CIRCULATING BLOOD and LYMPH
Circulating Blood
I. Classification
A. Connective tissue
1. Develops from embryonic tissue mesenchyme
B. Components of connective tissue
1. Cells
a. living component of connective tissue
b. have organelles and inclusions which constitute the intracellular components of the cell
2. Intercellular or extracellular material
a. non-living component of connective tissue
b. is the abundant material found between the cells and serves as the characteristic feature of connective tissue
C. Components of peripheral blood
1. Cells (formed elements)
a. erthyrocyte (RBC)
b. leukocyte (WBC)
c. platelets
d. "dust'' or hemoconia
e. chylomicrons
2. Intercellular material (plasma or fluid component)
a. composition of plasma:
i. water - 90%
ii. proteins (albumin; globulins - alpha, beta, gamma; fibrinogen) - 7%
iii. inorganic salts (Cl-, HC03-, P04-3, Ca+2 Na+, K+) 0.9%
iv. organic compounds (amino acids, vitamins, hormones, lipids, carbohydrates, etc.) - 2.1%
b. plasma vs. serum
i. plasma clots, serum does not clot
ii. serum lacks the protein fibrinogen and some clotting factors
II. Erthyrocytes (RBC)
A. General points
1. Life span of 120-130 days
2. Number present:
Male: 5 x 106/mm3
Female: 4.5 x 106/mm3
3. RBCs are 500-1000 times more numerous than leukocytes
B. Morphological features
1. Biconcave disk with a diameter of 7-8 um and width of 2 um. The shape is dependent on the spectrin-ankyrin-actin interaction.
2. Lacks a nucleus in mammals, but nucleated in other forms (birds, etc.)
3. Lacks a Golgi, centrioles, lysosomes, RER
4. Very few or no mitochondria , resulting in anaerobic glycolysis and pentose phosphate pathways being important for energy production.
5. Cytoplasm composed of:
a. water - 65%
b. organelles - 1%
c. hemoglobin - 34%
C. Function
1. transport of O2 and CO2
III. Platelets (thrombocytes)
A. General points
1. Life span of 8-10 days in the circulation
2. Number about 150,000 - 400,000/mm3
3. Derived from megakaryocytes in the bone marrow, and represent membrane bound fragments of the cell
B. Morphological features
1. Two to 4 um in diameter
Ultrastructure of platelets in cross section (above) and in longitudinal section (below). Infoldings of the plasma membrane give rise to numerous vacuoles. The large electron-dense granules are the azurophilic granules of light microscopy. Near the edge of the platelet, there is a marginal bundle of microtubules. Glycogen granules, mitochondria, and electron-dense tubules are present in the granulomere.
2. No nucleus, but a fragment of the megakaryocyte
3. Contains a central granulomere and a peripheral hyalomere
C. Function
1. platelets aggregate ---> platelet plug
2. clotting
IV. Leukocytes (WBCs)
A. Involved in both the cellular and humoral defenses of the organism.
B. Morphological features
1. Granulocytes/polymorphonuclear
a. have specific granules in the cytoplasm
b. non-mitotic cells in the blood stream and after leaving the vascular system
c. three types:
i. neutrophils
ii. eosinophils
iii. basophils
2. Agranulocytes/mononuclear
a. lack specific granules in the cytoplasm
b. differ from granulocytes in that they can reproduce by mitosis after leaving the vascular system
c. two types:
i. lymphocytes
ii. monocytes
C. Diapedesis
1. Diapedesis - the process by which WBCs leave the vascular system (post-capillary venules). Neutrophils circulate in the blood stream for 6- 12 hours and then migrate into the connective tissue where they perform their function.
