Transcription
After completing this section of the course, you should be able to describe the basic function and composition of a mature red cell, understand and describe the principles of normal red cell maturation, recognize the main morphological changes observed during the examination of the blood film, and associate these morphological changes to normal and pathological states.
Before we start, it's useful to go over some definitions. Some key terms are often used interchangeably. The word "erythroid" is a broad term that relates to red cells and their precursors. Likewise, a red cell can be referred to as an erythrocyte. Erythropoiesis is red cell production. The word "hematopoietic" involves anything related to or involved in the formation of blood cells. Anisocytosis denotes a variation in cell size, whilst poikilocytosis is a variation in cell shape. Anisopoikilocytosis is a combination of a variation in cell size and shape. Microcytosis refers to a reduction in cell size, whilst macrocytosis indicates an increase.
The red cell, or erythrocyte, is the most abundant cell found in the blood, with a lifespan of approximately 120 days. It is specialized at delivering oxygen collected from the lungs to maintain essential metabolic cellular activities. Red cells also transport carbon dioxide in reverse to help facilitate its excretion via the lungs. The transport of oxygen and carbon dioxide by red cells is undertaken by hemoglobin. Hemoglobin is an iron-containing protein. Adult hemoglobin is composed of two alpha and two beta-globin chains that encase the iron-containing heme molecule responsible for carrying oxygen. Iron is active in the ferrous state. The alternative ferric state is known as methemoglobin. Methemoglobin is incapable of binding oxygen and can develop as a result of exposure to certain drugs, such as dapsone, and conditions such as G6PD deficiency.
Like all other blood or hematopoietic cells, red cells are derived from a multipotent stem cell found in the bone marrow. This cell has the unique capacity to differentiate into myeloid or lymphoid cells, the two main branches of blood cell development or hematopoiesis. During the normal maturation of hematopoietic cells, a few immature cells found in the bone marrow develop into a large number of mature cells found in the blood. During the maturation process, the cells undergo a number of general changes to their size, cytoplasm, and nucleus. Of the most prominent of these are a reduction in size, loss of nucleoli, reduction in nuclear-cytoplasmic ratio, and chromatin condensation within the nucleus. The red cell is unique as its nucleus is expelled at the final stages of maturation.
We've seen how blood cells are derived from a multipotent stem cell and develop into either myeloid or lymphoid cells. Red cells are part of the myeloid compartment of hematopoiesis. The process of red cell maturation involves several intermediary stages before a mature red cell is released into the blood. These stages are distinct with a characteristic appearance, location, and biology. We'll now spend some time looking at these distinct stages, starting with the most immature cell, the pronormoblast.
The pronormoblast is the largest of all erythroid precursors and is also known as an erythroblast. It has a centrally placed nucleus with loose chromatin structure, often with the presence of nucleoli. The cytoplasm has a deep blue colour and lacks granules. A perinuclear halo may also be seen. Some cells may also exhibit cytoplasmic projections that resemble ears.
Next is the basophilic normoblast. This is smaller than the pronormoblast. Its nucleus has a more condensed chromatin composition, often described as fractured. Nucleoli are absent, and it maintains deep blue, agranular cytoplasm.
The polychromatic normoblast has a progressively more condensed chromatin structure to the nucleus, as opposed to its precursor, the basophilic normoblast. It has a mix of pink and blue cytoplasm. Pink refers to the start of hemoglobin formation and hence an acidophilic or pink colour. The blue denotes the residual presence of ribosomes.
The orthochromic normoblast is the last stage of erythroid development before the nucleus disappears. The nucleus has a very compact structure, known as pyknotic. It is ready to be expelled and hence often found close to the membrane. The cytoplasm is more red to pink in colour, looking more like a mature red cell.
In healthy adults and older children, the cells described so far are located only in the bone marrow. The reticulocyte, however, is the first cell to be found in low levels in the blood under normal conditions. It is also the first cell that lacks a nucleus. The name reticulocyte is derived from the reticular or web-like ribosomal RNA seen with supravital stains, such as methylene blue. It is larger or more blue than a mature red cell. An increase in the number of reticulocytes in the blood produces a heterogeneous colour to the red cells; this is known as polychromasia.
The number of reticulocytes found in the blood provides a crude indication of bone marrow function with regards to erythropoiesis. In the presence of anemia, a low reticulocyte count indicates bone marrow failure with regards to erythropoiesis and can be seen in conditions such as aplastic anemia. Where the reticulocyte count is high in the presence of anemia, it indicates bone marrow compensation with regard to erythropoiesis, and this can be seen in conditions such as bleeding and hemolysis.
