2Back to Basics Blood is a liquid tissue A mixture of cells and water The water containsProtein, glucose, cholesterol, calcium, hormones, metabolic waste and hundreds of other substancesPlasma is the liquid portion of the blood containing the blood clotting protein FibrinogenSerum is the fluid remaining after the blood clotsDoes not contain Fibrinogen
4Plasmaliquid part of bloodplasma transports:-soluble food moleculeswaste productshormonesantibodies
5Plateletsif you get cut:-platelets producetiny fibrin threadsthese form a web-likemesh that traps bloodcells.these harden forming a clot, or "scab."150,000 to 400,000 per mm3
6Physical Characteristics of Blood Average volume of blood:5–6 L for males; 4–5 L for females (Normovolemia)Hypovolemia - low blood volumeHypervolemia - high blood volumeViscosity (thickness) (where water = 1)The pH of blood is 7.35–7.45; x = 7.4Osmolarity = 300 mOsm or 0.3 OsmThis value reflects the concentration of solutes in the plasmaSalinity = 0.85%Reflects the concentration of NaCl in the bloodTemperature is 38C, slightly higher than “normal” body temperatureBlood accounts for approximately 8% of body weight
7Composition of Blood 2 major components Liquid = plasma (55%) Formed elements (45%)Erythrocytes, or red blood cells (RBCs)Leukocytes, or white blood cells (WBCs)Platelets - fragments of megakaryocytes in marrow
9Blood Plasma Blood plasma components: Water = 90-92% Proteins = 6-8% Albumins; maintain osmotic pressure of the bloodGlobulinsAlpha and beta globulins are used for transport purposesGamma globulins are the immunoglobulins (IgG, IgA, etc)Fibrinogen; a clotting proteinOrganic nutrients – glucose, carbohydrates, amino acidsElectrolytes – sodium, potassium, calcium, chloride, bicarbonateNonprotein nitrogenous substances – lactic acid, urea, creatinineRespiratory gases – oxygen and carbon dioxide
10Laboratory Assessment of Blood Cells Complete Blood Count (CBC) includesWhite Blood Cell Count (WBC)Red Blood Cell Count (RBC)Percentage of white cells that are neutrophils, eosinophis or basophils (white cell differential count)Amount of hemoglobinHematocritPercent of blood volume occupied by red blood cells
11Red Cell Indices Mean Cell Volume (MCV) Average size of a RBC Mean Cell Hemoglobin (MCH)Average amount of hemoglobin per RBCMean Corpuscular Hemoglobin Concentration (MCHC)Average concentration of hemoglobin in all RBCs
12Red Cell Indices Used to Diagnose Disease MacrocyticRed Blood Cells may be too largeMicrocyticRed Blood Cells may be too smallNormocyticRed Blood Cells are normal sizeHypochromicToo little hemoglobinNormochromicNormal amount of hemoglobinNormochromicNormal amount of hemoglobin
16The RBC's here are smaller than normal and have an increased zone of central pallor. This is indicative of a hypochromic (less hemoglobin in each RBC) microcytic (smaller size of each RBC) anemia. There is also increased anisocytosis (variation in size) and poikilocytosis (variation in shape).
18The most common cause for a hypochromic microcytic anemia is iron deficiency. The most common nutritional deficiency is lack of dietary iron. Thus, iron deficiency anemia is common. Persons most at risk are children and women in reproductive years (from menstrual blood loss and from pregnancy).
19the RBC size remains normal, whereas the overall volume of the haemoglobin is found below the healthy minimum of 11.
