Clinical Practice 4

(CP4)

HAEMATOLOGY HAEMATOLOGY HAEMATOLOGY HAEMATOLOGY IN IN IN IN

CLINICAL PRACTICECLINICAL PRACTICECLINICAL PRACTICECLINICAL PRACTICE

Course Handbook and

Guidelines for Undergraduates

Author: Dr C Dainty Basic Science Teacher/ Undergraduate Medical Education Postgraduate Tutor

CONTENT

1 Introduction Page

Haematology Handbook. This handbook has been designed to give you a basic understanding of science needed to understand haematological investigations and disease It is intended to cover the essentials to enable you to formulate further objectives and reading as appropriate. It is not intended to replace other texts and should be used alongside your clinical attachments.

Learning Outcomes.

1. List main constituents of blood and their functions. 2. Name conditions that shift oxygen dissociation curve to right and left 3. Compare differences between foetal and adult haemoglobin 4. To explain significance of differential WBC 5. Compare difference between primary and secondary haemostasis 6. List factors needed for coagulation process 7. Classify factors needed for intrinsic, extrinsic and final common pathway. 8. Recognise investigations needed to assess coagulation pathways 9. Name fibrin degradation products 10. List functions spleen and bone marrow. 11. Define and classify different anaemias 12. Read a routine blood count 13. Comment on reticulocyte count 14. Define MCV and discuss its diagnostic and patho physiologic relevance

Blood

Blood transports life-supporting food and oxygen to every cell of the body and removes their waste products. It also helps to maintain body temperature, transports hormones, and fights infections. The brain cells in particular are very dependent on a constant supply of oxygen. If the circulation to the brain is stopped,

death shortly follows.

Blood has two main constituents. The cells, or corpuscles, comprise about 45 percent, and the liquid portion, or plasma, in which the cells are suspended comprises 55 percent. Blood temp is approx 38 degrees C, 5X viscous as water, slightly alkaline Ph av 7.4. An adult male has approx 5-6 L of whole blood(woman 4-5L)

The blood cells comprise three main types:

1. Red blood cells, or erythrocytes; 2. White blood cells, or leukocytes, which in turn are of many different types; 3. and platelets, or thrombocytes.

Each type of cell has its own individual functions in the body.

The plasma is a complex colourless solution, about 90 percent water, that carries different ions and molecules including proteins, enzymes, hormones, nutrients, waste materials such as urea, and fibrinogen, the protein that aids in clotting.

Red Blood Cells

The red blood cells are tiny, round, biconcave disks, averaging about 7.5 microns (0.003 in) in diameter A normal-sized man has about 5 litres (5.3 qt) of blood in his body, containing more than 25 trillion red cells. Because the normal life span of red cells in

the circulation is only about 120 days; more than 200 billion cells are normally destroyed each day by the spleen and must be replaced. Red blood cells, as well as most white cells and platelets, are made by the bone marrow by process of erythopoiesis.

The main function of the red blood cells is to transport oxygen from the lungs to the tissues and to transport carbon dioxide, one of the chief waste products, it to the lungs for release from the body.

The substance in the red blood cells that is largely responsible for their ability to carry oxygen and carbon dioxide is haemoglobin, the material that gives the cells their red colour. It is a protein complex comprising many linked amino acids, and occupies almost the entire volume of a red blood cell. Essential to its structure and function is the mineral iron. It consists of two parts, haem and globin.

Haem is a complex of a porphyrin ring and iron in the ferrous state. Haem is conjugated with globin a polypeptide

The blood cell type responsible for the transport of oxygen and carbon dioxide is the ________.

platelet

lymphocyte

white blood cell

red blood cell

A. The blood cell type responsible for the transport of oxygen and carbon dioxide is the red blood cell (erythrocytes.)

Haemoglobin and transport of Oxygen

The amount of oxygen the blood carries is described as the oxygen content of the blood. Although vast amount of oxygen is carried bound to haemoglobin, a very small amount is dissolved in the plasma the total amount of oxygen carried by blood is given by adding these two amounts together. Under normal circumstances as blood leaves the lungs Hb is almost fully saturated with oxygen and each g of Hb carries 1.39mls of oxygen. As it reaches systemic circulation this falls because of addition of venous blood from lungs and heart. Therefore in arterial blood 1g of Hb is 98% saturated and contains 1.34mls of O2. The amount dissolved is proportional to partial pressure. At 37 Celsius 0.23 mls oxygen dissolves in each L of blood per kPa. Oxygen content t of blood calculated: O2 content = (Hb x 1.34 x SaO2) + (0.23 x Pa O2) = (150 x 1.34x 98%) + (0.23 x 13) = 20.3 ml/l Oxygen combines reversibly with ferrous ion in Hb molecule to form oxy haemoglobin. Hb + O2 >< HbO2 The no of haem units bound with O2 is called Hb saturation If fully loaded it is 100%. In lungs where partial pressure of O2 is high. Oxygenation of Hb is favoured. In tissues where PP is low. Reverse happens. Thus in tissues O2 are released. These are rapid reactions taking less than 0.01 seconds.

