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.