Articles on Medical Diseases and Conditions

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  • Red Blood Cell Membrane Abnormalities

    The major conditions in this category are congenital spherocytosis and hereditary elliptocytosis. Also included in this group are the uncommon condition abetalipoproteinemia and the extremely rare hereditary stomatocytosis.

    Congenital spherocytosis

    Congenital spherocytosis is one of the more common hereditary hemolytic anemias after the hemoglobinopathies and G-6-PD deficiency. Most patients are English or northern European. About 75% of cases manifest an autosomal dominant inheritance pattern, with about 25% apparently being sporadic but in most cases actually having a recessive inheritance pattern.

    The basic RBC defect is partial deficiency of a protein called spectrin that forms an important part of the RBC cell membrane cytoskeleton. Patients with the autosomal dominant form are said to have 60%-80% of normal spectrin levels; patients with the recessive form have 30%-70% of normal levels.

    Symptoms may develop at any time. Splenomegaly is found in approximately 50% of young children with the disease and in about 80% of older children and adults (literature range 72%-95%). About 50% develop jaundice, which is usually intermittent. Jaundice occurs in a substantial minority of patients in the first 48 hours of life but occasionally may appear after the first week. Gallstones develop in 55%-75% of patients by the time of old age and even during childhood in a few cases.

    Hematologic findings. Some degree of ongoing hemolysis is present in more than 90% of cases. However, about 50%-60% of patients are able to compensate by continued bone marrow hyperactivity and do not manifest anemia except during crises. When anemia is present, it is usually mild or moderate, and hemoglobin values are normally more than 8 gm/100 ml. Patients who are symptomatic and thus are diagnosed during childhood tend to have more pronounced anemia than those diagnosed in adulthood. Reticulocyte counts are elevated in approximately 90% of patients, with a mean count of approximately 9%. The MCV and MCH values are within reference range in about 80% of cases; in the remaining 20% these values may be increased or decreased. The MCHC is also more often within reference range, but 20%-50% of affected persons may have an increased value, and an increased MCHC is a finding that suggests the possibility of congenital spherocytosis. Peripheral blood spherocytes are the trademark of congenital spherocytosis. The number of spherocytes varies and in 20%-25% of cases are few in number and frequently not recognized. Spherocytes are not specific for congenital spherocytosis and may be found in ABO transfusion reactions as well as in some patients with widespread malignancy, Clostridium welchii septicemia, severe burns, some autoimmune hemolytic anemias, and after transfusion with relatively old stored blood.

    Patients with congenital spherocytosis may experience two different types of crises, which are self-limited episodes in which the anemia becomes significantly worse. The most common type is an increased degree of hemolysis (hemolytic crisis), which is frequently associated with viral infections and in which the decrease in hemoglobin level is usually not severe. The other type is the aplastic crisis, sometimes accompanied by fever and abdominal pain, lasting 6-14 days, in which bone marrow production of WBCs, RBCs, and platelets comes to a halt and during which the hemoglobin level drops to nearly one half previous values. Folic acid deficiency leading to megaloblastic anemia has also been described in congenital spherocytosis.

    The spherocytes are not destroyed in the bloodstream but are sequestered, removed, and destroyed in the spleen. Splenomegaly usually is present. Splenectomy satisfactorily cures the patient’s symptoms because increased marrow RBC production can then compensate for the presence of spherocytes, which have a shorter life span than normal RBCs.

    Diagnosis of congenital spherocytosis. The most useful diagnostic test in congenital spherocytosis is the osmotic fragility test. Red blood cells are placed in bottles containing decreasing concentrations of sodium chloride (NaCl). When the concentration becomes too dilute, normal RBCs begin to hemolyze. Spherocytes are more susceptible to hemolysis in hypotonic saline than normal RBCs, so that spherocytes begin hemolyzing at concentrations above normal range. This occurs when there are significant degrees of spherocytosis from any cause, not just congenital spherocytosis. Incidentally, target cells are resistant to hemolysis in hypotonic saline and begin to hemolyze at concentrations below those of normal RBCs. Osmotic fragility is not reliable in the newborn unless the blood is incubated at 37°C for 24 hours before testing. Incubation is also necessary in 20%-25% of adults, so that a normal nonincubated osmotic fragility result does not rule out congenital spherocytosis.

    In the great majority of patients with congenital spherocytosis, the nonincubated or the incubated osmotic fragility tests yield clear-cut positive results. In a few cases the results are equivocal, and in rare cases they are negative. In these few cases the autohemolysis test may be helpful. Congenital spherocytosis produces a considerable increase in hemolysis under the conditions of the test, which can be corrected to a considerable degree by addition of glucose to the test media. The autohemolysis test was originally proposed as a general screening procedure for genetic hemolytic disorders, but problems with sensitivity and specificity have led some investigators to seriously question its usefulness except when osmotic fragility tests fail to diagnose a case of possible congenital spherocytosis.

    Spherocytes are often confused with nonspherocytic small RBCs (microcytic RBCs). A classic spherocyte is smaller than normal RBCs, is round, and does not demonstrate the usual central clear area. (The relatively thin center associated with normal biconcave disk shape is lost as the RBC becomes a sphere.)

    Hereditary elliptocytosis (ovalocytosis)

    Hereditary elliptocytosis occurs in Europeans and African Americans, it is inherited as a dominant trait. Eighty percent to 90% of affected persons have mild compensated hemolysis either without anemia or with a mild anemia. Reticulocyte counts are usually slightly elevated but are not greatly increased. More than 40% of the RBCs are elliptocytes. These are oval RBCs that look like thick rods when viewed from the side. Normal persons reportedly may have up to 15% elliptocytes. There are several uncommon variants of hereditary elliptocytosis in which hemolysis is more pronounced, and moderate or severe anemia may be present; these include a variant of severe neonatal hemolytic anemia with jaundice in which anisocytosis and poikilocytosis are prominent but elliptocytosis is not. Infants with this variant slowly revert to the more usual mild elliptocytic clinical and hematologic picture by age 6-12 months. There is also a form called hemolytic hereditary elliptocytosis with spherocytosis in which there is mild anemia and another very similar form called homozygous hereditary elliptocytosis in which anemia is severe. Both variants demonstrate spherocytes as well as elliptocytes.

    Abetalipoproteinemia (Bassen-Kornsweig syndrome)

    Patients with abetalipoproteinemia totally lack chylomicrons, very low-density lipoproteins, and low-density lipoproteins (Chapter 22) and have only high-density lipoproteins in fasting plasma. There is associated fat malabsorption, various neuromuscular abnormalities (especially ataxia), retinitis pigmentosa, and presence of acanthocytes (Chapter 2) constituting 40%-80% of the peripheral blood RBCs. There is mild to moderate anemia with mild to moderate reticulocytosis. A peripheral smear picture similar to abetalipoproteinemia may be present in Zieve’s syndrome (hemolytic anemia with hypertriglyceridemia in alcoholic liver disease).

    Congenital stomatocytosis (stomatocytic elliptocytosis)

    Congenital stomatocytosis is found in certain Pacific Ocean populations, is inherited as an autosomal recessive trait, consists clinically of mild anemia, and demonstrates slightly elliptocytic stomatocytes on peripheral smear. Stomatocytes are RBCs with the central clear area compressed to a linear rodlike shape. Stomatocytes may also be found in association with alcoholism and liver disease.

  • Red Blood Cell Enzyme Deficiencies

    These conditions are sometimes called congenital nonspherocytic anemias. The RBC contains many enzymes involved in various metabolic activities. Theoretically any of these may be affected by congenital or possibly by acquired dysfunction. The most frequent congenital abnormalities are associated with enzymes that participate in metabolism of glucose. After glucose is phosphorylated to glucose-6-phosphate by hexokinase, about 10% of the original molecules follow the oxidative hexose monophosphate (pentose) pathway and about 90% traverse the anaerobic Embden-Meyerhof route. The only common defect associated with clinical disease is produced by G-6-PD deficiency, which is a part of the hexose monophosphate shunt. The primary importance of this sequence is its involvement with metabolism of reduced glutathione (GSH), which is important in protecting the RBC from damage by oxidizing agents. The next most frequent abnormality, pyruvate kinase defect, a part of the Embden-Meyerhof pathway, is very uncommon, and other enzyme defects are rare. The various RBC enzyme defects (plus the unstable hemoglobins) are sometimes collectively referred to as the congenital nonspherocytic anemias.

    Red blood cell enzyme defects of the hexose monophosphate shunt and others involved in glutathione metabolism are sometimes called Heinz body anemias. The term also includes many of the unstable hemoglobins and a few idiopathic cases. Heinz bodies are small, round, dark-staining, intraerythrocytic inclusions of varying size that are visualized only when stained by supravital stains (not ordinary Wright’s or Giemsa). In most cases, the Heinz bodies must be induced by oxidizing agents before staining.

