1. Leukocytes Formation, Function and Pathology Clinical Pathology
Kristin M. Canga, RVT

2. Leukopoiesis
All WBC production starts out in red bone marrow from the same _____________________ ______ _______ that produces RBCs.
The ____________ that act on the PPSC determine which cell type will be produced.
Each WBC has its own stimulus for production.
All WBCs differentiate and develop in the bone marrow except for some _____________________ which start out in bone marrow but develop elsewhere.
At the beginning of leukopoiesis, all WBCs ___________________ (in the bone marrow)
PPSC = Pluripotent stem cell

3. Leukopoiesis Granulopoiesis 1. Stem cell 2. Myeloblast
3. Promyelocyte Myelocyte
5. Metamyelocyte Band cell
7. Mature cell (segmented neutrophil, basophil or eosinophil)

4. Classification of Leukocytes
Leukocytes may be classified in three different ways:
____________________________
______________________________________ ______________________________________ __

5. Classification by Defense Function
____________________________
Phagocytic leukocytes include:
_________________ production and cellular ______________
_________________________

6. Classification by Shape of Nucleus
_____________________________
Means that the nucleus is _________________, or __________________
Neutrophils
Eosinophils
Basophils
Means that the nucleus is varying in ___________ and ______________________
Monocytes
Means that there is a _____________, rounded nucleus
Lymphocyte

7. Classification by Granules
____________________(Presence of granules)
________________– granules not attracted to stain
________________– granules attracted to acidic stain
________________– granules attracted to alkaline stain
____________________(Absence of granules)
________________

8. More on Granulocytes Originally no granules present
PPSC  _________________, _________________, __________________, __________________, and finally the various granulocytes based on _________________ granules.
_____________________ produces first set of granules as cells mature
First set are called _______________________granules
Golgi Apparatus also produces granules that are attracted to stain
__________________ granules are added as cell matures to determine ____________________ of the cell.

9. Functions of the Specific Granules
Specific Granules contain different substances depending on the cell:
Neutrophil granules contain ____________________
Aid the cell in killing microorganisms after ___________________
Eosinophil granules contain __________________________________________
De-granulate at site of allergic or anaphylactic reaction to reduce swelling
Basophil granules contain ___________ and _________
Histamines are released to initiate ___________________ at site of reaction
Inflammation draws ____________________ to site
_________________ acts as local anticoagulant to keep blood flowing to injured/damaged area.

10. Details of the Granulocytes
Recall that the granulocytes include:
_____________________

11. Granulocytes - Neutrophils
Also called “polymorphonuclear” cells
Lobes of the ____________ are connected by thin ________________
As cell ages, the chromatin of the nucleus ______________ (squeezes together) and changes shape. (Think “________ and __________”)
As cell becomes ____________, the segments break apart, losing all evidence of chromatin. (Indicates dead cell)

12. Neutrophil Formation
Adult neutrophils take ~___-___ days to be produced by the bone marrow
Are being produced for release as circulating neutrophils migrate to __________ to fight microorganisms or die of old age.
If high ___________ for neutrophils occurs, they can be produced in less time.

13. Neutrophil Characteristics
Granules are considered to be ________ because they stain neither red, nor blue
Often identified on blood smear based on shape of ______________.
PMN adult cells may contain between 2-4 lobes connected by thin filaments of _____________.
Sometimes the chromatin is so thin that segments look separated.
(your book says 2-5 lobes, either is fine.)

14. Neutrophil Function
Neutrophils only spend ~____ hours in circulation before migrating to __________.
Once in the tissue, neutrophils do not ________________ circulation
For this reason, the entire population of circulating neutrophils needs to be replaced ~________ times daily.
If demand is so high that bone marrow cannot supply enough adults, ________ cells will be released
If demand is still too high, progressively _______________ cells will be released.

15. Neutrophil Function First line of defense in the blood
Major function is __________________ of invaders
Can respond to foreign microorganisms very quickly.
Use the blood as _________________, squeezing through endothelium cells via ______________. (PLEASE review Figure 9-5 on pg 234 of A&P book)
Diapedesis = Pseudopod formation that pushes between endothelial cells of capillary, taking cytoplasm and rest of cell with it into tissue.

16. Neutrophil Function Attracted to site of infection by ______________
Chemotaxis is the chemical reaction created between the microorganisms and the tissues that ____________cells.
__________________ cells with granules that contain __________________.
Lysosomes contain _______________ enzymes capable of destroying bacteria and viruses upon phagocytosis.
Membrane flows around invader and encases it in vacuole that is created, allowing lysosomes to digest invader.
____________ is produced through increased _____ absorption, and is toxic to invaders AND ______________.
Cell rapidly absorbs O2 and forms “killing zone” resulting in H2O2

17. Neutrophil Function
Normally found in tissues that are constantly susceptible to invasion by microorganisms such as the ________ and _____________tract.
Dead or abnormal neutrophils are disposed of by tissue __________________.

