Articles on Medical Diseases and Conditions

Tag: Peripheral blood smear

  • Epstein-Barr Virus (EBV)

    The Epstein-Barr virus is a member of the herpesvirus group and is reported to infect 80% or more of the U.S. population. It is thought to be spread from person to person, most likely through saliva, with the majority of infections occurring in childhood, adolescents, and young adults. The EBV infects B-lymphocytes. In common with the other herpesviruses, once infection (with or without symptoms) takes place the virus eventually becomes dormant but can be reactivated later into clinical disease. Reactivation is said to occur in 15%-20% of healthy persons and in up to 85% in some groups of immunosuppressed patients. Epstein-Barr virus infection in young children is usually asymptomatic. Primary infection by EBV in older children, adolescents, or young adults produces the infectious mononucleosis syndrome in up to 50% of cases. The EBV is also strongly associated with Burkitt’s lymphoma in Africa and nasopharyngeal carcinoma in southern China.

    Infectious mononucleosis (infectious mono; IM)

    Infectious mononucleosis (IM) patients are most often adolescents and young adults, but a significant number are older children and middle-aged or even older adults. When IM is part of a primary infection, the incubation period is 3-7 weeks (range, 2-8 weeks). The acute phase of illness in those patients who are symptomatic lasts about 2-3 weeks (range, 0-7 weeks). Convalescence takes about 4-8 weeks. The most common features of the acute illness are fever, pharyngitis, and adenopathy, with lymph node enlargement occurring in 80%-90% of patients. The posterior cervical nodes are the ones most commonly enlarged. Soft palate petechiae are found in 10%-30% of cases. Jaundice, usually mild, is found in about 5%-10% (range, 4%-45%) of patients in large series. The spleen is mildly enlarged in about 50% of patients (range, 40%-75%) and hepatomegaly is present in about 10% (range, 6%-25%).

    Laboratory findings. Patients usually have normal hemoglobin values. Mild thrombocytopenia is reported in 25%-50% of patients (range, 15%-50%). Leukocytosis between 10,000 and 20,000/ mm3 (10 Ч 109-20 Ч 109/L) occurs in 50%-60% of patients (range, 40%-70%) by the second week of illness. About 10% (range, 5%-15%) of patients develop a leukocytosis over 25,000/mm3 (25 Ч 109/L). However, during the first week there may be leucopenia. About 85%-90% (range, 80%-100%) of patients with IM have laboratory evidence of hepatic involvement (Table 17-2). Peak values are reported to occur 5-14 days after onset of illness for aspartate aminotransferase (AST), bilirubin, and alkaline phosphatase (ALP); and between 7 and 21 days for gamma-glutamyltransferase (GGT). The AST and ALP levels return to normal in nearly all patients by 90 days, but occasionally there may be some degree of GGT elevation persisting between 3-12 months. Total LDH is elevated in about 95% of patients. LDH isoenzyme fractionation by electrophoresis can show three patterns: elevation of all five fractions; elevation of LDH 3, 4, and 5; or elevation of LDH-5 only.

