Articles on Medical Diseases and Conditions

Tag: Reticulocyte Count

  • 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.

  • Reticulocyte Count

    Reticulocytes occupy an intermediate position between nucleated RBCs in the bone marrow and mature (nonnucleated, fully hemoglobinated) RBCs. After the normoblast (metarubricyte) nucleus is extruded from the cell, some cytoplasmic microsomes and ribosomes remain for 1-2 days that are not ordinarily visible on peripheral blood smears using Wright’s or Giemsa stain but that can be seen by using vital staining techniques and dyes such as methylene blue or cresyl blue. The material then is seen microscopically in the form of dark blue dots or thin short irregular linear structures arranged in loose aggregates or reticulum. The reticulocyte count is an index of the production of mature RBCs by the bone marrow. Increased reticulocyte counts mean an increased number of RBCs being put into the peripheral blood in response to some stimulus. In exceptionally great reticulocyte responses, there may even be nucleated RBCs pushed out into the peripheral blood due to massive RBC production activity of the bone marrow. Except in a very few diseases, such as erythroblastosis, peripheral blood nucleated RBCs are usually few in number and of a later maturity stage when they do appear. Reticulocytes are not completely mature RBCs; therefore, when reticulocytes appear in the peripheral blood, they may be slightly larger than normal RBCs and may be sufficiently large to be recognizable as macrocytes. When present in sufficient numbers, these macrocytes may increase the MCV index. Early reticulocytes sometimes appear blue-gray or gray with Wright’s stain in contrast to the red-orange appearance of the normal RBC; this phenomenon is called polychromatophilia and is produced by immature bluish cytoplasmic material to which reddish staining hemoglobin is added. In some conditions a reticulocyte may display small, evenly distributed dotlike aggregates of cytoplasmic ribosomes visible with Wright’s stain, a phenomenon known as basophilic stippling.

    Reference limits for the reticulocyte count are usually considered to be 0.5%-1.5%. Some investigators have reported somewhat higher values, especially in women. There is a substantial problem concerning reproducibility of reticulocyte counts, with statistical variation on the order of ±1 reticulocyte percent unit at normal reticulocyte levels of 0.5%-1.5% standard manual (i.e., statistical variation of more than 50%) and somewhat greater variation at levels of 5% or more. More recently, it has become possible to count reticulocytes using a fluorescent nucleic acid stain in a flow cytometer. This method generally has statistical error less than 15%. Howell-Jolly bodies and medical parasites may interfere if large numbers are present. Some automated cell counters can be adapted to count reticulocytes in the red cell counting channel with preliminary reports suggesting accuracy comparable to flow cytometry. There may be differences in the reference range produced by different equipment and reports.

    Reticulocytes are traditionally reported as a percentage of total RBCs (total RBC includes both mature RBCs and reticulocytes). Automated meter methods are frequently reported as the absolute (quantitative) number of retics. In some clinical situations the number of mature RBCs may decrease while the absolute (total) number of reticulocytes remains the same; this increases the reticulocyte percentage and gives a false impression of increased reticulocyte production. Therefore, some authorities recommend that reticulocyte counts be corrected for effects of anemia. This may be done by multiplying the reticulocyte count (percent) by the patient hematocrit and dividing the result by an average normal hematocrit (47 for men and 42 for women). An alternate method is to obtain the absolute number of circulating reticulocytes by multiplying the patient RBC count by the reticulocyte count (after converting reticulocyte % to a decimal fraction). If polychromatophilic RBCs are present, some experts recommend that the (already) corrected reticulocyte count be divided by 2 to correct for the longer stay of younger reticulocytes in the peripheral blood.

    As noted previously, reticulocyte counts are used as an index of bone marrow activity. Any substantial change in bone marrow RBC production theoretically should be reflected in reticulocyte count change. A normal reticulocyte count has traditionally been considered evidence against a substantial degree of hemolytic anemia and can be used as an index of success in therapy for factor-deficiency anemia. One difficulty is that it usually takes 48-96 hours, sometimes even longer, to establish a reticulocyte count elevation following acute episodes of blood loss, onset of hemolysis, or beginning of factor therapy. Also, reticulocyte counts are not above reference range in some patients with hemolytic anemia (as many as 20%-25% in some studies, depending on the etiology of the disorder), with the degree of reticulocyte response having some correlation with the severity of the hemolytic process. In some cases, failure to obtain expected degrees of reticulocyte response by be due to superimposed factor deficiency (e.g., iron or folate). Another problem may be failure to suspect a hemolytic process or blood loss because the hemoglobin level may remain within population reference range if increased RBC production balances RBC loss or destruction.

    In certain hemolytic anemias such as sickle cell anemia and congenital spherocytosis, temporary aplastic “crisis” may develop in which the anemia worsens because of a halt in RBC production rather than an increase in rate of hemolysis. These crises are sometimes due to parvovirus B-19 infection. The reticulocyte count will become normal or decrease after time is allowed for reticulocytes already in the peripheral blood to disappear.

    In certain anemias caused by ineffective erythropoiesis, the reticulocyte count is normal or decreased unless therapy is given. Some examples include deficiencies of iron, folic acid, vitamin B12, or pyridoxine, or in many patients with anemia associated with chronic disease. In the case of factor deficiency, blood transfusion or hospital diet may contain a sufficient quantity of the deficient factor to increase RBC production.