Learning Objectives: Blood and Lymph WEEK 1 January 3, 2012 BL - Course Introduction and Overview of Hematology 1. Explain what a hematocrit is and how it is measured. a. Hematocrit: proportion of blood by volume made up of red blood cells, value determined by measuring the length of the RBC layer and dividing it by the total length of the column of blood (RBCs+buffy coat+plasma), always reported as percentage 2. Describe the basic shape and composition of an erythrocyte (red blood cell). a. Biconcave disc shape: i. Increases SA to volume ratio by 40% (compared to sphere) for greater gas exchange ii. Allows RBC to squeeze through tight places (sphere shape can’t compress like a biconcave disc) and RBCs are on average 25% wider than capillaries they travel. iii. Move through vessel easier with this shape b. Lacks nucleus: nucleus present in bone marrow, but then removed when it goes out into blood stream. c. Lacks mitochondria: they have these organelles in the bone marrow but lose them prior to being released into periphery, therefore RBCs required anaerobic glycolysis. d. Contains lots of hemoglobin, thus, a major function of RBC is to transport O2 from lungs to tissue, hemoglobin also makes RBCs and blood red. e. Membrane is highly elastic allow cell to deform while maintaining structural integrity. Has 2-D elastic network of cytoskeletal proteins that are tethered to sites on cytoplasmic domains to transmembrane proteins embedded in the plasma membrane. 3. Define the following: hematopoiesis, erythropoiesis, hemolysis, hemostasis, and thrombosis a. Hematopoiesis: formation of blood cellular components b. Erythropoiesis: process by which RBCs are produced c. Hemolysis: rupturing of erythrocytes and release of their contents (hemoglobin) d. Hemostasis: the arrest/stopping of bleeding e. Thrombosis: formation of a blood clot inside a blood vessel that obstructs the flow of blood 4. List the five types of white blood cells in the blood. a. Lymphocytes, Neutrophils, Monocytes, Eosinophils, Basophils 5. Explain what platelets are, where they come from, and what their basic function is. a. Platelets, thrombocytes, are small cell fragments produced from large cells in the bone marrow called megakaryocytes. 1 megakaryocyte=5000 platelets. Responsible for hemostasis (stop bleeding). Hemostasis results from interaction between platelets, endothelium and blood coagulation factors. 6. Compare and contrast leukemia vs lymphoma, acute leukemia vs chronic leukemia and lymphoid leukemia vs myeloid leukemia. a. Leukemia: cancer cells in blood and bone marrow b. Lymphoma: cancer cells predominantly outside of the bone marrow/blood 1 c. Acute: cells are immature in their degree of differentiation and that clinical course is usually rapid without intervention d. Chronic: cells are more mature in their differentiation and the disease follows a more indolent clinical course. e. Lymphoid: arising from lymphocytic lineage f. Myeloid: arising from one of the other cell types in the marrow BL - Overview: From Innate to Adaptive Immunity 1. Define and recognize their abbreviations a. Pattern-recognition receptor (PRR): proteins on the cell surface expressed by cells of innate immune system to identify pathogen-associated molecular patterns (PAMPs) b. Pathogen-associated molecular pattern (PAMP): foreign molecular structures on pathogens (bacterial cell wall protein, bacterial dsRNA) that are recognized by PRRs in the innate immune system c. Toll-like receptor (TLR): type of PRR, protein that recognize a foreign molecular structure that humans don’t have, there are a least 10 TLRs. i. Examples: TLR4 bines lipopolysaccharide (part of bacterial cell wall), TLR2 bind peptidoglycan (bacterial). d. Damage-associated molecular pattern: stress or damage indicators expressed by body cells (paintball example from Cohen). 2. Name some common foreign patterns recognized by TLR. a. TLR1: lipopeptide (bacterial), TLR2: zymosan (fungal), TLR3: dsRNA (viral), TLR4: endotoxin=lipopolysaccharide, TLR5: flagellin (bacterial flagellum), TLR6: lipopeptide (mycoplasma), TLR7: ssRNA (influenza), TLR8: ssRNA (viral), TLR9: unmethylated CpG (herpes virus), TLR10: asthma connection 3. Identify the final transcription factor that is most commonly activated in inflammation. a. Bad bug makes endotoxinendotoxin binds to a variety of things and then ultimately recognized by TLR Signal cascadeactivates NF-KB inflammation 4. Define cytokine and chemokine. a. The factors made by the PAMP-stimulated cell are called cytokines and chemokines. As cells get damaged and stressed, they release certain of their internal molecules (the DAMPs), and some TLRs bind them, too, increasing the local inflammation. Thus even before the “real” immune system gets involved the local area of damage is a hotbed of inflammatory mediators. Some of these mediators—especially the chemokines—are chemotactic for phagocytic white blood cells, which flow in from distant areas. i. Chemokines (Greek -kinos, movement) are a family of small cytokines, or proteins secreted by cells. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines. When these are released, WBC head towards it because they like areas of high concentration, like they like pizza. ii. Cytokines (Greek cyto-, cell; and -kinos, movement) are small cell-signaling protein molecules that are secreted numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins; the term "cytokine" encompasses a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin. Generally help inflammation. 5. Describe the function of the innate immune response. a. You are born with innate immunity, therefore it is very limited in its actions and NOT adaptive (can’t change with different threats). 2 b. It detects intruders via molecular motifs/chemical structures found in pathogens, for humans, such motifs would include components of bacterial cell walls (us eukaryotes don’t have cell walls, of course), and odd microbial nucleic acid patterns. This is achieved by pattern recognition receptors c. The intruders have ventured too deep into our body’s structures and then innate immune system arranges for their inactivation, destruction and removal. d. Most immune responses are innate, but if it becomes overwhelmed, then adaptive response kicks in. The innate response is required to activate the adaptive response. 6. Name the cell that forms the bridge between innate and adaptive immunity. a. Dendritic cells: phagocytic cells at the interfaces between the body and the world (skin, lung, mucous membranes—where bad stuff can enter). At wound site, immature DCs get activated by the cytokines and chemokines secreted from the innate immune response, and they take up anything they can including foreign molecules derived from invaders. Activated (mature) DCs leave the local wound area and travel in the lympatics to the nearest/first lymph node (known as the DRAINING lymph node). Here they “show” what they have eaten to lymphocytes (the present the antigen). In the lymph node, adaptive immune response develops with the presence of T cells, B cells, and DCs. Adaptive immune response CANNOT develop in the periphery. b. Immature DC: best phagocyte ever! c. Mature DC: best antigen presenting cell ever! (There is a change in phenotype that occurs with DC matures). 7. Discuss in principle the role T cells play in immunity. a. T lymphocytes: begin their development in the bone marrow, but mature in the THYMUS. 2 main classes: Helper T cells (5 subtypes of helpers) and Cytotoxic/Killer T cells. b. T lymphocytes recognize and remove foreign substances. T cells themselves survey the surfaces of the body’s cells, looking for ones that have parasites within them or that are dangerously mutated. T cells recognize antigens by means of their surface receptors which see antigens presented by the newly arrived dendritic cell that is traveling via lymphatics to a lymph node (antigen has been partially digested by DC and peptides derived from it are loaded into special antigen-presenting molecules called MHC Class II and go to cell surface). When helper T cell receptor recognizes foreign material, it becomes activated, proliferates and the daughter cells travel through the body until they reach where the antigen first invaded. They are then re-stimulated by local antigen-presenting cells and release short-range mediators called lymphokines (cytokines made by lymphocyte). These mediators call up a much augmented inflammatory response by attracting monocytes and macrophages (specialists in phagocytosis and destruction). c. Other T cells are specialized for killing any body cell that they identify as containing abnormal molecules. Cytotoxic T cells “killers” also examine surface of incoming DCs but they are looking for fragments on a different class of antigen-presenting molecule, MHC Class I, which is on ALL CELLS. Appropriate clones of cytotoxic T cells proliferate and daughter cells circulate. When daughter cell binds a cell showing the same peptide it delivers a lethal “hit” signaling target cell to commit suicide (apoptosis). 8. Discuss in principle the role B cells play in immunity a. B cells also recognize and remove foreign substances, but B cells protect the extracellular spaces of the body (tissues fluids, blood, secretions), by releasing antibodies into these fluids. b. B cells also arrange for phagocytosis and destruction of foreign material, they recognize antigens via surface receptors and become activated and proliferate (they DO NOT require the simultaneous 3 recognition of an associated MHC molecule—like T cells). Then, they release soluble versions of their receptors (antibodies) which go and do the work. c. Fully differentiated B cell is called a plasma cell and is an antibody production factory. 9. Distinguish briefly the chief functions of the 5 immunoglobulin classes. a. Immunoglobin G (IgG): most abundant antibody, 2 adjacent IgG molecules binding an antigen cooperate to activate COMPLEMENT, a system of proteins that enhances inflammation and pathogen destruction. Only antibody that passes mother to fetus, so its important in protecting newborn until it can get its own IgG synthesis. b. Immunoglobin M: Better at activating COMPLEMENT system than IgG, first antibody to appear in blood after exposure to new antigen. Replaced by IgG in 1-2 weeks. c. Immunoglobin D: antibody inserted into B cell membranes as their antigen receptor. d. Immunoglobin A: most important antibody in secretions (tears, saliva, intestinal fluid and milk). Associated with SECRETORY COMPONENT—makes antibody resistant to digestive enzymes and plays a role as the first line of defense against microorganisms. e. Immunoglobin E: designed to attached to mast cells in tissues. Once attached, when it encounters antigen, it will cause the mast cell to make prostaglandins, leukotrienes and cytokines and release its granules which contain mediators of inflammation (like histamine). Real role: resistance to parasites like worms. 10. Give examples of immunopathology. a. Type I immunopathology, immediate hypersensitivity i. Patient makes too much IgE to environmental antigen (pollen or food). 10% of population has allergic symptoms. b. Type II immunopathology, autoimmunity due to antibodies which react against self i. This can happen if foreign antigen looks like “self” molecule ii. Treat these diseases with immunosuppressives and anti-inflammatory drugs c. Type III immunopathology i. When patient makes antibody against a soluble antigen. Antigen-antibody complex is usually eaten, but if they are too small, they may get trapped in basement membrane of capillaries they circulate through. Trapped complex activated COMPLEMENT and inflammation occurs. ii. Symptoms: arthritis, glomerulonephritis, rash, pleurisy. Diseases: systemic lupus erythemtosus and rheumatoid arthritis. iii. Foreign antigens that cause Type III: penicillin (in large doses) and foreign serum, “serum sickness”. d. Type IV immunopathology, T-cell mediated i. Can be autoimmune or innocent bystander disease. Example: in TB most of the cavity formation in lungs is T cell mediated, not bacterium mediated (they are just doing their job). e. Chronic frustrated immune responses i. Antigen is not “self” but is something you can’t get rid of, ie: gut bacteria (inflammatory bowel disease) OR gluten (celiac disease). f. AIDS i. Caused by HIV-1 infection of the TH cells (binds to the CD4 molecules they have on their surface). Inside, it uses reverse transcriptase, to copy RNA to DNA and inserts its DNA into host 4 cell. Remains latent until reactivated when the T cell is activated by antigen, leading to progressive loss of Th cells. BL - Blood Cell Types 1. Understand the general principle of the Wright-Giemsa stain: describe how acidic components in the cell will stain and what the main acidic components of the cell are and how basic components in the cell will stain and what the main basic components of the cell are. a. The Wright-Giemsa stain is used to stain peripheral blood smear for light microscopy examination b. Eosin solution will stain BASIC cellular elements an ORANGE-RED color. This includes hemoglobin, basic proteins and some cytoplasmic granules. c. Methylene blue will stain ACIDIC cellular elements a PURPLE-BLUE color. This includes DNA, RNA, basophil granules and cytoplasm of mature lymphocytes and monocytes. 2. Recognize the types of white cells that may normally be found in the peripheral blood. a. Lymphocytes (T-cells, B-cells, NK cells) b. Granulocytes (WBCs that contain granules-neutrophils, eosinophils and basophils) c. Monocytes 3. Describe the difference between the white blood cell differential and the absolute count of a particular white cells count; given the white blood cell differential and the total white blood cell count, be able to calculate the absolute count of a particular white blood cell type. a. White blood cell count: total number of white blood cells (neutrophils, lymphocytes, monocytes, basophils and eosinophils) in a microliter or liter of blood. b. White blood cell differential: percentage of white cells in an individual that are neutrophils, lymphocytes, monocytes, basophils or eosinophils. c. Absolute count of a particular types of white blood cell: total number of particular types of white blood cell based on the white blood cell count AND the white blood cell differential d. Example: i. White blood cell differential= 50% neutrophils, total WBC count= 500/microL ii. Absolute neutrophil count= 250/microL (dangerously LOW) e. Example: i. White blood cell differential= 50% neutrophils, total WBC count= 8000/microL ii. Absolute neutrophil count= 4000/microL (within normal range) 4. Give a range for the absolute counts of the various white blood cells in a normal adult. Describe how the white cell counts fluctuate with age. WBC Type Facts Absolute CountChanges UCHSC with Age? Neutrophil Granulocyte (most abundant), nucleus 1.8-7.8x10^9/L Yes (most (12-15 µm) segmented into 2-5 lobes, protect body from (1800-7800/microL) abundant infection by phagocytosing and destroying WBC i0n bacteria adults nd Eosinophil Granulocyte (2 most abundant), defend 0-0.4x10^9/L Fairly (13 µm) against helminthic infection and mediate (0-400/microL) constant Type 1 hypersensitivity reaction Basophil Granulocyte (least abundant), central role in 0-0.2x10^9/L Very little 5 (10 µm) Monocyte (15-18 µm) Lymphocyte (6-9 µm or 12-15 µm) Type 1 hypersensitivity reactions Largest WBC, become macrophages in tissues which phagocytose foreign agents 2 types: small/round or large granular (0-200/microL) 0.2-0.9x10^9/L (200-900/microL) 1.0-4.8x10^9/L (1000-4800/microL) variation Yes Yes (most abundant WBC in children up to 8) 5. Describe the shape of a red blood cell and the significance of this shape. a. Biconcave disc increases SA for the O2 and CO2 transfer to hemoglobin is facilitated, 7-8 µm in diameter, lacks nucleus, RNA, ribosomes and other organelles. 