Laboratory Approach to Anemia PDF

Chapter 12 Laboratory Approach to Anemia Ebru Dündar Yenilmez and Abdullah Tuli Ebru Dündar Yenilmez and Abdullah Tuli Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.70359 Ab...

Chapter 12 Laboratory Approach to Anemia Ebru Dündar Yenilmez and Abdullah Tuli Ebru Dündar Yenilmez and Abdullah Tuli Additional information is available at the end of the chapter Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.70359 Abstract Anemia is a major cause of morbidity and mortality worldwide and can be defined as a decreased quantity of circulating red blood cells (RBCs). The epidemiological studies suggested that one-third of the world’s population is affected with anemia. Anemia is not a disease, but it is instead the sign of an underlying basic pathological process. However, the sign may function as a compass in the search for the cause. Therefore, the prediag- nosis revealed by thorough investigation of this sign should be supported by laboratory parameters according to the underlying pathological process. We expect that this review will provide guidance to clinicians with findings and laboratory tests that can be followed from the initial stage in the anemia search. Keywords: anemia, complete blood count, red blood cell indices, reticulocyte 1. Introduction Anemia, the meaning of which in Greek is “without blood,” is a relatively common sign and symptom of various medical conditions. Anemia is defined as a significant decrease in the count of total erythrocyte [red blood cell (RBC)] mass, although this definition is rarely used in clinical settings. According to the World Health Organization, anemia is a condition in which the number of red blood cells (RBCs, and consequently their oxygen-carrying capacity) is insufficient to meet the body’s physiologic needs [1, 2]. The individual variation such as a person’s age, gender, residential elevation above sea level (altitude), and different stages of pregnancy changes the specific physiologic requirements of the body. Anemia is not a dis- ease, but is instead the sign of an underlying basic pathological process. Nonetheless, the sign may function as a compass in the search for the cause, as well as function as a road marker © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2018 The Attribution Author(s). License Licensee InTech. This chapter is distributedwhich (http://creativecommons.org/licenses/by/3.0), underpermits the terms of the Creative unrestricted Commons use, distribution, Attribution and License reproduction (http://creativecommons.org/licenses/by/3.0), in any medium, provided the original work is properly which permits unrestricted use, cited. distribution, and reproduction in any medium, provided the original work is properly cited. 236 Current Topics in Anemia in the investigation of underlying pathological process. Hence, the diagnosis according to the symptoms obtained by history and physical examination of patients with anemia should be supported by laboratory parameters related to the underlying pathological cause. The first step in the diagnosis of anemia is detection with predictive, accurate tests so that important clues to underlying disease are not missed and patients are not subjected to unnecessary tests for and treatment of nonexistent anemia. Instead, clinicians rely on several other measures to identify the degree and the cause of anemia in a given patient. The purpose of this chapter is to discuss the clinical approaches with which a practicing physician is able to evaluate a patient with underlying anemia. 2. Classification of anemia Based on determination of the red blood cell mass, anemia can be classified as either relative or absolute. Relative anemia is characterized by a normal total red blood cell mass in an increased plasma volume, resulting in a dilution anemia, a disturbance in plasma volume regulation. However, dilution anemia is of clinical and differential diagnostic importance for the hema- tologist. Classification of the absolute anemias with decreased red blood cell mass is dif- ficult because the classification has to consider kinetic, morphologic, and pathophysiologic interacting criteria. Anemia of acute hemorrhage is not a diagnostic problem and is usually a genitourinary or gastrointestinal event, not a hematologic consideration. Initially, anemias should be classified into two groups as diminished production and increased destruction of RBCs. The number of reticulocytes is a remarkable parameter in the materialization of this classification. Then, diagnostic analysis is able to be based upon both morphologic and pathophysiological hallmarks. Anemias can morphologically be classified into three subgroups as macrocytic, normocytic, and