Finally, we have the mature red cell, or erythrocyte. This is the smallest of all the erythroid cells, approximately six to eight micrometers in diameter. It has a distinct ovoid, biconcave disc shape. When viewed in two dimensions in the blood film, this shape produces a pale central area. There is no RNA present, giving the cytoplasm an orange to red colour. A normal red cell should equate to the size of the nucleus of a small mature lymphocyte.
As we have already described, microcytosis and macrocytosis refer to a reduction in and increase in red cell size, respectively. Red cell size is reflected by the mean cell volume, or MCV, in the full blood count. When assessing red cell size, the blood film morphology must be interpreted along with laboratory data, including the MCV, red cell distribution width, and reticulocyte count, as well as clinical information like the patient's age, sex, and a thorough clinical history for factors such as alcohol intake and hemorrhage.
The blood film is vital in the morphological assessment of any red cell or blood cell abnormality. When assessing red cell size, the blood film provides confirmation of the MCV. It also allows assessments where there may be more than one red cell population present. This results in a falsely normal MCV but raised red cell distribution width. This may result in cases of mixed iron and vitamin B12 deficiency. The blood film also allows a method of assessment of additional morphological changes.
Let us now look at the definition, causes, and morphological changes seen in microcytosis and macrocytosis.
Microcytosis in adults is defined as an MCV of less than 80 centiliters or any red cell which is smaller than a mature lymphocyte nucleus. Microcytosis is more common in children between the ages of 3 months and 12 years, and hence the MCV reference range is lower in comparison to the adult range. Dietary factors leading to iron deficiency are the most common cause, including the early transition to cow's milk and insufficient nutritional iron intake. In adults, iron deficiency remains the most common global cause of microcytic anemia. Less common conditions that are also related with microcytosis include thalassemia and sideroblastic anemia.
Patients with iron deficiency present with variable anemia. The associated morphological features that can be seen include target cells, pencil cells, hyperchromia, and thrombocytosis. Hypochromia indicates an increase in the size of the central pallor exceeding one-third of the entire red cell diameter. A pencil cell is an elongated cell, or elliptocyte, which has a sharp pointed end to one pole, hence resembling a pencil. These morphological features should be correlated with the serum ferritin and clinical features of chronic blood loss, most commonly from gastrointestinal or gynecological cause. Gastrointestinal malabsorption and malnutrition are also common causes of iron deficiency.
Thalassemias are a spectrum of inherited red cell conditions characterized by an inability to produce the globin chains of hemoglobin. Alpha-thalassemia refers to an inability to make alpha-globin chains. Likewise, beta-thalassemia is an inability to produce beta-globin chains. The clinical consequences are dependent on the number of globin chains lost; the higher the number, the greater the instability of hemoglobin, resulting in red cell breakdown or hemolysis and therefore anemia. Mild thalassemia, or thalassemia trait, is associated with a number of morphological features. In addition to microcytosis, target cells and basophilic stippling can also be seen. There is usually an absence of anemia in uncomplicated cases. There is no morphological discriminator between alpha or beta thalassemia traits; hemoglobin and mutation analysis is required to distinguish alpha and beta origin.
Examples of severe thalassemia states include hemoglobin H and beta-thalassemia intermedia/major. The associated morphological features include anemia, asplenic features including Howell-Jolly bodies, target cells and acanthocytes, and the presence of nucleated red cells. In cases of the alpha-thalassemia condition hemoglobin H disease, unique golf ball-like inclusions can be seen, but only by using supravital stains such as methylene blue.
Sideroblastic anemia is a rare condition resulting from the inability to correctly localize iron in hemoglobin. Iron accumulates in the mitochondria of nucleated red cells found in the bone marrow. The iron is arranged in granules surrounding the nucleus, producing a ring-like appearance, and hence the resulting cell is referred to as a ring sideroblast. To fulfill the definition of a ring sideroblast, the ring must encircle a third or more of the nucleus and contain five or more iron granules. These cells can only be visualized using a specific iron stain, such as Prussian blue. Sideroblastic anemia can result either from rare congenital conditions or be acquired in conditions like myelodysplastic syndrome. Along with microcytosis, the associated morphological changes seen in the blood are basophilic stippling and Pappenheimer bodies. A dimorphic red cell picture is also seen; in other words, where two distinct red cell populations are present.
Macrocytosis in adults refers to an MCV of greater than 100 centiliters or a red cell larger than a small mature lymphocyte nucleus. It is useful to divide the causes of macrocytosis into two groups based on the shape of the red cell: either oval or round.
