VET 131

Leukocyte Cell Types and Functions

Required reading: 

 Hendrix, “Laboratory Procedures for Veterinary Technicians”, pages. 27-73

Voigt. “Hematology Techniques and Concepts for Veterinary Technicians”, Chapter 7, pages 55 – 66

Required website:

http://diaglab.vet.cornell.edu/clinpath/modules/


Objectives

 After completing this unit, you should be able to:

1. Describe the morphology and function of neutrophils, eosinophils, basophils, monocytes and lymphocytes. 
2. Describe the distribution of leukocytes in “pools” in the body.  
3. Describe species differences in the morphology of eosinophils. 
4. Relate changes in number or appearance of neutrophils, eosinophils, basophils, monocytes and lymphocytes to the health of an animal (i.e. neutrophilia, neutropenia, eosinophilia, eosinopenia, basophilia, basopenia, monocytosis, monocytopenia, lymphopenia, lymphocytosis. 
5. Define or describe the following:
a. Barr body
b. Dohle body
c. Toxic neutrophils
d. Left shift
e. Right shift
f. Reactive lymphocyte
g. Hypersegmentation

Sink and Feldman's  Laboratory Urinalysis and Hematology for the Small Animal Practitioner has excellent pictures of the cell types on the DVD included with the book.  Otherwise, please use the Cornell University website, or google the cell types to find pictures on the web to help with your identification.

Introduction

The leukocytes are responsible for the protecting the organism against foreign invaders.  This includes the mechanisms involved in identifying and removing bacteria and other foreign materials, and removing damaged or dead cells.  The leukocytes are extremely important in the inflammatory and immune responses of an animal. 

Animals have collections of leukocytes throughout their bodies.  These collections sites are called pools.  Leukocytes are produced in the bone marrow (proliferation pool) and stored in the bone marrow or spleen (storage pool) until needed.  When the leukocytes enter the blood many of them remain in the vicinity of the walls of the blood vessels (marginal pool).  The remaining leukocytes freely move in the blood and are available for specimen collection (circulating pool).  In most animals less than 50% of the leukocytes are available for blood collection.  Most leukocytes eventually leave the blood vessels and enter tissues (tissue pool) where they protect against foreign invaders. 

leukocyte pool.JPG (19309 bytes)

The life span of leukocytes varies depending upon the type of cell.  In general, it takes from 2 – 10 days for cells to develop in the bone marrow.  After being discharged into the blood stream the cells spend from six hours to a few days in the blood vessels before entering the tissues.  A majority of leukocytes live a few more days in the tissues, although one type can live for 20 years.   There is a constant movement of cells from the bone marrow to the circulating blood and then to the tissues.  The study of the number and kinds of leukocytes in circulating blood is a valuable diagnostic tool which helps a clinician determine the health of an animal.

There are five types of leukocytes - neutrophils, eosinophils, basophils, lymphocytes and monocytes.  Neutrophils, eosinophils and basophils contain cytoplasmic granules and are referred to as granuloctyes, whereas the lymphoctyes and monocytes do not contain cytoplasmic granules and are referred to as agranulocytes.  

In this unit you will study the structure and function of the five types of leukocytes

 

Neutrophils

Production

Neutrophils are produced from myeloblasts in the bone marrow in about 4-6 days.  A myelobast eventually differentiates into a neutrophil with a band nucleus that finally develops into a neutrophil with a segmented nucleus.   Healthy animals normally have a 5 day storage supply of mature neutrophils in the bone marrow.   Figure 13.1 on page 113 of Voigt illustrates the developmental stages of the neutrophil.

Once released into general circulation, neutrophils spend 6 to 7 hours in the blood vessels.  In dogs, approximately 50% of the neutrophils in blood vessels are in the circulating pool, whereas 25% of neutrophils in the blood vessels of cats are in the circulating pool.

After the neutrophils leave the blood vessels and migrate to tissues they survive another 1 to 4 days. 

Morphology 

Neutrophils are about twice the size of red blood cells.  The nuclear chromatin of neutrophils is dense, and stains dark purple.  The cytoplasm contains fine smooth granules and stains pale pink.  In normal animals most of the circulating neutrophils have a segmented nucleus with two to four lobes.  These cells are frequently called “segs.”   Very few of the circulating neutrophils (less than 5%) will be immature and will have a nucleus which is horseshoe shaped with large rounded ends.  These cells are frequently called “bands. As neutrophils age there is an increase in the number of lobes in the nucleus.  Hypersegmentation (five or more nuclear lobes) is evidence of increased aging of a cell.  

  neutrophil.JPG (42618 bytes)  "Seg"        Band.JPG (33695 bytes)   "Band"            Band and Seg.JPG (17864 bytes) "Band" and "Seg"

Barr bodies, tennis club shaped appendages from the nucleus, may be observed in female animals.  These appendages are inactive X chromosomes and their presence can be used to identify the gender of an animal from which the blood sample was obtained.  