    Glucose-6-phosphate dehydrogenase defect

    G-6-PD defect is a sex-linked genetic abnormality carried on the female (X) chromosome. To obtain full expression of its bad effects, the gene must not be opposed by a normal X chromosome. Therefore, the defect is most severe in males (XY) and in the much smaller number of females in whom both X chromosomes have the abnormal gene. Those females with only one abnormal gene (carrier females) have varying expressions of bad effect ranging from completely asymptomatic to only moderate abnormality even under a degree of stimulation that is greater than that needed to bring out the defect clinically in affected males or homozygous females.

    The G-6-PD defect is found mainly in sub-Saharan Africans (10%-14% of African Americans) and to a lesser extent in persons whose ancestors came from Mediterranean countries such as Italy, Greece, or Turkey; from some areas of India; and some Jewish population groups. The defect centers in the key role of G-6-PD in the pentose phosphate glucose metabolic cycle of RBCs. As RBCs age, they normally are less able to use the pentose phosphate (oxidative) cycle, which is an important pathway for use of glucose, although secondary to the Embden-Meyerhof (nonoxidative) glycolysis cycle. When defective G-6-PD status is superimposed on the older erythrocyte, use of the pentose phosphate shunt is lost. This cycle is apparently necessary to protect the integrity of the RBCs against certain chemicals. Currently it is thought that these chemicals act as oxidants and that reduced nicotinamide-adenine dinucleotide phosphate (NADPH) from the pentose cycle is the reducing agent needed to counteract their effects. At any rate, exposure to certain chemicals in sufficient dosage results in destruction of erythrocytes with a sufficiently severe G-6-PD defect. About 11% of African-American males are affected. Their defect is relatively mild, since the younger RBCs contain about 10% of normal enzyme activity, enough to resist drug-induced hemolysis. As the RBCs age they lose nearly all G-6-PD activity and are destroyed. In affected persons of Mediterranean ancestry, all RBCs regardless of age contain less than 1% G-6-PD activity.

    As previously noted, susceptible persons do not have anemia before drug exposure. After a hemolytic drug is given, acute hemolysis is usually seen on the second day, but sometimes not until the third or fourth day. All the classic laboratory signs of nonspecific acute hemolysis are present. The degree of anemia produced in African Americans is only moderate, because only the older cell population is destroyed. If the drug is discontinued, hemolysis stops in 48-72 hours. If the drug is continued, anemia continues at a plateau level, with only a small degree of active hemolysis taking place as the RBCs advance to the susceptible cell age. Whites have a more severe defect and therefore more intense hemolysis, which continues unabated as long as the drug is administered.

    Many drugs have been reported to cause this reaction in G-6-PD–defective persons. The most common are the antimalarials, sulfa drugs, nitrofurantoin (Furadantin) family, aspirin, and certain other analgesics such as phenacetin. Hemolysis induced by various infections has been frequently reported and may also occur in uremia and diabetic acidosis.

    Screening tests for G-6-PD deficiency. Several different tests are available, among which are methemoglobin reduction (Brewer’s test), glutathione stability, dye reduction, ascorbate test, and fluorescent spot tests. G-6-PD assay procedures can also be done. During hemolytic episodes in African Americans, dye reduction and glutathione stability tend to give false normal results. Blood transfusions may temporarily invalidate all G-6-PD deficiency tests in African Americans and Europeans.

    The same caution applies to G-6-PD that was applicable to the hemoglobinopathies. Hemolytic anemia in populations known to have a high incidence of G-6-PD defect should always raise the suspicion of its presence. However, even if a patient has the defect, this does not exclude the possibility that the actual cause of hemolysis was something else.

    Other red blood cell enzyme deficiencies

    There are numerous enzymes in both the anaerobic Embden-Meyerhof glycolytic pathway and the aerobic pentose phosphate shunt. Deficiency in any of these enzymes could result in clinical abnormality. After G-6-PD deficiency, by far the most common is pyruvate kinase deficiency, accounting for 90% of those hemolytic anemias from congenital RBC enzyme defects that are not produced by G-6-PD. Actually, pyruvate kinase deficiency is uncommon, and clinical abnormality from pyruvate kinase or other glycolytic enzymes is rare. Clinical abnormality from pyruvate kinase or other Embden-Meyerhof glycolytic enzyme deficiencies is usually manifest as a Coombs’-negative hemolytic anemia without the relationship to drugs or infections that is associated with G-6-PD.

  • Hemoglobin Synthesis Abnormalities

    Thalassemia

    Strictly speaking, there is no thalassemia hemoglobin. Thalassemia comprises a complex group of genetic abnormalities in globin chain synthesis. There are three major clinical categories: thalassemia major, associated with severe and often life-threatening clinical manifestations; thalassemia minor, with mild or minimal clinical manifestations; and a combination of the thalassemia gene with a gene for another abnormal hemoglobin.

    The genetic situation is much more complicated than arbitrary subdivision into the major clinical syndromes would imply.

    The globin portion of normal hemoglobin (Hb A1) is composed of two pairs of polypeptide (amino acid) chains, one pair (two chains) called alpha (a) and the other two chains called beta (b). All normal hemoglobins have two alpha chains, but certain hemoglobins have either one or both beta chains that have a polypeptide amino acid sequence different from usual beta chains. Thus, Hb A2 has two delta (d) chains, and Hb F has two gamma (g) chains in addition to the two alphas. All three of these hemoglobins (A1, A2, and F) are normally present in adult RBCs, but A2 and F normally are present only in trace amounts. One polypeptide chain from each pair of alpha and beta chains is inherited from each parent, so that one alpha chain and one beta chain are derived from the mother and the other alpha and beta chain from the father. The thalassemia gene may involve either the alpha or the beta chain. In the great majority of cases, the beta chain is affected; genetically speaking, it would be more correct to call such a condition a beta thalassemia. If the condition is heterozygous, only one of the two beta chains is affected; this usually leaves only one beta chain (instead of two) available for Hb A1 synthesis and yields a relative increase in Hb A2. This produces the clinical picture of thalassemia minor. In a homozygous beta thalassemia, both of the beta chains are affected; this apparently results in marked suppression of normal Hb A1 synthesis and leads to a compensatory increase in gamma chains; the combination of increased gamma chains with the nonaffected alpha chains produces marked elevation of Hb F. This gives the clinical syndrome of thalassemia major. It is also possible for the thalassemia genetic abnormality to affect the alpha chains. The genetics of alpha thalassemia are more complicated than those of beta thalassemia, because there are two alpha-globin gene loci on each of the two globin-controlling body chromosomes, whereas for beta-globin control there is one locus on each of the two chromosomes. In alpha thalassemia trait (“silent carrier”), one gene locus on one chromosome only is deleted or abnormal (one of the four loci). In alpha thalassemia minor, two of the four loci are affected. This may be produced either by deletion or abnormality in both loci on one of the two chromosomes (a genotype called alpha-thalassemia-1, more common in Asians), or by deletion of one of the two loci on each of the two chromosomes (a genotype called alpha-thalassemia-2, more common in Africans and those of Mediterranean descent). Hemoglobin A1 production is mildly curtailed, but no Hb A2 or F increase occurs because they also need alpha chains. Hemoglobin H disease results from deletion or inactivation of three of the four loci. All four globin chains of Hb H are beta chains. Hemoglobin H disease occurs mostly in Asians but occasionally is found in persons from the Mediterranean area. The most serious condition is that resulting from deletion or inactivation of all four alpha-globin gene loci; if this occurs, the hemoglobin produced is called Bart’s hemoglobin, and all four globin chains are the gamma type. In most cases functional hemoglobin production is curtailed enough to be lethal in utero or neonatal life. Another abnormal hemoglobin that should be mentioned is Hb-Lepore, which is called a fusion hemoglobin, and which consists of two normal alpha chains and two nonalpha fusion chains, each containing the amino terminal portion of a delta chain joined to the carboxy terminal portion of a beta chain.

    Thalassemia major (Cooley’s anemia). This globin variant is the homozygous form of beta thalassemia and consists of two alpha chains and two gamma chains. The condition generally does not become evident until substantial changeover to adult hemoglobin at about 2-3 months of age and clinically is manifest by severe hemolytic anemia and a considerable number of normoblasts in the peripheral blood. The nucleated RBCs are most often about one third or one half the number of WBCs but may even exceed them. There are frequent Howell-Jolly bodies and considerable numbers of polychromatophilic RBCs. The mature RBCs are usually very hypochromic, with considerable anisocytosis and poikilocytosis, and there are moderate numbers of target cells. The mean corpuscular volume (MCV) is microcytic. WBC counts are often mildly increased, and there may be mild granulocytic immaturity, sometimes even with myelocytes present. Platelets are normal. Skull x-ray films show abnormal patterns similar to those in sickle cell anemia but even more pronounced. Death most often occurs in childhood or adolescence.

