1. Leukocytes D.Rezazadeh Department of Medical Laboratory Science
Kermanshah,Faculty of Paramedical

2. RBC Membrane Proteins peripheral integral (lots of sialic acid)
alpha & beta spectrin
bands 1 & 2
actin
integral (lots of sialic acid)
glycophorin A
band 3
inorganic anion transport channel
cation pump regulates Na+/K+ at ratios 1:12/25:1

3. RBC Membrane Lipids phospholipids unesterified cholesterol
lipid bilayer
unesterified cholesterol
facilitates selective cation permeability
1:1 equilibrium w/plasma cholesterol
increases in membrane cholesterol = target cells!!

4. HEMATOPOIETIC FACTORS
NAME
CELLULAR SOURCE
CELL TYPES PRODUCED IN INCREASED NUMBERS
Erythropoietin (EPO)
Kidney cells, Kupffer cells
rbc
G-CSF
Monocytes, fibroblasts, endothelial cells n
M-CSF m
GM-GSF
T cells, monocytes, fibroblasts, endothelial cells
n, m, e, meg, rbc
IL-1
Macrophages, endothelial cells, fibroblasts
n, m, e, b, meg, rbc
IL-3
T cells
IL-4 b
IL-5 e
IL-6
n, neutrophils; m, monocytes; e, eosinophils; b, basophils; meg, megakaryocytes; rbc, red blood cells
IL – interleukin , CSF – colony stimulating factor

5. NEUTROPHILS
Myelopoiesis - the production and development of myeloid cells in the bone marrow
Growth factors (colony-stimulating factors - CSF’s) and interleukins regulate blood cell development by:
Mediating proliferation
Differentiation into cell lines
Maturation of hematopoietic progenitor cells

6. NEUTROPHILS
Three series of granulocytes (named for colors taken up by granules in Wright’s stain)
Neutrophils
Granules stain equally with eosin and methylene blue
Normal in circulation = %
Eosinophils
Granules stain mainly red
Normal in circulation = 0-4%
Basophils
Granules stain mainly with methylene blue
Normal in circulation = 0-1%

7. Maturation of the neutrophilic series
Myeloblast
Size: 10-20µm
Cytoplasm
Slight amount/blue
No granules
Nucleus
Round/Central or eccentric
Reddish purple
1-3 nucleoli
Fine meshlike chromatin with no clumping
N:C ratio = 4:1

8. Maturation of the neutrophilic series
Promyelocyte (progranulocyte)
Size: 10-20µm
Cytoplasm
Increased amount/blue
Fine, azurophilic, nonspecific granules present
Nucleus
Round/Central or eccentric
Reddish-purple
Fine, meshlike chromatin beginning to clump
1-2 nucleoli
N:C ratio = 3:1

9. Maturation of the neutrophilic series
Myelocyte (last myeloid cell capable of division)
Size: 10-18µm
Cytoplasm
Increased, bluish-pink (“dawning of neutrophilia”)
Fine azurophilic, specific granules
Nucleus
Round or oval/eccentric
Reddish-purple
Chromatin loosely clumped
No nucleolus
N:C ratio = 2:1 or 1:1

10. Maturation of the neutrophilic series
Metamyelocyte (also called “meta” or “juvenile”)
Size: 10-18µm
Cytoplasm
Moderate amount
Specific granules (neutrophilic, eosinophilic or basophilic)
Nucleus
Indented (kidney-bean shaped)
Central or eccentric
Purple
Clumped chromatin
No nucleolus
N:C ratio = 1:1

11. Maturation of the neutrophilic series
Band
Size: 10-16µm
Cytoplasm
Moderate amount
Specific granules (see meta)
Nucleus
Elongated, narrow band (sausage) shape with uniform thickness
Central or eccentric
Clumped, coarsely granular chromatin

12. WBC'S (LEUKOCYTES) NEUTROPHILS: Most abundant 50-70%
Function: phagocytosis
granules with hydrolytic enzymes
defensins - antibiotic proteins,

