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Blood Disorders: NormalStructure & Function

Retno Murwanti DVM, MSc, PhDFaculty of Pharmacy

Universitas Gadjah Mada2013


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Blood Disorders: Normal

Structure & Function

NORMAL STRUCTURE AND FUNCTION

OVERVIEW OF BLOOD DISORDERS


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NORMAL STRUCTURE

AND FUNCTION

Formed Elements of Blood

Coagulation Factors & the CoagulationCascade


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INTRODUCTION


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Blood Composition

Formed elements

red cells,

white cells,

platelets

Plasma.


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Red blood cells

The most common formed elements

Function : carrying oxygen to the cells of

the body via their main compoundhemoglobin.


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White Blood Cells

White blood cells are generally present at

about 1/700th the number of erythrocytes

Function : as mediator immune responsesto infection or other stimuli ofinflammation


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Platelets

Platelets are the formed elements thatparticipate in coagulation


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Plasma

Plasma is largely water, electrolytes, andplasma proteins

Most important plasma proteins in bloodclotting : coagulation factors.


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FORMED ELEMENTSOF BLOOD


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BONE MARROW AND

HEMATOPOIESIS Developed from progenitor cells or Stem

Cells

Hematopoiesis

Bone marrow

adults (ribs, vertebrae, sternum)

children (long bones)


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Hematopoiesis

normal peripheral blood


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Cytokines that Regulate

Hematopoiesis

Erythropoietin EPO

ThrombopoietinTPO

Colony-stimulating Factor CSF

Granulocyte G

Macrophage M

Interleukin IL

Stem Cell Factor SCF


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Erythropoiesis

Stimulated by erythropoietin (hormon).

Produced by the kidneys

Function: regulates red blood cell production by afeedback system:

When blood hemoglobin levels fall (anemia), delivery tothe kidneys falls, and they produce moreerythropoietin, causing the marrow to produce morered cells.

When hemoglobin levels rise, the kidney produces lesserythropoietin and the marrow fewer red cells.


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Myelopoiesis

Production of white blood cells (granulocyte)

Neutrophils

Affected by many cytokines

The most important :

interleukin-3 (IL-3)

granulocyte colony-stimulating factor (G-CSF)

granulocyte-macrophage co stimulating factor(GM-CSF)


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Thrombopoiesis

Thrombocytes are ligations of the cytoplasm from megakaryocytes (a singlemegakaryocyte can give rise to thousands of thrombocytes).

Thrombopoiesis refers to the process of thrombocyte generation.

Thrombopoietin stimulates megakaryopoiesis, process of megakaryocytematuration and differentiation. Thrombopoietin, upon release, binds to itsreceptor, c-mpl, found on megakaryocyte progenitor cells. Following binding,intracellular signalling leads to megakaryocyte growth, maturation,membrane stability, platelet granule formation and the demarcation of thecytoplasm into regions destined to fragment into mature platelets. These"proplatelet processes" further fragment into platelets. This last step of

proplatelet process and platelet formation, in vitro, has been shown to beindependent of thrombopoietin.


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Examination

Thin blood smear

Cell sorting (FACS, Magnetic beads, etc)

Wrights stain


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Blood smear

erythrocyte

neutrophil

lymphocyte

platelets


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Normal Values Obtained on AutomatedBlood CountFormed Elements of

Blood.Element Male Adult Female Adult

Hemoglobin 1418 g/dL 1216 g/dL

Hematocrit (percentage of bloodwhich is erythrocytes)

4250% 3747%

Red cell count 4.66 x 106/ L 4.25.4 x 106/ LMean corpuscular volume (MCV) 80100 fL 80100 fL

White blood cell (total) count 400011,000/ L 400011,000/ L

Neutrophils 25007500/ L 25007500/ L

Lymphocytes 15003500/ L 15003500/ L

Monocytes 200800/ L 200800/ L

Eosinophils 60600/ L 60600/ L

Basophils < 100/ L < 100/ L

Platelets 150,000400,000/ L 150,000400,000/ L


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Erythrocytes Mature red blood cells are biconcave disk-shaped cells filled with

hemoglobin, which function as the oxygen-carrying component of the blood.

