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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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Normoblast Maturation

7 Mar Normoblast Maturation

IMG_0354 Maturing Normoblast

The image above shows three polychromatophilic normoblasts. The one on the left is the least mature and the one on the right the most mature. Maturation is associated with

  1. Decrease in the size of the cell and nucleus: This is obvious.
  2. Clumping of chromatin: This is obvious.
  3. Increasing cytoplasmic acidophilia and decreased basophilia: The cells are flanked on each side by erythrocytes. The cytoplasm of the least mature cell (left most cell) is basophilic compared to that of the adjacent erythrocyte. The cytoplasm of the most mature cell (the right most cello) has lost basophilia and is almost the colour of the cytoplasm of the erythrocyte. 

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Laboratory diagnosis of Iron Deficiency

7 Mar

The investigation to diagnose iron deficiency include:

  1. Haemoglobin and red cell parameters
  2. Bone marrow iron staining
  3. Serum Iron, total iron binding capacity and transferrin saturation
  4. Serum ferritin
  5. Zinc Protoporphyrin
  6. Soluble transferrin receptor

Haemoglobin and Red cell Parameters

Patients undergo evaluation for iron deficiency because they are anaemic. Latent iron deficiency is characterized by a progressive decrease in bone marrow iron stores in patients who have yet not developed symptoms. These patients are asymptomatic and latent iron deficiency is not a clinical problem. Iron deficiency causes microcytic hypochromic anaemia. There are no reliable test to differentiate iron deficiency from other causes of microcytic hypochromic anaermia.  The red cell indices that have been proposed to be useful include

  1. Red cell distribution width (RDW): RDW is a measure of anisocytosis. Iron deficiency anaemia shows more anisocytosis than β-thalassaemia and is associated with a higher RDW. The promise held out by RDW to differentiate iron deficiency and thalassaemia has not fulfilled.
  2. Reticulocyte haemoglobn content: Changes in erythropoiesis are reflected earliest in the reticulocyte. Reticulocytes form a small fraction of erythrocytes. Change in reticulocyte indices do not change erythrocyte indices. Some automated counters are able to measure reticulocyte indices. Reticulocyte haemoglobin content falls with iron deficiency anaemia but the finding is not specific. It has been found to asses iron deficiency in patient of chronic renal failure being treated with erythropoietin accurately. It has not been found to be useful in diagnosing iron deficiency in patients with thalassaemia.

Bone Marrow Iron Staining

Bone marrow is stained for iron content by the Prussian blue reaction and graded in a semiquantative method. Bone marrow iron is the gold standard for diagnosis of iron deficiency. The test is invasive and suffers from an inter-observer variation. Bone marrow iron staining is resorted to only when diagnosis can not be reached by other methods.

Serum iron, total iron binding capacity and transferrin saturation

Iron deficiency is diagnosed by a transferrin saturation of less than 16%. The serum iron is low and the total iron binding capacity is usually increased. Patients with low total iron binding capacity have anaemia of chronic disease if the transferrin saturation is ≥16%  or iron deficiency along with anaemia of chronic disease if the transferrin saturation is <16%.

Serum ferritin

Ferritin is one of the iron storage proteins. Serum ferritin levels co-relates with body iron content. A ferritin level less than 12ng/mL is diagnostic of iron deficiency. Inflammation increases ferritin. Chronic inflammatory diseases like rheumatoid arthritis and ulcerative colitis have anaemia of chronic disease and may also have iron deficiency. A patient with iron deficiency in the setting of an inflammatory disease may not have a low ferritin. There is no consensus for diagnosing iron deficiency in patients with anaemia of chronic disease. Inflammation rarely increases the serum ferritin values more than 60-100ng/mL. Iron deficiency can be excluded in patients with ferritin above this cutoff.