This relationship between between saturation of Hb and PP of O2 is; Oxygen dissociation curve

The curve demonstrates that at a normal Pa O2 (13kPa) Hb is approaching 100% sat. Te curve has a sigmoid shape so at low levels of oxygen tension it is difficult for Hb to combine withO2. Steep section of curve is important as it demonstrates that below 92% saturation the drop in saturated Hb falls very quickly.

Other factors that effect Curve

Many factors influence the affinity of this binding and alter the shape of the curve:

right shift left shift

temperature high low

DPG high low

p(CO2) high low

p(CO) low high

pH (Bohr effect) low (acidosis) high (alkalosis)

type of haemoglobin adult haemoglobin fetal haemoglobin

Left shift of the curve is a sign of hemoglobin's increased affinity to take up oxygen (eg. at the lungs). Similarly, right shift shows decreased affinity ie releases it’s oxygen more easily, as would appear with an increase in body temperature, hydrogen ion(acidosis), 2,3-diphosphoglycerate or carbon dioxide concentration (the Bohr effect)De-oxy haemaglobin binds more easily with hydrogen ions than oxyHb thus as H ions rise, Ph falls Hb affinity for O2 falls

2,3diphosphoglycerate is formed in red blood cells as a product of glycolysis. It is a highly charged ion. It combines with Hb it displaces O2 and shifts curve to right.

Thus conditions that cause a right shift by encouraging unloading of O2 at relatively low PP of O2 can be thought of as an exercising muscle.

During exercise- Temp increases/ H ions increase/Co2 and DPG increase = more O2 to muscles

Carbon monoxide has a much higher affinity for haemoglobin than oxygen does. In carbon monoxide poisoning, oxygen cannot be transported and released to body tissues thus resulting in hypoxia.

Fetal Hb is structurally different from adult. This causes it to bind less with DPG. DPG reduces the amount of O2 that can combine so fetal Hb has a higher affinity for O2 than adult. This enables fetus to extract O2from maternal blood at placenta.

The oxygen dissociation curve for myoglobin exists even further to the left.

Carbon Dioxide transport

After entering blood CO2 is converted to carbonic acid and bound to Hb in RBCs(%) and some dissolved in plasma

CO2 + H2O <> H+ + HCO3-

Reversible reaction. Proceeds vigorously in peripheral capillaries tying up large numbers of CO2 molecules. H ions bind to HB = carboxy Hb HCO3 moves into plasma which exchanges another anion CL-. Resulting in chloride ions moving into RBCs = Chloride shift. Plasma becomes saturated rapidly and carries only 7% of absorbed CO2

Blood tests and RBCs

Haematocrit(Hct) % of formed elements in whole blood

High= polycythaemia Norm = 37-54 Low= anaemia Reticuloctye count % retics High=reticulocytosis Normal = 0.8% Low=anaemia Haemaglobin(Hb) Conc of Hb in blood Normal=12-18g/dl Low-anaemia RBC count no of RBCs per mu/l blood High-polycythaemia Normal= 4.2- 6.3 million Low= anaemia Mean Corpuscular av volume of RBC High= macrocytic Volume(MCV) Normal= 82-101 Low=microcytic Mean corpuscular Av amount Hb in RBC High= hyperchromic Normal=27-34 Low=hypochromic Haemaglobin(MCHC) Q. How would haematocrit change after a large haemorrhage?

White Blood Cells

The leukocytes, or white blood cells, are of three types; granulocytes, lymphocytes, and monocytes. All are involved in defending the body against foreign organisms.

There are three types of granulocytes: neutrophils, eosinophils, and basophils, with neutrophils the most abundant. Neutrophils seek out bacteria and phagocytise, or engulf, them.

The lymphocytes' chief function is to migrate into the connective tissue and build antibodies against bacteria and viruses.

White blood cells are almost colourless, considerably larger than red cells, have a nucleus, and are much less numerous; only one or two exist for every 1,000 red cells. The number increases in the presence of infection.