18. Normal Neutrophil Count in Blood
The neutrophil count in peripheral blood is kept within a specific range
(dogs: 3,000 – 11,400/µL; cats: 2,500 – 12,500/µL) in healthy animals and is controlled by three factors:
1) Release of mature neutrophils from the ______________ pool in the bone marrow into ________________________blood.
2) Rate of ______________ from peripheral blood into tissue.
3) Entrance of increase numbers of __________ into the neutrophil _________________ line.

19. Maintaining Normal Neutrophil Pools
1) Release of mature neutrophils from the ____________ pool in the bone marrow into the ________________ blood.
________________ supply of mature neutrophils are “on call” and ready for __________________ release.
Caused by sudden __________________ movement of neutrophils into _____________.

20. Maintaining Normal Neutrophil Pools
2) Rate of ___________ from peripheral blood into ____________.
Total neutrophil population in peripheral blood can enter the ___________ within a couple of hours
Triggered by massive acute _____________

21. Maintaining Normal Neutrophil Pools
3) Entrance of increased numbers of _________ into the neutrophil ______________ line.
_________ method of control; it takes 3 to 6 days for neutrophils to mature for release

22. Intravascular Pools of Neutrophils
The ______________________________(CNP)
Refers to the blood contained in the ____________________________.
Blood samples obtained for _______________ analysis contain neutrophils from this pool.
The normal range for neutrophils is based on those contained in this pool.
The _____________________________ (MNP)
Composed of neutrophils that line the _________ of small blood vessels (not _______________________).
These neutrophils are not contained in blood sampled for laboratory analysis.

23. Neutrophilia
Defined: An ______________ in neutrophils in blood circulation
If overall neutrophils increase, total ________ count increases causing __________________
To meet increased demand for neutrophils in tissue, the bone marrow releases its reserve stores of mature, and if necessary, _______________ neutrophils into the blood.
If a blood sample is drawn while these neutrophils are in transit, a ___________ than normal number of neutrophils will be included in the sample (__________________)

24. Neutrophilia in a Periperal Blood Sample

25. Neutrophilia and Stress
_____________________ leukocytosis:
Caused by exercise, ___________, or ________________
__________________ release results in a shift of neutrophils from the MNP to the CNP (_______________________)
No left shift occurs (no increase in _______ cells)
Count usually not more than ________ the normal value
Should return to normal within ___ minutes of removal of stimulus
Most common in _________ and animals <____mo.

26. Neutrophilia and Stress
____________________________
_____________________ -induced
Treatment with exogenous steroids
Steroids inhibit ______________
Endogenous steroids released in response to major _____________ illnesses, ________________ disturbances, and __________
Neutrophil count nearly _____________ – mature neutrophilia
No ____________/ Shift from MNP to CNP
Have students define diapedesis

27. Neutrophis and Stress Stress Leukogram – Continued
Expect to see _________________ , and _______________________.
Expect to see _________________ due to lysis of cells.
Expect to see ____________________.
Possible see ___________________ in dogs
Develops over several ________ and may last for several _________.

28. Neutrophilia Other causes of neutrophilia include:
____________________ – mild inflammation yields a leukocyte response similar to __________
severe inflammation yields neutrophilia with a _______________.
Bacterial infections
Conditions associated with extensive ________damage:
Burns, _______________, trauma, extensive ________, neoplasia
Extreme leukocytosis (with neutrophilia) may be associated with _____________________ that produce colony- stimulating factors:
Hepatozoon canis infections, leukemias, and closed cavity infections (i.e. _________________, _______________)
Tick borne blood parasite caused by ingestion of larval form of tick harboring parasite.

29. Leukemia Literally means “_______________ blood”
Caused by malignant proliferation of one of the WBC types
In response to some unknown stimulus, stem cells in bone marrow start producing _______________ cells in one cell line at an __________________ rate.
Abnormal cells show up in blood and bone marrow in large numbers, usually before they are _____________ and cause a dramatic _____________in total WBC count.
Leukemias are considered a form of ____________ and can be ___________ or _____________.
Classified by type of _________ involved
Leukemia and leukocytosis may resemble one another; sometimes the distinction is difficult.