6. Explain the difference between a red blood cell and a reticulocyte. Describe how a reticulocyte count is performed. a. Reticulocytes are young, anucleated red cells that retain RNA, ribosomes and other organelles that enable ongoing production of hemoglobin, retained in bone marrow for 3-4 days and then released into peripheral blood where they circulate for 1-2 days and then lose RNA, ribosomes and organelles which accumulating hemoglobin. When all RNA and protein-making machinery is lost, the reticulocyte is mature RBC. b. Reticulocyte count is done by supravital staining which causes the RNA, ribosomes and other organelles in these cells to aggregate into a reticulum, thus enabling their identification and enumeration. Can be done manually counting 100 red cells and determining the % of reticulocytes (subjective) OR automated—most objective and can count way more and way faster. Counts can be used to evaluate for anemia. 7. Give a range for the red blood cell count in a normal adult man and a normal adult woman. Describe how the red blood cell count fluctuates with age. Gender Adult RBC count Male 4.6-6.2x10^12/L Female 4.2-5.4x10^12/L Age 1-3 days 4-14 days 15-31 days 1-2 months 3-6 months 7-23 months 2-12 years 13-17 years RBC (x10^12/L) 4.1-6.1 3.8-5.6 3.8-5.6 3.8-5.2 3.9-5.3 4.2-5.4 4.2-5.4 4.2-5.4 8. Be able to recognize a platelet in a peripheral blood smear. 6 9. Give a range for the platelet count in a normal adult. Describe how the platelet count fluctuates with age. a. Platelet count does not vary with age or with gender b. 150,000-400,000/µL (150-400x10^9/L) BL - Blood Draw Demonstration and Blood Drawing 1. Explain what is contained in different colors of blood tubes (red, gold, purple, and blue tops) and why Color For collection of… Additive Mode of Action Uses Red Serum None Blood clots, and Chemistries, immunology serum separated by and serology, blood band centrifugation (crossmatch) Gold Serum None Serum separator Chemistries, immunology tube contains gel at and serology the bottom to separate blood from serum on centrifugation Purple Plasma EDTA liquid Forms Ca2+ salts to Hematology (CBC) and blood remove calcium for bank (Crossmatch), requires prevention of clotting full draw, invert 8 times Blue Plasma Sodium Forms Ca2+ salts to Coagulation tests, ful draw citrate remove calcium for needed to ensure proper prevention of clotting final concentration of citrate in plasma 2. Explain the difference between plasma and serum a. Plasma: fluid portion of blood in which the particulate components are suspended b. Serum: the clear liquid that separates from blood when its allowed to clot completely January 5, 2012 BL - Hematopoiesis, Hematopoietic Precursors, and the Bone Marrow 1. Describe where hematopoiesis occurs before birth, in childhood, and in adulthood. a. Hematopoiesis: process that results in the formation of the mature, functional RBCs, WBCs and platelets in the peripheral blood. b. Pre-natal hematopoiesis 7 i. Yolk sac period: 0 months-3 months gestation, primitive blood cells (especially RBCs) produced in yolk sac ii. Liver/spleen (lesser extent) period: 2 months-7 months gestation c. Post-natal hematopoiesis i. At birth, bone marrow is firmly established as site of hematopoiesis ii. As child ages, hematopoiesis becomes more and more localized to the axial skeleton iii. By age 18-20, 90% of hematopoietically active marrow is in vertebrae, pelvis, sternum, ribs and skull d. Hematopoeisis outside of the bone marrow after birth is abnormal, called extramedullary hematopoiesis. 2. List the types of myeloid cells found in the bone marrow. a. Myeloid: “pertaining to, derived from, or resembling bone marrow” b. Derived from bone marrow: granulocytes, red cells, platelets, monocytes, lymphocytes c. ***But term is usually restricted to non-lymphoid blood cells derived from bone marrow: granulocytes, red cells, platelets and monocytes. d. Lyphoid cells: lymphocytes (T-cells, B-cells and natural killer cells) 3. Define stem cell, progenitor cell, and precursor cell. a. Stem cells give rise to progenitor cells which give rise to precursor cells which mature into the mature cells found in the peripheral blood, lymphoid organs and reticuloendothelial system. i. Stem cell: the most primitive cell type, capable of self-renewal or differentiation/maturation 1. Pluripotential: mother of all blood cells, gives rise to both lymphoid and myeloid elements, CFU-LM (LM=lymphoid/myeloid), can self-renew or commit to becoming a multipotential stem cell 2. Multipotential: CFU-GEMM (GEMM=granulocyte/erythroid/monocyte/megakaryocyte), mother of all myeloid blood cells, some ability to self-renew or they commit to becoming progenitor cells 8 ii. Progenitor cell (aka: committed stem cells): limited ability to self-renew, irreversibly committed to differentiate along one or at most 2 lineages 1. Myeloid progenitors: CFU-GM (granulocytes/macrophage) CFU-G (granulocyte), CFU-M (monocyte), CFU-E (erythroid), CFU-Meg (megakaryocyte), CFU-Eo (Eosinophil), CFUBaso (Basophil), BFU-E (Burst forming unit, gives rise to CFU-E) iii. Precursor: recognizable, maturing cells (ie: that is an immature neutrophil, it will become a neutrophil), capable of cell division, but CANNOT self-renew, give rise to mature, functional cells in the peripheral blood, lymphoid organs and reticuloendothelial system 4. Describe how the phenomenon of self-renewal prevents the bone marrow from rapidly becoming depleted. a. 1% of nucleated cells in bone marrow are stem cells or progenitors, 1% of this 1% (0.01% of nucleated cells) are pluripotential cells. Thus most hematopoietic cells in marrow are precursor cells or mature blood cells waiting to be released. b. Self-renewal (the ability to go through numerous cycles of cell division while maintaining the undifferentiated state, produce a daughter cell that are completely unchanged—genetically and morphologically—from original cell) enables the bone marrow to have very FEW stem cells, but still give rise to billions of blood cells every day. Ensures that there is always a reserve of undifferentiated, uncommitted stem cells. Without this, the stem cells population would be exhausted. c. Self-renewed daughter cell ceases to proliferate, but can at a later time self-renew or commit to differentiation. 5. Discuss maturation and differentiation as they relate to hematopoiesis. a. Maturation: PROTEIN. The accumulation of protein products and refinement of cellular structure dictated by the pattern of gene expression in a cell committed to a particular lineage. b. Differentiation: Genes ON or OFF. As hematopoietic cells progresses from stem cell to functional cell in peripheral blood, it undergoes genetic changes that facilitate the expression of some genes and restrict the expression of other genes. The pattern of gene expression is what leads cells to become their respective committed cell types (ie: lymphocyte, monocyte). c. These 2 things are inextricably bound, so the terms will be used in tandem. 9 6. Discuss the role of hematopoietic growth factors in hematopoiesis. Include the names of the major hematopoietic growth factors, where hematopoietic growth factors are produced, and how they work. a. Hematopoietic growth factors (HGFs): glycoproteins i. Erythropoietin (Epo) ii. Interleukin-3 (IL-3) iii. Granulocyte colony-stimulating factor (G-CSF) iv. Granulocyte-macrophage colony-stimulating factor (GM-CSF) v. Macrophage colony-stimulating factor (M-CSF) b. HGFs regulate proliferation, differentitation and maturation of stem, progenitor and precursor cells, enhance survival and functional activites of mature blood cells, c. HGFs are produced by a number of cells: activated T and B-lymphocytes, macrophages, fibroblasts and endothelial cells. d. HGF interact with specific cell-surface receptors, receptor-ligand complex activates intracellular 2ndary messengers that regulate gene expression that leads to the maturation/differentiation of cells. e. HGFs have overlapping functions, they act synergistically f. Recombinant HGF protein examples: G-CSF (increases neutrophil production for AIDS and cancer patients), erythropoietin (anemic patient--chemo). 7. Using stem cells, progenitor cells, and precursor cells, draw the hierarchical scheme of hematopoiesis as it is understood today. 10 8. List and be able to recognize the granulocytic and erythoid precursors. Be able to recognize a megakaryocyte. a. Granulocytic precursors (in ascending order of maturity): i. Myeloblast: 15µm, high nuclear:cytoplasm ratio, fine nuclear chromatin, 2-5 nucleoli, cytoplasm is blue (basophilic) because it has a lot of RNA, NO CYTOPLASMIC GRANULES ii. Promyelocyte: 20 µm, chromatin is more condensed, presence of large # of large RED PRIMARY GRANULES, but cytoplasm is still blue. iii. Myelocyte: appearance of SECONDARY granules, impart a pinkish hue to the cytoplasm, fewer primary granulocytes in the myelocyte, last neutrophilic precursor with the ability to divide 11 iv. Metamyelocyte: 14-16 µm, ABUNDANT SECONDARY GRANULES, impart pinkish hueto cytoplasm, chromatin is condensed and coarse, nucleus is indented but INDENTATION IS LESS THAN HALF OF NUCLEUS DIAMETER v. neutrophil (band form): 13 µm, horseshoe-shaped nucleus, secondary granules outnumbers primary granules 8-10: 1, INDENTATION IS GREATER THAN ½ NUCLEUS DIAMETER vi. neutrophil (segmented form-MATURE): same size and properties as band form, nucleus segmented into 2-5 (usually 3) distinct lobes that are connected with thin chromatin strands. b. Erythroid precursors i. Pronormoblast: 18 µm, large nucleus, 1-2 nucleoli, cytoplasm has lots of RNA so stains blue ii. Basophilic normoblast: 12-14 µm, cytoplasm is still basophilic, although may be lighter colored perinuclear halo, coarse condensation of nuclear chromatin 12 iii. Polychromatophilic normoblast: hemoglobin accumulates in cell, hemoglobin+plentiful RNA= muddy purple-blue color to the cytoplasm, nucleus is smaller and chromatin has condensed to form chunks iv. Orthochromatic normoblast: “one color”, cytoplasm has distinct red-orange hue imparted by hemoglobin, small, shrunken pyknotic nucleus is soon to be extruded v. Reticulocyte: anucleate, contains ribosomes and mitochondria, blue-purple stain (polychromatophilic) vi. Erythrocyte: 7-8 µm, ribosomes and associated RNA are degraded and cell is now mature erythrocyte, biconcave disc, central area of pallor. 13 c. Megakaryocyte: through endoreduplication, the megakaryocyte contains a multi-lobulated nucleus with 16, 32, or 64 sets of chromosomes. Mature megakaryocyte hands out beside marrow sinues and extends strands of its membrane bound cytoplasm into the lumen or sinus, platelets (2-4 µm, anucleate) form when small chunks of this strand break off and float away. 9. Define cellularity as it relates to the bone marrow. a. Cellularity: the portion of the marrow that is hematopoietically active, non-hematopoietically active marrow is occupied by fat. If ½ of marrow is occupied by hematopoietic cells and ½ by fat, the cellularity is 50% 10. Describe how bone marrow cellularity changes with age. a. Cellularity decreases with age. After age 50, the cellularity is equal to: 100-age of patient. But, there is a lot of variability, and you are really only looking for extreme abnormalities (ie: kid with 20% cellularity or 75 yo male with 80% cellularity) 11. Define the M:E ratio and what this ratio should be in a normal person. a. Myeloid: Erythroid ratio (M:E ratio): assessment of the ratio of granulocytic precursors to erythroid precursors, estimated when bone marrow biopsy from patient is examined, ratio should be about 3:1. BL - Hemoglobin: Structure and Function 1. Describe the overall structure of hemoglobin, indicating the site of oxygen binding. 14 a. 68kD tetramer, with 2 pairs of globin polypeptide chains (1 pair of alpha-globin and 1 pair of non-alphaglobin). A heme prosthetic groups, consisting of a protoporphyrin ring bound to iron, is associated with each globin chain of the hemoglobin tetramer. It is the heme group that binds oxygen. 3. Explain the concepts of allostery and positive cooperativity as they relate to hemoglobin function. a. Allostery: when oxygen (substate) binds to hemoglobin at one site, hemoglobin has a change in configuration, which alters its binding affinity of additional oxygen molecules at another site. This enables hemoglobin to easily pick up oxygen in the lungs (high O2 levels) and unload it in the tissues (low O2 levels). If the allosteric changes lead to increased affinity for substrate at the other binding sites, it is termed positive cooperativity. b. Positive cooperativity: the phenomenon of binding to substrate (oxygen) leading to increased affinity for additional substrate (more oxygen!) is called positive cooperativity. When 1 oxygen binds, the configuration of hemoglobin changes so that the other 3 sites have a higher binding affinity. As the number of occupied sites increases, the affinity for the remaining sites continues to increase. 4. Compare the oxygenated and deoxygenated states of hemoglobin in relationship to taut (T) and relaxed (R) configurations of the molecule a. Taut (T): Deoxygenated, under conditions where the oxygen concentration is low enough that none of the 4 binding sites are occupied, the binding affinity to oxygen is relatively low, T-configuration is present due to inter-and intra-salt bonds, hydrogen bonding, and hydrophobic interactions within the molecule. b. Relaxed (R): Oxygenated, as oxygen becomes more available, 1 oxygen binds, the configuration changes and the other sites have higher binding affinity for oxygen. The sequential breaking of salt bonds leads to the R-configuration. 