Hematology Quick studyhttp://www.irvingcrowley.com/cls/maturation.htm Identify the sites of haemopoeitic tissue in fetal, neonate and adult life

Sites of haemopoiesis.

Fetus02 months (yolk sac)27 months (liver, spleen)59 months (bone marrow)

InfantBone marrow (practically all bones)

AdultsVertebrae, ribs, sternum, skull, sacrum and pelvis, proximal ends of femur

List the cell lines in the bone marrow that are derived from the pluripotential stem cells

Outline the development of red cells, granulocytes and platelets from the pluripotential stem cell to the end cell.

Identify the growth factors (including cytokines) which influence the development of various cell linesHaemopoietic tissue growth factors This is a diagram of the role of growth factors in normal haemopoiesis. Multiple growth factors act on the earlier marrow stem and progenitor cells. EPO, erythropoietin; PSC, pluripotential stem cell; SCF, stem cell factor; TPO, thrombopoietin.

Outline the role of connective tissues elements in Haemopoietic function

FUNCTIONS OF CONNECTIVE TISSUE Structural support The connective tissues serve several functions, of which the most prominent function is structural support to enable maintenance of anatomical form of organs and organ systems. Examples include the connective tissue capsules surrounding organs (such as the kidney, lymph nodes). The loose connective tissue acts to fill the spaces between organs. The tendons (connecting muscles to bone) and the elastic ligaments (connecting bones to bones) are examples of specialized orderly forms of connective tissue. The skeletal tissues (cartilage and bone) are special forms of connective tissue. Metabolic functions The connective tissues serve a nutritive role. All the metabolites from the blood pass from capillary beds and diffuse through the adjacent connective tissue to cells and tissues. Similarly waste metabolites from the cells and tissues diffuse through the loose connective tissue before returning to the blood capillaries. The adipose tissue (especially that of the hypodermis) serves as an energy store and also provides thermal insulation. Surplus calories can be converted into lipid and stored in adipocytes. Blood components and blood vessels The hematopoietic tissues (blood-forming tissues) are a further specialized form of connective tissue. These include the myeloid tissue (bone marrow) and the lymphoid (lymphatic) tissue. The lining of the blood and lymphatic vessels (endothelial cells) as well as the peripheral blood, are also specialized forms of connective tissue. Defensive functions Various components of the connective tissue play roles in the defense or protection of the body including many of the components of the vascular and immune systems (plasma cells, lymphocytes, neutrophils, eosinophils, basophils, mast cells). The various macrophages of the body are also categorized as connective tissue cells. These all develop from monocytes and are grouped as part of the Mononuclear Phagocyte System of the body. Macrophages are important in tissue repair as well as defense against bacterial invasion. The fibroblasts of connective tissue proliferate in response to injury of organs and migrate to and deposit abundant new collagen fibers, resulting in the formation of fibrous scar tissue.

Cell typeChief function

MesenchymeEmbryonic source of all connective tissue cells

Fibroblasts Chondroblasts OsteoblastsStructural support

Plasma cells Lymphocytes Neutrophils Eosinophils Basophils Mast cells MacrophagesDefense and immune

AdipocytesMetabolic Energy storage Thermal insulation

The formed elements of the blood. List the cellular components in peripheral blood.

Identify site of action of erythropoietin, GCSF and GMCSF

This is a diagrammatic representation of the bone marrow pluripotent stem cell and the cell lines that arise from it.Various progenitor cells can be identified by culture in semi-solid medium by the type of colony they form. Baso, basophil; BFU, burst-forming unit; CFU, colony-forming unit; E, erythroid; Eo, eosinophil; GEMM, granulocyte, erythroid, monocyte and megakaryocyte; GM, granulocyte, monocyte; Meg, megakaryocyte; NK, natural killer.

Outline factors which lead to increased production of erythropoietin

List the parameters in the CBC/FBC. Discuss the importance of the CBC/FBC.To determine general health status and to screen for a variety of disorders, such as anaemia and infection, inflammation nutritional status and exposure to toxic substances A CBC test usually includes: White blood cell (WBC, leukocyte) count. White blood cells protect the body against infection. If an infection develops, white blood cells attack and destroy the bacteria, virus, or other organism causing it. White blood cells are bigger than red blood cells but fewer in number. When a person has a bacterial infection, the number of white cells rises very quickly. The number of white blood cells is sometimes used to find an infection or to see how the body is dealing with cancer treatment.

White blood cell types (WBC differential). The major types of white blood cells are neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Immature neutrophils, called band neutrophils, are also part of this test. Each type of cell plays a different role in protecting the body. The numbers of each one of these types of white blood cells give important information about the immune system. Too many or too few of the different types of white blood cells can help find an infection, an allergic or toxic reaction to medicines or chemicals, and many conditions, such as leukemia.

Red blood cell (RBC) count. Red blood cells carry oxygen from the lungs to the rest of the body. They also carry carbon dioxide back to the lungs so it can be exhaled. If the RBC count is low (anemia), the body may not be getting the oxygen it needs. If the count is too high (a condition called polycythemia), there is a chance that the red blood cells will clump together and block tiny blood vessels (capillaries). This also makes it hard for your red blood cells to carry oxygen.

Hematocrit (HCT, packed cell volume, PCV). This test measures the amount of space (volume) red blood cells take up in the blood. The value is given as a percentage of red blood cells in a volume of blood. For example, a hematocrit of 38 means that 38% of the blood's volume is made of red blood cells. Hematocrit and hemoglobin values are the two major tests that show if anemia or polycythemia is present.

Hemoglobin (Hgb). The hemoglobin molecule fills up the red blood cells. It carries oxygen and gives the blood cell its red color. The hemoglobin test measures the amount of hemoglobin in blood and is a good measure of the blood's ability to carry oxygen throughout the body.