Monocytes, representing only 4 to 8 percent of white cells, attack organisms not destroyed by granulocytes and leukocytes.

The granulocytes, accounting for about 70 percent of all white blood cells, are formed in the bone marrow. The lymphocytes on the other hand are produced primarily by the lymphoid tissues of the body—the spleen and lymph nodes. They are usually smaller than the granulocytes. Monocytes are believed to originate from lymphocytes. Just as the oxygen-carrying function of red cells is necessary for our survival, so are normal numbers of leukocytes, which protect us against infection.

Fighting infection and foreign invaders is one of the primary functions of the

________.

erythrocytes (red blood cells)

leukocytes (white blood cells)

thrombocytes (platelets)

all of the above

Fighting infection and foreign invaders is one of the primary functions of the leucocytes.

Routine Hematological Tests

Differential WBC Count

There are five different kinds of WBCs 1.Eosinophils

1. Basophils 2. Neutrophils 3. Lymphocytes 4. Monocytes

Normal Values (Male or Female)

Neutrophils 40 - 75%

Eosinophils 1 - 4%

Basophils 0 - 1%

Lymphocytes 20 - 45%

Monocytes 2 - 8%

Significance Of The WBC Count

When there is an increase in eosinophils it is known as eosinophilia. It is observed in chronic inflammatory conditions, asthma, parasitic infestations, and in hypersensitivity reactions.

When there is a decrease in lymphocytes, it is known as lymphopenia. It is observed in acute stages of infections and where there is an excessive irradiation.

When there is an increase in neutrophils, it is known as neutrophilia. The common cause is pyogenic (pus forming) bacterial infections. When there is a decrease in neutrophils it is known as neutropenia. It is observed in bacterial infections such as

typhoid, viral infection such as measles, influenza etc. It is also found in anaemia (aplastic, megaloblastic, iron deficiency) and in suppression of bone marrow by various drugs and radiation.

When there is an increase in lymphocytes it is known as lymphocytosis. Lymphocytosis can be of two types, relative or absolute.

Relative Lymphocytosis: In this, the actual no of lymphocytes has not changed, but due to a decrease in neutrophils, the differential count shows an increase in lymphocytes. Absolute Lymphocytosis: It is seen in: Children. Also seen when there are infections such as tuberculosis, typhoid, mumps, measles, cough, influenza syphilis and other chronic infections. Infectious mononucleosis. Chronic lymphatic leukemia. When there is an increase in the number of monocytes, it is known as monocytosis. It is observed in tuberculosis, malaria, sub-acute bacterial endocarditis, typhoid and in Kala Azar.

A differential count is useful in identifying changes in the distribution of WBCs, which may be related to specific types of disorders. It also helps to know the severity of the disease and the degree of the response of the body

Platelet Count

Platelets are very small in diameter - around 3 mm. They help in clotting of blood.

said to be adequate. If it is less than that, it is said to be inadequate.

Abnormalities Of Erythrocytes (RBC)

In various types of anaemia and in other diseases such as thalassemia, malaria, kidney failure etc, the mature RBCs show significant changes. Various changed RBCs seen are termed as, Microcytes, Macrocytes,

Hypochromic, Spherocytes, Target cells, Stomatocytes,

Anisocytosis, Poilkilocytosis, sickle cells, Ovalocytes, Elliptocytes, Acanthocytes, Burr cells, Siderocytes, Basophilic stippling, Howell-Jolly Body, Cobot ring, Schi

stocytes, Crescent bodies and Creneted cells depending upon their morphology.

Abnormalities Of Leucocytes (WBC)

Most abnormalities are seen in Neutrophils. Different WBCs having abnormalities are termed as Myelocytes, Promyelocytes, Blasts, Metamyelocytes, Plasma Cell, Smudge cells etc.

Platelets

Platelets, or thrombocytes, are much smaller than the red blood cells. They are round or biconcave disks and are normally about 30 to 40 times more numerous than the white blood cells. The platelets' primary function is to stop bleeding. When tissue is damaged, the platelets aggregate in clumps to obstruct blood flow.

Blood platelets, or thrombocytes, are not true cells, but rather cytoplasmic fragments of a large cell in the bone marrow, the megakaryocyte. The central portion of a platelet stains purple with Wright's stain and is referred to as the granulomere. The peripheral portion stains clear and is called the hyalomere.

Platelet contents include glycogen granules, the open canalicular system (OCS), which is composed of canaliculi formed from invaginations of the platelet plasma membrane, mitochondria, occasional Golgi elements and ribosomes. Platelets have several types of membrane-bound granules which contain a number of constituents including fibrinogen and several growth factors (e.g., PDGF).