5. Draw a typical oxygen dissociation curve. Explain why it is sigmoidal in shape. Define the p50. Roughly estimate % oxygen saturation with pO2s of 100 mmHg, 60 mmHg, 40 mmHg, 30 mmHg, and 10 mmHg. a. The oxygen dissociation curve is sigmoidal in shape because of cooperativity: binding of substrate leads to increased affinity for additional substate. Allows oxygen to easily bind at higher pO2 level in lungs and unload at lower pO2 level in tissues. 15 b. p50: the partial pressure of oxygen at which the oxygen carrying protein (hemoglobin or myoglobin, usually) is 50% saturated. Normal conditions: p50 for hemoglobin is 27 mmHg and p50 for myoglobin is 2.75 mmHg. c. % O2 Saturation with pO2 of: 100 mmHg=98%, 60 mmHg= 90%, 40mmHg= 75%, 30mmHg=60%, 10mmHg= 10%. 6. Explain the effects of pH, CO2 concentration, temperature, and 2,3-BPG concentration on the hemoglobin oxygen dissociation curve. a. pH: low pH (more acidic)= decreased oxygen affinity, oxygen is unloaded, curve shifts to the right. Higher pH (more alkaline)= increased oxygen affinity, oxygen is held more tightly, curve shifts to the left. Known as the BOHR EFFECT. b. CO2 concentration: when CO2 is released into the bloodstream, the carbonic anhydrase converst CO2+H20 carbonic acid bicarbonate and H+ (drops the pH). This continues the Bohr Effect. Tissues with higher metabolic rate will release more CO2 and lactic acid, leading to a drop in pH, which then shifts the curve to the right, allowing greater release of oxygen to the tissues. Efflux of CO2: pH of blood rises, increased oxygen affinity, easier loading of oxygen to the hemoglobin. c. Temperature: Higher temperatures=more oxygen is unloaded to tissues and less is bound by hemoglobin (think: when you exercise your temperature rises because metabolic rates are higher, therefore increased need for oxygen). Curve shifts to the right. d. 2,3-BPG: by product of the anaerobic glycolytic pathway (normal level=5 mmol/L). When level is much higher (in states of increased O2 use, chronic HPX, chronic anemia), oxygen affinity of hemoglobin decreases and shifts the curve to the right, increasing delivery of O2 to the tissues. 2,3-BPG alters O2 16 affinity by binding to deoxyhemoglobin and stabilizing it in the T-configuration, leading to decreased affinity of the hemoglobin for oxygen. 7. Compare oxygen dissociation curves for myoglobin and hemoglobin and explain the reason for the differences. a. Hemoglobin is a tetramer, myoglobin is a monomer and therefore cannot undergo allosteric regulation or cooperativity. Myoglobin curve is shaped like a hyperbola, very high O2 affinity at very low O2 concentrations. Myoglobin would be a poor protein to use for O2 transport from lungs to tissues because it holds very tightly to O2 and does not release until O2 levels are extremely low. But, myoglobin is good to use for oxygen storage intracellularly where O2 levels are very low and where high oxygen affinity is needed to transfer the O2 from hemoglobin to myoglobin. 8. Describe the location and the general organization of the genes for alpha-like globin chains and beta-like globin chains. List and describe the typical hemoglobin proteins seen during fetal development and in adulthood, including fetal hemoglobin, hemoglobin A, and hemoglobin A2 and explain how amounts of these different hemoglobins change during development. a. Alpha-like genes are found on chromosome 16, 2 copies of alpha-globin from each parent (total of 4 gene copies) b. Beta-like genes are found on chromosome 11, 1 copies of beta-globin from each parent (total of 2 gene copies) c. Embryos (at 4-14 weeks) have 3 distinct hemoglobins with higher affinity for O2 than hemoglobin A (allows for O2 transfer from mom to baby) i. Hemoglobin Gower I ii. Hemoglobin Gower II iii. Hemoglobin Portland d. At 8 weeks, fetal hemoglobin predominates (HbF). HbF binds 2,3-BPG poorly, thereby stabilizing the Relaxed hemoglobin state and shifting the oxygen dissociation curve to the left. The Bohr effect also increases by 20% in HbF so that as fetal blood passes through the intravillous spaces of placenta, H+ ions are transferred to the maternal circulation and the pH rises, leading to increased O2 affinity and a further shift of the curve to the left. e. At birth: 65-95% HbF and 20% HbA f. Adults (actually around age 5, although HbF remains high in premature babies and infants of mothers with diabetes): 96-97% HbA, 2% HbA2, ˂1% HbF 17 g. HbA2 (α2/δ2): functions much like HbA, has same Bohr effect, cooperatitivity and response of 2,3-BPG but is more heat stable and has slightly higher O2 affinity. Can be used diagnostically for Betathalassesmias, sickle cell trait, hyperthyroidism and megaloblastic anemias. h. 9. Describe how the structural differences between fetal hemoglobin and hemoglobin A lead to differences in oxygen affinity and why it's important. a. After week 8 of gestation, fetal hemoglobin or hemoglobin F (α2γ2) predominates. The γ- chain differs from the β-globin chain by 39 amino acids. Fetal red cells have a higher oxygen affinity than adult red cells, primarily because hemoglobin F binds 2,3-BPG poorly, stabilizing the hemoglobin in the R state and shifting the oxygen dissociation curve to the left. The Bohr effect is also increased by 20% in fetal hemoglobin, so that as fetal blood passes through the intravillous spaces of the placenta, H+ ions are transferred to the maternal circulation and the pH rises, leading to increased oxygen affinity and a 18 further shift of the curve to the left. These changes favor transfer of oxygen from the maternal circulation to the fetal circulation. 10. Describe how unstable hemoglobins or hemoglobins with altered affinity can affect oxygen delivery to the tissues. a. 200 clinically significant hemoglobin variants, there are high-affinity and low-affinity hemoglobin variants b. Hemoglobin Chesapeake: First high-affinity hemoglobin variant discovered in 1966, single point mutation, symptoms of high-affinity hemoglobins: erythrocytosis (elevated RBC) because oxygen delivery to tissues is reduced increased erythropoiesis increase red cell count. Affected people are generally well. DX: measure p50 (shifts to left). c. Low-affinity hemoglobin variant: less common, presents as cyanosis +/- mild anemia, more oxygen is delivered to the tissues, DX: p50 value shifts to the right. d. Unstable hemoglobins: spontaneously denature, may or may not bind oxygen like HbA, Examples: Hemoglobin Zurich—single point mutation, doesn’t effect O2 binding, but increases CO binding. Hemoglobin Koln: most common in West, mutation in Beta-chain, increase O2 affinity, pts have 10-25% HbKoln and have mild anemia, reticulocytosis and splenomegaly. 11. Describe what methemoglobinemia is, what causes it, how to diagnose it, and how to treat it. a. Methemoglobinemia is when there is either too much methemoglobin production or because of decreased methemoglobin reduction. Methemoglobin forms when iron within hemoglobin is not reduced from its 2+ (ferrous—oxygen binding form) form to its 3+ (ferric) form. Usually NADPH methemoglobin reductase keeps the iron in its ferrous form in the erythrocyte. When iron is stuck in its +3 ferric form, it can’t bind oxygen, shifts the oxygen dissociation curve to the left and P50 drops. b. Can by acquired or genetic i. Acquired: exposure to drugs and chemicals that causes the oxidation of the heme by reaction with free radicals of hydrogen peroxide or NO or OH can generate methemoglobin. Examples: use of benzocaine in neonates, well water with nitrates. ii. Genetic (hereditary): most commonly due to homozygous deficiency of cytochrome b5 reductase or mutation in hemoglobin resulting in production of hemoglobin M. iii. Newborns are susceptible because HbF is more readily oxidized to ferric state, also decreased amount of cytochrome b5 reductase, may become cyanotic with well water, raw spinach, disinfectants, benzocaine. c. Diagnosis: person looks cyanotic but arterial partial pressure of O2 is normal, blood looks darkred/chocolate/brown-blue and doesn’t change with O2 exposure d. Treatment: i. Genetic: no treatment needed for hemoglobin M, cytochrome b5 deficient patients treated with methylene blue (for cosmetic reasons) ii. Acquired: methemoglobin levels below 30%=minimal symptoms (light headed, headache), 3050%= depress CV and CNS, rapid breathing, shortness of breath, 50%-70%= severe, stupor, low HR, respiratory depression, convulsions, 60%+ can be lethal and 70%+ is not compatible with life. Remove drug or chemical causing methemoglobinemia. Methylene blue can be given via IV to provide artificial electron acceptor from the reduction of methemoglobin via the NADPHdependent pathway. 19 12. Explain the pathophysiology of carbon monoxide poisoning and its treatment. a. CO binds heme with an affinity 240x that of oxygen, normally people have 3% CO (in smokers its 1015%), when heme binds CO, allosteric change occurs so that other 3 hemes unload oxygen less well, increasing the affinity of hemoglobin for oxygen and decreasing the delivery of O2 to the tissues (Curve shifts left). b. Symptoms: headache, malaise, nausea, dizziness, NOT cyanotic, “cherry red” appearance, higher levels: seizures, coma, MI, 40% of people have late neurological defects. c. Diagnosis: co-oximetry, Treatment: 100% O2 or hyperbaric O2 (competes with CO for binding sites on the heme) 13. Describe how a pulse oximeter works and what it measures. Describe situations where a pulse oximeter reading may inaccurately reflect a patient’s true oxygenation status. a. Pulse oximetry is based on Beer-Lamberts law which states the absorption of light of a given wavelength passing through a non-absorbing solvent, which contains an absorbing solute, is proportional to the product of the solute concentration, the light path length and an extinction coefficient. b. Pulse oximeter probe is a photo detector and 2 light emitting diodes (one at 660nm-red where deoxyhemoglobin absorbs maximally and one at 940nm-infrared where oxyhemoglobin absorbs maximally). The emitter and detector face each other through the tissue (placed on the finger), only PULSATILE FLOW (arterial blood flow) is measured, photodiodes switch on and off several hundred times/second and light absorption is measured. Displayed value is an average of 3-6 seconds. Used to measure oxygen saturation (not partial pressure of oxygen or carrying capacity of oxygen). c. Inaccurate is probe is placed wrong, if only 1 diode is working, shivering/seizing patient, nail polish, deeply pigmented ski, anemia, shock, abnormal hemoglobins (ie: carboxyhemoglobin absorbs at 660nm so will give a falsely high reading, methemoglobin absorbs at 660nm and 940nm so it will also give inaccurate results). Co-oximetry (measure absorbance at 4+ wavelengths) is needed to quantify carboxyhemoglobin and methemoglobin levels. BL - Anatomy and Physiology of the Immune System 1. Define: a. Leukocytes: nucleated cells of the blood, white blood cells, when you centrifuge anticoagulated blood, they sediment on top of the packed red cells forming buffy coat. b. Mononuclear cells: leukocytes whose nucleus has a smooth outline, monocytes (immaturemacrophages in tissue) and lymphocytes. Can be hard to distinguish macophages and lymphocytes. c. Polymorphonuclear cells: cells whose nucleus is lobulated, also called granulocytes because they usually have prominent cytoplasmic granules. They are eosinophils, basophils and neutrophils. d. Granulocytes: white blood cells that have cytoplasmic granules, also known as polymorphonuclear cells, they are eosinophils, basophils and neutrophils. e. Dendritic cells: cells that connect the innate immune response and adaptive immune response, act as antigen presenting cells to other cells in the immune system. f. Mast cells: granules full of histamine, role in allergy and anaphylaxis. Very similar to basophil granulocytes. g. Plasma: yellow fluid portion of blood in which the particulate components (blood cells) are suspended. 55% of blood volume. 20 h. Serum: the clear liquid that does not contain blood cells nor clotting factor, it is the blood plasma with fibrinogens removes, includes all proteins not used in blood clotting and electrolytes, antibodies, hormones, etc. 2. Sketch schematically a dendritic cell; neutrophil; eosinophil; basophil; small lymphocyte; lymphoblast; plasma cell; monocyte. Indicate the characteristic features which distinguish each cell type. a. Neutrophil has colorless granules, Eosinophil has red granules, and Basophil has blue granules. http://en.wikipedia.org/wiki/White_blood_cell 3. Name the major central and peripheral lymphoid organs. a. Central organs: ones in which lymphocytes deveolp, the bone marrow and the thymus b. Peripheral organs: mature cells are organized to trap and respond to foreign invaders, includes lymph nodes, spleen, Peyer’s patch and mesenteric lymph nodes of gut, tonsils, adenoids. 4. Describe the recirculation of lymphocytes from blood to lymph and back; include in your discussion the specialized features of lymph node endothelium that permit recirculation. a. Lymphocyte in the blood encounters the cells lining certain postcapillary venules in the peripheral lymphoid tissues (especially lymph nodes). These endothelial cells are unusual (not flat, but HIGH AND CUBODIAL). Recirculating lymphocytes may bind to and pass between the endothelial cells into the lymph node, where they may stay, or pass into the lymph which drains from that lymph node to the next node in the chain. Lymph goes to large lymph channels (thoracic duct, near heart)venous bloodcirculatory loop starts over again. Thus, there are 2 circulation: blood and lymphatic, in which lymphocytes cross from blood to lymph at nodes and from lymph back to blood at heart. 5. Define antigen, and compare it to immunogen. Define antigenic determinant and epitope. 