Diagnosis of Iron Deficiency Anemia Using the Abaxis VetScan HM2 Hematology System Contributing Authors: Mary Anna Thrall, BA, DVM, MS, DACVP; Barton Gillespie, BS, DVM Ross University School of Veterinary Medicine Basseterre, St. Kitts, West Indies 10 Feb/March 2011
CBC diagnostic case study O ne of the most important advantages of automated cell counters is the ability to determine erythrocyte size, reported as mean cell volume (MCV). Microcytosis as evidenced by a decreased MCV is almost always indicative of iron deficiency anemia; iron deficiency anemia in adult animals is almost always due to chronic blood loss. This case presentation illustrates the importance of the MCV in determining the cause of iron deficiency anemia. Case report Fluffy, a 5 year old mixed breed male dog weighing 6.5 kg, was presented for coughing. His Complete Blood Count (CBC) at presentation indicated a severe microcytic anemia (Table 1). The degree of microcytosis is illustrated by the RBC histogram generated by the HM2 on 1/20/2010 (Figure 1). The blood film contained many hypochromic erythrocytes, as well as keratocytes and schistocytes, erythrocyte shape changes and fragmentation that are very characteristic of iron deficient erythrocytes (Figures 2 and 3). The platelet concentration was markedly increased (Table 1 and Figure 2), as is often the case in patients with iron deficiency anemia. The diagnosis of iron deficiency anemia was confirmed by measuring serum iron, which was 27 µg/dl (Reference interval = 98-220 µg/dl). Table 1 01/20/2010 01/21/2010 01/28/2010 reference interval PCV (spun)(%) 14.0 16.0 30.0 37-55 Hematocrit(%) 12.4 11.0 21.3 37-55 RBC (x10 6 /µl) 4.26 3.75 5.77 5.5-8.5 Hgb (g/dl) 4.4 3.2 9.0 12-18 MCV (fl) 29.0 29.0 37.0 60-77 MCHC (g/dl) 35.2 29.0 42.1 31-34 RDW 23.8 24.8 31.7 12-15 Platelets (x10 3 /µl) 803.0 675.0 883.0 200-500 Reticulocytes (x10 3 /µl) 102.0 90.0 403.0 0-60 Total Protein (g/dl) 5.3 6.7 6.2 6-8 * sample moderately lipemic 2011 Feb/March 11
The source of chronic blood loss is usually the gastrointestinal tract. An occult blood test was performed on the feces on multiple occasions, and was consistently negative. An examination of feces for the eggs of parasites, such as hookworms, was also negative. Further history revealed that the owner had given Fluffy an anthelmintic two weeks prior to presentation, presumably eliminating a hookworm infestation. Fluffy was treated with 15 mg/kg iron dextran IM, and the owner was instructed to give him 162.5 mg Ferrous sulfate orally once daily for 4 weeks. The PCV markedly increased the week following iron therapy. A marked regenerative response is present, as evidenced by the marked reticulocytosis (Table 1). The MCV increased, and the new normal-sized erythrocytes are evident in the erythrocyte histogram on 1/28/10. They are represented by the additional curve on the right side of the histogram (Figure 4) as well as on the blood film made on 1/28/10 (Figures 5 and 6). The increased variation in erythrocyte size (anisocytosis) is also represented by the increased red cell distribution width (RDW), a numerical expression (coefficient of variation) that correlates with the degree of anisocytosis and which the instrument determines by dividing the standard deviation of the red cell size by the MCV. This case also nicely illustrates spurious results that can arise due to lipemia and platelets interfering with the counting and sizing of erythrocytes. There is a marked discrepancy in the calculated hematocrit (HCT) and the spun packed cell volume (PCV). The HCT is calculated by the instrument using the following formula: (MCV x RBC)/10 = HCT(%). In the case of the sample from 1/28/10, the RBC count may be under-reporting the sample s RBC contents since some small RBCs are likely appearing in the PLT channel. The lower reported RBC count is also likely due to the fact that two overlapping RBC peaks are now present in the RBC histogram from Figure 1 Figure 2: Note the hypochromasia (increased central pallor). Figure 3: Note the obvious small size of the erythrocytes when compared to the leukocytes. 1/28/10; the two overlapping peaks will be underestimated since the software expects only one RBC peak to be present and will not apply curve fitting algorithms here. Since the RBC count is underestimated, the HCT calculated here is lower than the reported PCV. The MCHC is also miscalculated to be high based on the undercounted RBCs. The MCHC is probably erroneously increased on 1/20/10 as well. The MCHC is calculated from the hemoglobin concentration and the HCT using the following formula: HGB (g/dl) divided 12 Feb/March 2011
