2523  HEMATOLOGY  I

                                    WBC:   Morphology and Physiology

This teaching syllabus discusses the physiology and morphology of the leukocytes appropriate for basic hematology. All objectives are in the cognitive domain unless otherwise noted. The student, at the end of the instructional period, is responsible for meeting these objectives by achieving a cumulative score of 70% or better on all problem sets, case studies, major exams, quizzes, and library assignments.

Objectives are listed in numerical order. The student, upon completion of the classroom component of this syllabus will be responsible to successfully:

01   BRIEFLY DESCRIBE THE CONCEPT OF LEUKOPOIESIS.

Leukopoiesis is the development of all types of white blood cells. Most leukocyte production occurs in the bone marrow along with the erythrocytes. The lymphocytes are ubiquitous. They are produced in the lymph nodes, spleen, thymus, and bone marrow.

02    LIST THE THREE TYPES OF GRANULOCYTES.

[1] neutrophils, [2] basophils, and [3] eosinophils.

03    EXPLAIN THE CONCEPT OF THE GRANULOCYTE POOL.

The “granulocyte pool” is a generic term that describes four storage sites (called compartments) containing elevated concentrations of neutrophils. The first compartment is the bone marrow pool. Three levels can be identified in this compartment: proliferation, maturation, and storage. The proliferation role is characterized by the presence of myeloblasts, promyelocytes, and myelocytes. The maturation level is evidence by metamyelocytes and bands. The actual storage pool consists of bands and neutrophils, which are ready to be moved immediately into circulation.

The second compartment is the peripheral blood pool. The predominate leukocyte is the segmented neutrophil, making up to 65% of the total number of WBC’s. Bands may be found, but will not exceed 3% -6% of the total count in normal conditions. The third compartment is the marginal pool and will contain about 50% of the total number of neutrophils in the body. These are those WBC’s that adhere to the blood vessel walls and those that migrate (diapedesis) into the tissues. The fourth pool is the tissue pool and consists only of those neutrophils found in the body tissues.

04   DISCUSS THE LINE OF PRECURSOR CELLS FOR THE GRANULOCYTE

The original or parent cell is the pleuripotential cell. This cell may be designated as the uncommitted stem cell and designated as Colony Forming Unit (CFU)- Spleen (S) cell = CFU-S.. It has also been called the pleuripotential stem cell (PSC). This cell cannot be identified via Wright’s stain. The next cell in the maturation sequence is the CFU-GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte) cell. It is relatively uncommitted stem cell and can undergo mitosis to form additional stem cells. It will differentiate into the CFU-GM (granulocyte, monocyte) line which is committed to form either neutrophils or monocytes, when stimulated with interleukin-3 and the GM(CSF) cytokine. The CFU-GM line, when stimulated by G(CSF), GM(CSF), and IL-3 will cause the maturation into the precursor (CFU-G) for the neutrophil line of WBC’s. The myeloblast will rise from the CFU-G cell. The CFU-M cell differentiates from the CFU-GM cell when stimulated with IL-3, GM(CSF) and M(CSF). Further stimulation with IL-3, GM(CSF), and M(CSF) produces the monoblast. The CFU-GEMM cell gives rise to the CFU -MEG when stimulated with IL-3 and GM(CSF). Additional stimulation with IL-3, GM(CSF) and thrombopoietin gives rise to the megakaryoblast.

05   DISCUSS THE MYELOBLAST.

This begins a sequence of developmental events that produces the neutrophil. (NOTE: It will not be unusual to see this series prefixed with the term granulocytic.) The myeloblast ranges from 15 to 20 μM in diameter. The cytoplasm is characterized by [1] small in volume, [2] smooth and a non-granular appearance, and [3] stains a moderate blue color in Wright’s stain. The nucleus (with its N/C ratio of 4/1) occupies about 75% of the cell. It is round to slightly oval in shape. It stains a reddish purple color in Wright’s stain and has a very fine chromatin pattern. The nucleus will contain 1 to 5 nucleoli and fewer is the rule. This cell make up less than 1% of the bone marrow population.
           
06    DISCUSS THE PROMYELOCYTE.

This cell is slightly larger than the myeloblast. Its size ranges from 15 to 21 μM in diameter. The cytoplasm mass is increased over than of the myeloblast and is characterized by few to many granules, thus appear larger than the myeloblast. These granules are large and will stain from blue to reddish-purple. They are designated as non-specific, primary, or azurophilic granules which contain a myeloperoxidase enzyme. Other substances found in these primary granules are [1] acid phosphatase, [2] acid hydrolases, [3] lysozyme, [4] sulfated mucopolysaccharides, and [5] other basic proteins. The production of these granules is confined to this stage, but will dilute out as the cell undergoes mitosis and transforms to the myelocyte stage. The dilution of these granules results in their being less obvious with the Wright’s stain, but may be identified by a peroxidase stain. The cytoplasm stains from pale blue to basophilic dependent upon the amount of cytoplasmic RNA present.. The nucleus is round to oval in shape and occupies over one-half of the cell. The N/C ratio ranges from 3/1 to 4/1. The nuclear chromatin pattern continues to be fine, but is coarser than the myeloblast. It is the usual rule to observe 2 to 3 nucleoli, but they will be “fading” in this stage. The promyelocyte will make up about 2 to 5% of the bone marrow population.
               
07    DISCUSS THE MYELOCYTE.

The size of the myelocyte ranges from 12 to 18 μM in diameter. The cytoplasm volume is designated as a moderate amount with N/C ratio of 1:1. The cytoplasm is no longer producing primary granules, although there are a few large granules present. It has started the production of secondary granules, which first appear around the nucleus in the region of the Golgi body to form a slight arc. These granules contains enzymes (acid hydroxylases, lysozyme, etc.). The cytoplasm stains less intensely, but patches of blue may be observed (usually around the edges of the cytoplasm) where RNA is still present. The eccentric or centric nucleus will be oval to round, contain a denser and more coarse chromatin pattern. No nucleoli are usually observed. Caution. The nucleus may be indented and resemble a metamyelocyte. The nucleus to cytoplasm ratio is about equal. This cell will make up about 10% to 20% of the bone marrow population. It is non-functional, but it does appear to have the necessary constituents for phagocytosis based upon the enzyme content of the granules. This is the last stage in which this cell line can undergo mitosis.
              
08    DISCUSS THE METAMYELOCYTE.

Active DNA synthesis has ceased and the cell has shrunk to a diameter of 10 to 15 μM. The cytoplasm is described as being moderate to abundant, having a few primary granules present. The fine secondary granules are evenly distributed in the cytoplasm and will stain a pink to pinkish purple color with Wright’s stain. The granules contains enzymes, but the cell is not fully functional at this stage. The cytoplasm has lost all traces of blue color and may contain a few light blue-pink areas. The nucleus is indented or kidney shape with its chromatin being coarser and more clumped. The nucleus takes on a dark blue-purple color. The nuclear indentation will be less than ½ the diameter of the nucleus and may resemble Dutchman’s breeches. The cell will make up about 15 to 30% of the total bone marrow population and it will take from six to fourteen days to mature to the neutrophil stage.
          
09     DISCUSS THE BAND.

The band, also known as a “stab” is similar in size to the metamyelocyte having a similar cytoplasmic characteristics. Look for fine lilac-colored granules with no blue-pink areas. Pinkish coloration should be rule. The nucleus has undergone additional indentation to take on a long and narrow “C” or “S” shape appearance. The nuclear material is a coarse clumped pattern. The name of the cell takes it name from the “band” shape of the nucleus. The concentration of bands in peripheral blood will range to a high of 18%. Most normal values sets the upper limits at 6%. In the bone marrow, the band may make up 40% of the total cells. The band has some phagocytic ability. To identify use the following guidelines: [1] No distinct filaments. [2] There is chromatin material present in the bridge between the lobes. [3] The nucleus is so twisted that filaments cannot be see, but the nucleus continues to retains a “C” or “S” shape characteristic.
           