21Erythrocytes (RBCs) Biconcave disc Folding increases surface area (30% more surface area)Plasma membrane contains spectrinGive erythrocytes their flexibilityAnucleate, no centrioles, no organellesEnd result - no cell divisionNo mitochondria means they generate ATP anaerobicallyPrevents consumption of O2 being transportedFilled with hemoglobin (Hb) - 97% of cell contentsHb functions in gas transportHb + O HbO2 (oxyhemoglobin)Most numerous of the formed elementsFemales: 4.3–5.2 million cells/cubic millimeterMales: 5.2–5.8 million cells/cubic millimeter
23Erythrocyte FunctionErythrocytes are dedicated to respiratory gas transportHemoglobin reversibly binds with oxygen and most oxygen in the blood is bound to hemoglobinComposition of hemoglobinA protein called globinmade up of two alpha and two beta chainsA heme moleculeEach heme group bears an atom of iron, which can bind to one oxygen moleculeEach hemoglobin molecule thus can transport four molecules of oxygen
25Hemoglobin Oxyhemoglobin – hemoglobin bound to oxygen Oxygen loading takes place in the lungsDeoxyhemoglobin – hemoglobin after oxygen diffuses into tissues (reduced Hb)Carbaminohemoglobin – hemoglobin bound to carbon dioxideCarbon dioxide loading takes place in the tissues
27Fate and Destruction of Erythrocytes The life span of an erythrocyte is 100–120 daysTravels about 750 miles in that time (LA to Albuquerque)Old erythrocytes become rigid and fragile, and their hemoglobin begins to degenerateDying erythrocytes are engulfed by macrophagesHeme and globin are separatedIron is removed from the heme and salvaged for reuseStored as hemosiderin or ferritin in tissuesTransported in plasma by beta-globulins as transferrin
28Fate and Destruction of Erythrocytes Heme is degraded to a yellow pigment called bilirubinLiver secretes bilirubin into the intestines as bileIntestines metabolize bilirubin into urobilinogenUrobilinogen leaves the body in feces, in a pigment called stercobilinGlobin is metabolized into amino acids which are then released into the circulation
30Production of Erythrocytes Hematopoiesis – blood cell formationOccurs in the red bone marrow (myeloid tissue)Axial skeleton and girdlesEpiphyses of the humerus and femurMarrow contains immature erythrocytesComposed of reticular connective tissueHemocytoblasts give rise to ALL formed elementsLymphoid stem cells - give rise to lymphocytesMyeloid stem cells - give rise to all other blood cells
31Production of Erythrocytes: Erythropoiesis A hemocytoblast is transformed into a committed cell called the proerythroblastProerythroblasts develop into early erythroblastsThe developmental pathway consists of three phasesPhase 1 – ribosome synthesis in early erythroblastsPhase 2 – hemoglobin accumulation in late erythroblasts and normoblastsPhase 3 – ejection of the nucleus from normoblasts and formation of reticulocytesReticulocytes then become mature erythrocytesReticulocytes make up about 1 -2 % of all circulating erythrocytes
33Regulation and Requirements for Erythropoiesis Circulating erythrocytes – the number remains constant and reflects a balance between RBC production and destructionToo few red blood cells leads to tissue hypoxiaToo many red blood cells causes an undesirable increase in blood viscosityErythropoiesis is hormonally controlled and depends on adequate supplies of iron, amino acids, and B vitamins
34Hormonal Control of Erythropoiesis Erythropoietin (EPO) released by the kidneys is triggered by:Hypoxia due to decreased RBCsDecreased oxygen availabilityIncreased tissue demand for oxygenEnhanced erythropoiesis increases the:RBC count in circulating bloodOxygen carrying ability of the blood
35Erythropoietin Mechanism ImbalanceStartNormal blood oxygen levelsStimulus: Hypoxia due to decreased RBC count, decreased availability of O2 to blood, or increased tissue demands for O2ImbalanceIncreases O2-carrying ability of bloodReduces O2 levels in bloodErythropoietin stimulates red bone marrowKidney (and liver to a smaller extent) releases erythropoietinEnhanced erythropoiesis increases RBC count
37Dietary Requirements of Erythropoiesis Erythropoiesis requires:Proteins, lipids, and carbohydratesIron, vitamin B12, and folic acidThe body stores iron in Hb (65%), the liver, spleen, and bone marrowIntracellular iron is stored in protein-iron complexes such as ferritin and hemosiderinCirculating iron is loosely bound to the transport protein transferrin