Red blood cell indices. There are three red blood cell indices: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). They are measured by a machine and their values come from other measurements in a CBC. The MCV shows the size of the red blood cells. The MCH value is the amount of hemoglobin in an average red blood cell. The MCHC measures the concentration of hemoglobin in an average red blood cell. These numbers help in the diagnosis of different types of anemia. Red cell distribution width (RDW) can also be measured which shows if the cells are all the same or different sizes or shapes.

Platelet (thrombocyte) count. Platelets (thrombocytes) are the smallest type of blood cell. They are important in blood clotting. When bleeding occurs, the platelets swell, clump together, and form a sticky plug that helps stop the bleeding. If there are too few platelets, uncontrolled bleeding may be a problem. If there are too many platelets, there is a chance of a blood clot forming in a blood vessel. Also, platelets may be involved in hardening of the arteries (atherosclerosis).

Mean platelet volume (MPV). Mean platelet volume measures the average amount (volume) of platelets. Mean platelet volume is used along with platelet count to diagnose some diseases. If the platelet count is normal, the mean platelet volume can still be too high or too low

Give the specimen tube/ anticoagulant used to collect a specimen for a complete blood count / full blood count (CBC/FBC).

Blood Collection Tubes

Complete Blood Count (CBC): EDTA tubes (purple top): Purple top tubes should be 50-60% full. Do not overfill tubes. Gently mix specimen by inverting 5-10 times and place it on a rocker for up to 30 minutes, then refrigerate at 2-8C. When a differential is required as part of a CBC, slides must be prepared within 12 hours of blood collection. Refrigerated EDTA blood is stable for CBC for up to 24 hours. Clotted or hemolyzed specimens are unacceptable. Check for clots by using a clean wooden applicator stick and gently swirling blood in tube. EDTA microtainers must be shaken 10-15 times to overcome the surface tension within the tube Serum Chemistry: Plain or serum separator tube (red top or SST, red/camouflage top): SST tubes should be at least 50-80% full and should be mixed. No mixing is necessary with plain tubes. Both plain and SST tubes must be allowed to clot at 4C for 30 minutes - 60 minutes. Then the samples must be centrifuged at 7000rpm for 10 minutes and the serum decanted and frozen at -80C for clinical chemistry analysis.

Heparin tube (green top chemistry/blood gas): Tubes should be at least 50% full. Gently mix specimen by inverting 8-10 times and place immediately on at 4C for 30 minutes - 60 minutes before spinning in a centrifuge, although the exact time allowable may be protocol specific. Coagulation: Sodium citrate tubes (blue top): Citrate tubes should be filled to the top. Gently mix specimen by inverting at least 5-6 times and place immediately at 4C for up to 30 minutes before spinning in a centrifuge. Blood Glucose: Sodium fluoride tubes (gray top): Tubes should be at least 65-80% full. Gently mix specimen by inverting 5-6 times and place immediately at 4C for up to 30 minutes before spinning in a centrifuge If blood is placed into tubes after removing the tube tops, care must be taken not to cross contaminate tubes containing anticoagulant. It is best to remove the needle before filling the tubes. Capillary tubes: Take capillary tube and place finger on the end of tube that has the blue or red fill line mark on it. Place other end of the capillary tube into the tip of a blood filled syringe, in a drop of blood on Parafilm, or along the edge of a tube of blood (being careful not to spill it!). Release finger and allow blood to fill the capillary tube (If possible to the blue or red line) by capillary action. If less blood is available, fill as full as is possible.

Green-Top Tube (Sodium Heparin): This tube contains sodium heparin -- used for collection of heparinized plasma or whole blood for special tests. Note: After tube has been filled with blood, immediately invert tube several times to prevent coagulation.Grey-Top Tube (Potassium Oxalate/Sodium Fluoride): This tube contains potassium oxalate as an anticoagulant and sodium fluoride as a preservative -- used to preserve glucose in whole blood and for some special chemistry tests. Note: After tube has been filled with blood, immediately invert tube several times to prevent coagulation.Lavender-Top Tube (EDTA): This tube contains EDTA as an anticoagulant -- used for most hematological procedures. Note: After tube has been filled with blood, immediately invert tube several times to prevent coagulation.Light Blue-Top Tube (Sodium Citrate): This tube contains sodium citrate as an anticoagulant -- used for drawing blood for coagulation studies. Note: It is imperative that the tube be completely filled. The ratio of blood to anticoagulant is critical for valid prothrombin time results. Immediately after draw, invert tube 6 to 10 times to activate the anticoagulant.Red-Top Tube: This tube is a plain VACUTAINER containing no anticoagulant -- used for collection of serum for selected chemistry tests as well as clotted blood for immunohematology.Royal Blue-Top Tube: There are two types of royal bluetop Monoject tubes -- one with the anticoagulant EDTA and the other plain. These are used for collection of whole blood or serum for trace element analysis. To determine tube type necessary, refer to individual metals in individual test listings.Serum Gel Tube: This tube contains a clot activator and serum gel separator -- used for various laboratory tests. Note: Invert tube to activate clotting; let stand for 20 to 30 minutes before centrifuging for 10 minutes. If frozen serum is required, pour off serum into plastic vial and freeze. Do not freeze VACUTAINER(S).Special Collection Tubes: Some tests require specific tubes for proper analysis. To obtain correct tubes for metal analysis or other tests as identified in individual test listings. Yellow-Top Tube (ACD): This tube contains ACD -- used for drawing whole blood for special tests

List the functions of the erythrocytes in relation to its structureThe majority of normal red cells or erythrocytes are disciform in shape (Fig. 3.12) [4]; a minority are bowl-shaped. On a stained peripheral blood film they are approximately circular in outline and show only minor variations in shape and moderate variations in size (Fig. 3.13). The average diameter is about 7.5 m. In the area of a film where cells form a monolayer, a paler central area occupies approximately the middle third of the cell.The normal shape and flexibility of a red cell are dependent on the integrity of the cytoskeleton to which the lipid membrane is bound. An abnormal shape can be caused by a primary defect of the cytoskeleton or membrane or be secondary to red cell fragmentation or to polymerization, crystallization or precipitation of haemoglobin.

