ANSWER
Abstract
The morphology of blood cells is an essential tool in laboratory hematology. In several primary and secondary haemopathies, erythrocyte morphology suggests possible etiopathogenic events. Despite advancements in medical technology and laboratory automation, red cell morphology remains a fundamental aspect of hematological assessment. Human erythrocytes are discoid (bi-concave), about 7-8 m (the size of a small lymphocyte’s nucleus) in diameter, with a central area of pallor (which occupies one-third of the red cell diameter), and are well hemoglobins in the outer two-thirds of the red cell diameters, with no inclusions. Abnormalities in size, shape, color, distribution, or presence of inclusion bodies suggest possible disease processes. This chapter is thus devoted to the morphologic description of human erythrocytes and a study of possible abnormalities, underlying pathophysiology, and the associated differential diagnosis in humans.
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morphology of red cell erythrocytes

hematology

Disorders of the red blood cells

film of peripheral blood
Show Author Information + 1. Introduction
Erythrocytes are the most numerous cellular components of circulating blood. The number of erythrocytes in circulation is approximately 5 million cells per cubic millimeter of blood. With an average life span of 100-120 days, erythrocyte production and senescence are constantly balanced. Any imbalances in the production or destruction of red cells cause red cell disorder. In essence, various factors keep red cells at a constant volume in the body. Physiologic factors such as age, gender, altitude, smoking status, and pregnancy account for minor inter- and intra-individual differences. Red cell counts are typically measured using red cell mass, volume, count, hematocrit, and hemoglobin concentration. For a person’s age, gender, and race, red cell volume or group is expected to fall within a mean 2 SD interval within a specified population.

Beyond count abnormalities (quantitative abnormalities), morphologic abnormalities (qualitative abnormalities) are significant in diagnosing red cell diseases. A red cell is usually round, shaped like a disc, with a well-haemoglobinised cytoplasmic rim and a central pallor covering the inner third of the red cell. Morphological variations (size, shape, color, contents/inclusion, or distribution) may be associated with or diagnostic of disease entities. A blood picture with a lack of red cells, numerous red cell fragments, and an increase in polychromatic red cells, for example, suggests a microangiopathy or fragmentation syndrome.

This chapter aims to discuss the principles of red cell morphology, describe red cells in terms of morphology, and identify morphologic abnormalities associated with various disease conditions.

Advertisement 2. Erythrocyte Morphology Principles
Bone marrow stem cells generate circulating red cells. Pluripotent stem cells self-replicate and differentiate into specialized cells as they circulate through different lineages. The myeloid stem cell lineage gives rise to red cells (colony-forming unit—granulocytes, erythroid, myeloid, and megakaryocytes). The pronormoblast is the first recognizable red cell precursor in the bone marrow. The pronormoblast matures into the orthochromatic normoblast through a series of steps. The late normoblast transforms into shift reticulocytes after its nucleus is extruded and released into circulation. Finally, the pitting action of the spleen removes DNA remnants and other chromatin materials in the reticulocytes, resulting in mature red cells.

Erythrocytes are invisible to the naked eye. When there is an indication, the morphology of red cells is typically performed on peripheral blood smears. A clinical request or laboratory flags indicate erythrocyte morphology. Table 1 shows clinical manifestations of peripheral blood film/erythrocyte morphology.

The following indications may prompt a clinical request for a PBF:
Unknown cause of anemia, leucopenia, or thrombocytopenia

Unknown cause of leukocytosis, lymphocytosis, or monocytosis

Unknown cause of jaundice or hemolysis

Splenomegaly, jaundice, and bone pains are characteristics of congenital hemolytic anemias.

Chronic or acute myeloproliferative disease, such as chronic myeloid leukemia, is suspected.

Organ failures, such as renal disease or liver failure, are suspected.

Characteristics of hyperviscosity syndrome include leukemic hyperleukocytosis and polycythemia.

Infections caused by parasites and severe bacterial sepsis

Advanced cancers that may involve the bone marrow

Cases of nutritional anemia are suspected.

Chronic lymphoproliferative disorders, such as chronic lymphocytic leukemia, are suspected.

Leukemic spills in advanced lymphoma

Disseminated intravascular coagulopathy and other red cell fragmentation syndromes are assessed.

Table 1 summarizes the clinical indications for peripheral blood film.