10     DISCUSS THE NEUTROPHIL.

Known as a segmented neutrophil or polymorphonuclear leukocyte, synonyms have been developed that include: seg, poly, and PMN. The diameter is similar to that of the “band” and is reported to be from 9 to 15 μM. The cytoplasm is abundant, characterized by fine pinkish to rose-violet granules. A few primary granules may be present. The nucleus is lobed, each lobe connected by a thin filament. The number of filaments normally range from two to five. (Note: Textbooks state that if 40% or more of the “segs” have 5 lobes, the cells should be designated as hypersegmented. Realistically, labs will set this value lower.) The lobes are designated as being pyknotic, having very dense, clumped chromatin. These cells make up about 60% of the peripheral population of WBC’s. About one-half of these cells are in the peripheral pool and the other half are in the marginated pool where they are freely, continuously, and rapidly exchanged. It is estimated that “segs” remain in peripheral circulation for about 7 hours, then they pass into the tissues. They may return to peripheral circulation. The life span of a neutrophil is estimated to average about 5 days. The body contains up to a 10 day supply of these WBC’s. There is little information about the neutrophil’s function after they migrate into the tissues.

These cells are fully phagocytic. If “large stuff” can be demonstrated in the light microscope, the phagocytic process is termed phagocytosis. Small stuff cannot be demonstrated in the light microscope and this is denoted as pinocytosis. The neutrophil will migrate to the infection by a process called chemotaxis. Chemotaxic factors include antibodies and complement fixation products.
       
11    BRIEFLY DISCUSS NECROBIOSIS.

This is a disintegrating WBC, generally considered to be a granulocyte. It can occur in any WBC at any stage of its development. It appears when the nucleus coalesces to form a single globule or several small globules. These liquified nucleus material is also called drops or droplets. If the disintegrating cell continues to contain the liquified basophilic chromatin, this cell is called necrobiotic cell. If the basophilic chromatin absent, it is more properly referred to as necrotic cell. Once necrobiosis has occurred to a certain stage, the identifying features of the cell will disappear and the cell cannot be identified according to its cell type. The cell undergoing necrobiosis may break apart to form remnants. See following illustration.
               
Example of necrobiosis in the promonocyte:
[1]   In the initial stages the cell size remains the same.
[2]   The cytoplasm will become basophilic
[3]   The nucleus becomes structureless and markedly basophilic.
[4]   As necrobiosis progresses, areas of light and moderate basophilia
         become evident

Note the following examples of necrobiosis.
                
12    LIST THE FIVE STEPS FOR PHAGOCYTOSIS IN THE NEUTROPHIL.

[1] MOTILITY. This occurs under the guidance of chemoattractants. The neutrophil takes on a flattened and ruffled appearance characterized by pseudopods. This leukocyte will move with repetitive wavelike motions until they reach the site of infection. The neutrophil can attach to endothelial receptors and migrate through the junctions between cells by a process called diapedesis.
[2] RECOGNITION. Neutrophils recognizes pathogens by the coating of immunoglobulins and activated compliment components coating the surface of the pathogen. Such “flagged” pathogens are referred to as opsonins and plasma membrane receptors of the neutrophil can attach to these opsonized surfaces and ingest them.
[3] INGESTION. This is part of the phagocytosis process and takes place as the plasma membrane of the neutrophil surrounds the pathogen and engulfs to form an isolated vacuole within the cytoplasm which is now called a phagosome.
[4] DEGRANULATION. The cytoplasmic granules that attach to the phagosome will rupture and empty their contents into the vacuole . This process is called degranulation.
[5] KILLING. This takes place as the enzymes initiate the formation of superoxides and hydrogen peroxide to create a respiratory burst that is seriously injurious to the pathogen. Other enzymes (myeloperoxidases) promote formation of other toxic agents against the pathogen to cause their death.

13    DEFINE HYPERSEGMENTATION IN THE GRANULOCYTES AND EXPLAIN IT SIGNIFICANCE.

Hypersegmentation indicates changes of abnormal leukopoiesis in granulocytes. This is the presence of increased nuclear lobes in neutrophils, eosinophils, or basophils. For the neutrophil, the appearance of cells with five or more lobes indicates abnormal DNA synthesis which may indicate megaloblastic anemia due to a vitamin B12 and/or folic deficiency. It has been associated with a benign hereditary hypersegmentation disorder. Patients under rhG-CSF (recombinant human granulocyte colony stimulating factor) therapy have demonstrated hypersegmentation. NOTE: There is disagreement as to what constitutes hyper-segmentation. Textbooks typically describe hypersegmentation as the presence of six lobes. In the “real world” it is not unusual for pathologists to consider the presence of 5-lobed neutrophils as being hypersegmented. Hypersegmentation does appear in patients with long term chronic infections. In aged neutrophils, as nuclear deterioration takes place, there may be a temporary pseudohypersegmentation phenomenon. Generally hypersegmentation is considered to be an indicator of megaloblastic anemia.

Eosinophils normally characterized by a two lobed nucleus. Eosinophilic hypersegmentation occurs when three or more lobes are present. Up to five lobes have been reported. The causes of this hypersegmentation is probably due to a vitamin B12 and/or folic deficiency.

Basophil hypersegmentation is similar to that describe for eosinophils. The causes are probably similar. There is little information in the textbooks about basophil hypersegmentation.

14    REVIEW TOXIC GRANULATION.

Toxic granules are found in the cytoplasm of neutrophils and must be included in the differential report. It represents a transient change in the cytoplasmic morphology of the neutrophils due to infectious or toxic agents. These are medium to large size dark blue-black granules formed from primary granules which suggest a nonspecific reactive change. Primary granules, in the neutrophils of normal healthy individuals, are not visible with Wright’s stain. Toxic granules tend to cluster in the cytoplasm and not all neutrophils will be affected. If there is a greater number of neutrophils presenting with toxic granulation, this may indicate a more serious prognosis. Toxic granules are graded on a scale of 1+ to 4+. If the neutrophil is demonstrating evenly distributed larger dark granules and all cells seem to be affected, then this is more likely a staining artifact or the patient may have Alder-Reilly anomaly. The true toxic granule is composed of peroxidases and acid hydrolases. Toxic granulation is seen in bacterial infections, toxemia of pregnancy, vasculitis, burns, malignancy, or chemotherapy.
            

15   EXPLAIN THE SIGNIFICANCE OF VACUOLATION IN NEUTROPHILS.

Vacuolation in the neutrophil may be a better indicator of a bacterial infection than toxic granulation. Also called toxic vacuolation, these are clear, unstained, round areas in the cytoplasm. They are usually most obvious in severe infections.

16      DEFINE NEUTROPHILIA.

Neutrophilia is any increase in the numbers of neutrophils (usually >7000/μL). It may be due to inflammation, certain medications, extreme exercise, stress, burns, acute or chronic infections, malignant, or nonmalignant conditions.

17   DISCUSS NEUTROPENIA.

Neutropenia is the reduction in the number of neutrophils occurs when the absolute count drops below 1000/μL. If the count drops below 500/μL, then there is a serious risk of severe infections. Suppression of the neutrophil count may be due to some medications (penicillin, ibuprofen, phenytoin, and chlorpropamide), severe infections that cause the outflow of neutrophils into the tissue spaces to exceed the body’s ability to replace them), or increased loss of WBC’s (example: as seen in a splenectomy).