microcytic hypochromic anemias. This classification is based on mean corpuscular vol- ume (MCV) and mean corpuscular hemoglobin concentration (MCHC) of complete blood count (CBC) and aids the physician to the diagnosis and monitoring of anemias that can be easily cured, such as deficiency of vitamin B12, folic acid, and iron. Pathophysiologic classification is best suited for relating disease processes to potential treat- ment (Figure 1). In addition, anemia resulting from vitamin- or iron-deficiency states occurs in a significant proportion of patients with normal red blood cell indices. Each step indicated in Figure 1 can be disrupted and cause anemia. Identifying the affected step is important for therapeutic intervention and specific treatment. The limitation of pathophysiologic classification is that pathogenesis involves several steps in most anemias. Therefore, the provided chapter is a guideline for the practical understanding of the processes underlying the production and destruction of RBCs. Despite all these morphological clas- sification is more useful in terms of convenience and clinical usage. Hence, morphological classification serves to support the diagnosis and indirectly treatment in connection with the laboratory and clinic. The major limitation of such a classification is that it tells nothing about the etiology or reason for the anemia.  Laboratory Approach to Anemia 237 http://dx.doi.org/10.5772/intechopen.70359 Figure 1. Classification of anemia according to pathophysiologic characteristics (figure has been modified from Ref. ). 3. Laboratory evaluation A comprehensive laboratory evaluation is required for definitive diagnosis and treatment for any anemia, although the anamnesis (history of patient) and physical examination of the patient may indicate the presence of anemia and propose its cause. As appropriate to this aim, the various tests for the diagnosis of anemia are done with routine hematological tests such as CBC and reticulocyte counts as well as studies of iron status that serve as a leaping point to the diagnosis (Figure 2). When the diagnosis of specific anemic conditions is confirmed, a large number of other specific tests are used. Laboratory tests used in the diagnosis of ane- mia are roughly summarized in Figure 2. The laboratory investigation of anemias involves the quantitative and semiquantitative measurements of RBCs and supplementary testing of blood and body fluids. The laboratory results obtained from these parameters are important arguments in the diagnosis, treatment, and monitoring of the anemias. 3.1. Complete blood count Prior to the development of modern hematology blood analyzers, blood counts included hemoglobin (Hb) concentration, white blood cell (WBC) count, and manual platelet count. The other parameters like mean corpuscular volume (MCV) had to be mathematically calculated by using the measured parameters such as Hb, RBC count, and hematocrit (Hct). Modern ana- lyzers provide CBC indices by using various physical and chemical methods such as electronic impedance, laser light scattering, light absorption, and staining properties. How will CBC parameters such as Hb concentration, Hct, RBC count, MCV, MCHC, WBC count, platelet count, and other parameters related to formed elements of blood measured by modern blood analyzers help the diagnosis or management of the patient? CBC identifies 238 Current Topics in Anemia Figure 2. Laboratory tests used in anemia diagnosis (figure has been modified from Ref. ). several different parameters and can provide a great deal of information. Hematologic and biochemical variations of red blood cells determine whether the patient is anemic or not. If anemia is present, MCV is likely to provide clues about the cause of anemia. While an infection can lead to increased WBC, lymphocytosis can be seen in viral infections (but not always so). Abnormal size or number of platelets may be either due to the direct effect of any underlying blood disease or may simply be the reflection of the presence of some other underlying pathol- ogies. Because of all these, CBC parameters obtained as a result of clinical evaluation should be reassessed more carefully and curiously. Therefore, the fundamental parameters of CBC such as Hb concentration, RBC, Hct, MCV, mean corpuscular hemoglobin (MCH), MCHC, and red blood cell distribution width (RDW) which plays an important role in the diagnosis, treatment, and monitoring of the anemic patient will be explained below. 