V. Neutrophils (Polymorphonuclear neutrophils, polys, PMNs, polymorphs)
A. General points
1. Characterized by having a many lobed nucleus and specific granules in the cytoplasm
2. Comprise 50-70% of differential count
3. 4,400/mm3
B. Morphological features
1. Diameter of 10 to 15 um
2. Nucleus
a. generally segmented, consisting of 2 to 5 lobes held together by chromatin
b. young or immature cells are non-segmented (band or stab); may comprise 1% of cells
3. Cytoplasm
a. very little RER, Golgi, free ribosomes, and few mitochondria
b. three types of granules (formed by the Golgi apparatus): 50-200 granules/cell
(1) azurophilic or primary granules (20% of granules)(reddish- purple color)
i. 400-700 nm (0.4 m) in diameter
ii. membrane bound granules containing lysosomal enzymes and myeloperoxidase (peroxidase)
iii. appear early in cell development (promyelocyte stage) and progressively decrease in number
(2) neutrophilic or specific or secondary granules (80% of granules) (lavender or lilac color)
i. less than 400 nm in diameter
ii. membrane bound granules containing alkaline phosphatase (hydrolases that function at a neutral or alkaline pH) and bacteriocidal substances (lysozyme, lactoferrin)
iii. appear later in cell development (myelocyte stage) and progressively increase in number
(3) tertiary granules
i. contain gelatinase; cathepsins; glycoproteins that can be inserted into the plasma membrane (aid in the phagocytic processes)
4. Plasma membrane
a. contains NADPH oxidase bound to the extracellular surface
(1) faces lumen of phagosome after phagocytosis of bacteria
i. catalyzes formation of reactive oxygen compounds (superoxide; hydrogen peroxide; hypochlorous acid) within the phagosomes of neutrophils
C. Functions
1. Neutrophils serve as a first line of cellular defense against invasion of microorganisms. PMNs are chemotactically attracted by devitalized tissue, bacteria, and other foreign bodies and factors produced by antigen- antibody interactions with certain blood proteins (complement) and they migrate to the site of infection
2. Killing of bacteria is thus accomplished via two different mechanisms:
a. enzymatic (via fusion of specific and azurophilic granule contents with the phagosome) which involves:
(1) phagocytosis of the foreign material, thus forming a phagosome,
(2) the specific granules fusing with the phagosome, inactivating the material,
(3) the azurophilic granules fusing with the phagosome, digesting the material, and
(4) finally the digested material expelled from the cell.
b. via formation of reactive oxygen compounds within the phagosome
3. Neutrophils die and become the pus of an abscess.
4. Not all bacteria destroyed by neutrophils. For example, the tubercle bacillus survives phagocytosis by the PMN, and must be contained by the macrophage which is derived from the monocyte
VI. Eosinophil (polymorphonuclear eosinophil)
A. General points
1. Characterized by its large eosinophilic granules in the cytoplasm
2. Comprise 1-4% of the differential count
3. 200/mm3
B. Morphological features
1. Diameter of 12 to 17 um
2. Nucleus
a. generally consists of two lobes held together by a strand of chromatin
b. more than two lobes is not common
3. Cytoplasm
a. Golgi and mitochondria seen with the electron microscope, with other organelles having minimal representation
b. two types of granules present in mature cells (eosinophilic or specific granules and azurophilic granules)
c. specific granules
(1) specific granules very large (0.5 to 1.5 um wide)
(2) granules eosinophilic due to the large amount of arginine in the protein
(3) crystalline structure in granule core seen with TEM (contains major basic protein, eosinophilic cationic protein, and eosinophil-derived neurotoxin): major basic protein and eosinophilic cationic protein are very effective agents in combating parasites
c. azurophilic granules
(1) these are lysosomes and contain hydrolytic enzymes and peroxidase
(2)contents help in the destruction of parasitic worms and in the hydrolysis of antigen-antibody complexes internalized by the eosinophils
C. Functions
1. Eosinophils leave the vascular system by diapedesis, and locate especially in the connective tissue beneath the epithelium of the respiratory and gastro-intestinal tract.