        Barr body.JPG (30992 bytes)   Barr body on nucleus of neutrophil on the right

 Dohle bodies are small, irregular blue-gray granular cytoplasmic inclusions which may occasionally be seen in neutrophils.  They are the remains of rough endoplasmic reticulum and are more frequently seen in cats than in other animals.  Dohle bodies are more prevalent with severe inflammation or toxemia.  A Dohle body is illustrated on page 58 of Voigt.

Function

Neutrophils are the most common leukocyte in the blood of dogs, cats, horses and humans.  Their primary function is to phagocytose bacteria and subsequently kill them with the enzymes present in the neutrophil’s lysosomes. 

When a tissue is inflamed or invaded by bacteria, the injured cells release chemicals which increase blood flow to the area and increases capillary permeability causing more neutrophils to leave the marginal pool and enter the tissue pool.  Neutrophils migrate from the blood vessels to the tissue by squeezing between the cells lining the capillaries.  Once in the inflamed or infected area, the neutrophils, along with tissue macrophages, proceed to ingest the foreign invaders (bacteria, fungi, algae, viruses).  Most of the neutrophils “die for the cause”.  Pus is the accumulation of dead neutrophils and macrophages.

When neutrophils are exposed to bacteria or their products, they may secrete endogenous pyrogens which stimulates metabolic activity and results in fever. 

Neutrophils in Disease

An increase in the number of neutrophils in the circulating pool is called neutrophilia.  It is the most frequent cause of an increased leukocyte count (leukocytosis).  Neutrophilia may be induced by adrenaline, corticosteroids or as a result of an inflammatory response. 

Adrenaline release associated with fear, excitement, vigorous exercise or seizure activity causes an increased movement of neutrophils from the marginal pool to the circulating pool.   This effect lasts approximately one hour.  

Corticosteroids cause an increased movement of neutrophils from the storage pool to the circulating pool and a decrease in the movement of cells into the tissue pool.  This neutrophilia can occur from endogenous release of corticosteroids because of painful conditions resulting from injury, boarding, transport or other conditions.  Exogenous administration of corticosteroids results in neutrophilia within a few hours of administration, but the animal will return to normal within a few days.

Acute inflammation causes increased movement of neutrophils from the marginal pool to the tissue pool, a release of neutrophils from the storage pool to the circulating pool and a subsequent release of additional neutrophils from the proliferative pool into the circulating pool.  In attempting to keep up with the demand for neutrophils, immature cells may eventually find their way into circulation, resulting in an increased number of "baby" neutrophils or bands.  Neutrophilia may intensify after the abrupt removal of an abscess because there is no longer a tissue demand for neutrophils and the bone marrow has not slowed down its production of neutrophils. 

A decrease in the number of neutrophils in circulation is called neutropenia.  Neutropenia is the most frequent cause of leukopenia and can be caused by increased demand and usage, decreased production and increased margination. 

When there is an acute infection or inflammation tissue demand for neutrophils suddenly increases and neutrophils leave the storage pool to meet the tissue demands.  When usage exceeds production, neutropenia results with band cells becoming more abundant in the circulating pool.

If the bone marrow decreases production of neutrophils, neutropenia can result since there will be fewer cells in the proliferating pool.  Marrow production can be impacted by drugs (e.g. chemotherapeutic agents), toxic chemicals and plants, some infectious agents (e.g. Ehrlichia) and certain diseases (e.g. feline leukemia)

Anaphylactic shock or endotoxemia can cause a movement of neutrophils from the circulating pool to the marginal pool (margination).  The resulting neutropenia usually lasts a few hours.

Changes in the supply and demand of neutrophils in an animal lead to the presence of cells in the circulating pool that are in different stages of development and aging.  In a healthy animal, 95-100% of the neutrophils have normal segmented nuclei with an occasional band or hypersegmented cell being found.  An excess of immature cells in the circulatory pool is called a left shift, or immature neutrophilia, whereas an excess of older, hypersegmented cells in the circulatory pool is called a right shift, or mature neutrophilia. 

                lymph distr.JPG (21224 bytes)

When an animal has a severe inflammatory disease or toxemia, the neutrophils change and are called toxic neutrophils. Toxic changes are scored as mild, moderate or sever on a scale of 1+ to 3+.   Toxic neutrophils have the following characteristics:  

1. blue-grey cytoplasm
2. Dohle bodies in the cytoplasm
3.  “foamy” cytoplasm because of the presence of vacuoles
4. abnormal nuclear shapes
5. if severe toxemia – purple toxic granules may be found in the cytoplasm of the neutrophil

    

Hypersegmented nuclei are present when animals are keeping neutrophils in circulation longer than normal.  Hypersegmentation can be associated with chronic infections or with the presence of excess corticosteroids.  When blood samples are stored too long the prolonged exposure to EDTA may also result in hypersegmentation. 