3.2. Hemoglobin concentration Determination of Hb is a part of CBC. Hemoglobin is intensely colored, and this property has been used in methods for estimating its concentration in the blood. Erythrocytes contain a mixture of hemoglobin, oxyhemoglobin, carboxyhemoglobin, methemoglobin, and minor amounts of other forms of hemoglobin. Monitoring the response to treatment of anemia and to evaluate polycythemia, Hb concentration is used to screen for diseases associated with anemia and to determine the severity of anemia. Finding an increased Hb concentration requires a systematic clinical approach for differential diagnosis and further investigation. The conditions such as polycythemia vera, congestive heart failure, chronic obstructive pulmonary disease, etc., can cause Hb levels to rise.  Laboratory Approach to Anemia 239 http://dx.doi.org/10.5772/intechopen.70359 Decreased Hb levels are found in anemia. Hb must be evaluated along with the RBC and Hct. In iron deficiency, hemoglobinopathies, pernicious anemia, liver disease, hypothyroidism, hemorrhage (chronic or acute), hemolytic anemia (caused by transfusions, reactions to chemi- cal or drugs, infectious and physical agents), and various systemic diseases (e.g., Hodgkin’s disease, leukemia, etc.), decrease in Hb levels can be observed. Variations in Hb levels occur after hemorrhages, transfusions, and burns (Hb and Hct are both high during and immediately after hemorrhage). Hb and Hct supply valuable information in an emergency situation. Excessive fluid intake, pregnancy, and drugs, etc., which cause increase in plasma volume and decrease the Hb values, are interfering factors. Drugs such as methyldopa and extreme physi- cal exercise can give rise to increased Hb levels. In addition, people living in high altitudes have increased Hb concentration, Hct, and RBC count. 3.3. Red blood cell count The quantification of the percentage of microcytic and hypochromic RBCs has proved its clinical usefulness in the differential diagnosis of microcytic anemia. RBC count has been recognized as the most efficient single classical measurement in the differential diag- nosis of microcytic anemia. Iron-deficient erythropoiesis is characterized by the pro- duction of RBC with a decrease in Hb content, so a high percentage of hypochromic cells are present. In β-thalassemia cases, increased RBC count is a characteristic as a result of chronic increase in erythropoiesis. Therefore, MCV and MCH are lower in beta thalassemia than in iron deficiency anemia. 3.4. Hematocrit The word hematocrit, also called packed cell volume (PCV), means “to separate blood,” which underscores the mechanism of the test, because the plasma and blood cells are separated by centrifugation. Decreased Hct values are an indicator of anemia, in which there is a reduction in the Hct. An Hct ≤30% means that the patient is severely anemic. Decreased values also occur in leukemias, lymphomas, Hodgkin’s disease, adrenal insufficiency, chronic diseases, acute and chronic blood loss, and hemolytic reactions (transfusions, chemical, drug reactions, etc.). Increased Hct values are observed in erythrocytosis, polycythemia vera, and shock (when hemoconcentration rise). Interfering factors such as pregnancy, age, sex, and dehydration have different effects in Hct. People living in high altitudes have increased Hct values and RBC count. Hct decreases in the physiologic hydremia of pregnancy. Hct varies with age and gender. Hct levels are lower in men and women older than 60 years of age. Severe dehydration from any cause falsely increases the Hct value [8, 12]. 240 Current Topics in Anemia 4. Red blood cell indices The size and hemoglobin content of erythrocytes (red blood cell indices), based on popu- lation averages, have traditionally been used to assist in the differential diagnosis of ane- mia. Some red blood cell parameters (for instance, RBC count, Hb concentration, MCV, RDW) are directly measured, while the others (e.g., Hct, MCV, MCHC) are derived from these primary measurements. These measurements are provided by any of the common automated instruments. Instruments vary somewhat in their technologies. The most com- monly used method is either a combination of a highly focused light source, an electric field, and a laser-based flow cytometry or a radiofrequency wave to discriminate between cells. Automated instruments are not only fast but extremely accurate. The coefficient of variation (measurement error) of an automated counter is usually less than 2%, and each of the major measurements, including the hemoglobin level, red blood cell count, and mean corpuscular volume, can be standardized independently with commercial red blood cell and hemoglobin standards [4, 6, 12]. 