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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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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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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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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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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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Megaloblasts

25 Jul Megaloblasts

Development of erythrocytes involves coordinated changes in the nucleus and the cytoplasm of erythroid processors (see Morphology of Erythroid Precursors) . The proerythroblast is a large cell with a fine chromatin, the earliest forms not being different from other blasts. As the cell matures the chromatin becomes more clumped and the nucleus reduces in size and the cytoplasm becomes acidophilic. Finally a dark pyknotic nucleus is extruded from the orthchromatophilic normoblast to give a reticulocyte.

Figure 1. Basophilic Normoblast

A group of basophilic normoblast are shown above. The cytoplasm is basophilic and the chromatin more clumped than a proerythroblast. The two cells on the right are less mature than the two on the left.

Figure 2. A group of orthochromatophilic normoblasts

The figure above show a group of  orthochromatophilic normoblasts. The cells of the left are more mature. The nucleus is reduced to a dense body in the more mature forms. The cytoplasm still has a blue tinge. which contrasts from the megaloblasts shown below.

Figure 3. Basophilic, polychromatophilic and orthochromatophilic normoblasts

The figure above shows three stages of erythroid maturation. The cell on the top right is a basophilic normoblast, bottom right is a orthochromatophilic normoblast and the bottom right is an orthochromatophlic normoblast. Note the evolution of nuclear and cytoplasmic changes.

Figure 4. Megaloblasts

The figure above shows a group of megaloblasts. Cells in the right lower corner have a cytoplasm which is fully haemoglobinized and resembles the mature erythrocyte. These cell still have a nucleus. The cell on the left upper corner has a cytoplasm resembling a orthochromatophilc normoblast (see figure 2 and 3) but nuclear features resembling a basophilic normoblast (figures 1 and 2). Megaloblastic anaemia results from conditions that hamper DNA synthesis (B12 deficiency, folate deficiency, Chemotherapy drugs). The nucleus of a megaloblast thus matures slower than the cytoplasm resulting in cells having a nuclear morphology resembling a previous stage. This, known as nucleo-cytoplasmic dissociation, is the characteristic feature of megaloblastic anaemia.

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Large Granular Lymphocyte

24 Jul

Typically lymphocytes are small (10-12μm) cells with a scanty agranular cytoplasm with a round slightly indented nucleus. About 10% of lymphocytes are larger (12-16μ), have a nucleus which has a slight less compact chromatin and azurophilic granules in the cytoplasm. These are known as large granular lymphocytes. 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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Bleeding Time

1 Jun

Figure 1. Components of the homeostatic system Haemeostasis involves an interaction between the coagulation system, platelets and the vessel wall. Coagulation defects are screened by the prothrombin time, activated partial thromboplastin time and thrombin time. If activation of coagulation is prevented (by blotting blood with a filter paper) and vessels are not allowed to contract by occluding venous flow hemostasis will depend on vessel wall and platelet function. The bleeding time  that is based on this principle was proposed as a screening test for defects in homeostatic functions of the vessel wall and platelets.

Though bleeding time was described by Milan in 1901, Duke, who described the earlobe test in 1910,  demonstrated that the bleeding time was prolonged by thrombocytopenia and corrected by transfusion of whole blood that increased platelet count. Ivy, a surgeon observed that bleeding time by the Duke’s method was normal in many patients with jaundice. He attributed this to variability in the “tone” of capillaries. He modified the bleeding time by applying a standard pressure with a sphygmomanometer cuff proximal to the test wound to counter the interpatinet variability in capillary “tone”. Ivy’s method eventually became the standard method. The bleeding time appeared to be a clinically useful test. One of the greatest concerns of a surgeon is peri-operative bleeding. The purpose of bleeding time was to asses the platelet and vessel wall function and provide the surgeon with safety net. It was shown that the bleeding time correlated with platelet count. The incidence of bleeding in patient with idiopathic thrombocytopaenic purpura is less for the platelet count and patients with uraemia may bleed even with normal counts. Bleeding time was shown to be shorter than expected form platelet count in idiopathic thrombocytopaenic purpura and longer than expected form platelet counts in patients with uremia suggesting that it could mirror in vivo platelet function. The simplicity and the apparent usefulness of bleeding time made it a popular test for screening and diagnosis of bleeding disorders.

Bleeding time was never systemically evaluated. Studies evaluating bleeding time began to appear 1984. The characteristics of a test include sensitivity, specificity, positive predictive value and negative predictive value. These have never been determined for bleeding time. Bleeding time failed to predict bleeding leading to discontinuation of the practice of performance of pre-opertive bleeding time. In more damming evidence the discontinuation of bleeding time has not been found to increase the incidence of post-operative bleeding. An important assumption of bleeding time is  that skin bleeding time co-relates with tissue bleeding time. No relation has however been  found between skin and brain bleeding time in rats.

How is Bleeding Time Performed?
Bleeding time is performed by the Ivy’s method. The sphygmomanometer cuff is inflated to a presure of 40mm of Hg and maintined at that throughout the test. Using a standardized divice a cut is made on the dorsal aspect of the forarm avoiding scars and veins. The blood is blotted every 30 seconds using a filter paper avioding touching the wound with the filter paper. Bleeding time is the time taken for the bleeding to stop. If bleeding does not stop by 20 minutes the cuff is deflated and haemostasis is achieved by applying pressure. The bleeding time leaves a scar on the forearm and this is an issue in patients with a tendency to form keloids. The bleeding time is often performed by measuring the time taken for bleeding to stop following puncture of fingertip by a hypodermic needle. This not standardized and is inappropriate.

Limitations of the Bleeding Time
Bleeding time has not been found to be useful for screening for bleeding or for the diagnosis of bleeding disorders. Bleeding time is of limited value in von Willebrand’s disease, a common bleeding disorder. Half the patinets with von Willebrand’s disease have a normal bleeding time.  Even when prolonged, the bleeding time shows a day to day variability in the same patient. The coefficient of variation of the test is 15%. Given the numerous interactions between platelet, vessel wall and the coagulation system, the seperation between the components of haemostasis is artificial. Patients with hemophilia, a coagulation disease, have a prolonged bleeding time and heparin may increase bleeding time.

Place of Bleeding Time in Clinical Practice
Bleeding time should not be performed to asses haemostasis. The starting point to investigate a bleeding patient is a combination of prothrombin time, activated partial thromboplastin time, thrombin time and platelet count. Platelet count, morphology and function tests should be used to asses platelet function. The concept behind bleeding time is appealing better methods are needed to test the concept.

 

 

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