    Liver function tests in EBV-induced infectious mononucleosis

    Table 17-2 Liver function tests in EBV-induced infectious mononucleosis

    Peripheral blood smear. The first of three classic findings is a lymphocytosis, with lymphocytes making up more than 50% of the total white blood cells (WBCs). Lymphocytosis is said to be present in 80%-90% of patients (range, 62%-100%), peaks during the second or third week, and lasts for an additional 2-6 weeks. The second classic criterion is the presence of a “significant number” of atypical lymphocytes on Wright-stained peripheral blood smear. There is disagreement as to whether greater than 10% or greater than 20% must be atypical. These atypical lymphocytes are of three main types (Downey types). Type I has vacuolated or foamy blue cytoplasm and a rounded nucleus. Type II has an elongated flattened nucleus and large amounts of pale cytoplasm with sharply defined borders and often some “washed-out” blue cytoplasm coloring at the outer edge of the cytoplasm. Type III has an irregularly shaped nucleus or one that may be immature and even may have a nucleolus and resemble a blast. All three types are larger than normal mature lymphocytes, and their nuclei are somewhat less dense. Most of the atypical lymphocytes are activated T-lymphocytes of the CD-8 cytotoxic-suppressor type. Some of the Downey III lymphocytes may be EBV-transformed B lymphocytes, but this is controversial. These atypical lymphocytes are not specific for IM, and may be found in small to moderate numbers in a variety of diseases, especially cytomegalovirus and hepatitis virus acute infections. In addition, an appearance similar to that of the type II variety may be created artificially by crushing and flattening normal lymphocytes near the thin edge of the blood smear. IM cells are sometimes confused with those of acute leukemia or disseminated lymphoma, although in the majority of cases there is no problem.

    Although most reports state or imply that nearly all patients with IM satisfy the criteria for lymphocytosis and percent atypical lymphocytes, one study found only 55% of patients had a lymphocytosis and only 45% had more than 10% atypical lymphocytes on peripheral smear when the patients were first seen. Two studies found that only about 40% of patients with IM satisfied both criteria.

    Serologic tests. The third criterion is a positive serologic test for IM either based on heterophil antibodies or specific anti-EBV antibodies. The classic procedure is the heterophil agglutination tube test (Paul-Bunnell test). Rapid heterophil antibody slide agglutination tests have also been devised. Slide tests now are the usual procedure done in most laboratories. However, since the basic principles, interpretation, and drawbacks of the slide tests are the same as those of the older Paul-Bunnell tube test, there are some advantages in discussing the Paul-Bunnell procedure in detail.

    Serologic tests based on heterophil antibodies.

    Paul-Bunnell antibody is an IgM-type antibody of uncertain origin that is not specific for EBV infection but is seldom found in other disorders (there are other heterophil antibodies that are not associated with EBV infection). Paul-Bunnell antibodies begin to appear in the first week of clinical illness (about 50% of patients detectable; range, 38%-70%), reaching a peak in the second week (60%-78% of patients positive) or third (sometimes the fourth) week (85%-90% positive; range, 75%-100%), then begin to decline in titer during the fourth or fifth week, most often becoming undetectable 8-12 weeks after beginning of clinical illness. However in some cases some elevation is present as long as 1-2 years (up to 20% of patients). In children less than 2 years old, only 10%-30% develop heterophil antibodies; about 50%-75% of those 2-4 years old develop heterophil antibodies. One report states that these antibodies are rarely elevated in Japanese patients of any age. Once elevated and returned to undetectable level, heterophil antibodies usually will not reelevate in reactivated IM, although there are some reports of mild heterophil responses to other viruses.

    The original Paul-Bunnell test was based on the discovery that the heterophil antibody produced in IM would agglutinate sheep red blood cells (RBCs). In normal persons the sheep cell agglutination titer is less than 1:112 and most often is almost or completely negative. The Paul-Bunnell test is also known as the “presumptive test” because later it was found that certain antibodies different from those of IM would also attack sheep RBCs. Examples are the antibodies produced to the Forssman antigen found naturally in humans and certain other animals and the antibody produced in “serum sickness” due to certain drug reactions. To solve this problem the differential absorption test (Davidsohn differential test) was developed. Guinea pig kidney is a good source of Forssman antigen. Therefore, if serum containing Forssman antibody is allowed to come in contact with guinea pig kidney material, the Forssman antibody will react with the kidney antigen and be removed from the serum when the serum is taken off. The serum will then show either a very low or a negative titer, whereas before it was strongly positive. The IM heterophil antibody is not significantly absorbed by guinea pig kidney but is nearly completely absorbed by bovine (beef) RBCs, which do not significantly affect the Forssman antibody. The antibody produced in serum sickness will absorb both with beef RBCs and guinea pig kidney.

    The level of Paul-Bunnell titer does not correlate well with the clinical course of IM. Titer is useful only in making a diagnosis and should not be relied on to follow the clinical course of the disease or to assess results of therapy.