21 a. Antigen: substance that can be recognized by the immune system b. Immunogen: an antigen in a form that can give rise to an immune response, that is, which can immunize c. Antigenic determinant and epitope: small part of a large antigenic molecule, fits (lock and key) into lymphocytes receptor and activates lymphocyte, an isolated antigenic determinant is not usually an immunogen; it can be recognized by antibody, but is too small to trigger an immune response. d. Tolerogen: antigen delivered in a form, or by a route, which does not give rise to an immune response, and which furthermore prevents an immune response to subsequently administered immunogen which has the same epitopes 7. Discuss lymphocyte activation by antigen with respect to: role of dendritic cells, receptor binding, proliferation, differentiation. Draw a graph showing relative time on one axis and relative lymphocyte numbers on the other, in response to antigen administration. a. Each lymphocyte has receptors, there are many copies on each cell, but all are identical (ie: each cell has single specificity). T cell receptors are alpha and beta chains. B cell receptors are samples of the antibodies that the cell will eventually secrete. The antigenic determinant (presented by the dendritic cell) fits into the lymphocyte receptor. To activate T or B cell: 1. The fit between receptor and antigen must be good enough, 2. Several nearby receptors must be simultaneously bound by antigen, 3. For Tcells only, other cell surface molecules must be involved too. Activated cell proliferates and differentiates. b. Lymphocytes can divide every 6 hours, 1 can give rise to 64,000 at the end of 4 days. 8. Distinguish between "humoral" (antibody-mediated) and cell-mediated immunity in terms of: the types of lymphocytes involved, the nature of the molecules they release when activated, the types of inflammatory cells they preferentially involved. State which of these immunities can be transferred by serum. a. Humoral: this is the antibody mediated response, and this occurs extra-cellularly where all the bacteria etc. live. B lymphocytes are the main cells involved. B cells transform into plasma cells which secrete antibodies. Cytokines are also released. Can be transferred by serum. b. Cell mediated immunity: T lymphocytes become activated. These in turn activate macrophages, NK cells, and cytotoxic T lymphocytes. Cytokines are released when the T cells become activated. Not transferred by serum. 9. List the normal adult white blood cell count and differential percentages. From these calculate the absolute counts for the different cell types. a. Normal total white blood cell count: 4,500-10,500/µL of blood (4.5-10.5x10^9/L) White Cell Type Neutrophils Eosinophils Basophils Monocytes Lymphocytes Differential 40-60% 1-4% (higher in developing countries) 0.5-1% 2-8% 20-40% Absolute Count 2250-5250 per µL 113-263 per µL 34-79 per µL 225-525 per µL 1350-3150 per µL BL - Anemia: Overview of the Approach to a Patient 1. Define anemia and discuss the laboratory tests used to determine its existence in an individual. Explain the influence of age and gender on the definition. 22 a. Anemia: insufficient red call mass to adequately deliver oxygen to peripheral tissues b. Measurements to determine anemia: hemoglobin concentration, hematocrit and red blood cell count. Also, mean corpuscular hemoglobin concentration, mean corpuscular volume, red cell distribution width, white blood cell count and differential, and platelet count. Red blood cell morphology via blood smear and Reticulocyte count (%), reticulocyte production index. c. Reference ranges provide values below which anemia is defined, variation based on age, gender and geography (Denver vs. sea level) 2. Define reticulocyte count, absolute reticulocyte count, and reticulocyte index and discuss how these measurements are used in assessing the rate of RBC production. a. Reticulocyte count: percentage of reticulocytes when 1,000 red blood cells are counted i. Normal range: 0.4-1.7% ii. Increased RBC production 3.5-5 fold increase from normal range b. Absolute reticulocyte count: percentage of reticulocytes x red cell count i. Helps determine the relevance of the reticulocyte count ii. ˃50,000/µL is considered an increase from baseline maintenance of RBC production c. Reticulocyte index: measurement of production of red cells, way to correct reticulocyte count for red cell concentration and stress reticulocytes (marrow pushes reticulocytes out before they are fully matured) i. RI= reticulocyte count x (patient Hgb/Normal Hgb) x (1/stress factor) 1. Stress factor = 1.5 (mild anemia), 2.0 (moderate anemia), 2.5 (severe anemia) ii. RI= 1.0-2.0 in a healthy patient. RI ˂1 with anemia indicates decreased production of reticulocytes and therefore RBCs. RI ˃2 with anemia indicates loss of RBCs (hemolysis, bleeding) leading to increased compensatory production of reticulocytes to replace lost RBCs. 3. Draw a general classification scheme of anemias based on mean corpuscular volume (MCV) and reticulocyte count. a. Question 1: are there any additional hematological abnormalities besides anemia (ie: are WBCs or platelets abnormal, thrombocytopenia, leukopenia, neutropenia)? b. Question 2: Is there an appropriate response to anemia? i. Increased reticulocytes indicates possibility of increased red cell destruction or blood loss ii. NON-increased reticulocytes (no evidence for hemolysis), then consider the types of anemia based n MCV and size (ie: normo-, macro- or microcytic) c. Question 3: What are the red blood cell indices? i. MCV ˃ 100, Macrocytic ii. MCV 80-100, Normocytic iii. MCV ˂ 80, Microcytic 23 4. Recognize critical findings in the history and physical examination important in determining the cause of anemia. a. History: time of onset (acute or chronic), family history of gallstones, jaundice, splenomegaly, splenectomy or cholecystectomy (predisposition for hemolytic anemias), PMH of occupation, toxin exposure, travel or drugs. Dietary information re: intake of vitamins and iron. ROS: hair, skin, nails, stool, menstrual flow, fever, urine color. PMH of renal, liver, endocrine disease, malignancy, infection or inflammation (important for sorting out etiologies of anemia). b. Physical exam: vital signs, color of skin, conjunctiva, lymph nodes, size of liver and spleen, cardiovascular and pulmonary findings. c. Symptoms: shortness of breath, fatigue, rapid heart rate, dizziness, pain with exercise (claudication) and pallor. 5. Describe iron metabolism and the iron cycle, and describe where iron is distributed in the body. Describe the route by which iron from the diet becomes incorporated into hemoglobin (including absorption, transport, delivery, storage, and loss of iron in humans). List factors that increase or decrease iron absorption. a. GENERAL IRON i. Fe exists in two valence states and activity may depend on a specific state. ii. In aqueous solutions, Fe forms insoluble hydroxides unless bound (protein, heme, etc.). Iron is never FREE. iii. Fe salts are more soluble at low pH. iv. Fe balance: controlled by absorption; no active excretion/secretion mechanism. v. Losses each day are small: loss from exfoliation of skin and mucosal surfaces (GI, skin); in the urine or with menstruation. b. IRON CYCLE i. Allows most of the recyclable (red cell) iron to be re-used and minimizes the amount of iron required from absorption each day. ii. Once iron is through mucosal cell and bound to transferrin (84kDa plasma protein, main transport protein for iron, binds 2 moles of iron in ferric 3+ form, specificity and affinity are very high), it enters the cycle. Transferrin bound with iron goes to bone marrow/maturing normoblasts and binds to cell surface receptors called “transferrin receptors”. Transferrintransferrin receptor complex enter cell through invagination of clathrin coated pits to form endosome. Endosome becomes acidified through entry of protons releasing iron from 24 endosome through the divalent metal ion transporter DMT1. Receptor directs iron into the maturing normoblast and is incorporated into hemoglobin mature RBC into circulation for 120 days. After 120 days, RBCs removed by macrophages in spleen, macrophages sequester iron in intracellular ferritin (24 subunit coat--alternating heavy and light chains—can contain 4500 atoms of iron) molecule stores. Iron from stores can be released fron cell and bound by transferrin again. Hemosiderin is another iron storage molecule but the iron is not complete soluble and bioavailable. 25 c. IRON ABSORPTION i. Iron from diet enters stomachgastric pH and gastroferrin maintain solubility and availability of iron until it reaches duodenum absorption occurs at duodenum (at mucosal surface). Nonheme bound iron enters duodenum as ferric iron (enters cell through action of divalent mental iron transporter DMT1) and is concerted to ferrous iron by surface reductase (mediated by duodenal cytochrome b-like protein). In cell, some iron stored in ferritin, some transported across baso-lateral membrane by ferroportin transporter. Hephaestin (a ferroxidase) facilitates basolateral iron export. ii. Two types of iron: elemental (better understood process) and heme-bound d. INCREASED ABSORPTION i. Presence of protein (amino acids), vitamin C (maintains iron is appropriate valence state), increased amount of iron presented to the duodenum, increased erythropoietic activity (nonspecific increases absorption). e. DECREASED ABSORPTION i. phytates, oxalates and other food constituents (precipitate iron making it less available), decreased amount of iron presented to duodenum. 26 f. HEPCIDIN i. 25 aa peptide in liver, produced in response to high iron intake, inflammation/infection. Production of hepcidin is reduced by anemia and/or hypoxia. LOW hepcidin= increased iron absorption, plasma transferrin is saturated with iron and iron accumulated in liver stores. HIGH hepcidin=plasma iron is decreased limiting erythropoiesis because hepcidin binds to ferroportin causing its degradation, loss of export of iron out of cell and increased accumulation of iron in ferritin. ii. Negative regulator of iron absorption by the intestinal epithelial cells, transport by the placenta, and release from macrophages (RE system) 6. Describe the hematologic changes associated with the development of iron deficiency and the timeline by which they occur. Recognize some of the major causes for iron deficiency. a. Hematologic changes: decrease in hemoglobin, decrease in cell proliferation, mild hemolytic component caused by an increase in the rigidity of the cells produced under iron deficient conditions. b. Systemic changes: defective muscle performance, neuropsychological dysfunction, ridges on nails, koilonychia (flat or concave nails), papillary atrophy of tongue. Dysphasia, esophageal webs, gastritis, immune dysfunction. c. Major causes: excessive losses (bleeding), failure to accumulate iron to replace the small on-going losses, inability to gain iron required during excessive demand (growth/pregnancy). d. Iron depletion (stage 1): iron from stores decreases (diminished ferritin levels), serum-normal, transferrin saturation—normal, hemoglobin stores—normal, erythropoiesis—normal. Iron absorption may increase slightly. e. Iron deficient (stage 2): iron stores depleted, decrease in serum iron, increase in iron binding capacity, iron loading of normoblasts starts to become impaired, erythrocytes—normal. f. Iron deficiency anemia (stage 3, final): transferrin is increased, serum iron is very low, saturation is so low it cannot meet erythropoiesis needs, cells produced are microcytic and hypochromic. 7. Describe the symptoms, signs, and laboratory findings associated with iron deficiency anemia. 27 a. Epidemiology: most common nutritional deficiency. Iron deficiency in 9% in infants and toddlers (3% are anemic). 11% in adolescent females, 7% in post-menopause females, 1% in males less than 50yo, 2-4% in males over 50 (due to GI problems/bleeding) b. Symptoms: pallor, fatigue, loss of exercise tolerance, irritability, behavioral changes. c. Lab tests: decrease in oxygen carrying capacity (Hgb, Hct), decrease in production (ie: low retic count and index). Early-on: mild normochromic, normocytic anemia observed. Later-on: CBC shows microcytic, hypochromic cells, also see elliptocytes, fragmented RBCs, spherocytes. d. Additional studies: decreased serum iron, increase in total iron binding capacity, low serum ferritin, increase in erythrocyte protoporphyrin. e. Treatment: iron salts orally, iron by IM or IV route. 8. Describe the effects of over accumulation of iron in the body and describe two treatments for iron overload. a. CAUSES i. Increase in iron intake from diet ii. Increase in absorption of iron: hemochromatosis 1. Mutation in HLA-H gene which encodes from protein that acts as co-factor for absorption results in an increase in absorption of iron iii. Repeated transfusions of iron for chronic anemia: hemosiderosis b. EFFECTS 28 i. Accumulation of iron damages organs like the heart (arrhythmia and CHF), liver (dysfunction/failure), endocrine organs (diabetes, pancreatic function). c. TREATMENTS i. Depends on cause. Increased absorption tx=therapeutic phlebotomy to reduce iron burden to body until ferritin levels are in normal range. For repeat transfusions: iron chelators used to return iron to normal levels. 29 WEEK 2 BL - CBC/Peripheral Smear and Bone Marrow Large Group Presentation 1. Understand the basic principles of flow cytometry. a. Primary method for characterizing normal and abnormal blood cells (automated hemotology machines that perform the CBC are in large part flow cytometers). b. Method of cellular analysis that involved passing cells in a signle files line past a probe of some sort. When a cell passes through a hole, it causes a voltage drop, and the size of the voltage drop is related to the size of the cell. c. Depending on the type of probe, a number of different things can be determined. The probe is usually a source of monochromatic light. Intrinsic parameters assessed using flow cytometry: cell size/shape (forward angle light scatter-0.5-15 degree), cytoplasmic granularity (right angle light scatter-90 degree, more granules=more right angle light scatter). 2. Describe the things that should be scrutinized when a peripheral smear is examined. a. 3. Define and give the units for the hemoglobin (HGB) measurement. a. In vitro measurement of the concentration of hemoglobin released by lysed red cells into whole blood, red cells are lysed and converted into a form that can be measured (cyanmethemoglobin). Units: g per dL or g per L. 4. Define and give the units for the hematocrit (HCT) measurement. a. Measure of how much of a given volume of whole blood is occupied by red blood cells. Can be expressed as a percentage (%) or in terms of liters per liter (HCT of 0.5L/L means that there are 0.5 L of red cells per 1 L of whole blood). Blood is centrifuged and separated into 3 layers: bottom layer (RBCs), middle layer (buffy coat-WBCs and platelets), top layer (plasma). HCT is determined by dividing the height of the entire column by the height of the red cell layer. Indirectly you can find HCT with the formula: HCT%= (RBCxMCV)/10 30 5. Given the red blood cell count (RBC), the HGB and the HCT be able to calculate the MCV, MCH, and the MCHC. Define the MCV, the MCH, and the MCHC and relate these definitions to the units for these indices. Describe how the MCV, the MCH, and the MCHC are used (or not used) in evaluating a patient’s red blood cells. a. MCV=(HCT in L/ L ÷ RBC in 1012/L) x 10 i. Mean size of red cells counted, units: femtoliters (fL)—10-15 L. Used often to see the relative number of red cells of a particular size vs. size of red cells. In patients with anemia, it is the MCV measurement that allows classification as either a microcytic anemia (MCV below normal range), normocytic anemia (MCV within normal range) or macrocytic anemia (MCV above normal range). b. MCH= (HGB in g per L/RBC in 1012/L) x 10 i. Mean quantity of hemoglobin in a single red cell, unit: picograms—10-12 g. NOT used frequently since it parallels the MCV (when MCV goes up, MCH goes up). diminished in hypochromic anemias c. MCHC=(HGB in g per L/HCT in L/L)x 100 i. Average concentration of hemoglobin in red cells, units: g per dL or g per L. NOT used frequently, increased value ONLY seen in hereditary spherocytosis. Decreased value seen in moderate – severe microcytic anemia. 