a. binding of histamine, leukotrienes, and esoinophil chemotactic factor (released by mast cells, basophils, and neutrophils) to eosinophil plasma membrane receptors results in the migration of the eosinophils to the site of the allergic reaction, inflammatory reaction, or parasitic worm invasion
2. The differential count of eosinophils increases with parasitic infections (trichinosis, schistosomiasis, ascaris)
a. major basic protein and eosinophil cationic protein bore holes in the pellicles of parasitic worms, facilitating access of reactive oxygen compounds (e.g. superoxides; hydrogen peroxide) to the parasite
3. Differential count increases in allergic conditions such as hay fever and asthma.
4. Cells play a role in the phagocytosis and hydrolysis of antigen-antibody complexes.
5. Eosinophils degrade chemical mediators such as leukotrienes and histamine released by mast cells and basophils, thus regulating local inflammatory responses
VII. Basophil (Polymorphonuclear basophil)
A. General points
1. Comprise about 0.5% of differential count
2. 40/mm3
B. Morphological features
1. Diameter of 8 to 12 um
2. Nucleus
a. irregular shape
b. does not appear to be lobated
3. Cytoplasm
a. organelles have minimal representation
b. two types of membrane bound granules (produced by the Golgi) are present
c. specific granules
(1) vary in size, but may be as large as those of eosinophils
(2) are basophilic and metachromatic
(3) contain:
i. heparin, histamine, eosinophil chemotactic factor, neutrophil chemotactic factor, and peroxidase
d. azurophilic granules
(1) lysosomes: similar to the azurophilic granules of neutrophils
C. Functions
1. Increase in number along with other leukocytes with leukemia.
2. Increase in number in smallpox, chicken pox, and sinus inflammations.
3. Functions are appear to be involved in mediating allergic and inflammatory reactions (functions are similar to mast cells)
a. antigens can bind to IgE molecules whose Fc portion is bound to Fc receptors on the basophil surface; this may cause the basophils to release the specific granule contents into the extracellular spaces
(1) release of histamine causes smooth muscle contraction (in the bronchial tree), vasodilation of the microvasculature, and leaking of blood vessels
b. begin to produce and release leukotrienes
(1) similar effects to histamine, but actions are slower and more persistent
VIII. Lymphocytes
A. General points
1. Produced in lymphatic nodules, lymph nodes, spleen, thymus, tonsils, and bone marrow, and endow the body with its immunological defense.
2. Comprise 20-40% of differential count
3. 2,500/mm3
B. Morphological features
1. Two size categories:
a. Small lymphocytes
(1) comprise 90% of the lymphocytes
(2) diameter of 6 to 12 m
b. Large lymphocytes
(1) comprise 10% of the lymphocytes
(2) diameter of 12 to 18 m
(3) significance of these cells is unknown, but may be partially differentiated plasma cell precursors
(4) differ from small lymphocyte in that nucleus is slightly bigger, and there is slightly more cytoplasm and organelles
(5) appear similar to monocytes, but smaller and nucleus is never kidney shaped
2. Nucleus
a. spherical or slightly indented on one side
b. chromatin is densely packed with a hill and valley pattern
c. nucleolus present but not seen because of clumped chromatin
3. Cytoplasm
a. frequently appears as a thin rim
b. many free ribosomes and polysomes, some azuorphilic granules, Golgi, RER, and mitochondria
C. Subdivision of small lymphocytes based on their function, not on morphologic features.
1. B-lymphocytes
a. compose about 15% of circulating lymphocytes
b. called B-cells because in chick develop in bursa of Fabricius
c. in humans develop in bursa equivalent (gut) or bone marrow
d. cells may leave the circulation and enter lymphatic tissue where by the process of mitosis they give rise to clones of B-cells
e. function or fate of B-lymphocytes:
(1) Plasma cells - produce antigen-specific circulating immunoglobins (humoral antibody response)
(2) Memory cells - found in lymphatic tissue and stimulated by re-exposure to antigen; reaction termed the secondary response
2. T-lymphocytes
a. compose 80-90% of circulating lymphocytes
b. originate embryological from the yolk sac and seed the thymus by way of the liver and bone marrow
c. multiply and differentiate into T-lymphocytes in the thymus - each developing lymphocyte develops an individual antigenic specificity.