13. EM:
specific granule:
70%
small, um
ovoid or irregular in shape
contain: lysozyme, defensin
azurophilic granule:
30%
large, round or ovoid
electron dense
lysosome: acid phosphatase, peroxidase, acidic hydrolase

14. ---function: phagocytose bacterium:
specific g.-kill B
azurophilic g.- digest B
---life span: in blood 1 day, in Tissue 2-3 days

15. Granule type
Protein
specific granules (or "secondary granules")
alkaline phosphatase, lysozyme, NADPH oxidase, collagenase, Lactoferrin and Cathelicidin
azurophilic granules (or "primary granules")
myeloperoxidase, bactericidal/permeability-increasing protein (BPI), Defensins, and the serine proteases neutrophil elastase and cathepsin G
tertiary granules
cathepsin and gelatinase

16. WBC'S (LEUKOCYTES) EOSINOPHILS: 2-4% of WBC's
Function: Allergies, parasitic worms
granules w/lysosomes and digestive enzymes ( enzymes for bacteria)
attack parasitic worms - release enzymes onto the worms
lessen allergies by phagocytizing immune (Ab/antigen) complexes
inactivate certain inflammatory chemical

17. EM: granules:
round or ovoid
with cube-liked electron dense crystal
contain:
-ACPase
-histaminase
-peroxidase
---function:
counteract the infection of parasite
reduce allergic reaction
---life span: in blood 18 hour, in Tissue 6-12 days

18. WBC'S (LEUKOCYTES) BASOPHILS: 0.5% of WBC'S
Function: liberate heparin, histamines
histamine containing granules = inflammatory chemical that vasodilates
attracts other WBC's to site of inflammation
mast cells - like basophils are found in Tissue = tissue basophils?

19. WBC'S (LEUKOCYTES) LYMPHOCYTES: 25-40% of WBC'S
Function: immune responses and memory, mainly found in lymph tissue
T cells - immune response to viral infected and tumor cells
B cells - give rise to plasma cells which produce Ab's
more later

20. EM: /free ribosome
/mito.
/RER
---classification:
TC: thymus dependent lymphocyte, 75%, involve in cellular immune reaction and regulate immune response
BC: bone marrow dependent lymphocyte, 10-15%, become into plasma cell, involve in humoral immune response
large granular cell: KC (killer cell) and NKC( natural killer cell), 10%
---function: involve in immune response

21. WBC'S (LEUKOCYTES) MONOCYTES: 3-8% of WBC's Function: phagocytosis
in tissues differentiate into macrophages
evident in chronic infections - Tuberculosis
defense vs. viruses and certain bacteria
activate lymphocytes

22. EM:
azurophilic granule: lysosome- contain peroxidase, ACPase,non-spacial esterase and lysozyme
---function: actively mobile and chemotaxis
---life span: in blood 1-5 days, in Tissue which become into macrophage- mononuclear phagocytic system, MPS

23. ---normal number: 150-450X109/L ---structure: LM:
3) blood platelet: cytoplasmic fragment of megakaryocyte in bone marrow
---normal number: X109/L
---structure:
LM:
disc-liked → irregular in shape
Stimulation

24. EM:
/cell coat: glycosaminoglycan and glycoprotein
/tubular systems:
opening tubule system- increasing the exchange area, facilitate the intaking and releasing
dense tubule system- SER, collecting Ca2+ and synthesizing prostaglandin
/granules:
specific granule:, with dense core, contains clotting factor, fibrinogen, acidic hydrolase
dense granule: contains serotonin(5-hydroxytryptamine), ADP,ATP, Ca2+ and adrenalin

25. ---function:
involve in clotting and stopping the bleed:
-aggregation
-release components of granule, make protrombin become into thrombin, then later make fibrinogen become into fibrin

26. * stromal cells:
---including reticular cell, fibroblast, macrophage, endothelial cell and lipid-laden cell
---function:
supporting
secrete cellular factors, regulate the proliferation and differentiating of hemopoietic cells

27. Maturation of the neutrophilic series

28. Function of the neutrophilic series
Neutrophils - defense against bacterial infections
Eosinophils
Regulate inflammation
Respond to antigenic stimulation in chronic allergies
Interact with larval stages of some helminthic parasites
Basophils
Histamine in granules plays a role in acute, systemic allergic reactions (sudden release of histamine can cause anaphylactic shock)
Granules also contain heparin

29. MONOCYTES
Monopoiesis - the production and development of monocytes, macrophages and their precursors. Monocytes are produced in the bone marrow. Their primary function is phagocytosis.