Oval macrocytosis is most commonly seen in megaloblastic anemia. The most common causes for megaloblastic anemia include severe vitamin B12 deficiency, folate deficiency, and anti-metabolite medication such as methotrexate and hydroxycarbamide. It can also be seen in myelodysplastic syndrome. The key features seen in the blood film include hyper-segmented or right-shifted neutrophils, red cell fragments or schistocytes, and reticulocytopenia manifesting as an absence of polychromasia in the presence of anemia.
Round macrocytosis, however, has a broad differential diagnosis. This includes liver disease, alcohol excess, and an increase in reticulocytes, often secondary to hemolysis. The associated morphological features are dependent on the underlying cause; for example, polychromasia and spherocytosis will also be seen in acquired hemolytic anemia.
Let us now look at changes in the mature red cell shape. The red cell can vary from its normal biconcave shape to a range of different shapes. These include the spherocyte, target cell, stomatocyte, echinocyte, acanthocyte, sickle cell, teardrop cell, schistocyte, and elliptocyte. Let us look at all these in more detail.
As the name suggests, a spherocyte is a sphere-shaped cell. They are seen as small red cells lacking a central pallor when viewed in two dimensions in the blood film. It is often seen in association with polychromasia and late nucleated red cells. It is seen in hereditary spherocytosis, autoimmune hemolytic anemia, and transfusion incompatibility.
A target cell is characterized by a central round stained area with a peripheral rim of cytoplasm. It is also referred to as a codocyte. It is seen in chronic liver disease, iron deficiency anemia, hemoglobinopathies such as hemoglobin C disease, hemoglobin H disease, and homozygous HbE, as well as the post-splenectomy state.
In a stomatocyte, the normal central pallor of the red cell is replaced by a slit-like shape. It is seen in liver disease, excess alcohol intake, and in the rare red cell membrane disorder Southeast Asian ovalocytosis.
An echinocyte derives its name from the Greek word "echinos," meaning prickly, and the echinocyte has small, evenly spaced, sharp, and blunt projections around its periphery. It can be referred to as a burr cell. Echinocytes can be seen in patients with uremia and liver disease. Echinocytes are most commonly seen as a result of prolonged storage of blood samples.
An acanthocyte also derives its name from a Greek word; the word "akantha" meaning thorn, and an acanthocyte has large, irregularly spaced, and coarse projections around its periphery. They are seen in end-stage liver disease and in the post-splenectomy state. Rare conditions can also result in acanthocytes. These include pyruvate kinase deficiency, hereditary abetalipoproteinemia, and McLeod syndrome.
Sickle cells are crescent-shaped red cells with sharp pointed ends. They are the hallmark feature of sickle cell disease. Associated morphological features include polychromasia, nucleated red blood cells, and asplenic features including Howell-Jolly bodies and target cells.
As the name suggests, a teardrop cell is a red cell with a teardrop shape. It is often a sign of bone marrow fibrosis and infiltration. It is also known as a dacrocyte. It can be seen in myelofibrosis. In cases of bone marrow infiltration, small numbers are also seen in pregnant women.
A schistocyte is a fragmented red cell formed by direct mechanical damage and high shear. It is also known as a red cell fragment. It often reflects an emergency presentation. When schistocytes are found in high numbers, high clinical concern should always be raised. Schistocytes can be seen in microangiopathic hemolytic anemias such as TTP, DIC, and HUS, as well as preeclampsia. Due to the wide differential diagnosis, it is important to consider both additional laboratory and clinical features in making a diagnosis of a microangiopathic hemolytic anemia. Schistocytes can also be found in patients with prosthetic heart valves in a phenomenon known as cardiac valve hemolysis.
An elliptocyte is also known as an ovalocyte. Elliptocytes are elongated and thin, often described as rod or cigar shaped. They are seen in conditions such as hereditary elliptocytosis and Southeast Asian ovalocytosis, myelodysplastic syndrome, and thalassemia. As we have already described, pencil cells are a type of elliptocyte, but differ in that they have a sharp-pointed end to one of their poles.
Changes in how mature red cells are arranged can also be seen, of which the two most common features are rouleaux and agglutination. Red cell rosetting will also be described but is rarely seen.
The name rouleaux is derived from the French for "rolls." Red cells usually have a negative charge, known as the zeta potential, on their surface, allowing them to repel each other. Positively charged plasma proteins, such as fibrinogen and immunoglobulins, can cancel this negative charge. In such scenarios, the zeta potential maintaining red cell repulsion is lost. Red cells come into proximity to each other, producing an arrangement akin to a collection of stacked coins. You should not confuse rouleaux with irregular red cell clumping, known as agglutination, described later, and in the thick part of the blood film. More pronounced rouleaux formation causes prolongation in the rate of red cell sedimentation when whole blood is centrifuged. This principle mirrors that used in determining the erythrocyte sedimentation rate. Rouleaux is most commonly seen in multiple myeloma, infection, inflammation, and malignancy.