Erythrocyte morphology may also be indicated when significant deviations from normal are observed in the laboratory during blood work (complete blood count), regardless of a clinical request. For example, a significantly lower hemoglobin level combined with a low MCV and a high RDW may indicate iron deficiency anemia. This is a red cell morphology and other ancillary investigation for iron deficiency indication.

A venipuncture is used to collect blood for a peripheral blood film. The preferred anticoagulant is potassium EDTA. Specimens should be tested as soon as possible, preferably within 2 hours of being collected. Samples that are not immediately analyzed should be stored in a refrigerator at 2-6°C, or a blood smear should be made, dried, and fixed for subsequent staining with Romanowsky dyes.

Aside from automated slide makers, the slide ‘wedge’ or push technique is the most commonly used method for preparing peripheral blood film. This technique usually necessitates using microscope slides, a pipette/blood dropper, a spreader slide, and the blood specimen to be examined. To prevent the spread of infectious pathogens such as the human immunodeficiency virus and hepatitis viruses, standard precautions must be taken.

Quality control measures will ensure that the anticoagulant: blood ratio is correct, that samples are processed/analyzed within the sample viability period, and that the blood is adequately mixed before smearing. Each slide must bear at least two patient identifiers, such as name and laboratory, as well as the date of the procedure. Fixation of the blood tissue occurs after the smear has air-dried for about 5-10 minutes. Fixation aids in the preservation of cell architecture, which ensures good morphology. A dried slide should be fixed within 4 hours, preferably within the first hour.

The glass slides are stained with Romanowsky dyes for normal morphology. Romanowsky dyes are stains that contain both acidic and basic components. Eosin is the acidic component, and azure B, polychrome methylene blue, is the essential component. Leishman, Jenner, Wright, May-Grunwald-Giemsa, and Giemsa stains are all Romanowsky stains. In general, the eosin component of the dye binds to the cell’s essential element, such as the hemoglobin molecules in the red cell, and stains it pink. The dye’s basophilic component attaches to the acidic components of cells, such as the nucleus, and stains it blue. Other cell components appear in a variety of color shades that contrast and contrast with the dye. A neutral to sky-blue color shade is described as azurophilic. A neutrophil’s cytoplasm, for example, is described as azurophilic in color. Table 2 also shows the characteristic staining quality of various red cell components.

Chromatin, a cell component of color (including H-J bodies)

Purple
Cytoplasm containing RNA and nuclear remnants (for example, polychromasia and basophilic stippling)
RNA produces a blue color in polychromatic red cells, which offsets the pink color, imparting a purple tinge.
Basophilic stippling appears as blue granules scattered throughout the cytoplasm in a punctate pattern.
mature red blood cells
Pink
Table 2.
The Romanowsky staining properties of red cell components.

The staining procedure and stain contact time are determined by the type of dye used. Standard laboratory texts and reagent manuals contain staining protocols. The morphology of red cells should be studied in the monolayer region of the film, which is 2-4 10 fields from the feathered edge. Red cells are randomly distributed in this location, with the majority lying singly and only a few overlapping. RBCs will appear flat with no central pallor if the area is too thin. Because red cells are packed, false rouleaux may be reported, and morphology may be challenging to evaluate.

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3. Red cell morphologic abnormalities
Under the compound microscope, the haematomorphologist examines the red cell morphology and notes any significant abnormalities for reporting/diagnosis in light of the patient’s clinical context. The morphology of red blood cells is assessed in size, shape, color, distribution, and intracytoplasmic inclusions. In general, red cells vary in size in a relatively uniform manner, with a red cell distribution width of 11-15% in normal individuals. Anisocytosis and poikilocytosis are abnormal variations in size and shape [1].