    In suspected IM, the presumptive test is performed first; if necessary, it can be followed by a differential absorption procedure.

    “Spot” tests were eventually devised in which the Paul-Bunnell and differential absorption tests are converted to a rapid slide agglutination procedure without titration. Most of the slide tests use either horse RBCs, which are more sensitive than sheep RBCs, or bovine RBCs, which have sensitivity intermediate between sheep and horse RBC but which are specific for IM heterophil antibody and therefore do not need differential absorption. Slide test horse cells can also be treated with formalin or other preservatives that extend the shelf life of the RBC but diminish test sensitivity by a small to moderate degree.

    Heterophil-negative infectious mononucleosis.

    This term refers to conditions that resemble IM clinically and show a similar Wright-stained peripheral blood smear picture, but without demonstrable elevation of Paul-Bunnell heterophil antibody (see the box) About 65% (range, 33%-79%) are CMV infection, about 25% (15%-63%) are heterophil-negative EBV infections, about 1%-2% are toxoplasmosis, and the remainder are other conditions or of unknown etiology.

    Diagnosis of infectious mononucleosis. When all three criteria for IM are satisfied, there is no problem in differential diagnosis. When the results of Paul-Bunnell test or differential absorption test are positive, most authors believe that the diagnosis can be made, although there are reports that viral infections occurring after IM can cause anamnestic false positive heterophil reelevations. When the clinical picture is suggestive of IM but results of the Paul-Bunnell test, differential absorption procedure, or the spot test are negative, at least one follow-up specimen should be obtained in 14 days, since about 20%-30% of IM patients have negative heterophil test results when first seen versus 10%-15% negative at 3 weeks after onset of clinical symptoms (although the usual time of antibody appearance is 7-10 days, it may take as long as 21 days and, uncommonly, up to 30 days). About 10% (range, 2%-20%) of patients over age 5 years and 25%-50% or more under age 5 years never produce detectable heterophil antibody. Another potential problem is that several evaluations of different heterophil kits found substantial variation in sensitivity between some of the kits. If the clinical picture is typical and the blood picture is very characteristic (regarding both number and type of lymphocytes), many believe that the diagnosis of IM can be considered probable but not established. This may be influenced by the expense and time lapse needed for specific EBV serologic tests or investigation of CMV and the various other possible infectious etiologies.

    Some “Heterophil-Negative” Mononucleosis Syndrome Etiologies

    Viruses

    EBV heterophil-negative infections
    Cytomegalovirus
    Hepatitis viruses
    HIV-1 seroconversion syndrome
    Other (rubella, herpes simplex, herpesvirus 6, mumps, adenovirus)

    Bacteria

    Listeria, tularemia, brucellosis, cat scratch disease, Lyme disease, syphilis, rickettsial diseases

    Parasites

    Toxoplasmosis, malaria

    Medications

    Dilantin, azulfidine, dapsone, “serum sickness” drug reactions

    Other

    Collagen diseases (especially SLE, primary or drug-induced)
    Lymphoma
    Postvaccination syndrome
    Subacute bacterial endocarditis (SBE)

    In summary, the three classic criteria for the diagnosis of IM are the following:

    1. Lymphocytes comprising more than 50% of total WBC count.
    2. Atypical lymphocytes comprising more than 10% (liberal) or 20% (conservative) of the total lymphocytes.
    3. Significantly elevated Paul-Bunnell test and/or differential absorption test result. A positive slide agglutination test result satisfies this criterion.