30. Left shifts
_______________________ Left Shift - Neutrophilia with some ________ cells present; however, mature, _____________ neutrophils predominate.
________________________ Left Shift – Neutropenia where ____________ neutrophils outnumber _____________neutrophils
Usually result of extreme migration of cells into tissues and/or detrimental effects of toxins.
Degenerative Left Shift

31. Left Shifts
Characterized by an increase in _________ (immature) neutrophils in the blood.
Note: It is normal for 0-300/µL band cells to be present in the blood of a healthy canine/feline.
Usually associated with _________________ conditions
Demand for neutrophils is ______than bone marrow pool.
Left shifts vary from ___________ (slightly increased number of bands) to _____________ (metamyelocytes, myelocytes, and rarely- even promyelocytes present in blood)
___________ cytoplasm often present during left shifts
Toxic = One that has gone thru an accelerated rate of production. This is seen with inflammation, which results in the persistence of ribosomes.

Notice: increased basophilia of cytoplasm, presence of dohle bodies, and cytoplasmic vacuolation.

32. Toxic Neutrophils Normal Neutrophils Toxic Neutrophils
A/C are normal; B/D = Toxic. Less dense nucleus and purple cytoplasm Cell is less contained when toxic
Dohle bodies may also be present

33. Toxic Neutrophils
Dohle Bodies may be noted in cytoplasm of toxic neutrophils.
Dohle Bodies are __________ and appear similar to _____________ seen in monocytes

34. Left Shift
Degenerative or Regenerative? = Degenerative b/c of lack of mature cells.

35. Right shift
___________________________ – refers to the presence of ___ or more distinct nuclear lobes within neutrophils.
A right shift reflects prolonged ___________time of neutrophils in blood and can occur as a result of:
Resolving chronic ___________________
Glucocorticoid administration
Hyperadrenocorticism (Cushing’s disease)
___________________________________ disorders
May develop _____________ when blood film preparation is delayed for more than a few hours.

36. Hyper-segmented Neutrophil

37. Neutropenia
Neutropenia will cause the total ________ count to decrease (_____________________)
If an infection is out of control, all the reserves of ____________________ can be used up faster than the bone marrow can replace them.
Such a condition signifies that the body is _____________ against the invading microorganisms.
Prognosis is ________ for a critically ill animal that has __________________ and ___________________________

38. Neutropenia Neutropenia can occur due to:
_______________________ of neutrophils (pseudoneutropenia)
Excessive ___________ demand or destruction of neutrophils
Destruction can be _______________-mediated
Reduced or ineffective granulopoiesis
Conditions that cause neutropenia:
Overwhelming _____________ infections (ex: septicemia)
Idiosyncratic drug reactions may result in neutropenia or pancytopenia (e.g. penicillins, cephalosporins, sulfonamides in dogs and chloramphenicol in cats)
Feline ______________ Virus
Canine ____________ Neutropenia (a.k.a. Gray Collie Syndrome)
Gray Collie Syndrome = dogs appear lighter than others
Every days, dog will experience drastic reduction in neutrophil presence. High risk of infection during these times.
Poor prognosis, average life span ~3 years

39. Neutropenia in a Peripheral Blood Smear

40. Eosinophils
Formation: It takes ___ to ___ days to produce an eosinophil from a PPSC.
Population in circulation ~__-__% of total count
_______________ and ____________ pools are also found in peripheral blood (like __________________)
Bone marrow contains good supply of mature cells for release as needed.
Eosinophils migrate into tissue in just a few _______ where they spend the rest of their lives and undergo the same aging process as neutrophils
Granules’ color and size vary depending on the ____________.

41. Eosinophil Granules
Feline
Canine
Equine
Bovine

42. Eosinophils
Eosinophils are attracted to, and inhibit local ____________ and hypersensitivity-related reactions.
Their granules contain anti-________________ substances that are released at the site of the allergic reaction.
They ingest substances associated with the _______________ immune response (antigen- antibody reaction complexes)
They have some ________________ and bactericidal functions
They are especially effective in phagocytosis of ______ pathogenic organisms, such as ____________, and some parasitic worms but are not protective against most _______________ infections.

44. Eosinophils Normal Eosinophil Values:
dogs: /µL; cats: 0 – 750/µL)
___________________ is usually a result of:
___________________: Skin, respiratory, GI tract
___________________________: Anaphylaxis, Fleas, food, grasses, or non-specific allergens. Feline asthma, Chronic ______________ (skin, GI, respiratory, urogenital), Tumor associated (fibrosarcoma, mast cell tumor, lymphoma, etc.)
____________________is difficult to detect and evaluate because their numbers are normally ________.

45. Feline eosinophilic leukemia
Feline eosinophilic leukemia. Two neutrophils (arrows) are adjacent to several eosinophils at various stages of development that include segmented, band, metamyelocyte, and myelocyte forms

46. Basophils Formation: Characteristics: Function:
Basophils are produced in the bone marrow from the same PPSCs as other cells
Characteristics:
The granules of basophils are ________ soluble and often washed out during the staining procedure
_____________ are not always visible on a stained smear.
Function:
Basophils are the _________ phagocytic of the granulocytes.
Their granules contain ____________ and _____________ which are responsible for at least part of their function (not much is known about basophil production or function).

