Histology of Hematopoiesis
Blood is one of the most integral components of the cardiovascular system. Between 4 to 5 litres of blood circulates through the body of the average (70 kg) human. It has a multi-factorial role in the survival of each individual. It not only shuttles much needed oxygen to different organ systems, but it also facilitates the removal of waste products from these sites as well.
Furthermore, there are constituent cells whose principal function is to protect the host from invasion from foreign organisms as well as keeping the host cells in check to avoid undesired mutations. The process by which these cells develop throughout extra-uterine life is known as haematopoiesis; the Greek translation of which simply means “to make blood”.
The bone marrow is comprised of a myriad of haematopoietic stem cells that respond to a variety of biochemical cues that promote their differentiation of mature blood cells.
All the mature blood cells have a relatively limited lifespan. For example, normally developed red blood cells remain in circulation for approximately 120 days before being decommissioned. Therefore, it is necessary that a renewable supply of progenitor cells exists to replace old cells. The pluripotent stem cells formed during the embryonic period have the capacity to differentiate into common myeloid and common lymphoid cell lineages.
The common myeloid cell line further differentiates into granulocytes, erythrocytes, and thrombocytes, while the common lymphoid cells give rise to thymic and bone marrow lymphocytes.
This article aims at discussing the embryology and histology of blood cells, additionally evaluating the post-embryonic development of these cells. Disorders of cellular differentiation and their clinical consequences will also be mentioned.
- Histology of Myeloid Cell Lineage
- Histology of Lymphoid Cell Lineage
- Clinical Significance
- Related Atlas Images
It has long been accepted that extra-embryonic haematopoiesis (formation of blood cells outside of the embryo) precedes intra-embryonic blood cell development. Around week two of gestation, mesodermal cells congregate within the yolk sack of the developing embryo to form well defined cell clusters. These clusters of blood cells possess both vasculogenic and haematopoietic potential and are therefore classified as hemangioblasts.
The peripheral cells differentiate into endothelium, and by extension will form blood vessels. However, the remaining are pluripotent cells that will differentiate into granulocyte, erythrocyte, lymphocyte, thrombocyte, and monocyte cells lines subsequently. These primitive mesodermal cells move more peripherally to facilitate the creation of the vessel lumen, and are attached to the vascular endothelium. These clusters of cells, are referred to as blood islands, begin to appear around day 16 of development and form the basis of extra-embryonic haematopoiesis. Subsequently they are completely missing by the 8th gestational week.
Pluripotent granulocyte erythroid macrophage megakaryocyte colony forming units (CFU-GEMM), erythroid blast and colony forming units (BFU-E and CFU-E) as well as granulo-macrophage forming units (CFU-GM) are seen in the embryo half way through the 4th gestational week.
Most of the blood cells formed in the yolk sac are primitive erythrocytes (occasionally primitive megakaryocytes and macrophages are also present in very small quantities). Morphologically, the primitive erythrocytes are megaloblasts (contain a nucleus) that express the glycophorin A surface markers and produce embryonic haemoglobin (i.e. Hb Portland-1 [ζ2γ2], Hb Portland-2 [ζ2β2], Hb Gower-1 [ζ2ɛ2] and Hb Gower 2 [α2ɛ2]). It should be noted that embryonic haemoglobin has a short life span and is no longer detectable by around the 12th week of gestation. Thankfully, the synthesis of foetal haemoglobin (Hb F; α2γ2) begins simultaneously with embryonic haemoglobin. Hb F is more durable outlives embryonic haemoglobin and persists into extra-uterine life.
Foetal circulation coincides with the onset of the heart beating. This important event also allows for yolk sac derived blood cells to enter embryonic tissue. Conveniently, haematopoietic stem cells are able to begin colonizing future sites of haematopoiesis. The liver – which is derived from both endoderm (the foregut diverticulum) and mesoderm (septum transversum) – is the first intra-embryonic site of haematopoiesis.
As blood cells move from the yolk sac to the liver, the morphology also changes from megaloblastic primitive erythrocytes to definitie macrocytic erythrocytes (nucleus removed). Consequently, there is also a switch from embryonic haemoglobin to foetal haemoglobin. There is a proposition that the liver is colonized in two phases. Initially around the 3rd gestational week the hepatic sinusoids contain cluster of differentiation 34 negative (CD34-) erythro-myeloid cells.