Neutropenia may be observed in young children as a transient disorder due to viral causes (influenza A and B, rubella, rubeola, hepatitis A, hepatitis B, and respiratory syncytial virus. It can be an acquired secondary disorder associated with aplastic anemia, bone marrow malignancy, or vitamin B12 and/or folic acid deficiency.

18     EXPLAIN HYPOSEGMENTATION.

Hyposegmentation is a condition in which the nucleus contains only two lobes or no lobes at all. This condition is characteristic of Pelger-Huët anomaly, a benign autosomal dominant (homozygous) or a heterozygous disorder. The heterozygous condition is characterized by neutrophils whose nuclear shape appears as a dumbbell or pair of eyeglasses (hence the term ‘pince-nez’). The chromatin clumping and cytoplasmic characteristics are similar to that in the mature normal lobed neutrophil. If the neutrophil, with the hyposegmented nucleus in an infectious state, the chromatin material appears coarser and more heavily clumped. In the homozygous state, the nucleus does not form lobes but appears as a oval or round nucleus. There is a pseudo-Pelger-Huët condition in which the hyposegmented neutrophil appears bilobed. This is an acquired condition in which trilobed neutrophils are present. This acquired condition occurs due to drug ingestion or a malignancy. A word of caution is in order, do not confuse Pelger-Huët cells with bands or metamyelocytes as seen in ‘shifts-to-the-left’ in patients with an infection. Remember that the chromatin in the Pelger-Huët cell is more condensed and coarser than the band or metamyelocyte.

19    DESCRIBE THE DÖHLE BODY.

The Döhle body is a cytoplasmic inclusion (from 1 to 5 μM diameter) composed of ribosomal RNA (aggregates of rough endoplasmic reticulum) arranged in parallel rows found in neutrophils and bands. These inclusions stain readily with Wright’s stain. In a stained blood film prepared from a non-anticoagulant blood sample, the inclusion tends to stain with a bluer coloration. If the smear is made from an EDTA preserved blood sample, the inclusion takes on a more gray color. The Döhle body tends to be found in close proximity to the cell membrane. These inclusions are sen in pregnancy, bacterial infections, burns, cancer, aplastic anemia, and toxic conditions. If toxic granulation is present then look for Döhle bodies. These inclusions may be found in eosinophils. They are similar in morphological appearance to the neutrophil inclusions found in May-Hegglin anomaly.  Note the Pelger-Huet cell on the right from a heterozygous patient.
         

20    DEFINE THE FOLLOWING GLOSSARY TERMS.

A.   Endocytosis: A generic term to describe phagocytosis and pinocytosis.
B.   Respiratory burst: A process in phagocytosis in which the enzyme “myeloperoxidase” is released from secondary granules and combines with peroxidase to form a peroxide halide which can kill bacteria, viruses, and fungi.
C.  Degranulation: After the WBC ingests the foreign particle, the particle is enclosed in a vacuole to be digested/phagocytized. Specific neutrophil granules attach to the vacuole (now called a phagosome) and empty their contents (enzymes) into the phagosome. This process is called degranulation.
D.   Chemotaxic factors: Chemical substances (includes: secretions from lymphocytes and monocytes, endotoxins from bacteria, activated compliment, metabolites from damaged cells) that can attract WBC’s into the infectious/damaged site.
E.   Chemokinesis: Movement of WBC’s activated by chemical stimuli.
F.   Chemotaxis: Directional movement of WBC’s to the site of infection by chemical stimulus.
G.   Locomotion: Random and nondirectional movement by the leukocyte.
H.   KL: A new stimulating factor system that acts on early progenitors of WBC’s.
I.   Undifferentiated: Non-specialized structures, embryonic, not developed, primitive.

21  BRIEFLY DISCUSS THE DEVELOPMENT OF THE EOSINOPHIL.

The eosinophil arises from it own committed stem cell. At the CFU-GEMM stage, under the appropriate cytokines, the GEMM stage will differentiate to the CFU-EO and from this stage it is stimulated by GM(CSF), IL-3 and IL-5. It will then maturate to the eosinophilic myeloblast. The maturation sequence is very similar to that for the neutrophil. The stages of development to the promyelocyte stage are visually indistinguishable from that for the neutrophil.

22    DISCUSS THE MYELOCYTE-EO.

The diameter of this stage is from 12 to 18 μM as is the neutrophilic myelocyte. It is considered to be visually distinguishable from the basophilic or neutrophilic myelocyte. Small specific secondary granules are beginning to appear. Larger granules are beginning to appear that stain a “dirty” orange to blue color.
             

23    DISCUSS THE METAMYELOCYTE-EO.

This developmental stage is similar to that of the neutrophilic metamyelocyte, but more gradual. The specific granules are more numerous and larger with the granular color becoming more red-orange. It takes about six days for the developing EO to reach this stage.
            

24   DISCUSS THE BAND-EO.

This development stage is about the same as for the neutrophilic band. This stage is rarely seen in peripheral circulation. This cell type makes up about 1% of the bone marrow cell population.
           
25   DISCUSS THE MATURE EOSINOPHIL.

This cell is slightly larger than the neutrophil, its diameter measuring from 10 to 17 μM. The cytoplasm contains many large bright red-orange granules. The nucleus is pyknotic, with one to three lobes. The most commonly appearing nucleus is the bi-lobed form. For every eosinophil seen in the bone marrow, an estimated 300 are circulating in peripheral circulation and another 200 are in the tissue spaces. The eosinophil makes up about 3% of the total WBC count. In the tissues, the EO tends to accumulate in the intestines and lungs. Eosinophilic facts include:
[1] Defense against parasitic infections. The granules contain “Major Basic Protein” (MBP), toxic to parasites and host tissues. A metabolite of this protein is a constituent of the Charcot-Leyden crystals.
[2] Eosinophil granules also contain acid hydrolases, histaminase, aryl-sulphatase B (inactivates the slow-reacting substance of basophils and mast cells), phospholipase D (inactivates platelet activating factors).
[3] EO cell density in peripheral blood is the highest at night (a.m. hours)
[4] Steroids tend to block eosinophil release from bone marrow.
[5] Eosinophils can neutralize heparin from mast cells.
              
The eosinophil will spend up to eight hours (some textbooks cite 7 days) in the peripheral blood before migrating to the tissues where they will reside for several weeks. To obtain the highest concentrations of eosinophils in the blood, collect the specimen during the morning hours. Afternoon collections produce the lowest eosinophil counts.

26   BRIEFLY DISCUSS THE DEVELOPMENT OF THE BASOPHIL.

The basophil development parallels that of the eosinophil. Development stages are indistinguishable from the CFU-GEMM cell type to the basophil promyelocyte. Under the proper cytokine stimulus [GM(CSF) and IL-3] the GEMM cell differentiates to the CFU-BASO cell. The cell is recognizable as a undifferentiated blast at this stage.
       
27    DISCUSS THE MYELOCYTE-BASOPHIL.

This is the first visually distinguishable stage in the development of this cell type. All cell features are the same except that the specific granules appear larger and take on a dark blue stain.
       

28    DISCUSS THE METAMYELOCYTE-BASOPHIL.

This developmental stage is characterized by the appearance of more numerous and larger specific granules that stain a more intense dark blue color. All other features are similar to that of the neutrophilic and eosinophilic metamyelocyte.
      

29      DISCUSS THE BASOPHIL BAND.

There is no difference in development and appearance from that of the neutrophilic and eosinophilic band. The cytoplasm stains a pink color or it may be colorless. The large specific granules appear black. There are fewer granules in the basophil than in the eosinophil.
      

