The Haemopoietic Stem Cell

17 May

The Discovery of Haemopoietic Stem Cell

 

Blood was regarded as an important tissue, but till the mid 1800s it was not known how blood cells were made. Three discoveries lead to the modern concept of haemopoietic stem cell.

  1. Neumann and Bizzozero separately, in 1868, proposed that the bone marrow was the site of blood production throughout the postnatal life.
  2. Improvement in staining techniques lead to the discovery of a spectrum of cells in the bone marrow.  Pappenheimer organized these cells into a tree with the most mature cells being at the leaves. He proposed that at the trunk was a cell that was so primitive that it could not be typed into any lineage. Ehrlich and Schilling differed from Pappenheimer in the existence of a single precursor for all blood cells. They proposed that there were two (Ehrlich) and three (Schilling) different precursor for blood cells respectively.
  3. The stem cell defies morphological definition. It is possible to define populations that are rich in stem cells but not all the cells thus defined are stem cells. The next chapter in the definition of haemopoietic stem cell was written when the focus shifted from what stem cells looked to what they could do.  Two discoveries, both by scientists not intending to looking for the haemopoietic stem cell, laid the foundation for the modern concepts of haemopoietic stem cells
    1. Lineage is important in cattle. It provides assurance to a farmer that the calf he is rearing will give a good amount of milk. Calves of good lineage command a high price. In the 1930s and 40s blood groups seemed to be reliable way of determining paternity of an animal. In 1944 a breeder reported twin calves of different sexes but identical blood groups. In 1945 Ray Owen in an attempted to resolve why calves that appeared to have two fathers had identical groups. He discovered that the calves actually had two erythrocyte populations (Genetics 1996; 144:855-59).  Each calf had blood from both the calves. The cattle uterine anatomy allows communication between extra-embryonic circulation of twins.  The only explanation for two types of erythrocytes in each calf was that

i.     Cells capable of producing both types of blood cells were present in the bone marrow of both the calves

ii.     The bone marrow of each calf had been seeded by cells from calls that could produce blood cells and arose from the other calf.

iii.     These cells could produce blood indefinitely

  1. Till and McCoulloch were interested in developing an assay for radiosensitivity of marrow cells. Lethally irradiated animals that survive acute radiation sickness succumb to bone marrow failure. Till and McCoulloch transplanted lethally irradiated mice with bone marrow cells exposed to different doses of radiation. They discovered the following

i.     Animals survive the radiation and develop islands of hematopoiesis in the spleen in the form of nodules.

ii.     The number of the nodules is related to the dose of bone marrow cell infused.

iii.     The colonies contain cells of erythroid and myeloid. The cells show different degree of maturity

iv.     Using chromosomal markers the colonies were shown to arising from a single cell

v.     When cells from the colonies were transplanted to another lethally irradiated mouse cells some colonies were able to establish haemopoietic splenic colonies.
These studies eastablished that the bone marrow had cells that that could self-renew, have a high capacity to proliferate and have capacity to differentiate into multiple lineages defining a haemopoietic stem cell.

Stem-Cell Renewal

 

Identification of the Pleuripotent haemopoietic

Isolation of a pure population of stem cells has not been possible.

The definition of stem cell is a functional definition. The stem cell is defined as a cell that has the capacity to

  1. Self-renewal: Self-renewal is the phenomena of stem cells giving rise to more stem cells. Self-renewal is essential to maintain the stem cell pool and ensure adequate blood production. The stem cells population can be maintained at a constant levels only if half the cell produced as a result mitosis retain the characters of a stem cell and the other half differentiate into blood cells.  If more than half the stem cells differentiate, the stem cell pool will eventually deplete. If less than half the stem cells differentiate there would be an unnecessary accumulation of stem cells. The details of the self-renewal and commitment processes are not completely understood.
  2. Indefinite proliferation: The bone marrow stem cells maintain haematopoiesis for a lifetime.
  3. Capacity to differentiate into many cell types: The haemopoietic stem cell gives rise to the erythrocytes, myelocytes, lymphocytes, monocytes, megakaryocytes and the dendritic cells.

Stem cells are identified by their ability to reconstitute haematopoiesis in a lethally irradiated animal.

Haemopoietic stem cell transplant became a reality in 1968.  Initially the bone marrow was the source of stem cells. Today the peripheral blood and occasionally the cord blood serve as the source of stem cells. The number of stem cell infused determines the success of the transplant. Animal transplantation take too long to be clinically useful.