    Diagnosis. Diagnosis is suggested by a severe anemia with very hypochromic RBCs, moderate numbers of target cells, many nucleated RBCs, and a family history of Mediterranean origin. The sickle preparation is negative. Definitive diagnosis depends on the fact that in thalassemia major, Hb F is elevated (10%-90% of the total hemoglobin, usually >50%). Hemoglobin F has approximately the same migration rate as Hb A1 on paper electrophoresis but may be separated by other electrophoretic techniques. In addition, Hb F is much more resistant to denaturation by alkali than is Hb A1. This fact is utilized in the alkali denaturation test. The hemoglobin solution is added to a certain concentration of sodium hydroxide (NaOH), and after filtration the amount of hemoglobin in the filtrate (the undenatured hemoglobin) is measured and compared with the original total quantity of hemoglobin. One report cautions that if the RBCs are not washed sufficiently before the hemoglobin solution is prepared, reagents of some manufacturers may produce a false apparent increase in Hb F.

    The gene producing the Mediterranean type of homozygous beta thalassemia does not synthesize any beta chains; sometimes referred to as b°. When homozygous beta thalassemia occurs in African Americans, the gene is apparently slightly different because small amounts of beta chains may be produced as well as the predominant gamma chains. This gene is referred to as b+, and the degree of anemia tends to be less severe than in the Mediterranean b° type.

    Thalassemia minor. This clinical subgroup of the thalassemias is most frequently the heterozygous form of beta thalassemia (beta thalassemia trait). Besides a relatively high incidence (1%-10%) in Americans of Mediterranean extraction, there is an estimated frequency of 1% (0.5%-2%) in African Americans. About 75% of patients have anemia, which is usually mild; fewer than 10% of patients with anemia have hemoglobin levels less than 10 gm/100 ml. Patients with beta thalassemia trait have microcytic MCV values in a great majority of cases (87%-100%) whether or not anemia is present. The mean corpuscular hemoglobin (MCH) value is also decreased below reference range in almost all cases. Peripheral blood smears typically contain hypochromic and somewhat microcytic RBCs, usually with some target cells and frequently with some RBCs containing basophilic stippling. Nucleated RBCs are not present. The reticulocyte count is frequently elevated (50% of patients in one study had reticulocytosis >3%).

    The main laboratory abnormality in beta thalassemia trait is an increased amount of Hb A2 (A2 is not elevated in alpha thalassemia trait). As noted previously, A2 is a variant of adult Hb A and is normally present in quantities up to 2.5% or 4%, depending on the method used. In beta thalassemia trait, A2 is elevated to some degree with a maximum of approximately 10%. (If >10% is reported using cellulose acetate electrophoresis, this suggests another Hb migrating in the A2 area, such as Hb C.) Hemoglobin F is usually normal but can be present in quantities up to 5%. Hemoglobin A2 cannot be identified on paper electrophoresis, and demonstration or quantitation necessitates cellulose acetate or polyacrylamide gel electrophoresis or resin column methods. DNA probe tests are available in some university centers for diagnosis of beta thalassemia.

    Thalassemia minor due to alpha thalassemia trait is probably more common in the United States than previously recognized. There is a relatively high incidence in persons of Southeast Asian origin and in African Americans. (Limited studies have detected 6%-30% affected persons from African Americans.) There is also a significant incidence in persons of Mediterranean extraction. The majority of affected persons do not exhibit any clinical symptoms of any anemia, and of the minority that do, symptoms of anemia are most often relatively mild. In one limited study of African Americans in Los Angeles, the majority of affected persons had decreased MCV but only about 10% were anemic. The average MCH value was about 2% less than the average value for normal persons, and the average MCH concentration (MCHC) was about the same as that of normal persons. Hemoglobin H disease can be detected by appropriate hemoglobin electrophoretic techniques. Hemoglobin H disease is mostly restricted to Asians and is manifest by a chronic hemolytic anemia of moderate degree (which may, however, be mild rather than moderate). There is also an acquired form of Hb H disease, reported in some patients with myelodysplastic or myeloproliferative syndromes.

    Currently there is no easy laboratory method to diagnose genetic alpha-globin abnormality. However, in the newborn’s cord blood, Bart’s hemoglobin is generally elevated (by electrophoresis) in rough proportion to the severity of the alpha thalassemia syndrome. Bart’s hemoglobin thus constitutes about 25% (range 20%-40%) in Hb H disease, about 5% (range 2%-10%) in alpha thalassemia trait, and about 1%-2% in the silent carrier state. After about 4 months (range 4-6 months) of age, Bart’s hemoglobin has mostly disappeared. Thereafter, globin chain synthesis studies or DNA probe techniques are the current methods used, but these techniques are available only in research laboratories. Hemoglobin H inclusions in RBCs can be seen using standard reticulocyte count methods, but their sensitivity is disputed (especially in alpha thalassemia trait), possibly due in part to differences in methodology.

    Thalassemia minor vs. chronic iron deficiency. Thalassemia minor must sometimes be differentiated from iron deficiency anemia because of the hypochromic-microcytic status of the RBCs. Certain guidelines have been suggested to increase suspicion for thalassemia minor or to presumptively rule it out in patients with microcytic anemia. The RBC distribution width (RDW; Chapter 2) is usually normal in uncomplicated thalassemia minor and elevated in chronic iron deficiency anemia. Uncomplicated thalassemia minor typically has an RBC count greater than 5 million/mm3 in spite of decreased MCV. Uncomplicated thalassemia minor very uncommonly has a hemoglobin level less than 9.0 gm/100 ml (90 g/L) and usually has a MCHC of 30% or greater (adult reference range, 33%-37% or 330-370 g/L), whereas 50% of patients with chronic iron deficiency anemia have a hemoglobin level less than 9.0 gm (90 g/L), and 50% or more have an MCHC less than 30% (300 g/L). There are also several formulas to segregate thalassemia trait from chronic iron deficiency, of which the best known is the discriminant function of England and Frazer. Unfortunately, there is enough overlap to severely limit the usefulness of these formulas for any individual patient. Serum iron is usually decreased in uncomplicated chronic iron deficiency anemia or anemia of chronic disease and is usually normal in uncomplicated beta thalassemia trait. An elevated total iron-binding capacity (TIBC) suggests iron deficiency, and a decreased TIBC suggests chronic disease. Serum ferritin is decreased in uncomplicated chronic iron deficiency and is normal in uncomplicated beta thalassemia trait. Bone marrow iron is absent in iron deficiency and normal or increased in thalassemia trait. The word “uncomplicated” is stressed because patients with beta thalassemia trait may have concurrent chronic iron deficiency and because anemia of chronic disease may be concurrent with either condition. The anemia of chronic disease may itself be microcytic and hypochromic in about 10% of cases.

    Definitive diagnosis of beta thalassemia trait involves measurement of Hb A2, which is increased in uncomplicated beta thalassemia trait but not in chronic iron deficiency or chronic disease. However, chronic iron deficiency decreases Hb A2 levels so that iron deficiency coexistent with beta thalassemia trait could lead to falsely normal Hb A2 results. In one study, 15% of children with beta thalassemia minor initially displayed normal A2 levels that became elevated after 2 months of therapy with oral iron. Conditions other than beta thalassemia that raise Hb A2 levels include folate or B12 deficiency, increase in Hb F level, and the presence of certain abnormal hemoglobins that migrate with A2, the particular variety of interfering hemoglobin being dependent on the A2 assay method used.

    Sickle thalassemia. This combination produces a condition analogous to SC disease, clinically similar in many respects to SS anemia but considerably milder. Sickle cell test results are positive. There frequently are considerable numbers of target cells. Approximately 60%-80% of the hemoglobin is Hb S. In the S-thalassemia b° (S-Thal-b°) type, most of the remaining hemoglobin is Hb F, so that the pattern resembles SS anemia. However, S-Thal-b° is clinically milder, and the peripheral smear may display RBCs that are more hypochromic than one would expect with SS disease. Also, Hb A2 is increased. The S-thalassemia b+ (S-Thal-b+) pattern might be confused with the SA pattern of sickle trait. However, in sickle trait Hb A predominates rather than Hb S, so that an electrophoretic SA pattern in which more than 50% of the total Hb is Hb S suggests S-thalassemia.

    Screening for thalassemia. Several reports indicate that a decreased MCV detected by automated hematology cell counters is a useful screening method for thalassemia as well as for chronic iron deficiency anemia. One report suggests an 85% chance of thalassemia when there is a combination of MCV of less than 75 femtoliters (fL) with a RBC count of more than 5 million/mm3 (5 Ч 106/L). A hemoglobin level less than 9.0 gm/100 ml (90 g/L) plus an MCHC less than 30% suggests chronic iron deficiency rather than thalassemia. As noted previously, cord blood has been used for detection of the alpha thalassemias. It is now becoming possible to screen for homozygous hemoglobinopathies and severe forms of thalassemia in utero employing DNA analysis of a chorionic villus biopsy at 8-10 weeks or amniotic fluid cells from amniocentesis at 16-18 weeks.

