bongo drums. The of GSHPX. other situation is chronic and iatrogenic (it has been called microan- PYRIMIDINE 5’-NUCLEOTIDASE (P5N) DEFICIENCY P5N is a key giopathic hemolytic anemia). It takes place in patients with prosthetic enzyme in the catabolism of nucleotides arising from the degradation heart valves, especially when paraprosthetic regurgitation is present. of nucleic acids that takes place in the final stages of erythroid cell mat- If the hemolysis consequent on mechanical trauma to the red cells uration. How exactly its deficiency causes HA is not well understood, is mild, and if the supply of iron is adequate, the loss may be largely  786 TABLE 100-6 Diseases and Clinical Situations in Which Hemolysis Is Largely Intravascular ONSET/TIME APPROPRIATE COURSE MAIN MECHANISM DIAGNOSTIC PROCEDURE COMMENTS Mismatched blood Abrupt Nearly always ABO Repeat cross-match transfusion incompatibility Paroxysmal nocturnal Chronic with acute Complement (C)-mediated Flow cytometry to display a Exacerbations due to C activation through any hemoglobinuria (PNH) exacerbations destruction of CD59(−) red cells CD59(−) red cell population pathway Paroxysmal cold Acute Immune lysis of normal red Test for Donath-Landsteiner Often triggered by viral infection hemoglobinuria (PCH) cells antibody Septicemia Very acute Exotoxins produced by Blood cultures Other organisms may be responsible Clostridium perfringens Microangiopathic Acute or chronic Red cell fragmentation Red cell morphology on Different causes ranging from endothelial damage to blood smear hemangioma to leaky prosthetic heart valve March hemoglobinuria Abrupt Mechanical destruction Targeted history taking Has been reported after extreme ritual dancing Favism Acute Destruction of older fraction of G6PD assay Triggered by ingestion of large dish of fava beansa G6PD-deficient red cells a The trigger of acute hemolytic anemia, often with hemoglobinuria, can be infection or a drug (see Table 100-5) rather than fava beans. Hemoglobinuria may or may not be reported by patient; but it is often macroscopic, i.e., recognizable by simple inspection of urine. Abbreviation: G6PD, glucose 6-phosphate dehydrogenase. compensated; if more than mild anemia develops, reintervention to anemia (Hb 1 cm in length) is superior for determination of cellularity and shows ASXL1 occur in both diseases. mainly fat under the microscope, with hematopoietic cells occupying