30     DISCUSS THE BASOPHIL.

The following are the essential differences compared to the neutrophil. [1] the chromatin is less coarse, [2] nucleus may be unsegmented or bilobed (3 to 4 lobes are rarely observed), and [3] dark granules are water soluble and if over washed or rinsed, the granules may decolorize.

The basophil granules contain [1] heparin, [2] chondroitin sulfate, [3] proteoglycans, [4] histamine, [5] serotonin, and [6] peroxidase. The basophil is the least common of the leukocytes, making up from 0.5% to 1.0% of the total peripheral blood WBC’s. It forms up to 0.3% of the cells in bone marrow. It is not unusual to NOT see a basophil in the average differential. Basophils, once released into blood circulation, will circulate about 10 hours and then migrate into the tissues.
        
31   DISCUSS THE RELATIONSHIP OF THE BASOPHIL TO THE MAST CELL.

Basophils are thought by some hematologists to be related to mast cells, even though mast cells are larger, have a more rounded nucleus, and abundant cytoplasm. Mast cells granules are more numerous, closer packed, and smaller. The relationship of the basophil to the mast cells is controversial. There is a consensus that states that the mast cell descends from it stem cell, independent of the basophil. The mast cell is characterized by a cytoplasm that appears packed with granules which tend to overlay the nucleus, obscuring it. The mast cell nucleus does not segment nor is it consistent in its shape. It has displayed spindle shapes and presented with ragged cytoplasmic margins. It is not a constituent of peripheral blood but is seen in connective tissue, bone marrow, and the mucosal areas of the serous membranes. The chemical make-up of the granules is the same as for the basophils.
         
32     DISCUSS THE DEVELOPMENT OF THE MONOCYTE.

The monocyte rises from the CFU-C stem cell. This cell differentiates into the CFU-GEMM line of cells. Stimulation with the cytokines (IL-3, KL, and GM-CSF) causes differentiation into the CFU-GM line. Differentiation to form the monoblast requires stimulation with the IL-3, CSF-GM, and CSF-M cytokines.

33   DISCUSS THE MONOBLAST.

The diameter of the monoblast ranges form 12 to 20 μM. The cytoplasm is nongranular and has moderate basophilic to blue-gray coloration. The nucleus is ovoid, round, or irregular with a light, purplish-blue color. The nucleus may be centric or eccentric. The chromatin has a fine and lacy appearance. A single, large nucleolus is the rule and two nucleoli are not uncommon. The N/C ratio ranges from 3/1 to 4/1. The monoblast can be differentiated at this stage, but it may be extremely difficult. CAUTION: The monoblast can be confused with the myeloblast and to differentiate, take note of the following points:
[1] The monoblast nucleus tends to be more irregular in shape.
[2] The myeloblast is characterized by 2 to 5 nucleoli.
[3] The chromatin pattern of the myeloblast is more fine than the monoblast
[4] Examine the area. If there is an increase of monocytes, this will help to facilitate the identification of the monoblast.
[5] Look for indenting and folding of nucleus.
[6] Mature monocytes with blunt pseudopods, vacuoles, and phagocytized particles in the cytoplasm.
[7] Absence of granules in the cytoplasm.

This cell stains positive with α-naphthyl butyrate esterase, α-naphthyl acetate esterase, or chloroacetate esterase stains.
        
34    DISCUSS THE PROMONOCYTE.

The promonocyte (also called an immature monocyte) has a diameter of 14 to 18 μM (some authorities quote diameters of up to 22 μM). The cytoplasm is more abundant and continues to be nongranular, but has a more ground glass appearance. Look for fine, dust-like red particles (these are some times absent). Fine azurophilic granules are present.  Azurophilic implies a reddish-bluish coloration.

The nucleus tends to be ovoid in shape, but folded, twisted, indented, convoluted, and irregular shapes may be observed. Indentations will be deeper. More nucleoli may be observed in this stage than in the blast stage, with up to five being observed. The chromatin pattern is more condensed, but still retains it fine and lacy features. Look for a prominent central crease in the nucleus. This is called the “peaked cap” appearance and is a distinguishing characteristic. Monocytes remain in this stage for up to 60 hours.
   
35    DISCUSS THE MONOCYTE.

The monocyte with a diameter of 14 to 20 μM is the largest of the circulating leukocytes. The mature form is about the same size as the immature form. Some sources reports diameters up to 30 μM. The cytoplasm is abundant with a blue-gray color and tends to be more equally proportioned about the nucleus. The cytoplasmic outline is irregular and may form blunt pseudopods. Fine dust-like red or azurophilic granules are present and evenly distributed throughout the cytoplasm. The cytoplasm continues it ground glass appearance. Vacuoles may be present and are numerous in phagocytic active cells. The nucleus to cytoplasm ratio ranges from 2:1 to 1:1. The nucleus is characterized by [1] increased folding, [2] tendency to a more narrow, elongated, horseshoe shape. Also the nucleus is unusual in that it appears in a variety of shapes, from round to kidney, lobulated, or convoluted in shape. The chromatin is denser, blue-purple color, and with a fine reticular pattern, with skein like strands are still present. Nucleoli are present in 50% of the nuclei, but remain invisible. The following are examples of the variations in the mature monocyte nucleus.
         
The mature monocyte is present in peripheral circulation for up to 70 hours. Some reference sources suggest up to 100 hours and then it enters the tissues. The monocyte is a transition stage to the macrophage. Note the following facts about the monocyte:
[1] It takes about 55 hours for a monocyte to differentiate to its mature form.
[2] The marginal pool contains 3.5 times more monocytes that the peripheral blood.
[3] It is a highly mobile and phagocytic cell.
[4] They give a positive reaction with the esterase stains.
[5] Mature monocytes contain a variety of enzymes: acid phosphatase, β-glucuronidase, lysozyme, lipase, and peroxidase are examples.
[6] Monocytes may make up to 9% of the peripheral blood leukocytes. References cite maximum values of 6%, 7%, and 8%.
[7] They secrete such chemicals as: interleukin 1, interferon, plasminogen activator, plasmin inhibitor, platelet activation factor, and a tissue thromboplastin-like procoagulant.
[8] They phagocytize hemoglobin, old and degenerating cells, cellular debris, activated clotting factors, denatured proteins, antigen-antibody complexes, and kill several types of tumor cells.
[9] Some researchers refer to the monocyte as an immature macrophage.
[10] When contrasting the mature monocyte with the immature forms, there is an increase in the number of nuclear folds, no visible nucleoli, and increased numbers of reddish dust-like particles in the cytoplasm.

      

36     DISCUSS THE MACROPHAGE.

The macrophage is a large cell with a diameter ranging up to 80 μM, however most such cells fall in the range of 30 μM. It is characterized by abundant cytoplasm that appears “sky” blue to gray in color and may contain azurophilic granules that vary in size and number.. Vacuoles are common. Phagosomes may be observed with ingested matter. Note the spreading outline of the macrophage on the slide on a stained blood film. It will be irregular with pseudopods. The nucleus is usually eccentric and may be indented, oval, or elongated. The chromatin material appear spongy and will take on lilac to reddish purple color with Wright’s stain. One or more nucleoli may be seen. Macrophages tend to be found on the edge of the blood film due their large size.