  • The Unstable Hemoglobins

    The unstable hemoglobins are characterized by a focal amino acid mutation that permits hemoglobin denaturation under certain conditions (e.g., heat or oxidation), with the formation of Heinz bodies. There are several different abnormalities, and either the alpha or beta hemoglobin chain may be affected. Unstable hemoglobin variants are rare; the best known are Hb-Kцln, Hb-Zurich, and Hb-Gun Hill. They are usually inherited as autosomal dominant traits. Depending on the hemoglobin variant, clinical and laboratory evidence of hemolytic anemia varies from normality to severe hemolytic disease. In some types that normally are subclinical, such as Hb-Kцln and Hb-Zurich, hemolysis may be precipitated by infection or oxidizing medications. Laboratory diagnosis is accomplished with a Heinz body test; if the results are positive for hemolytic disease, a heat stability test or isopropanol precipitation test is called for. In those patients with acute hemolytic episodes, glucose-6-phosphate dehydrogenase (G-6-PD) deficiency may have to be differentiated. Heinz body formation is not specific for the unstable hemoglobins but may appear in patients with G-6-PD or certain other RBC enzyme defects when hemolysis is precipitated by chemicals and in alpha or beta thalassemia trait.

  • The Hemoglobinopathies

    At birth, approximately 80% of the infant’s hemoglobin is fetal-type hemoglobin (Hb F), which has a greater affinity for oxygen than the adult type. By age 6 months, all except 1%-2% is replaced by adult hemoglobin (Hb A). Persistence of large amounts of Hb F is abnormal. There are a considerable number of abnormal hemoglobins that differ structurally and biochemically, to varying degrees, from normal Hb A. The clinical syndromes produced in persons having certain of these abnormal hemoglobins are called the hemoglobinopathies. The most common abnormal hemoglobins in the Western Hemisphere are sickle hemoglobin (Hb S) and hemoglobin C (Hb C). Hemoglobin E is comparably important in Southeast Asia. All the abnormal hemoglobins are genetically transmitted, just as normal Hb A is. Therefore, since each person has two genes for each trait (e.g., hemoglobin type), one gene on one chromosome received from the mother and one gene on one chromosome received from the father, a person can be either homozygous (two genes with the trait) or heterozygous (only one of the two genes with the trait). The syndrome produced by the abnormal hemoglobin is usually much more severe in homozygous persons than in heterozygous persons. Less commonly, a gene for two different abnormal hemoglobins can be present in the same person (double heterozygosity).

    Sickle hemoglobin

    Several disease states may be due to the abnormal hemoglobin gene called sickle hemoglobin (Hb S). When Hb S is present in both genes (SS), the disease produced is called sickle cell anemia. When Hb S is present in one gene and the other gene has normal Hb A, the disease is called sickle trait. Hb S is found mostly in African Americans, although it may occur in populations along the Northern and Eastern Mediterranean, the Caribbean, and in India. In African Americans the incidence of sickle trait is about 8% (literature range 5%-14%) and of sickle cell anemia less than 1%. The S gene may also be found in combination with a gene for another abnormal hemoglobin, such as Hb C.

    Tests to detect sickle hemoglobin. Diagnosis rests on first demonstrating the characteristic sickling phenomenon and then doing hemoglobin electrophoresis to find out if the abnormality is SS disease or some combination of another hemoglobin with the S gene. Bone marrow shows marked erythroid hyperplasia, but bone marrow aspiration is not helpful and is not indicated for diagnosis of suspected sickle cell disease.

    Peripheral blood smear. Sickled cells can be found on a peripheral blood smear in many patients with SS disease but rarely in sickle trait. The peripheral blood smear is much less sensitive than a sickle preparation. Other abnormal RBC shapes may be confused with sickle cells on the peripheral smear. The most common of these are ovalocytes and schistocytes (burr cells). Ovalocytes are rod-shaped RBCs that, on occasion, may be found normally in small numbers but that may also appear due to another genetically inherited abnormality, hereditary ovalocytosis. Compared with sickle cells, the ovalocytes are not usually curved and are fairly well rounded at each end, lacking the sharply pointed ends of the classic sickle cell. Schistocytes (schizocytes, Chapter 2) may be found in certain severe hemolytic anemias, usually of toxic or antigen-antibody etiology. Schistocytes are RBCs in the process of destruction. They are smaller than normal RBCs and misshapen and have one or more sharp spinous processes on the surface. One variant has the form of a stubby short crescent; however, close inspection should differentiate these without difficulty from the more slender, smooth, and regular sickle cell.

    Screening tests. When oxygen tension is lowered, Hb S becomes less soluble than normal Hb A and forms crystalline aggregates that distort the RBC into a sickle shape. A sickle preparation (“sickle cell prep”) may be done in two ways. A drop of blood from a finger puncture is placed on a slide, coverslipped, and the edges are sealed with petrolatum. The characteristic sickle forms may be seen at 6 hours (or earlier) but may not appear for nearly 24 hours. A more widely used procedure is to add a reducing substance, 2% sodium metabisulfite, to the blood before coverslipping. This speeds the reaction markedly, with the preparation becoming readable in 15-60 minutes. Many laboratories have experienced difficulty with sodium metabisulfite, since it may deteriorate during storage, and occasionally the reaction is not clear-cut, especially in patients with sickle trait.

    DIFFERENTIAL HEMOGLOBIN SOLUBILITY TESTS. A second sickle prep method involves deoxygenation of Hb S by certain chemicals such as dithionate; Hb S then becomes insoluble and precipitates in certain media. These chemical tests, sold under a variety of trade names (usually beginning with the word “sickle”), are easier to perform than the coverslip methods and have replaced the earlier coverslip methods in most laboratories. These tests are generally reliable, but there are certain drawbacks. False negative results may be obtained in patients whose hemoglobin level is less than 10 gm/100 ml (or hematocrit 30%) unless the hematocrit reading is adjusted by removing plasma. Instead of this, the National Committee on Clinical Laboratory Standards recommends using packed RBC rather than whole blood. Reagents may deteriorate and inactivate. Dysglobulinemia (myeloma, Waldenstrom’s macroglobulinemia, or cryoglobulinemia) may produce false positive results by creating turbidity in the reaction.

    Hb F also interferes with the turbidity reaction, and therefore dithionate chemical sickle preps may yield false negative results in infants less than 4-6 months old because Hb F is not yet completely replaced by Hb A. Neither the coverslip nor the chemical tests quantitate Hb S and therefore neither test can differentiate between homozygous SS disease and heterozygous combinations of S with normal A Hb (sickle trait) or with another hemoglobin. Although theoretically these methods are positive at Hb S levels greater than 8%, proficiency test surveys have shown that as many as 50% failed to detect less than 20% Hb S. Neither the coverslip nor the chemical tests are completely specific for Hb S, because several rare non-S hemoglobins (e.g., C-Harlem) will produce a sickle reaction with metabisulfite or dithionate. None of the tests will detect other abnormal hemoglobins that may be combined with Hb S in heterozygous patients. In summary, these tests are screening procedures useful only after 6 months of age; not reliable for small amounts of Hb S; and abnormal results should be confirmed with more definitive techniques.

    Immunoassay. A third commercially available sickle screening method is enzyme immunoassay (JOSHUA; HemoCard Hb S) using an antibody that is specific for Hb S; sensitive enough for newborn screening and not affected by Hb F or the hematocrit level.

    Definitive diagnosis. Hemoglobin electrophoresis produces good separation of Hb S from Hb A and C. In cord blood, sickle cell anemia (SS) infants demonstrate an Hb F plus S (FS) pattern, with Hb F comprising 60%-80% of the total. A cord blood FSA Hb mixture suggests either sickle trait or sickle thalassemia (S-thalassemia). After age 3-6 months the SS infant’s electrophoretic pattern discloses 80%-90% Hb S with the remainder being Hb F. Sickle trait patients have more than 50% Hb A with the remainder Hb S (therefore more A than S), whereas S-(beta) thalassemia has 50% or more Hb S with about 25% Hb A and less than 20% Hb F (therefore more S than A and more A than F). Hemoglobin electrophoresis is most often done using cellulose acetate or agarose media at alkaline pH. Some hemoglobins migrate together on cellulose acetate or agarose under these conditions; the most important are Hb C, E, and A2 together and Hb S and D together (see Fig. 37-2). In some systems, Hb A and F cannot be reliably separated. Citrate agar at a more acid pH has separation patterns in some respects similar to cellulose acetate, but Hb D, E, and G migrate with A on citrate, whereas they travel with S on cellulose acetate. Likewise, Hb C and A2 migrate separately on citrate, whereas they migrate together on cellulose acetate. Thus, citrate agar electrophoresis can be used after cellulose acetate for additional information or as a confirmatory method. In addition, citrate agar gives a little better separation of Hb F from A in newborn cord blood, and some investigators prefer it for population screening. Isoelectric focusing electrophoresis is available in some specialized laboratories. This procedure gives very good separation of the major abnormal hemoglobins plus some of the uncommon variants. No single currently available method will identify all of the numerous hemoglobin variants that have been reported. Hemoglobin F can be identified and quantitated by the alkali denaturation procedure or by a suitable electrophoretic method.