Platelet activation occurs when injury to the vessel wall exposes sub-endothelial components, especially collagen. Platelets adhere to the damaged area and become cohesive to other platelets. This aggregation leads to the formation of a platelet plug, which prevents further blood loss and allows the repair process to begin. The micrograph shown below shows activated platelets adhering to some damaged cells.

Severe reduction in the number of circulating platelets results in a condition known as thrombocytopenia.

It is a condition which causes spontaneous bleeding as a reaction to minor trauma. This is due to failure of the platelets to seal over microscopic breaches in blood vessel walls.

In the skin this is manifest by a reddish-purple blotchy rash. This can be either small blotch called purpura or larger bruise like areas called ecchymoses.

Cytotoxic drugs used in treatment of cancers may cause this condition. It is also seen associated with acute leukaemias.

Haemostasis is a complex process which changes blood from a fluid to a solid state. Intact blood vessels are central to moderating blood's tendency to clot. The endothelial cells of intact vessels prevent thrombus formation by secreting tissue plasminogen activator (t-PA) and by inactivating thrombin and adenosine diphosphate (ADP). Injury to vessels overwhelms these protective mechanisms and haemostasis ensues. Haemostasis proceeds in two phases: primary and secondary haemostasis.

• Primary haemostasis is characterized by vascular contraction, platelet adhesion and formation of a soft aggregate plug. It begins immediately after endothelial disruption. Injury causes temporary local contraction of vascular smooth muscle. Vasoconstriction slows blood flow, enhancing platelet adhesion and activation.

o Adhesion occurs when circulating von Willebrand factor(vWf) attaches to the subendothelium. Next, glycoproteins on the platelet surface adhere to the "sticky" von Willebrand factor(vWf). Platelets collect across the injured surface. These platelets are then "activated" by contact with collagen. Collagen-activated platelets form pseudopods which stretch out to cover the injured surface and bridge exposed fibers. The collagen-activated platelet membranes expose receptors which bind circulating fibrinogen to their surfaces. Fibrinogen has many platelet binding sites. An aggregation of platelets and fibrinogen build up to form a soft plug. Platelet aggregation occurs about 20 seconds after injury.

o Primary haemostasis is short lived. The immediate post injury vascular constriction abates quickly. If flow is allowed to increase, the soft plug could be sheared from the injured surface, possibly creating emboli.

• Secondary haemostasis is responsible for stabilizing the soft clot and maintaining vasoconstriction. Vasoconstriction is maintained by platelet secretion of serotonin, prostaglandin and thromboxane. The soft plug is solidified through a complex interaction between platelet membrane, enzymes, and coagulation factors.

o Coagulation factors are produced by the liver and circulate in an inactive form until the coagulation cascade is initiated. The cascade occurs in steps. The completion of each step activates another coagulation factor in a chain reaction which leads to the conversion of fibrinogen to fibrin.

The Blood Coagulation Process

Blood coagulation is a process that changes circulating substances within the blood into an insoluble gel. The gel plugs leaks in blood vessels and stops the loss of blood. The process requires coagulation factors, calcium and phospholipids.

• The coagulation factors (proteins) are manufactured by the liver. • Ionized calcium ( Ca++ ) is available in the blood and from intracellular

sources. • Phospholipids are prominent components of cellular and platelet

membranes. They provide a surface upon which the chemical reactions of coagulation can take place.

•

Coagulation can be initiated by either of two distinct pathways.

• The Intrinsic pathway can be initiated by events that take place within the lumen of blood vessels. The Intrinsic pathway requires only elements (clotting factors, Ca++, platelet surface etc.) found within, or intrinsic to the vascular system.

• The Extrinsic pathway is the other route to coagulation. It requires Tissue Factor (tissue thromboplastin), a substance which is "extrinsic to", or not normally circulating in the vessel. Tissue Factor is released when the vessel wall is ruptured.

Regardless of whether the Extrinsic or Intrinsic pathway starts coagulation, completion of the process follows a common pathway. The common pathway involves the activation of factors: X, V, II, XIII and I. Both pathways are required for normal hemostasis and there are positive feedback loops between the two pathways that amplify reactions to produce enough fibrin to form a lifesaving plug. Deficiencies or abnormalities in any one factor can slow the overall process, increasing the risk of haemorrhage.