Fig. 3.12 Scanning electron micrograph of a normal redcell (discocyte). Courtesy of Professor A. Polliack,Jerusalem, from Hoffbrand and Pettit [4].

Fig. 3.13 Peripheral blood film of a healthy subjectshowing normal red cells and platelets. The red cells showlittle variation in size and shape. Some of the plateletsshow granules dispersed through the cytoplasm whileothers have a granulomere and a hyalomere.

Functions of Red Blood CellsApart from carrying oxygen, which is the main function of red blood cell, it can also conduct the following functions.1. Release the enzyme carbonic anhydrase which allows water in the blood to carry carbon dioxide to the lungs where it is expelled.2. Control the pH of the blood by acting as an acid-base buffer.Shape and Size of a Red Blood CellA red blood cell is a biconcave disc. Simply it is a round ball that is squeezed from two opposite ends to appear, widest at the sides and narrowest in the middle.A red blood cell measures about 6 to 8 micrometers in diameter (average = 7.8 um) with an average thickness of 2 micrometers (2.5 um at the thickest point and less than 1um at the center). Although a red blood cell is wider than some capillaries, its flexibility allows it to become distorted as it squeezes through narrow passages and then restores to its original shape.There shape confers them a degree of flexibility as some capillaries are smaller than the diameter of a RBC. Secondly, the biconcave shape of the RBC gives it an ideal surface area to volume ratio for maximum gas exchange.

List the major blood group antigens (e.g. ABO), discuss rhesus blood group systems in humans and just make note of other systems

RHESUS BLOOD GROUPING SYSTEMThe Rh system was named after rhesus monkeys, since they were initially used in the research to make the antiserum for typing blood samples. If the antiserum agglutinates your red cells, you are Rh+ . If it doesn't, you are Rh- . Despite its actual genetic complexity, the inheritance of this trait usually can be predicted by a simple conceptual model in which there are two alleles, D and d. Individuals who are homozygous dominant (DD) or heterozygous (Dd) are Rh+. Those who are homozygous recessive (dd) are Rh- (i.e., they do not have the key Rh antigens).Clinically, the Rh factor, like ABO factors, can lead to serious medical complications. The greatest problem with the Rh group is not so much incompatibilities following transfusions (though they can occur) as those between a mother and her developing fetus. Mother-fetus incompatibility occurs when the mother is Rh- (dd) and her fetus is Rh+ (DD or Dd). Maternal antibodies can cross the placenta and destroy fetal red blood cells. The risk increases with each pregnancy. Europeans are the most likely to have this problem--13% of their newborn babies are at risk. Actually only about of these babies (6% of all European births) have complications. With preventive treatment, this number can be cut down even further. Less than 1% of those treated have trouble. However, Rh blood type incompatibility is still the leading cause of potentially fatal blood related problems of the newborn. In the United States, 1 out of 1000 babies are born with this condition.Rh type mother-fetus incompatibility occurs only when an Rh+ man fathers a child with an Rh- mother. Since an Rh+ father can have either a DD or Dd genotype, there are 2 mating combinations possible with differing risks as shown below. Regardless of the father's genotype, if he is Rh+ and the mother is Rh-, doctors assume that there will be an incompatibility problem and act accordingly.

Human fetus in a mother's uterus(the umbilical cord and placentaconnect the fetus to its mother)