Macrophages form an effective defense system against bacterial organisms, fungi, viruses, and tumor cells. Their functions include:
a.   removing damaged and old blood cells, plasma proteins, and plasma lipids.
b.   iron metabolism. Storage iron may be found in the macrophage in anemia of chronic disease, blood transfusions, and sideroblastic anemia.
c.  processing antigen information for lymphocytes. The macrophage will ingest the antigen, modify it, and present it to the lymphocyte. They will also secrete interleukin -1 (an lymphocyte activating factor).
d.   synthesize and secrete lysosomal enzymes, colony stimulating factors, erythropoietic factor, cytokines that differentiate T-type and B-type lymphocytes, prostaglandins that inhibit lymphocytes, platelet activating factor, and complement factors.

37   DISCUSS THE MEGAKARYOBLAST.

This cell constitutes the largest of the hematopoietic cells within the bone marrow being derived from the CFU-S pleuripotential stem cell. It line of development includes the CFU-C, then to the CFU-GEMM. The GEMM, stimulated by GM-CSF (also KL, IL-3, and IL-11) will develop into the CFU-Meg cell. This cell then proliferates and differentiates into the megakaryoblast.

The megakaryoblast is also known as the MK1 or Levine stage I cell. It diameter ranges form 20 to 50 μM. The cytoplasm is basophilic with varying shades of blue. Its cytoplasm has been described as resembling that of the lymphocyte. The cytoplasm is usually seen as a thin round band surrounding the nucleus and is non-granular in appearance. The nuclear to cytoplasm (N/C) ratio is about 10:1. The nucleus can be round, oval, or kidney shaped. It has a fine chromatin pattern. The nucleus is characterized by multiple nucleoli (up to six). The megakaryoblast has been confused with the myeloblast.

At the megakaryoblast stage, the cell will begin to undergo a process of endomitosis (also called endo-reduplication) in which the nucleus does not divide but doubles itself. Endomitosis bypasses the telophase stage allowing the nucleus to reduplicate itself without forming a duplicate cell. If the cell undergoes a single endomitosis, the nucleus goes from a 2n to a 4n state (the chromosome number has doubled) and the nucleus contains 2 lobes. It ploidy value is designated as four, which means that the nucleus contains 4 single sets of chromosomes. If the cell undergoes a second or double endomitosis, the nucleus goes to the 8n stage and is characterized by four lobes (implying that there is one lobe for each set of chromosomes). In a third or triple endomitosis, the nucleus goes to the 16n stage and eight nuclear lobes can be counted. This is usually as far as endomitosis is carried out, however the literature reports that a 64n stage has been observed. As the megakaryoblast maturates and undergoes endomitosis, these stages are referred to as polyploidy. Endomitosis ceases when the megakaryocyte is formed. The endomitosis event is why the megakaryocyte can attain a diameter up to 120 μM.

       

38 DISCUSS THE PROMEGAKARYOCYTE.

The megakaryocyte ranges in size up to 60 μM, however there are reports of diameters up to 80 μM. The cytoplasm increases over the megakaryoblast stage with it color becoming less basophilic. This lighter coloration is referred to as being orthochromic. Granules begin to appear in the Golgi body region and azurophilic granules may be present. The nucleus is characterized by a coarser chromatin and usually two nucleoli are present, however the nucleoli number may vary. The nucleus is typically lobulated, but shape irregularities may be present. The nuclear to cytoplasm ratio ranges form 4:1 to 7:1 dependent upon the amount of polyploidy (number of endomitotic divisions). This stage of megakaryocyte development is also known as the Levine stage II or MK2. This stage is characterized by increasing nuclear and cytoplasmic volumes. This will continue to occur until the MK4 stage.

Glycoproteins begin to form on the cytoplasm membrane surface and will eventually constitute the granules of the platelets. As the promegakaryocyte matures into its subsequent stages, the granules, as they form in the cytoplasm will disperse and collect in small aggregates of 10 to 12 granules that are surrounded by a clear area of cytoplasm called the hyalomere.

   

39   DISCUSS THE GRANULAR MEGAKARYOCYTE.

The granular megakaryocyte (MK3 or Levine stage III) presents with diameters up to 90 μM. The cytoplasm is more abundant and stains a pinkish-blue color with Wright’s stain. The peripheral border of the cytoplasm is characterized by an irregular appearance. If this cell is stained with the Periodic Acid-Schiff stain, glycogen stores will be demonstrated in the cytoplasm. The nucleus will be characterized additional lobes (due to further polyploidy). Nucleoli are present but are not usually visible. The chromatin is more coarse due to additional condensing. This stage is included to maintain congruency with the MK or Levine nomenclature. Most texts only distinguish the megakaryocyte series as [1] megakaryoblast, [2] promegakaryocyte, and [3] mature megakaryocyte.

40   DISCUSS THE MATURE MEGAKARYOCYTE.

This is the platelet forming stage. Synonyms are: MK4, Levine state IV, and platelet-forming megakaryocyte. The diameter of this cell type ranges up to 120 μM. It contain numerous coarse clumps of granules. Each of these clumps will contain a “packet” of glycogen. The clumps are predestined to fragment from other clumps to form the platelets (thrombocytes). The glycogen packet is capable of supporting the platelet for up to 12 days. The nuclear to cytoplasm ratio is <1:1. The nuclear appearance multi-lobulated with no evidence of nucleoli and a coarser chromatin than the previous stage. The cytoplasm of each mature megakaryocyte will fragment, producing from 2,000 to 4,000 platelets. This fragmentation process will occur over a period up to 12 hours.
  
41   DISCUSS THE THROMBOCYTE.

The thrombocyte or platelet will measure from 1.0 to 4.0 μM in diameter. The cytoplasm will stain with a light blue to purple color and contain a centromere. The centromere consists of centrally located granular material. The non-granular area of the platelet is colorless to light blue and is known as the hyalomere.

In the fragmentation of the megakaryocyte (MK4) cytoplasm, the cytoplasmic membrane tubules will fuse to form fissures that spreads across the membrane and into the interior. These are called the “Demarcation Membrane System”. This will ultimately form margins and the plasma membrane of the platelets.

The mature megakaryocyte lies next to the sinus walls of the bone marrow and as fragmentation occurs, the platelets will enter circulation through fenestrations of the sinus walls. The platelets are initially captured by the spleen and held for 2 to 4 days, after which they are released. The spleen forms the splenic pool that contains about 1/3 of the platelets in peripheral circulation.
                   

42   DISCUSS THE LYMPHOBLAST.

Maturation begins with the CFU-S stem cell. The maturation events take place in the bone marrow and lymph system (spleen, thymus, and lymph nodes). If the maturation begins in the bone marrow, the lymphocyte is destined to be the B-type. If the cell maturates in the thymus, it is a T-type.

                    THE THYMUS GLAND
The thymus, at birth is a well-developed lymphatic organ located in the mediastinum and in the vicinity of the great vessels of the heart. Like the adrenal gland, it is divided into a cortex and medulla. The vascular network of the thymus consists of capillaries. Both regions of the thymus contains the same types of cells: lymphocytes, reticular cells, mesenchymal cells, and macrophages. The lymphocytes in the cortex region appear to have originated in the bone marrow and have been captured by the thymus. The cortex is considered to be a holding zone for lymphocytes. There are no identifiable surface antigens on the cortical lymphocytes. The lymphocytes will move into the medulla region and are colonized with “surface antigens”, after which they will move into peripheral circulation and migrate to specific regions of other lymphoid tissue and set up colonies. It is in the thymus that the non-marked lymphocytes are differentiated into the mature T-type lymphocyte. The thymus gland contains thymosin and other thymic factors (examples: CD4, CD8, TCR, CD3).

The thymus, so well developed at birth, will begin remain a viable structure until the individual enters into puberty, after which the organ will begin to atrophy until it is almost nonexistent in old age.

