1. Practical Hematology Lab Normal Cell Maturation

2. Blood Cells Maturation
Blood cells go through several stages of development; progression from one stage to the next is not rapid, so frequently the cell being studied may be between stages.
When this occurs the cell is generally given the name of the mature stage).
As a cell transforms from the primitive blast stage to the mature form found in the blood, there are changes in the cytoplasm, nucleus, and cell size.

3. Normally, all three of these changes occurs gradually and simultaneously.
In some disease states, however, changes will take place at different rates  

4. Cytoplasmic Maturation
The immature cytoplasm usually stain a deep blue color (basophilic) because of the high content of RNA present as the cell matures, there is a gradual loss of cytoplasmic RNA and therefore a lessening of blue color.
In some cells (for example, the myelogenous cells), granules appear in the cytoplasm as the cell matures. At first, these granules are few and nonspecific. As the cell matures further, these granules increase in number and take on specific characteristics and functions.
The amount of cytoplasm in relationship to the rest of the cell usually increases as the cell matures.

5. Nuclear Maturation
The nucleus of the immature cell is round oval and is large in proportion to the rest of the cell. As the cell matures the nucleus decreases in relative size and may or may not take on various shapes (depending on the cell type).
The nuclear chromatin transforms from a fine, delicate pattern to become more coarse and clumped on the mature form, and the staining properties change from a reddish purple to a bluish purple.
Nucleoli present in early stages of cell development usually disappear gradually as the cell ages.

6. Cell Size
As a cell matures, it usually becomes smaller in size. (for the new student, this change may be difficult to detect.
The normal mature red blood cells or small lymphocytes are generally of relatively constant size and may be used as a guide for comparison) the student should know the relative size of each cell type.

7. Identification of cells
In identifying a cell we should think of the following terms:
What is the size of the cell?
Small
Medium
Large
What are the characteristics of the cytoplasm?
Granular or nongranular; specific or nonspecific granules.
Color (staining properties).
Relative amount.

8. Chromatin pattern: smooth or coarse.
What are the characteristics of the nucleus?
Shape.
Relative size.
The size of the nucleus compared to the amount of cytoplasm is expressed as the nuclear to cytoplasmic ratio (N/C ratio).
Chromatin pattern: smooth or coarse.
Presence of or absence of nucleoli; number and size.

10. Erythrocyte Maturation
4 mitotic divisions between pronormoblast and orthochromic normoblast stage.
Thus giving rise 16 RBCs.
But not all of the 16 will be good RBCs, some are bad and will be destroyed, these destroyed cells is called ineffective erythropoiesis.
2 – 7 days for pronormoblast to mature into orthochromic normoblast
1 more day to extrude the nucleus from the orthochromic normoblast.
Reticulocyte further matures for 2 – 3 days in bone marrow before it is released into the peripheral blood.
Red cell has life span of 120 days in peripheral blood.

11. Erythrocyte Maturation
The overall trend in RBC maturation is large, pale nucleus to darker, smaller nucleus to loss of nucleus; increase in cytoplasm; gradual decrease in size; cytoplasm from intensely blue (full of RNA) to grayish (mixture of RNA and hemoglobin) to reddish (full of hemoglobin, no RNA). Identify the following cells.
Maturation

12. Erythrocyte Maturation
1. Pronormobast (Rubriblast)
14-19 µm
Large nucleus 12 Microns occupies 3/4th of the cell volume.
Nucleus has fine stippled reticulum & many nucleoli.
Cytoplasm is scant and basophilic (deep violet-blue staining).
Has no Hemoglobin.

13. 2. Early Normoblast , Basophilic (Prorubriblast)
Smaller in size µm
Shows active Mitosis.
Slightly smaller nucleus with slight chromatin condensation
Increased cytoplasm and intensely blue (RNA abundance); no granules and no nucleoli present.

14. 3. Intermediate normoblast, Polychromatophilic Normoblast (Rubricyte)
Shows active Mitosis.
Lighter, grayish cytoplasm.
Increased Hemoglobin content in the cytoplasm hence, cytoplasm is Polychromatophilic
Color of the cytoplasm is due to coloring by both acidic and basic components of the stain.
Basophilia is from staining of ribosomes and acidophilia from hemoglobin.
The nucleus is condensed and intensely basophilic with moderately coarse heterochromatin granules .

16. Orthochromatophilic Normoblast (Metarubricyte)
4. Late Normoblast
Orthochromatophilic Normoblast
(Metarubricyte)
8-12 µm
Dark, opaque nucleus; gray-red cytoplasm (trace blue)
Cytoplasm has an eosinophilic appearance.
The nucleus has become pyknotic (a homogenous blue-black mass with no structure) and there is abundant acidophilic hemoglobin.
Appears like a “Cartwheel”
In some instances you can detect the nucleus in the process of extrusion.