Do not have nuclei at maturity . Erythrocytes with nuclei in the peripheralblood smear suggests an underlying disease state.

Biconcave shape gives them enough flexibility to slip through small capillaries

and deliver oxygen to the tissues. Individual erythrocytes function for about 120 days before they are removed

from the circulation by the spleen.

Hemoglobin is the most important substance in the erythrocyte.

This protein is actually a tetramer, made of two -protein subunits andtwo -protein subunits (in normal adult hemoglobin, called hemoglobinA). Each - or -subunit contains the actual oxygen-binding portion of the

complex, heme ( a compound whose centrally important atom is iron; itis this atom that actually binds oxygen in the lungs and subsequentlyreleases it in the tissues of the body.)

A low level of hemoglobin in the blood, from a variety of causes (seelater discussion), is anemia, the most common general blood disorder.


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Granulocytes: Neutrophils, Eosinophils,and Basophils

The most common white blood cells; of these (neutrophilsare most abundant, followed by eosinophils and basophils).

Development :

As they mature, their nuclei become more convoluted and multilobed,and each develops a cytoplasm filled with granules.

These granules contain a variety of enzymes, prostaglandins, andmediators of inflammation, with specific factors dependent on the celltype.

Early progenitor cells for each type of granulocyte ("blasts") areindistinguishable on microscopic examination of the bone marrow, butunder the influence of different cytokines, they become morphologicallydistinct cell types.


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Basophils

contain very dark blue or purple granules (rGiemsa's or Wright's stain).

Basophil granules are large and usually obscure

the nucleus because of their density.

Function in hypersensitivity reactions. Theirnumbers can be increased in diseases notassociated with hypersensitivity, such as chronicmyelogenous leukemia.


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Eosinophils

contain large, strikingly "eosinophilic" granules(staining red with Wright's or Giemsa's stain).

Eosinophil nuclei are usually bilobed.

Function as part of the inflammatory responseto parasites too large to be engulfed byindividual immune cells and involved in someallergic reactions.


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Neutrophils

Major function is actually in the tissues; they must leave the blood byinserting themselves between the endothelial cells of the vasculature to

reach sites of injury or infection.

Granules contain: highly active enzymes such as myeloperoxidase, which,along with the free radical oxygen ions produced by membrane enzymessuch as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,kill bacteria that neutrophils ingest via endocytosis or phagocytosis.

They are the "first line of defense" against bacterial pathogens, and lownumbers of them (leukopenia) lead directly to a high incidence ofsignificant bacterial infections

Evidence of their importance and their short survival is commonlymanifested, because examination of the blood smear under themicroscope in a patient with an active infection may show not onlyincreased numbers of mature, multilobed neutrophils (neutrophilia) butalso increased numbers of less mature cells.

These less mature cells, released from a large storage pool in the bonemarrow, are called bands and have a characteristic horseshoe-shapednucleus that is not yet fully lobulated. The phenomenon of finding thesecells in the peripheral blood is called a left shift of the granulocytelineage.


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Platelets

Platelets are fragments of larger, multinucleated cells(megakaryocytes), butplatelets have no nuclei of their own.

Most platelets remain in the circulation, but a substantial minority aretrapped in the spleen; this phenomenon becomes important in a variety ofimmune-mediated decreases in platelet count (thrombocytopenia).

In the setting of a normal platelet count, they have a circulatory half-life ofabout 10 days. In cases of thrombocytopenia, their half-life decreases, as theyare consumed in the routine maintenance of vascular integrity.

Platelets are integral components of the coagulation system. Theirmembranes provide an important source of phospholipids, which are

required for the function of the coagulation system proteins), and containimportant receptors that allow attachment to endothelial cells (plateletadhesion) so that a platelet plug can be formed in response to blood vesselinjury. This prevents further blood loss after trauma and limits thecoagulation response to the site of injury rather than letting coagulationproceed inappropriately.