Keep in mind that only the Rh+ children (Dd) are likely to have medical complications. When both the mother and her fetus are Rh- (dd), the birth will be normal.The first time an Rh- woman becomes pregnant, there usually are not incompatibility difficulties for her Rh+ fetus. However, the second and subsequent births are likely to have life-threatening problems for Rh+ fetuses. The risk increases with each birth. In order to understand why first born are normally safe and later children are not, it is necessary to understand some of the placenta's functions. It is an organ that connects the fetus to the wall of the uterus via an umbilical cord. Nutrients and the mother's antibodies regularly transfer across the placental boundary into the fetus, but her red blood cells usually do not (except in the case of an accidental rupture). Normally, anti-Rh+ antibodies do not exist in the first-time mother unless she has previously come in contact with Rh+ blood. Therefore, her antibodies are not likely to agglutinate the red blood cells of her Rh+ fetus. Placental ruptures do occur normally at birth so that some fetal blood gets into the mother's system, stimulating the development of antibodies to Rh+ blood antigens. As little as one drop of fetal blood stimulates the production of large amounts of antibodies. When the next pregnancy occurs, a transfer of antibodies from the mother's system once again takes place across the placental boundary into the fetus. The anti-Rh+ antibodies that she now produces react with the fetal blood, causing many of its red cells to burst or agglutinate. As a result, the newborn baby may have a life-threatening anemia because of a lack of oxygen in the blood. The baby also usually is jaundiced, fevered, quite swollen, and has an enlarged liver and spleen. This condition is called erythroblastosis fetalis . The standard treatment in severe cases is immediate massive transfusions of Rh- blood into the baby with the simultaneous draining of the existing blood to flush out Rh+ antibodies from the mother. This is usually done immediately following birth, but it can be done to a fetus prior to birth. Later, the Rh- blood will be replaced naturally as the baby gradually produces its own Rh+ blood. Any residual anti-Rh+ antibodies from the mother will leave gradually as well because the baby does not produce them.Erythroblastosis fetalis can be prevented for women at high risk (i.e., Rh- women with Rh+ mates or mates whose blood type is unknown) by administering a serum (Rho-GAM) containing anti-Rh+ antibodies into the mother around the 28th week of pregnancy and again within 72 hours after the delivery of an Rh+ baby. This must be done for the first and all subsequent pregnancies. The injected antibodies quickly agglutinate any fetal red cells as they enter the mother's blood, thereby preventing her from forming her own antibodies. The serum provides only a passive form of immunization and will shortly leave her blood stream. Therefore, she does not produce any long-lasting antibodies. This treatment can be 99% effective in preventing erythroblastosis fetalis. Rho-GAM is also routinely given to Rh- women after a miscarriage, an ectopic pregnancy, or an induced abortion. Without the use of Rho-GAM, an Rh- woman is likely to produce larger amounts of Rh+ antibodies every time she becomes pregnant with an Rh+ baby because she is liable to come in contact with more Rh+ blood. Therefore, the risk of life-threatening erythroblastosis fetalis increases with each subsequent pregnancy.Anti-Rh+ antibodies may be produced in an individual with Rh- blood as a result of receiving a mismatched blood transfusion. When this occurs, there is likely to be production of the antibodies throughout life. Once again, Rho-GAM can prevent this from happening.Mother-fetus incompatibility problems can result with the ABO system also. However, they are very rare--less than .1% of births are affected and usually the symptoms are not as severe. It most commonly occurs when the mother is type O and her fetus is A, B, or AB. The symptoms in newborn babies are usually jaundice, mild anemia, and elevated bilirubin levels. These problems in a baby are usually treated successfully without blood transfusions.NOTE: Identifying someone as being Rh+ or Rh- is a simplification. There are many variations of Rh blood types depending on which of the 45 Rh antigens are present. The most important of these antigens for mother-fetus incompatibility and transfusion problems apparently are D, C, c, E, and e. When an individual is identified as being Rh+ or Rh-, it is usually is in reference to the D antigen. In other words, the individual is RhD+ or RhD-.Other blood grouping systemshttp://faculty.matcmadison.edu/mljensen/BloodBank/lectures/other_blood_group_systems.htm Explain the importance of blood group antigens in blood transfusion

Blood TransfusionsBlood that has antibodies on it that is not recognized by the body will be attacked by your immune systemO is the Universal Donor because a person with this type of blood does not have antigens on the surface of the blood cells - hence will not cause an immune reaction in the patient.AB is the universal Acceptor because this person will not have an immune reaction to A, B, AB, or O*Just remember, the antigens on the surface of your cells (or donated cells) will cause a reaction if your immune system does not recognize them as being part of you. Hence, if you are Type A, and transfused with Type B, your body will mobilize a massive immune response against the "invading" blood. This will cause coagulation of blood and death.----- AGGLUTINATION (the clumping of red blood cells following a transfusion reaction; likely fatal)

Blood SafetyBlood can carry diseases and health care professionals must be careful when working with blood. A bloodborne pathogen is any disease causing agent that is present in the blood and can be transferred from one person to another. HEPATITIS B (HBV)HEPATITIS C (HCV)HUMAN IMMUNODEFICIENCY VIRUS (HIV)MALARIATesting Your BloodA test kit can be used to test your blood type. It involves pricking your finger and placing a drop of blood on a card that will react to a serum on the card that contains antibodies. You will be given the opportunity to test your blood type using this technique.

List the white blood cells present in peripheral blood giving their functions and identify them on a blood filmNormal peripheral blood The usual diagnostic approach to blood disorders is blood counting and blood film examination. Blood films on glass slides are stained with a Romanowsky stain (usually Wright's, Giemsa, or May-Grnwald). Red cells in normal peripheral blood are circular and fairly uniform in size. Mild variation in shape (poikilocytosis) and size (anisocytosis) is seen. Platelets appear as small bluish-purple discs. During blood film examination, the individual types of white blood cells are enumerated; this is referred to as the differential count. Slide Listings Band neutrophils Basophil Eosinophil Erythrocyte (red blood cell) Lymphocyte Monocytes Platelets Segmented neutrophilsBand neutrophil Band neutrophils comprise approximately 1 to 3% of the peripheral leukocytes. They are usually 9 to 15 m in diameter. The nucleus forms a "U" or curled rod prior to segmentation. The chromatin pattern is coarse and clumped. The cytoplasm is moderate to abundant with a few nonspecific granules and many specific granules.

Basophil Basophils are granulocytes that contain purple-blue granules that contain heparin and vasoactive compounds. They comprise approximately 0.5% of the total leukocyte count. Basophils participate in immediate hypersensitivity reactions, such as allergic reactions to wasp stings, and are also involved in some delayed hypersensitivity reactions. Basophils are the smallest circulating granulocytes, averaging 10 to 15 m in diameter. The nucleus to cytoplasm ratio is about 1:1, and the nucleus is often unsegmented or bilobed, rarely with three or four lobes. The chromatin pattern is coarse and patchy, staining a deep blue to reddish-purple. The cytoplasm is a homogenous pale blue, but this is often obscured by the large dark granules.

Eosinophil Eosinophils are the mature granulocytes that respond to parasitic infections and allergic conditions. Eosinophils comprise about 1 to 4% of the peripheral leukocytes. They are usually 9 to 15 m in diameter. Granules stain a bright reddish-orange with Wright's or Giemsa stains. The nucleus contains one to three lobes. The chromatin pattern is coarse and clumped. The cytoplasm is abundant with a full complement of bright reddish-orange specific granules.