30. MONOCYTES Other names for macrophages
Histiocytes in the loose connective tissue
Kupffer cells in the liver
Osteoclasts in the bone
Microglial cells in the nervous system
Also scattered throughout the body in all organ tissues (spleen, lung, abdomen, etc.) One monocytes have grown into macrophages, they do not normally reenter the bloodstream except in cases of inflammation

31. Maturation of the monocytic series
Monoblast
Size: 12-20µm
Cytoplasm
Blue, moderate amount
No granules
Nucleus
Round, oval or slightly folded
Eccentric
Reddish-purple
Chromatin fine and thready
Vacuoles may be present
1-2 nucleoli
N:C ratio = 4:1

32. Maturation of the monocytic series
Promonocyte (difficult to discern)
Size: 21-20µm
Cytoplasm
Paler gray-blue and abundant
“Bleb-like” pseudopods at periphery
May contain fine red “dust-like” particles
Vacuoles may be present
Nucleus
Round with chromatin creases or brain-like folding
Central
Reddish-purple with light clumping of chromatin
0-2 nucleoli
N:C ratio = 3:1 or 2:1

33. Promonocyte

34. Maturation of the monocytic series
Mature monocyte (Normal in circulation: 2-9%)
Size: 15-18µm
Cytoplasm
Pale gray-blue and abundant
Bleb-like pseudopods at periphery, variable shape
Numerous fine pale red dustlike granule scattered throughout
Vacuoles common
Nucleus
Increased folding or elongated
Central
Purplish with finer clumped chromatin
No nucleoli
N:C ratio = 2:1 or 1:1

35. Monocyte

36. LYMPHOCYTES Normal adult value: 20-45%
Produced in the bone marrow (primary lymphoid tissue)

37. LYMPHOCYTES
Growth factors cause lymphs to differentiate. They proliferate and mature in the primary lymphoid organs:

38. LYMPHOCYTES Thymus - T cells Function
Cellular immune response (they do not produce antibodies)
Regulate antibody reactions by helping or suppressing activation of B lymphs. T cells act as “helper cells” (HIV or AIDS virus causes a destruction of these helper cells), which are instrumental in aiding B cells in antibody production, and “suppressor cells”, which act as a “thermostat” to shut off the system or keep it under control. The helper to suppressor ratio is very important and is normally 2:1.
Protect against viral, bacterial, fungal or protozoan infection
Responsible for chronic organ graft rejection. (T cells have many unique antigens on their cell surfaces, some of which are the HLA - human lymphocyte antigen - markers. Cytotoxic and helper T cells act against these HLA markers if they are foreign to the host organism. HLA markers are instrumental in chronic graft rejection and that is why tissue typing is done to establish a good donor match.)

39. LYMPHOCYTES Bone marrow - B cells Function
Humoral (antibody) immune response by transforming into antibody-producing plasma cells
Defense against encapsulated bacteria such as streptococci
Mediate hyperacute organ rejection

40. Secondary lymphoid tissue
Lymph node, spleen, Peyer’s patches in intestine and mucosal tissues such as tonsils
Active immune response occurs in above tissues where lymphs communicate and interact with antigen-presenting cells, phagocytes and macrophages

41. Maturation sequence of lymphocytes
Lymphoblast (which is preceded by a lymphoid stem cell)
Cell size: 10-20µm
Cytoplasm
Blue/scanty
No granules, Auer rods are never present
Nucleus
Purple, smooth chromatin
Round, central or eccentric
1-2 nucleoli
N:C ratio = 4:1

42. Maturation sequence of lymphocytes
Prolymphocyte (difficult to distinguish)
Size: 9-18µm
Cytoplasm
Blue, scanty
Usually granules are absent, but a few azurophilic granules may be present
Nucleus
Purple, condensed chromatin
Round or indented, eccentric
0-1 nucleoli
N:C ratio = 4:1