6. Describe the cause of platelet clumping and what should be done when platelet clumping is suspected as the cause of a spuriously low platelet count a. Platelet clumping on a peripheral smear is caused by the presence of EDTA anticoagulant. This artifact causes a decrease in platelet count. Use a different anticoagulant (Sodium citrate) to get a better platelet count next time. b. The automated hematology analyzer perceives this clump as a large platelet. Thus the rise from the baseline – the analyzer thinks it is seeing larger platelets, counts them, and displays them on the PLT histogram. However the analyzer is programmed to know that this histogram is abnormal and will alert the operator. This alert is blacked out on the displayed printout in Slide 11. 7. Describe the advantages and disadvantages of a manual white blood cell differential. Describe the advantages and disadvantages of an automated white blood cell differential. a. Manual white blood cell differential i. Advantages: more accurate (depending on the user) ii. Disadvantages: time consuming, can’t count as many cells as quickly b. Automated white blood cell differential i. Advantages: very good at recognizing NORMAL blood cells, can count 1000’s of cells rather than hundreds (therefore more statistically accurate under normal conditions) ii. Disadvantages: not good as identifying abnormal cells, may still require a manual differential 8. Be able to identify normal white blood cells in a peripheral smear. 31 A: small round lymphocyte (top), segmented neutrophil B: monocyte C: small round lymphocyte D: large granular lymphocyte A: monocyte B: large granular lymphocyte C: large granular lymphocyte D: monocyte (left), band (right) 32 A: Band B: segmented neutrophil (right), monocyte (bottom) C: eosinophil D: basophil BL - Antibody Structure 1. DEFINE a. H chain: heavy chain, MW=50,000, each antibody has 2 H chains, each H chain has 1 variable domain (VH) and 3-4 constant domains (CH1, CH2, CH3, (CH4)), 5 kinds of H chains (gamma, alpha, mu, epsilon, delta—each corresponds to the appropriately named antibody: IgA has alpha chains 33 b. L chain: light chain, MW= 25,000, each antibody has 2 L chains, each L chain has 1 variable domain (VL) and 1 constant domain (CL) c. Kappa and lambda chains: 2 varieties of L chain, each cell that makes antibody has a choice, but it uses only one kind. d. hinge region: allows for flexibility so that when bound to antigen, the constant part of the antibody can change conformation e. Fab: S--S bonds between the H chains fully reduced f. F(ab')2: 2 Fabs still joined by S—S bond g. Fc: non-antigen binding region of the antibody, makes antibody participate in complement h. complementarity-determining regions (CDRs): light chain and heavy chain variable region is NOT uniformly variable, most of the variability is in 3 regions called hyper-variable regions of CDR’s, amino acids in this region comprise the actual antigen-binding site. i. hypervariable regions: areas on the variable region of light/heavy chain with amino acid availability j. variable (V) domain: at N-terminal of antibody, differences in amino acid sequences between antibodies of different specificities k. constant (C) domains: region that is essentially identical on the antibodies, made up of 1 (in L chains and 4 (in epsilon and mu) compact, structurally similar domains called C domains. l. VL and CL: variable domain of light chain and constant domain of light chain m. VH and CH: variable domain of heavy chain and constant domain of heavy chain 2. Name the 5 antibody classes, and their characteristic heavy chains. a. IgG: 2 light and 2 gamma (heavy) chains b. IgE: 2 light and 2 epsilon (heavy) chains c. IgD: 2 light and 2 delta (heavy) chains d. IgA: 4 light, 4 alpha (heavy) chains, 1 Joining chain and 1 Secretory component e. IgM: 10 light, 10 mu and 1 joining chain 3. Draw a diagram of the structure of typical molecules of each class. Do not bother with the exact number of CH domains. Label the heavy and light chains; Fc and Fab parts; J chains if any; antibody combining sites; main interchain disulfide bonds; secretory component. 34 4. Distinguish the 5 immunoglobulin classes in terms of size, and for IgG, IgM, and IgA, their approximate concentration in serum. Antibody Size [Serum] IgG 150,000 1000 mg/dL (dL=100mL) IgE 190,000 (extra long CH4) (0.02 mg/dL) IgD 180,000 (extra long hinge) (5 mg/dL) IgA Secreted 400,000 (monomer is 160,000, J chain is 200 mg/dL 15,000 and SC is 70,000) IgM 900,000 (5x 180,000, an extra CH4 domain + J chain) 100 mg/dl 5. Describe the structure of antibody combining sites. a. Combining site is where the antibody binds to the antigen, is made up of V domains of both the H and L chain (VH and VL) 6. Explain why complementarity-determining regions are also called hyper variable regions. a. There are 3 hyper variable regions on the variable domain of light and heavy chains. These regions have amino acid variability. It is more functionally significant to call them complementarity-determining regions because the amino acids in the hyper variable region comprise the actual antigen-binding site 7. Give an example of a subclass, an allotype, an idiotype. a. Subclass: immunoglobulins are divided into subclasses because of slight differences in the amino acid sequences of their H chain C regions. IgG1, IgG2, IgG3, IgG4. IgA1, IgA2. IgM1, IgM2. IgD and IgE. b. Allotype: minor allelic differences in the sequence of immunoglobulins between individuals, determined by allotypes of your parents, useful in determining relatedness c. Idiotype: unique combining region, made up of the CDR amino acids of its L and H chain, that each antibody has. Anti-idiotype: antibodies made that recognize the unique sequence of that combining site. 35 8. Diagram an electrophoretic separation of human serum. Label the anode and cathode. Identify the albumin, alpha1, alpha2, beta and gamma peaks. BL - Antibody Function and Complement 1. Define: a. Valence: The valency of antibody refers to the number of antigenic determinants that an individual antibody molecule can bind. The valency of all antibodies is at least two (divalent) and in some instances more (multivalent). b. Affinity: The strength with which an antibody molecule binds an epitope (= antigenic determinant) is called its affinity. c. Precipitation: large immune complexes that are formed at or near equivalence tend to become insoluble and fall out of solution, when the antigen is a molecule, it is called precipitation. d. Agglutination: large immune complexes that are formed at or near equivalence tend to become insoluble and fall out of solution, when the antigen is a cell or cell-sized particle, it is called agglutination. e. Epitope: the part of the antibody that actually interacts with the antigen, usually 10-20 amino acids long. Also known as antigenic determinant. Proteins have several epitopes which bind to different antibodies. 2. Distinguish the five classes of immunoglobulins in terms of: passage across the placenta, ability to activate complement by the classical pathway, ability to activate complement by the alternative pathway, involvement in allergic diseases, “first line of defense”, most resistant to enzymatic digestion a. IgG: most abundant immunoglobulin in blood, only class that passes the placenta (mother fetus, requires active transport), comes up later than IgM after primary immunization, but levels go higher and last longer, plasma life= 3 weeks, phagocytic cells have receptors for the Fc of bound IgG opsonizing. It takes 2 IgG’s close together to activate complement. b. IgM: first immunoglobulin seen in blood after immunization, decavalent, but shape rarely allows more than 2 of its 10 binding sites to interact with antigenic determinants. Best at complement because it always has 2 adjacent Fc’s to begin complement cascade. IgM is viscous in solution (because of its size), so if we only had IgM we couldn’t pump our blood. There are no useful IgM receptors on phagocytes. IgM is the only antibody made in the fetus. c. IgA: made by plasma cells in lymphoid tissues near mucous membranes, assembled into dimer by the adition of the J chain while in plasma cell and then secreted into interstitial space. Adjacent epithelial cells have receptors for IgA which binds to them and is taken up and moved to the luminal side. IgA is exocytosed, still bound to receptor which is now called Secretory Component (SC). SC protects IgA from digestion in the gut and works as our first line of immunological defense against invading organisms. d. IgD: only important role is as a B cell receptor. 36 e. IgE: its Fc adheres to mast cells and basophiles trigger these histamine loaded cells, causes immediate hypersensitivity or allergy. Important for resistance to parasites when it triggers mast cells to release eosinophil chemotactic factor eosinophils come and kill parasites. 3. Describe a quantitative precipitin test where amount of antigen/tube is varied while antibody/tube is constant. Draw a graph which compares, on the ordinate, amount of precipitate obtained, with amount of antigen added/tube. Identify the zones of antigen and antibody excess, and equivalence. a. Quantitative precipitin test: mix the antigen and antibody in different ratios and see how much precipitate is form. Relative antigen or antibody excess the amount of precipitate is less because the complexes are smaller and not every molecule may get bound. 4. Sketch the lattices obtained in antigen or antibody excess, and at equivalence, using Y as antibody and + as antigen. 5. Discuss why a line of precipitate may form in agar gel when antigen and antibody diffuse towards each other. a. Precipitate forms in the agar when the antigen and antibody meet at optimal proportions (more immune complex forms and precipitates out of solution). This technique is called Immunodiffusion. 37 6. Compare and contrast precipitation and agglutination in terms of the nature of the antigens involved, and sensitivity of the tests. a. Precipitation: large immune complexes that are formed at or near equivalence tend to become insoluble and fall out of solution, when the antigen is a molecule, it is called precipitation. b. Agglutination: large immune complexes that are formed at or near equivalence tend to become insoluble and fall out of solution, when the antigen is a cell or cell-sized particle, it is called agglutination. More readily detected than precipitation so the agglutination test is MORE sensitive. 7. Discuss how complement plays roles in both innate and adaptive immunity. a. The alternative pathway seems to be a more primitive, early, less-specific sort of defense, since it can work even without waiting for antibody to be made. There is also the lectin pathway of complement activation, truly part of innate immunity. The lectin pathway is mediated by mannose-binding protein, MBP or MBL, a lectin. Lectins are proteins that bind (usually foreign4) carbohydrates. MBP binds certain mannose–containing structures found in carbohydrates of bacteria but not humans. 8. List the components of complement in the order in which they become activated in the classical pathway. Name those that are also activated in the alternative pathway. a. Classical pathway: activated by complexes of IgG or IgM antibody with antigen. Fc portion of antibodies change which allows binding and activation of C1q. C1q must interact with the 2 Fcs simultaneously (2 IgGs close together or IgM). C1 activates C4 and then C2, together they activate C3, which activates C5C6-C7-C8-C9. Activating C3 and C5 is a vital part of complement because they are responsible for opsonizing, chemotaxis and anaphylastoxic. b. Alternative: activated by IgA-antigen complexes, bacteria may activate C this way even in the absence of antibody. This pathway is also part of the innate immune response. Complement is activated by C3 (always breaking own at a low rate to C3a and C3b). IgA and the cell wall structures provide a surface for binding of C3b (thus stabilizing it) and factor B, properin and factor D forms which can activate C5 (and thus C6-C7-C8-C9). 9. 10. Describe how the lectin pathway of complement activation is triggered, and whether it is part of innate or adaptive immunity. a. Lectin pathway is part of the innate immunity. Pathway is mediated by mannose-binding protein (MBO or MBL), a lectin (protein that binds carbohydrates). MBP binds certain mannose-containing structures 38 found in carbohydrates of bacteria but not humans. MBP is similar to C1q so the lectin pathway goes MBP-4-2-3-5-6-7-8-9. 11. Discuss the different ways in which complement is activated by IgG and IgM. a. C1q must interact with 2 IgG Fcs close together or a single IgM (reinforces that IgM is for efficient at activating complement than IgG) 12. Identify the complement components which are: a. Opsonizing: C3b adheres to membranes, phagocytic cells have C3b receptors and so they get a firm grip on an antigen if opsonized with C3b. IgG is also opsonizing because phagocytes have receptors for its Fc end called FcR. b. Lytic: if the membrane attack complex (MAC) is activated when C5 activates C6-C7-C8-C9. C8 and C9 form a lesion on the target cell membrane which looks like a hold and the cell loses ability to regulate osmotic pressure and lyses or pops. c. Anaphylatoxic: C3a, C4a and C5a can all release histamine from mast cells by binding. This leads to increase blood flow to the area of antigen deposition and a better chance for inflammatory cells to get out of the blood and into tissues. d. Chemotactic: the C5 activation product, C5a, is chemotactic for phagocytes, especially neutrophils. 