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The cytoplasm is also important for platelet function, particularly the

intracellular dense granules and alpha granules. The phenomenon of plateletactivation is also called "degranulation" and can be initiated by exposure ofplatelets to the activated blood coagulation factor thrombin,adenosine 5'-diphosphate (ADP), or collagen. This last reaction is probably the mostimportant, occurring when collagen, normally in the basement membranebelow the endothelial cells, is exposed to the blood after injury. Plateletactivation can also be induced by exposure to platelet-activating factor(PAF), a neutrophil-derived phospholipid cytokine.

During platelet activation, the dense and alpha granules release furtheractivators of platelet activity, such as ADP, and platelet factor 4, which canalso bind to endothelial cells. It is important because it binds to the mostcommonly used therapeutic anticoagulant, heparin (see later discussion). Thelast step in platelet activity is platelet aggregation, where platelets stick toeach other, firming up the platelet plug. On examination of the blood smear,

platelets are small, irregularly shaped blue or purple granular bodies. Inconditions in which platelet numbers are rising as a result of increasedmarrow activity, more immature platelets can be identified by their largersize.


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COAGULATION FACTORS &

THE COAGULATIONCASCADES


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Coagulation Factors of Plasma.

Name Production Source

Procoagulant factorsFactor I (fibrinogen) Liver

Factor II (prothrombin) Liver

Factor III (tissue thromboplastin) Tissue

Factor IV (calcium) . . .

Factor V (proaccelerin) Liver

Factor VI (obsolete = factor Va) . . .

Factor VII (proconvertin) Liver

Factor VIII (antihemophilic factor) Endothelial cells

Factor IX (Christmas factor) Liver

Factor X (Stuart-Prower factor) Liver

Factor XI (plasma thromboplastin antecedent) Liver

Factor XII (Hageman factor) Liver

Factor XIII (fibrin-stabilizing factor) Platelets

Anticoagulant factorsAntithrombin Liver

Protein C Liver

Protein S Liver

Plasminogen Liver

Tissue factor pathway inhibitor Endothelial cells


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The coagulation system

The coagulation system is remarkably complex in bothstructure and function.

The coagulation system provides for immediate activationwhen there is blood loss that needs to be stemmed but alsoconfines its activity to the site of blood loss.

There are two major components of the coagulation system:

platelets

the coagulation factors (plasma proteins.)

The end result of coagulation factor activity:

the formation of a complex of cross-linked fibrinmolecules and platelets that terminate hemorrhageafter injury.


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The coagulation factors do not generally circulate in activeforms.

Most of them are enzymes (serine proteases) and remaindormant until they are needed.

Other enzymes (the other proteases in the cascade) cleavethe inactive factors into active ones.

Presumably, the many interactions in the cascade allow asmall increase in the activity of two key early enzymes,factors VII and XI, to be amplified.

This results in a timely change in the availability of thrombin,which cleaves fibrinogen, leaving fibrin to form the clot.


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Coagulation and thrombolytic systems


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Laboratory Testing of the

Coagulation Process

In vitro tests of coagulation function, "seconds required to form a clot":

the prothrombin time (PT)

the test used clinically to monitor the effects of warfarin.

The activated partial thromboplastin time (aPTT)

Additional if there is an abnormality regarding the increasedose

ThePTT is prolonged most easily when there are reducedlevels of factor VIII or factor IX activity,

The aPTT is also very sensitive to the presence of heparin

bound to antithrombin and is used to monitor theanticoagulant effects of unfractionated heparin.

The tests are designed in such a way that the results will be prolonged outof the normal range in different pathologic states, but significant alterationsin the coagulation pathway inevitably lead to changes in both tests becauseof the multiple interactions of the involved factors.


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Questions

What are the different formed elements of blood and how can they andtheir subtypes be distinguished?

Name the vitamin Kdependent clotting factors and the organ in which theyare synthesized.

The extrinsic and intrinsic coagulation pathways converge with theactivation of which clotting factor?

Describe the two anticoagulant systems that participate in clottinghomeostasis.


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APLASTIC ANEMIA

Peripheral blood pancytopenia and ahypocellular marrow in which normalmarrow is replaced by fat cells.

Abnormal cells are not found.


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History of Aplastic anaemia.

Paul Ehrlich (1854-1915) described the first

case of aplastic anaemia in a pregnantwoman who died of marrow failure in1888.