39Causes of Anemia Decreased erythrocyte production Decreased erythropoietin productionInadequate marrow response to erythropoietinErythrocyte lossHemorrhageHemolysis
40Erythrocyte Disorders PolycythemiaAbnormal excess of erythrocytesIncreases viscosity, decreases flow rate of bloodAnemiaAbnormally low hemoglobin in bloodCaused by decreased numbers of RBC’s, decreased amount of hemoglobin in RBC’s, or both
41Anemia Anemia – blood has abnormally low oxygen-carrying capacity It is a symptom rather than a disease itselfDue to some underlying conditionBlood oxygen levels cannot support normal metabolismSigns/symptoms include fatigue, paleness, shortness of breath, and chills
42Morphological Approach (big versus little) First, measure the size of the RBCs:Use of volume-sensitive automated blood cell counters, such as the Coulter counter. The red cells pass through a small aperture and generate a signal directly proportional to their volume.Other automated counters measure red blood cell volume by means of techniques that measure refracted, diffracted, or scattered lightBy calculation from an independently-measured red blood cell count and hematocrit:MCV (femtoliters) = 10 x HCT(percent) ÷ RBC (millions/µL)
43Diagnosis of Anemia CBC and Determination of Red Blood Cell Indices Different types of Anemia are generally characterized by red blood cells of a certain sizeFor Example, small (microcytic, low MCV) RBCs occur with iron deficiencyRBCs contain less hemoglobin and are pale (hypochromic, low MCHC)
44Underproduction (morphological approach) MCV>115B12, FolateDrugs that impair DNA synthesis (AZT (Zidovudine, chemo)MDS (myelodysplastic syndromes)Ineffective production (or dysplasia) of the myeloid class of blood cellsMCV =DittoEndocrinopathy (hypothyroidism)ReticulocytosisIncreased number of immature RBCs
45Underproduction Microcytic Iron deficiency Thalassemia trait abnormal form of hemoglobinAnemia due to chronic disease (30-40%)Sideroblastic anemiabone marrow produces ringed sideroblasts rather than healthy RBCsNormocyticAnemia from a chronic diseaseRenal failure
51Thalassemia Genetic defect in hemoglobin synthesis synthesis of one of the 2 globin chains ( or )Imbalance of globin chain synthesis leads to depression of hemoglobin production and precipitation of excess globin (toxic)“Ineffective erythropoiesis”Ranges in severity from asymptomatic to incompatible with life (hydrops fetalis)Found in people of African, Asian, and Mediterranean heritage
52Alpha ThalassemiaThis is alpha thalassemia major. There have been two major variations of alpha thalassemia arise in human history. One variation, most prevalent in Southeast Asia, is known as alpha thalassemia 1. In this variant, two alpha globin genes are deleted on one chromosome 16. The other variant, known as alpha thalassemia 2, is most common in Africa and the Mediterranean region, and differs in that a single alpha globin gene is missing from one chromosome 16. Alpha thalassemia 1, with alpha globin gene deletions on a single chromosome 16, can give rise to alpha thalassemia major in the homozygous state, when both chromosomes are affected. Affected persons become anemic in utero, because even fetal hemoglobin cannot be produced, and severe hydrops fetalis results, which leads to stillbirth, or death soon after birth from pulmonary hypoplasia or cardiac failure. Hemoglobin electrophoresis will reveal affected fetuses or neonates to have about 80% hemoglobin Barts (a tetramer of gamma chains) and about 20% hemoglobin Portland (or sometimes hemoglobin Gower 1) normally present only in embryonic life in the first trimester. RBCs that contain mostly hemoglobin Barts have marked anisocytosis and poikilocytosis, and there is expansion of erythropoiesis with many immature RBCs in the peripheral blood, as evidenced by polychromasia, nucleated RBCs and even erythroblasts as shown here.
53Thalassemias Dx: Smear: microcytic/hypochromic, misshapen RBCs -thalassemia will have an abnormal Hgb electrophoresis (HbA2, HbF)The more severe -thalassemia syndrome can have HbH inclusions in RBCsFe stores are usually elevated
54ThalassemiaThe oxygen depletion in the body becomes apparent within the first 6 months of life.If left untreated, death usually results within a few years.Note the small, pale (hypochromic), abnormally-shaped red blood cells. The darker cells likely represent normal RBCs from a blood transfusion.
55ThalassemiaThe only treatments are stem cell transplant and simple transfusion.Chelation therapy to avoid iron overload has to be started early.