Anisocytosis 3.1
Normal red cells (normocytes) have a diameter of about 7-8 m [2]. Microcytosis refers to a reduction in size. Macrocytosis is defined as an increase in red cell diameter above normal. The size of red blood cells is used to classify anemia morphologically or cytometrically. Anaemia can be classified as microcytic, normocytic, or macrocytic based on red cell size. Typically, the size of a normal red cell is determined by comparing it to the nucleus of a small lymphocyte. The normal range for mean red cell volume (MCV) is 80-95 fl [3, 4]. MCV greater than 95 fl is referred to as macrocytic. Microcytic red cells have a diameter of 6 m and an MCV of 80 fl [5]. Microcytic anemias are distinguished by iron deficiency, thalassemias, sideroblastic anemia, and anaemia of chronic inflammation (20% of cases). Serum ferritin, total iron binding capacity (TIBC), and hemoglobin electrophoresis with quantification are additional tests that can help differentiate microcytic anemia [4, 6]. Iron deficiency, for example, is characterized by low serum ferritin, increased TIBC, and increased RDW. A thalassemia is indicated by normal or elevated red cell counts with slight red cell size variation (RDW) in macrocytosis.

Normocytic anaemia occurs in acute blood loss, marrow aplasia, chronic disease anaemia (80% of cases), and endocrine anaemia. Macrocytosis can be oval or round, with distinct casual relationships. Oval macrocytes are found in megaloblastic anemias (folate/cobalamin deficiencies), myelodysplastic syndrome, and drug therapies like hydroxyurea [7]. Round macrocytes are found in liver disease and excessive alcohol consumption. MCV may appear falsely normal in combined substrate deficiency states on a hematology analyzer. However, the blood test will show significant anisopoikilocytosis. The red cell distribution width (RDW) is a calculated parameter that measures the variability in red cell size (heterogeneity). RDW is the percentage coefficient of variation of the particle counter-enumerated individual red cell volumes [8]. RDW is typically between 11.5 and 15.5%. RDW is elevated in iron deficiency anaemia, megaloblastic anaemia (folate and cobalamin deficiency), hemolytic anaemia, recent blood transfusion, hereditary spherocytosis, and sickle cell syndromes [8, 9]. RDW helps interpret seemingly normal MCV because it will be high in combined micronutrient deficiency.

Poikilocytosis (3.2)
Shape abnormalities, also known as poikilocytes, can help narrow down a diagnosis. It is important to note that poikilocytosis can also occur in vitro (artefactual causes). As a result, adequate precautions must be taken to reduce pre-analytic and intra-analytic errors that affect morphology. In blood film morphology, the following quality control measures must be followed:

Blood specimens for PBF are best collected via venipuncture in EDTA bottles.

The optimal blood-anticoagulant ratio should be followed.

Samples should be sent to the hematology laboratory as soon as possible. Prolonged analysis delays allow cellular degeneration, pseudo-thrombocytopenia, and artifacts [10].

Blood specimens for morphology should be analyzed within 2 hours of being collected.

Poikilocytes are classified as spiculated or non-spiculated. At least one pointed projection from the cell surface is present in spiculated red cells. Burr cells, schistocytes (red cell fragments), irreversibly sickled red cells (drepanocytes), acanthocytes, and teardrop red cells are all examples of spiculated poikilocytes (dacrocytes). Target cells, ovalocytes, and stomatocytes are examples of non-spiculated poikilocytes. Pathologic changes in red cell shape are caused by various mechanical, biochemical, and molecular mechanisms. Some occur as a result of hematopoietic system disruptions. Target cells have a central area of haemoglobinization (dubbed hyperchromic bull eyes) surrounded by a pallor halo. Target cells have a higher surface area to volume ratio due to their redundant membrane, which gives rise to the targetoid shape. Target cells are seen in sickle hemoglobinopathies, thalassemias, iron deficiency, and post-splenectomy states (Figure 1). Teardrop red cells (Figure 2) are caused by abnormal spleen or bone marrow pathology, such as primary myelofibrosis, when red cells stretch out to navigate their way into the periphery, or by splenic pitting action when red cells with inclusions such as Heinz bodies guide the splenic cords into the sinuses [5].

(1) Nucleated red cell, (2) target cell, and (3) irreversibly sickled red cell are depicted in Figure 1.

Figure 2. (4) Red teardrop cell.
Stomatocytes resemble fish mouths (slit-like central pallor). They are primarily caused by increased red cell permeability, which results in increased volume. Stomatocytes can be inherited or developed. Rh null phenotype has hereditary stomatocytosis. Acquired stomatocytosis is most commonly associated with recent excessive alcohol consumption and usually resolves within two weeks of alcohol withdrawal. Stomatocytes are typically 10% of the red cell population when artefactual. Sickle syndromes are characterized by irreversibly sickled red cells (Figure 1). The primary event is intra-erythrocytic hemoglobin precipitation (gelation), which leads to tactoids forming, which deforms the discoid red cell into a sickle or crescent shape [11]. Burr cells are observed in renal failure and may be fictitious. Poor fixation and high humidity in the laboratory environment may result in artefactual red cells. If the tails of the teardrop cells line up in the same direction, artefactual tear drop cells should be suspected. Table 3 lists common poikilocytes and their variants [1, 5, 12, 13, 14, 15].