The lymphoblast has a diameter that ranges from 10 to 15 μM. The cytoplasm is non-granular and smooth appearing with a moderate to dark blue color. Look for a periphery that stains more darkly and the region around the nucleus stains lighter. The volume of cytoplasm tends to be more abundant than in the myelocyte. The nucleus is round to oval and is situated eccentrically in the cell. One or two nucleoli are typical. The chromatin pattern tends to be slightly coarser than other blast cells. The nuclear to cytoplasmic ratio is 4/1.
   
   
43    DISCUSS THE PROLYMPHOCYTE.

The prolymphocyte is about the same size of the lymphoblast. The changes in this cell may be very subtle, leaving the prolymphocyte indistinguishable from the lymphoblast. The cytoplasm is agranular and very similar to that of the lymphoblast, but tends to be more abundant. Coloration is similar to that of the lymphoblast and an occasional azurophilic granule may be observed. The coarseness of the nuclear chromatin is increased in a nucleus that may be round, oval, or indented. Nucleoli, when visible, are almost indistinguishable and never more than one is the usual rule. The nuclear membrane is thicker. If it is certain that the cell being observed is either a lymphoblast or prolymphocyte, but there is uncertainty as to how to classify it, it is usually appropriate to refer to the immature cell as a “pre-lymphocyte” or “immature lymphocyte.”

       
 

44    DISCUSS THE LYMPHOCYTE.

This cell actually exists as subpopulations consisting of a small, medium, and large lymphocytes. Some professionals classify the lymph cell into only two subpopulations (small and large).

The small lymphocyte measures from 8.0 to 10.0 μM, somewhat larger than a RBC. The cytoplasm is characterized as a thin rim about the nucleus. The color of the cytoplasm is dependent upon the properties of the stain being used and will range from a sky blue color to a dark blue coloration. Occasional azurophilic granules may be observed. The nucleus to cytoplasm ration ranges from 5:1 to 3:1. The nucleus is about the size of a normal RBC and may be used to estimate the size of the RBC. The nucleus is characterized by a clumped, dense chromatin pattern (designated as the block type). The chromatin of the nucleus is designated as pachychromatic, its parachromatin appearing smudged and indistinct. This characteristic is consistent in the larger lymphocyte forms, but may less obvious than in the small lymphocyte. The nucleus is usually round or oval, but may be indented. There are no visible nucleoli. The nucleus stains a dark reddish-purple color with Wright’s stain. Comment: The nucleus of the small lymphocyte tends to be consistent in its size. For this reason, it may be used as a reference for sizing RBC’s.                                      

   

The moderate or medium lymphocyte measures from 10 to 12 μM, although some texts cites diameters up to 14 μM. There is more abundant cytoplasm than in the small lymphocyte. The cytoplasm often stains lighter than that of the small lymphocyte, from a sky blue to a medium blue. A few azurophilic granules may be seen in the cytoplasm. The nucleus will be round, oval, or indented; with the chromatin appearing less dense than in the small lymphocyte. The chromatin will have a clumped appearance. There are no visible nucleoli.

               

The large lymphocyte is characterized by a diameter of 12 to 16 μM. Some textbooks will cite values up to 18 μM. The cytoplasm is very abundant, tending to be clear or pale blue in color. Basophilic colors have been reported. Non-specific azurophilic granules may be seen. The cytoplasmic outline may be irregular. The nucleus is usually round, oval, or indented. The chromatin pattern is coarse. Nucleoli are not visible. The nucleus is eccentric.
       

45    DISCUSS THE B-TYPE LYMPHOCYTE.

The “B” used to describe the B-type lymphocyte comes from the research originally conducted in studying the role of the bursa of Fabricius in chickens. This bursa was essential to the development of antibodies and its absence left the chicken immunocompromised. The term “B-lymphocytes” implies this lymphocytes is equivalent to the bursa-dependent cells of the chicken and produces antibodies.

The cell that develops into the mature B-lymphocyte passes through three stages: [1] early pre-B cell, [2] pre-B cell, and [3] mature B-cell. As the maturing lymphocyte passes through each of these stages, the membrane surface markers changes as indicated by examples in the following table:

                HLA-DR CD19 CD10 Cμ  sIg  Pan-T  CD7  TdT CD20
early pre-B  +            +      -      -    -      -       +      +      +
pre-B          +            +      +      +   -      -        +     +       +
mature B     +            +     -/+    -    +     -         -     -       +

Once the B-lymphocyte reaches it mature or resting stage, the CD markers (membrane surface molecules will not change. The B-lymphocytes life span is measured in days (usually 3 to 4).

The B-lymphocyte develops in the bone marrow (which is considered to be the bursa-equivalent tissue of the chicken) from the pluripotent stem cell, after which it migrates to the peripheral lymphoid organs to remain in a resting state until they are stimulated by antigens to further development. The B-lymphocyte can also be formed in the lymphoid patches of the small intestine (designated as Gut Associated Lymphoid Tissue or GALT). This stage of maturation is designated as antigen-independent development. The B-lymphocyte will make up from 10 to 20% of the lymphocytes in peripheral circulation and is the antibody producing line of leukocytes. The plasma cell (a short lived cell) is the final state of maturation for the B-lymphocyte. When the B-lymphocyte is stimulated by specific antigens, it can undergo clonal expansion, producing a long-lived, identical or daughter cells that recognize the same antigen and are designated as memory cells. These memory cells appear morphologically similar to the small lymphocyte. The functional role of the B-lymphocyte is humoral immunity.

46    DISCUSS THE T-TYPE LYMPHOCYTE.

The T-lymphocyte development occurs in the thymus, hence its designation “T-lymphocyte”. There are several stages of development of this cell. This lymphocyte forms first from the pluripotent stem cell in the bone marrow and then migrates to the thymus for further development. At this stage of development, this lymphocyte contains a TdT marker. Further lymphocyte development begins in cortex of the thymus as an early thymocyte with CD2, CD5, and CD7 markers added. As the “lymph” matures in the cortex of the thymus, it becomes a common thymocyte with the addition of CD4 and CD8 markers. The common thymocyte then migrates to the medulla of the thymus and further differentiation occurs. Those thymocytes that lose the CD8 marker and take on the TCR and CD3 markers become “helper” T-cells. Those thymocytes that lose the CD4 marker and acquire the TCR and CD3 markers become “suppressor” T-cells. The presence of the CD3 marker indicates a mature T-type lymphocyte. These cells can survive for several months and/or years (up to 10, although the average life span appears to be about 4 years.)

T-lymphocytes are identified by the type of antigen marker on the membrane which is designated by a CD label. There are several types of T-lymphocytes. These will be discussed in the objectives to follow. T-lymphocytes will make up from 60% to 80% of the circulating lymphocytes.

47    DISCUSS THE CD NOMENCLATURE.

CD is an acronym that means Cluster Designation or Clusters of Differentiation. This is a classification system designed for identifying the cell surface antigens on leukocytes. Over 125 CD antigens have been identified. Examples are as follows:
[1] Myelocytes: CD13, CD15, CD34, CD35. More than 24 antigens known.
[2] Eosinophils: CD 23, CD33, CD44,CD48. Over 11 antigens identified.
[3] Basophils: CD44, CD 45, CD46, CD47. Over 15 antigens known.
[4] Monocytes: CD13, CD14, CD15, CD31. Over 24 known antigens.
[5] Megakaryocytes: CD36, CD44, CD51, and CD61. Over 13 known.
[6] Erythrocytes: CD35, CD55, CD71. Only a few such markers reported.
[7] Lymphocytes:
     a. B-cell: CD10, CD19, CD20, and CD21. Over 21 known.
     b. T-cell: CD2, CD3, CD4, and CD5. Over 22 known.
    c. NK-cell: CD2, CD5, CD16, and CD18. Over 13 known.