Erythrocytes (red blood cells) The mature red blood cell (rbc) consists primarily of hemoglobin (about 90%). The membrane is composed of lipids and proteins. In addition, there are numerous enzymes present which are necessary for oxygen transport and cell viability. The main function of the red cell is to carry oxygen to the tissues and return carbon dioxide from the tissues to the lungs. The protein hemoglobin is responsible for most of this exchange. Normal red blood cells are round, have a small area of central pallor, and show only a slight variation in size. A normal red cell is 6-8 m in diameter. As the relative amount of hemoglobin in the red cell decreases or increases, the area of central pallor will decrease or increase accordingly.

Lymphocyte Lymphocytes in the peripheral blood have been described on the basis of size and cytoplasmic granularity. Small lymphocytes are the most common, ranging in size from 6 to 10 m. The nucleus is usually round or slightly oval, occasionally showing a small indentation due to the adjacent centrosome. Except in the smallest cells, the nucleus is about 7 m in diameter, a size that has been convenient for estimating the size of the surrounding erythrocytes. Nuclear chromatin stains a dark reddish-purple to blue with large dark patches of condensed chromatin. The nuclear cytoplasm ratio is 5:1 to 3:1, and the cytoplasm is often seen only as a peripheral ring around part of the nucleus.

Platelets Platelets are the cytoplasmic fragments of megakaryocytes, circulating as small discs in the peripheral blood. They are responsible for hemostasis (the stoppage of bleeding) and maintaining the endothelial lining of the blood vessels. During hemostasis, platelets clump together and adhere to the injured vessel in this area to form a plug and further inhibit bleeding. Platelets average 1 to 4 m in diameter. The cytoplasm stains light blue to purple, and is very granular. There is no nucleus present. Normal blood concentrations range from 130,000 to 450,000/L.

Segmented neutrophil (seg) Segmented neutrophils (polymorphonuclear leukocytes, or segs) are the mature phagocytes that migrate through tissues to destroy microbes and respond to inflammatory stimuli. Segmented neutrophils comprise 40-75 % of the peripheral leukocytes. They are usually 9 to 16 m in diameter. The nuclear lobes, normally numbering from 2 to 5, may be spread out so that the connecting filaments are clearly visible, or the lobes may overlap or twist. The chromatin pattern is coarse and clumped. The cytoplasm is abundant with a few nonspecific granules and a full complement of rose-violet specific granules.

List the features of the blood count and blood film that are associated with: acute bacterial infection, viral infection, parasitic infestations and chronic infection.White cells Acute Bacterial infection: increase neutrophils Parasitic infestation: increase eosinophils Acute Viral infection: increase lymphocytesNormal values vary with age. White counts are highest in children under one year of age and then decrease somewhat until adulthood. The increase is largely in the lymphocyte population. Adult normal values are shown below. WBC count: 4,500-11,000/L polymorphonuclear neutrophils: 1800-7800/L; (50-70%) band neutrophils: 0-700/L; (0-10%) lymphocytes: 1000-4800/L; (15-45%) monocytes: 0-800/L; (0-10%) eosinophils: 0-450/L; (0-6%) basophils: 0-200/L; (0-2%)

Acute lymphocytic leukemia (ALL) Acute lymphocytic leukemia (ALL) usually strikes children between the ages of 2 to 10. A second peak in incidence is seen in elderly patients.Only half of all patients with ALL have increased leukocytes and may not have lymphoblasts in their peripheral blood. Neutropenia, thrombocytopenia, and anemia are usually present. Patients have symptoms of fatigue, fever and bleeding. There is often lymph node enlargement. Enlargement of the spleen and of the liver may be seen. The cells depicted in the image below are two lymphoblasts and a neutrophil.

Acute myelocytic leukemia (AML) Acute myelocytic leukemia (AML), is the most common leukemia in children less than 1 year of age. It is rare in older children and adolescents, but a second peak of incidence occurs among adults 40 years of age. The patient usually has an elevated white blood cell count, and myeloblasts are present.Anemia, thrombocytopenia, and neutropenia give rise to the clinical findings of pallor, bruising and bleeding, fever with infections, and fatigue. Bone pain and joint pain are seen as the first symptoms in 25% of patients. Enlarged spleen is seen in 50% of all AML patients, but lymph node enlargement is rare. The cell depicted in the image below is a myeloblast. Chronic granulocytic leukemia (CGL) Chronic granulocytic leukemia or chronic myelogenous leukemia can occur at any age, but is most common after the age of 45 years. Weight loss and fatigue are often the initial symptoms. There is usually massive spleen enlargement, which may cause left upper abdominal pain. There is anemia, markedly elevated levels of leukocytes, thrombocytosis, eosinophilia, basophilia, and a predominance of myelocytes in the peripheral blood. Myeloblasts constitute fewer than 10% of circulating leukocytes. Occasional nucleated red blood cells are seen. The cells depicted in the image below are two myeloblasts and a hypersegmented neutrophil.

Chronic lymphocytic leukemia (CLL) Chronic lymphocytic leukemia is the most common type of leukemia and usually occurs in older patients; it is rare in patients less than 40 years of age. The disease is usually discovered when other medical problems are present. Weakness, fatigue, and weight loss are usually seen. The malignant cell in CLL is usually a small, mature-appearing lymphocyte.

Hairy cell leukemia Hairy cell leukemia, or leukemic reticuloendotheliosis, is a rare malignant disorder. It usually occurs in middle-aged patients over 50. The first symptoms of disease include weakness and lethargy. Enlarged spleen occurs in 80% of patients.Hairy cells are characterized by their fine, irregular pseudopods and immature nuclear features. Bone marrow aspiration is often unsuccessful because of complete infiltration by hairy cells, resulting in a dispersed spongy web of cells in an increased meshwork of reticulin fiber.