17. Reticulocyte
(7-10 µm)
Nucleus has been extruded; cytoplasm is reddish-pale blue. RNA is still present.
The penultimate stage cell.
Has a fine network of reticulum like a heavy wreath or as clumps of dots
This is the remnant of the basophilic cytoplasm, comprising RNA.
In the Neonates, Count is 2 – 6/Cu.mm.
Falls to <1 in the first week of life.

18. Mature Erythrocyte Biconcave disc. No nucleus.
About One-third filled with Hemoglobin.

20. Factors regulating erythropoiesis
Single most important regulator: “tissue oxygenation”
Burst promoting activity
Erythropoietin
Iron
Vitamins:
Vitamin B12
Folic acid
Miscellaneous

21. Erythropoietin A hormone produced by the Kidney.
A circulating Glycoprotein
Nowadays available as Synthetic Epoietin
Acts mainly on CFU – E.
Increases the number of:
Nucleated precursors in the marrow.
Reticulocytes & Mature Erythrocytes in the blood.

22. Vitamins B12: Cyanocobalamine & Folic Acid:
Is also called Extrinsic Factor of Castle.
Needs the Intrinsic Factor from the Gastric juice for absorption from Small Intestine.
Deficiency causes Pernicious (When IF is missing) or Megaloblastic Anemia.
Stimulates Erythropoiesis
Is found in meat & diary products.

23. Iron Essential for the synthesis of Hemoglobin.
Deficiency causes Microcytic, Hypochromic Anemia.
The MCV, Color Index & MCH are low.

24. Regulation of Erythropoiesis
Stimulates
CFU – E
Proerythroblasts
Mature Erythrocytes
Tissue Oxygenation
Factors decreasing:
Hypovolemia
Anemia
Poor blood flow
Pulmonary Disease
ERYTHROPOIETIN
An example of a
Negative feed back
mechanism
Decreases

25. Erythropoietin (Epo)
As its name suggests, EPO stimulates growth of Erythrocytes, and its function include:
Activates stem cells of bone marrow to differentiate into pronormoblasts.
Shortening cell cycle.
Decrease maturation time.
Increases rate of mitosis and maturation process.
Increases rate of hemoglobin production.
Causes increased rate of reticulocyte release into the peripheral blood, (normally the reticulocyte when it is released to the peripheral blood it need only one day to mature to RBC, but here they will be released prematurely into peripheral blood, thus they need more than one day to mature to mature RBC.

26. EPO receptors
Found on surface on bone marrow erythroid progenitor and precursor cells.
The highest number of EPO receptors is seen on the CFU-E and the pronormoblasts.
The number of EPO receptors per cell gradually decreases during erythroid cell differentiation, and studies have shown that the reticulocyte and mature erythrocyte do not contain EPO receptors

27. EPO Receptor
Divided into extracellular, transmembrane, and cytoplasmic domains.
The cytoplasmic domain has terminal that contains both positive and negative growth-regulatory domains.
After EPO binding, EPOR homodimarizes and JAK-2 phosphorylates itself, so EPOR and other proteins like STAT-5 initiate the cascade of proliferative signals.

28. Myeloid Maturation MATURATION myeloblast promyelocyte myelocyte
metamyelocyte
band
neutrophil
MATURATION

30. Maturation and Morphology of Immature Granulocytes

31. 1. Myeloblast
Cells in the BM proliferation pool take hours for a single cell cycle
The first and earliest granulocyte
Is a large cell (15 μm)
High nucleus to cytoplasm (N:C) ratio (5:1)
Round or oval nucleus with loose light staining euchromatin
1-2 nucleoli
Has minimal light blue cytoplasm
Contains no cytoplasmic granules

32. 1. Myeloblast Comprises 1% of the nucleated cells in the bone marrow
Cytochemical staining shows presence of myeloperoxidase which is required for intracellular kills
Killing function is the first to be operational in the neutrophil cell line
Myeloblast is incapable of motility, adhesion and phagocytosis and is therefore nonfunctional

33. 2. Promyelocyte Larger than a myeloblast (20 μm) High N:C ratio (3:1)
Loose chromatin with nucleoli
Dark blue cytoplasm
Contains large nonspecific cytoplasmic granules
Containing myeloperoxidase (MPO)
Comprises 3-4% of nucleated bone marrow cells