    Sickle cell anemia. Homozygous sickle cell (SS) disease symptoms are not usually noted until age 6 months or later. On the other hand, a significant number of these patients die before age 40. Anemia is moderate or severe in degree, and the patient often has slight jaundice (manifest by scleral icterus). The patients seem to adapt surprisingly well to their anemic state and, apart from easy fatigability or perhaps weakness, have few symptoms until a sickle cell “crisis” develops. The painful crisis of sickle cell disease is often due to small-vessel occlusion producing small infarcts in various organs, but in some cases the reason is unknown. Abdominal pain or bone pain are the two most common symptoms, and the pain may be extremely severe. There usually is an accompanying leukocytosis, which, if associated with abdominal pain, may suggest acute intraabdominal surgical disease. The crisis ordinarily lasts 5-7 days. In most cases, there is no change in hemoglobin levels during the crisis. Patients may have nonpainful transient crises involving change in level of anemia. Children 6 months to two years of age may have episodes of RBC splenic sequestration, frequently associated with a virus infection. There may be bone marrow aplastic crises in which marrow RBC production is sharply curtailed, also frequently associated with infection (e.g., parvovirus B-19). Uncommonly there may be crisis due to acceleration of hemolysis.

    Infection, most often pneumococcal, is the greatest problem in childhood, especially in the early age group from 2 months to 2 years. Because of this, an NIH Consensus Conference (1987) recommended neonatal screening for SS disease in high-risk groups, so that affected infants can be treated with prophylactic antibiotics. After infancy, there is still some predisposition toward infection, with the best-known types being pneumococcal pneumonia and staphylococcal or Salmonella osteomyelitis.

    Other commonly found abnormalities in sickle cell disease are chronic leg ulcers (usually over the ankles), hematuria, and a loss of urine-concentrating ability. Characteristic bone abnormalities are frequently seen on x-ray films, especially of the skull, and avascular necrosis of the femoral head is relatively common. Gallstone frequency is increased. There may be various neurologic signs and symptoms. The spleen may be palpable in a few patients early in their clinical course, but eventually it becomes smaller than normal due to repeated infarcts. The liver is palpable in occasional cases. Obstetric problems are common for both the mother and the fetus.

    HEMATOLOGIC FINDINGS. As previously mentioned, anemia in SS disease is moderate to severe. There is moderate anisocytosis. Target cells are characteristically present but constitute less than 30% of the RBCs. Sickle cells are found on peripheral blood smear in many, although not all, patients. Sometimes they are very few and take a careful search. There are usually nucleated RBCs of the orthochromic or polychromatophilic normoblast stages, most often ranging from 1/100-10/100 white blood cells (WBCs). Polychromatophilic RBCs are usually present. Howell-Jolly bodies appear in a moderate number of patients. The WBC count may be normal or there may be a mild leukocytosis, which sometimes may become moderate in degree. There is often a shift to the left in the WBC maturation sequence (in crisis, this becomes more pronounced), and sometimes even a few myelocytes are found. Platelets may be normal or even moderately increased.

    The laboratory features of active hemolytic anemia are present, including reticulocytosis of 10%-15% (range, 5%-30%).

    Sickle cell trait. As mentioned earlier, sickle cell trait is the heterozygous combination of one gene for Hb S with one gene for normal Hb A. There is no anemia and no clinical evidence of any disease, except in two situations: some persons with S trait develop splenic infarcts under hypoxic conditions, such as flying at high altitudes in nonpressurized airplanes; and some persons develop hematuria. On paper electrophoresis, 20%-45% of the hemoglobin is Hb S and the remainder is normal Hb A. The metabisulfite sickle preparation is usually positive. Although a few patients have been reported to have negative results, some believe that every person with Hb S will have a positive sickle preparation if it is properly done. The chemical sickle tests are perhaps slightly more reliable in the average laboratory. The peripheral blood smear rarely contains any sickle cells.

    Sickle Hb–Hb C disease (HbSC disease). As previously mentioned, in this disease one gene for Hb S is combined with one gene for Hb C. About 20% of patients do not have anemia and are asymptomatic. In the others a disease is produced that may be much like SS disease but is usually milder. Compared with SS disease, the anemia is usually only of mild or moderate degree, although sometimes it may be severe. Crises are less frequent; abdominal pain has been reported in 30%. Bone pain is almost as common as in SS disease but is usually much milder. Idiopathic hematuria is found in a substantial minority of cases. Chronic leg ulcers occur but are not frequent. Skull x-ray abnormalities are not frequent but may be present.

    Hemoglobin SC disease differs in some other respects from SS disease. In SC disease, aseptic necrosis in the head of the femur is common; this can occur in SS disease but not as frequently. Splenomegaly is common in SC disease, with a palpable spleen in 65%-80% of the patients. Finally, target cells are more frequent on the average than in SS disease (due to the Hb C gene), although the number present varies considerably from patient to patient and cannot be used as a distinguishing feature unless more than 30% of the RBCs are involved. Nucleated RBCs are not common in the peripheral blood. Sickle cells may or may not be present on the peripheral smear; if present, they are usually few in number. WBC counts are usually normal except in crises or with superimposed infection.

    Sickle preparations are usually positive. Hemoglobin electrophoresis establishes a definitive diagnosis.

    Hemoglobin C

    The C gene may be homozygous (CC), combined with normal Hb A (AC), or combined with any of the other abnormal hemoglobins (e.g., SC disease).

    Hemoglobin C disease. Persons with Hb C disease are homozygous (CC) for the Hb C gene. The C gene is said to be present in only about 3% of African Americans, so homozygous Hb C (CC) disease is not common. Episodes of abdominal and bone pain may occur but usually are not severe. Splenomegaly is generally present. The most striking feature on the peripheral blood smear is the large number of target cells, always more than 30% and often close to 90%. Diagnosis is by means of hemoglobin electrophoresis.

    Hemoglobin C trait. Persons with Hb C trait have one Hb C gene and the normal Hb A gene. There is no anemia or any other symptom. The only abnormality is a variable number of target cells on the peripheral blood smear.

    Definitive diagnosis. Diagnosis of Hb C is made using hemoglobin electrophoresis. As noted previously, on cellulose acetate or agar electrophoresis, Hb C migrates with Hb A2. Hemoglobin A2 is rarely present in quantities greater than 10% of total hemoglobin, so that hemoglobin migrating in the A2 area in quantity greater than 10% is suspicious for Hb C.

    Comments on detection of the hemoglobinopathies

    To conclude this discussion of the hemoglobinopathies, I must make certain observations. First, a sickle screening procedure should be done on all African Americans who have anemia, hematuria, abdominal pain, or arthralgias. This should be followed up with hemoglobin electrophoresis if the sickle screening procedure is positive or if peripheral blood smears show significant numbers of target cells. However, if the patient has had these studies done previously, there is no need to repeat them. Second, these patients may have other diseases superimposed on their hemoglobinopathy. For example, unexplained hematuria in a person with Hb S may be due to carcinoma and should not be blamed on the hemoglobinopathy without investigation. Likewise, when there is hypochromia and microcytosis, one should rule out chronic iron deficiency (e.g., chronic bleeding). This is especially true when the patient has sickle trait only, since this does not usually produce anemia. The leukocytosis found as part of SS disease (and to a lesser degree in SC and S-thalassemia) may mask the leukocytosis of infection. As mentioned, finding significant numbers of target cells suggests one of the hemoglobinopathies. However, target cells are often found in chronic liver disease, may be seen in any severe anemia in relatively small numbers, and are sometimes produced artifactually at the thin edge of a blood smear.

  • Hemoglobin Structure Abnormalities

    Hemoglobin consists of one heme unit (a complex of one iron atom within four protoporphyrin structures) plus one globin unit (consisting of two pairs of polypeptide chains, one pair known as alpha and the other pair called beta). The heme units are identical in each hemoglobin molecule; changes in amino acid sequence in the globin chains result in different hemoglobins.

    The major hemoglobin structural abnormalities can be divided into two groups: the hemoglobinopathies, consisting of amino acid substitutions in the globin beta chain; and the unstable hemoglobins, which have focal structural abnormality in the globin chains of a different type than the hemoglobinopathies, predisposing the hemoglobin molecule to denaturation.

  • Laboratory Tests in Hemolytic Anemias

    Certain laboratory tests are extremely helpful in suggesting or demonstrating the presence of hemolytic anemia. Which tests give abnormal results, and to what degree, depends on the severity of the hemolytic process and possibly on its duration.