Infectious mononucleosis Infectious mononucleosis is caused by the Epstein-Barr virus, a DNA herpes-type virus that infects B lymphocytes. Patients present with mild to severe adenopathy, hepatosplenomegaly, fever, malaise, pharyngitis, and a characteristic peripheral blood smear demonstrating reactive lymphocytes.

Malaria Malaria is a disease caused by the parasite Plasmodium. The four species most commonly found in man are vivax, malariae, falciparum, and ovale."Malaria is mainly transmitted from person to person through the bite of the female Anopheles mosquito. Other means of transmission are through the use of contaminated needles, by congenital means, and through blood transfusions.When the infected Anopheles mosquito bites a human, sporozoites are injected into the peripheral blood of the individual. The sporozoites then invade the liver. When the red blood cell has been penetrated by the merozoite, the parasite develops into the trophozoite ring form and thence to a mature schizont. The merozoites rupture from the mature schizonts and penetrate other red blood cells. Fever and chills are associated with the rupture of the red blood cells.Pelger-Hut Anomaly Pelger-Hut anomaly is a benign hereditary condition characterized by decreased segmentation in the neutrophils. These neutrophils usually contain two lobes, but appear to function normally.

Describe the role of platelets in haematostasis

The platelet Role in Hemostasis

In hemostasis, platelets play three main roles: 1. They maintain the endothelial surface. Loss of circulating platelets quickly results in morphologic changes in the endothelial cells of the capillaries. These changes cause intravascular material to leak into the capillary bed. 2. They initially arrest bleeding in severed blood vessels. 3. They provide phospholipid, which acts as the catalytic surface for initiation of the coagulation process.

When platelets encounter a break in the endothelial surface, several important actions cause the bleeding to cease.

A. Adhesion occurs when they encounter collagen, membranes or non-collagenous microfibrils beneath the basement membranes. Adhesion is mediated by GP-Ib and von Willebrand Factor (vWF).a. Receptors (especially for glycoprotein-Ib) bind vWF and facilitate adhesion. b. vWF is an extremely large molecule (a multimer) that bridges the gap between different cells, the platelet, and subendothelial surfaces. c. vWF is a very sticky protein and binds readily. d. Besides binding GP-Ib, vWF also binds to GP-IIb-IIIa to facilitate adhesion. e. Fibrinogen, binding to the GP IIb-IIIa complex on two separate platelets, can bridge the gap between those two platelets (this is important to a subsequent functionaggregation). f. In the absence of these factors, (or their receptors) both adhesion and aggregation are abnormal and certain types of bleeding problems can occur.

B. Release reaction: Platelets then undergo a "release reaction". This process involves change from a disc shape to a spherical shape, constriction of the microtubules toward the center of the platelet, and the release of contents of the granules (primarily ADP, catecholamine, and serotonin) into the open canalicular system. These platelets thus become activated. a. During the release reaction, the granules within the platelets release their contents into the canalicular system.b. These granule components then leak into the plasma around the platelet.c. The released ADP binds other circulating platelets in close proximity to activated platelets and this binding to surface receptors initiates the release reaction in these recruited platelets. d. The release reaction is mediated by means of Thromboxane A2. Arachidonate is integrated in the phospholipids in the cell wall and is freed by a phospholipase, activated during the process of adhesion or by binding of certain ligands to receptors on the platelet surface. e. Cyclo-oxygenase converts arachidonate to an intermediate, prostaglandin H2. f. In the platelets, PGH2 is acted upon by thromboxane synthetase to form thromboxane A2. Thromboxane A2 promotes the release reaction, change in shape, and aggregation. In the endothelial cell, the pathway is different from that of the platelet.g. Following the formation of PGH2, prostacyclin synthetase produces PGI2, which inhibits adhesion, aggregation and the release reaction, forces that oppose those of Thromboxane A2. h. Aspirin blocks cyclo-oxygenase and therefore the pathway that leads to both Thromboxane A2 and PGI2. In the platelet, the block is permanent for the life of the platelet, because the platelet does not have a nucleus to direct the formation of more cyclo-oxygenase. This yields a platelet that cannot function. Since the life of the platelet is about 7-10 days, the effect of the aspirin on bleeding will gradually decrease over a week, as new platelets replace those that were exposed to aspirin. In the endothelial cell, however, cyclo-oxygenase is regenerated.

C. Platelet aggregation occurs as platelets are "recruited" from the immediate area by the released contents, for example, ADP. This process is accomplished by fibrinogen, binding to the GP IIb-IIIa complex on separate platelets, and bridging the gap between platelets When the release of ADP, or other aggregating agents, is minimal, the local concentration of these agents do not reach a high level, this aggregation may be reversible; with higher concentrations, aggregation is irreversible.

Associated with the change of shape of the platelet and the release reaction, is the appearance of clotting promoting sites (historically referred to as platelet factor 3) on the platelet membranes. The receptor sites for the coagulation proteins serve as a catalytic site for the clotting proteins and assists in initiating the clotting mechanism. Important coagulation proteins that are bound to the surface include factors V and VIII among others.

E. Clot retraction occurs when platelets are trapped within the enlarging blood clot. During the release reaction, pseudopodia like structures extend some distance from the surface of the platelet, and attach to similar structures on adjacent platelets. With time, these structures retract, pulling the body of the clot together, and sealing the vessel wall at the site of injury.