    Serologic tests based on specific antibodies against EBV. The other type of serologic test for IM detects patient antibodies against various components of the EBV (Table 17-3). Tests are available to detect either viral capsid antigen-IgM or IgG (VCA-IgM or IgG) antibodies. VCA-IgM antibody is usually detectable less than one week after onset of clinical illness and becomes nondetectable in the late convalescent stage. Therefore, when present it suggests acute or convalescent EBV infection. Rheumatoid factor (RF) may produce false positive results, but most current kits incorporate some method to prevent RF interference. VCA-IgG is usually detectable very soon after VCA-IgM, but remains elevated for life after some initial decline from peak titer. Therefore, when present it could mean either acute, convalescent, or old infection. Tests are available for Epstein-Barr nuclear antigen (EBNA) IgM or IgG antibody, located in nuclei of infected lymphocytes. Most kits currently available test for IgG antibody (EBNA-IgG or simply EBNA). EBNA-IgM has a time sequence similar to that of VCA-IgM. The more commonly used EBNA-IgG test begins to rise in late acute stage (10%-34% positive) but most often after 2-3 weeks of the convalescent stage. It rises to a peak after the end of the convalescent stage (90% or more positive), then persists for life. Elevated EBNA/EBNA-IgG is suggestive of nonacute infection when positive at lower titers and older or remote infection at high or moderately high titer. A third type of EBV test is detection of EBV early antigen (EA), located in cytoplasm of infected cells. There are two subtypes; in one the antigen is spread throughout the cytoplasm (“diffuse”; EA-D) and in the other, the antigen is present only in one area (“restricted”; EA-R). The EA-D antibody begins to rise in the first week of clinical illness, a short time after the heterophil antibody, then peaks and disappears in the late convalescent stage about the same time as the heterophil antibody. About 85% (range, 80%-90%) of patients with IM produce detectable EA-D antibody, which usually means EBV acute or convalescent stage infection, similar to VCA-IgM or heterophil antibody. However, EA-D may rise again to some extent in reactivated EBV disease, whereas VCA-IgM does not (whether heterophil antibody ever rises is controversial, especially since it may persist for up to a year or even more in some patients). EA-D is typically elevated in EBV-associated nasopharyngeal carcinoma. EA-R is found in about 5%-15% of patients with clinical IM. It is more frequent (10%-20%) in children less than 2 years old with acute EBV infection and is typically elevated in patients with EBV-related Burkitt’s lymphoma. Expected results from the various serologic tests in different stages of EBV infection are summarized in Table 17-3 and Fig. 17-9.

    Antibody tests in EBV infection

    Table 17-3 Antibody tests in EBV infection

    Tests in EBV infection

    Fig. 17-9 Tests in EBV infection.

    Specific serologic tests for EBV are relatively expensive compared to heterophil antibody tests and are available predominantly in university centers and large reference laboratories. Such tests are not needed to diagnose IM in the great majority of cases. The EBV tests are useful in heterophil-negative patients, in problem cases, in patients with atypical clinical symptoms when serologic confirmation of heterophil results is desirable, and for epidemiologic investigations. If the initial heterophil test is nonreactive or equivocal, it is desirable to freeze the remainder of the serum in case specific EBV tests are needed later.

    Summary of Epstein-Barr Antibody Test Interpretation
    Never infected (susceptible) = VCA-IgM and IgG both negative.
    Presumptive primary infection = Clinical symptoms, heterophil positive.
    Primary infection: VCA-IgM positive (EBNA-IgG negative; heterophil positive or negative)
    Reactivated infection: VCA-IgG positive; EBNA-IgG positive; EA-D positive (heterophil negative, VCA-IgM negative)
    Old previous infection: VCA-IgG positive; EBNA-IgG positive; EA-D negative (VCA-IgM negative, heterophil negative).

  • 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.

  • Laboratory Tests for Chronic Iron Deficiency

    Several laboratory tests are commonly used to screen for or establish a diagnosis of chronic iron deficiency. The sequence in which abnormal test results appear is given in the box below. In a normal adult on a normal diet made iron deficient by repeated phlebotomy (for experimental reasons), it takes about 3 months before significant anemia (Hb more than 2 gm/dl below normal) appears. The first laboratory indication of iron deficiency is lack of marrow iron on bone marrow aspiration. The next test to become abnormal is the serum iron level. When anemia becomes manifest, it is moderately hypochromic but only slightly microcytic; marked hypochromia and microcytosis are relatively late manifestations of iron deficiency. When the anemia is treated, results of these tests return to normal in reverse order. Even with adequate therapy it takes several months before bone marrow iron appears again.