It is not until early in the 5th gestational week that CD34+ precursors are found in the liver. The latter cells are capable of carrying out extended haematopoiesis. As BFU-E cell lines become more abundant in the liver and embryonic circulatory system, there is a dramatic reduction of these cells in the yolk sac. Eventually, midway through the 8th week of gestation, all haematopoietic activity ceases in the yolk sac. By the 12th week of gestation, high proliferative potential colony forming cells (HPP-CFC) as well as CFU-GEMM are detectable in the liver, and absent from the yolk sac.
In addition to the classical notion that all blood progenitor cells originate in the yolk sac then migrate into the embryo and persist into adulthood, evidence supports the notion that some progenitor cells actually develop within the embryo. The abdominal arterial system has been noted to house haematopoietic stem cells in the ventral region of the endothelium.
Clusters of about 3 cells are observed in the rostral part of the duplicated aorta around the end of the 4th gestational week. These cells undergo significant replication and by the end of the 5th week of gestation, they number in the thousands and extend caudally toward the umbilical portion of the abdominal aorta. This process is usually seen between the first and second hepatic colonisations. Evidence supports the concept that the endothelial lining of the abdominal arteries possess haematopoietic capabilities and is able to differentiate into haematopoietic stem cells that can both replicate and differentiate.
Coincidentally, the final haematopoietic site to develop in utero, takes over haematopoietic activity into extra-uterine life. The bone marrow is colonized by haematopoietic stem cells in the 11th week of gestation; long after blood cells production has ceased in the yolk sac, and is well underway in the thymus and liver.
The osteoblasts (which are also mesodermal in origin) are thought to aid in haematopoiesis by sustaining the bone marrow stroma that supports the haematopoietic stem cells. A mixture of dense fibrils and loose mesenchyme surrounding a central artery is the point at which marrow haematopoiesis begins. These areas are known as the primary logettes and are curiously located as far from the ossifying trabecular bone as possible.
To summarize the temporal sequence of the embryological appearance of haematopoietic stem cells:
- Within the third week of gestation (day 17) the yolk sac begins to show haematopoietic activity.
- Early in week 4 (day 23) the first wave of haematopoietic cells colonize the liver. This coincides with a tapering off of yolk sac blood cell activity.
- The abdominal arterial clusters show haematopoietic activity towards the end of week 4 (day 27).
- The second weave of haematopoietic cells colonize the liver early in week 5 (day 30).
- The bone marrow is colonized in week 11 of gestation.
Histology of Myeloid Cell Lineage
Megakaryocyte & Erythroid Progenitor Cell
Red blood cells are the most abundant of the formed haematological elements within the circulation. Its primary role is to carry oxygen to target tissue and return carbon dioxide to the lungs for excretion. A bi-progenitor cell line known as the megakaryocyte-erythroid progenitor is formed from the common myeloid progenitor series. The cells then give rise to both megakaryocyte and erythroid progenitor cells.
During erythropoiesis, the erythroid progenitor cells arise after erythropoietin promotes the differentiation of erythroid blast forming units (BFU-E) to erythroid colony forming units (CFU-E). All the progenitor cells (across the board) resemble large lymphocytes (mononuclear cells with a uniformed, basophilic nucleus) and are therefore difficult to distinguish from each other on a morphological basis.
The CFU-E differentiates into proerythroblasts. These cells are relatively large with basophilic cytoplasm and contain loosely arranged chromatin in a single nucleus with a visible nucleolus. Under the influence of erythropoietin, proerythroblast transforms into basophilic erythroblast. The cytoplasm of these cells is more intensely stained with basophil as there are more polysomes (site of haemoglobin synthesis) present in the cytoplasm. Additionally, the nucleolus is no longer visible.
As basophilic erythroblasts differentiate, the number of polysomes decreases and the size of the cells are reduced. The new polychromatophilic erythroblast has a characteristic patchy appearance with areas of basophilia (remaining polysomes) and acidophilia (areas of haemoglobin deposition).
Further differentiation results in further decrease in the cell volume and total absence of polysomes. Therefore, the resulting erythroblast (also known as the normoblast) has no cytoplasmic basophilia and appears strongly acidophilic.
As the normoblast continues to mature, it extrudes its nucleus, thus forming a reticulocyte. Reticulocytes are rich in polyribosomes. However, these are also extruded once the reticulocyte enters the peripheral circulation, forming the terminally differentiated erythrocyte.
Blood outside of the circulatory system can have catastrophic effects on the human body. The endothelial lining of the blood vessels are so equipped that it keeps blood in the intravascular compartment. However, in the event that the endothelium is damaged it is important to be able to occlude the opening. Platelets are a part of this intricate system that provides haemostasis following an injury.
