Articles on Medical Diseases and Conditions

Tag: Laboratory Tests

  • Coagulation Pathway Factors

    In this section the various coagulation pathway factors are discussed individually, with emphasis on abnormalities for which laboratory tests are useful.

    Factor I (fibrinogen)

    Fibrinogen is a glycoprotein that is synthesized in the liver, the liver being a major source of many coagulation factors. Conversion of fibrinogen to fibrin under the influence of thrombin is the final major step in coagulation. Besides its role in coagulation, fibrinogen is a risk factor for coronary heart disease and stroke. Increased plasma fibrinogen most often is temporary and involves the important role it plays as part of the body’s “acute-reaction” response to trauma or to onset of a variety of severe illnesses. This initial response to illness results in fibrinogen production increase and therefore serum level increase. Fibrinogen levels also can be increased by cigarette smoking and by genetic influences. Decreased plasma fibrinogen levels may occurfrom decreased liver production, from the action of fibrinolysins (enzymes thatdestroy fibrin and may attack fibrinogen), and from conversion of fibrinogen tofibrin that is too extensive to permit adequate replacement of the fibrinogen. Decreased fibrinogen production is usually due to liver cell damage, as occurs in acute hepatitis or cirrhosis. However, production mechanisms are efficient, and a severe degree of liver damage is required before significant hypofibrinogenemia develops. Fibrinolysins may be primary or secondary. Primary fibrinolysins are rare and usually attack only fibrinogen. Secondary fibrinolysins are more common and attack primarily fibrin but can attack fibrinogen. Fibrinolysins are discussed in more detail in the section on anticoagulants. The major etiology of hypofibrinogenemia other than severe liver disease is fibrinogen depletion caused by DIC.

    Current therapeutic sources of fibrinogen are fresh frozen plasma or cryoprecipitate (Chapter 10).

    Factor II (prothrombin)

    Prothrombin is a proenzyme synthesized by liver cells. That portion of the prothrombin molecule that permits the molecule to function in the coagulation pathway is formed with the help of vitamin K. Without vitamin K a variety of prothrombin molecules are produced that are inactive. Vitamin K exists in two forms, one of which is preformed in green vegetables and certain other foods and oneof which is synthesized by GI tract bacteria. Both forms are fat soluble and depend on bile salts for absorption. A deficiency of vitamin K may thus result from malabsorption of fat (as seen with lack of bile salts in obstructive jaundice, or primary malabsorption from sprue; see Chapter 26) or from failure of GI tract bacteria to synthesize this substance (due to prolonged oral antibiotic therapy). It usually takes more than 3 weeks before the body vitamin K stores are exhausted and the deficiency of available vitamin K becomes manifest. Dietary lack may be important but ordinarily does not causea severe enough deficiency to produce clinical abnormality unless other factors(e.g., the anticoagulant vitamin K inhibitors) are present.

    Neonatal hemorrhage due to vitamin K deficiency may occur during the first 24 hours of life, from 1-7 days after birth, and uncommonly, 1-3 months later. Neonatal deficiency may be idiopathic or associated with certain maternal medications (anticonvulsants, warfarin, antibiotic therapy). It has been reported that up to 3% of infants at birth are vitamin K deficient. Thereafter, dietis important. Breast milk and certain infant milk formulas are relatively low in vitamin K and predispose to clinical deficiency. One dose of vitamin K at birth is frequently used as a prophylactic measure.

    Assuming that normal supplies of vitamin K are available, the other main limiting factor in prothrombin formation is the ability of the liver to synthesizeit. In severe liver disease (most often far-advanced cirrhosis), enough parenchyma is destroyed to decrease prothrombin formation in a measurable way, eventually leading to a clinical coagulation defect. Whereas a deficiency of availablevitamin K responds promptly to administration of parenteral vitamin K, hypoprothrombinemia due to liver parenchymal disease responds poorly, if at all, to parenteral vitamin K therapy.

    Vitamin K is also necessary for synthesis of factors VII, IX, and X. The greatest effect seems to be on prothrombin and factor VII.