Zinc Portoporphyrin

Iron is added to propoporphyrin in the final step of heme synthesis. Zinc takes the place of iron in patients with iron deficiency. A rise in the concentration of zinc protoporphyrin is the earliest manifestations of iron deficiency. Zinc protoporphyrin levels rise in about 2 weeks from the onset of iron deficiency and need more than a month to normalize after restoration of normal iron levels.

Soluble transferrin Receptor

Iron deficiency results in an increase in soluble transferrin receptor (sTfR). Inflammation impacts serum ferritin but not sTfR making it a potentially useful investigation for differentiating anaemia of chronic disease and iron deficiency. The assay has been difficult to standardize. A ratio of sTfR/log ferritin is more useful.

The sTfR levels reflect the density of transferrin receptors cells and number of cells. sTfR increases when there is erythroid hyperplasia due to any cause like haemolytic anaemia and may not reflect iron deficiency in these disorders.

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Orthochromatophilic Normoblast

7 Mar IMG_0286 - Orthochromatophilic Normoblast

Image

Orthochromatophilic normoblast

An orthochromatophilic normoblast is the most mature of nut;elated red cell precursors. The cytoplasm has lost almost all it’s basophilia and is the same colour as that of the erythrocyte. The nucleus is small pyknotic with densely condensed chromatin.

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The Reticulocyte Count

10 Oct

Anaemia induces production of erythropoietin which promotes differentiation of the hemopoietic stem cell along the erythroid lineage. With maturation, the erythroid precursors shrink in size, loose nucleus and haemoglobinize (see Morphology of Erythroid Precurssors). A newly released erythrocyte contains ribosomal RNA that it looses over 24-36 hours. These young erythrocytes are called reticulocytes because the ribosomal RNA gives a reticular appearance when stained by certain supravital stains like new methylene blue.

Anaemia may result from an increased destruction or impaired production of erythrocytes. The former is characterised by an increased erythrocyte production and the latter by an inappropriately low erythrocyte production. The normal life span of the erythrocyte is about 120 days. About 0.8% of the erythrocytes are destroyed everyday. If erythrocyte production ceases completely, a 10% fall in erythrocyte count (also in haemoglobin) would take about two weeks. Though a greater fall would indicate a loss of erythrocytes in the form of haemolysis or acute blood loss, the erythrocyte count is of little in diagnostic value in assessing bone marrow activity accompanying anaemia. The number of the reticulocytes indicates the bone marrow activity in a short period preceding assessment (a day or two) and allows classification anaemia into those resulting from decreased production and increased destruction.

The reticulocyte

On Romanowsky staining, other than polychromasia and a slightly larger size, there are no morphological features to differentiate reticulocytes from other erythrocytes. Differentiation on the basis of size is difficult and all reticulocytes do not show polychromasia. When reticulocytes are stained with new methylene blue or brilliant cresyl blue the ribosomes precipitate as a blue staining reticulum (hence the name reticulocytes). As the staining is done on cells that are not fixed (are live) it is knows as supravital staining. New methylene blue stains the cytoplasm blue-green obviating the need for a cytoplasmic counterstain. The density of the reticulum falls with age of the reticulocyte. The most mature reticulocytes may show only one or two dots. The majority of reticulocytes have a few dots and the precise definition of a reticulocyte has a bearing on the reticulocyte count. The definition of a reticulocyte is any non-nucleated erythroid cell containing two or more bluish–stained material corresponding to ribosomal RNA. The reticulocyte needs to be differentiated from other intracellular inclusions viz. Pappenheimer bodies, Heinz bodies, Howell-Jolly Bodies and Hb H inclusions.