Forms of red cells

Diagnosis differentiation
Red blood cells that have become irreversibly sickled (drepanocytes)
Sickle cell disease (SS, SC, and S—thalassemia)
Red blood cells should be targeted (codocytes)
Cells of interest (codocytes, Mexican hat cells)
Sickle cell disease, hemoglobin C trait, hemoglobin CC disease, thalassemia, iron deficiency, liver disease (cholestasis), and asplenia are all examples of blood disorders.
Red cells that have been fragmented (schistocytes, helmet cells, keratocytes)
Disseminated intravascular coagulopathy (DIC), thrombotic thrombocytopenic purpura, and hemolytic uraemic syndrome are examples of thrombotic microangiopathic hemolytic anemias.
Cells with pencils
a deficiency in iron
Stomatocytes
Artefact (due to slow drying in a humid environment), liver disease, alcoholism, Rh-null disease, and obstructive lung disease are all possibilities.
Elliptocytes
Hereditary elliptocytosis (>25% of cases)
Cell bites (degmacytes)
G6PD deficiency, oxidative stress, unstable hemoglobins, and congenital Heinz body anemia are all factors to consider.
Basket cells (also known as half-ghost cells or butterfly cells)
oxidative stress, G6PD deficiency, and unstable hemoglobin
Spherocytes
Hereditary spherocytosis, ABO incompatibility, autoimmune hemolytic anemia (warm antibody type), and severe burns are all possibilities.
Red teardrop cells (dacrocytes, macrocytes)
Table 3: Red cell shape anomalies and associated diseases in idiopathic myelofibrosis, myelophthisic anemia, and thalassemia.

3.3 Polychromasia/Anisochromia
Anisochromia is characterized by increased or decreased haemoglobinization of red blood cells. The central pallor of hypochromic red cells exceeds one-third of their diameter. In iron deficiency states, hypochromic usually follows microcytosis. Shape abnormalities such as (micro)-spherocytes and sickled red cells are associated with hyperchromasia (increased haemoglobinisation). Central pallor is eliminated by increased haemoglobinization. Severe hypothermia is occasionally related to macrocytic red cells known as leptocytes. Leptocytes are found in patients with severe iron deficiency, thalassemia, and liver disease [14]. Polychromasia on PBF suggests reticulocytosis in vivo. Polychromasia means “many colors,” implying that red cells bear a color other than pink (eosinophilic). Polychromatic red cells have a bluish tinge and are macrocytic (young red cells). The blue tinge indicates the presence of rRNA, which is eventually pitted by the spleen to form mature circulating red cells [1]. Typically, polychromatic red cells on PBF are not visible—the adult reticulocyte population is around 0.5-2.5% [3]. Iridescent red cells over 1-2% in the periphery, on the other hand, should be considered significant because the average daily rate of red cell turnover is about 1-2% [16]. In acute hemorrhage, hemolysis, or high altitude, hypoxia causes increased erythroid activity, resulting in polychromasia. Polychromasia occurs in extramedullary hemopoiesis due to myeloid metaplasia in reticuloendothelial tissue. Polychromatic red cells are seen as a response to micronutrient deficiency treatment after haematinic therapy [1].

Similarly, when there is marrow stress, nucleated red cells (erythroblastosis) leave the bone marrow prematurely to compensate. Severe anemia, asplenic/hyposplenism state as in sickle cell disease, severe hypoxia, marrow replacements or infiltrations, and extramedullary hemopoiesis are all notable causes of erythroblastosis (or normoblastemia) [17, 18]. Nucleated red cells are typically seen in the periphery of neonates [15].