    Reticulocyte count. Reticulocyte counts are nearly always elevated in moderate or severe active hemolytic anemia, with the degree of reticulocytosis having some correlation with the degree of anemia. The highest counts appear after acute hemolytic episodes. Hemolytic anemia may be subclinical, detected only by RBC survival studies, or more overt but of minimal or mild intensity. In overt hemolytic anemia of mild intensity the reticulocyte count may or may not be elevated. Studies have found reticulocyte counts within reference range in 20%-25% of patients with hemolytic anemia, most often of the idiopathic autoimmune type. In one study of 35 patients with congenital spherocytosis, reticulocyte counts were normal in 8.5% of patients; and in one study of patients with thalassemia minor, reticulocyte counts were less than 3% in one half of the patients. Nevertheless, the reticulocyte count is a valuable screening test for active hemolytic anemia, and reticulocyte counts of more than 5% should suggest this diagnosis. Other conditions that give similar reticulocyte response are acute bleeding and deficiency anemias after initial treatment (sometimes the treatment may be dietary only). It usually takes 2 to 3 days after acute hemolysis or bleeding for the characteristic reticulocyte response to appear, and occasionally 4 or 5 days if the episode is relatively mild.

    Lactic dehydrogenase. Total serum lactic dehydrogenase (LDH) consists of a group of enzymes (isoenzymes) that appear in varying amounts in different tissues. The electrophoretically fast-migrating fraction LDH-1 is found in RBCs, myocardial muscle fibers, and renal cortex cells. RBC hemolysis releases LDH-1, which elevates LDH-1 values and usually increases total LDH values. The LDH measurement is a fairly sensitive screening test in hemolytic disease, probably as sensitive as the reticulocyte count, although some investigators believe that LDH results are too inconsistent and unreliable in mild disease. Other conditions that increase LDH-1 levels include artifactual hemolysis from improper venipuncture technique or specimen handling, megaloblastic anemia, and acute myocardial infarction. In addition, the total LDH value may be elevated due to an increase in one of the other LDH isoenzymes, especially the liver fraction. Therefore, nonspecificity has limited the usefulness of total LDH values in the diagnosis of hemolytic anemia. The LDH-1 assay is more helpful. A normal total LDH value, however, would assist in ruling out hemolytic anemia if the degree of anemia were substantial. The LDH-1/LDH-2 ratio is reported to be reversed in about 60% of patients with hemolytic anemia when the lab uses electrophoresis on cellulose acetate and may or may not occur using agarose gel, depending on the method. According to one study, reversed LDH-1/LDH-2 ratio is more likely to occur in hemolytic episodes if there is a substantial degree of reticulocytosis.

    Serum haptoglobin. Haptoglobin is an alpha-2 globulin produced by the liver that binds any free hemoglobin released into the blood from intravascular or extravascular RBC destruction. Haptoglobin can be estimated in terms of haptoglobin-binding capacity or measured by using antihaptoglobin antibody techniques (Chapter 11). Under ordinary conditions a decreased serum haptoglobin level suggests that hemolysis has lowered available haptoglobin through binding of free hemoglobin. Total haptoglobin levels decrease within 8 hours after onset of hemolysis.

    The usefulness of serum haptoglobin levels in the diagnosis of hemolytic conditions is somewhat controversial, although the haptoglobin level is generally considered to have a sensitivity equal to or better than that of the reticulocyte count. The actual sensitivity for minimal or mild hemolytic disease is not well established. There are reports that haptoglobin values may be normal in 10%-20% of cases. Serum haptoglobin levels have been used to differentiate reticulocytosis due to hemolytic anemia from reticulocytosis due to acute bleeding or iron deficiency anemia under therapy. However, some reports indicate that occasionally haptoglobin levels may be mildly decreased in patients with iron deficiency anemia not known to have hemolytic anemia. Most patients with megaloblastic anemia have decreased haptoglobin levels. Haptoglobin levels also may be decreased in severe liver disease, from extravascular hematomas (due to absorption of hemoglobin into the vascular system), and with estrogen therapy or pregnancy. Congenital absence of haptoglobin occurs in approximately 3% of African Americans and about 1% (range, less than 1%-2%) of Europeans. About 80%-90% of newborns lack haptoglobin after the first day of life until 1-6 months of age. Haptoglobin is one of the “acute-phase reaction” serum proteins that are increased in conditions such as severe infection, tissue destruction, acute myocardial infarction, and burns, and in some patients with cancer; these conditions may increase the haptoglobin level sufficiently to mask the effect of hemolytic anemia or a hemolytic episode.

    Plasma methemalbumin. After the binding capacity of haptoglobin is exhausted, free hemoglobin combines with albumin to form a compound known as methemalbumin. This can be demonstrated with a spectroscope. The presence of methemalbumin means that intravascular hemolysis has occurred to a considerable extent. It also suggests that the episode was either continuing or relatively recent, because otherwise the haptoglobins would be replenished and would once again take over the hemoglobin removal duty from albumin.

    Free hemoglobin in plasma or urine. Circulating free hemoglobin occurs when all of the plasma protein-binding capacity for free hemoglobin is exhausted, including albumin. Normally there is a small amount of free hemoglobin in the plasma, probably because some artifactual hemolysis is unavoidable in drawing blood and processing the specimen. This is less when plasma is used instead of serum. If increased amounts of free hemoglobin are found in plasma, and if artifactual hemolysis due to poor blood-drawing technique (very frequent, unfortunately) can be ruled out, a relatively severe degree of intravascular hemolysis is probable. Marked hemolysis is often accompanied by free hemoglobin in the urine (hemoglobinuria). In chronic hemolysis, the urine may contain hemosiderin, located in urothelial cells or casts.

    Direct Coombs’ test. This test is helpful when a hemolytic process is suspected or demonstrated. It detects a wide variety of both isoantibodies and autoantibodies that have attached to the patient’s RBCs (see Chapter 9). The indirect Coombs’ test is often wrongly ordered in such situations. The indirect Coombs’ test belongs to a set of special techniques for antibody identification and by itself is usually not helpful in most clinical situations. If antibody is demonstrated by the direct Coombs’ test, an antibody identification test should be requested. The laboratory will decide what techniques to use, depending on the situation.

    Serum unconjugated (indirect-acting) bilirubin. The serum unconjugated bilirubin level is often elevated in hemolysis of at least moderate degree. Slight or mild degrees of hemolysis often show no elevation. The direct-acting (conjugated) fraction is usually elevated to less than 1.2 mg/100 ml (2.05 µmol/L) and less than 30% of total bilirubin unless the patient has coexisting liver disease. Except in blood bank problems, serum bilirubin is not as helpful in diagnosis of hemolytic anemias as most of the other tests and often shows equivocal results.

    Red blood cell survival studies. RBC survival can be estimated in vivo by tagging some of the patient’s RBCs with a radioactive isotope, such as chromium 51, drawing blood samples daily for isotope counting, and determining how long it takes for the tagged cells to disappear from the circulation. Survival studies are most useful to demonstrate low-grade hemolytic anemias, situations in which bone marrow production is able to keep pace with RBC destruction but is not able to keep the RBC count at normal levels. Low-grade hemolysis often presents as anemia whose etiology cannot be demonstrated by the usual methods. There are, however, certain drawbacks to this procedure. If anemia is actually due to chronic occult extravascular blood loss, radioisotope-labeled RBCs will disappear from the circulation by this route and simulate decreased intravascular survival. A minor difficulty is the fact that survival data are only approximate, because certain technical aspects of isotope RBC tagging limit the accuracy of measurement.

  • Depletion Anemia

    Two types of depletion anemia are possible: (1) abnormal loss of red blood cells (RBCs) from the circulation and (2) abnormal destruction of RBCs within the circulation. RBC loss due to hemorrhage has been covered elsewhere (blood volume, Chapter 10; iron deficiency anemia, Chapter 4). Intravascular or intrasplenic RBC destruction is called hemolytic anemia. There are two clinical varieties of hemolytic anemia. In one type, RBC destruction is relatively slow. Although RBC survival is shortened, the only laboratory test that demonstrates this fact is radioisotope study using tagged RBCs. In the other variety, hemolysis or shortened RBC life span is sufficient to cause abnormality on one or more standard laboratory test results.

    Two etiologic groups comprise most of the hemolytic anemias: those due primarily to intra corpuscular RBC defects and those due primarily to extracorpuscular agents acting on the RBCs. This provides a rational basis for classification of the hemolytic anemias, as follows.