    Peripheral blood smear. The peripheral blood smear in chronic iron deficiency anemia typically shows RBC hypochromia. There is also microcytosis, but lesser degrees of microcytosis are more difficult to recognize than hypochromia. Some of the peripheral blood changes may appear before actual anemia. However, examination of the peripheral blood smear cannot be depended on to detect iron deficiency, since changes suggestive of chronic iron deficiency either may not be present or may be missed. In one study the peripheral blood smear did not show typical RBC changes in as many as 50% of patients with chronic iron deficiency anemia. This happens more often in patients with mild anemia. Even if hypochromia is present, iron deficiency must be differentiated from other conditions that also may produce hypochromic RBC. In severe anemia there is anisocytosis and poikilocytosis in addition to microcytosis and rather marked hypochromia. The anisocytosis means that the RBCs may not all be microcytes. The microcytes of iron deficiency must be differentiated from spherocytes; such distinction is usually not difficult, since in chronic iron deficiency even the microcytes are hypochromic. Bone marrow aspiration reveals mild erythroid hyperplasia and no marrow iron (using iron stains).

    Hypochromic anemias

    Table 3-1 Hypochromic anemias

    Sequence of Test Abnormalities in the Evolution of Chronic Iron Deficiency

    EARLY PRECLINICAL CHANGES
    Negative iron balance
    Decreased bone marrow hemosiderin
    Decreased serum ferritin

    LATER PRECLINICAL CHANGES
    Increased RBC protoporphyrin levels
    Increased total iron-binding capacity
    Decreased serum iron

    RELATIVELY LATE CHANGES
    RBC microcytosis
    RBC hypochromia
    Anemia

    Red blood cell indices. The mean corpuscular volume (MCV) typically is decreased below reference range lower limits, and the RBC distribution width (RDW) is increased by the time iron deficiency anemia has appeared. However, the few studies available indicate that the MCV is normal in about 30%5% (range 24%-55%) of patients. The mean corpuscular hemoglobin (MCH) value is normal in about 20%. There is considerable disagreement on MCH concentration (MCHC) values, with a decrease reported in 21%-81% of patients. Although various factors could have biased these studies, it is probable that chronic iron deficiency will not be detected by RBC indices in a significant number of patients with chronic iron deficiency anemia. Some of these patients, but not all, may have other superimposed conditions that mask the morphologic effects of iron deficiency. Even if the MCV is decreased, iron deficiency must be differentiated from various other conditions that also produce microcytosis.

    Reticulocytes. The reticulocyte count is normal in uncomplicated chronic iron deficiency anemia. Superimposed acute blood loss or other factors, such as adequate iron in the hospital diet, may cause reticulocytosis. For a short time following recent (acute) hemorrhage, the Wintrobe MCV may be normal or even increased due to the reticulocytosis. The reticulocyte response to iron therapy (3%-7%) is somewhat less than that seen with treatment of megaloblastic anemia.

    Serum iron. Serum iron levels fall sometime between depletion of tissue iron stores and development of anemia. Therefore, the serum iron value should be a sensitive indicator of possible iron deficiency by the time a patient has anemia. Unfortunately, about 10%-15% (literature range 0%2%) of serum iron measurements in patients with iron deficiency anemia remain in the lower half of the reference range.