The coagulation factors are numbered in the order of their discovery. There are 13 numerals but only 12 factors. Factor VI was subsequently found to be part of another factor. The following are coagulation factors and their common names:

• Factor I - fibrinogen • Factor II - prothrombin • Factor III - tissue thromboplastin (tissue factor) • Factor IV - ionized calcium ( Ca++ ) • Factor V - labile factor or proaccelerin • Factor VI - unassigned • Factor VII - stable factor or proconvertin • Factor VIII - antihemophilic factor • Factor IX - plasma thromboplastin component, Christmas factor • Factor X - Stuart-Prower factor • Factor XI - plasma thromboplastin antecedent • Factor XII - Hageman factor • Factor XIII - fibrin-stabilizing factor

The liver must be able to use Vitamin K to produce Factors II, VII, IX, and X. Dietary vitamin K is widely available from plant and animal sources. It is also produced by normal intestinal flora. A deficiency is rare but may occur:

• in newborns because they must first develop normal flora to produce Vitamin K, or

• when the flora is disturbed by broad-spectrum antibiotics.

At birth and throughout childhood, Factor VIII levels are the same as adult values. Many other factor levels are below adult levels at birth, some as low as 10% of adult levels. Theses levels increase toward the adult levels by age 6 months, although they may remain mildly below adult normal range throughout childhood. Despite lower levels, newborns and children do not normally experience bleeding. This confers some level of antithrombolic protection in youth. During pregnancy Factor XI can decrease, but fibrinogen and factor VIII increase.

Instant Feedback Of the factors below, which is not produced in the liver?

Factor IV

Factor V

Factor X

Factor VIII

Prothrombin time

The prothrombin time or PT is an assessment of the extrinsic and common pathways. The PT is the specific and only lab test used to measure the effectiveness of coumarin-type anticoagulant drugs, such as warfarin sodium (Coumadin). The most common methods to report the PT are time (in seconds) and the International Normalized Ratio (INR).

• The PT reported as time in seconds, represents how long a plasma sample takes to clot after a mixture of thromboplastin and calcium are added. If the patient's blood has less prothrombin than the normal ( or "control"), or a decrease in other clotting factors that affect the prothrombin time, the PT time in seconds will be longer than the control values. Normal values for PT time reported in seconds is between 11 and 13 seconds, depending on the method used by the laboratory.

• To give PT values a consistent basis of comparison from laboratory to laboratory, the World Health Organization instituted the INR, a uniform value in which PT is expressed as a ratio. In the last few years, the INR is becoming a more common method of measuring and reporting the PT. Targets for the INR vary, depending on the reason for anticoagulation. For example, a patient having hip surgery, who is being anticoagulated to prevent deep vein thrombosis, may have a target INR of between 2 and 3. To prevent arterial thrombosis in a patient with an artificial heart valve, the INR therapeutic goal may be in the 2.5 to 3.5 range.

As oral coumadin has a relatively long onset of action, it takes several days of therapy to achieve a therapeutic level. When a patient will be managed on oral coumadin, he or she may be given both heparin and coumadin, and then heparin is discontinued. If a PT is increased beyond a therapeutic level, the coumadin dose may be reduced, or the patient may be given parenteral Vitamin K. Drugs that increase or prolong the PT time include antibiotics, cimetidine (Tagamet), salicylates, and sulfonamides. Barbiturates, oral contraceptives, and Vitamin K in multivitamin preparations or in liquid nutritional supplements decrease the PT time.

When therapy with coumadin is begun, the dose is guided by monitoring the prothrombin time. Therapeutic levels are generally between 1 1/2 and 2 times normal, depending on the patient's need for anticoagulation. Expressed in terms of the INR, the range is between 2.0 and 3.0. A usual dose requirement is between 5 to 7.5 mg. daily. In some patients with coumadin resistance, the dosage may be much higher. If a patient with a prolonged PT must have surgery, it is important that the PT be brought within a normal range before surgery. This is often done with Vitamin K injections. Whole blood or fresh frozen plasma should be available for the surgical patient with an abnormal PT.

Instant Feedback: The INR is increasingly becoming an important way to measure and report prothrombin time.

True

False

Activated Partial Thromboplastin time (aPTT)

The activated partial thromboplastin time (aPTT) is a common screening test done to evaluate function of the intrinsic clotting system.

• It has largely replaced the older PTT, which was unable to incorporate variables in surface/contact time.

• The aPTT now measures the clotting time of plasma, from the activation of factor XII by a reagent (a negatively charged activator such as silica and a phospholipid) through the formation of a fibrin clot.

• If a patient's aPTT is abnormal, additional tests will be done to determine the exact cause of the coagulation problem.