The term aplastic anaemia first used byAnatole Chauffard in 1904.


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Diagnosis of Pancytopenia

Diagnosis of pancytopenia.

Suspect from signsand symptoms

Made by check of fullblood count, FBC

Male Female

Hb 13.5-17.5g/

dl

11.5-16g/dl

PCV 38-50% 36-45%

MCV 80-100fl 80-100fl

MCH 27-34pg 27-34pg

WBC 4-11x109/l 4-11x109/l

Neut 2.0-7.5 2.0-7.5

Lymph 1.5-4 1.5-4

Platelet 135-450 135-450

PCV = packed cell volume (persentase eritrosit dalamvolume tertentu darah)MCV = Mean corpuscular volume (ukuran atauvolume rata-rata eritroit)MCH = Mean corpuscular hemoglobin (jumlah rata-rata hemoglobin dalam eritrosit)


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Normal Erythropoiesis

Red cell life span 120 days.

Platelet life span 6 days.

Granulocyte life span < 24 hours.

Constant marrow activity needed to replace dead cells.


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Ethiology Aplastic anemia can be caused by exposure to chemicals, drugs, radiation,

infection, immune disease, and heredity; in about half the cases, the cause isunknown

exposure to toxins such as benzene, or with the use of certain drugs, includingchloramphenicol, carbamazepine, felbamate, phenytoin, quinine, andphenylbutazone.

Exposure to ionizing radiation from radioactive materials or radiation-producing

devices is also associated with the development of aplastic anemia. Marie Curie,

Aplastic anemia is present in up to 2% of patients with acute viral hepatitis.

One known cause is an autoimmune disorder in which white blood cells attackthe bone marrow.

Short-lived aplastic anemia can also be a result of parvovirus infection.

In some animals, aplastic anemia may have other causes. For example, in theferret (Mustela putorius furo), it is caused by estrogen toxicity, because female

ferrets are induced ovulators, so mating is required to bring the female out ofheat. Intact females, if not mated, will remain in heat, and after some time thehigh levels of estrogen will cause the bone marrow to stop producing red bloodcells


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Inherited Marrow Failure

Important diagnosis to make.

Implications for treatment.

Detailed history and examination.

Nail dystrophy, skin pigmentation and leukoplakia may

suggest dyskeratosis. Short stature, metaphyseal dysarthrosis, pancreatic

exocrine deficiency or family history of cytopenia inShwachman-Diamond syndrome.

Check for gene mutations can help.


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Incidence of Acquired Aplastic Anaemia

Rare condition; 2-5/million per year

Incidence is higher in East, environmental as migrants haveincidence of local population.

male to female incidence = equal

Disease of young adults, 2nd peak in 4th -5th decade oflife.


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Pathogenesis

Primary defect or damage to haematopoietic stem cell.

possible Immunological attack on stem cells.

HLA-D2 (Human leukocyte antigen D2) isoverrepresented in patients, suggests a role for antigen

recognition. Defective microenvironment (i.e. marrow stromal defect)


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Clinical Features

Anaemia;tiredness &fatigue

Low white count;recurrentinfections, flu-likeillness.

Low platelets;easy bruising andbleeding


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Investigations

Bone marrow aspirate and biopsy: to rule out other causes of pancytopenia(i.e. neoplastic infiltration or significant myelofibrosis).

History of exposure to cytotoxic chemotherapy: can cause transient bonemarrow suppression

X-rays, computed tomography (CT) scans, or ultrasound imaging tests:enlarged lymph nodes (sign of lymphoma), kidneys and bones in arms andhands (abnormal in Fanconi anemia)

Chest X-ray: infections

Liver tests: liver diseases

Viral studies: viral infections

Vitamin B12 and folate levels: vitamin deficiency

Blood tests for paroxysmal nocturnal hemoglobinuria

Test for antibodies: immune competency


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Marrow Trepine


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Treatment of aplastic anaemia

Supportive with blood products.

Prophylactic antibiotics.

Growth factor support.

Androgens. Immunosuppressive therapy with antilymphocyte globulin

& cyclosporin.

Allogeneic stem cell transplantation.


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