The Red Blood Cell

The red blood cell (RBC), or erythrocyte, is an anucleate biconcave disk, approximately 7 µm in diameter. The outer membrane of the RBC consists of a lipid bilayer linked to a cytoskeleton by various transmembrane proteins [Reference Narla and Gallagher2,Reference Daniels3]. The content within the RBC membrane consists primarily of hemoglobin (Hb), but also includes enzymes such as glucose 6 phosphate dehydrogenase (G6PD) and other components of intermediary energy metabolism [Reference Prchal and Gregg4,Reference van Wijk and van Solinge5]. The physiological function of the RBC is to exchange Hb-bound carbon dioxide for oxygen while in the pulmonary circulation, to exchange Hb-bound oxygen for carbon dioxide in the peripheral tissue and to carry Hb-bound carbon dioxide back to the lungs where the cycle begins again. All aspects of RBC structure, content, and chemistry are aligned to optimize that function. For example, the biconcave shape of the RBC optimizes passage of the cell through the circulation and facilitates gas exchange across the capillary endothelium. The generation of 2,3-diphosphoglycerate (2,3-DPG) in the course of RBC metabolism regulates Hb oxygen affinity [Reference Yonetani, Park, Tsuneshige, Imai and Kanaori6], while other metabolic mechanisms support the red cell structural integrity by maintaining optimal intracellular redox conditions and osmotic stability [Reference Siems, Sommerburg and Grune7,Reference Joiner and Lauf8].

RBC Development

A detailed discussion of erythropoiesis, the regulated process by which RBCs develop and differentiate, is beyond the scope of the present work. A pluripotent hematopoietic stem cell gives rise to a more restricted but still multipotent progenitor cell, the colony forming unit (CFU) – granulocyte, erythroid, monocyte, megakaryocyte (GEMM) [Reference Nakahata and Ogawa9]. Through a regulated process of proliferation and progressive differentiation to lineage restricted progenitors [Reference Leary, Ogawa, Strauss and Civin10], CFU-GEMM give rise to erythroid (E) burst-forming units (BFUs) and subsequently CFU-E. These two stages of erythroid progenitors are distinguished in vitro by the quantity of erythroid precursors to which they give rise [Reference Eaves and Eaves11]. Erythroid precursors, which are terminally differentiated and lack the capacity to divide, then progress through distinct and morphologically identifiable stages (basophilic erythroblast, polychromatophilic erythroblast, orthochromatic erythroblast) to the nucleated RBC. These stages are distinguished by progressive increases in cellular Hb synthesis, giving rise to the characteristic changes in cytoplasm color (from blue to red) on Wright–Giemsa stained preparations that are reflected in the names of the precursor stages. Simultaneously, the nuclei of the erythroid precursors undergo progressive maturation and become pyknotic. Nucleated RBCs then extrude their nuclei through an active process [Reference Koury, Koury and Bondurant12]. The RBC immediately after enucleation still contains precipitated RNA, which can be identified by supravital staining or flow cytometric techniques, and is referred to as a reticulocyte. As these residua of the former nucleus degrade, the cell is defined as a mature erythrocyte. Many of the nutritional factors giving rise to anemia are involved in the processes of red cell differentiation. For example, B12 is required for nuclear maturation, and iron is a key component of Hb.

Mature RBCs circulate in the peripheral blood for 100–120 days, and approximately 1% of the body's red cells are lost and replaced each day. Red cells recognized as being old are removed from the circulation by macrophages in the spleen, liver, and bone marrow.

Erythropoietin

Erythropoietin (Epo) is a glycoprotein hormone produced in the kidney. Although other growth factors are required for erythropoiesis, particularly in the earlier stages [Reference Muta, Krantz, Bondurant and Wickrema13], Epo is dominant from the late BFU-E stage forward and is the primary positive physiological regulator of erythropoiesis. Epo production is regulated by a classic feedback loop; anemia is perceived as hypoxia by the Epo-producing cell, leading to upregulation of hypoxia-inducible factor (HIF). HIF then upregulates Epo production, leading to increased RBC production. As anemia resolves, the Epo-producing cell is adequately oxygenated and Epo production is turned off.