    Factor III (tissue thromboplastin)

    Tissue thromboplastin is composed of phospholipids and lipoproteins and can be extracted from most tissues. Tissue thromboplastin forms a complex with factor VII and calcium ions that initiates the extrinsic coagulation pathway. This complex is used as the reagent for the PT test.

    Factor IV (calcium ions)

    Calcium is necessary as a cofactor in several steps of the coagulation pathway. The common hematology and blood bank anticoagulants oxalate, ethylenediamine tetraacetic acid (EDTA), and citrate exert their anticoagulant effect by blocking calcium function through chelation of the calcium ions.

    Factor V (labile factor)

    Factor V is synthesized by the liver and is decreased in severe liver disease. Factor V is found in plasma but not in serum. Factor V is unstable and becomes markedly reduced in 2-3 days when refrigerated anticoagulated blood is stored in the blood bank. Refrigeration helps preserve factor V in laboratory specimen plasma, but even at 4°C factor V is considerably reduced within 24 hours, and freezing soon after the specimen is obtained is necessary to maintain activity. In factor V deficiency the PT is abnormal; the APTT test is abnormal in severe deficiency but may be normal in mild deficiency.

    Factor VII (stable factor)

    Factor VII is synthesized in the liver and is dependent on vitamin K for itsactivity. The biologic half-life of factor VII is only 4-6 hours, making it theshortest lived of the vitamin K–dependent coagulation factors. The coumarin vitamin K antagonists produce a more rapid and profound depressive effect onfactor VII than on any of the other vitamin K–dependent factors (discussed at greater length in the section on Coumadin anticoagulants). Factor VII is present in plasma and in serum and is stable in both. In factor VII deficiency the PT is abnormal; the APTT and bleeding time are normal.

    Factor VIII/von Willebrand factor complex

    Nomenclature of Factor VIII/von Willebrand factor (factor VIII/vWF) complex has been confusing. Until recently, the entire complex was known as “Factor VIII.” In 1985 the International Committee on Thrombosis and Haemostasis proposed a new nomenclature system that is adopted here. Research onFactor VIII over many years demonstrated that the factor was not a single molecule but a complex with at least two components. One is the substance whose deficiency produces classic hemophilia. This component previously was called antihemophilic globulin (AHG) and is now called factor VIII (or antihemophilic factor). For coagulation test purposes, factor VIII (antihemophilic factor) is separated into (1) the factor protein molecule, and (2) its coagulant activity. Antibodies have been produced against the factor VIII protein, and this antigen is now called factor VIII antigen, or VIII:Ag (formerly this was designated VIII C:Ag). The coagulant activity of factor VIII is now designated VIII:C. The secondcomponent of the complex is associated with von Willebrand’s disease and is called von Willebrand factor, or vWf (formerly, this was called factor VIII-related antigen, or VIIIR). Antibodies have been produced against von Willebrandfactor protein, and this antigen is now called von Willebrand factor antigen, or vWf:Ag (formerly, this was called VIII R:Ag). In addition, the von Willebrandfactor has at least two coagulant actions or activities: an effect on platelet adhesion demonstrated by the glass bead column retention test and an effect on platelet aggregation demonstrated by the ristocetin test. The von Willebrand–ristocetin effect on platelets was formerly called the von Willebrand ristocetin cofactor and designated VIIIR:RC; but in the new nomenclature system no formal name or abbreviation is assigned to any vWf function such as the ristocetin cofactor or the vWf effect on platelet adhesion. The site where factor VIII is synthesized in humans is not known, but the von Willebrand factor is apparently synthesized in endothelial cells and megakaryocytes.

    The hemorrhagic disease known as hemophilia is usually associated with factor VIIIC deficiency. A similar disease may result from factor IX deficiency (anda similar, but milder, disease from factor XI (deficiency), so factor VIIIC deficiency is sometimes referred to as hemophilia A and factor IX deficiency as hemophilia B. About 90% of clinical hemophilia cases are due to hemophiliaA and about 9% to hemophilia B.