47. Basophils
Above: feline basophil (left); canine basophil (right)

48. Basophil Function Histamine and heparin granules
_______________ helps initiate __________________ and acute ___________ reactions.
_____________________ are attracted to the site of an allergic reaction by ____________________________________ released from the granules.
_____________ acts as a localized _____________________ to keep blood flowing to an injured or damaged area.
Eosinophilic chemotactic factor is what draws the eosinophils to the site of the reaction. It is a chemical that tells the eosinophil where to go.

49. Basophilia and Basopenia
Normal Basophil Values: ________ in both dogs and cats
___________________ can be associated with an __________ or _________________ reaction in the tissue.
Sometimes ______________ and _______________________ are seen at the same time.
_______________________: is not clinically significant. Basophils should make up less than ___% of all WBCs in peripheral blood.

50. Agranulocytes Recall that agranulocytes include
_________________________

51. Details of Agranulocytes
Monocytes are formed in the bone marrow from the PPSC population.
(Maturation: 1. ______________– 2. __________________– 3. ______________)
Lymphocytes originate from the PPSC in the bone marrow; however some travel to ________________ organs to develop and mature before settling into their permanent home in ________________ lymphoid tissue. (More on these later.)

52. Monocytes Formation: Description
Mature much ____________ and stay in the blood _____________ than neutrophils.
Total development time is ___-____ hours then they stay in the blood for ___-____hours before entering tissue where they carry out their function.
Description
Monocytes are slightly __________than a neutrophil
Monocytes have a _______-_______cytoplasm, ______________nucleus, and a “_________” chromatin pattern.

53. Monocytes
Function:
Major _________________cells; are known as tissue ________________________once in the tissues.
Monocytes in the blood are considered _______________ tissue macrophages and are less effective ________________ than they are in the tissue.

54. Monocytes
Monocytes enter tissue by the process of _________________ in response to tissue damage caused by trauma or invading microorganisms.
_________________ respond more quickly to tissue damage, but ________________ stay around longer once they reach the damaged site and have become ________________.
Monocytes can also function in circulating blood to ___________________ damaged blood cells or microorganisms found in the blood (septicemia).

55. Monocytes M
Notice the looser chromatin in the Monocyte compared to the Segmented Neutrophil S

56. Monocytes n M
M- Monocyte, BC= Band Cell, n = nRBC
Film is on 100x BC

57. Tissue Macrophages _____________ than monocytes in bloodstream
Are most prevalent in “__________” organs such as the _________, spleen, lungs, and ____________________________.
Some tissue macrophages are free and wander through tissue, while others become ___________ in specific tissues and remain there for the rest of their life span (e.g. ____________________ cells of liver).
Macrophages are often associated with _________________________ since they have a longer life span than neutrophils.
Collectively, the tissue macrophages and monocytes are known as the ____________________________ phagocyte system.
Specialized macrophages that stay in liver and help break down RBCs in order to form Bilirubin

58. The Mononuclear Phagocyte System
Functions:
Clean up __________ debris that remains after _________________ or infection is ______________
____________ processing cells. The MPS can ingest antigens and present them on their cell membranes to the lymphocytes, which will destroy them.
Have ability to form multinucleated __________cells in the tissue in response to foreign bodies (ex: granulomas).
Ingest foreign substances.
Capable of _________________and _________________. Can engulf structures beyond the phagocytic capacity of neutrophils such as fungi, protozoa, viruses, and dead neutrophils.
Are a major source of colony stimulating factors, _______________ that regulate inflammatory responses.

59. Fine needle aspirate of a granulomatous skin lesion
Fine needle aspirate of a granulomatous skin lesion. Neutrophils are adjacent to large macrophages that have abundant vacuolated cytoplasm and round to oval nuclei. Macrophages are derived from blood monocytes

60. Buffy coat film from a dog
Buffy coat film from a dog. A large macrophage contains phagocytozed Histoplasma organisms.
Histoplasma capsulatum is a fungus commonly found in bird and bat feces that can cause a potentailly fatal condition.

61. Monocytosis & Monocytopenia
Normal Monocyte Values: (dogs: 150-1,350/µL; cats: 0-850/µL)
____________________ is often associated with:
Chronic inflammatory conditions (particularly mycotic and other granulomatous infections)
________________________
Bacteriemia
Corticosteroid or ____________ responses (especially in dogs)
_________________________is occasionally seen but usually has no __________________ significance.

62. Lymphocytes
…To be continued.