Hemoglobin

Hb is the molecule responsible for the actual function of gas exchange. Hb is a spherical molecule composed of two pairs of dissimilar globin chains, with a heme group, ferriprotoporphyrin IX, bound covalently at a specific site in each chain [Reference Hsia14]. The heme group consists of an iron-containing porphyrin ring. The configuration of Hb shifts with oxygenation and deoxygenation in order to optimize oxygen release to tissue or uptake from the lung, as is required [Reference Yonetani, Park, Tsuneshige, Imai and Kanaori6,Reference Henry, Bettati, Hofrichter and Eaton15].

Definition of Anemia

In a physiological sense, anemia can be defined as an insufficient RBC mass to adequately deliver oxygen to peripheral tissues. Since methods to assess effective peripheral oxygen delivery at a tissue level are largely reserved to the intensive care or research settings (and in any event would require correction for individual patient circumstances such as tissue perfusion pressure), anemia is defined for practical purposes as a decrease of red cell mass from the population norm. Red cell mass is estimated by any of the three concentration measurements performed on whole blood: Hb concentration, typically expressed as grams Hb per deciliter (g/dL) in the United States and as grams per liter (g/L) in countries using Systeme Internationale units; the hematocrit (Hct; also called the packed cell volume [PCV]), which represents the proportion of blood volume occupied by RBCs, expressed as a percent or as a decimal; and the RBC concentration in cells per liter (10¹²/L). The last is least commonly used in the definition of anemia.

In the past, these parameters were measured using manual physical and chemical techniques. The term hematocrit originally referred to the graduated tube in which the PCV was measured following centrifugation of whole blood [Reference Means16]. In the more developed world, these parameters are usually determined using electronic cell counters with automated Hb determination function. In most of the current analyzers, RBC concentration, Hb concentration, and mean corpuscular volume (MCV, reported in femtoliters [fL]) are directly measured; Hct/PCV and various red cell indices (discussed below) are then calculated automatically. For this reason, many physicians prefer to define anemia using the Hb concentration, although in most cases the Hct is comparably reliable. In developing countries, individual physician offices, small freestanding clinics, or in field situations, may assess Hb using a handheld portable electronic Hb photometer [Reference Nkrumah, Nguah and Sarpong17]; or the Hct can be determined on a centrifuged capillary tube of blood. Simple and inexpensive centrifugation techniques have been described [Reference Riegger, Grumann and Steigert18]. In both cases, fingerstick blood specimens may be used, obviating the need for venipuncture.

The mean normal Hb and Hct values and the lower limits of the normal ranges of these parameters depend on the age and gender of the subjects, as well as their altitude of residence. The World Health Organization (WHO) defines the lower limit of normal for Hb concentration at sea level to be 12.0 g/dL in women and 13.0 g/dL in men [Reference Khusun, Yip, Schultink and Dillon19]. Although many laboratories in the United States report somewhat higher ranges of normal (>14.0 g/dL in men) based on older survey data [Reference Wintrobe20], a more representative sample studied during the second National Health And Nutrition Examination Survey (NHANES II) indicated that the lower limit of normal was 13.2 g/dL in men and 11.7 g/dL in women [Reference Dallman, Yip and Johnson21], consistent with WHO ranges. Values for African-American subjects were approximately 0.5–0.6 g/dL lower than those of white/Caucasian subjects.

Anemia is more common after the age of 50. In the NHANES III study, 10% of men or women over age 65 were anemic, with the frequency rising to more than 20% of subjects over age 85 [Reference Guralnik, Eisenstaedt, Ferrucci, Klein and Woodman22]. There is ongoing debate as to whether this is a normal consequence of aging, resulting from phenomena like decreased androgen secretion in men or age-related decreases in stem cell proliferation; and that, therefore, the Hb definition of anemia in the elderly should be defined downward [Reference Lipschitz, Udupa, Milton and Thompson23,Reference Yip, Johnson and Dallman24]. Others argue that rather it anemia in the elderly reflects an increased frequency of disorders associated with anemia, such as chronic inflammation or renal insufficiency [Reference Ferrucci, Guralnik and Woodman25,Reference Ble, Fink and Woodman26]. The latter position appears more strongly supported at present, although it is the author's experience that many physicians believe that at least slightly lower limits of normal Hb concentration may be applicable in evaluating the elderly.