13. Discuss how complement is important in immunity to bacteria even if the bacteria are resistant to lysis by C9. Identify the family of bacteria for which lysis is necessary for their clearance. a. Not all bacteria need to complete the complement pathway all the way to C9, many can be killed via opsonizing with just C5 activation. The most susceptible family of bacteria to lysis is Neisseria (gonorrhea and meningitis) which may require the activation of membrane attack complex. BL - Anemia Due to Decreased RBC Production 1. Compare and contrast the clinical and lab features of anemia due to the following causes: iron deficiency, chronic inflammation and infection, renal disease/failure, and lead intoxication. Describe general approaches to their treatment. Cause Iron deficiency Chronic inflammation and infection Pathophysio Clinical Features In previous learning objectives! TNF decreases Dependent on iron availability underlying disease, 39 Lab Features Treatment Mild-mod anemia (Hgb 8-12 gm/dL)—severity is Tx underlying disease to decrease (maliganancy) Lead intoxication Renal disease/failure Endocrine from stores and ↓EPO production, INF-beta inhibits erythropoiesis. Infection/Inflamm: IL-1 diminishes iron mobilization and EPO production, INFgamma inhibits prolif of erythroid precursors Lead inhibits synthesis of protoporphyrin and the enzyme that ligates iron to the porphyrin ring. EPO cannot be produced (its made in the kidneys) may include: fever, arthralgia, arthritis, fatigue. For infection, symptoms and signs relate to the focus (pain, cough, swelling) Personality changes, irritiable, headache, weakness, wt loss, adb pain, vomiting proportional to underlying disease, may be normochromatic/normocytic or microcytic with some hypochromia. ↓serum Fe, total iron binding capacity (TIBC), EPO for Hct, retic count. Nrml to ↑ ferritin (Fe stores) Mild-mod anemia, ↓ retic count, microcytosi and mild hypochromia, basophilic stippling, ↑ zinc protoporphyrin, may see concurrent iron def (“inner city triple whammy”), ↑ lead levels Related to those Don’t see anemia until with renal kidney function is ˂40%. deficiency: fatigue, Mod-sev anemia. Hgb: 5-9 pallow, low exercise mg/dL. tolerance, dyspnea, Normochromic/normocytic. tachypnea ↓retic, ↓EPO, ↓production THYROID: Hyper or ↓retic count and index in all hypo activity, weight Hypothyroid: mild anemia, gain or loss, normochromic/normocytic. systemic skin, nail, May be microcytic or hair changes. macrocytic. Hyperthryroid: ADRENAL: nausea, usually normocytic, may be vomiting, microcytic. Adrenal: mild dehydration, anemia, normocytic weakness, circulatory collapse cytokines and interleukins. Tx comorbid conditions (ie: Fe deficiency), EPO has been shown to be effective in some cases. Chelation therapy to relive the lead toxicity Administer EPO, treat co-morbid conditions (ie: Fe def, vitamin def) Hormone replacement (thyroid hormone, cortisol, etc). 2. Identify other causes for underproduction anemia, including sideroblastic anemia, protein malnutrition, hypothyroidism, hypopituitarism, and decreased affinity hemoglobins. 40 3. Describe the rationale and indications for the use of erythropoietin and transfusion in the management of underproduction anemia. a. Erythropoietin: used in specific conditions when there is an absolute deficiency or where EPO levels are decreased out of proportion to the degree of anemia and administration is known to induce a response. b. Transfusions: only when severity of anemia has resulted in severe cardiovascular decompensation 4. Explain the biochemical basis for B12 deficiency and folate deficiency leading to a macrocytic anemia. a. Both are critical co-factors for normal hematopoiesis (in the synthesis of methionine from homocysteine). Deficiencies of folic acid and Vit B12 affect the maturation process of red cell precursoes in the marrow. The cells increase in size, arrest in S phase of mitosis, undergo destruction, resulting in ineffective erythropoiesis and anemia. 5. Identify the dietary sources of vitamin B12 and folate and describe their associated sites and mechanisms of absorption, means of transport, and duration and location of storage. a. Folate is widespread in food: cereals, breads (1/3), fruits and veggies (1/3), meats and fish (1/3). Human milk has enough folate for infants. Overcooking leads to loss of folates from food. Folate absorbed in the JEJUNUM. It is hydrolyzed, reduced and methylated before distribution to tissues or liver for storage (as methyltetrahydrofolate). Liver stores undergo turnover, secretion in the bile and reabsorption (enterohepatic circulation). 41 b. B12: 6 months of B12 stored in liver, therefore disease doesn’t progress as rapidly. Originally synthesized by bacteria and algae (works it way up the food chain), we eat it through eggs, meat and milk (NO PLANTS). Once ingested, released in the acid environment of stomach. Intrinsic factor (IF) secreted by gastric parietal cells, is a protein carrier and binds VitB12. Absorbed in ILEUM and released from IF, bound to transcobalamin binding protein II and transported to liver for storage or to other tissues for use. 6. Describe the findings in the peripheral blood and bone marrow in a patient with B12 or folate deficiency. a. Hematologic Changes: both will show erythroid hyperplasia, cytoplasmic maturation normal, Peripheral blood, Macrocytosis (MCV > 97 fl in adults), Ovalocytes, Hypersegmented nuclei of neutrophils, As anemia progresses, poikilocytes and fragmentation, In severe cases, see neutropenia and thrombocytopenia, ↑ bilirubin (and other evidence of hemolysis, destruction in marrow), Retic index < 1.0. Anemia (may also see neutropenia and thrombocytopenia in severe cases).↑ MCV. Low reticulocyte count and index. ↑ unconjugated bilirubin and LDH. 7. Describe the differences between vitamin B12 deficiency and folate deficiency with respect to: their most common causes, time to development of the clinical state, presence of neurologic and neuropsychiatric abnormalities, and laboratory studies used to make a diagnosis. a. Most common cause of folate deficiency is inadequate dietary intake megaloblastic anemia. Other causes of folate deficiency: malabsorption (tropical sprue or parasite), inborn errors of folate metabolism, increased demands (hemolysis, pregnancy, rapid growth, psoriasis), alcohol consumption 42 (decreased dietary intake and disruption of cycling from liver to tissues). Leads to megaloblastic anemia. Pace of folate deficiency is weeks to months (rapid) b. Causes of Vit B12 deficiency: autoimmune disease (#1 cause: pernicious anemia: autoimmune destruction of paretial cells that produce intrinsic factor), intrinsic factor deficiency (congenital, atrophic gastritis, gastrectomy—no paretial cells= no IF), malabsorption (pancreatic insufficiency, bacterial overgrowth, parasites, AIDS), defective transport/storage (transcobalamin II deficiency), metabolic defect. Vit B12 deficiency develops slowly (over years) and is most likely associated with malabsorption. Classical neurologic features: sensory losses first (numbness/tingling), loss of proprioception, ataxia, spasticity, gait disturbances, + Babinski signs, cognitive/emotional changes. Neuro defects may be nonreversible. c. To diagnose: measurement of serum cobalamin levels and serum or red cell folate levels. Measurement of plasma homocysteine levels has been used as a more sensitive marker. Methylmalonic acid will be ↑ in B12 deficiency ONLY. TO determine etiology of B12 deficiency use Schilling Test. Test: 1 µg of radiolabeled cobalamin given orally, IF produced in stomach binds radiolabeled cobalamin and it is absorbed at terminal ileum. Tagged colabamin is then bound to transcobalamin II and enters blood. A dose of unlabeled colabamin is given IM 2 hrs later, causing some radiolabeled to be excreted via urine (5-35%). If patient isn’t absorbing cobalamin given orally, less radiolabeled cobalamin will be excreted. 8. Describe the clinical, laboratory, and autoimmune findings associated with pernicious anemia. a. Common cause of vitamin B12 deficiency because autoimmune disorder kills IF-producing parietal cells. No IF= no binding of VitB12. b. Clinical: Bleeding gums, diahhrea, fatigue, neurological symptoms, pallor, personality and memory changes, SOB c. Laboratory: Measurement of serum autoantibodies against intrinsic factor, the cobalamin-intrinsic factor complex, and parietal cells is now commonly used to diagnose pernicious anemia, with positivity in more than 60% of adults with the disease. d. Autoimmune: autoantibodies against intrinsic factor 43 9. Describe the appropriate therapies for B12 deficiency and folate deficiency. a. Cobalamin deficiency: IM or SQ injections of B12 (daily for 2 wks, weekly until HCT is normal, then monthly for life). b. If absorption is normal, oral replacement works. c. Folate deficiency: 1 mg/day orally or parenterally BL - Sickle Cell Disease 1. Explain the molecular bases for sickle cell disease and how specific mutation leads to the phenotype. Describe its mode of inheritance. a. SCD is a autosomal recessive genetic disorder of hemoglobin in which both of the Beta-globin genes are mutated, at least 1 with the characteristic single amino acid substitution of sickle cell (Beta6 glu val) and 1 that is abnormal. If the other gene also has the sickle cell mutation, the disease is called Sickle Cell Anemia (HbSS). Sickle cell disease also occurs if the other gene is abnormal (HbC) or there is a underproduction of normal Beta globin chain (B-thalassemia). 2. Describe the geographic distribution of sickle cell disease. Describe a situation where people heterozygous for sickle cell disease may have a survival advantage. a. Most common in African, Indian, Middle Eastern and Mediterranean populations (around equatorial region of the world). When carried as a genetic TRAIT (heterozygous state- 1 gene normal, 1 gene mutated) the presence of the abnormal hemoglobin likely reduces the morbidity and mortality of malaria, providing carriers with survival advantage. 3. Describe the findings on the CBC and peripheral blood smear in patients with sickle cell disease. a. Blood smear: sickle cells, schistocytes (broken cells), polychromasia (blue colored retic’s), anisocytosis (size of RBCs varies), poikilocytosis (shape of RBCs varies). Howell-Jolly bodies seen in pts without a spleen (purple dots in RBCs). Target cells and Hgb C crystals (seen in HbSC disease). Microcytosis (with low MCV) and target cells found in HbSβ0 thalassemia and HbSβ+ thalassemia. Sickled RBC, Howell-Jolly body, nucleated RBC, polychromasia. 4. Describe what “sickle trait” is. Describe the consequences of having sickle cell trait. 44 a. Sickle cell trait occurs in a person with 1 sickle gene and one NORMAL gene. The normal gene produces Beta-globin chains in normal quantities and protects against the development of sickle cell disease. People with sickle cell trait do NOT develop sickle cell disease. 5. Describe major variants of sickle cell disease, including sickle beta-thalassemia and SC disease. 6. Describe the precipitating factors and pathophysiologic process by which hemoglobin S causes sickling, as well as the signs and symptoms, both acute and chronic, of the consequences of sickling. a. Deoxygenated state: sickle hemoglobin polymerizes into 14-strand helical fibers which distore the shape of the RBC into a sickle form and membrane is damaged. When reoxygenated, the polymers dissolve and the RBC returns to its normal shape. After several deoxy-reoxy cycles, the cell becomes IRREVERSIBLY sickled and is lysed/destroyed. The presence of other hemoglobins (ie: HgbC or some normal HgbA) interferes with the polymerization process and lessens the tendency for RBC sickling and membrane injuryattenuates disease. b. Signs and Symptoms are related to underlying pathology: i. Chronic hemolytic anemia: sickle RBC is fragile/rigid chronic RBC destruction. Sickle RBC’s survive only 20 days. Labs show: anemia, increase in retic count, increase in WBC and platelet count (due to exuberant bone marrow response to hemolytic anemia), increased red cell distribution width (due to sickle RBCs transiting from sickle to non-sickle, changing shape and because retic’s are young and large). Abnormal blood chemistry with increase bilirubin, lactate dehydrogenase and aspartate aminotransferase (released from lysed RBCs). Can also have aplastic crisis (sudden drop in hemoglobin due to bone marrow’s inability to produce RBCs (ie: infection, medications, vitamin deficiency-folic acid), presentation is low retic count. Growth retardation/delay (related to anemia and increased metabolic rate from increased RBC production). Bilirubin gallstones (from chronic bilirubin elevation). ii. Chronic RBC adhesion/vascular occlusion: even when not sickled, the RBCs in sickle cell disease are extra sticky due to membrane injury and retention of adhesion molecules of the surface. Adhesion of cells to microvasculature transient vaso-occlusion, vessel wall injury, endothelial remodeling vessels narrow chronic organ damage especially to 45 1. spleen—when large # of RBCs are trapped: splenic sequestration, chronic occlusion of spleen’s microcirculation: autoinfarction (destruction) of spleen by age 5. This also compromises the spleens ability to kill organisims (pneumococcus, meningococcus, hemophilis) which can lead to sepsis. Reduced risk if children use prophylactic penicillin. 2. CNS—large blood vessels of CNS can be damaged by sickle RBCs. 10% of children with HbSS has a large vessel stroke. Risk detected by transcranial Doppler (increased blood velocity= increased risk for stroke). Risk reduced with blood transfusions. Adults are more likely to have hemorrhages from progressive weakening and rupture of vessels. 3. Lung—damage to vessels in lungs increase pressure in pulmonary arteries (PAH), which strains the R side of the heart (30-40% of patients). Most common cause of death in adults with sickle cell disease. 4. Kidney—Tubules damaged by chronic vaso-occlusion resulting in the inability to concentrate urine to avoid dehydration, blood in urine, enlargement of the glomerulus and protein in urine. 10% of pts develop renal insufficiency. 5. Retina—retinal vessel damage retinal detachment and blindness 6. femoral/humeral heads—avascular necrosis chronic pain and progressive join deterioration (requires hip and shoulder replacement). 7. Skin ulcers—microvascular ischemia and poor healing (most often on ankles). iii. Acute RBC adhesion/occlusion: sickle cell crisis: under conditions of hypoxia, dehydration, inflammation, infection or other stresses, the RBCs sickle and blood cells become acutely damaged and constricted, which promotes sudden vaso-occlusion “pain crisis” (pattern of pain is unique to each individual, but in arms, legs, chest and ABD). Pain is due to reversible ischemia and resolves when inciting factors improve. 1. Splenic sequestration 2. Hand-foot syndrome: in infants, swelling of hands and feet, early manifestation of HbSS 3. Acute chest syndrome: sickled RBCs trapped in lung circulation, damages vessels, fluid leaks out, cannot oxygenate blood. 