    CONDITIONS THAT AFFECT SERUM IRON LEVELS. The first is transferrin levels. Serum iron measurement predominantly reflects iron bound to serum proteins. Under usual conditions, most iron is bound to transferrin. Normally, transferrin is about one-third saturated. Therefore, serum iron values depend not only on the quantity of iron available but also on the amount of transferrin present. (If transferrin is increased, the serum iron measurement reflects not only the quantity of iron bound to the normal amount of protein but also the iron bound to the additional protein. The opposite happens when transferrin is decreased.) Second is the time of day. There is a 20%0% diurnal variation in serum iron levels (literature range 2%-69%); the time of day at which the peak value appears is most often in the morning, but it may occur in the early or late afternoon. In one study the peak was found at 8 A in 72% of 25 patients and at 4 P in 28%. Therefore, in some patients the time of day that the specimen is obtained can materially influence whether a result is interpreted as mildly decreased or still within the lower reference range. Third, it has also been found that serum iron displays considerable day-to-day variation among individuals, with changes averaging 20%0% but in some cases varying over 100%. Finally, in some cases there may be some degree of iron contamination of laboratory materials.

    SERUM IRON DECREASE IN VARIOUS CONDITIONS. Serum iron levels may be decreased in other conditions besides iron deficiency; the most frequent is probably the anemia associated with severe chronic disease such as the rheumatoid-collagen diseases, extensive malignancy, uremia, cirrhosis, and severe chronic infection (Table 3-1). There is usually a slight increase in serum iron levels in the first trimester of pregnancy, since increased estrogens tend to increase transferrin. However, by the third trimester the effect of estrogens is reversed, partially by hemodilution but also from utilization of maternal iron by the fetus. This leads to a decrease in serum iron in the third trimester. Severe stress (surgery, infection, myocardial or cerebral infarction) frequently produces a considerable decrease in serum iron (in one study by an average of 65% with a range of 38%-93%), which begins within 24 hours of the onset of the stress (sometimes as early as 4-6 hours). Its nadir occurs between 24 and 48 hours, and recovery begins toward baseline about 6-7 days after the original decrease.

    SERUM IRON INCREASE. Serum iron levels may be increased in hemolytic anemia, iron overload conditions, estrogen therapy (due to an increase in transferrin), acute hepatitis, and parenteral iron therapy. The effects of intramuscular iron-dextran (Imferon) administration persist for several weeks. The serum iron level is normal or increased in thalassemia minor without coexisting iron deficiency(Table 3-2 and Table 37-2).

    Serum iron not total iron-binding capacity patterns

    Table 3-2 Serum iron not total iron-binding capacity patterns

    SERUM IRON IN MEGALOBLASTIC ANEMIA. When megaloblastic anemia is treated, the serum iron level temporarily falls resulting from marked utilization of previously unused available iron. On the other hand, a significant minority of patients with megaloblastic anemia (20%-40%) have coexisting iron deficiency that eventually will be unmasked by correction of the folate or B12 deficiency. Since megaloblastic anemia can interfere with interpretation of tests for iron deficiency, it has been recommended that follow-up studies be done 1 months after the beginning of folate or B12 therapy to rule out iron deficiency.

    Serum total iron-binding capacity. Serum total iron-binding capacity (TIBC) is an approximate estimate of serum transferrin. Assay is usually performed by adding an excess of iron to serum to saturate serum transferrin, removing all iron not bound to protein, and then measuring the serum iron (which is assumed to be mostly bound to transferrin under these conditions). Since transferrin is not the only protein that can bind iron, the TIBC is not an exact measurement of transferrin and tends to be even less representative in cases of iron overload and certain other conditions.

    Serum TIBC is increased in uncomplicated chronic iron deficiency, most studies indicating abnormality at the same time as a decrease in serum iron levels or even before. Unfortunately, the TIBC is not elevated above reference limits in 30%-40% (29%-68%) of patients with chronic iron deficiency anemia. In the best-known study published, 69% of iron deficiency anemia patients with low serum iron levels had an elevated TIBC, 11% had a TIBC within reference limits, and an additional 21% had decreased TIBC values. Transferrin is a “negative” acute-phase reaction protein and decreases both with various acute diseases and with severe chronic diseases (the same chronic diseases that decrease serum iron levels). Decrease in transferrin depresses TIBC to low or low-normal levels. Hypoproteinemia and iron overload conditions are also associated with a decreased TIBC. Unfortunately, conditions that decrease TIBC can mask the TIBC elevation of coexisting chronic iron deficiency. Some conditions increase transferrin levels and therefore increase TIBC; these include pregnancy, estrogen therapy, alcoholism, and acute hepatitis (Table 3-2 and Table 37-2).