3.4 Additional red cell abnormalities
Other morphologic abnormalities include inclusion bodies and pathologic red cell distribution on the smear. Inclusion bodies are absent in mature erythrocytes. Nuclear products RNA/DNA, hemoglobin, and iron pigments are examples of red cell inclusion bodies. Some, such as hemoglobin H inclusions and Heinz bodies, require supravital staining to be seen. Oxidative stress, severe infections, and dyserythropoietic cause red cell inclusions (maturation defects). Denatured RNA fragments dispersed within the cytoplasm cause basophilic stipplings or punctuate basophilia. Fine, blue stipplings or coarse granules are examples of basophilic stipplings. They are non-specific and are generally associated with haem biosynthetic pathway disorders [1, 19]. Differentials include hemoglobinopathies (thalassemias), lead or arsenic poisoning, unstable hemoglobins, severe infections, sideroblastic anaemia, megaloblastic anaemia, and pyrimidine 5′ nucleotidase deficiency, a rare inherited condition [1, 10, 20].

Fine basophilic stippling, which is clinically insignificant, may be associated with polychromasia/accelerated erythropoiesis/reticulocytosis. Coarse stipplings are clinically significant and indicate impaired hemoglobin synthesis, which is seen in megaloblastic anaemia, thalassemias, sideroblastic anemias, and lead poisoning [1, 19]. Basophilic stipplings are diffusely distributed throughout the red cell cytoplasm, unlike other basophilic inclusions such as Howell jolly bodies and Pappenheimer bodies. Howell jolly bodies (Figure 3) are DNA remnants found in patients with anatomical or functional asplenia after splenectomy. Siderotic granules, also known as Pappenheimer bodies, appear purple on Romanowsky stain and blue on Perl’s stain and are found in iron utilization disorders such as sideroblastic anemia.

Figure 3: Howell. Jovial physique (in a 36-year-old lady with sickle cell disease).
Plasmodium spp. and Babesia spp. can also be found on peripheral blood smears [21]. Both parasites infiltrate red blood cells. Their identification necessitates some knowledge and experience. Plasmodium spp. is divided into several species. Plasmodium spp. can exist in various forms, including trophozoites, gametocytes, and schizonts. Babesia spp. Appear in small ring forms (similar to Plasmodium falciparum), but no schizonts or gametocytes form [1, 21]. Babesia spp., unlike Plasmodium spp., does not produce pigments. Babesia spp., on the other hand, can be found in groups other than erythrocytes. Clinical and travel history are also helpful in distinguishing the two parasites. Supravital staining is required to see other red cell inclusions, such as Heinz bodies and hemoglobin H inclusions (reticulocyte preparations). Heinz bodies are hemoglobin denatured (seen in oxidant injury, G6PD deficiency). In alpha-thalassemias, hemoglobin H inclusions are seen, giving rise to the characteristic ‘golf ball’ appearance of the erythrocytes [1, 11, 12].

Rouleaux formation is stacking red cells in a single file like coins. Rouleaux can be found in hyperproteinaemias. Increased plasma fibrinogen or globulin levels reduce the zeta potential (repulsive force) between circulating red cells, allowing them to stack. Rouleaux has been linked to myeloma/paraproteinaemias, other plasma cell disorders, and B-cell lymphomas. Agglutination, on the other hand, is the clumping or aggregation of red cells into clusters or masses that are usually antibody-mediated [1]. Red cell agglutination can occur in cold haemagglutinin disease and Waldenstrom’s macroglobulinemia [1, 11]. Agglutination is linked to a falsely low red cell count and a high MCV. Warming the specimen with a heating block before performing a blood smear and automated cell counts help to disperse the red cells.

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4. Finally,
Red cell morphology is critical in diagnosing anemias and other blood disorders. A good quality smear with proper Romanowsky/special staining, combined with the expertise of a haemato-morphologist (hematologist/pathologist of hematology), is still extremely valuable in patient care.

QUESTION
1.Sickle cell disease is an adaptive response against malaria. Discuss and identify the pathogen and transmission of malaria.

2. Where and why do you think an adaptation occurred that led to the development of sickle cell disease and what is the pathophysiology of the adaptation?

3. Why do people who no longer live in parts of the world where malaria is prevalent still develop sickle cell disease and how and why does it occur?

4.How can we advise families with this debilitating disease?

RUBRIC

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4. Please expand your ideas and add your thought to your writing. Why did you use that resources? What will this quote add to the discussion?

You are now at the master’s level, you can agree or disagree with the author. Your opinion and experience counts!

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