4. Acute multi-organ failure syndrome: acute renal failure and liver failure. 5. Priapism: RBCs trapped in penis, painful sustained erections 6. Bone infarction: ischemia of bone that can lead to necrosis. 7. Explain the relationship between aplastic crisis and parvovirus B19. a. In children, an important cause of aplastic crisis is Parvovirus B19, which causes “fifth disease” and infects RBC precursors, arresting their development into mature RBC’s. This infection is usually transient, but patients may require transfusion of RBCs if hemoglobin falls significantly. 8. Describe some of the therapies to treat patients with sickle cell disease and the rationale for therapies such as folic acid, penicillin, exchange transfusion, hydroxyurea, and bone marrow transplantation a. Folic acid: could be used in response to developmental delays caused by anemia b. Penicillin: Sepsis (overwhelming blood infection) due to spleen death is a common cause of death for infants and young children with sickle cell disease, is significantly reduced by the use of prophylactic penicillin and prompt treatment of fever with additional antibiotic therapy. c. Bone marrow transplantation: transplantation done with HLA-matched full sibling unaffected by sickle cell disease with a greater than 90% disease free survival. Only 20% of eligible patients have such a donor available. 46 d. Hydroxyurea therapy: oral chemotherapy agent that induces production of HbF which interferes with sickle hemoglobin polymerization. Improves anemia, reduces frequency of acute pain crises and reduces mortality. e. Transfusion therapy: most people don’t require transfusions. But, if there is worsening anemia, transfusion of RBCs may reverse life-threatening process. Transfusion can be simple or exchange (remove pts RBC’s when normal RBCs are given). Transfusions are associated with transmission of infectious agents and antibody formation and iron overload. 9. Explain iron chelation therapy, its indications, and its drawbacks. a. Administer chelating agents to bind up excess iron, indicated for people who receive multiple simple transfusions because excess iron can lead to organ damage (especially liver and heart). The most common chelation agent, deferoxamine, is infused subcutaneously over 8-12 hours, usually in the abdominal area, 5 to 7 times a week. Compliance with this therapy is challenging for some patients. 10. Explain how newborn screens can be used to diagnose sickle cell disease. a. Virtually all infants born in the United States are screened at birth for hemoglobinopathies, using the blood from a heel stick placed on filter paper, as is done for the PKU and other sponsored newborn screening tests. The purpose is early identification of sickle cell disease, so that parental education and prophylactic penicillin can be provided to prevent early mortality. However, it is also an opportunity to identify children with other forms of hemoglobinopathies, including Bthalassemia major. BL – Thalassemia 1. Review the normal structure of hemoglobin and indicate the globin chains that typically make it up. Describe how the composition of globin chains in hemoglobin changes during fetal development and after birth. a. Hemoglobin is a heterodimer made up to 2 alpha-globin chains and another pair of different globin chains (usually beta and delta). Majority of hemoglobin in human RBCs after 4-6 months of age is Hemoglobin A1: 2 alpha chains and 2 beta chains (95%), A2: 2 alpha chains and 2 delta chains (3.5%) and HbF: 2 alpha chains and 2 gamma chains (2%). 2. Describe what thalassemia is and explain in a general way the molecular basis for it. Describe the basic genetic differences between alpha-thalassemia and beta-thalassemia. 47 a. Thalassemia: underproduction of a hemoglobin chains due to a variety of mutations that result in poor or absent function of the globin gene. Imbalance of the chains leads to: free excess chains binding to RBC membrane, membrane oxidative injury, increased membrane rigidity, decreased membrane stability. Two common types: alpha-thalassemia and beta-thalassemia. i. Alpha-thalassemia: alpha-globin is under produced, due to an absence of 1+/4 genes that control production on chromosome 16. ii. Beta-thalassemia: beta-globin is under produced most often due to point mutations which result in a dysfunctional gene on chromosome 11. 3. Explain the meaning of the terms thalassemia major, thalassemia intermedia, and thalassemia minor 4. Describe the genetic, hematologic, and clinical differences between alpha-thalassemia trait, hemoglobin H disease, and hydrops fetalis. 48 5. Describe clinical manifestations and findings on the CBC and peripheral blood smear in patients with thalassemia. a. Chronic hemolytic anemia: fragile RBC has short half-life and is destroyed in marrow or culled by the spleen from circulation i. Anemia with some increase in retic count: varies with severity of thalassemia, Cooley’s anemia severe anemia (Hgb ˂7 g/dL) that develops within first year of life and requires transfusions to sustain life beyond 2-3 years. Milder thalassemia may require transfusions later in life. ii. Abnormal peripheral smear: microcytosis (small RBC’s), target cells, polychromasia (blue cells that represent retic’s), mild anisocytosis (variation in RBCs size). RDW is nrml-minimally elevated. Low MCV, low MCHC, sombrero shaped RBC’s iii. Abnormal chemistry profile increased total/indirect bilirubin, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) as they are released from lysed RBCs. iv. Splenomegaly: enlarged from removing so many damaged RBCs. b. Expanded bone marrow and extramedullary hematopoiesis: bone marrow expands to try and produce adequate RBC mass, it fills with RBC precursors (but they are fragile and destroyed—ineffective erythropoiesis). Leads to frontal bossing, osteopenia, enlargement of liver and spleen. c. Increased iron absorption: increase absorption of iron from diet, but these patients usually are getting transfused high iron burden and iron overload. Must use iron chelation therapy. d. Delayed growth and development: anemia, increased metabolism and endocrinopathiesshort stature, delayed puberty. e. Endocrinopathies: 2/3 of Cooley’s anemia patients have abnormal endocrine function. Pituitary gland is often effected and can lead to hypogonadotrophic hypogonadism. Hypothyroidism is present in 40-60% of patients with Beta-thalassemia major. f. Pulmonary HTN: chronic hemolytic anemia increases risk of pulmonary HTN. 6. Describe the geographic distribution of thalassemia. Describe a situation where people heterozygous for thalassemia may have a survival advantage. a. Thalassemia: most common in SE Asian, African and Mediterranean descent. May have an heterozygote advantage for malaria. 49 7. Explain why Southeast Asians with alpha-thalassemia are more likely than Africans with alpha-thalassemia to have a child with hydrops fetalis. a. The genotype --/αα (both genes on the SAME chromosome are missing) is more common in the SE Asian populations. This person can pass on a chromosome with no functional alpha genes (--) and if the other parent contributes a similar chromosomes theoffpsring will have no alpha genes. The child will be unable to amek any normal hemoglobin (alpha chain is required for all types of Hgb), and the baby will die in utero (Hydrops fetalis). The –α/-α genotype (each chromosome has 1 intact alpha gene) is more common in the African populations and therefore the offspring will inherit a least 1 alpha gene. Less likely to see Hydrops fetalis is this population. 8. Describe approaches to treatment for thalassemia. a. Transfusion support: in severe thalassemia, transfusions are started in the first 2 years of life to maintain normal Hgb levels and avoid excess bone marrow expansion and extramedullary hematopoiesis. Chelation therapy must be paired with transfusions to prevent iron overload. Chelation agent: deferoxamine (infused SQ over 8-12 hours, in ABD area, 5-7x/wk, compliance is challenging). b. Increase fetal Hgb production: Hydroxyurea, butyrate and decitabine can induce the gamma chain to produce providing a pool of globin chains for the excess alpha chains to combine with, thus reducing their negative impact on RBCs. c. Bone marrow transplant: thalassemia can be cured with BMT, 70% thalassemia-free survival at 20 years for people who receive a HLA-identical unaffected sibling match. Only 30% of patients have a matched sibling. 9. Explain how newborn screens can be used to diagnose thalassemia. a. Using a heel prick, Virtually all infants born in the United States are screened at birth for hemoglobinopathies, using the blood from a heel stick placed on filter paper, as is done for the PKU and other sponsored newborn screening tests. The purpose is early identification of sickle cell disease, so that parental education and prophylactic penicillin can be provided to prevent early mortality. However, it is also an opportunity to identify children with other forms of hemoglobinopathies, including Bthalassemia major. BL - Antibody Genes 1. Define a. Toxoid: bacterial toxin (usually an exotoxin) whose toxicity has been weakened or suppressed either by chemical (formalin) or heat treatment b. DNA recombination: Changing the relative positions of 2 pieces of DNA c. RNA splicing d. mRNA e. somatic mutation 2. Define cross-reactivity. Give an example of a non-self antigen which cross-reacts with a self antigen. Explain, in terms of lymphocyte activation, how a self antigen might not itself elicit antibody, but might react with antibody elicited by a cross- reacting antigen. a. Cross-reactivity: refers the tendency of one antibody to react with more than one antigen. Other antigenic determinants might also fit into the complementary-determining region (CDR) and if they did so detectably, we would say that the antibody cross reacted with those determinants. Example: if you 50 immunize a person with cow pox, the antigenic determinants of small pox will also be recognized and the person will be immunized. b. B cells are typically activated when the antigen binds to the B cell’s receptor (membrane-bound versions of the antibodies it will release), if the binding is strong enough, the B cell will be activated. If the binding is with a low affinity, the B cell is not activated, but if another antigen comes along which binds AND activates, then the product of the cell (secreted antibody) may combine with the low affinity antigen well enough to be “inconvenient”. i. Example: the valves in the heart contain an antigen called laminin which cross reacts with Group A streptococci. The antigen in the valves does not normally activate the corresponding B cells. But, when people get streptococcal infection, the strep antigens DO activate the B cells because they bind with high affinity. The release antibody reacts with the heart values with low affinity and sometimes cause rheumatic heart disease. 3. Discuss the Clonal Selection Theory in term of: the number of different receptor specificities it postulates per cell; the role antigen plays in the initial expression of receptors; the role of antigen in clonal selection; one experiment which provides strong evidence for the theory; how it differs from an instructional theory; whether it is Darwinian or Lamarckian. a. Instructional Theory: old theory, antigen told the immune system in some way to make antibodies of appropriate conformation. Lamarckian theory because they implied that the outside world instructed the cell to change its genetic information. WRONG. b. Clonal Selection Theory: each cell of the immune system is programed to make only one antibody, the choice of which antibody the cell makes is random, NOT dependent on outside information and that the entire population PREEXISTS in a normal individual BEFORE contact with the antigens. When a new antigen enters the body, it contacts many lymphocytes. When it encounters a lymphocyte whose receptors bind it with a high affinity, the lymphocyte is activated and makes closes and antibodies. The best fitting clones are SELECTED by the antigen. Clonal selection is Darwinian because it has to do with the survival of the fittest. c. Experiment to prove theory: Antigen X linked with radioactive label, such that any cell bound to it would die of radiation. If instructional theory was true, enough antigen would bind all the B cells, because they were “non-specific” until instructed, so no antibody could be made. If selection theory was correct, only pre-existing B cells with randomly expressed receptors for X would bind and die, all other would survive. After receiving radioactive X, animal immunized with nonradioactive X and Y, unrelated antigen. It responded normally to Y, but not to X. Selection theory was proven. 4. Define allotypic exclusion. Demonstrate your knowledge of the concept by first stating the number of chromosomes in a cell which bear H or L genes, and then the number that actually contribute to a particular B cell's antibody product. a. Allotype: minor allelic differences in the sequence of immunoglobulins between individuals, determined by allotypes of your parents, useful in determining relatedness. b. The lambda, kappa and H chain gene families are all on different chromosomes. Each cell has 2 copies of each gene, but only 1 H chain (maternal or paternal) and 1 L chain (either kappa or lambda) are synthesized in any 1 B cell. All other genes are silenced. Though a person can make 2 allotypes, an individual B cell can only make 1. 51 5. Draw a diagram of the heavy and light chain gene regions of human DNA. Indicate V, D, J and C subregions. Show how a heavy or light chain gene is assembled out of these subregions during the differentiation of a B cell. a. Many genes encode for the variable domains, only one of each set of constant domain genes (1 gene for the constant part of the delta chain, 1 gene for mu) b. DNA rearranges in developing B cells, to bring one of the many V’s together with the correct C so that the unit can be made into mRNA. Changing the relative positions of 2 pieces of DNA is called recombination. This is occurring in SOMATIC cells, which is unusual. c. Assembling a Heavy Chain gene: variable domain is code for by V, D, and J segments. Developing B cell brings one random D segment close to 1 J, the DNA is cut, and intervening DNA is excised and ends are joined. Then, V segment is brought up to the recombined DJ, cutting is repeated, and ends are joined. The entire region from the assembled VDJ unit through to the end of the delta constant region gene is transcribed into nuclear RNA. These primary RNA transcripts are alternatively spliced to make VDJC(mu) and later to make both VDJ-C(mu) and VDJ-C(delta). IgM from VDJ-C(mu) is always produced first, hangs out in the bone marrow for 24 hours and if nothing-self binds to IgM it makes IgD and IgM simultaneously, matures, and enters the circulation. 