Performing the Reticulocyte Count

The count is performed by mixing 2-3 drops of new methylene blue (or brilliant cresyl blue) with 2-4 volumes of EDTA-anticoagulated blood, allowing the mixture to stand for 15-20mins, making films on a glass slide and examining these when dry under 100X oil immersion. The number of erythrocytes and reticulocytes is counted till at least 100 reticulocytes and a total of 10 oil immersion fields are counted. The reticulocyte count is expressed as follows:

Reticulocyte count = [number of reticulocytes counted]/[number of erythrocytes counted]

The reticulocyte count may also be expressed as absolute reticulocyte count as follows:

Absolute reticulocyte count = [RBC count X Reticulocyte count]/100

The normal reticulocyte count is 0.5-2.5% and the normal absolute reticulocyte count is 50-100X109/L

Correcting the reticulocyte count for anaemia

Anaemia decreases the amount of time the reticulocyte spends in the marrow. The reticulocytes of patients with haematocrits in the range of 45% are estimmated to spend 3.5 days in the marrow and about 1 day in the peripheral blood. At a haematocrit 15% these times are 1.5 and 2.5 days respectively. Decreases RBC count gives a false elevation in reticulocyte count. The haematological parameters of four patients given in the table below highlight the limitations of a reticulocyte count.

Patient A

Patient B

Patient C

Patients D
Haemoglobin

13g/dL

5 g/dL

7.5 g/dL

7.2 g/dL
Erythrocyte Count

4.4 Million/mm3

1.2 Million/mm3

2.5 Million/mm3

3.5 Million/mm3
Haematocrit

40.48%

16.2%

23.25%

25.2%
MCH

29.55 pg

41.67 pg

30 pg

20.57 pg
MCV

92 fl

135 fl

93 fl

72 fl
MCHC

32 g/dL

31 g/dL

32 g/dL

29 g/dL
Reticulocyte Count

1.3%

3%

15%

2%
Absolute Reticulocyte count

57200 /mm3

36000/mm3

375000/mm3

70000/mm3
Corrected Reticulocyte Count

1.17

1.08

7.75

1.12
Reticulocyte Production Index

0.78

0.43

3.1

0.56
  • Low erythrocyte count can cause an false impression of reticulocytosis: The patient B has an erythrocyte count of 1.2 million/mm3, a hematocrit of 16.2% and a reticulocyte count of 3%. The absolute reticulocyte count is 36000/mm3. Patient A has a erythrocyte count of 4.4 million/mm3, a haematocrit of 40.48% and a reticulocyte count of 1.3%. The absolute reticulocyte count is 57200/mm3. Despite having a higher reticulocyte count patient B actually has a lower absolute reticulocyte count and a less active marrow than patient A. Corrected reticulocyte count corrects reticulocyte count for low erythrocyte count and is calculated as follows:

Corrected Reticulocyte Count = Reticulocyte count X (Patinets Haemotocrit/Normal Haemotocrit)

(Haemoglobin or erythrocyte count may be used for the correction instead of haematocrit)

The corrected reticulocyte count in both patients is almost identical.

  • In addition to correcting for erythrocyte count one must correct for a premature release of erythrocytes: Patinet A and patient B have almost identical corrected reticulocyte count. Does this mean they are producing the same number of erythrocytes per day? The reticulocyte spends about 2.5 days in peripheral blood in the patient A and about 1.5 days in the patient B. Only 14,400/mm3 (36,000/number of days reticulocyte spends in peripheral blood) reticulocytes are produced every day in patient B and about 38,000/mm3 produced in patient A. Despite having an almost identical corrected reticulocyte count as patient B, patients A is producing twice as many reticulocytes as patient B. The corrected reticulocyte needs to be further corrected for an early release of erythrocyte in anaemia. This gives the reticulocyte production index as follows

    Reticulocyte Production Index = Corrected Reticulocyte Count X Correction Factor

    The correction factor is 1 for haemotocrit of 40-45%, 1.5 for haemotocrit of 35-40%, 2 for haematocrit of 25-35% and 2.5 for haematocrit of 15-25%.

    A reticulocyte production index of <2 in the presence of anaemia indicates a bone marrow pathology. Patient C, a patient of haemolytic anaemia, and has a RPI of 3.1 indicating a normally responding bone marrow. Patient D is a patients with a hypochromic n=microcytic anaemia with a low RPI. This indicates a impaired erythropoiesis which in the case is most likely to be an iron deficiency anaemia.