34. 3. Neutrophilic Myelocyte
Medium cell size (12 μm)
High N:C ratio (3:1)
Round, oval, or slightly indented nucleus with darker
Blue heterochromatin
Last stage of cell division
Has active RNA, therefore, the cytoplasm is blue
Comprises 10% of bone marrow nucleated cells

35. 3. Neutrophilic Myelocyte
Production and accumulation of secondery granules is characteristic of the myelocyte
Myelocytes demonstrate morphologic variability as this development stage lasts from 4-5 days and cause alterations in the staining characteristics of the cell
Contains MPO and secondary granules containing leukocyte alkaline phosphatase
The cell acquires some motility

36. Granulopoiesis
Notice change in
granules color

37. 4. Neutrophilic Metamyelocyte
N:C ratio (2:1)
Last mononuclear stage, no mitosis
Nucleus is kidney or horseshoe shaped, and has condensed heterochromatin
Has a prominent Golgi apparatus – clear area located at the indentation site of the nucleus
Cytoplasm is similar to the mature cell
Comprises 18% of bone marrow cells
Not seen in normal PB
Not fully functional, part of the maturation component of the marrow

38. 5. Band Neutrophile Same size as a mature neutrophil (10-12 μm)
N:C ratio has reversed (1:2)
Nucleus is band- or sausage-shaped without segmentation
Cytoplasm is filled with small neutrophilic granules
Last immature stage
Comprises 11% of bone marrow cells and 0-3% of peripheral WBCs Stored in the bone marrow and released when there is an increased demand for neutrophils

39. Band Neutrophile
The band is a transitional form that exists in both the PB and the BM and considered part of both the maturation and storage pools
Represents the “almost mature” neutrophil having full motility, active adhesion properties, and some phagocytic ability
Membrane maturity shows changes in cytoskeleton, surface charge and presence of receptors for complement

40. Morphology of Mature Granulocytes

41. Neutrophils
This cell’s nucleus continues to indent until thin strands of membrane and heterochromatin form into segments, hence it is also called a “seg”
Polymorphonuclear means “many-shaped nucleus”, describing the varied nuclear shapes
Cell is completely functional and spend time in the storage pool of the BM as well as marginating and circulating pools of the PB
N:C ratio is 1:3, and the size is μm
Average nucleus contains 3-5 segments connected by narrow filaments
Cytoplasm contains very small neutral granules

42. Neutrophils
Granules can become larger upon bacterial infection producing toxic granulation, which are numerous, large, basophilic granules
PMNs spend their life performing phagocytosis and pinocytosis
Phagocytosis involves larger material and can be observed with light microscopy, pinocytosis involves small material (liquids) and is observed with EM
Both of these function can be performed in the circulation of the blood stream or in the tissues
50-70% of circulating WBCs of PB

43. Neutrophil kinetics

44. 2. Eosinophils Average size is 13 μm Nucleus is generally bilobed
Cytoplasm is bright red or orange which is due to large specific, secretory granules containing peroxidase, acid phosphatase, aryl sulfatase, beta-glucuronidase, etc. that stain red with the eosin component of Wright’s stain
Makes up 3% of WBCs in the peripheral blood

45. 3. Basophils Is the smallest granulocyte at 10 μm
The nucleus is difficult to see due to heavy granulation
Cytoplasm contains large specific, secondary granules that contain heparin and histamines, which stain purple with Wright’s stain.
These granules are water soluble and sometimes appear as holes in the cell if the cells are not fixed well during staining.
Makes up to 0.5% of peripheral WBCs
Note: Tissue mast cells are similar to basophils but are larger and have no developmental relationship with basophils. Mast cells have a mesenchymal (connective tissue) origin and have granules containing serotonin (basophilic granules contain no serotonin).

46. 4. Lymphopoiesis
The only human WBCs whose site of development is not just BM, but also tissues referred to as primary and secondary lymphoid organs
In humans, the primary lymphoid organs are the thymus and bone marrow, the secondary organs include the spleen, Peyer’s patches of the GI tract, the Waldermyer ring of the tonsils and adenoids, the lymph nodes and modules scattered throughout the body
Lymphocytes circulate throughout the body in both PB and lymph which act as carrier streams to bring the lymphocytes to sites of activity

47. Lymphopoiesis
Lymphocytes migrate from thoracic duct through vessel endothelium to lymph nodes to blood stream and back.
Lymphocytes are categorized in a variety of ways and may be short-lived or long-lived cells
Lymphocytes may produce antibodies or lymphokines and have different surface charges, densities and antigen receptors.
Lymphocyte % in the PB varies, depending on age.
Children under the age of 4 have a higher proportion of lymphocytes in the PB than do adults
Lymphocytes are the second most common WBC of the PB making up 20-40% of WBCs.
20-35% of circulating lymphocytes are B cells

48. Lymphopoiesis

49. A. Lymphoblast Size : 10 to 18 µm
Cytoplasm : no granules present. Appears smooth. Moderate to dark blue. May stain deep blue at the periphery and a lighter blue near the nucleus. More abundant than in the myeloblast.
Nucleus : chromatin pattern is somewhat coarse. Round or oval in shape.
Generally contain one to two distinct nucleoli.
N/C ratio is 4:1.