    The gene for factor VIII is located on the long arm of the female sex chromosome (Xq28) and is transmitted as a sex-linked recessive trait. Females with the hemophilia A gene are usually carriers (heterozygous, xX), and males with thegene have clinical disease (xY, the hemophilic x gene not being suppressed by anormal X gene as it is in a carrier female). However, about one third of cases are due to fetal gene mutation, not gene inheritance. The gene for vWf is usually transmitted as an autosomal incomplete dominant trait and uncommonly as an autosomal recessive trait. Rare cases of x-linked recessive inheritance have also been reported.

    The VIII/vWf complex is found in plasma but not in serum. Both VIII:C and vWf activity (or vWf:RC) are unstable. Refrigeration helps preserve factor VIII activity (VIII:C), but even at 4°C the coagulant activity decreases considerably within 24 hours in laboratory plasma specimens. Freezing maintains factor VIII:C activity if it is done within a short time after the specimen is obtained.

    Differentiation of hemophilia A and von Willebrand’s disease. In hemophilia A, male patients have decreased factor VIII coagulant activity, whereas vWf coagulant activity, as measured by platelet function tests, is normal. Factor VIII:Ag is decreased by antibody techniques, whereas vWf: Ag levels are normal. The majority of female hemophilia A carriers have also been shownto have reduced VIII:Ag levels, although usually not depressed to a marked degree (since one chromosome has a normal X gene and apparently can ensure some production of factor VIII). In contrast to hemophilia, patients with classic von Willebrand’s disease have reduced VIII:C activity and vWf activity, both reduced about equal in degree, and demonstrate corresponding decreases in both antigens using antibody techniques. Further discussion of von Willebrand’s disease will be deferred to the section on platelet disorders.

    CLINICAL ASPECTS OF HEMOPHILIA A. Hemophilia A exists in varying degrees of factor VIII:C activity deficiency that roughly correlate with clinical severity. Severe (classic) hemophiliacs have 1% or less of normal factor VIII:C activity levels on assay, moderate hemophiliacs have 2%-5%, and mild hemophiliacs have 5%-25% of normal levels. Hemophilia with levels between 25% and 50% of normal is sometimes called subhemophilia or borderline hemophilia. In one study, 55% (literature range, 40%-70%) of factor VIII:C–deficient male patients had classic (severe) hemophilia, about 25% had moderately severe disease, and about 20% had mild disease. Most carrier females have about 50% of normal factor VIII:C activity. About one third of carrier females have factor VIII:C activitylevels similar to those in the borderline decreased group, although usually thecarriers are asymptomatic. About 5% of female carriers have been reported to have mild clinical disease with factor VIII:C activity also in the mildly decreased range; (both X chromosomes affected) and rare homozygous females withclassic disease have been reported.

    The major symptom of hemophilia is excessive bleeding. Most bleeding episodes follow trauma. In classic cases the trauma may be very slight. In mild cases the trauma must usually be more significant, such as a surgical operation or dental extraction. Bleeding into joints (hemarthrosis) is a characteristic finding in classic and moderate factor VIII:C activity deficiency, with the more repeated and severe hemorrhagic episodes being found in the more severely factor-deficient patients. Bleeding from the mouth, urinary tract, and GI tract and intracranial hemorrhage are also relatively common in severe cases. In mild cases there may be only an equivocal history of excessive bleeding or no history at all before severe trauma or a surgical procedure brings the condition to light. Another complication of severe hemophilia is hepatitis virus infection from factor replacement therapy. Before laboratory screening of donor blood was possible, transmission of human immunodeficiency virus-I (HIV-I) infection through blood product transfusion affected many hemophiliacs.

    The biologic half-life of factor VIII:C activity is approximately 8-12 hours. Factor VIII:C activity is unstable, and in freshly drawn and anticoagulated blood stored in the refrigerator the activity decreases to 40% by 24 hours. The current therapeutic sources of factor VIII:C include fresh frozen plasma, factor VIII:C concentrate, and cryoprecipitate (Chapter 10). Factor VIII:C in therapeutic material is measured in terms of units that equal the amount of factor VIII:C activity in 1 ml of normal plasma. As a rule of thumb, one factor VIII unit in therapeutic material per kilogram of body weight should increase factor VIII activity levels by 2% of normal with a half-life of 12 hours.