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Morphology of Erythroid Precursors

12 Apr 041211_0144_Morphologyo3.jpg

Figure 1. Maturation of erythroid cells in the bone marrow

Pleuripotent stem cells give rise to erythrocytes by the process of erythropoiesis. The stem cell looks like a small lymphocyte and lacks the functional capabilities of the erythrocyte. The stem cells have the capacity of infinite division something the mature cells lack. Some of the daughter cells arising from the stem cell acquire erythroid characters over generations and time. Most of the erythroid cells in the bone marrow have a distinct morphology but commitment to erythroid maturation is seen even in cells that have not acquired morphological features distinctive of the erythroid lineage. These cells are recognized by the type of colonies they form in vitro. Two such cells are recognized. Burst-forming unit erythroid (BFU-E) arise from the stem cell and gives rise to colony-forming unit erythroid (CFU-E). CFU-E gives rise to pronormoblast, the most immature of erythroid cells with a distinct morphology (figure 1). BFU-E and CFU-E form a very small fraction of bone marrow cells and are not important in diagnosis. Examination of Romanovsky stained (Giemsa, Wright’s) bone marrow smears is central to the haematological diagnosis. Morphologically five erythroid precursors are identifiable in the bone marrow stained with Romanovsky stains. The five stages from the most immature to the most mature are the proerythroblast, the basophilic normoblast (early erythroblast), polychromatophilic normoblast (intermediate erythroblast), orthochromatophilic normoblast (late erythroblast) and reticulocyte (figure 1). As the cell matures the following morphological changes take place

  1. Cell becomes smaller
  2. Nucleus becomes smaller, chromatin more clumped and the nucleoli disappear
  3. Cytoplasm shrinks
  4. The cytoplasmic basophilia decreases: Haemoglobin is a major constituent of the red cell takes a pink to red colour on staining with Romanovsky stains. The machinery to synthesize haemoglobin (ribosomes) must appear before haemoglobin. Ribosomes make the cytoplasm basophilic (blue) because of their RNA content. As the haemoglobin content approaches the desired levels the number of ribosomes decreases. The cytoplasm of the maturing erythroid cell captures these changes and changes from deep blue (mainly ribosomes) in basophilic normoblast to polychromatophilic (ribosomes and haemoglobin) in polychromatophilic normoblast and resembling that of a erythrocyte (mainly haemoglobin) in orthochromatophilic normoblast.
  5. The earliest nucleated stages are least numerous and the later stages the most numerous

Figure 2. Proerythroblasts. The nucleus has multiple nucleoli a feature that distinguishes this stage.

Proerythroblast: Proerythroblast (figure 2) is a large cell (12-20μm in size or about 1.5-3 times a normal erythrocyte) with a large nucleus that occupies almost 80% of the cell and a blue cytoplasm that forms a thin rim around the nucleus. The chromatin is finely granular and stripped. The nucleus shows multiple nucleoli (the multiple pale staining areas in the nucleus). The cytoplasm may show a small pale area that corresponds to the Golgi apparatus and may have a pale perinuclear halo. While it is usually possible to tell a proerythroblast from other blasts (myeloblasts, lymphoblasts and monoblasts) by it’s more homgenous of cytoplasm, larger size ,a chromatin that coarser and the perinuclear halo in very immature cells this distinction may be impossible.

Figure 3. Basophilic Normoblasts. All four cells have basophilic cytoplasm but lack nucleoli. Nucleoli are a feature of proerythroblasts. Out of the four cells seen the two on the left are larger, have a less clumped chromatin. The cytoplasm of all four cells shows no polychromasia.