    Due Primarily to Intracorpuscular Defects

    1. Hemoglobin structure abnormalities (e.g., sickle cell and Hb C disease)
    2. Hemoglobin synthesis abnormalities (e.g., thalassemia)
    3. RBC enzyme deficiencies (e.g., glucose-6-phosphate dehydrogenase deficiency)
    4. RBC membrane abnormalities (e.g., congenital spherocytosis)

    Due Primarily to Extracorpuscular Defects

    1. Isoimmune antibodies (e.g., ABO transfusion reactions)
    2. Autoimmune antibodies (e.g., cold agglutinins)
    3. Drug-induced (e.g., a-methyldopa–induced hemolytic anemia)
    4. Traumatic (“microangiopathic”) (e.g., disseminated intravascular coagulation)
    5. Abnormal interaction with activated complement (e.g., paroxysmal nocturnal hemoglobinuria)
    6. Toxins (e.g., lead, bacterial toxins)
    7. Parasites (e.g., malaria)
    8. Hypersplenism

  • Anemia Associated With Systemic Disease

    As noted in Chapter 3, anemia associated with various chronic diseases is usually normocytic and either normochromic or hypochromic. The serum iron and total iron-binding capacity (TIBC) are typically both decreased. In 100 consecutive patients in our hospital who had chronic disease and red cell or iron-related biochemical abnormalities, 68 had anemia with normal mean corpuscular volume (MCV), decreased serum iron, and decreased TIBC; 7 had no anemia; 9 had normal serum iron levels; 6 had normal TIBC; and 7 had decreased MCV (with normal serum ferritin levels). Others have reported that decreased MCV may occur in up to 25% of cases.

    Chronic renal disease

    Anemia of moderate degree is frequently found in association with uremia. Some investigators claim it is almost always present when the blood urea nitrogen (BUN) level is persistently more than twice normal, and it often appears before this level is reached. Patients with prolonged but potentially reversible azotemia (e.g., acute renal failure) often develop anemia until the kidneys recover. Transient types of azotemia usually do not produce anemia unless azotemia is prolonged or due to the underlying cause itself. The anemia of actual renal insufficiency develops regardless of the cause of the uremia.

    The peripheral blood RBCs are usually normocytic-normochromic; there is often mild to moderate anisocytosis. Varying numbers of burr cells (triangular shrunken RBCs with irregular pointed projections from the surface (Chapter 2) are found in some patients. In some cases there is mild hypochromia and, occasionally, some degree of microcytosis. On the other hand, mild macrocytosis may be present in a few patients.

    Bone marrow usually shows normal cellularity, although in some cases there is mild RBC hypoplasia. Marrow iron is adequate. The serum iron level is usually normal, but about 20%-30% of patients have low serum iron levels even though they do not have iron deficiency. Most of these patients also have a low or low-normal TIBC typical of chronic disease anemia (Chapter 3). Reticulocyte counts are usually normal; occasionally, they may be slightly elevated.

    The pathophysiology involved is not well understood. The primary known abnormality is a lack of incorporation of iron into RBCs within the bone marrow. There is depression both of hemoglobin synthesis and of formation and release of mature RBCs into the peripheral blood. In 10%-15% of patients there is also decreased RBC survival in the peripheral blood, although the hemolytic aspect is usually not severe. There is, however, a rare condition known as the hemolytic-uremic syndrome that features a severe microangiopathic (RBC fragmentation) hemolytic anemia. Patients in the late stages of uremia may have a bleeding tendency due to coagulation defects, most commonly thrombocytopenia. Platelet function may be abnormal even with normal numbers of platelets. The effect of hemorrhage, if it occurs, is separate and additional to the anemia of chronic renal disease.

    Anemia of neoplasia

    Anemia develops in 60%-90% of patients with moderate or far-advanced cancer. The anemia of neoplasia is usually normocytic with normal reticulocyte counts, unless there is hemorrhage or chronic blood loss. Cytotoxic chemotherapy is accompanied by a macrocytic MCV in 30%-40% (12%-82%) of patients. A hemolytic component is present in a considerable minority of patients, but hemolysis is generally mild and is not detectable except with radioisotope RBC survival procedures. Occasionally, hemolysis may be severe, especially in patients with chronic lymphocytic leukemia and malignant lymphomas. In one series, anemia was ascribed to a combination of decreased RBC survival and decreased marrow production in 56% of patients, to blood loss in 29%, and to marrow metastases by the tumor in 13%. Thrombocytopenia may be found in certain types of leukemia and in myelophthisic anemias. Fibrinolysins appear in occasional cases of widespread malignancy, most often prostate carcinoma.

    Anemia of infection

    Mild to moderate anemia is frequently associated with subacute or chronic infection. The mechanism of this anemia is not well understood, but there seems to be a decreased rate of erythropoiesis, coupled in some patients with slightly shortened RBC survival time and failure to use iron normally. The anemia of infection usually does not develop unless the infection lasts 1 month or more, although it may develop rapidly in patients with severe acute infection such as septicemia. Chronic infection producing anemia generally is of at least moderate severity. Infections in which anemia is likely to develop include bronchiectasis, salpingitis, abscess of visceral organs or body cavities, and severe pyelonephritis. Anemia is a common finding in subacute bacterial endocarditis and in the granulomatous diseases such as tuberculosis and sarcoidosis. The anemia is usually normocytic and normochromic, but sometimes it is hypochromic. Reticulocyte counts are usually normal, although occasionally they may be slightly increased. Bone marrow aspiration shows either normal marrow or hyperplasia of the granulocytes. The serum iron level is usually low or low-normal, and plasma TIBC is reduced (in iron deficiency anemia the TIBC is elevated).

    Aplastic anemia is a rare complication of type C (non-A, non-B) hepatitis virus infection.

    Rheumatoid-collagen disease group

    Rheumatoid-collagen diseases are frequently associated with mild to moderate normocytic anemia. In one study 40% of males and 63% of females with rheumatoid arthritis were anemic. Active disease is more likely to produce anemia. Incidence of coexistent iron deficiency ranges from 10%-30%. Reticulocytes are usually normal, and the bone marrow is unremarkable. In many patients there apparently is decreased erythropoiesis with a slightly shortened RBC survival time, but there is some disagreement regarding frequency of decreased RBC survival. About 5%-10% of patients with rheumatoid arthritis have splenomegaly, which may be associated with cytopenias.

    Chronic liver disease

    The type and frequency of anemia in liver disease vary with the type and severity of hepatic dysfunction, but anemia has been reported in up to 75% of patients. It is most frequently seen in far-advanced cirrhosis. Extensive metastatic carcinoma of the liver may produce the same effect, although it is difficult to say whether the liver involvement or the neoplasm itself is the real cause. About 30%-50% (8%-65%) of patients with anemia have macrocytosis; about one third are normocytic. Some have hypochromia due to GI blood loss. Target cells in varying numbers are a frequent finding on peripheral blood smear.

    Macrocytic anemia in liver disease is most often found in severe chronic liver damage; this type of anemia is not frequent in acute liver disease, even when severe, or in chronic disease of only slight or mild extent. A small but significant percentage of hepatic macrocytic anemias are megaloblastic, usually secondary to folic acid dietary deficiency, although most are not megaloblastic and are not corrected by folic acid treatment. A peripheral blood smear may be macrocytic even when there is a normal hemoglobin or hematocrit reading, and sometimes even with a normal MCV.

    GI bleeding occurs in a considerable number of cirrhotic patients; often it is very slight and intermittent. Esophageal varices are present in some. Other lesions may be demonstrated in other patients. In a considerable proportion of cases the source of bleeding cannot be located.

    Hypersplenism occurs in some patients with portal vein hypertension and its resulting splenic congestion. Thrombocytopenia, usually mild, is reported to occur in up to 50% of patients with cirrhosis, and other cytopenias may sometimes develop. In severe chronic (or massive acute) liver disease, coagulation problems may result from insufficient hepatic synthesis of several blood coagulation factors.

    Some liver-diseased patients have shortened RBC survival demonstrated only by using radioactive isotope studies and show no evidence of GI bleeding. There is no clinical or laboratory evidence of hemolysis otherwise. About 3%-5% develop Zieve’s syndrome, a combination of hyperlipemia, cirrhosis, and microangiopathic hemolytic anemia. This hemolytic anemia is associated with reticulocytosis and the other classic features of hemolysis.

    Unless blood loss is a factor, and excluding megaloblastic anemia, the bone marrow is unremarkable in liver disease and the reticulocyte count is usually close to normal. Not all cases of anemia associated with liver disease can be explained.

    Hypothyroidism

    Anemia is found in 30%-50% (21%-60%) of hypothyroid patients. About 15% (8%-20%) of the anemic patients have macrocytosis, most of the remainder having either normocytic-normochromic or normocytic-hypochromic indices. A small percentage have hypochromic-microcytic RBCs.