    About 15% (range, 5%-25%) of patients with hemophilia Adevelop antibodies (acquired inhibitors) against transfused factor VIII, whether obtained from pooled or synthetic (recombinant) plasma. In addition, some persons without factor deficiency develop inhibitors against a coagulation factor (usually factor VIII) for unknown reasons. The most common associated conditions are rheumatoid-collagen diseases, medications, pregnancy or postpartum, malignancy, and Crohn’s disease. Whereas hemophilics typically hemorrhage into joints, factor VIII inhibitors in nonhemophiliacs tend to produce hemorrhage in nonjoint areas.

    In either hemophiliacs or nonhemophiliacs, factor VIII inhibitors elevate the APTT just as factor VIII does; and similar to factor VIII deficiency, a 1:1 mixture of patient plasma and fresh normal plasma should decrease the APTT into or nearly into APTT reference range. Incubation of the mixture at 37°C for 1 hour shows relatively little change (less than 8 seconds) in factor VIII deficiency but a significant increase (8 seconds or more) when an inhibitor is present. A 2-hour incubation is necessary with weak inhibitors. About 10%-15% of patients with antiphospholipid antibodies (“lupus anticoagulant,” discussed later in detail) have the same response as a factor VIII inhibitor. The strength of the inhibitor can be quantitated by testing its effect on a standardized preparation of factor VIII, with the result reported in Bethesda units. This test is available mostly in large reference laboratories, university medical centers, and hemophiliac care centers. Factor VIII inhibitors may interfere with those factor VIII assays and other factor assays that use theAPTT as the assay endpoint. Elevation of the APTT due to action of the inhibitor similates the result due to factor deficiency.

    Factor IX (prothrombin complex) concentrate has been used with some success in factor inhibitor patients on an empirical basis.

    Mild hemophilia A (factor VIII levels >5%) can also be treated with desmopressin (DDAVP), which was found to act as a stimulant to factor VIII and to vWf production. Intravenous injection of desmopressin produces peak action in about 30 minutes, resulting in average twofold increases in factor VIII that last about as long as the effect of cryoprecipitate. Since desmopressin is synthetic, the possible infectious complications of blood products are avoided. However, patients with severe hemophilia A cannot respond sufficiently.

    Laboratory tests in hemophilia A.— The PT test results are normal in all factor VIII:C activity–deficient groups. The bleeding time is usually normal; but it will be elevated in about 20% of patients, more often those with severe deficiency. The major screening test for hemophilia A is the APTT. The APTT becomes abnormal when factor VIII:C activity is <30%-35% of normal (range, <20%-<49%). The APTT detects 99%-100% of patients with severe and moderate factor VIII:C activity deficiency. In mild cases, reported detection rate varies from 48%-100% (depending on the particular reagents used and the population selected). Therefore, some of the milder (“subhemophilia”) cases produce borderline or normal results. Definitive diagnosis of hemophilia A usually requires assay of factor VIII:C activity. This used to require the thromboplastin generation test but now is ordinarily carried out with the APTT test and commercially obtained factor VIII-deficient plasma reagent. There is a substantial variation in results in average laboratories, and the reference range is usually considered to be 50%-150% of a normal pooled plasma. The APTT alone detects patients with factor VIII:C less than 30%-35% of normal. This will miss some mild and borderline hemophiliacs and most carriers. Factor VIII:C assay can detect severe, moderate, and mild male hemophiliacs; a considerable number of borderline male hemophiliacs; and about 35% of female carriers. Studies using a combination of factor VIII:C activity assay and vWf protein assay (obtaining a ratio of the two results) are more sensitive than standard factor VIII activity assays alone in detection of hemophilia A, with results of the combined studies (ratio) positive in approximately 50%-75% of female carriers (literature range, 48%-94%). Deoxyribonucleic acid (DNA) probes using multiple targets have been reported to detect 90%-95% of female carriers, making it the most sensitive screening or diagnostic method.