57Marrow Production - Myelodysplasia Used to be referred to as “Preleukemia”Most commonly in the elderly.Occurs when something goes wrong in your bone marrow
58Signs and Symptoms of Myelodysplasia FatigueShortness of breathUnusual paleness (pallor) due to anemiaEasy or unusual bruising or bleedingPinpoint-sized red spots just beneath your skin caused by bleeding (petechiae)Frequent infections
59CausesCaused by poorly formed or dysfunctional blood cells due to eitherUnknown causesChemical exposure
61Marrow Production - Myelophthisic Myelophthisic anemia is a normocytic-normochromic anemia that occurs when normal marrow space is infiltrated and replaced by nonhematopoietic or abnormal cells.Causes:Most often due to replacement of the bone marrow by metastatic cancers such as breast or prostate; less often, kidney, lung, adrenal, or thyroid.Marrow fibrosis often occurs.Splenomegaly may develop.
63Marrow Production - Aplastic Anemia The body stops producing enough new blood cells.Signs and symptoms may include:FatigueShortness of breath with exertionRapid or irregular heart ratePale skinFrequent or prolonged infectionsUnexplained or easy bruisingNosebleeds and bleeding gumsProlonged bleeding from cutsSkin rashDizzinessHeadache
65Here we see a sample of bone marrow in a patient with Aplastic Anaemia Here we see a sample of bone marrow in a patient with Aplastic Anaemia. Notice there are very few cells except for the fat cells
66Factors that can temporarily or permanently injure bone marrow and affect blood cell production include:AcquiredRadiation and chemotherapy treatmentsExposure to toxic chemicals.Exposure to benzeneUse of certain drugs even some antibiotics.Autoimmune disordersViral infectionsEpstein Barr, CMV, Parvovirus B19, HIVPregnancy.Unknown factors. This is called idiopathic aplastic anemia.
67Marrow Production - Aplastic Anemia HereditaryFanconia rare, inherited blood disorder that leads to bone marrow failure.Diamond-Shwachmana rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction
68Marrow Production - Aplastic Anemia TreatmentMost patients require red cell transfusions.Bone MarrowTransplant when possible.Stem Cell TransplantMedication: Bone Marrow StimulantsSargramostim (Leukine)Filgrastim (Neupogen)Pegfilgrastim (Neulasta)Epoetin alfa (Epogen, Procrit)
69Hemolytic Anemia – RBC Destruction Hemolytic anemias are either acquired or congenital.Hemolytic anemia is a condition in which there are not enough RBCs in the bloodDue to premature RBC destructionHemolytic anemia can result from:infectioncertain drugsautoimmune disorders in which the body attacks and destroys its own red blood cellsinherited disorders such as sickle cell anemia or thalassemia.
71Symptoms of Hemolytic Anemia Dark UrineEnlarged spleenFatigueFeverPale skins colorRapid heart rateShortness of breathYellow skin color (jaundice)Chills
72Sickle Cell AnemiaSingle base pair mutation results in a single amino acid change.Under low oxygen, Hgb becomes insoluble forming long polymersThis leads to membrane changes (“sickling”) and vasoocclusion
76Red Blood Cells from Sickle Cell Anemia Deoxygenation of SS erythrocytes leads to intracellular hemoglobin polymerization, loss of deformability and changes in cell morphology.OXY-STATEDEOXY-STATE
77Transfusion in Sickle Cell (Controversy!) Used correctly, transfusion can prevent organ damage and save the lives of sickle cell disease patients.Used unwisely, transfusion therapy can result in serious complications.
78Transfusion in Sickle Cell (Controversy!) Simple transfusion – give bloodPartial exchange transfusion - remove blood and give bloodErythrocytapheresis – use apheresis to maximize blood exchangeWhen to use each method?
79Transfusion in Sickle Cell In general, patients should be transfused if there is sufficient physiological derangement to result in heart failure, dyspnea, hypotension, or marked fatigue.Tends to occur during an acute illness or when hemoglobin falls under 5 g/dL.
80Transfusion in Sickle Cell (exchange transfusion) Bleed one unit (500 ml), infuse 500 ml of salineBleed a second unit and infuse two units.Repeat. If the patient has a large blood mass, do it again.