The method by which they are made is known as thrombopoiesis. The megakaryoblast is the first identifiable precursor in this lineage. It is a large cell with an oval or kidney shaped nucleus. Multiple nucleoli can be visualized in the nucleus and a homogenous basophilic cytoplasm can be seen.
Megakaryoblasts are modified in a process known as endomitosis under the influence of thrombopoietin, where there is DNA replication without cellular division. The resultant cells are therefore polyploid promegakaryocytes.
At the end of differentiation, the resulting megakaryocyte is characterized by an abundance of mitochondria, rough endoplasmic reticula, and large Golgi bodies. The Golgi bodies are responsible for the granules observed in platelets.
Megakaryocytes have cytoplasmic extensions known as proplatelets. These broad, long branches protrude through the sinusoidal endothelium, leaving the terminal aspect of the branch exposed to blood circulating in the sinusoid. Proplatelets also contain terminal actin filament and microtubules along with the vesicles. The distal end subsequently tapers off and detaches from the branch to release the platelet in circulation.
Granulocytes (also known as polymorphonuclear leukocytes) are a series of white blood cells characterized by their uniquely granular cytoplasm as well as their uniquely lobulated nucleus. There are three subtypes of granulocytes:
- neutrophils (most abundant)
- basophils (least abundant)
- eosinophils (moderately abundant)
The process by which these cells are formed is therefore known as granulopoiesis. During this process, granulocyte macrophage colony stimulating factor (GM-CSF) promotes the differentiation of the common myeloid precursor to the myeloblast. The fine, widely spread chromatin particles and pale nucleus makes the myeloblast the earliest identifiable cell in the series.
An increase in azurophilic granules (containing hydrolytic and digestive enzymes) as well as a basophilic cytoplasm signals the change from myeloblast to promyelocyte. This change is mitigated by granulocyte colony stimulating factor (G-CSF).
At this point, the promyelocyte will differentiate into one of the three myelocyte forms of the granulocyte (i.e. neutrophilic myelocyte, eosinophilic myelocyte or basophilic myelocyte) depending on which genes are activated. The key feature of the myelocytes is the appearance of granules specific to the granulocyte subtype. These new granules will be present in addition to the azurophilic granules of the myeloblast phase.
Neutrophils are most important in fighting bacterial cells. As a result, the granules found in neutrophils are rich in proteolytic enzymes to destroy bacterial proteins, lysozyme to attack the cell walls, and myeloperoxidase to generate toxic oxygen species that will destroy the invading bacteria.
Granules of basophils contain histamine and other pro-inflammatory agents that facilitate the inflammatory response at the site of injury or infection.
Eosinophil granules are filled with cathepsin and other toxic proteins that facilitate the destruction of parasitic infections. The difference between the myelocyte and metamyelocyte stage of the granulocyte series is the size of the cytoplasm (with the latter stage being larger). Additionally, each granulocyte has a unique nucleus that allows them to be distinguished morphologically. The nucleus of neutrophils are segmented into about 2 to 3 lobes, eosinophils have a single kidney-shaped nucleus, while a basophil has a bi-lobulated nucleus.
The monopoiesis gives rise to monocytes and their peripheral progeny – the macrophages. Monoblasts, which are also derivatives of the GM-CFU, are morphologically similar to myeloblast (described above). Under the influence of monocyte colony stimulating factor (M-CSF) monoblasts differentiate into promonocytes. These cells are typified by basophilic cytoplasm and a large nucleus with a minor indentation. Nucleoli can be visualized along with loosely arranged chromatin. An increase in the number of rough endoplasmic reticulum and increase in the size of the Golgi complex signals the change to mature monocytes.
Once the monocytes leave the bloodstream to enter specific tissues (lungs, kidneys, etc.) they become macrophages.
Histology of Lymphoid Cell Lineage
Lymphopoiesis refers to the formation of agranular nucleocytes that either mature or reside in peripheral lymphoid tissue. There are two major types of lymphoid cells – T-lymphocyte and B-lymphocytes. Although lymphocytes have secondary sites of maturation, all these cells originate in the bone marrow.
Lymphoblasts are the earliest identifiable lymphoid cells. They are large, mononuclear and undergoes division at least twice before forming prolymphoblasts.
Prolymphoblast & Lymphocyte
These prolymphoblasts then become lymphocytes. As these cells mature, the entire cell becomes smaller. This is associated with difficulty visualizing the nucleoli and shrinkage of the nuclei. Some lymphocytes will leave the bone marrow and undergo further ‘education’ in the thymus. After this process of thymic education, the cells are referred to as T-lymphocytes.