CD34 is a trans-membrane protein expressed on haemopoietic progenitors till the stage of committed progenitor cells. CD34 expression can rapidly be assessed by flow cytometery. CD34+ cells show the characters of haemopoietic stem cells

  1. Autologous CD34 enriched population has been shown to protect against myeloablative doses of radiation and chemotherapy. CD34- negative cell fail to do so.
  2. Allogenic CD34+ cell are able to establish haematopoiesis. This chemerism remains stable over a prolonged period of time.

Haemopoietic stem cell is a CD34+ cell that lacks lineage commitment markers. These include

  1. T cells: CD2
  2. B cells: CD19
  3. Monocytes: CD14
  4. Granulocytes: CD15
  5. NK Cells: CD16
  6. Erythroid cells: Glycophorin A
  7. Others: CD24, CD56 and CD66b, DR

Not all the CD34+ cells are pleuripotent stem cell but CD34 is a useful marker for estimating the number of stem cells infused during a haemopoietic stem cell transplant.

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Pomalidomide Approved for Refractory Myeloma

16 Apr 20120619_204106-Skull-AP-MM-SC

Pomalidomide has been accelerated approved by US FDA for multiple myeloma that has progressed on at least two previous therapies including bortezomib and lenalidomide, within 60 days of the last therapy. The approval was based on the multicenter, randomized, open-label study CC-4047-MM-002. The approval is based on response rates. Pomelidomide has not been as yet shown to improve survival. The combination of pomalidomide and low dose dexamethasone was resulted in a 29% response in 113 patients with one complete response and a median duration of response of 7.4 months.

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Toxic Granules in Neutrophils

23 Mar IMG_1153 - Toxic Granules

IMG_1153 - Toxic GranulesGranulocytes have two types of granules primary and secondary. Primary granules are azurophilic, most numerous and prominent at the promyelocyte stage (see morphology of myeloid precursors) and diminish in number with further maturation. As the granulocyte matures the staining characters of the primary granules changes. They initially become violet and then became inapparent because they fail to take up stain. The secondary granules appear in the myelocytes and persist for the rest of the ice of the granulocyte. Neutrophils have fine pink secondary granules.

In conditions of intense stimulation of neutrophil maturation the primay granules may continue to take up stain in mature neutrophil because of a higher concentration of acid mucosubstances. These are called as toxic granules and the change called toxic change. The toxic granules are so called because they were first described in patients with gram negative sepsis and endotoxemia but may be found under conditions of intense stimulation of neutrophil production. The may be seen in

  1. Infection
  2. Inflammatory diseases
  3. Pregnancy
  4. Use of haemopoietic growth factors (G-CSF or GM-CSF)

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Photomicrography with a Canon 7D SLR

19 Mar

English: Canon EOS 7D with EF 28mm f/2.8 日本語: ...

English: Canon EOS 7D with EF 28mm f/2.8 日本語: キヤノン EOS 7D レンズ：EF 28mm F2.8 (Photo credit: Wikipedia)

“Necessity is the mother of invention (or actually discovery in this case)”

This blog is developed from a manuscript of a haematology book. No haematology book can be complete without photomicrographs. As I did not have access to a microscope camera I had to look for other ways to photograph peripheral blood and bone marrow slides. The first digital camera I had was the Nikon Coolpix 4500. The lens of this camera had a ring equal to that of a microscope eyepiece and the focusing mechanism, unlike most cameras was internal. These features gave me the confidence that if I hold the camera against the eyepiece of a microscope I will not be damaging either. It worked! I have since then used Canon IXUS 105, Canon 400D and Canon 7D for photomicrography. This post is about photomicrography by holding a camera close to the eyepiece of a microscope.

Nikon Coolpix 4500: Nikon Coolpix 4500 is a 4 megapixel camera that was the first camera I tried for photomicrography. It gave usable photographs but the images had many concentric rings. To overcome the “ring” problem one had to ensure that the area to be photographed was at the periphery, where the rings were absent/less prominent. The image below is that with megaloblastic anaemia where the rings were seen

002

Photomicrograph of a the bone marrow of a patient with megaloblastic anaemia taken with Nikon Coolpix 4500 showing concentric rings

One could apply post processing to some of the photomicrographs to get images acceptable for displaying on a webpage. The same image with post processing is shown below.

Megaloblasts

Cameras Canon IXUS 105: Canon IXUS 105, a much later model, was free of concentric rings. It gave good photographs but gave six dots (I am not sure if this was an issue with the model or the piece I had). The images were large enough to be cropped and I have used these in the post morphology of myeloid precursors. All the images in this post are taken by holding a Canon IXUS against the eyepiece of a microscope.