    Increased estrogen levels (pregnancy or estrogen-containing contraceptive medication) increase factor VIII:C activity and could mask a mild deficiency. Factor VIII protein is one of the so-called “acute reaction” proteins (Chapter 22) whose synthesis is increased in response to acute illness or stress. Surgery, acute bleeding, pregnancy, severe exercise, inflammation (infectious or noninfectious), and severe stress of other types may raise factor VIII:C activity levels somewhat (within 1-2 days after stress onset) and may temporarily mask a mild factor deficiency state if testing is delayed that long. Another problem is therapy given before test specimens areobtained. There are some technical details that affect tests for factor VIII:C activity. The specimen must be fresh or preserved by short-term refrigeration or by freezing. Most methods quantitate results by comparing the patient resultsagainst those of a reference plasma. Commercially available lyophilized reference plasma gives more reliable results than fresh “normal” plasma. Photooptical instruments in general give better results than mechanical clot timers.

    Factor IX (plasma thromboplastin component)

    Factor IX deficiency is inherited as an X-linked recessive trait. Deficiencyoccurs in variable degree, and clinical severity varies accordingly, similar tohemophilia due to factor VIII deficiency. Factor IX deficiency produces a hemophilia syndrome comparable to that of factor VII:C deficiency, but hemorrhage into joints is not as frequent as in factor VII:C deficiency, even in the severe form of factor IX disease. The factor IX deficiency syndrome is also called hemophilia B, or Christmas disease (named after the first patient studied in detail). Factor IX is found in plasma or serum and is stable in either.

    The APTT test is the standard screening method; however, some reagents only detect deficiency when <20% of normal. It is also used for diagnosis and quantitative activity assay with factor IX-deficient plasma reagent. As in factor VIII deficiency, the PT is normal. Quick differentiation of factor IX deficiency from factor VIII deficiency can be provided by addition of normal serum or normal fresh plasma to patient serum; the APTT abnormality is corrected byserum in factor IX deficiency, whereas fresh plasma but not serum is required to correct a factor VIII deficiency. Increased estrogens may falsely increase factor IX. Diagnosis can also be made by DNA probe methods, similar to hemophilia A.

    Current therapeutic sources of factor IX include ordinary blood bank plasma, fresh frozen plasma, and factor IX (prothrombin complex) concentrate. Cryoprecipitate is not useful because it does not contain any factor IX. As a rule of thumb, one unit of factor IX in therapeutic material per kilogram of body weightshould increase factor IX levels by 1% of normal with a half-life of 24 hours. Occasionally, patients develop antibodies against transfused factor IX.

    Factor X (Stuart factor)

    This factor is a part of both the extrinsic and the intrinsic coagulation pathways. Deficiency is rare and is inherited as an autosomal recessive trait. The clinical syndrome produced is usually mild compared with classic hemophilia A. Factor X is present in plasma and in serum and is stable in both. Both the APTT and the PT are abnormal; the bleeding time is normal.

    Factor XI (plasma thromboplastin antecedent)

    This factor deficiency is uncommon and is inherited as an autosomal incompletely recessive trait. Deficiency varies in degree according to whether the patient is homozygous or heterozygous. Clinical symptoms from either degree of deficiency are milder than those of comparable cases of hemophilia A or B. Factor XI is present in plasma and serum and is stable in both. The APTT is abnormal; the PT and bleeding time are normal.

    Factor XII (Hageman factor)

    Factor XII deficiency is very uncommon and is inherited as an autosomal recessive trait. Clinical manifestations (bleeding) are rare, and most of the interest in this disorder derives from the role of factor XII as a surface contact activator of the intrinsic coagulation pathway. The extrinsic pathway is not affected. Factor XII deficiency produces clotting deficiency in laboratory tests that is particularly marked when activation enhancement materials are included in coagulation test reagents (such as the APTT) or glass tubes are used (glass ordinarily would strongly activate factor XII). The APTT and whole blood clotting time are abnormal; the PT and the bleeding time are normal. Diagnosis can be confirmed by means of the APTT with commercial factor XII–deficient plasma reagent. It has been reported that patients with factor XII deficiency have an increased incidence of myocardial infarction and thrombosis.