d. activation of T-cells (needed for activation)
(1) appropriate antigen
(2) macrophages must process the antigen for presentation
e. subsets of T-lymphocytes
(1) Cytotoxic T cells
(2) T helper cells
(3) T suppressor cells
f. function (s) of T-lymphocytes
(1) cell mediated immunity; assist in humoral immunity
3. Null cells
a. neither B- nor T-lymphocytes
b. example: NK (natural killer) cells
IX. Monocyte
A. General points
1. Characterized partly by its large size
2. Comprise 2-8% of the differential count
3. 300/mm3
4. Cells originate in the bone marrow
B. Morphological features
1. Diameter of 12 to 20 um
2. Nucleus
a. eccentrically placed and may be oval, indented, kidney or horseshoe shaped
b. chromatin less condensed than in lymphocyte, and therefore, nucleus in lighter staining
3. Cytoplasm
a. stains a light or pale blue
b. vacuoles may or may not be present in the cytoplasm
c. some azurophilic granules may be present (contain lysosomal enzymes)
d. well-developed Golgi, some ribosomes and polysomes, mitochondria, and little RER
C. Functions
1. Monocytes exhibit diapedesis (continually extend and withdraw pseudopodia) and reach full development outside the blood stream where they are known as macrophages. Macrophages fuse to form foreign body giant cells and osteoclasts; In the CNS the macrophages form microglia; in the liver, Kupffer cells; and in the lungs, alveolar macrophages.
2. Serve as the second line of defense against invading organisms. Found in areas of chronic inflammation.
3. After leaving the vascular system, the macrophage (monocyte) plays a role along with the T-lymphocyte in the differentiation of the B-lymphocyte into the plasma cell, which produces immunoglobulins.
4. Some macrophages are particularly good at processing and presenting antigen and are called antigen presenting cells
5. Monocytosis - increased monocyte count due to infectious and inflammatory diseases, tuberculosis, and leukemia.
X. Chylomicrons
A. Found in the plasma
B. Consists of fat combined with plasma proteins
C. Can be visualized with the light microscope after eating a fatty meal
XI. Hemoconia ("dust")
A. Best described as junk in the blood stream
B. Consists of broken down RBCs, endothelial cells, and ingested material not filtered out in the spleen
Lymph
I. Components of lymph
A. Plasma (fluid)
B. Cells
1. Lymphocytes - chief element
2. Granulocytes - very few
II. Lymph plasma
A. Carries carbonic acid, but very little oxygen
B. Intestinal lymphatics after a meal become filled with a large number of chylomicrons, resulting in a white colored lymph known as chyle
C. Coagulates more slowly than blood, the fibrin forming a colorless clot
Circulating Blood Appendix
Clinical Notes
NB: The clinical notes that follow are for your general information; you will not be tested on this information unless it appears elsewhere in the course.
CLINICAL NOTES
A. Red Blood Cells
1. Hematocrit - refers to the volume of packed RBCs expressed at a %; determined by centrifugation of blood in a heparinized capillary tube.
Newborn - 45 to 60%
Children up to10 yrs. of age -35%
Adult male -42 to 52%
Adult female - 37 to 47%
2. Cyanosis - a deficient oxygenation of the blood resulting in an increased amount of reduced or deoxygenated hemoglobin which imparts a bluish color to the skin.
3. Isosmotic or isotonic plasma - normal plasma (0.85% NaCl)
4. Crenation - a shrinkage observed in RBCs when placed in hypertonic solutions (> 0.85% NaCl)
5. Hemolysis - refers to the lysis or breakdown of the RBC with the release of hemoglobin. The remaining cell membrane is referred to as the cell ghost. Will occur when RBC placed in hypotonic solution (< 0.85% NaCl) due to an intake of water and bursting of the cell membrane, with the escape of hemoglobin and the formation of the cell ghost.