Six Dots of Canon IXUS 105 001

The image taken with a Canon IXUS 105. The six dots are evident above and to the right (11 o’clock) of the myeloid cells.

Canon 400D with a 50mm f/1.8 II lens: The images of a digital SLR offers are of a better quality than compact cameras. The SLR also gives images that can be processed better. I had tied to use the 400D with the 18-55 kit lens, before using the Canon IXUS 105, but never succeeded. The photograph never filled the entire frame. Then the internet came to the rescue. The 400D set to manual focus took pictures. The 50mm f/1.8mm lens is an inexpensive lens of extremely good quality. I switched to a 50mm f/1.8 II lens. The 400D allows shooing in the RAW format allowing greater post-processing. The Low ISO of the 400D means shooting a slow sutter speeds (1/20-1/40 seconds) which resulted in blurring of images because of camera shake. The 400D does not have alive view mode. The Canon 7D overcomes these limitations.

Canon 7D with 50mm f/1.8 II lens: The Canon 7D allowed fast shutter speed and has a better image capturing device that has given the best images I have taken. The high ISO (unto 3200) allows shutter speeds of 1/100 and eliminates blurring because of camera shake. To take pictures by holding a digital camera close to the eyepiece of a microscope on has to

  1. Set the focus to manual
  2. Set the camera to aperture priority and set the aperture wide open
  3. I prefer setting the exposure to expose such that the histograms is snuggled to the right. This reduces the noise.
  4. Focus the images using the adjustment on the microscope. No focusing is done by the camera. Photographs are taken using one of the eyepieces of a binocular microscope. The two eyepieces may have different focus planes. A slide may appear to be in sharp focus when seen through both objectives but may still not be in sharp focus when through the eyepiece used for photography. Look and focus the slide though the eyepiece you intend to use for photography.
  5. Adjust the white balance. You may use auto white balance, custom white balance or adjust the white balance shift (add blue/green if the microscope lamp is too yellow). you can also adjust the temperature and tint on the image processing software after taking the picture.
  6. Sometimes the image has a bright spot (shown in the picture below). This has happen with the 400D and 7D. Images are framed with the 400D by looking through the viewfinder. The bright spot can not be seen on looking through the viewfinder. It can however be seen in the live view mode of the 7D. It is best to shoot using the live view mode. The spot can be eliminated by slightly tilting the camera. The problem of the bright spot has occurred with all brands of microscopes (local brands to Olympus). I have tried eliminate the spot by adjusting the condenser and changing the illumination but with little success. This has occurred only when seeing the slide under oil immersion objective (100X) never under 40X or 10X.

bright Spot Final

Why the 7D? Photomicrography does not use all the features of a 7D. A 600D or 650D would perform as well. SLR cameras by other manufacturers like Nikon or Sony should give the same results but my experience is limited to Canon SLRs. If you intend to use a camera for photomicrography you need to

  1. Preferably have an SLR – They have a superior image quality than compact cameras. Compact cameras can be used if an SLR is not available.
  2. Those using Canon SLRs may find the 50mm f/1.8 II lens most useful. Some believe this is the best value for money lens Canon has produced. It is incredibly sharp and incredibly inexpensive. ALWAYS USE A UV FILTER. The lens must never touch the eyepiece. I have found the performance better the the kit 18-55 lens.
  3. High ISO capabilities to allow high shutter speeds to eliminate blurring due to camera shake
  4. Live View gives a better view and helps in identifying and eliminating the bright spot.

I have used the 7D because I happen to purchase one for photography which is my hobby. What the 7D offers over a 600D or a 650D is never used in photomicrography. You may use any of the above mentioned cameras and get results.

ISO – The Exposure Triangle

ISO – The Exposure Triangle (Photo credit: abanakas)

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Myelocyte and Neutrophils – Band and Segmented

18 Mar Myelo Band Neutro Final

Myelo Band Neutro Final

Neutrophilic Cells. The images shows myelocyte (1), band neutrophils (2), two lobed neutrophil (3) and neutrophils (4)

The image about shows morphology of cells of the neutrophilic series. The first cell to show commitment to a particular granulcoytic series (eosinophilic, basophilic or eosinophilic) is the promyelocyte. The promyelocytes of the three series can however only be differentiated by electron microscopy. The earliest cell showing features of neutrophilic differentiation on staining with Romanowsky staining is the myelocyte. Myelocytes may be neutrophilic, eosinophilic and basophilic. Myelocyte is a round cell with a round to oval nucleus that may be eccentrically placed. The cytoplasm shows two types of granules.