48     DISCUSS THE NK T-LYMPHOCYTE.

The NK or Natural Killer T-type lymphocyte are morphologically large lymphocytes with azurophilic granules in their cytoplasm. These cells are also designated as Large Granular Lymphocytes (LGL). Their functional role is designated as destroying tumor cells, microbial agents, and virus-infected cells through lytic activity (cellular immunity). These cells are characterized by the presence of CD2, CD8, CD11b, CD16, and CD56 antigens. These cells are non-adherent and non-phagocytic. This is a sub-population of lymphocytes that make up 5% to 10% of the circulating lymphs. The NK type lymphocyte destroys the target cell through a non-phagocytic, extracellular mechanism designated as a cytotoxic reaction. This cell produces interferon and interleukin-2. These cells have been referred to as null cells. This population of lymphocytes may be referred to as suppressor cells by some clinical professionals.

49    DISCUSS THE HELPER T-TYPE LYMPHOCYTE.

The “helper” T-type lymphocyte (TH) contains the cell surface membrane antigen designated as OKT4. This lymphocyte type makes up from 55% to 70% of the total T-lymphocyte population. The function of the “helper” cell is to signal B-lymphocytes to produce antibodies, control the production of the antibodies, and to change the types of antibodies being formed. The “helper” cells also activated cytotoxic-type lymphocytes. The helper cell can recognize an infected cell through its antigens or membrane markers. This causes the “helper” cell to activate the B-lymphocyte and/or cytotoxic cell.

The “helper” cell also influences the activity of the monocyte and macrophage. If the “helper” cell function is lost, then the body’s ability to fight infections from viruses, bacteria, fungi, and parasites is severely impacted. The normal ratio of “helper” cells to cytotoxic cells is two to one.

This example is an overview describing how the “helper” T-lymphocyte functions.
[1] The TH cell received antigen information from a monocyte or macrophage.
[2] This process requires the two cells to bind through the TH receptor (Ia) to the monocyte’s receptor (MHC-II).
[3] The TH cell is stimulated to reproduce itself so that the information can be transmitted to additional types of lymphocytes (TH cells, B-lymphocytes, T-suppressor cells, and cytotoxic T lymphocytes.)

50    DISCUSS THE K T-TYPE LYMPHOCYTE.

The “K” or “killer” type lymphocytes may be related to the population of “natural killer” type of lymphocytes and contains CD8. They differ from the NK cells in that they demonstrate a different type of cytotoxic mechanism for destroying cells. The K-type lymphocyte depends on the target cell being coated with a low concentration of IgG antibody. Because of this, the mechanism is designated as an Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) reaction. It is reported that these cells appear to be morphologically similar to the small lymphocyte, however be aware that these cells may be similar in appearance to the NK-type cell.

51   DISCUSS THE CYTOTOXIC T-TYPE LYMPHOCYTE.

This is a third type of killer cell, with direct cytotoxic activity, that also contains the membrane marker, CD8. It lyses its target cell by cell-to-cell contact.

52   DISCUSS THE SUPPRESSOR T-TYPE LYMPHOCYTE.

This cell type is designated as TS. It function is to keep the immune process under control. This cell has the CD4 membrane marker as does the TH cell. This cell affects the responses of the T-lymphocytes that effect/cause cell-mediated immunity of the T-lymphocytes and the humoral immunity of the B-lymphocytes. The suppressor T-type lymphocyte may exist in three sub-types: [1] an inducer TS, [2] a mediator TS, and an effector TS.

53   DISCUSS THE DOWNEY CELL CLASSIFICATION.

The Downey cell is (for all practical purposes) an obsolete classification scheme proposed by Hal Downey and McKinlay (1923). This classification method was implemented to describe the lymphocytes observed in infectious mononucleosis. This group structured the reactive lymphocyte into three categories. When classifying lymphocytes under the Downy classification, be sure to note the appearance of the cytoplasm and the quantity and distribution of the chromatin structure.

[1] Downey Cell Type I. The nucleus is irregularly shaped. If the nucleus is round or oval, it may be indented. The nuclear chromatin resembles that of a mature lymphocyte. The cytoplasm is basophilic and may be foamy appearing at times. Vacuoles are present.
[2] Downey Cell Type II. The nucleus is characterized by coarser appearing chromatin, but is less so than in the type I cell. The cytoplasm is characterized by an irregular border (scalloped appearance) and is increased in quantity. The cytoplasm is usually characterized by light blue staining cytoplasm around the nucleus but a more intense blue color at the periphery, giving the appearance of basophilia. Radial basophilia may be present. Vacuolation will less than in type I and azurophilic granules may be seen on occasions..
[3] Downey Cell Type III. The nucleus may resemble the immature lymphocyte and nucleoli (one to four) may be visible. There is an increased in cytoplasmic basophilia. The cytoplasm is abundant and tends to flow around the red blood cells. This cell is larger than types I and II.

54   DISCUSS THE REACTIVE LYMPHOCYTE.

The reactive lymphocyte represents a morphological variation in the lymphocyte that has been exposed to antigenic stimulus. It is characterized by size variation and often demonstrates an increase in cytoplasmic mass. This is due to an increase in DNA and RNA synthesis. The reactive lymphocyte is often larger than large lymphocytes. They are T-lymphocytes that become cytotoxic to infected B-lymphocytes.

The nucleus may be oval, round, indented, or irregular in shape. The chromatin pattern is less pachychromatic, being designated as intermediate. It is possible to observe nuclei in reactive lymphocytes that have a “blast” appearance. Nucleoli may be faintly visible.

The cytoplasm is usually abundant and demonstrates varying shades of basophilia. The basophilia may distribute in variable patterns within the cytoplasm. Azurophilic granules may be present. Scalloped margins is the rule as the lymphocyte flows around the edges of other cells. The Golgi apparatus may be observed. The presence of vacuoles is not unusual and may even give a “bubbly” or “foamy” appearance to the cell.

The reactive lymphocyte is seen in infectious mononucleosis, cytomegalovirus infections, HIV, infectious hepatitis, organ transplants, and serum sickness. If the problem is serum sickness, then symptoms should include fever, splenomegaly, swollen lymph nodes, skin rash, and/or joint pain. Serum sickness is an immunological disorder that appears about 2-3 weeks after administration of an antiserum. Caution should be exercised when identifying reactive lymphocytes. These cells are vulnerable to the effects of anticoagulant and delay. Be sure that blood films are prepared at time of collection and no longer than 30 minutes after collection.

Comment: The term “atypical” is often used to describe reactive lymphocytes. This is an old term used to describe any lymphocyte that is larger or more basophilic or has nuclear variation that distinguishes it from the normal and mature lymphocyte. Other terms that have been used to describe the reactive lymphocyte are: Turk cell, plasmacytoid lymphocyte, and Downey cell.

An additional comment about lymphocytes. Lymphocytes are capable of blast cell formation and mitotic activity. This cell type is capable of serving as its own stem cell when replacement lymphocytes are needed.
   
55   DESCRIBE HOW TO DISTINGUISH BETWEEN THE REACTIVE LYMPHOCYTE AND THE MALIGNANT LYMPHOCYTE.

This will require careful observation since both types of lymphocytes may demonstrate immature appearances. When differentiating, examine the specimen for the presence of [1] large and small cells, [2] non-basophilic and basophilic cells, [3] nuclei with immature chromatin or containing pachychromatin. Reactive lymphocytes exhibit wide variation among themselves, whereas the malignant lymphocyte tends to be clonal in origin and appear similar to each other.