Children also exhibit age-dependent differences in red cell parameters compared with healthy adults. The lower limit of normal Hb concentration at birth is 13 g/dL, and this decreases to approximately 11 g/dL by 1 year of age [Reference Saarinen and Siimes27]. This change, called physiological anemia of infancy, reflects the normal physiological adaptation from the relatively hypoxic intrauterine existence to the well-oxygenated extrauterine environment and results from a rapid reduction in Epo production. This is enhanced by the higher Hb oxygen affinity of neonatal RBCs, which reflects both the higher oxygen affinity of residual fetal Hb and the effects on oxygen affinity of the increased 2,3-DPG concentration in these cells [Reference Card and Brain28]. Also, as fetal erythropoiesis is replaced, the MCV decreases from being elevated at birth (100–130 fL) to slightly low by 1 year of age (70–85 fL) [Reference Hows, Hussein, Hoffbrand and Wickramasinghe29]. While the changes in Hb concentration and MCV can be exacerbated by nutritional deficiencies [Reference Gofin, Palti and Adler30], they are not eliminated by nutritional supplemental since they reflect normal physiological processes. The lower limit of normal Hb concentration in both male and female children in the United States, ages 1–2 years, is 10.7 g/dL, and the value rises with advancing age until adult levels are reached near the onset of puberty [Reference Dallman, Yip and Johnson21,Reference Bao, Dalferes, Srinivasan, Webber and Berenson31].

Basic Studies in the Evaluation of Anemia

Specific studies used to identify specific anemia syndromes will be discussed in detail in the chapters dealing with those disorders. In this section, readily available tools used in the initial assessment of anemia will be introduced.

RBC Indices. As mentioned before, electronic blood counters directly measure the RBC concentration, Hb concentration, and MCV. These values are used to calculate the Hct (MCV × RBC concentration), the mean corpuscular Hb (MCH; Hb concentration/RBC concentration), and the MCH concentration (MCHC; MCH/MCV). MCV, MCH, and MCHC are referred to as red cell indices. Anemia with a low MCV is described as microcytic; anemia with an elevated MCV is described as macrocytic; and an MCV in the normal range indicates normocytic anemia. Differences in MCV provide the basis for a popular classification of anemia (discussed below). Anemia with a decreased MCHC is referred to as hypochromic. Iron deficiency is the classic example of a hypochromic anemia. The electronic counters also generate an index of red cell size, the red cell distribution width (RDW). The RDW is a quantitative measure of the variation in red cell size, and the higher the values, the more heterogeneous the RBC population size. Elevated RDW values are common in adult anemias. Highly elevated RDW may be seen in iron deficiency or B12 deficiency, but the discriminatory power of this finding is limited [Reference Bessman, Gilmer and Gardner36]. In microcytic anemias, a normal RDW favors a diagnosis of thalassemia trait over that of iron deficiency [Reference Bessman, Gilmer and Gardner37].