6. Agglutination - clumping of RBCs; an immunological reaction occurring from mismatched blood; has severe consequences.
7. Reticulocytes or reticulated erythrocytes - youngest erythrocytes in the circulating blood, containing groups of ribosomes (polysomes); comprise 1 to 2% of peripheral blood erythrocytes; increase in number under conditions which require an increased influx of erythrocytes from the bone marrow.
8. Rouleaux formation - rows of RBCs adhering to each other due to surface tension (like stack of coins or donuts)
9. Poikilocyte - an abnormally shaped cell (e.g. tear drop, target cell, sickle cell, helmet cell, spherocyte)
10. Anisocytosis - an abnormal variation in RBC size
11. Terms (based on staining) referring to the amount of hemoglobin in RBC (hemoglobin is acidophilic because of the positive charges associated with histidine, arginine and lysine):
a. Normochromic - a cell which has the normal staining characteristics (middle 1/3 lighter staining)
b. Hyperchromic - a cell more intensely stained, not due to more hemoglobin/unit volume, but due to a larger cell; term should no longer be used
c. Hypochromic - a pale staining cell containing a decreased amount of hemoglobin; occurs in iron deficiency anemia
12. Terms referring to the size of RBC:
a. Normocyte - a normal size (7 to 8 um) RBC
b. Microcyte - a RBC smaller than 6 um; occurs in iron deficiency
c. Macrocyte - a RBC larger than 9 um (9-12 um); occurs in pernicious anemia (fail to secrete intrinsic factor & absorb vit B12)
13. Anemia - refers to a reduction in the amount of hemoglobin due either to a lack of erythrocytes or to a lack of hemoglobin
a. Normochromic anemia - anemia characterized by a reduction in the number of erythrocytes; occurs with bleeding (e.g. injury, ulcers).
b. Macrocytic anemia - fewer cells than normal, but those present are larger than normal and are well filled with hemoglobin; occurs in pernicious anemia or where there is a deficiency of folic acid or vitamin B12, compounds needed for DNA synthesis. Also seen in alcoholics and in patients with liver disease and hypothyroidism.
c. Sickle cell anemia - abnormal hemoglobin (HbS) because the amino acid valine substituted for glutamic acid in the hemoglobin molecule. The RBC assumes the shape of an elongated cresent (sickle) as they yield up oxygen. Because of damage to the RBC membrane, the cells are prematurely destroyed.
d. Thalassemia - a hemolytic anemia due to an abnormal polypeptide chain in hemoglobin (fetal Hg); a hereditary anemia.
14. Polycythemia vera - a disease characterized by excessive production of red cells by the bone marrow. Because of the unregulated character of the red cell production, the disorder is regarded as neoplastic. The white cell counts and the platelet number may also be increased. This condition must be distinguished from secondary or physiological polycythemia, which occurs in persons living at high attitudes, where the oxygen concentration is low.
15. Hematology Report Slip
a. WBC - 4.8 to 10.8 x 103/mm3
b. RBC - 4.6 to 6.2 x 106/mm3 (male); 4.2 to 5.4 x 106/mm3 (female)
c. HGB (hemoglobin) - 14 to 18 gm/dl (male); 12 to 16 gm/dl (female)
d. HCT (hemotocrit) - 42 to 52% (male); 37 to 47% (female)
e. MCV (mean corpuscular volume)
- 80 to 94um3 (male)
- 79 to 97um3 (female)
This is the average volume of the RBC and is obtained by dividing the volume of the RBCs in 1,000 ml of blood (HCT x 10) by the RBC count in millions.
MCV in um3 = HCT x 10
RBCs (in millions)
e.g. HCT = 45; RBC = 5 x 106
450/5 = 90um3
A low MCV means microcytosis and a high MCV means macrocytotis
f. MCH (mean corpuscular hemoglobin) (23 to 31uug)
This is the average weight of hemoglobin in the RBC and is obtained by dividing the weight of hemoglobin in 1,000 ml of blood (HGB x 10) by the RBC count in millions:
MCH in uug = HGB x 10
RBCs (in millions)
e.g. HGB = 15gm/lOOml; RBC = 5 x 106
15/5 x 10 = 30uug
A low MCH can mean microcytosis, hypochromia, or both. A high MCH is evidence for macrocytosis.