    Transferrin saturation. The textbook pattern of iron tests in chronic iron deficiency shows a decrease in serum iron levels and an increase in TIBC. This will increase the unsaturated binding capacity of transferrin and decrease the percent of transferrin that is bound to iron (percent transferrin saturation, or %TS). A %TS of 15% or less is the classic finding in chronic iron deficiency anemia. The %TS is said to be a more sensitive screening test for chronic iron deficiency than either serum iron levels or the TIBC, since a decreased serum iron level that still remains in the lower end of the reference range plus a TIBC still in the upper end of the TIBC reference range may produce a %TS below 15%. A decrease in %TS is also found in many patients with anemia of chronic disease, so that decreased %TS is not specific for iron deficiency. Also, about 15% (10%4%) of patients with iron deficiency have a %TS greater than 15%, especially in the early stages or when iron deficiency is superimposed on other conditions. The %TS is increased in hemolytic or megaloblastic anemia, sideroblastic anemia, and iron overload states and is normal or increased in thalassemia minor (Table 3-2; a more complete list of conditions that affect TIBC and %TS is included in Table 37-2).

    Serum ferritin. Ferritin is the major body iron-storage compound. Routine tissues or bone marrow iron stains, however, detect hemosiderin but not ferritin. Ferritin in serum can be measured by radioassay or enzyme immunoassay. A serum ferritin level decrease accompanies a decrease in tissue ferritin level, which, in turn, closely mirrors decrease of body iron stores in iron deficiency. The decrease in tissue ferritin occurs before changes in serum iron tests, changes in RBC morphology, or anemia. Except for bone marrow iron stains, serum ferritin is currently the most sensitive test available for detection of iron deficiency. The major factors that modify its efficacy as an indicator involve the technical aspects of present-day ferritin immunoassay kits, some of which have less than desirable reproducibility and accuracy at the low end of the reference range. A major reason for this is the fact that the lower edge of the reference range (20-150 ng/ml or µg/L) is not far from zero. Another problem is the extreme difficulty most laboratories have in establishing their own ferritin reference range, since there is no good way to exclude subclinical iron deficiency from the clinically “normal” population without performing bone marrow aspiration. A third problem, partially arising from inadequately validated reference ranges, is disagreement in the literature as to what cutoff level should be used to confirm or exclude iron deficiency. The majority of investigators use 12 ng/ml as the cutoff level (literature range 10-20 ng/ml). A fourth problem (discussed later) is increase in ferritin levels by various conditions that may coexist with iron deficiency.

    Ferritin levels at birth are very high and are the same for boys and girls. Ferritin values decrease rapidly by age 3 months and reach their lowest point at about age 9 months. At some time during the teenage years the reference ranges for boys and girls being to diverge somewhat, with the lower limit of the reference range for girls being approximately 10 ng/100 ml lower than that for boys. The upper limit in men tends to increase slowly until old age, whereas the upper limit in women tends to remain relatively stationary until menopause and then slowly increases. The lower limits of reference ranges for both sexes are affected only to a small degree by age. There is approximately a 10%-15% average daily variation in ferritin values in the same individual; about one half the variation is due to fluctuation in serum iron values.

    INTERPRETATION OF SERUM FERRITIN RESULTS. A serum ferritin level less than 12 ng/ml is considered almost diagnostic of iron deficiency. Presumably false positive results in the literature based on bone marrow iron stains (displaying decreased serum ferritin levels with bone marrow iron present) range from 0%-4% of cases. False negative results have been reported in 2.6% of bone marrow-proven uncomplicated iron-deficient cases. However, if iron deficiency coexists with a condition that raises the serum ferritin, the ferritin value in a substantial number of patients may be higher than the cutoff value for iron deficiency. Serum ferritin level is decreased to a variable degree during pregnancy; the amount of decrease may be reduced as much as 50% if iron supplements are given.