    High molecular weight kininogen (HMWK; Fitzgerald factor)

    This factor is a part of the kallikrein system, which affects factor XII in the coagulation intrinsic pathway and also plays a role in the body inflammatory response. Deficiency of HMWK is rare, and no clinical bleeding is induced. The APTT and whole blood clotting time are abnormal; the PT and bleeding time are normal.

    Prekallikrein (Fleher factor)

    This proenzyme is the central part of the kallikrein system. Prekallikrein deficiency does not predispose to abnormal bleeding. The whole blood clotting time is abnormal; the PT and bleeding time are normal. The APTT is abnormal if particulate activators such as silica or kaolin are used but is normal if solubleactivators such as ellagic acid are used. The APTT abnormality with particle activators may be corrected by prolonged incubation (10-15 minutes vs. the usual 1-3 minutes).

    Factor XIII (fibrin stabilizing factor)

    Factor XIII deficiency is transmitted as an autosomal recessive trait. Bleeding frequently is first noted in newborns. In later life bleeding episodes are usually mild except when related to severe trauma or surgery. Secondary deficiency may occur in a variety of conditions, especially malignancy or severe liverdisease, but abnormality is usually subclinical. All of the usual coagulation tests (PT, APTT, and bleeding time) are normal, even in congenital deficiency. Diagnosis can be made because fibrin clots from factor XIII–deficient persons are soluble in certain concentrations of urea, whereas clots from normal persons are not. Several other assay methods have been reported.

  • 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.

  • 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.

  • Laboratory Tests and the Medical Literature

    One of the more interesting phenomena in medicine is the scenario under which new tests or new uses for old tests are introduced. In most cases the initial reports are highly enthusiastic. Also in most cases there is eventual follow-up by other investigators who either cannot reproduce the initial good results or who uncover substantial drawbacks to the test. In some cases the problem lies in the fact that there may not be any way to provide an unequivocal standard against which test accuracy can be measured. An example is acute myocardial infarction, because there is no conclusive method to definitively separate severe myocardial ischemia from early infarction (i.e., severe reversible change from irreversible change). Another example is acute pancreatitis. In other cases the initial investigators may use analytical methods (e.g., “homemade” reagents) that are not identical to those of subsequent users. Other possible variances include different populations tested, different conditions under which testing is carried out, and effects of medication. Historical perspective thus suggests that initial highly enthusiastic claims about laboratory tests should be received with caution.

    Many readers of medical articles do not pay much attention to the technical sections where the materials and methods are outlined, how the subjects or patient specimens are selected and acquired, and how the actual data from the experiments are presented. Unfortunately, rather frequently the conclusions (both in the article and in the abstract) may not be proven or, at times, even may not be compatible with the actual data (due to insufficient numbers of subjects, conflicting results, or most often magnifying the significance of relatively small differences or trends). This often makes a test appear to give clear-cut differentiation, whereas in reality there is substantial overlap between two groups and the test cannot reliably differentiate individual patients in either group. Another pitfall in medical reports is obtaining test sensitivity by comparing the test being evaluated with some other procedure or test. While there usually is no other way to obtain this information, the reader must be aware that the gold standard against which the new test is being compared may itself not be 100% sensitive. It is rare for the report to state the actual sensitivity of the gold standard being used; even if it is, one may find that several evaluations of the gold standard test had been done without all evaluations being equally favorable. Therefore, one may find that a new test claimed to be 95% sensitive is really only 76% sensitive because the gold standard test against which the new test is being compared is itself only 80% sensitive. One should be especially wary when the gold standard is identified only as “a standard test” or “another (same method) test.” In addition, even if the gold standard were claimed to be 100% sensitive, this is unlikely because some patients would not be tested by the gold standard test due to subclinical or atypical illness; or patients could be missed because of interferences by medications, various technical reasons, or how the gold standard reference range was established (discussed previously).