  1. Primary granules: The primary granules are azurophilic (reddish-purple or burgundy color) and are remnants of the granules of  the promyelocyte stage. The myelocyte does not synthesize primary granules. As the cell matures the number of primary granules declines and they disappear by the cells matures to a polymorphonuclear neutrophil.
  2. Secondary granules: The the size and staining of secondary granules are specific to the type of granulocytes. In neutrophils these granules are fine and pink staining

As the myelocyte matures the nucleus becomes more indented and finally becomes lobed.

The image above captures these changes

  1. Cells labels (1) are myelocytes. These show a pink cytoplasm with few primary (azurophilic) and many secondary (fine pink) granules and have a oval eccentrically placed nucleus with clumped chromatin without a nucleolus.
  2. The myelocyte develops and indentation of the nucleus and matures to a metamyelocyte. The metamyelocyte matures to a band neutrophil (cells labeled 2). which is called so because the nucleus is band shaped. With maturation the nucleus develops lobulation. The differentiation between a band neutrophil and a two lobed neutrophil is arbitrary and of little practical importance. A band cell becomes a two lobed neutrophil when its nucleus develops a constriction that is more than half to two third of the nuclear width (cell labeled 3).
  3. A neutrophil usually has 2-5 nuclear lobes (cells labeled 4)

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Microcytic Hypochromic Anaemia

13 Mar hypochromic Micro Anaemia

hypochromic Micro Anaemia

Hypochromic Microcytic Anaemia

Shown above is an image of hypochromic microcytic anaemia. This patient had iron deficiency anaemia. Microcytosis is presence of small erythrocytes and hypochromia presence of erythrocytes that are poorly haemoglobinized. The nucleus of a small lymphocyte, the cell in the centre of the image, is a good guide to the size of erythrocytes on a peripheral smear. The nucleus has a diameter of 8.5 µm and a normal erythrocyte a diameter of 7.5 µm. The erythrocytes in in the image are substantially smaller than the lymphocyte nucleus. Erythrocytes have a central pale staining area which occupies about one third of erythrocyte diameter. As the cells get less haemoglobinized the central pale staining area increases. Many of the erythrocytes in the image above have a pale staining area occupies all but a thin rim at the periphery. In others, the pale staining area is increased. The erythrocyte sizes vary. Anisocytosis, increased variation in erythrocyte size, a feature of iron deficiency anemia, is evident in the image.

 

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Sites of Haematopoiesis

12 Mar

Normal Haematopoiesis

The bone marrow is the only site of blood production in extrauterine life. The yolk sac and the liver produce blood in the intrauterine life. The two sites have distinct precursors. The yolk sac haematopoiesis, known as primitive haematopoiesis, is transient haematopoiesis that serves the purpose of rapidly providing blood to the growing embryo. Haematopoietis in the liver in utero and subsequently extrauterine haematopoiesis in the bone marrow are known as definitive haematopoiesis. The haemopoietic precursors of primitive haematopoesis disappear once the definitive haematopoiesis establishes.

The first evidence of haematopoiesis in the developing embryo is in the yolk sac. Yolk sac erythropoiesis produced nucleated erythrocytes and macrophages but no granulocytes. The yolk sac erythrocytes are large, nucleated and have embryonic haemoglobins. The yolk sac remains the site for erythropoieis from 19 days to 8 weeks of intrauterine life. The yolk sac haematopoiesis ceases once the liver becomes the dominant site of haematopoiesis.

Liver is the main site for erythropoiesis through intrauterine life. Haematopoiesis taking place in the liver differs from the yolk sac haematopoiesis in the following ways

  1. Liver haematopoiesis produced erythrocytes, moncytes and lymphocytes, yolk sac only produces erythrocytes and monocytes
  2. Erythrocytes produced by the liver are smaller
  3. The erythrocytes enucleate before release into blood. Yolk sac erythrocytes are released with a nucleus that enucleates in circulation.
  4. The erythrocytes have have fetal haemoglobin whereas the yolk sac erythrocytes have embryonic haemoglobin.

The precursors involved in definitive haematopoiesis originate in the mesodermal tissue of the the aorta-gonad-mesonephros region and migrate to the liver from there (The Lancet 2013; 381:S12). From the liver, haemopoietic progenitors migrate the bone marrow. The contribution of the bone marrow increases with gestational age and the bone marrow becomes the only site for normal haematopoiesis in the extrauterine life.