Agglutination describes red cells that are irregularly arranged in clumps. This phenomenon can cause a falsely low hemoglobin level and elevated MCV in the full blood count. Agglutination is most often due to cold agglutinin disease or CHAD. CHAD is a type of autoimmune hemolytic anemia caused by high concentrations of circulating cold-sensitive autoantibodies. These antibodies can be monoclonal or polyclonal and are active at temperatures below 30 degrees Celsius. They are directed against red blood cells, causing them to agglutinate and undergo lysis. CHAD can be secondary to infections, such as Mycoplasma pneumoniae, and lymphoma, or be referred to as primary disease arising from an unknown cause. In the cold peripheries of the body, free IgM autoantibodies in the plasma transiently bind to the red cell, activating complement. When the red cell, bound with antibody, enters the warm core of the body, the antibody dissociates from the red cell, and extravascular hemolysis occurs. In CHAD, the direct antiglobulin test, or DAT, is usually positive for C3d.
Red cell rosetting is a rare feature of blood films. It is characterized by red cells arranged around a central white cell. The significance of this phenomenon is unclear. It can also be seen in malaria, secondary to Plasmodium falciparum infection, where a single infected red cell is surrounded by non-infected red cells.
Let us now consider the intracellular changes and inclusions that can be seen in mature red cells. We will look at Howell-Jolly bodies, basophilic stippling, Pappenheimer bodies, Heinz bodies, and Cabot rings.
Howell-Jolly bodies are single dark inclusions located at the periphery of a mature red cell. It is formed from remnant DNA, usually removed by the spleen. It is one of the main features as a result of complete or partial absence of splenic function, hence it is associated after surgical splenectomy and in functional hypo- or asplenic states associated with conditions such as sickle cell disease, thalassemia, and celiac disease. The associated morphological features that may also be seen include target cells, thrombocytosis, and possibly a panleukocytosis.
Basophilic stippling is also known as punctate basophilia. This is fine, multiple granular inclusions formed from abnormal RNA. They are most commonly seen in lead poisoning, thalassemia, and myelodysplasia.
Pappenheimer bodies are abnormal basophilic or blue-purple granules seen on routine stain. They are made of iron found inside red blood cells and visible due to co-precipitation of iron-containing organelles with clumps of ribosomes. The number of granules ranges from one to two and rarely exceeds five, and are located in the cell periphery.
A Heinz body is the accumulation of denatured hemoglobin. This can arise either from oxidative damage to the red cell or, as seen in thalassemia, from an imbalance of alpha and beta globin chains. Heinz bodies are removed by splenic macrophages and form a bite cell or degmacyte through conformational changes. The bite cell then adopts the shape of a spherocyte. Heinz bodies are visible using supravital stains, such as methylene blue (see blue inclusions around the periphery of the red cell). Heinz bodies are seen in G6PD deficiency and NADPH deficiency, as well as liver disease and alpha-thalassemia.
A Cabot ring is a thin structure resembling a figure of eight. It is formed by abnormal mitotic spindle formation. It is seen in myelodysplastic syndrome, megaloblastic anemia, and where patients have received drugs that disrupt the normal mitotic spindle formation, such as vincristine.
Up until now, we have concentrated on the main morphological changes related to the mature red cell. In this section, we will focus on the conditions related to the proliferation of erythroid precursors in the blood.
In the normal adult state, nucleated red cells are found almost exclusively in the bone marrow. Although reticulocytes can be seen in the blood, they do not exceed two percent of the total number of red cells. Hence, the presence of nucleated erythroid cells is always abnormal in adults. Nucleated red cells, however, can be found in newborns and pregnant women.
As we have already seen, the presence of a significant number of reticulocytes produces polychromasia. This is due to the fact that reticulocytes are larger and have a blue tinge compared to mature erythrocytes. It is useful to remind ourselves of the importance of the presence of reticulocytes and hence polychromasia in cases of anemia with or without other cytopenias. The number of reticulocytes found in the blood provides a crude indication of bone marrow function and particularly erythropoiesis.
In the presence of anemia, where there is an absence of polychromasia and a low reticulocyte count, it suggests that the bone marrow has failed with regards to erythropoiesis, and this can be seen in conditions such as aplastic anemia. Where the reticulocyte count is high, and hence the presence of polychromasia, erythropoiesis within the bone marrow is functional and is an appropriate compensatory mechanism for the anemia seen in conditions such as bleeding and hemolysis.
