Tag Archives: India

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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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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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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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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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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Iron Storage Compounds

12 Feb

Ferritin

Ferritin is made of apoferritin and iron. Apoferritin is a protein shell made of 24 subunits that is 13nm in diameter. The shell has six channels for entry of ions. The core of ferritin holds ferric-hydroxyl phosphate. Ferritin can hold 4500 atoms of iron but usually close to 2000 atoms are present.

Apoferritin is made of two units, the H and L units. About twenty types of ferritins (isoferritins) having different ratios subunits are been found in humans. The H (heavy or heart) subunit has a molecular weight of 21000 Da. The gene for the subunit is present at 11q12.3. It has a ferroxidase centre that is responsible for oxidation of Fe2+ ion to Fe3+ ion before incorporation into ferritin. The L (Liver or light) subunit has a molecular weight of 19000 Da. The gene for the L subunit is at 19q13.33. Ferritins rich in H unit acquire iron rapidly where as those rich in the L subunits are more stable.

Synthesis of ferritin begins with the apoferritin shell. Iron in the ferrous form then passes to the core through the channels, is oxidized by the ferroxidase to the ferric form and incorporated in the ferritin. Fully assembled ferritin has molecular weight of 400-500kDa. Iron promotes ferritin synthesis.

Mitochondrial ferritin is transcribed from a different gene situated on chromosome 5q23. Its function is not fully understood. As mitochondrial ferritin is highly conserved, it must have an important role.

Hemosiderin

Hemosiderin, unlike ferritin, is not s defined chemical entity. It is formed by the partial degradation of ferritin. It contains more iron than ferritin and is less soluble than ferritin. This iron however is not as freely available as iron from ferritin. Hemosiderin only releases iron after prolonged or severe iron depletion.

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Case 2 – Thrombocytopenia Due to Large Platelets

3 Nov Large-Platelets-600px

A 52 year old patient presented with thrombocytopenia discovered on a haemogram performed about 2 years before presentation. The first haemogram performed as a part of a health checkup showed a platelet count of 81 X 109/L with an MPV of 13.1fl. The haemogram had been performed on multiple occasions since then and the platelet count has been reported by different laboratories to be between 63 X 109/L and 103 X 109/L. The haemoglobin, red cell indices, total and differential WBC count was normal.

The patients had no personal or family history of bleeding. He had undergone an appendicectomy and a tooth extraction without excessive bleeding.

The first step in the evaluation of a patient with thrombocytopenia is to confirm if the platelet count is really low. The causes of pseudothrombocytopenia include

  1. Presence of large platelets
  2. Platelet satellitism
  3. EDTA (or rarely other anticoagulant) induced platelet aggregation

The figure below shows the peripheral smear and the platelet histogram of the patients compared to a normal individual.

The platelet count performed manually was 130 X 109/L. The patients was a native of North India. Mild thrombocytopenia (platelet count between 100 X 109/L and 150 X 109L) is found in 26% of blood donors from eastern India and 18% blood donors from northern india (HVK Naina et al. Journal of Thrombosis and Haemostasis 2005; 11:2581-2).

Automated counters have limitations in evaluation of thrombocytopenia. The peripheral smear must be examined in every patient with thrombocytopenia to exclude giant platelets, platelet aggregation and platelet satellitism that may cause thrombocytopenia.

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Case No 1 – Severe Microcytic Anaemia

1 Nov

A 17-year-old woman from a farming community presented with the history of severe weakness, fatigue and dyspnoea on moderate exertion.

She was well till about 4 months before presentation when she complained of fatigue that gradually became worse. Though progressively symptomatic she was able to attend school till 3 days before presentation.

There was no history of fever, weight loss, abnormal bleeding or melena. There was no increase in the menstrual blood loss. The patient was living with her parents and two sisters both of whom were healthy.

Her appetite had been normal through most of the illness though off late it had diminished.

On examination the patient was comfortable but tachypnic  in bed. She had severe pallor of the skin, conjunctiva and mucous membranes.  There was platonychia and glossitis. The jugular venous pressure was elevated. There was no icterus, clubbing, lymphadenopathy. The pulse was 108/min and the blood pressure 110/60. The third heart sound was audible at the apex. She had bilateral fine crepitation in the bases of both lungs. The was no hepatomegaly. There was no splenomegaly. Examination of the neurological system revealed no abnormality.

