lecture 1 stem cells tipparat 2015.pdf
TRANSCRIPT
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Stem cell
Normal bone marrow and blood
Tipparat Penglong, Ph.D.
Division of Hematology, Department of Pathology
Faculty of Medicine, Prince of Songkhla University
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After today’s lecture, you should be able to explain:
1. The unique properties, types, and sources of stem cells
2. The potential applications and limitations of stem cells in medicine
3. Structure and function of normal bone marrow
4. Developmental stages and functions of cells in bone marrow
5. Normal morphologies and functions of hematopoietic cells
Learning objectives
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What are stem cells and their unique properties?
• Definition: Stem cells are undifferentiated biological cells that can differentiate into specialized cells and can divide to produce more stem cells.
1) Self-renewal
2) Differentiation potential
Stem cell
Catherin Twomey , the National Academies, Understanding Stem Cells
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Stem cells can be discriminated based on their differentiation potential:
• Totipotent stem cells
• Pluripotent stem cells
• Multipotent stem cells
• Unipotent stem cells
can give rise to an entire viable organism
can give rise to every type of cell derived from the three germ layers except trophoblast
can only produce a limited number of cell types such as hematopoietic stem cell
can’t produce any cell type other than itself
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Self-renewal
1) Somatic cell nuclear transfer(Cloning)
2) Induce pluripotent stem cells
Mitalipov and Wolf, Adv Biochem Eng Biotechnol, 2009
Egg cell
Developmental potential and reprogramming of stem cells
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ESCs
Types and sources of stem cells
1) Embryonic stem cells(pluripotent stem cells)
• The inner cell mass of blastocysts
2) Adult stem cells(multipotent and unipotent stem cells)
• Found in various tissues such as
- Fat tissue
- Bone marrow
- Brain
- Muscle
- Etc.
• Multiply by cell division to replenish dying cells
- Skin renewal at 20 days
- Heart remodeling at 5 years
- All bones rebuilt at 10 years
- Brain tissue remodelled at 20 years
Leeb C et al., Stem Cell Reviews and Reports, 2010
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• Cell therapy
• Models of disease for new drug testing
• Models of disease for basic knowledge
The potential applications of stem cellsin medicine
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Therapeutic applicability of stem cells
1) Embryonic stem cells(pluripotent stem cells)
• Up to date, there is no single approved medical treatment derived from ESCs
• In clinical trial
- Spinal cord injury
- Diabetes
- Severe myopia
- Etc.
2) Adult stem cells(multipotent and unipotent stem cells)
• Current clinic uses
- Skin grafts
- Hematopoietic stem cells transplantation
• In clinical trials
-Spinal cord injury
- Corneal regeneration
- Heart damage
- Etc.
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Limitation of stem cells therapies
1) Embryonic stem cells(pluripotent stem cells)
1. Tumors formation
2. Incompatible with the immune system of patients
3. Ethically problematic cell source
2) Adult stem cells(multipotent and unipotent stem cells)
1. Reduce the problem of tumors formation
2. Reduce the problem of severe
immune reactivity by autologoustreatment.
3. Reduce the problem of ethically controversial
4. Difficulties in isolation and purification
5. Limit in proliferation capability
Pluripotent stem cells can develop teratoma
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Answer: HSCs is multipotent stem cell that give rise to all red and white blood cells and platelets.
The therapeutic applications of hematopoietic stem cells
What is hematopoietic stem cell?
Resemble lymphocyte
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Michał Komorniczak (Poland)
Development of hematopoiesis
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Sources of hematopoietic stem cells for therapeutic applications
3) Umbilical cord1) Bone marrow 2) Mobilizedperipheral blood
pelvis
sternum
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Sources of hematopoietic stem cells for therapeutic applications
3) Umbilical cord1) Bone marrow 2) Mobilized peripheral blood
• Limited number of cells
available from a single unit
• Non-invasive
• Graft Versus Host Disease (GVHD) risk : +
• The main source of HSCs in adult
• Invasive
• Graft Versus Host Disease (GVHD) risk : ++
• Require granulocyte colony-stimulating factor-induced peripheral stem cell mobilization
• Less-invasive
• Graft Versus Host Disease (GVHD) risk : +++
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Hematopoietic stem cell therapies
Autologous stem cell transplantation
-Transplantation using the patient’s own stem cells
-Transplantation using the patient’s own modified stem cells
Allogeneic stem cell transplantation
-Transplantation using stem cells from
a matched donor carrying the normal gene
Hematopoietic stem cell transplantation is the intravenous infusion of hematopoietic stem cells to establish marrow and immune function in patients with a variety of acquired and inherited malignant and nonmalignant disorders.