     Hyperseg.JPG (13864 bytes)     Hypersegmented neutrophil

  

Eosinophils 

Production

Eosinophils are produced in 2-6 days in the bone marrow in a manner similar to the development of neutrophils. A myelobast eventually differentiates into an eosinophil with a band nucleus that finally develops into an eosinophil with a segmented nucleus.   Many mature eosinophils are stored in the bone marrow.  Figure 13.1 on page 113 of Voigt illustrates the developmental stages of the eosinophil.

Once released into general circulation, neutrophils spend 6 to 10 hours in the blood vessels.   After the eosinophils leave the blood vessels and migrate to tissues they survive for several days. 

Morphology

Eosinophils are about the same size as neutrophils.  The nucleus is lobulated or partially segmented with chromatin that stains dark purple.  The cytoplasm stains a light blue color and contains red granules.  The size, color intensity and shape of the granules vary with the species.   Feline eosinophils have small, rod-shaped granules, whereas the granules in canine eosinophils vary in size and have a less intense color.  Equine eosinophils have large orange to red granules and appear like a ball of grapes, whereas ruminant eosinophils have small, intense red to lilac granules.

        Eosinophil.JPG (13477 bytes)   Eosinophil             Eo and Neut.JPG (20985 bytes)     Eosinophil and Neutrophil

 

Function

Eosinophils appear to have a number of different functions.  They play an important role in immediate hypersensitivity responses of an organism.  Mast cells (tissue macrophages) produce histamines when they come in contact with allergens or parasite antigens.  The histamines subsequently attract eosinophils to the site. 

 Eosinophils have parasiticidal properties and kill parasites (worms and flukes) that have invaded the organism.  They also have some phagocytic and bactericidal properties similar to neutrophils.

Eosinophils in Disease

An increase in the number of circulating eosinophils is called eosinophilia.   Eosinophilia may be induced by allergies, parasite infestation and other inflammatory or neoplastic conditions. 

A decrease in the number of circulating eosinophils is called eosinopenia.  Since there are not many eosinophils in the circulatory pool of healthy animals, eosinopenia is a difficult condition to detect.  Glucocorticoids cause a decreased movement of eosinophils from the marrow to the circulating pool and an increase migration of cells to the tissue, resulting in eosinopenia.  Since glucocorticoids increase with stress, a stressed animal with a normal number of eosinophils may be experiencing eosinophilia. 

 

Basophils

 Production

Basophils are produced in the bone marrow in a manner similar to the development of neutrophils.  Within a few hours after being released into the circulatory pool, basophils migrate to tissues where they survive for a few weeks.  Mast cells (tissue macrophages) share a common ancestry with basophils.  Figure 13.1 on page 113 of Voigt illustrates the developmental stages of the basophil. 

Morphology

Basophils are about the same size as neutrophils.  The nucleus is lobulated or partially segmented and often appears coiled.  The cytoplasm stains a blue to grey color and contains granules that vary in color from lavender to black.  The color and quantity of the granules vary with the species.   Feline eosinophils have numerous lavender granules, whereas canine eosinophils have a few discrete dark staining granules.  

basophil.jpg (29609 bytes)     Basophil

 Function

Basophils are functionally similar to mast cells.  They are involved with hypersensitivity and inflammatory reactions.  When basophils are exposed to allergens, they release histamine and heparin.   The histamine is involved with hypersensitivity reactions and heparin inhibits coagulation.  After the basophils and mast cells release their granular contents (degranulation), they can resynthsize their granules and continue to function.

Basophils in Disease

Basophilic response to disease is similar to the response of eosinophils.  Basophilia may be induced by allergies, parasite infestation and other inflammatory or neoplastic conditions.    Since there are very few basophils in circulating blood it is difficult to determine if there is basopenia.  Glucocorticoids cause a decrease in the number of circulating basophils.

 

 

Monocytes

Production

Monocytes are produced in the bone marrow from monoblasts in 2-4 days.  They are immediately dumped into the circulatory pool where they spend 1-2 days before they migrate to the tissues and become macrophages. Monocytes are not stored in bone marrow. Figure 13.1 on page 113 of Voigt illustrates the developmental stages of the monocyte.

Morphology

Monocytes are the largest leukocytes and are usually about 25% larger than a neutrophil.  The nucleus can be round, oval or kidney-shaped with diffuse, lacy appearing chromatin.  The cytoplasm is abundant, has a foamy appearance and stains grey to blue-grey.  Vacuoles are frequently seen in the cytoplasm, especially in EDTA treated blood. 

mono.JPG (10011 bytes)       Monocyte

 Function

The major function of monocytes is phagocytosis.  They engulf foreign materials (bacteria, fungi, protozoans) which cannot be controlled by neutrophils.   Monocyte and tissue macrophage system work with the neutrophils in combating disease by phagocytosis and the inflammatory response.