A haemogram was performed and the results were as follows:

  • Haemoglobin: 1.7g/dL
  • RBC Count: 0.9 X 1012/L
  • Haematocrit: 5.9%
  • WBC Count: 4.1 X 109/L
  • Differential Count:
    • Neutrophils: 35%
    • Lymphocytes 49%
    • Monocytes 10%
    • Eosinophils: 6%
  • Platelet Count: 505 X 109/L
  • RBC Indices
    • MCV: 66.1 fl
    • MCH: 19.4 pg
    • MCHC: 29.3 g/L
    • RDW: 44%
  • Reticulocyte Count: 8%

What are the probable causes of anaemia in this patient?

Presence of platelet and/or leucocyte anomalies in a patients with anaemia suggests that the pathology lies in the bone marrow. Anaemia without leucocyte or platelet anomalies may be caused by increased erythrocyte loss or decreased erythrocyte production. Anaemias caused by decreased production of erythrocytes are further divided according to erythrocyte size into microcytic, normocytic and macrocytic  (see Evaluating anaemia).

This patient does not have platelet or leucocyte anomalies. The anaemia is either a result of a decreased production or increased loss of erythrocytes. Both erythrocyte loss and decreased production are difficult to quantify in clinical practice and are diagnosed by indirect methods. Increased eryrthrocyte production should increase haemoglobin unless there is an equal or greater loss. Increased erythrocyte production result in reticulocytosis. In patients with anemia the reticulocyte production index is a more accurate index of erythrocyte production. A patient with normal or low haemoglobin but an increased reticulocyte production index is presumed to be loosing erythrocytes  (see diagnosis of haemolytic anaemia).  Others are presumed to have decreased erythrocyte production.

Reticulocyte production index is calculated as follows:

RPI = [Reticulocyte count X HCTPatient] / [HCTNormal X CF]

where

RPI is the Reticulocyte Production Index. RPI values less than 2 indicate inadequate erythrocyte production

HCTPatient is the patient’s haematocrit

HCTNormal is the normal haematocrit

CF is the correction factor. The correction factor depends on the haematocrit. It’s values are as follows:

  • Haematocrit 40-45%: 1
  • Haematocrit 35-40%: 1.5
  • Haematocrit 25-35%: 2
  • Haematocrit 15-25%: 2.5

The reticulocyte production index in this patient is as follows

RPI = [8 X 5.9 ] / [45*2.5]  =  0.41

RPI of 2 indicates an adequate marrow response. This patient, despite having a high reticulocyte count, has decreased erythrocyte production.

Anaemias due to inadequate erythrocyte production are classified according to erythrocyte size as microcytic, normocytic and macrocytic (see red cell indices). This patient has microcytosis. The causes of microcytic anaemia include:

  • Iron deficiency
  • Thalassaemia
  • Sideroblastic anaemia
  • Anaemia of chronic disease

Chronic inflammation is associated with mild to moderate anaemia that is proportional to the degree of inflammation. Most of the patients have normochromic anaemia but about 30-50% of the patients may develop mild microcytosis with a normal RDW.  This patient has no evidence of chronic inflammatory process. She has severe anaemia, pronounced microcytosis and a high RDW. She is unlikely to have anaemia of chronic disease.

Iron deficiency and thalassaemia are the commonest causes of microcytic anaemia. β-Thalassaemia has three clinical forms, major, minor and intermedia. Thalassaemia minor does not cause symptoms. Symptomatic β-thalassaemia (thalassaemia major and intermedia) usually present early. The transfusion dependent patients present by 8.4±9.1months and non-transfusion dependent patients by 17± 11.8 months (Cao A Birth Defects 1988;23;219) . This patent has a late presentation making thalasaemia unlikely.

The haemogram of patients with iron deficiency shows anaemia, microcytosis, hypochromia and anisocytosis. Thrombocytois is not uncommon. The platelet counts in patients with iron deficiency are twice those without deficiency (Dan K Intern Med 2005; 10: 1025). The thrombocytosis associated with iron deficiency has an inverse relationship with serum iron but no relationship with TIBC and transferrin saturation. (Park MJ et al Platelets 2012 Jun 27. [Epub ahead of print]). This patients has anaemia, microcytosis, hypochromia and thrombocytosis. The high red cell distribution width (RDW) indicated the presence of anisocytosis. The most likely diagnosis in this patient is iron deficiency anaemia.

Is there a haemoglobin value that should trigger a blood transfusion?

