Granulocytes 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
- Inflammatory diseases
- Use of haemopoietic growth factors (G-CSF or GM-CSF)
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
- Deep purple, small, round, usually indented
- Has no nucleoli
- Has a densely clumped deep chromatin
The cytoplasm of the lymphocyte is
- Moderately basophilic (blue)
- Scanty forming a thin rim around the nucleus
- 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
The old/middle english term faggot means a bundle of sticks bound together as fuel (faggot is derived the latin term fascis which is also the root for the term fasiculus). Patinets with acute promyelocytic leukemia have cells with bundles of auer rods. These are known as faggot cells and are virtually diagnostic of acute promyelocytic leukemia. faggot cells can be seen in more mature cells following treatment of acute promyelocytic leukemia with ATRA. The have rarely been reported in patients with other from of acute myeloid leukemia.
Normal erythrocytes are round disks about 7.5μm in diameter.The central one third is paler than the periphery because of the discoid shape of the erythrocyte. The picture above is a 40X image that shows the uniformity of size and staining of normal erythrocytes. Can one say that the
se cells are 7.5μm in diameter? They could all be 10μm or be 6μm. Is it possible to known about the size of erythrocytes using an unsophisticated laboratory microscope?
The small lymphocyte comes to the rescue! The size of the small lymphocyte nucleus is approximately 8.5μm and it does not vary significantly with disease. The picture above is a 100X image comparing the normal erythrocyte with a small lymphocyte. The cells are slightly smaller than a small lymphocyte. The uniformity of size and that of the pale staining area is evident. Microcytes are smaller erythrocytes and macrocytes larger erythrocytes. Hypochromia is increase in the pale staining area and indicates decreased content haemoglobin. Anisocytosis is increased variability in erythrocyte shape.
The neutrophil nucleus is segmented. The nucleus of the most immature neutrophil, band neutrophil lacks segmentation. It differs from a metamyelocyte in that the concave and convex surfaces of the nucleus are parallel. It may be coiled as is the case of the cell next to the basophil in figure 1.
The Arneth believed that the number of lobes increase as the neutrophil ages. This is not entirely true. The number of lobes in a neutrophil nucleus is decided at the band stage or earlier. After release the neutrophil nuclear segmentation may continue till the cell achieves the number of lobes it is programmed to reach. A cell that is programmed to become a three lobed neutrophil and a cell that is programmed to become a five lobed neutrophil both start as band forms but segmentation in the former stops at three lobes. The average number of neutophil lobes in a normal peripheral smear is 2.5-3.3. The definition of lobes differs from observer to observer (figure 2 and 3)
Infection and inflammation result in increased neutrophil production. Young neutrophils have fewer lobes and the average neutrophil lobulation decreases (shift to left, smaller numbers occupy a left position on the X axis of a graph). Other inflammatory changes including leucocytosis, toxic granules, Döhle bodies and neutrophil vacuolation may also be seen in patients showing “shift to left” because of infection/inflammation. Patients with chronic myeloid leukaemia (CML) and other myeloproliferative diseases or myelodysplastic/myeloproliferative disease (MDS/MPD) also show a shift to left but it is usually possible to differentiate these conditions from infection/inflammation. Peripheral smear of patients of CML (typical and atypical) and chronic myelomonocytic leukaemia show the entire spectrum of myeloid cells (myeloblasts, promyelocytes, myelocytes, metamyelocytes, band forms and segmented neutrophils). CML shows more than 10% immature forms and without monocytosis. Chronic myelomonocytic leukaemia usually shows less than 10% immature forms and shows an absolute monocyte count more than 1 X 109/L. The BCR-ABL translocation can be detected in patients with CML, but not in atypical CML. Band forms and cells with few nuclear segments dominate the peripheral smear in infection/inflammation. An occasional myelocyte or metamyelocyte may be seen. A promyelocyte or myeloblast is almost never seen. When these cells are seen in seen in substantial numbers a diagnosis of MDS/MPD should be considered.
In women one of the X chromosomes is inactivated and appears as the drumstick appendage. It is seen in 0.5-2.6% of the neutrophils. The prevalence of the drumstick increases with segmentation of the nucleus.
The exact mechanism and purpose of segmentation of the granulocyte nucleus is not known. It may aid in making the cell more deformable and allow it to squeeze from the vessels into the tissue. It is rare to find a neutrophil with more than five nuclear lobes. Increased nuclear lobulation is a feature of megaloblastic anaemia, iron deficiency anaemia (Br J Haematol 107:512;1999), uraemia, infection, myelodysplastic syndromes and hereditary neutrophil hypersegmentations. Hypersegmentation increases the average neutrophil lobe count and hence the term shift to right. A ratio of five lobed to four lobed neutrophil of 17% or more is the most sensitive indicator of shift to right. Hypersegmentation due to megaloblastic anaemia recovers in about two weeks after initiation of therapy (Ann Intern Med 90:757). Hypersegmentation can be used for diagnosis in patients who have been empirically treated with vitamin B12 and/or folate as other disease related changes rapidly revert to normal. Macropolycytes are large neutrophils often with a hypersegmented nucleus. They are tetraploid (have 96 chromosomes instead of 48 chromosomes). Hypersegmentation in macropolycytes reflects increased DNA.
One X chromosome of women is inactivated. In nrutrophils this may appear in one of three forms. Drumsticks are nuclear appendages 1.5μ in diameter. They are seen in 0.5-2.6% of neutrophils. The inactivated X chromosome may also appear as sessile nodules or as a condensation under the nuclear membrane. The frequency of drumsticks increases with nuclear segmentation. They may be seen in eosinophils but are uncommon as eosinophils have fewer lobes. Racquet forms have a central clearing and should not be confused with drumsticks. They are not inactivations of X chromosomes. The X chromosome is only inactivated in an individual with more than one chromosome. Drumsticks are not seen in individuals having only one X chromosomes [males (XY), Turner's syndrome (XO) and testicular feminization (XY)]. Contrary to expectations individuals who are XXX rarely have cells with two drumsticks. They have an increased incidence of sessile nodule. XXX is also characterized by fewer neutrophil segments. Drumsticks in XXX are less common than normal women. The incidence of drumsticks in patients with Klienfelter’s Syndrome (XXY) is lesser than normal women. Shift to left, CML and Down’s syndrome is characterized by a decreased drumstick count. It returns to normal in CML following treatment. Drumsticks are more frequent in women with isochromosome of the long are of X. Patients with megaloblastic anaemia and congenital hypersegmentation have a higher frequency of drumsticks in the peripheral smear.