Reference values for aPTT vary among laboratories, but generally range between 25 and 38 seconds. The aPTT of a newborn will usually be prolonged and may be up to 55 seconds at birth. It decreases to the adult range by 6 months of age. (Note: each lab has its own reference values based upon the equipment and reagents used.)

The aPTT is the most commonly used test to monitor heparin therapy. The therapeutic goal for a patient being anticoagulated with heparin, is an aPTT approximately 1.5 to 2.5 times the mean normal value. Heparin is most often administered as an initial intravenous bolus followed by a continuous intravenous infusion. The aPTT is evaluated every 6 hours during the first day of heparin therapy and 6 hours after any dosage change. If the aPTT is therapeutic, it can be checked once daily while patients are on heparin. (Note: If low molecular weight heparin is given for anticoagulation, a prolonged aPTT does not occur, so another test(s) may be indicated to monitor therapy.)

If the aPTT is increased beyond the therapeutic range, the physician may order the heparin IV flow slowed or briefly discontinued. As the half-life of heparin is quite short, these measures will often allow the aPTT to rapidly return to a therapeutic range. Protamine sulfate may also be given to block the action of heparin. In some serious situations, such as active bleeding, the physician will order a transfusion of whole blood or plasma to increase clotting factors. Surgery may be postponed in a patient who has an increased aPTT, unless it is an emergency procedure.

A prolonged aPTT in non-heparinised patients can occur due to:

• salicylates

• inherited or acquired intrinsic clotting factor deficiency or abnormality (XII, XI, X, IX, VII, V, II, I)

• massive blood replacement • hemophilia A • lupus anticoagulant • excessive coumarin dosage

A decreased aPTT can occur due to:

• digitalis • tetracyclines • antihistamines • nicotine • elevated factor VIII • tissue inflammation or trauma

Instant Feedback:

The aPTT is a measurement of the intrinsic coagulation system.

True

False

Fibrinogen, and fibrin degradation products

Fibrinogen is a circulating plasma protein manufactured by the liver. Thrombin converts fibrinogen to fibrin in the final stage of blood coagulation. Low fibrinogen levels can occur as a result of severe liver disease or due to a disorder such as disseminated intravascular coagulation (DIC). Fibrinogen is quantified by adding thrombin to a series of successively more dilute plasma samples and comparing clotting time to a control series. A coagulation analyzer is used to determine clotting time which will be inversely proportional to the concentration of fibrinogen. Normal values are approximately 200-400 mg/dl. Fibrinogen can also be measured directly by immunoassay. In this test a fibrinogen antibody is added to a plasma sample and then fibrinogen marked with the antibody is measured.

Fibrin degradation products (FDP), also known as fibrin slpit products, are present in blood when the thrombolytic enzyme plasmin cleaves fibrin or fibrinogen. Plasmin is produced when the thrombolytic system is activated. D-dimers are an

FDP produced when fibrin is cleaved by plasmin. The presence of D-dimers or FDP may be used to assist with the diagnosis of DIC, Deep Venous Thrombosis (DVT) or Pulmonary Embolism (PE). However, because they are produced under a variety of circumstances their presence alone is not diagnostic.

Instant Feedback:

Plasmin produces D-dimers when which substance is cleaved?

Fibrinogen

Fibrin

Plasma

The plasma is more than 90 percent water and contains a large number of substances, many essential to life. Its major solute is a mixture of proteins. The most abundant plasma protein is albumin. The globulins are even larger protein molecules than albumin and are of many chemical structures and functions. The antibodies, produced by lymphocytes, are globulins and are carried throughout the body, where many of them fight bacteria and viruses.

An important function of plasma is to transport nutrients to the tissues. Glucose, for example, absorbed from the intestines, constitutes a major source of body energy. Some of the plasma proteins and fats, or lipids, are also used by the tissues for cell growth and energy. Minerals essential to body function, although present only in trace amounts, are other important elements of the plasma. The calcium ion, for example, is essential to the building of bone, as is phosphorus. Calcium is also essential to the clotting of blood. Copper is another necessary component of the plasma.

A major source of body energy, transported to the cells by the plasma, is ________.

adenosine triphosphate (ATP)

glucose

oxygen

coenzyme Q-10

A major source of body energy, transported to the cells by the plasma, is glucose.