4.1. Mean corpuscular volume (MCV) MCV has been used to guide the diagnosis of anemia in patients, for example, testing patients with microcytic anemia for iron deficiency or thalassemia and those with macrocytic anemia for deficiency of folate or vitamin B12 [4, 15]. The reference value of MCV ± 2 SD is 90 ± 9 fL and generally coincides with the peak of the Gaussian distribution of RBC size. Although MCV is both accurate and highly reproducible, errors may be introduced by RBC agglutination, distortions in cell shape, the presence of very high numbers of WBCs, and sudden osmotic swelling. MCV results are the basis of the classification system used to evaluate an anemia (Table 1, Figure 3). Increased reticulocytes and marked leukocytosis can also increase MCV. The mixed popula- tion of microcytes and macrocytes results in normal MCV values and is an interfering factor in evaluating MCV. 4.2. Mean corpuscular hemoglobin (MCH) MCH, the amount of hemoglobin per red blood cell, increases or decreases in parallel with MCV and generally provides similar diagnostic information. Because this parameter is affected by both hypochromia and microcytosis, it is least sensitive as MCV in detecting iron deficiency states. The reference value of MCH is 32 ± 2 pg. This is an excellent measure of the amount of hemo- globin in individual red blood cell. Patients with iron deficiency or thalassemia who are unable to synthesize normal amounts of hemoglobin show significant reductions in the MCH [8, 17]. An increase of MCH is associated with macrocytic anemia; a decrease of MCH is associated with microcytic anemia.  Laboratory Approach to Anemia 241 http://dx.doi.org/10.5772/intechopen.70359 Microcytic anemias (MCV 50–79 fL) Disorders of iron metabolism Iron deficiency anemia, anemia of chronic disease, congenital hypochromic-microcytic anemia with iron overload Disorders of porphyrin and heme synthesis Acquired sideroblastic anemias, idiopathic refractory sideroblastic anemia Disorders of globin synthesis Thalassemias, hemoglobinopathies, characterized by unstable hemoglobins Normocytic normochromic anemia (MCV 80–98 fL) Anemia with appropriate bone marrow Acute posthemorrhagic anemia, hemolytic anemia response Anemia with impaired marrow response Aplastic anemia, pure red blood cell aplasia, myelofibrosis Macrocytic anemias (MCV 99–150 fL) Cobalamin (B12) deficiency Lack of animal products, intrinsic factor deficiency, pernicious anemia, hyperthyroidism, pregnancy, enzyme deficiencies Folate deficiency Lack of vegetables, celiac disease, hypothyroidism, folic acid antagonists, hemodialysis Unresponsive to cobalamin or folate Metabolic inhibitors (i.e., 6-mercaptopurine), inborn errors (Lesch- Nyhan syndrome) Table 1. Classification and possible diagnosis of anemia according to MCV in clinical use. Figure 3. Flowchart to follow in the diagnosis of anemia according to MCV. 242 Current Topics in Anemia Hyperlipidemia is one of the interfering factors of MCH because it falsely increases MCH values. WBC counts >50,000/mm3 also falsely provide increased level for MCV as well as for Hb. In addition, high heparin concentrations also falsely elevate MCH value. 4.3. Mean corpuscular hemoglobin concentration (MCHC) MCHC is not used frequently for diagnostic purpose, but is primarily useful for quality con- trol purposes, such as detecting sample turbidity. Because MCHCs are average quantities in the blood with mixed-cell populations, it is difficult for these red blood cell indices to detect abnormalities in the blood. The reference value of MCHC is 33 ± 3 g/dL. The principal purpose of MCHC is to detect patients with hereditary spherocytosis who has very small, dense spherocytes in the circula- tion. These spherocytes represent cells that have lost considerable intracellular fluid because of a membrane defect. In situations such as sideroblastic anemia, recently transfused patients, patients with severe pernicious anemia with red blood cell fragmentation, and in conditions where both folate and iron deficiency are present, both large and small red blood cells are observed, which compromise the value of MCV. When present in significant numbers, they will cause MCHC to increase to levels in excess of 36 g/dL [4, 6, 15]. Decreased MCHC indicates that packed RBCs (a unit volume) contain less Hb than normal. MCHC is decreased in hypochromic anemia (MCHC < 30 g/dL) observed in iron deficiency, microcytic anemias, chronic blood loss anemia, and some thalassemias. Increased MCHC levels (RBCs cannot accommodate more than 37 g/dL Hb) occur in sphero- cytosis, in newborns and infants. Because of falsely elevating MCHC, lipemia, cold agglutinins or rouleaux, and high heparin concentrations may be among the interfering factors. MCHC cannot be greater than 37 g/dL because the RBC cannot accommodate more than 37 g/dL Hb. 4.4. Red blood cell distribution width (RDW) RDW is an estimate of the variance in the volume within the population of red blood