    The hypochromic anemia of hypothyroidism responds to a combination of iron and thyroid hormone preparation. The iron deficiency component is frequently produced by excessive menstrual bleeding. In patients without demonstrable blood loss it is speculated that decreased intestinal iron absorption may occur, since thyroid hormone is known to affect intestinal carbohydrate absorption. Most of the macrocytic cases respond only to thyroid hormone. In these patients the bone marrow is not megaloblastic and is sometimes slightly hypocellular. The reticulocyte count is usually normal. Isotope studies reportedly show normal RBC survival time in most cases. Lack of thyroid hormone seems to have a direct effect on erythropoiesis, since thyroid hormone therapy cures both the myxedema and the anemia (unless there is superimposed iron deficiency). A minority of patients with macrocytic anemia have folic acid or vitamin B12 deficiency, presumably secondary to decreased intestinal absorption. Thyroid hormone is required in addition to folic acid or vitamin B12. About 5% have actual pernicious anemia, with megaloblastic bone marrow.

    Comments on chronic disease anemia

    To conclude this discussion, it should be noted that the normocytic-normochromic anemia of systemic disease has often been called“simple chronic anemia,” although the pathophysiology is far from simple. The disease categories listed in this chapter are only the most common. In many cases, the diagnosis is one of exclusion; the patient has anemia for which no definite etiology can be found, so whatever systemic disease he or she has is blamed for the anemia. Some investigators restrict the diagnosis of chronic disease anemia to those who have decreased serum iron and TIBC. Regardless, it is important to rule out treatable serious diseases. This is especially true for hypochromic anemias (in which blood loss might be occurring) and macrocytic anemias (which may be due to vitamin B12 or folic acid deficiency). A normocytic-normochromic anemia may be due to an occult underlying disease, such as malignant lymphoma or multiple myeloma.

  • Hypoplastic Marrow

    Anemia due to inadequate erythropoiesis without factor deficiency may be classified in several ways. One system is based on the mechanism involved, including (1) marrow failure to incorporate adequate supplies of hematopoietic raw materials (e.g., iron) into red blood cell (RBC) precursors, (2) failure to release mature RBCs from the marrow, or (3) destruction of RBC precursors in the marrow. From a clinical point of view, it is easier to divide production-defect anemias into two categories: those due to a hypoplastic bone marrow and those with normally cellular marrow that are associated with certain systemic diseases.

    Conditions that produce a hypoplastic marrow affect the bone marrow directly either by actual replacement or by toxic depression of RBC precursors. Bone marrow examination is the main diagnostic or confirmatory test.

    Replacement of marrow by fibrosis. This condition, commonly termed myelofibrosis, is usually idiopathic and leads to a clinical syndrome called myeloid metaplasia. The peripheral blood picture is similar in many ways to that of chronic myelogenous leukemia. Many include this condition with the myeloproliferative syndromes.

    Replacement of marrow by neoplasm. The types of tumors most commonly metastatic to bone marrow, the laboratory abnormalities produced, and the main hematologic findings are described in Chapter 33. The anemia of neoplasia is usually normocytic and normochromic. Iron deficiency anemia secondary to hemorrhage may be present if the tumor has invaded or originated from the gastrointestinal (GI) tract. Besides extensive marrow replacement (myelophthisic anemia), neoplasia may produce anemia with minimal bone involvement or even without any marrow metastases; in these patients, there seems to be some sort of toxic influence on the marrow production and release mechanism. In occasional cases of widespread neoplasm, a hemolytic component (shortened RBC life span) has been demonstrated.

    Multiple myeloma is a neoplasm of plasma cells that is difficult to distinguish for classification purposes from leukemia on one hand and malignant lymphoma on the other. Myeloma initially or eventually involves the bone marrow and produces a moderate normocytic-normochromic anemia. Despite proliferation of plasma cells in the bone marrow, appearance of more than an occasional plasma cell in the peripheral blood is very uncommon. Peripheral blood RBCs often display the phenomenon of rouleau formation, a piling up of RBCs like a stack of coins. This is not specific for myeloma and is most often associated with hyperglobinemia.

    Aplastic anemia. Aplastic anemia is defined as peripheral blood pancytopenia (decrease in RBCs, white blood cells [WBCs], and platelets below population reference range) due to below-normal numbers and function of bone marrow cell precursors without cytologic marrow abnormality or marrow replacement by fibrosis or malignancy. Among the various etiologies are agents that predictably damage the bone marrow (e.g., radiation, certain chemicals such as benzene, and certain cytotoxic antitumor drugs). Another category, sometimes called idiosyncratic or acquired aplastic anemia, includes medications or chemicals that ordinarily do not produce cytopenia. Effects of some medications in this group are dose-related (e.g., chloramphenicol) and in others occur completely unpredictably. A third category of aplasia appears to have some autoimmune component. This includes aplasia (usually temporary) that uncommonly occurs in association with certain viral infections (e.g., parvovirus B-19, Epstein-Barr, rubella, herpes zoster-varicella) and a permanent type rarely seen in non-A, non-B (type C) hepatitis virus infection. A fourth category, probably related to category 3, might include aplasia associated with pregnancy or thymoma (the latter most often affecting RBCs only). The aplastic“crisis” of sickle cell anemia might also fit here. Some of these temporary aplastic crises may be due to parvovirus B-19 infection. A fifth category includes congenital diseases in which aplasia appears with varying frequency, of which the best known are Fanconi’s syndrome and the Diamond-Blackfan syndrome. Finally, some investigators create a more controversial category into which they place certain conditions involving bone marrow that frequently, but not always, develop into typical hematopoietic malignancies. Even more controversial is the status of other hematopoietic or nonhematopoietic malignancies that affect bone marrow function without actual marrow involvement.

    About 50% (in some reports, up to 70%) of aplastic anemia cases are unexplained or the cause is unproven. To make matters even more difficult, in some cases marrow aplasia may develop days or weeks after beginning treatment or exposure to the causative agent; and in some cases it may appear some time after exposure has ceased (in the case of radiation, even years later). Also, certain other conditions, such as hypersplenism, megaloblastic anemia, or marrow replacement by tumor, can simulate aplastic anemia.

    A great variety of drugs and chemicals have been reported to cause idiosyncratic reactions. The effects range from pancytopenia to any combination of single or multiple blood element defects. Bone marrow aspiration usually shows a deficiency in the particular cell precursor involved, although, especially with megakaryocytes, this is not always true. Patients most often recover if they can be supported long enough, although a considerable number die of superimposed infection.

    The drugs most often implicated in idiosyncratic reaction aplastic change are listed here according to blood element defect:

    Pancytopenia. Chloramphenicol (Chloromycetin), phenylbutazone (Butazolidin), indomethacin, mephenytoin (Mesantoin), gold preparations, nitrogen mustard compounds (e.g., busulfan [Myleran]) and other antileukemic drugs. In addition, chloramphenicol may produce the“gray syndrome” in premature infants and newborns.

    Leukopenia. Chlorpromazine (Thorazine), promazine (Sparine), phenylbutazone, thiouracil, antileukemic drugs, sulfonamides.

    Thrombocytopenia. Quinidine, nitrofurantoin (Furadantin), sulfonylureas, chlorothiazide.

    Aplastic anemia is most often normocytic-normochromic. Reticulocyte counts are usually low (although they sometimes are slightly elevated if the patient is in a recovery phase). About one third of aplastic anemia patients have a macrocytic peripheral blood smear.

    As noted, bone marrow aspiration is usually essential for diagnosis and can be used to follow any response to therapy. However, certain problems are associated with this method of diagnosis and must be taken into account. A false impression of marrow hypocellularity may be produced by hemodilution of the marrow specimen, by aspiration at a place that has unusually large amounts of fatty tissue, and by poor slide preparation technique. An occasional completely dry puncture may occur in normal persons due to considerable variability in the bone marrow distribution. Therefore, the diagnosis should never be made on the basis of a single failure to obtain marrow. Also, a bone marrow biopsy specimen, or at least a clot section (clotted marrow aspirate, processed as an ordinary histologic specimen), is more reliable than a smear for estimating cellularity. This is especially true for megakaryocytes. On the other hand, a smear is definitely more valuable for demonstrating abnormal morphology. Both can usually be done at the same time.

    Certain conditions may be associated with episodes of transient bone marrow RBC hypoplasia. These include congenital spherocytosis, sickle cell anemia, and RBC hypoplasia associated with thymoma. Aplastic pancytopenia may occur in paroxysmal nocturnal hemoglobinuria, either preceding onset of the disease or after onset as a transient episode.

    Pancytopenia in children may be caused by Fanconi’s anemia or Diamond-Blackfan congenital hypoplastic anemia. Fanconi’s anemia is an autosomal recessive disorder characterized by pancytopenia and congenital abnormalities such as short stature, web neck, cleft lip, mental retardation, and renal anomalies. More than 10% of peripheral blood lymphocytes display chromosome abnormalities. Anemia may appear in children up to age 10 years with the disease. Diamond-Blackfan syndrome also has an autosomal recessive inheritance pattern and displays congenital anomalies, but it consists of pure RBC aplasia, and onset of anemia occurs either at birth or by age 6 months.

    In children, apparent aplastic anemia or pancytopenia must be differentiated from acute leukemia.