43. Maturation sequence of lymphocytes
Mature lymphocyte
Cell size: 7-10µm (a typical normal lymph has a nucleus that is the size of a normal RBC)
Cytoplasm
Light blue, scanty to moderate
Few azurophilic granules may be present
Nucleus
Purple, dense, clumped chromatin
Round or indented, eccentric
No nucleoli

44. Large lymphocytes versus monocytes
Size
Large lymph: µm
Mono: µm
Nucleus
Large lymph: clumped, condensed
Mono: lacy, brainlike folds
Granules
Large lymph: large azurophilic, easy to count
Mono: red, fine

45. Maturation sequence of lymphocytes
Variant lymphs
Other terms used are reactive, atypical, Downey cell, transformed, virocyte, plasmacytoid
Caused by antigenic stimulants such as viruses, post-transfusion reactions and organ transplants

46. Reactive lymphocyte

47. Plasma cells Plasma cells
Function is the synthesis and excretion of antibodies (immunoglobulins)
Normally not present in the peripheral blood; comprise 2% of bone marrow cells. (May be seen in the peripheral blood in the disease called multiple myeloma, a disease of uncontrolled production of immunoglobulins.)
End stage of the B lymphocyte
Appearance
Size: 10-18µm
Cytoplasm is dark blue with perinuclear halo and may contain vacuoles indicating antibody synthesis
Nucleus is round, eccentric, dark purple with dense clumped chromatin

48. Plasma cells

49. Thank you

50. Sites of Haemopoiesis Yolk sac Liver and spleen Bone marrow
Gradual replacement of active (red) marrow by tissue inactive (fatty)
Expansion can occur during increased need for cell production
Embryonic haemopoietic stem cells-mesenchymal cells in yolk sac
After 12 week fetal liver and spleen becomes the main site
From week 20, bone marrow starts to become important and by the time of birth it is the main haemopoietic organ

52. Extravascular Erythrocyte Destruction
Normal end-of-life event
Inherited RBC Defects
Membrane abnormalities
Enzyme deficiencies
Hemoglobinopathies
Thalassemias
Acquired RBC Defects
B12 Deficiency
Immune-mediated destruction
Drug-induced
Autoimmune

53. Intravascular Erythrocyte Destruction
Intravascular hemolysis
Normal end-of-life event
Complement Activation
PNH
PCH
Physical/mechanical Trauma
DIC
Prosthetic heart valves
Toxins
Arsenic poisoning
Bacterial Infections

54. Lab Evidence of Hemolysis
Extravascular
Urine urobilinogen
Fecal urobilin
Yellow-colored plasma
Intravascular
Increased Reticulocyte Index
Decreased haptoglobin
Decreased hemopexin
Presence of Methemoglobin/methalbumin
Increased indirect bilirubin
Increased urobilinogen
Decreased RBC
Increased iron stores
Hemosiderinuria
Ferritin

55. نوتروفیل G.CSF C/EBPα,ε IL5 C/EBPα,ε,β بازوفیل IL3,Kit ligand مونوسیت
ائوزینوفیل
IL5
C/EBPα,ε,β
بازوفیل
IL3,Kit ligand
مونوسیت
M.CSF
PU.1 , C/EBPβ
لنفوسیتB
IL4
PAX5,IKAROS
لنفوسیتT
IL7,IL2
GATA_3,notch1
NKسل
SCF,IL15
C/EBPγ
اریتروئید
EPO,GMCSF,IL3
GATA_1,NFE2
مگاکاریوسیتیک
TPO,IL3,IL11,IL6
GATA_1,GATA_2

56. Lymphocytes and Lymphoid Tissues
Primary lymphoid tissues
(thymus and bone marrow),
Secondary lymphoid tissues
(lymph nodes, Peyer’s patches, and spleen)
Tertiary lymphoid tissues
(skin and mucosal)

58. Thymus
Cortex
Medulla

59. Thymic
processing of
T-cells
See notes