Haematopoiesis sites in disease 

Haematopoiesis can take place in the liver, spleen, lymph node, kidney and posterior mediastinum when the bone marrow function is insufficient. Rarely, involvement of other organs including adrenal gland, central nervous system, skin and spine have been described. Extramedullary erythropoiesis is seen where bone marrow is diseased or overwhelmed.

  1. Diseased bone marrow: Bone marrow diseases like myelofibrosis and bone marrow infiltrations limit the marrow available for haematopoiesis forcing haematopoiesis to extramedullary sites.
  2. Overwhelmed bone marrow: Patients with chronic haemolytic anaemia and thalassaemias have bone marrow expansion to cope up with increased demand of erythrocytes. Extramedullary erythropoiesis may be seen in patients whose needs are not met with full marrow expansion.

Extramedullary haematopoiesis can cause symptoms including

  1. Leukoerythroblastic anaemia and the presence of tear drop erythrocytes.
  2. Symptoms caused by splenomegaly:
  3. Spinal cord compression (usually thoracic)
  4. Rare manifestation include effusion due to involvement of the serious membranes and compression by masses arising from the skull and the paranasal sinuses

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Lymphocytes

11 Mar Lymphocytes

Lymphocytes

Lymphocytes – Large and Small

There are two types of lymphocytes small (10-12µm) and large (12-16µm).

Most of the lymphocytes in the peripheral blood are small. The nucleus of he lymphocytes is small, round, usually indented. The chromatin of the lymphocyte nucleus

  1. Deep purple, small, round, usually indented
  2. Has no nucleoli
  3. Has a densely clumped deep chromatin

The cytoplasm of the lymphocyte is

  1. Moderately basophilic (blue)
  2. Scanty forming a thin rim around the nucleus
  3. Devoid of granules

Large lymphocytes have a more abundant cytoplasm with a few azurophilic granules. Some of these are T supressor lymphocytes (Cd3+ Cd8+) while others are NK cells (Cd3 – CD8+). The picture above shows a large granular lymphocyte

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Monocyte

11 Mar Two Monocytes

Two Monocytes

Figure 1. Two Monocytes

Monocytes are the largest leucocytes. They vary considerable in size and shape and may measure about 12-20 µm in diameter. The have a lobulated nucleus that is centrally placed with a fine chromatin. The nucleus has been classically described as kidney shaped but may take other lobulated forms. The cytoplasm is abundant, grey or light-blue grey.  Fine azurophilic granules that are seen on staining on wright’s stain giving the cytoplasm a ground glass appearance (evident in the monocyte on the left in figure 1). The granules may become prominent in patients with marrow stimulation (bacteraemia, marrow recovery from aplasia or following the use of G-CSF)

Monocytes show typical granules in some inherited disorders. They are phagocytic and may show ingested red cells, malarial pigment or microorganisms.

Myelocyte and Metamyelocyte

Figure 2. Neutrophilic Myelocyte and Metamyelocyte- The cell on the left is a myelocyte and the right is a metamyelocyte. Compared to a monocyte (figure 1) a metamyelocyte has a coarser chromatin and pink cytoplasm with fine pink granules. The monocyte nucleus is lobulated and that of a metamyelocyte indented.

The monocyte needs to be differentiated from a neutrophilic metamyelocyte, a cell with a similar size and indented nucleus. The monocyte has a fine chromatin. The chromatin of the metamyelocyte is coarser and clumped. The cytoplasm of a monocyte is grey or light grey blue cytoplasm and fine azurophilic granules whereas the cytoplasm of a metamyelocyte has is pink with fine pink granules.

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Iron Studies in Microcytic Anaemia

8 Mar

Diagnosis Serum Iron Total Iron Binding Capacity Transferrin1 Serum Ferritin2
Iron deficiency anaemis Low High or Normal <16% <12ng/mL
Anaemia of Chronic Disease Normal Low or Normal ≥16% High or normal
β-Thalasaemia Trait, HbE, HbC Normal Normal ≥16% Normal
Sideroblastic Anaemia High Normal High High
  1. Two causes of microcytic anaemia may co-exist e.g. thalassaemia trait with iron deficiency anaemia or anaemia of chronic disease with iron deficiency anaemia. When iron deficiency exists with other forms of microcytic anaemia the transferrin saturation is <16%
  2. Patients with low ferritin (<12ng/ml) always have iron deficiency. Higher values of ferritin do not exclude iron deficiency particularly in patients with anaemia of chronic disease. There are no guidelines about the ferritin levels that exclude iron deficiency in patients of anaemia of chronic disease. The reported values vary between 60-100ng/ml.

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