Spleen The spleen is an organ that is not essential for life. It can be divided into red sinuses lined by macrophages and the white pulp which is similar in structure to follicles. It has 4 main functions; 1. Sequestrartion and phagocytosis of aged RBCs. RBcs pass through red pulp and are phagocytosed. 2. Immunological. Macrophages within spleen can remove antibody coated red cells from blood 3. Blood reservoir. Up to 1/3 of platelets are sequestrated in spleen. In addition an enlarged spleen can accommodate RBCs. 4. Extra-medullary haemapoiesis Spleen is an organ of haemapoiesis for foetus. It also undertakes this role in adult life at times of haemapoietic crises e.g. haemolytic anaemia, thallassaemia and myelofibrosis. A spleen only becomes palpable when it is approximately three times normal size

Hypersplenism An imprecise term commonly used to refer to a clinical state of; Reduced RBCs, platelets and granulocytes in any combination An enlarged spleen from any cause An adequate cellular bone marrow indicating thee is sufficient cells

Totally or partially correctable by splenectomy. Symptoms include abdo discomfort, anaemia, infection and bleeding

Bone marrow (or medulla ossea) is the soft tissue found in the hollow interior of bones. In adults, marrow in large bones produces new blood cells.

Marrow types

There are two types of bone marrow: red marrow (also known as myeloid tissue) and yellow marrow. Red blood cells, platelets and most white blood cells arise in red marrow; some white blood cells develop in yellow marrow. The color of yellow marrow is due to the much higher number of fat cells. Both types of bone marrow contain numerous blood vessels and capillaries.

At birth, all bone marrow is red. With age, more and more of it is converted to the yellow type. Adults have on average about 2.6 kg (5.7 lb) of bone marrow, with about half of it being red. Red marrow is found mainly in the flat bones such as hip bone, breast bone, skull, ribs, vertebrae and shoulder blades, and in the cancellous ("spongy") material at the proximal ends of the long bones femur and humerus. Yellow marrow is found in the hollow interior of the middle portion of long bones.

In cases of severe blood loss, the body can convert yellow marrow back to red marrow in order to increase blood cell production.

Types of stem cells

Bone marrow contains two types of stem cells:

• Haematopoietic stem cells give rise to the three classes of blood cell that are found in the circulation: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes).

• Mesenchymal stem cells are found arrayed around the central sinus in the bone marrow. They have the capability to differentiate into osteoblasts, chondrocytes, myocytes, and many other types of cells. They also function as "gatekeeper" cells of the bone marrow.

Diseases of the Blood

Anaemia

Objectives.

Students must be able to; 1.Define anaemia 2. Read a routine blood report and indicate if anaemia is present 3. Comment on reticulocyte count and indicate if result indicates likelihood of haemolysis/ loss versus RBC underproduction 4.Define MCV and discuss its diagnostic and pathophysiological relevance 5. Classify anaemia on the basis of MCV into- normocytic/ microcytic/ macrocytic.

Appproach to the diagnosis of anaemia.

Anaemia is a deficiency of haemoglobin in the blood. It can be caused by blood loss, abnormal destruction of the red cells (haemolysis), and inadequate red cell formation by the bone marrow.

There are 3 important diagnostic principles:

1.FBC tells you if an anaemia is an isolated abnormality, or it is associated with another condition.

2.Reticulocyte count tells you whether an anaemia is likely to be due to underproduction of RBC or haemolysis/loss

3. Mean cell volume(MCV) allows you to classify anaemia by RBC size into microcytic/ macrocytic/ normocytic.

It is size of RBC that directs your subsequent history and investigations

Anaemia is a reduction in Hb below normal level.Age and sex are important in consideration of level

.Look at FBC.

Check WBC and platelets and differential WBC.

Abnormally high or low WBC or platelets may be a clue of an underlying blood disease.

Pancytopenia suggests marrow replacement or hypersplenism

An erythroleukoblastic blood smear(circulating immature RBCs such as myelocytes or nucleated RBCs) could indicate marrow replacement with tumour,fibrosis or leukaemia.

When anaemia is only problem look at MCV.

MCV suggests pathophysiology

Mean corpuscular volume is the mean size of RBC as determined by automated cell counter.

LOW MCV- indicates Hb synthesis is impaired from no of causes.

HIGH MCV- indicates DNA synthesis is impaired or an increase in RBC membrane.

NORMAL MCV- means no impairment in HB or DNA synthesis.

Microcytic Anaemia- low MCV

Indictes impaired Hb synthesis

1. Iron deficiency- cause to be ascertained

2. Iron unavailabilty as in chronic diseases

3. Impaired globin chain synthesis as in thalassemia

4. impaired haem synthesis as in lead poisoning ad other rare diseases.

Macrocytic Anaemia- high MCV

Classifed as with and without megaloblastosis.