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Enrichment and purification methods for hematopoietic stem cells
Magnetic enrichment
Fluorescence Activated Cell Sorting (FACS)
plasma
mononuclear cell
Ficoll 1.077
Packed red cell +
polymorphonuclear cell
Density-gradient centrifugation
CD34+ cells
CD34+ cells
Domen J et al., Chapter 2: Bone marrow (hematopoietic) stem cells. National Institutes of Health, 2006
CD34+ cells
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Question
What do you think about stem cell beauty treatments?
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Bone marrow
• BM is a highly cellular structure present within the hollow cavities of hard bone tissue.
pelvis sternum
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Normal bone marrow
• Children: bone marrow in all bones is red bone marrow.
• Adulthood: bone marrow cells in long bones of hand and leg become non-functional and are replaced by fat cells to form yellow bone marrow. (100-age = % cellularity)
• The only bones to carry red bone marrow throughout life are the vertebrae, sternum, rib, hip bone, and skull bones.
• In cases of severe blood loss, the body can convert yellow marrow back to red marrow to increase blood cell production.
• On average, bone marrow constitutes 4% of the total body mass of humans.
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Normal cytologic pattern in BM
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Normal cytologic pattern in BM
2015 Sysmex Middle East
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Hematopoietic cells and non-hematopoietic cells in bone marrow
2001 Terese Winslow (assisted by Lydia Kibiuk)
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Hematopoietic cells: Erythroid, Myeloid, Lymphoid, and Megakaryocytic series
Pronormoblast Myeloblast Megakaryoblast
Basophilic Normoblast
Polychromatophilic N.
Orthochromatophilic N.
Reticulocyte(Polychromasia)
Promyelocyte
Myelocyte
Metamyelocyte
Band form
Neutro, Eo, Ba
Megakaryocyte
Lymphoblast
Prolymphocyte
Rbc Platelet(Thrombocyte)
Lymphocyte
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Non-hematopoietic cells in bone marrow
• Non-hematopoietic cells (BM stromal cells) are connective tissue cells that support the function of parenchymal cells (hematopoietic cells)
• BM stromal cells release colony stimulating factors, provide hematopoietic environment that facilitate hematopoiesis– Adipocyte (storing energy as fat)
– Osteoblast (synthesize bone)
– Osteoclast (resorb bone)
– Fibroblast (maintain the structural integrity of connective tissues)
– Endothelial cell (create the thin-walled endothelium that lines the inner surface of blood vessels and lymphatic vessels)
– Macrophage (deliver iron for hemoglobin production)
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BM stromal cells
Osteoblast Osteoclast
Adipocyte Fibroblast Endothelial cell
Macrophage
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Bone remodeling
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Bone remodeling
https://www.youtube.com/watch?v=78RBpWSOl08#
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Indication for BM examination
ICSH guidelines for the standardization of bone marrow specimens and reports
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Disease of bone marrow
• Aplastic anemia
• Myelodysplastic syndrome
• Multiple myeloma
• Infections-tuberculosis
• Various forms of leukemia
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Bone marrow aspiration and biopsy
• Aplastic anemia
• Myelodysplastic syndrome
• Multiple myeloma
• Infections-tuberculosis
• Various forms of leukemia
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Composition of blood
• Blood transports materials through out the body inside blood vessels.
• Average blood volume is 4-6 litters
Blood
Hematocrit
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Plasma
• Liquid part of blood (90% Water)
• Helps to regulate body temperature
• Contains electrolytes
• Plasma transports blood cells, products of digestion and hormones throughout the body.
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Blood cells
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Erythropoiesis
Early erythropoiesis Late erythropoiesis
HSC CMP MEP BFU-e CFU-eProerythroblast
Orthochromaticerythroblast
Reticulocyte
ErythrocytePolychromaticerythroblast
Basophilicerythroblast
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Proerythroblast = Pronormoblast
• Biggest in erythroid lineage
~ 20-25 µm
• Large central nucleus with nucleoli
• Fine chromatin
• Basophilic cytoplasm
• No granule
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Basophilic erythroblast = Basophilic normoblast
• Smaller than proerythroblast ~ 16-18 µm
• Heterochromatin
• Intense basophilia (lots of ribosomes!)