The monocyte/macrophage population is frequently called the reticuo-endothelial system.  Examples of tissue macrophages are the histiocytes in connective tissue and the Kupffer cells in liver

 Monocytes in Disease

An increase in the number of circulating monocytes is called monocytosis.  This condition usually indicates that the animal has a chronic infection or is in the recovery phase of an acute infection.  When there is necrotic tissue and demand for its removal, monocyte numbers will increase.  A mild, stress induced monocytosis can occur, especially in dogs, because of the presence of corticosteroids.

 Monocytopenia, a decrease in the number of circulating monocytes, is not clinically significant.

 

 

Lymphocytes

Production

Lymphocytes are produced in the bone marrow, lymphoid organs (lymph nodes, spleen and thymus), and GALT (gut-associated lymphoid tissues - Peyer’s patches, tonsils and appendix.)  Lymphocytes live much longer than other leukocytes and are able to return to general circulation, by way of the lymphatic circulation, after spending time in the tissues.

Most lymphocytes are produced in the bone marrow and in the thymus of young animals (the thymus degenerates and becomes non functional in older animals).  After entering the circulatory pool, a majority of lymphocytes enter lymphoid tissues (lymph nodes, spleen, tonsils and GALT) where they continue to produce new cells.  It normally takes 2 -5 days for lymphocyte to be produced in the bone marrow, but antigenic stimulation can decrease production and maturation time to 6-8 hours.  Lymphocytes can live for over 20 years.

In healthy dogs and cats, about 30% of the lymphocytes originate from bone marrow (B lymphocytes) and 70% of the lymphocytes are derived from cells originating from the thymus gland (T lymphocytes.)  Figure 13.1 on page 113 of Voigt illustrates the developmental stages of the lymphocyte.

Morphology

Lymphocytes have a roundish or slightly indented nucleus with dark staining chromatin and cytoplasm which stains a pale blue.   Lymphocytes vary in size with the majority being mature, smaller cells that are 1.5 to 2 times the size of red blood cells.  Mature lymphocytes have a very thin rim of cytoplasm.  Medium sized and larger lymphoctyes have more cytoplasm and are more common in ruminants. 

 lymphocyte.JPG (9007 bytes)  Lymphocyte                             mono and lymph.JPG (22981 bytes)  Size comparison of lymphocyte on left and
                                                                                                             monocyte on right

Reactive lymphocytes, also called plasma cells, are large lymphocytes with an egg shaped nucleus tending to locate at one end of the cells.  They are about the same size as monocytes.  The cytoplasm of a reactive lymphocyte stains a deeper blue and the chromatin is clumped and may appear as spokes of a wheel.  These cells are produced in response to an antigenic stimulus. 

        plasma cell.JPG (15438 bytes)    Reactive lymphocyte

Lymphoblasts are immature lymphocytes.  They contain a light blue nucleolus which may be surrounded by a ring of chromatin.   These cells are not found in normal circulating blood and should be recorded if found in a differential smear.

Function

Lymphocytes are the primary cells involved in the immune responses of an animal.  The B lymphocytes produce antibodies (humoral response); whereas the T lymphocytes attach to target cells and kill them (cell mediated response.)  Once activated by an antigen, B and T lymphocytes become programmed to react to future antigen attacks, by producing B and T memory cells.  The B and T memory lymphocytes provide continued immunity for the animal.  There are some circulating lymphocytes that are not B or T cells.  These cells are called null lymphocytes and may be the natural killer (NK) cells involved in an animal’s nonspecific immune response. 

Lymphocytes in Disease

A reduction in the number of circulating lymphocytes is called lymphopenia.  This condition may occur in an animal with a high level of corticosteroids (stress, Cushing’s disease).  Chemotherapy and radiation treatments usually interfere with lymphocyte production and produce lymphopenia.  Since lymphocytes are critical for an immune response, an absolute, long term lymphopenia is life threatening.

An increase in the number of circulating lymphocytes is called lymphocytosis.   This condition may occur when there is an inflammatory condition associated with the introduction of an antigen.  Lymphosarcomas or leukemias usually produce an increase in the number of large lymphoblasts in the circulating blood.  Finally, in cats, an increase in adrenalin can cause a marked increase in the number of circulating lymphocytes.

 

Assignment: 

Perform a UA and CBC on two patients and report your findings on Lab Report 5.   Ask your mentor to check your results.

Answer the questions on Unit 4 - Leukocytes

Email your UA's, CBC's and answers to Dr. Durham