Megaloblastosis; implies impared DNA synthesis as in B12 and folic aid deficiency.

Absence of megaloblasts; implies normal DNA synthesis. There is probably increased RBC membrane formation as in alcoholism, liver disease and hypothyroidism.

MCV is also increased in haemolytic anaema due to an increase in reticulocyes which are larger than erythrocytes.

Normocytic anaemia

Could be underproduction or RBC loss by bleeding or haemolysis.

Reticulocyte count will indicate which.

LOW retic count= underproduction

HIGH retic count= blood loss or haemolysis

Note; response to therapy(b12 or fe) is associated with reticulocytosis

Anaemia caused by acute or chronic blood loss, or abnormal bleeding, results from the inability of the bone marrow to make new cells as fast as they are needed. In acute or massive bleeding, the red blood cells and their haemoglobin are normal but are not abundant. Chronic slow bleeding leads to a deficiency in iron stores needed for haemoglobin. This results in smaller red blood cells that are paler than normal.

Abnormal destruction of red cells (the haemolytic anaemias) leads to a shorter than normal red cell survival. For example, in the hereditary disease Sickle-cell Anaemia the haemoglobin is built erroneously. Such cells are more fragile and break more readily in circulation.

Anaemias caused by bone-marrow failure include aplastic anaemia, in which the bone marrow lacks adequate numbers of some or all types of blood cells. Another anaemia caused by failure of production of red cells is pernicious anaemia. In this

disease, the person's stomach fails to produce "intrinsic factor," which is necessary for the normal absorption of vitamin B-12 from the intestines. Because vitamin B-12 is essential for normal bone marrow function, red cells are not formed normally. Sublingual vitamin B-12 supplements are available, from which the vitamin is absorbed through the blood vessels under the tongue, thus avoiding the potential problems associated with intestinal absorption.

Pernicious anaemia results when the person's stomach fails to produce intrinsic factor, which is necessary for the normal absorption of vitamin ________.

B-1

B-6

B-12

C

Red Blood Cells - Heads and Tails

Heads..... .....tails

There are numerous red blood cells in the body

.....because a large amount of oxygen must be carried from the lungs to all body cells.

Red blood cells are tiny .....because they must fit through extremely narrow capillaries.

Red blood cells are flexible .....because they must bend and squeeze through capillaries without being broken.

Red blood cells are packed with haemoglobin

.....because this protein binds oxygen in the lungs and releases it where it's needed in the body.

Red blood cells have no nucleus .....because their job is very simple and more haemoglobin can be packed in.

Red blood cells have a flat disc shape .....because this provides a large surface area for oxygen to diffuse across.

Haemoglobin molecules contain iron .....because ions of this metal can bind and release oxygen.

Red blood cells are broken down in the liver after only about four months

…..but they are continuously made by the red marrow in certain bones.

Diagnosis of an Abnormal WBC

Given a patient with abnormal WBC, your approach should be very straightforward.

Basically you want to determine

1. Which cell type or types are abnormal? Is the abnormality reactive (due to infection) if not is it neoplastic? The differential WBC is essential

2. Is maturation normal? Are immature cells present? Are there any blasts?

3. Are cell marker studies needed? 4. Are indicators of clonality needed 5. Is there an emergency problem eg hyperleukocytosis or

agranulocytosis?

Indicators of clonality.

In B cells – kappa or lambda chain or immunoglobulin heavy chain rearrangents

In T cells T cell receptor gene rearrangements

Plasma cells- serum rotein electrophoresis

Granulocytes- karyotypes.

Causes of Neutropenias

1. Margination 2. Impaired production 3. impaired maturation 4. sequestration/destruction/loss 5. chemotherapy 6. chronic benign neutropenia 7. severe congenital neutropenia

Lymphocytosis and Lymphopenia

Put it into clinical context. Take a history with knowledge of lymphocytosis and lymphopenia. Do a physical examination

Determine if;

Lymphadenopathy present, location, size and texture of glands

Presence of local inflammatory or neoplastic disase

Is splenomegaly present? Test for monoclonality of lymphocytes.

Causes of abnormal lymphocyte count

Proliferation of normal cells responding to antigen

Chronic leukaemia

\Acute leukaemia

Splenectomy

Causes of Lymphopenia

AIDS

Lymphoma

Splenomegaly

Chemotherapy

Protein losing enteropathy

Further Reading. Useful website is www.hemeteam.com A collection of tutorials on basic principles of diagnosis, pathogenesis and treatment of common blood disorders. It gives you information and option to go deeper if you wish. Many tutorials have MCQs with instant feedback.