Other lymphocytes undergo their ‘education’ in the bone marrow and are subsequently called B-lymphocytes. The differences between these two cell lines are mostly immunohistochemically detected. As such, the morphological distinction is much more obscure.
Haematological disorders can be discussed based on there is a decrease, increase or abnormal function of individual or multiple mature cells or entire cell lines. These abnormalities may be dependent on one of the following:
- There is an insult to the haematopoietic stem cell progeny or their microenvironment that impedes the stem cell's ability to replicate.
- Nutrient deficiencies will suppress the bone marrow’s ability to carry out cellular replication.
- Cells may not be able to differentiate even with normally functioning stem cells and adequate nutrition.
The suffix –penia refers to a decrease in a given cell line below the age and gender appropriate lower limit. For example, a thrombocytopenia refers to a decrease in the total number of platelets in circulation. Either the suffix –osis, or –philia, refers to the opposite end of the spectrum where there is an increase in a given cell line above the age and gender appropriate lab reference range. For example leucocytosis refers to an overall increase in the white blood cell count, while a neutrophilia means that the neutrophils in particular are elevated.
Complete Blood Count
Hematopoietic homeostasis has wide reaching implications on numerous physiological processes in the body. One very insightful test that clinicians often order to assess the balance of blood cells is a complete blood count (CBC). This test requires that a peripheral venous blood sample is drawn from the patient and analyzed. While there are healthy individuals whose baseline lab values deviate significantly from the lab reference levels, other deviations may either be physiological or pathological. For this and other reasons, a diagnosis of a disease should never be made with just the lab results. When the lab results are received the patient’s historical account of the illness, as well as the physician’s clinical examination findings of the patient must be taken into account.
Majority of the parameters in the complete blood count look at the red blood cells. The red cell count assesses the total number of red cells within the patient’s body. Derangement in the red cell count may also be associated with changes in the haematocrit (also known as the packed cell volume) and haemoglobin. The haematocrit is the percentage of the total blood volume that is made up of erythrocytes; while the haemoglobin reflects the oxygen carrying capacity of erythrocytes. An increase in these indices is referred to as polycythemia, while a decrease in the same values is called anemia.
Athletes who exercise vigorously may have an increase in these red cell parameters when compared to a non-athletic individual. This would be due to the increased physiological demand of their bodies for more oxygen and a subsequent increase in the release of erythropoietin (EPO) that would stimulate red cell production; therefore this would be considered as a normal deviation. However, a patient with hypoxemia subsequent to an interstitial lung disease or cyanotic heart disease (or any other cause of hypoxemia) may also have increased erythropoietin secretion due to the low oxygen saturation of the blood cells. Therefore, this would be a pathological cause of polycythemia.
Patients who are dehydrated may appear to have a normal haemoglobin, red cell count and hematocrit levels when truly they could be anemic; for example, as observed in patients with sickle cell disease. The dehydration results in a decrease in the blood volume; therefore, when the sample is taken, the lab technician will perceive relatively normal parameters. However, once the patient is rehydrated and the complete blood count is repeated, the decrease in quantity of cells as a result of haemodilution, will become apparent.
Anemia can also result from nutritional deficiencies (iron, folate, vitamin B12). At this point, it is necessary to evaluate the size and colour of the cells to better hone in on a diagnosis. Iron deficiency anemia is associated with a mean corpuscular volume less than 80 fL (a measure of the average volume of the red cells calculated in femtolitres = x10-15L) and a mean corpuscular haemoglobin less than 27 pg (a measure of the amount of haemoglobin within a red blood cell calculated in picograms = x10-15 kg). A sample falling within these parameters would be classified as a microcytic (i.e. small cells) hypochromic (pale colour) anemia. Other causes of this picture includes thalassaemias, sideroblastic anemias and lead poisoning.
On the flip side, vitamin B12 and folate deficiencies will result in a mean corpuscular volume above 95 fL and a mean corpuscular haemoglobin greater than 27 pg; thus producing a macrocytic (large cell size) normochromic (normal colour; note that there is no such thing as a hyperchromic red cell) anaemia. Alcohol abuse and liver disease can also produce this picture. There are also cases of anemia where the mean corpuscular volume and the mean haemoglobin concentration values are normal. This would be a normocytic normochromic anemia and is typically seen in anaemia of chronic disease or following an acute haemorrhagic episode.