Identify the difference between plasma and serum. List the components of plasma. Describe the functions of the different plasma proteinsBlood serum; the clear liquid that separates from blood when it is allowed to clot completely, and is therefore blood plasma from which fibrogen has been removed during clotting.Blood plasma; the fluid portion of the blood in which the particulate components are suspended.BLOOD PLASMA COMPONENTSWaterThe primary component of blood plasma is water. The American Association of Blood Banks (AABB) estimates that water makes up 90 percent of blood plasma. Blood plasma can provide water to body tissues that are in need of additional fluid or it can absorb excess water and pass it along to the kidneys to be excreted as urine.AlbuminAlbumin is the predominate protein present in blood plasma. This protein prevents blood fluid from inadvertently leaking out of blood vessels, explains the Merck Manual: Home Edition, a medical reference guide for patients and caregivers. Albumin also helps carry certain substances, such as hormones and ingested drug products, through the body to the appropriate cells and tissues.AntibodiesBlood plasma contains a number of additional proteins called immunoglobulins or antibodies. Antibodies are part of your body's immune system and are produced by the white blood cells, explain health professionals at the National Blood Service in the United Kingdom. If bacteria or viruses invade your body, antibodies within your blood plasma help to identify and fight off infection.Blood Clotting FactorsIf you sustain an injury to your body, you can begin to bleed. The body uses blood clotting factors---which are major components of blood plasma---to prevent you from losing excessive amounts of this vital body fluid. Blood clotting factors, such as fibrinogen or Factor VIII, help the body seal damaged blood vessels and stop bleeding.

BLOOD PLASMA PROTEINS

Draw a diagram of the intrinsic, extrinsic and final common pathways of coagulation and complement systemsThe coagulation mechanismThe blood clothing system or coagulation pathway, like the complement system, is a proteolytic cascade. Each enzyme of the pathway is present in the plasma as a zymogen, in other words in an inactive form, which on activation undergoes proteolytic cleavage to release the active factor from the precursor molecule. The coagulation pathway functions as a series of positive and negative feedback loops which control the activation process. The ultimate goal of the pathway is to produce thrombin, which can then convert soluble fibrinogen into fibrin, which forms a clot. The generation of thrombin can be divided into three phases, the intrinsic and extrinsic pathways that provide alternative routes for the generation of factor X, and the final common pathway which results in thrombin formation (Figure 1.3).

Figure 1.3: The intrinsic, extrinsic, and common pathways of the coagulation (clotting) cascade The intrinsic pathway is activated when blood comes into contact with sub-endothelial connective tissues or with negatively charged surface that are exposed as a result of tissue damage. Quantitatively it is the most important of the two pathways, but is slower to cleave fibrin than the extrinsic pathway. The Hageman factor (factor XII), factor XI, prekallikrein, and high molecular weight kininogen (HMWK) are involved in this pathway of activation. Thus this pathway provides a further of the interrelationship between the various enzyme cascade systems in plasma. The first step is the binding of Hageman factor to a sub-endothelial surface exposed by an injury. A complex of prekallikrein and HMWK also interacts with the exposed surface in close proximity to the bound factor XII, which becomes activated. During activation, the single chain protein of the native Hageman factor is cleaved into two chains (50 and 28 kDa), that remain linked by a disulphide bond. The light chain (28kDa) contains the active site and the molecule is referred to as activated Hageman factor (factor XIIa). There is evidence that the Hageman factor can autoactivate, thus the pathway is self-amplifying once triggered (compare with the alternative pathway of complement). Activated Hageman factor in turn activates prekallikrein. The kallikrein produced can then also cleave factor XII, and a further amplification mechanism is triggered. The activated factor XII remains in close contact with the activating surface, such that it can activate factor XI, the next step in the intrinsic pathway which, to proceed efficiently, requires Ca2+ . Also involved at this stage is HMWK, which binds to factor XI and facilitates the activation process. Activated factors XIa, XIIa, and kallikrein are all serine proteases, like many of the enzymes of the complement system. Eventually the intrinsic pathway activates factor X, a process that can also be brought about by the extrinsic pathway. Factor X is the first molecule of the common pathway and is activated by a complex of molecules containing activated factor IX, factor VIII, calcium, and phospholipid which is provided by the platelet surface, where this reaction usually takes place. The precise role of factor VIII in this reaction is not clearly understood. Its presence in the complex is obviously essential, as evidenced by the serious consequences of factor VIII deficiency experienced by haemophiliacs. Factor VIII is modified by thrombin, a reaction that results in greatly enhanced factor VIII activity, promoting the activation of factor X. The extrinsic pathway is an alternative route for the activation of the clothing cascade. It provides a very rapid response to tissue injury, generating activated factor X almost instantaneously, compared to the seconds or even minutes required for the intrinsic pathway to activate factor X. The main function of the extrinsic pathway is to augment the activity of the intrinsic pathway. There are two components unique to the extrinsic pathway, tissue factor or factor III, and factor VII. Tissue factor is present in most human cells bound to the cell membrane. The activation process for tissue factor is not entirely clear. Once activated, tissue factor binds rapidly to factor VII which is then activated to form a complex of tissue factor, activated factor VII, calcium, and a phospholipid, and this complex then rapidly activates factor X. The intrinsic and extrinsic systems converge at factor X to a single common pathway which is ultimately responsible for the production of thrombin (factor IIa). Clot formation. The end result of the clotting pathway is the production of thrombin for the conversion of fibrinogen to fibrin. Fibrinogen is a dimer soluble in plasma. Exposure of fibrinogen to thrombin results in rapid proteolysis of fibrinogen and the release of fibrinopeptide A. The loss of small peptide A is not sufficient to render the resulting fibrin molecule insoluble, a proces that is required for clot formation, but it tends to form complexes with adjacent fibrin and fibrinogen molecules. A second peptide, fibrinopeptide B, is then cleaved by thrombin, and the fibrin monomers formed by this second proteolytic cleavage polymerize spontaneously to form an insoluble gel. The polymerized fibrin, held together by noncovalent and electrostatic forces, is stabilized by the transamidating enzyme factor XIIIa, produced by the action of thrombin on factor XIII. These insoluble fibrin aggregates (clots), together with aggregated platelets ( thrombi), block the damaged blood vessel and prevent further bleeding. There is an interrelationships between the coagulation pathway and other plasma enzyme systems. Contact activation of the coagulation pathway, in addition to promoting blood clotting, results in the generation of plasminogen activator activity, which is involved in fibrinolysis or clot removal. Activated Hageman factor and its peptides can also initiate the formation of kallikrein from plasma prekallikrein, and this triggers the release of bradykinin from kininogens in the plasma. Kinins are responsible for dilating small blood vessels, inducing a fall in blood presssure, triggering smooth muscle contraction, and increasing the permeability of vessel walls. In addition, activation of the coagulation pathway produces a vascular permeability factor, as well as chemotactic peptides for professional phagocytes.