g. MCHC (mean corpuscular hemoglobin concentration) - 32 to 36%
This is the weight of hemoglobin per volume of cells, and is therefore an indicator of the hemoglobin concentration of the average cell independent of cell size. It is calculated by dividing the hemoglobin value by the hematocrit and multiplying by 100:
MCHC in % or gm/lOOml RBC = HGB x 100
HCT
e.g. HGB = 15; HCT = 45
15/45 x 100 = 33%
A low MCHC means hypochromia (microcytic cells with a normal hemoglobin concentration will have a low MCH but a normal MCHC). A high MCHC is found only with marked spherocytosis. In becoming spherocytes, red cells lose cell volume without proportionate loss of hemoglobin.
B. Platelets
1. Thrombocytopenia - a deficiency in platelets (< 60,000/mm3). May occur with certain viral diseases.
2. Role in clot formation:
a. With trauma, platelets release seratonin which causes contraction of vascular smooth muscle, reducing blood loss.
b. When vessel ruptures, platelets agglutinate, forming a plug which helps close the gap
c. Platelets release the enzyme thromboplastin
Fibrinogen
thromboplastin
Thrombin
Fibrin (composes thrombus)
3. Functions in the maintenance of endothelial cells - with thrombocytopenia, blood vessels loose their competence and blood seeps out into the tissue, resulting in the condition of thrombocytopenia purpura.
C. Leukocytes
1. Leukocytosis - refers to an increased number of leukocytes to over 12,000 WBCs/mm3; occurs with infections (appendix, etc.) (40,000-100,000 WBCs/mm3); not used to refer to leukemic conditions
2. Leukopenia - refers to a decreased number of WBCs to less than 4,000 WBCs/mm3; seem to occur with viral infections, rickettsiae, protozoal infections
3. Differential count - refers to a count made by observation on the stained blood smear of the proportion of the different leukocyte types expressed in percentages.
a. Count 100 to 200 WBCs and determine the percentage of various WBC types
b. Pitfalls to avoid in counting:
i. damaged or degenerating cells
ii. platelet clumps
iii. "difficult" leukocytes
4. Normal values for leukocytes in the American population
5. Leukemia - a progressive proliferation of abnormal leukocytes in the hematopoietic tissues, with an increased number of the abnormal cells in the peripheral blood.
6. Summary: Most hematologic phenomena will be the result of either too few cells (anemia, leukopenia, thrombocytopenia) or excessive, occasionally neoplastic, overproduction of cells (polycythemia - 1o or 2o, leukocytosis and leukemia, thrombocytosis and 1o thrombocythemia). Abnormal cell morphology often accompanies the pathological increases or decreases in cell numbers. In clinical practice, the two most common phenomena you'll see will be patients with below normal hemoglobin values (anemia) and patients with elevated WBCs secondary to infection/ inflammation. Despite the frequent press and journal coverage of the leukemias, they are, fortunately, uncommon diseases.
D. Neutrophils
1.Microphage - a PMN in the tissue destroying a foreign substance
2. Symptoms of an acute inflammatory response:
a. redness - due to vasodilation and increased blood flow
b. heat - due to increased blood flow
c. swelling - due to leakage of fluid from vessels
d. pain - due to pressure on nerves
3. Neutrophilia - an increase in the proportion of PMNs in the peripheral blood
4. Neutropenia - an abnormally small number of neutrophils in the circulating blood
5. Toxic granulation - coarse black or purple granules in the cytoplasm, indicative of infections or inflammatory disease.
E. Lymphocyte
1. Lymphocytosis - occurs with mononucleosis, pertussis, viral infections, chronic lymphocytic leukemia, syphilis
2. Atypical - these lymphocytes may have a vacuolated cytoplasm, and others may contain a lobulated nucleus and sometimes nucleoli. Occurs with infectious mononucleosis, viral hepatitis and other viral infections, tuberculosis, drug (e.g., penicillin) sensitivity