Another very important red cell parameter is the reticulocyte count. Reticulocytes are immature red blood cells that are not expected to be in the peripheral circulatory pool. If a patient is anemic and the reticulocyte count is high, then it is most likely that red cells are being destroyed in the periphery (i.e. haemolytic anemia) and the bone marrow attempts to compensate for the fall in red cells by increasing production. However, the cells do not fully mature because of the high demand for them in the periphery so immature cells are released. In contrast, if the reticulocyte count is low, then it is most likely that the cause of the anaemia is a decrease in cellular production and as such the problem is at the level of the bone marrow (i.e. bone marrow suppression or nutritional deficiency).
While red blood cells carry oxygen, white blood cells fight infections and cellular debris from the body. Changes in white blood cell counts can result from isolated or global deviations in individual cell lines. Decreases in white cell count coincide with an immunosuppressed state that may be reversible (i.e drug induced damage to the bone marrow) or irreversible (i,e, HIV infected patient not responding to therapy). An increase in white cell count - leukocytosis - is most often associated with an underlying infection. Of course, if the patient is immunocompromised, then they would not have a leukocytosis in the presence of an infection as there is a problem with leukocyte production. Additionally, inflammatory processes, seizures, myeloproliferative disorders, and leukemias can also increase the white cell count.
Blood Film Analysis
An important adjunct to the complete blood count is a blood film analysis. This is a histological assessment of the cellular morphology of white and red cells that indicates particular pathologies. For example, sickled cells are pathognomonic of the SS genotype of sickle cell disease, tear drop poikilocytes are found in patients with myelofibrosis and extramedullary haematopoiesis are typical of megaloblastic anemia, and target cells are seen with some haemoglobinopathies, liver disease as well as iron deficiency. There are also very specific morphological changes seen in leukopathies that are telltale signs of disease processes. Smudge cells suggest increased fragility of the white cells and is a hallmark sign of the Epstein Barr virus. Also diagnostic of acute myeloid leukemia are auer rods.
The components of the test, their significance as well as their reference ranges are outlined below. Please note that for paediatric cases, the reference ranges vary based on the age of the child, not the gender. However, there are gender differences after the patient passes about twelve years of age.
- Test - Haemoglobin
- Abbreviation - Hb
- Reference Range - 130 to 180 g/L for males and 120-160 g/L for females
- Purpose of Test - Measures the oxygen carrying capacity of the cell
- Test - Red Cell Count
- Abbreviation - RCC
- Reference Range - 4.2 to 6.9 x 10^12/L for both sexes
- Purpose of Test - Absolute count of the number of erythrocytes
- Test - Haematocrit
- Abbreviation - HCT
- Reference Range - 40 to 52 % for males and 36 -48 % for females
- Purpose of Test - A percentage of the total blood volume that consists of erythrocytes
- Test - Red Cell Distribution
- Abbreviation - RDW
- Reference Range - 11.0 to 15.0 % for both sexes
- Purpose of Test - Variation in size of RBCs
- Test - Mean Corpuscular Volume
- Abbreviation - MCV
- Reference Range - 80 to 100 fL for both sexes
- Purpose of Test - Average size of RBCs (calculated)
- Test - Mean Corpuscular Haemoglobin
- Abbreviation - MCH
- Reference Range - 27-32 pg/cell for both sexes
- Purpose of Test - Average amount of Hb within the RBCs (calculated)
- Test - Mean Corpuscular Hb Concentration
- Abbreviation - MCHC
- Reference Range - 32-36 % for both sexes
- Purpose of Test - Average concentration of Hb within a single RBC
- Test - Platelet
- Abbreviation - PLT
- Reference Range - 150-400 x10^9/L for both sexes
- Purpose of Test - Number of PLT in a blood sample
- Test - White Blood Cell
- Abbreviation - WBC
- Reference Range - 4.0-11 x 10^9/L for both sexes
- Purpose of Test - Total WBC count in a host’s body
- Test - White Blood Cell Differential
- Abbreviation - N
- Reference Range - 1.8 - 7.5 x 10^9/L for both sexes
- Abbreviation - L
- Reference Range - 0.7 - 4.5 x 10^9/L for both sexes
- Abbreviation - M
- Reference Range - 0.1 - 1.0 x 10^9/L for both sexes
- Abbreviation - E
- Reference Range - 0.0 - 0.4 x 10^9/L for both sexes
- Abbreviation - B
- Reference Range - 0.0 - 0.2 x 10^9/L for both sexes
- Purpose of Test - Levels of specific WBC components. Abnormalities of each component may support the diagnosis of a particular pathology
- Test - Reticulocytes
- Abbreviation - Retic
- Reference Range - 50-150 x10^9/L for both sexes
- Purpose of Test - Count of young RBCs in circulation