Describe the vascular epithelium and its role in haematostasis as a pro-coagulant and anti-coagulant surface

Platelet pro-coagulant activityAfter platelet aggregation and release, the exposed membrane phospholipid (platelet factor 3) is available for two reactions in the coagulation cascade. Both phospholipid-mediated reactions are calcium ion dependent. The first (tenase) involves factors IXa, VIIIa and X in the formation of factor Xa(Fig. 22.7). The second (prothrombinase) results in the formation of thrombin from the interaction of factors Xa, Va and prothrombin (II). The phospholipid surface forms an ideal template for the crucial concentration and orientation of these proteins.

Growth factorPDGF found in the specific granules of platelets stimulates vascular smooth muscle cells to multiply and this may hasten vascular healing following injury.Natural inhibitors of platelet functionNitric oxide (NO) is constitutively released from endothelial cells and also from macrophages and platelets. It has a short half-life of 3-5 s. It inhibits platelet activation and promotes vasodilatation. Prostacyclin synthesized by endothelial cells also inhibits platelet function (Fig. 22.8) and causes vasodilatation by raising cyclic guanosine monophosphate (GMP) levels. The transmembrane protein PECAM-1 is expressed also on endothelial cells. It is its own ligand and inhibits platelet activation by collagen.

Define bleeding time (BT), prothrombin time (PT) and partial thromboplastin time (PTT). State the importance of each test and the relevant specimen tube/ anticoagulant.Bleeding Time is a crude test of hemostasis (the arrest or stopping of bleeding). It indicates how well platelets interact with blood vessel walls to form blood clots.

Bleeding time is used most often to detect qualitative defects of platelets, such as Von Willebrand's disease. The test helps identify people who have defects in their platelet function. This is the ability of blood to clot following a wound or trauma. Normally, platelets interact with the walls of blood vessels to cause a blood clot. There are many factors in the clotting mechanism, and they are initiated by platelets. The bleeding time test is usually used on patients who have a history of prolonged bleeding after cuts, or who have a family history of bleeding disorders. Also, the bleeding time test is sometimes performed as a preoperative test to determine a patient's likely bleeding response during and after surgery. However, in patients with no history of bleeding problems, or who are not taking anti-inflammatory drugs, the bleeding time test is not usually necessary.

Prothrombin time (PT) The rate at which prothrombin is converted to thrombin in citrated blood with added calcium; used to assess the extrinsic coagulation system of the blood The PT test is used to monitor patients taking certain medications as well as to help diagnose clotting disorders.DiagnosisPatients who have problems with delayed blood clotting are given a number of tests to determine the cause of the problem. The prothrombin test specifically evaluates the presence of factors VIIa, V, and X, prothrombin, and fibrinogen. Prothrombin is a protein in the liquid part of blood (plasma) that is converted to thrombin as part of the clotting process. Fibrinogen is a type of blood protein called a globulin; it is converted to fibrin during the clotting process. A drop in the concentration of any of these factors will cause the blood to take longer to clot. The PT test is used in combination with the partial thromboplastin time (PTT) test to screen for hemophilia and other hereditary clotting disorders.MonitoringThe PT test is also used to monitor the condition of patients who are taking warfarin (Coumadin). Warfarin is a drug that is given to prevent clots in the deep veins of the legs and to treat pulmonary embolism. It interferes with blood clotting by lowering the liver's production of certain clotting factors.

Partial Thromboplastin Time A test for detecting coagulation defects of the intrinsic system by adding activated partial thromboplastin to a sample of test plasma and to a control sample of normal plasma. The time required for the formation of a clot is compared with the normal plasma. It is also used to monitor the activity of heparin in patients who are being treated for a variety of cardiovascular disorders.

The partial thromboplastin time (PTT) test is a blood test that is done to investigate bleeding disorders and to monitor patients taking an anticlotting drug (heparin).DiagnosisBlood clotting (coagulation) depends on the action of substances in the blood called clotting factors. Measuring the partial thromboplastin time helps to assess which specific clotting factors may be missing or defective.MonitoringCertain surgical procedures and diseases cause blood clots to form within blood vessels. Heparin is used to treat these clots. The PTT test can be used to monitor the effect of heparin on a patient's coagulation system.

Light blue: A reversible anticoagulant Sodium citrate in measured amount is present. Used for coagulation assays (Prothrombin time, Partial Thromboplastin Time). Full draw is essential since dilution factor with liquid citrate should be maintained.

List the anticoagulants used in the laboratory and state their mechanisms of action

Describe naturally occurring anticoagulants and outline their role in health and disease

Describe the fibrinolytic system

b