81Transfusion in Sickle Cell (chronic transfusion therapy) StrokeChronic debilitating painPulmonary hypertensionSetting of renal failure and heart failure
82Transfusion in Sickle Cell (chronic transfusion therapy) Controversial uses:Prior to contast media exposureSub-clinical neurological damagePriapismLeg UlcersPregnancy
83Pernicious AnemiaPernicious anemia is a decrease in red blood cells that occurs when the body cannot properly absorb vitamin B12 from the GI TractCommon causes include:Weakened stomach lining (atrophic gastritis)The body's immune system attacking the cells that make intrinsic factor (autoimmunity against gastric parietal cells) or intrinsic factor itself
84Symptoms Diarrhea or constipation Fatigue Loss of appetite Pale skin Shortness of breath, mostly during exerciseSwollen, red tongue or bleeding gumsNerve damage
90Review red blood cell disorders Red cell destruction – membrane disorders
91Review red blood cell disorders Red cell destruction – enzymopathies G6PD deficiencyPyruvate kinase deficiencyOther very rare deficiencies
92Deoxyhemoglobin S Polymer Structure A) Deoxyhemoglobin S14-stranded polymer(electron micrograph)B) Paired strands ofdeoxyhemoglobin S(crystal structure)C) Hydrophobic pocketfor 6b ValD) Charge and size prevent6b Glu from binding.Dykes, Nature 1978; JMB 1979Crepeau, PNAS 1981Wishner, JMB 1975
93Transfusion in Sickle Cell In severely anemic patients, simple transfusions should be used.Common causes of acute anemia:acute splenic sequestrationtransient red cell aplasiaHyperhemolysis (infection, acute chest syndrome, malaria).If the patient is stable and the reticulocyte count high, transfusions can (and should) be deferred.
94Transfusion in Sickle Cell (exchange transfusion) A comprehensive transfusion protocol should include accurate records of the patient’s red cell phenotype, alloimmunization history, number of units received, serial Hb S percentages, and results of monitoring for infectious diseases and iron overload.Transfusions are used to raise the oxygen-carrying capacity of blood and decrease the proportion of sickle red cells.
95Transfusion in Sickle Cell (exchange transfusion) Transfusions usually fall into two categories:episodic, acute transfusions to stabilize or reverse complications.long-term, prophylactic transfusions to prevent future complications.
96Transfusion in Sickle Cell (exchange transfusion) episodic, acute transfusions to stabilize or reverse complications.Limited studies have shown that aggressive transfusion (get Hgb S < 30%) may help in sudden severe illness.May be useful before general anesthesia.Vichinsky et al., NEJM 1995
97Transfusion in Sickle Cell Inappropriate uses of transfusion:Chronic steady-state anemiaUncomplicated pain episodesInfectionMinor surgeryUncomplicated pregnanciesAseptoic necrosis
98Transfusion in Sickle Cell (exchange transfusion) Except in severe anemia, exchange transfusion offers many benefits and is our first choicePhenotypically matched, leukodepleted packed cells are the blood product of choice.A posttransfusion hematocrit of 36 percent or less is recommended.Avoid hyperviscosity, which is dangerous to sickle cell patients.
99Iron overload and chelation Can occur in any patient requiring chronic transfusion therapy or in hemochromatosis.Liver biopsy is the most accurate test though MRI is being investigated.Ferritin is a good starting test.120 cc of red cells/kg of body weight is an approximate point at which to think about iron overload
100Iron overload and chelation Chelator, deferoxamine25 mg/kg sq per day over 8 hours.Supplementation with vitamin C may aid excretion.Otooxicity, eye toxicity, allergic reactions.Discontinue during an infection.Oral chelators are in development.
101ConclusionsTransfuse for any severe anemia with physiologic compromise.Decide early whether transfusion will be rare or part of therapy.Avoid long-term complications by working with your blood bank and using chelation theraoy.
102Anemia: Insufficient Erythrocytes Hemorrhagic anemia – result of acute or chronic loss of bloodHemolytic anemia – prematurely ruptured erythrocytesAplastic anemia – destruction or inhibition of red bone marrow