Reticulocyte Count. As noted earlier, the RBC immediately post enucleation contains residual RNA that can be identified by either supravital staining or by flow cytometry methods and is called a reticulocyte. The reticulocyte count is the percentage of RBCs that are reticulocytes and is an indicator of erythropoiesis. A low reticulocyte count indicates an inadequate erythropoietic response and is referred to as an underproduction or hypoproliferative anemia. The reticulocyte count must be interpreted in the context of the degree of anemia. A reticulocyte count of 2% is normal in a patient with a normal Hct of 45% (0.45), but would be inadequate in a patient with significant anemia and an Hct of 20% (0.20). For this reason, many prefer to use the corrected reticulocyte count, which is the reticulocyte count multiplied by the ratio between the observed Hct and some standard normal Hct, usually 45%. In the example cited above, the corrected reticulocyte count is 2% in the patient with Hct 45% and is 0.88% in the anemic patient. The maturation time of reticulocytes to mature RBCs also varies with the degree of anemia [Reference Ganzoni, Hillman and Finch38]. The reticulocyte production index (RPI) takes this into account by adjusting the corrected reticulocyte count by a maturation factor specific to the degree of anemia [Reference Wiktor-Jedrzejczak, Szczylik, Siekierzynski and Rychowiecka39]. In the example above, the RPI for the nonanemic patient is 2.0 (the normal value); the RPI for the anemic patient is low at 0.39. Tables of maturation factors and online tools for calculating RPI (http://cpsc.acponline.org/enhancements/227rpiCalc.html) are readily available. Anemia with an appropriate or elevated reticulocyte count is typically associated with blood loss, either by bleeding or hemolysis.

Peripheral Blood Smear. The microscopic examination of a peripheral blood smear stained with Wright–Giemsa or some comparable staining is a powerful technique in the evaluation of anemia. A normal RBC is approximately the diameter of the nucleus of a small lymphocyte; by making this comparison, macrocytosis (elevated MCV) or microcytosis (low MCV) can be identified. The biconcave nature of the RBC means that there appears to be a translucent area in the center of the RBC, referred to as central pallor. if the area of central pallor is greater than one-third the area of the cell, that indicates hypochromia (low MCHC). RBCs under normal circumstances are relatively uniform in size: increased size variation indicates an elevated RDW. In a number of disorders, red cells or other blood cells exhibit characteristic morphological features (such as the macrocytic oval RBCs and hypersegmented neutrophils of B12 deficiency [Reference Stabler, Allen, Savage and Lindenbaum40,Reference Thompson, Cassino and Babitz41]), which will be discussed in individual chapters.

Bone Marrow Examination. Another morphological approach to the evaluation of anemia involves the microscopic examination of aspirates or needle biopsies of the bone marrow, which are easily obtainable from the posterior iliac crest or (less commonly) from the sternum. This technique allows the assessment of hematopoietic maturation and the quantification of reticuloendothelial iron stores, and, like the examination of the blood smear, permits the identification of diagnostically useful morphological features, such as ring sideroblasts in pyridoxine deficiency [Reference Weintraub42], plasma cell iron deposition in alcohol abuse [Reference Budde and Hellerich43], gelatinous marrow necrosis in malnutrition [Reference Smith and Spivak44], or vacuolated myeloid and erythroid precursors in copper deficiency [Reference Koca, Buyukasik and Cetiner45].

Diagnostic Classification of Anemia

As noted earlier, anemia may be classified in a variety of ways to direct the diagnostic evaluation. One of the most common approaches is to categorize anemia by MCV, as microcytic (MCV usually <80 fL), normocytic (MCV 80–100 fL), or macrocytic (MCV > 100 fL). Specific MCV cutoffs will vary with individual laboratory standards. Microcytic anemias typically result from defects in cellular Hb synthesis. Since Hb consists of globins linked with the iron-containing heme group ferroprotoporphyrin IX, these include globin defects, iron deficiency, as well as syndromes that interfere with the synthesis of the porphyrin component of the heme group, like lead poisoning. Macrocytic anemias include syndromes of asynchronous nuclear/cytoplasmic maturation such as B12 or folate deficiency, but also syndromes of abnormal RBC membrane synthesis like liver disease. Normocytic anemias tend to reflect a mixed bag, including blood loss anemias, most cases of hemolytic anemia, as well as early iron deficiency and the anemia of chronic disease/inflammation, the anemia of renal failure, and most anemias due to endocrine deficiencies. The simultaneous presence of both a microcytic anemia mechanism and a macrocytic anemia mechanism may lead to a falsely normocytic anemia [Reference Spivak46].

Common causes of microcytic and macrocytic anemia are summarized in Boxes 1.1 and 1.2.