It was common to use the 10/30 (haemoglobin 10g/dL or PCV 30%) trigger for transfusion. Transfusion is associated with morbidity and mortality (Marik et al. Crit Care Med 2008; 36:2667-74). Following the 10/30 rule results in overtransfusion. The case highlights poor relation between haemoglobin levels and symptoms. Anaemia is compensated by increasing cardiac output, redistributing blood from non-critical to critical circulations and increasing oxygen extraction (see Pathophysiology of anaemia). Older individuals may have one or more of these mechanisms compromised hampering their ability to withstand anaemia. Younger individuals can compensate efficiently and can withstand lower haemoglobin values. Compensation takes time and is more complete in anaemias that have a slow onset. Nutritional deficiencies develop slowly. Patients with nutritional anaemia compensate better and become symptomatic at lower haemoglobin values than anaemia with a more acute onset.

She was in cardiac failure and was transfused. But would she have been transfused if she was not in cardiac failure and had a haemoglobin of 7g/dL? The threshold for transfusion according to most current guidelines is 7-8 g/dL. Trials evaluating transfusion threshold have been performed in hospitalized, surgical or intensive care patients. Transfusion guidelines are most appropriate when used for indications the were developed. There are few trials addressing the issue of transfusion in patients with nutritional anaemia. Clinical experience shows that patients without respiratory or cardiovascular co-morbidities are able to tolerate severe nutritional anaemia. One needs to balance the risk of anaemia against risk of transfusion in patients with nutritional anaemias. The transfusion trigger for patients of nutritional is not established. Symptoms guide transfusion in patients with nutritional anaemia.

How should the patient be investigated?

As iron deficiency is the commonest cause of microcytic anaemia, patients should be evaluated for iron deficiency. The serum iron, total iron binding capacity and serum ferritin are estimated and the transferrin saturation calculated . The results of these investigation are diagnostic for iron deficiency and often allow the diagnosis of other causes of microcytic anaemia. The interpretation these tests are as follows:

Serum Iron Total Iron Binding Capacity Transferrin Saturation Serum Ferritin
Iron Deficiency Anaemia  Low  Normal/High  Low (<16%)  Low (<12ng/ml)
Thalassaemia Normal Normal Normal Normal/High
Sideroblastic
Anaemia		 High Normal High (>50%) High
Chronic Disease low/Normal Low Normal Normal/High

This patients was diagnosed as iron deficiency anaemia because of a low serum iron, high total iron binding capacity and a low transferrin saturation. Does that complete the diagnosis?

The diagnosis of iron deficiency is incomplete without finding out its cause. The following populations are prone to iron deficiency.

  1. Preterm infants: Most of the iron transfer to the foetus takes place in the third trimester. Premature infants have less time to absorb iron from the mother.
  2. Infants and Toddles: Dietary iron is not able to keep up with the requirements of rapid growth.
  3. Women in the childbearing age group: Losses due to menstruation and pregnancy cause iron deficiency.
Iron deficiency is due to inadequate iron intake or increased iron/blood loss. Blood/iron is lost because of diseases/conditions causing exacerbation of physiological blood/iron loss or diseases causing physiological loss. Multiple pregnancies cause exacerbation of physiological iron loss. The only physiological blood loss is menstrual blood loss.
This patient gave no history of increased menstrual blood loss. Hookworm (Ancyclostoma duodenale and Necator americanus) infestations infestations affect over 500 million individuals in the hot and humid areas of the world. Infestations are common in farming communities. The stool examination of this patients showed hookworm ova.
Finally, does the diagnosis of iron deficiency rule out other causes of microcytic anaemia? No. Iron deficiency is very common that it may co-exit with another cause of microcytic anaemia particularly anaemia of chronic disease and thalassaemia trait. Anaemia of chronic disease is characterized by a low iron, low total iron binding capacity and normal transferrin saturation. When such patients become iron deficient the transferrin saturation falls below 16%. Ferritin is increased in the presence of inflammation/infection. While a low ferritin is diagnostic of iron deficiency in the presence of infection/inflammation, a normal ferritin does not exclude iron deficiency. β-Thalassaemia is diagnosed by an elevated HbA2. HbA2 has been shown to fall with iron deficiency. This may not have any clinical significance (Madan N et al. Ann Hematol 1998; 77; 93-6, Passarello C et al 2012; 97:472). If the haemoglobin and/or microcytosis fail to correct after therapy the patients must be evaluated for thalassaemia.