56   DEVELOP A TABLE THAT COMPARES THE MONOCYTE TO THE REACTIVE LYMPHOCYTE.

                        MONOCYTE                 REACTIVE LYMPHOCYTE
Chromatin         Lacy, loosely            Variable, fine and dispersed
                        woven, or ropy        to clumped (expecially at
                                                       membrane periphery)

Cytoplasm        Blue-grey                    Pale blue to deeply
                                                        basophilic, it tends to
                                                        stain unevenly.

Granules            Numerous very            A few obvious azurophilic
                        fine red granules          granules may be present.
                        that give a ground
                        glass appearance

Nucleolus            Absent                        May be present

Nucleus             Convoluted,                   Variable shapes from
                        horseshoe, kidney-        round, elongated,
                        shaped, oval, or            irregular to stretched
                        round                            out

Shape                Pleomorphic,                Pleomorphic and is
                        pseudopodia may           easily indented by the
                        may be present,             surrounding cells
                        resists indentation
                        by surrounding cells

Size                12 to 20 uM                       10 to 30 uM

Vacuoles          Absent to numerous,          Occasionally present
                      small to large

57    DISCUSS THE CONVERSION OF THE LYMPHOCYTE TO THE PLASMA CELL.

The conversion of the lymphocyte to the plasma cell begins when the B-lymphocyte is antigenically stimulated. One of the first steps in the transformation is the enlargement of the B-lymphocyte. The nucleus will manifest visible nucleoli. The cytoplasm becomes basophilic. A clear halo-like area will appear close to the nucleus indicating enlargement of the Golgi body. This clear area is designated as the perinuclear halo or hof. At this stage, the B-lymphocyte is a reactive lymphocyte. The transformation continues and the plasmablast is formed, to produce a cell that is more efficient at the production of antibodies. The plasma cell will be characterized by the synthesis and release of specific immunoglobulins.
 

58   DISCUSS THE PLASMABLAST.

The diameter ranges from 18 to 25 μM. The cytoplasm is abundant, basophilic, and non-granular. The perinuclear halo may be present representing the active region of the Golgi body. The nucleus will be characterized by clumps of chromatin somewhat similar to the lymphocytes, but the clumping is moderately greater. The generally round nucleus is eccentric with a round to oval shape. Nuclear to cytoplasm ratio is 4:1 Several nucleoli are present but may be difficult to see. This stage may not seen in the bone marrow.
        

59    DISCUSS THE PROPLASMACYTE.

This cell has a diameter ranging from 15 to 25 μM. The cytoplasm continues to be strongly basophilic (usually more blue than the ‘blast’ form). The volume of cytoplasm is greater than the plasma blast and is non-granular. Edges of the cytoplasm may appear ragged. The perinuclear halo is obvious. The reddish-purple nucleus continues it eccentric position and is round to oval in shape. The nuclear to cytoplasm ration is 3:1 or higher. Nucleoli may be observed. This cell is usually not observed in bone marrow. If seen, then it may be an indicator of multiple myeloma.
       
 

60    DISCUSS THE PLASMA CELL.

The plasma cell or plasmacyte is characterized by a diameter that measure from 8 to 20 μM. The cytoplasm is moderately abundant, less than that of the promyelocyte. The perinuclear halo is adjacent to the nucleus and distinctive in appearance. The cytoplasm is non-granular as a rule. The nucleus is characterized by a more dense chromatin in coarser clumps. The nucleus is eccentric with a round to oval shape. Nucleoli are not visible.
     

61   DESCRIBE SEVEN CELLS THAT MAY BE FOUND IN A PERIPHERAL BLOOD SMEAR.

[1] Megakaryocyte Fragments. This is a nucleus void of its cytoplasm and can be observed in healthy newborns. In this case it is not abnormal. The nucleus may have a few small platelet like fragments attached. When seen in other patients, it may be an indicator of essential thrombocythemia, myelofibrosis, or some other platelet disorder. These have been reported in severe hypoxia.

[2]  Basket Cells. These have been equated to smudge cells, but are considered to be the remnants of granulocytic cells that are formed during the preparation of the smear. It is normal to see a few in normal conditions. They can be increased in some leukemic conditions.

[3] Nucleated Red Blood Cells. These may be seen in newborns but should not be seen in normal healthy children or adults. When these are observed on a blood smear, they are identified as NRBC’s without any attempt to classify the stage of development. Keep a count of how many are seen during a WBC diff and report as # NRBC’s/100 WBC’s. They are found in a variety of abnormal conditions such as anemia’s, hemolysis, leukemia, myeloproliferative disorders, hemorrhage, etc.

[4] Smudge Cells. These are the structureless remnants of lymphocytes that are formed during the preparation of the blood smear. A few smudge cells on a blood smear is not clinically significant. They can be increased during disorders involving lymphocyte proliferation, especially chronic lymphocytic leukemia.

[5] Phagocytic Cells. Can be neutrophils or monocytes that have engulfed foreign material such as bacteria or fungi. These are not found during normal conditions. They can be observed during severe infections. Occasionally, the leukocyte can be observed engulfing an erythrocyte, in which case it is known as erythrophagocytosis.

[6] Endothelial Cells. These are more likely to be observed in smears made from capillary punctures but may be seen in smears made from venipunctures. This is a contaminant. It is a cell with a somewhat oval appearance with a single centric nucleus. The chromatin is dense and the cytoplasm abundant without granules. These cells may be seen in small clusters. Because of their larger size, they are more apt to be seen in the feathered edge or at the lateral edges of the smear. They are not to be confused with malignant cells.

[7] Necrotic Cells. These can be any of the leukocytes but most often it will be a granulocyte that is breaking down and filaments are missing between the nuclear segments. These are usually not seen but can be in a blood film that is prepared from a specimen that has prolonged exposure to EDTA. They have been reported in blood film preparations of patients on chemotherapy.

62    DISCUSS THE LEUKEMOID REACTION.

Leukemoid reactions are characterized by highly elevated leukocyte count in any of the white blood cell lines. In most cases, this elevated count is greater than 50,000/μL and involves the neutrophil granulocytes. With a neutrophilic leukemoid reaction, the stained blood film will have a distinctive shift-to-the-left with immaturity in two or more cells. An occasional blast cell may be seen but is not typical for this reactive response. The extreme proliferation of cells closely resembles that of chronic myelocytic leukemia and must be differentiated. Generally, if the WBC count is over 100,000/μL with increased numbers of blast cells, then a myeloproliferative disorder is likely. Acute infections (septicemia), chronic infections (tuberculosis), severe metabolic inflammatory processes, acute alcoholic hepatitis, and neoplastic disorders are causes of a leukemoid response. Review the following chart to differentiate between a leukemoid and chronic myelocytic leukemia (CML).

                        Leukemoid reaction         Chronic Myelocytic Leukemia
WBC Count                increased                                 increased
Differential            shift-to-the-left                     shift-to-the-left
LAP Score                  increased                                 decreased
Philadelphia
   chromosome              absent                                      present
Platelets                 usually normal                          may be increased
                                                                               or decreased
RBC count                      normal                              usually decreased

If the leukemoid reaction involves the lymphocyte, then the peripheral blood picture resembles that of leukemia. Illnesses involving viruses or Bordetella pertussis may result in elevated lymphocyte counts. If the patient is presenting with a lymphocytic leukemoid reaction, the lymphocytes on the stained blood film will exhibit immature appearing cells but with polymorphism. If it is a malignancy, then the cells are usually clonal and tend to appear very similar to each other.

 

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This page last updated 07/28/08