CBC diagnostic case study Figure 4 Figures 5 and 6: Note the large immature erythrocytes that are now present: these larger erythrocytes are represented by the additional curve on the right of the erythrocyte histogram in figure 4. Discussion of iron deficiency anemia The hallmark of iron deficiency anemia is a decreased MCV. Microcytosis occurs supposedly because erythrocyte precursors continue to divide in an attempt to reach their full hemoglobin content. Additional divisions result in smaller than normal erythrocytes. Examination of the erythrocyte histogram generated by the electronic cell counter is often useful, because sub-populations of microcytic erythrocytes can be observed, even when the MCV is within the reference interval. In Fluffy s case, a subpopulation of normal-sized and macrocytic erythrocytes can be observed on the histogram generated on 1/28/10. The red cell distribution width (RDW) is usually increased when sub-populations of microcytic or macrocytic erythrocytes are present, and will often be increased before the MCV decreases below the reference interval. Although one might expect the MCHC to be decreased in these patients, since the cells contain less hemoglobin than normal, it is commonly within the reference interval, and erroneously high MCHCs may be observed for reasons explained above. by the HCT (%) x 100 = MCHC (g/dl). It provides an index for the quantity of hemoglobin relative to the volume of packed erythrocytes. It may be erroneously increased due to lipemia, intravascular hemolysis, or an erroneously low HCT. On 1/28/10, it is likely erroneously high due to the erroneously low HCT. On 1/20/10, it may be erroneously high due to lipemia increasing the HGB. 2011 Feb/March 13
Blood film examination is diagnostically useful, particularly in the late stages of iron deficiency anemia. Erythrocytes of most species usually appear pale, with increased central pallor, and sometimes only a thin rim of hemoglobin is present. Membrane abnormalities are common, including keratocyte and schistocyte formation, presumably due to increased susceptibility to oxidative damage. Initially the RBC develops what appears to be a blister or vacuole where inner membrane surfaces are cross-linked across the cell. These lesions subsequently enlarge and break open to form apple-stem cells and keratocytes, spiculated red cells with two or more pointed projections. The projections from the keratocytes then fragment from the cell, forming schistocytes. The anemia is usually regenerative, but may become non-regenerative in the late stages. Occasionally, the bone marrow response may be inappropriate due to underlying anemia of inflammatory disease, since many of these animals have concurrent inflammation related to bleeding lesions. Thrombocytosis is present in approximately 50% of iron deficient patients. The mechanism for the increased platelet concentration is not well understood, but may be due to increased erythropoietin or other cytokines. Approximately one-third of animals with chronic blood loss become hypoproteinemic, as protein production sometimes cannot keep pace with blood loss. staining cells and tissues. Thus, storage iron can be evaluated by measuring serum ferritin, or by examining a bone marrow aspirate and noting lack of hemosiderin in macrophages. Serum ferritin is difficult to measure, is species-specific, and since it is an acute phase reactant protein, tends to increase when inflammation or liver disease are present. Special iron stains, such as Prussian blue, are not necessary in order to visualize hemosiderin in the bone marrow. For practical purposes, low serum iron in a patient with a decreased MCV and anemia is usually adequate to diagnose iron deficiency anemia, and to trigger additional diagnostic procedures to determine the source of blood loss, such as testing the feces for occult blood. Treatment consists of finding and treating the source of blood loss. Iron supplementation with intramuscular injectable iron in iron deficient patients is useful, as evidenced in Fluffy s case. Although oral iron supplementation is commonly used to treat iron deficiency, it is likely of little value, because commercial pet food usually contains adequate iron. Although intestinal absorption of iron has been shown to increase dramatically when animals are iron deficient, there is some evidence that iron absorption from the intestine is impaired in animals that are iron deficient, and that treatment with injectable iron is necessary for oral iron to be absorbed. Other laboratory findings in patients with iron deficiency include decreased serum iron concentration, decreased transferrin (a glycoprotein in plasma that transports iron between compartments) saturation, and low storage iron. Total iron binding capacity, a test for measuring the amount of transferrin available to transport iron, is usually normal in iron deficient dogs and cats, although it is usually increased in other species with iron deficiency. Iron is stored as either ferritin or hemosiderin. Although ferritin is primarily an intracellular iron storage compound, it can be detected in serum. Hemosiderin, on the other hand, is insoluble, and can only be detected by 14 Feb/March 2011