50. B. Prolymphocyte
Size: May be the same size as the lymphoblast or smaller.
Cytoplasm: Moderate to dark blue.
Usually nongranular, but may contain occasional azurophilic granules. More abundant than in the lymphoblast.
Nucleus: Round, oval, or slightly indented. Chromatin pattern is more clumped than in the lymphoblast. May contain one to two nucleoli.

51. C. Small Lymphocyte Size : 7 to 18 μm N:C : 4:1
Chromatin : Oval nucleus with coarse lumpy chromatin with specific areas of clumping, a compact cell
Cytoplasm : Usually just a thin border, with few azurophilic, red granules.

52. D. Large Lymphocyte Size : 9 to 12 μm N:C : 3:1
Chromatin : Looser chromatin pattern, more transparent
Cytoplasm : Larger amount of cytoplasm, lighter in color
Distinguishing characteristic : Cytoplasm is more abundant with tendency for azurophilic granules.

54. Monocyte/Macrophage Maturation

55. 5. Monopoiesis A. Monoblast Size : 12 to 20 µm
Cytoplasm : moderately basophilic to blue-gray. Nongranular.
Nucleus : Ovoid or round in shape. Light blue-purple in color. Fine, lacey chromatin. One to two nucleoli.
N/C ratio is 4:1 to 3:1.

56. B. Promonocyte (immature monocyte) Size : 14 to 18 µm
Cytoplasm : Blue-gray. May contain fine dustlike azurophilic granules. Ground glass appearance. Moderate amount.
Nucleus : oval, may have a single fold or fissure. One to five nucleoli. Fine chromatin pattern.
N/C ratio is 3:1 to 2:1.

57. C. Monocyte
Size : 14 to 20 µm
Cytoplasm : Abundant. Blue-gray. Outline may be irregular because of the presence of pseudopods. Many fine azurophilic granules, giving a ground glass appearance. Vacuoles may some times be present.
Nucleus : Round, kidney shaped, or may show slight lobulation. It may be folded over on top of itself, thus showing brain-like convolution. No nucleoli are visible. Chromatin is fine and lacey (not clumped), arranged in skin-like strands.

58. 6. Megakaryocytopoiesis
A. Megakaryoblast
(stage 1)
Cytoplasm : varying shades of blue usually darker than the myeloblast.
May have small, blunt pseudopods.
Small to moderate amount. usually a narrow band around the nucleus. As the cell matures, the amount of cytoplasm increases. Usually nongranular.

59. Megakaryoblast (stage 1)
Nucleus : Round, oval, or may be kidney shaped.
Fine chromatin pattern.
Multiple nucleoli that generally stain blue.
N/C ratio is about 10:1.

60. B. Promegakaryocyte (stage 2)
Cytoplasm : more abundant than previous stage.
Lass basophilic than in the blast. Granules begin to form in the Golgi region.
Nucleus : chromatin becomes more coarse. Multiple nucleoli are visible. Irregular in shape; may even show slight lobulation.
N/C ratio is 4:1 to 7:1 depending on the ploidy.

61. C. Granular Megakaryocyte (stage3)
Cytoplasm : abundent. Pinkish blue in color. Very fine and diffusely granulat. Usually has an irregular peripheral border.
Nucleus : small in comparison to cell size. Multiple nuclei may be visible or the nucleus may show multilobulation.
Chromatin is coarser than in the previous stage. No nucleoli are visable. N/C ratio is 2:1 to 1:1.

62. D. Mature Megakaryocyte (stage IV)
Cytoplasm: contain coarse clumps of granules aggregating into little bundles, which bud off from the periphery to become platelets.
Nucleus: multiple nuclei are present, or the nucleus is multilobulated. No nucleoli visible. N/C ratio is less than 1:1.

63. E. Platelet (Thrombocyte)
Size: 1 to 4 µm in diameter.
Cytoplasm: light blue to purple.
Very granular. Consists of two parts:
The chromomere, which is granular and located centrally, and
The hyalomere, which surrounds the chromomere and is nongranular and clear to light blue.

65. Hematopoiesis
Just notice
general trends,
Don’t memorize