• No granule
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Polychromatophilic erythroblast = Polychromatophilic normoblast
• Smaller than basophilic erythroblast ~ 9-12 µm
• Smaller intensely heterochromatic nucleus
• Purple/lilac cytoplasm mix of basophilia from ribosomes and growing eosinophilia from hemoglobin
• Last mitotic stage!
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Orthochromatic erythroblast = orthochromatic normoblast
• Smaller than polychromatophilicerythroblast ~ 7-10 µm
• Small, compact, intensely staining nucleus that is getting ready to be extruded
• Eosinophilic cytoplasm due to abundant hemoglobin
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Polychromasia=Reticulocyte
• Immature rbc that contained polyribosomes
• Appear as polychromatophilicerythrocyte on blood smearwhen stained with a special (supravital) stain 0.5% New methylene blue
Normal range = 0-2%
Wright stain
Methylene blue stain
Polychromasia
Reticulocyte
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Erythrocyte (Red blood cell)
• Size: 7-7.5 uM
• Morphology: biconcave
• Composition: hemoglobin
• Function: transport oxygen
• Normal value: 4-6x106/uL
• Life span: ~120 days
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http://www.slideshare.net/raghuveer
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Normal rbc count in blood
• Remains remarkably constant although there are some
variations.
• MALE : 5.2 ± 0.3 x 106 /uL
• FEMALE : 4.7 ± 0.3 x 106 /uL
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Abnormal red blood cells
Hematology Outlines: HH Rashidi MD & JC Nguyen MD
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Abnormal red blood cells
Hematology Outlines: HH Rashidi MD & JC Nguyen MD
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Abnormal red blood cell sizes
Normochromic normocytic rbc Microcytic rbc Macrocytic rbc
Anisocytosis = increased variation of rbc sizes
Example-Anisocytosis 2+ with microcyte 2+-Anisocytosis 3+ with microcyte 2+ macrocyte 1+
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Abnormal red blood cell staining
Hypochromic rbcPolychromasiaNumber of Polychromasiaper OPF
Grad
1-4 Few
5-10 1+
11-15 2+
16-20 3+
>20 4+
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Abnormal shape of red blood cell
Normal rbc Ovalocyte Spherocyte
Crenated cellTear drop Target cell
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Abnormal shape of red blood cell
Keratocyte
Bite cell Blister cell
schistocyte
Poikilocytosis = increased variation of rbc shapes
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Inclusion in red blood cell
Hewell-Jolly bodies Basophilic stippling
Pappenheimer bodiesCabot’s ring
-Microtubules of mitotic spindle-Found in severe anemia
-Clusters of DNA-Found in splenectomy, hemolytic anemia
-Clusters of RNA-Found in hemolytic anemia, lead poinsoning
-Abnormal granules of iron-Found in sideroblasticanemia,
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Abnormal red blood cell distribution
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Leukocytes (white blood cells)
Granulocytes Agranulocytes
Normal value of WBC in peripheral blood : 4.5-10x103/uL
40-60% 0-6% 0-1% 20-40% 2-6%
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Maturation of granulocytes
GM-CSFG-CSF
UMN Hematography Plus, Labeled by J. Levine
specific granules
azurophilic granules
no granules
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Granulocytes: Neutrophil
• Diameter: 10-12 μm• Polymorphonuclear or PMN cells (3-5 lobes)• Most numerous WBC in blood (40-60%)• Granules:
– Azurophilic granules– Specific granules– Tertiary granules
The contents of these granules have antimicrobial properties, and help combat infection.