cells. RDW, provided by automated counters, is an index of the distribution of RBC volumes. RDW is derived from pulse height analysis and can be expressed as an SD (fL) or as a coefficient of vari- ation (%) of the red cell volume. Automated counters use two methods to calculate RDW. The first is referred to as RDW-CV. RDW-CV is the ratio of the width of the red blood cell distribution curve at 1 SD divided by MCV (normal RDW-CV = 13 ± 1%) (Figure 4). Since it is a ratio, changes in either the width of the curve or MCV will influence the result. In micro- cytosis, any changes in the RDW-CV simply reduce the denominator of the ratio. Conversely, in macrocytosis the change in the width of the curve will minimize the change in RDW-CV. A second method of measuring the RDW is RDW-SD and is independent of MCV. RDW-SD is measured by calculating the width at the 20% height level of the red blood cell size distribu- tion histogram (normal RDW-SD = 42 ± 5 fL) [6, 8, 15].  Laboratory Approach to Anemia 243 http://dx.doi.org/10.5772/intechopen.70359 Figure 4. Red blood cell distribution width. Automated counters provide measurements of the width of the red blood cell distribution curve. RDW-CV is calculated from the width of the histogram at 1 SD from the mean divided by MCV. Both measurements of RDW are essentially mathematical statement of anisocytosis. Increases in the RDW suggest the presence of a mixed population of cells. Double populations, whether microcytic cells mixed with normal cells or macrocytic cells mixed with normal cells, will widen the curve and increase the RDW. The RDW-SD is more sensitive to the appearance of minor populations of macrocytes or microcytes since it is measured lower on the red blood cell volume-distribution curve (Figure 4) [4, 8]. The RDW can be used to distinguish thalassemia (normal RDW) from iron deficiency anemia (high RDW). Also, it can be used to distinguish chronic disease anemia (normal RDW) from early iron deficiency anemia (elevated RDW). RDW increases in iron deficiency anemia, vita- min B12 or folate deficiency (pernicious anemia), abnormal Hb (S, S-C, or H), S-β thalassemia, immune hemolytic anemia, marked reticulocytosis, and posthemorrhagic anemia. The RDW may be an alternate marker for systemic inflammation and/or oxidative stress; however, the predictive value of RDW is independent of other inflammatory markers. This suggests that this biomarker also follows other nonempirical processes [8, 17]. The determina- tion of the physiological and biological mechanisms that associate RDW to adverse clinical results is important in using these prognostic biomarkers to therapeutic decisions. 4.5. Stained peripheral blood smear Peripheral blood smears can provide important additional information about RBC morphology in anemia and are easily prepared manually using glass slides. The hematology laboratory usu- ally examines a peripheral blood smear if the patient’s indices are abnormal (unless there has been no major change from previous CBCs). If an underlying blood disorder is suspected, a film should be requested. Automated instruments ensure accurate RBC counts and indices and WBC counts and differentials in both healthy and diseased individuals [8, 19]. 244 Current Topics in Anemia The peripheral blood smear complements the automated countermeasurements of MCV and MCH. Visible changes in cell diameter, shape, and hemoglobin content can be used to distinguish both microcytic and macrocytic cells from normocytic/normochromic RBCs (Table 2). In clinical cases, the variation such as staining, color, shape, and inclusion bodies in the blood smear of RBCs is not only an indication of RBC abnormalities but also a diagnosis of diseases. 4.6. Reticulocyte count Reticulocyte count is an essential component of CBC and has a substantial role in initially clas- sifying any anemia. Reticulocytes are newly formed red blood cells with residual strands of nuclear material called “reticulin” that remain following extrusion of the nucleus from bone mar- row normoblasts. The reticulocyte is a young red blood cell containing residual ribosomal RNA that can be stained with a supravital dye such as acridine orange or new methylene blue. The reticulocyte count can be used in differentiation of the patients with a functionally nor- mal marrow response to anemia/hypoxia and those with a failed marrow response. Whenever the reticulocyte production index (RPI) increases to levels greater than three times normal in response to an anemia (hematocrit 8.5 μm diameter) Microcyte Smaller than normal (40 mg/dL, the patient probably has/had an acute hemorrhage or is respond- ing to hematinic. Patient should be evaluated for external or internal bleeding. If Haptoglobin is