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Red Cell Indices

13 Oct

Estimation of haemoglobin concentration, erythrocyte count, haematocrit, total and differential leucocyte count and platelet count along with examination of the peripheral smear are a part of evaluation of anaemia. Examination of the peripheral smear detects abnormalities in size (microcytosis or microcytosis, anisocytosis), shape and haemoglobnization. Reporting a peripheral smear need experience and is subjective. Red cell indices evolved to bring objectivity and increase reproducibility reporting of red cell characteristics. Red cell indices have allowed a reproducible algorithmic approach to the diagnosis of anaemia.

Normal red cell counts were described in 1852 . This measurement made on four subjects was the normal standard for the next 70 years. Wintrobe in the 1930s determined the normal red cell count described the method for calculating red cell indices (Wintrobe MM Am J Med Sci 1929;177:513–23) and classified anaemia into four groups, macrocytic, normocytic, microcytic and microcytic hypochromic (Wintrobe MM Arch Intern Med. 1934. 54:256-280). Though the methods of determining red cell indices has changed, the concept of classification of red cells by size and the classes of anaemia described by Wintrobe still hold. The three indices are

  1. Mean corpuscular volume (MCV)
  2. Mean corpuscular haemoglobin (MCH)
  3. Mean corpuscular haemoglobin concentration (MCHC)

Mean Corpuscular Volume (MCV)

MCV is the average volume of erythrocytes and is defined as follows:

MCV = [Haematocrit (in percentage) X 10]/RBC count (in millions/mm3)

The normal value is 92±9fl

At the time MCV was described it was calculated as described above. Erythrocyte counts performed manually were cumbersome, technician dependent and inaccurate limiting the value of MCV. Automated haematology counters may be based on impedance or light scatter. Sensors of counters, impedance as well as those based on light scatter, generate signals according to cell size and number. The size of each particle is calculated by the pulse it generates and the MCV reported as an average of these measurements. The standard deviation of observation of MCV is reported as red cell distribution width. When automated counters are used MCV is a measured parameter rather than a calculated parameter. These measurements are accurate and reproducible.

Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC)

Mean corpuscular haemoglobin is defined as follows.

MCV = [Haemoglobin (in g/dL) X 10]/RBC count (in millions/mm3)

The normal value for MCH is 29.5±2.5pg.

Mean corpuscular haemoglobin concentration is calculated as follows

MCHC = [Haemoglobin (in g/dL) X100]/[Haematocrit (in percentage)]

Normal 33±1.5g/dL

The MCV and MCHC are of limited value in diagnosis of anaemias. A low MCH has the same significance as a low MCV. MCHC is low in microcytic anaemias. Macrocytic anaemias may have a high MCH but the MCHC is normal in these case. Some anaemias like hereditary spherocytosis, sickle cell anaemia and haemoglobin C disease are characterized by a high MCHC indicating cellular dehydration

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Red Cell Distribution Width

1 Oct

Features of peripheral smear like erythrocyte size, haemoglobinization and variability in size have long been useful in evaluation of patients with anaemia. The definitions of these parameters before the advent of haematological counters were subjective. The haematological counters have allowed an accurate measurement of erythrocyte size and numbers allowing reliable determination of mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cellular haemoglobin concentration (MCHC). Mean cellular volume is used to classify anaemias (see Evaluating Anaemias). MCV is of little value in differentiating two of the commonest causes of anemia, iron deficiency and β-thalassaemia trait as both are microcytic hypochromic.

Anisocytosis is and abnormal variation in erythrocyte size. It is a feature of nutritional deficiencies, myelofibrosis, bone marrow infiltrations, microangiopathic haemolytic anaemia and in the presence of erythrocyte aggregates. Thalassaemias do not show anisocytosis. The erythrocyte is a disc approximately the diameter of the nucleus of a small lymphocyte and the central one-third is pale. The assessment of anisocytosis is subjective. Red cell distribution width(RDW) is quantitation of aniscytosis.

Standard deviation of a parameter is a measure of its scatter from the mean. MCV is an average of erythrocyte volumes measured by the counter. RDW is the standard deviation of these observations. It is expressed as a percentage of MCV. The normal values are 11.5-14.5%. High RDW means more anisocytosis.

Despite the apparent promise RDW has a limited role in diagnosis. A normal RDW in a microcytic anaemia can suggest the presence of on thalssaemia but can not be relied on as a sole criteria for separating iron deficiency from thalassaemia. The presence of a high RDW should alert one to the presence of one of the causes of anaemia listed above but again RDW can not be relied on for making a final diagnosis of any of these conditions. Blood transfusion in an anaemic patient increases the RDW.

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