The conditions where nucleated red cells are seen in the blood can be divided into two groups depending on the dominant stage of maturity. The first group is the later red cell precursors, namely polychromatic normoblasts, orthochromic normoblasts, and reticulocytes. The second group is made up of early red cell precursors, comprising of pronormoblasts and basophilic normoblasts.
Significant polychromasia is often accompanied by the presence of late nucleated erythroid precursors. Late nucleated red cells are found in term newborns, continuing in the first few days of life. An increase in the number of these cells is often exacerbated by physiological stress. This includes prematurity, fetal blood loss, intrauterine hypoxia, and maternal diabetes mellitus. In adults, the main pathological states that result in the presence of late nucleated red cells include leukoerythroblastosis, thalassemia, sickle cell disease, and hemolysis.
Leukoerythroblastosis denotes the presence of both immature red cells and granulocytes, namely neutrophils, in the blood. It indicates bone marrow infiltration, bone marrow fibrosis, or severe bone marrow stress induced by conditions such as infection. In the adult, it is always abnormal and should prompt clinical correlation and, if deemed appropriate, bone marrow examination.
The presence of high numbers of nucleated red cells are a distinct feature of beta-thalassemia intermedia and major. These cells are often dysplastic with abnormal nuclear shape, reflecting ineffective erythropoiesis. Features include microcytosis, hyperchromia, and significant red cell anisopoikilocytosis, such as target cells. Nucleated red cells are also seen in severe alpha-thalassemia, in particular hemoglobin H disease, although in fewer numbers in uncomplicated alpha and beta thalassemia trait. Nucleated red cells are rarely seen in sickle cell disease. The presence of nucleated red cells reflects the background hemolysis caused by defective erythropoiesis.
The absence of polychromasia or reticulocytopenia and nucleated red cells indicates red cell aplasia. This is often caused by infection with parvovirus B19. In warm autoimmune hemolytic anemia, polychromasia and spherocytosis are the most prominent morphological features. The number of late nucleated precursors is variable.
Hemolytic disease of the fetus and newborn, or HDFN, secondary to fetal-maternal RhD incompatibility produces a marked polychromasia and an increase in late nucleated erythroid cells. Erythroblastosis fetalis is the term used to describe this morphological phenomenon. It is mediated by IgG alloantibodies between an RhD-negative mother and RhD-positive fetus. Other alloautoantibodies produce different morphological features. In HDFN, marks spherocytosis rather than an increase in nucleated red cells, as seen. Cold-induced HDFN causes inhibition of erythroid progenitors in the bone marrow. This leads to reticulocytopenia, an absence of spherocytosis, and nucleated red cells.
The presence of pronormoblasts and basophilic normoblasts in the blood are rare occurrences and should raise significant concern of an underlying hematological disorder. The conditions most commonly seen in this scenario include erythroid leukemia, megaloblastic anemia, and congenital dyserythropoietic anemia.
Pure erythroleukemia is an aggressive subtype of acute myeloid leukemia. It is caused by the malignant proliferation of erythroid precursors. It accounts for less than one percent of all acute myeloid leukemia cases, often with a very poor prognosis. In pure erythroleukemia, the blasts are predominantly at the pronormoblast stage. Cytoplasmic vacuolation and blebs are a common finding, and blasts account for 20% or more of the total nucleated cells in the bone marrow. If there are less than 20% erythroid blasts, you should consider the presence of such genetic abnormalities and dysplasia. These states will be classified as either myelodysplastic syndrome or other non-erythroid acute myeloid leukemia.
Megaloblastic anemia causes a maturation arrest at the early stages of erythroid development. Hence, there is an excess of erythroblasts in the bone marrow with dyserythropoiesis. Erythroblasts can rarely spill over into the blood and be seen in the blood film. This, however, is not the predominant feature, and you should not forget to look out for the classical features, including oval macrocytes and hyper-segmented neutrophils.
Congenital dyserythropoietic anemia, or CDA, are a rare heterogeneous group of inherited conditions characterized by anemia and iron overload. A distinct feature is multinuclearity. Three types of CDAs are recognized: type 1, type 2, and type 3. Of these types, type 3 is the rarest form, caused by mutations of the CDAN3 gene. Early erythroblasts are most commonly found in the blood of patients with type 3 CDA. Multinucleated erythroblasts are more commonly found in the bone marrow of the other two CDA groups.
You have now completed this section of the course. In order to progress further, you must complete and successfully pass the red cell assessment found on your course dashboard. In the next section, our focus will move on to platelets.