    Many conditions can elevate ferritin levels. Serum ferritin is one of a group of proteins that become elevated in response to acute inflammation, infection, or trauma; elevation begins between 24 and 48 hours, peaks in about 3 days, and lasts 5 days to 5 weeks. In addition, a more sustained increase in ferritin levels may be produced by various chronic diseases (see Table 3-1), including those that decrease serum iron and serum TIBC values. Fortunately, some patients with coexisting chronic disease and iron deficiency still have decreased serum ferritin levels. Ferritin values may also be increased in some patients who have had blood transfusions, in megaloblastic anemia, and in hemolytic anemias. Ferritin is greatly increased in iron overload states such as hemochromatosis and acute iron poisoning. One study reports that about one third of patients with chronic hepatitis virus had elevated serum ferritin and some also had elevated serum iron and TIBC, simulating hemochromatosis.

    The serum ferritin level has been used in chronic renal failure to monitor iron status. Because chronic disease raises serum ferritin levels, the ferritin lower limit used for this purpose (approximately 100 ng/ml) is much higher than the lower limit of reference range used for the general population.

    Free erythrocyte protoporphyrin (zinc protoporphyrin). This test is discussed in detail in Chapter 35. The last step in heme synthesis occurs when the heme precursor protoporphyrin IX forms a complex with an iron atom with the help of the enzyme ferrochelatase (see Fig. 34-1). If iron is not available, or if ferrochelatase is inhibited (as occurs in lead poisoning), a zinc ion becomes complexed with protoporphyrin IX (zinc protoporphyrin; ZPP) instead of iron. When ZPP is assayed using manual biochemical techniques, the zinc ion is removed during acid extraction of RBC hemoglobin, and the metal-free substance measured is then called free erythrocyte protoporphyrin. Zinc protoporphyrin can be measured directly and quickly using one or two drops of whole blood by means of a small commercially available instrument called a hematofluorometer.

    ZINC PROTOPORPHYRIN IN IRON DEFICIENCY. Zinc protoporphyrin levels are elevated in iron deficiency and in lead poisoning. In iron deficiency, ZPP levels become elevated after several weeks of deficient iron stores and return to normal only after 2 to 3 months of iron therapy. In two studies, elevated ZPP levels detected 83%-94% of patients who were iron deficient on the basis of low serum ferritin levels.

    PROBLEMS WITH ZINC PROTOPORPHYRIN ASSAYS. Some hematofluorometers report ZPP per unit of whole blood; this reporting system may be affected by changes in hematocrit values. This problem is avoided with instruments that report results as a ZPP/heme ratio. Another potential difficulty is falsely decreased results due to a shift in the protoporphyrin fluorescent maximal absorption peak if the Hb is not fully oxygenated. This can be avoided in several ways. More troublesome is ZPP elevation by acute or chronic infections, noninfectious inflammation, various malignancies, chronic liver disease, and moderate or severe hemolytic anemias. Therefore, ZPP levels are elevated in many of the same conditions that falsely elevate serum ferritin levels. Although ZPP is a good screening method for iron deficiency and lead poisoning, most laboratories do not own a hematofluorometer.

    Bone marrow iron stain. The gold standard for chronic iron deficiency has been bone marrow aspiration or biopsy with Prussian blue chemical reaction for iron (hemosiderin). Although there is some disagreement, a clot section is generally considered more reliable for iron staining than an aspiration smear. Bone biopsy specimens must be decalcified, and some decalcifying reagents (but not others) may destroy some iron. The major problem with bone marrow aspiration has been reluctance of patients to undergo the procedure. Occasionally, bone marrow aspiration may be necessary to diagnose patients with hypochromic anemia without clear-cut evidence from other tests for or against iron deficiency. However, a therapeutic trial of iron might provide the same information.