Basophilic Normoblast: The basophilic normoblast (figure 3, 4 and 5) is a smaller (12-17μm) cell. The nucleus is round like that of a proerythroblast but lacks nucleoli. Condensation of chromatin with the appearance of heterochromatin begins at this stage giving the nucleus a coarse and granular appearance. The number of ribosomes peak at in basophilic normoblasts and this reflects in the cytoplasmic colour. The basophilic normoblast has one of the bluest cytoplasm amongst the bone marrow cells. It may have a perinuclear halo. The nucleus may assume a wheel spoke arrangement like a plasma cell. The spoke wheel arrangement, the blue cytoplasm and similar size makes the basophilic normoblast resemble a plasma cell (figure 4). The plasma cell is elliptical with an eccentric nucleus while the basophilic normoblast is round with a central nucleus.

Figure 4. A basophilic normoblast (1) with a polychromatophilic normoblast (2) which is smaller with a cytoplasm more like a mature erythrocyte and a nuclear chromatin that is more clumped

Figure 5. A basophilic normoblast with two plasm cells. The plasma cells have an eccentric nucleus and are elliptical

Polychoromatophilic Normoblast: The polychromatophilic normoblast (figure 4) is a smaller (12-15μm). The distinguishing feature of this stage is the appearance of haemoglobin which reduces the basophilia of the cytoplasm. The chromatin shows a greater degree of clumping and irregular dense areas of staining are seen in the nucleus. Nucleoli are nor seen. Figure 4 shows adjacent basophilic and polychromatophilic normoblasts to contrast the size, the clumping of the chromatin and cytoplasmic staining (see normoblast maturation for more images of maturing polychromatophilic normoblasts).

Figure 6. Orthochromatophilic Normoblast. The cell slightly larger than an erythrocyte, nucleus is condensed and the cytoplasm is almost the colour of the erythrocyte

Orthochromaphilic Normoblast: The process of haemoglobinization is almost complete by the stage of orthochromatophilic normoblast (figure 6). “Ortho-” in Greek means straight, upright or correct. Though orthochromatophilic suggests that the colour of the cytoplasm is the same as mature erythrocyte, this is not the case. The orthochromatophilic normoblast is the nucleated erythroid precursor that is closest to a mature erythrocyte in terms of size (8-12μm) and cytoplasmic staining. The cytoplasm however retains a blue tinge much like a reticulocyte. The nucleus is greatly condensed, shrunk and assumes a variety of bizarre shapes (buds, clover leaves, double spheres). The chromatin is greatly condensed and almost completely homogenous.

Figure 7. The Reticulocyte. Reticulocytes (blue arrows) are larger than the normal erythrocytes. Some

Reticulocyte: The Orthochromatophilic normoblasts finally extrudes the nucleus and reticulocyte (figure 7)is formed. The reticulocyte is about 20% larger (7-9μm) than the mature erythrocyte. The lifespan of the reticulocyte is about 3 days. It spends 2 of these in the bone marrow. A day after appearing in the peripheral blood the reticulocyte looses the blue colour and becomes a erythrocyte.

Table 1. The summary of morphological features of erythroid cells
Cell Size Nucleus Cytoplasm
BFU-E, CFU-E The two cells are indistingushible from blasts of other series. They posses no morphological characters that indicates their erythroid origin
Pronormoblast 12-20 μ Fine chromatin, many nucleoli Blue, a perinuclear halo and a small pale area (Golgi apparatus) may be seen
Basophilic Normoblast 12-17 μ Granular chromatin, no nucleoli Very deep blue, perinuclear halo may be seen.
Polychromatophilic Normoblast 12-15 μ Cromatin is visibly clumped with dark staining areas Basophilia reduced but still not as pink as an erythrocyte
Orthochromatophilic normoblast 8-12 μ A featureless nucleus with dense chromatin Almost the colour of a reticulocyte
Reticulocyte 7-9μ No Nucleus Slightly blue compared to an erythrocyte
The nucleus of the small lymphocyte and the normoblast are about 7.5μ

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