• Function– 1st wave of cells in acute inflammation;
can phagocytose bacteria• Life span ~ 5.4 days
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Granulocytes: Eosinophil
• Diameter: 10-12 μm• Bilobed nucleus• Bright red/pink granules contain
– Arginine rich major basic protein, peroxidase, histaminase, arylsulfatase
• 0-6% in blood• Function:
– Important in allergic rxns, parasitic infections
– Phagocytosis of Ab-Ag complexes, and a variety of organisms, including complement-coated Candida albicansand bacteria
• Life span 8–12 days
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Granulocytes: Basophil
• A small percentage (0.5%) of the cells in circulation
• Lobulated nucleus often obscured by granules
• Dark Blue Granules contain
– Heparin: anticoagulant
– Histamine: vasodilator
• 0-1% in blood
• Function
– Play a role in inducing and maintaining allergic reactions
– Role in hypersensitivity and anaphylaxis
• Life span ~ 60-70 hours
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Mast cell
• Very similar in both appearance and function to the basophil
• Mast cell ≠ Tissue basophil
NIH Publication No. 03–5423, September 2003
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Maturation of agranulocytes
GM-CSF
UMN Hematography Plus, Labeled by J. Levine
Interleukines
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Jean E. Goasguen et al. Haematologica 2009;94:994-997©2009 by Ferrata Storti Foundation
Agranulocytes: Monocyte
Monoblast Promonocyte MonocyteImmature monocyte
Migrate through blood to the tissues; once in tissues they differentiate into phagocytes (macrophages)
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Macrophage or histiocyte
• Macrophage or histiocyte is a type of immune cell that destroys foreign substances in an effort to protect the body
from infection.
Erythroblastic islandPhagocytes
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“eat-me” signals
Charles N. Serhan et al. Nature review immunology 2008
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Agranulocytes: Lymphocytes
• Main functional cells of immune system
– T (Thymus) lymphocyte is involved in cell-mediated immunity. (80%)
– B (Bone marrow or Bursa derived) lymphocyte is primarily responsible for humoral immunity (relating to antibodies). (15%)
– Natural killer cell plays a major role in defending the host from both tumors and virally infected cells. (<5%)
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Plasma cell
• A plasma cell is a mature B lymphocyte that is specialized for antibody production.
• Eccentric nucleus , perinuclear halo cytopalsm
• Rarely found in the peripheral blood. (0.2%-2.8% of the bone marrow white cell count.
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Lymphocyte markers
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Thrombocyte (Platelet)
• 2–3 µm in diameter
• Derived from megakaryocyte in bone marrow – formed from small bits of megakaryocyte cytoplasm
• Function in blood clotting
• Life span 5-9 days
• Platelets normally circulate at concentration of
150–400 × 103/uL
Megakaryocyte Platelet
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Thrombopoiesis
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Platelet examination
• Platelet count: 150-400 x103/uL
• Mean platelet volume (MPV): 7.5-11.5 fL
platelet number platelet size granule content platelet distribution
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Platelet examination
Thrombocytopenia
Giant platelet Pale stained platelet Platelet aggregation
5-25/OPF=Adequate >25/OPF=Increase<5/OPF=Decrease
Thrombocytosis
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Thank you for your attention
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Post test1. What is this cell type?
F
G
A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte
E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet
11
2
3
4
5
6
7
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Post test2. What is this cell type?
F
G
A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte
E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet
1
2
1
2
3
4
5
6
7
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Post test3. What is this cell type?
F
G
A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte
E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet
1
2
3
4
5
6
7
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Post test4. What is this cell type?
F
G
A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte
E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet
1
2
3
4
5
6
7
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Post test5. What is this cell type?
F
G
A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte
E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet
1
2
3
4
5
6
7
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Post test6. What is this cell type?
F
G
A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte
E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet
1
2
3
4
5
6
7
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Post test7. What is this cell type?
F
G
A. NeutrophilB. BasophilC. EosinophilD. Lymphocyte
E. MonocyteF. Band form neutrophilG. Red blood cellH. Platelet
1
2
3
4
5
6
7
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8. Which of the following cell type would be expected to be increased in number with a bacterial infection?
C
G
Post test
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9. Which of the following cell type would be expected to be increased in number with a viral infection?
C
G
Post test
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10. Which of the following cell type would be expected to be increased in number with a parasitic infection?
C
G
Post test
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11. Please report red cell morphology of this blood smear.
Post test
A. Normochromic normocytic rbcB. Hyperchromic normocytic rbcC. Hypochromic normocytic rbcD. Normochromic microcytic rbcE. Hyperchromic microcytic rbcF. Hypochromic microcytic rbc
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12. Please report this platelet smear.
Post test
A. IncreaseB. AdequateC. DecreaseD. Increase with clumpingE. Adequate with pale stainF. Decrease with giant platelet