6. 52 7. Describe the somatic recombination model which explains how antibodies of the same specificity (idiotype) can be found in two or more different classes (“class switching”). a. Class switching: single mature B cell starts by making IgM and IgD. Later, it may switch to IgG, IgE or IgA. In all cases, the V domain stays the same but the C region of the H chain changes. The cell with its Hchain VDJ combination together with its mu and delta, puts VDJ next to the C-region gene of gamma or epsilon or alpha and excises the intervening DNA. The new mRNA, the may be VDJ (alpha), VDJ(epsilon), VDJ(gamma). A cell which is making IgM can go on to make IgG. A cell making IgG CANNOT go back to making IgM because the mu gene is physically GONE. A cell can switch heavy chains, but cannot switch light chains. 8. Calculate the minimal number of genes required to code for a million different antibody molecules, based on the (outdated) concept of "one gene, one H or L chain". Show how breaking the variable region gene up into V, D and J subregions requires fewer genes. a. 2000 genes (1000 L chains and 1000 H chains, 1000x1000= 1,000,000 antibody combining sites. b. V-D and D-J joins are “sloppy”. #1: Exonucleases chew away a few nucleotides after the DNA is cut but before the 2 gene segements (D to J, V to DJ) are joined. #2: terminal deoxynucleotidyl transferase (TdT) adds a few nucleotides without using a template, so its additions are random. Thus you CANNNOT predict the sequence at the joining area, called the “N region”. This produces a lot of diversity, but 2/3 times the N region will not be the correct length (incorrect # of bases added) and the frame-shift mutation occurs (nonsense codon that terminates transcription). c. A lot of diversity in the germ line (the individual V, D, J segements you are born with), a lot of diversity in the “slopping” variable V-J and V-D joining. Also, the recombined V(D)J unit is hypermutable: each time a B cell divides after antigenic stimulation, there is a good chance that one of the daughter cells will amek a slightly different antibody (selection of the best fitting mutants gradual increase of affinity during immune response). 9. Define somatic hypermutation and distinguish it from the somatic mutation mechanism that produces N-region diversity. a. Hypermutation: each time a B cell divides after antigenic stimulation, there is a good chance 1 of the daughter cells will make a slightly different antibody mutants that are better (or worse) affinities during immune response. This happens via activation-induced (cytidine) deaminase (AID) which converts a random Cytosine in CDR gene regions to uracil. C:G becomes U:G mismatch. Uracil removed by repair enzyme and error-prone DNA polymerases fill in the gap creating mostly single-base substitution mutations so at the end of cell division the daughter may be making a different antibody. b. N-region diversity: created by the “sloppiness” of V-D and D-J joining. 1: exonucleases chew away a few nucleotides after the DNA is cut but before the gene segments (D to J and V to DJ) are joined. 2: enzyme TdT adds nucleotides without a template (random!), you can’t predict the sequence at the joining area “N region”. Lots of diversity produced, but the price is frame shift mutations that occur frequently. 10. Define somatic mutation and describe the essential differences between the somatic mutation and germ line hypotheses of immunological diversity. a. Somatic mutation: Alterations in DNA that occur after conception. Somatic mutations can occur in any of the cells of the body except the germ cells (sperm and egg) and therefore are not passed on to children. 53 b. There used to be two schools of thought about antibody diversity: one said that all the V genes were in the germ line; if you looked at a fertilized ovum you could predict all potential antibodies that potential individual could potentially make. The other said that only a few were there. It postulated that during embryonic lymphoid development these genes underwent repeated (somatic) mutation until a full complement of antibodies was produced. Both theories, it turns out, were right. As we’ve just discussed, a lot of our diversity is in the germ line (that is, in the individual V, D, and J segments you’re born with). Even more diversity is also generated by variable (“sloppy”) V/J and V/D joining. c. 11. Explain why we commonly write V(D)J instead of VDJ. a. The variable domain region of heavy chain genes is composed of multiple V, multiple D and multiple J gene segments, the V region of light chains into V and J segements. The cell will choose one of its Vs, 1 D and 1 J to make the variable heavy chain domain gene region. BL - Anemia Due to Hemolysis 1. Provide a definition for hemolysis and describe the two main mechanisms of increased destruction of RBCs, intravascular hemolysis and extravascular hemolysis. a. Hemolysis: decrease in red cell survival or increase in turnover beyond standard norms. b. Intravascular hemolysis: turnover within the vascular space, red cells undergoing this will release hemoglobin into the circulation dissociates into dimer alpha/beta binds to haptoglobin removed by liver. If haptoglobin is overwhelmed, hemoglobin will have the iron oxidized to methemoglobin. Dissociation of globin releases metheme which binds albumin or hemopexin converted to bilirubin. c. Extravascular hemolysis: through ingestion and clearance by macrophages of the reiculoendothelial (RE) system. Red cell ingested by macrophage heme separated from globin, iron removed and stored in ferritin and porphyrin ring converted to bilirubin which is released from cell. Bilirubin converted to water-soluble compound with addition of glucuronic acid. After ecretion into biliary tract/small bowel, glucuronic acid is removed and bilibuin is converted to urobilinogen (this cycles between gut and liver or is excreted by kidney into urine. 54 2. Describe the biochemical pathways of breakdown of hemoglobin and the relevant clinical lab tests for hemolysis, including indirect bilirubin concentration, serum LDH level, reticulocyte count, and serum haptoglobin concentration. a. The CBC will determine whether anemia is present or not, the mean size of the red cells and whether the size varies significantly. In most cases, the short red cell life ↑reticulocyte count and index. ↑bilirubin (if hemolysis is brisk enough to overcome the bilirubin processing system of the liver ↑unconjugated fraction. ↓serum haptoglobin levels. ↑metheme or methemalbumin. 3. List the major hereditary and acquired causes of hemolytic anemia. a. Hereditary spherocytosis: characterized by anemia, jaundice (intermittent), splenomegaly, and responsiveness to splenectomy. Hallmark: loss of plasma membrane and formation of the microspherocyte. Pathophysiology: spectrin, ankyrin or band 3 defects weaken the cytoskeleton and destabilize the lipid bilayer. Clinically: anemia, jaundice, splenomegaly. Inherited as autosomal dominant (75%), 25% are autosomal recesseive. Treatment: supportive care for anemia, splenectomy. Lab features: variable HCT and HGB, ↑ retic count/index, ↓ MCV, spherocytes on smear, unconjugated hyperbilirubin. Clinical complications: aplastic crisis (rapid, severe, lifethreatening anemia) and bilirubin stones (increased bilirubin in biliary tree leads to stones in gall bladder). 55 b. Enzyme disorder: i. most commonly glucose-6-phosphate dehydrogenase (G6PD) deficiency. Inherited as Xlinked recessive. Highest incidence in tropical and sub-tropical areas because it may be associated with selective resistance of MALARIA. G6PD deficiency results in inability to restore reduced glutathione, with oxidant stress, denatured HGB attaches to the membrane and spectrin may be damageddecrease in deformability and abnormal membrane splenic trapping and extravascular hemolysis. Present as intermittent episodes of acute hemolytic anemia and hyperbilirubinemia associated with oxidant stress. Smear occasionally shows microspherocytes, blister or bite cells. TX: avoid oxidant foods and drugs. ii. Pyruvate Kinase Deficiency: 2nd most common enzyme deficiency, presentation: variable chronic anemia, hemolysis, splenomegaly, gallstones, aplastic crises. Labs: mild-severe anemia, ↑ retic, no specific morphology. TX: supportive care, folate, transfusions. Splenectomy may help with disorder. c. Autoimmune Hemolytic Anemia i. Antibodies to universal red cell antigens can cause hemolysis. Cold antibodies of IgG or IgM transiently bind red cell membrane in cooler areas of body and as thye move back to central circulation they avidly activate complement and create holes in plasma membrane. When cells move more centrally, antibody dissociates and complement is left to destroy the cell (intravascular hemolysis). Warm antibodies, IgG, bind the red cell with high affinity and have no complement activating capacity inciting the splenic macrophage to antibodymediaed phagocytosis through complement receptors (extravascular hemolysis). ii. Lab test: antiglobulin or Coombs test used to detect IgG and/or complement on cell surface. AIHA: acute or chromic anemia, pallor, jaundice, dark urine. Smear shows spherocytes, teardrop or “bite” cells. 56 4. Describe the major constituents of the RBC membrane and cytoskeleton; identify the major defects in hereditary spherocytosis. Relate these to the clinical and laboratory findings of the disorder. Recognize other erythrocyte membrane disorders (elliptocytosis, pyropoikilocytosis). a. The basic pathophysiology is that spectrin, ankyrin or band 3 defects weaken the cytoskeleton and destabilize the lipid bilayer. Loss of membrane and formation of the spherocyte leads to decreased deformability and entrapment in the spleen. Conditioning in the red pulp leads to further loss of red cell membrane and, ultimately, removal by the macrophage. b. Clinically, patients present with a variable degree of anemia as well as jaundice and splenomegaly. One third have hyperbilirubinemia as neonates. Most inherit the condition as autosomal dominant, although inheritance is sometimes autosomal recessive. Treatment includes supportive care for chronic anemia and intermittent complications and splenectomy, which usually resolves the clinical manifestations. c. Hereditary pyropoikilocytosis is a severe form of congenital hemolytic anemia. It is clinically similar to, and now considered a subtype of, homozygous hereditary elliptocytosis. Hereditary pyropoikilocytosis is an autosomal recessive disorder that produces a molecular defect in spectrin and a partial spectrin deficiency. It manifests as a severe hemolytic anemia with thermal instability of the red blood cells. 5. Interpret an osmotic fragility test for diagnosis of hereditary spherocytosis. a. Osmotic fragility test is a formal laboratory test used in the diagnosis of HS. The test measures the in vitro lysis of RBCs suspended in solutions of decreasing osmolarity. Normal RBCs swell in hypotonic solutions and burst when a critical cellular volume is reached. Spherocytes lyse in solutions of higher osmolarity than normal RBCs. Spherocytes are also more sensitive to a decrease in osmolarity. When graphed, compared to normal RBCs, if spherocytes are present, then the curve will be shifted to the left. 6. Explain when splenectomy is indicated for treatment of hereditary spherocytosis. a. The basic pathophysiology is that spectrin, ankyrin or band 3 defects weaken the cytoskeleton and destabilize the lipid bilayer. Loss of membrane and formation of the spherocyte leads to decreased deformability and entrapment in the spleen. Thus, if the spleen is taken out - no more entrapment. b. The pathophysiology of many hemolytic disorders, including hereditary spherocytosis, involves destruction by the spleen, so management of chronic severe anemia sometimes includes splenectomy as a specific strategy. After splenectomy, spherocytes are present on the peripheral blood smear, but RBC survival is relatively normal. c. Splenectomy is not indicated for children under 5 because their immune system is not fully developed. 7. Describe the major energy and antioxidant pathways in the RBC and explain how G6PD deficiency and pyruvate kinase deficiency affect these pathways, leading to hemolysis. Describe the inheritance patterns and discuss the clinical and laboratory findings in patients with these syndromes. a. Embden-Meyerhof pathway: energy is generated through the breakdown of glucose in RBCs due to the absence of mitochondria. Metabolism of glucose to lactate and pyruvate provides ATP necessary to maintain the plasma membrane and cytoskeleton, and energize metabolic pumps to control intracellular sodium, potassium and calcium. ATP generation is critical for RBC survival. 57 b. Rapoport-Leubering pathway: produces 2,3-diphosphoglycerate which stabilizes the deoxy form of hemoglobin and maximizes transport of O2to tissues. c. Hexose monophosphate shunt (phosphogluconate or pentose pathway): produces pyridine nucleotide which reduces glutathione and provides protection from oxidant stress. d. Methemoglobin reductase pathway: maintains the iron in hemoglobin in the ferrous state required for reversible oxygen binding by hemoglobin. e. G6PD deficiency: Inheritance: sex-linked recessive (female carriers, male affected). G6PD is an enzyme in the hexose monophosphate shunt (phosphocluconate or pentose pathway) which protection against oxidant stress. Loss of enzyme activity in the red cell results in inability to restore reduced glutathione. Clinically, G6PD deficiency presents as intermittent episodes of acute hemolytic anemia and hyperbilirubinemia associated with oxidant stress (infection, drugs, ingestion of specific foods—fava beans). May be characterized as a chronic hemolytic anemia punctuated by episodes of acute exacerbation anemia. Cause of neonate hyperbilirubinemia No specific morphologic features are associated with G6PD deficiency. Although occasionally, the smear will show microspherocytosis and “blister” or “bite” cells. f. PK deficiency: Inheritance: autosomal recessive. PK is an enzyme which catalyzes the conversion of phosphoenolpyruvate to pyruvate in the Embden-Meyerhof pathway responsible for ATP production within the RBC. PK deficiency results in reduced ATP production, 2,3-DPG (right shift in the O2 binding curve), loss of membrane plasticity, rigidity and destruction in the spleen. Patients present with variable chronic anemia, hemolysis, splenomegaly, gallstones, and aplastic crisis. Lab features: mild to severe anemia, reticulocytes and no specific morphology. 8. List some of the major foods, drugs, or other chemicals which can induce hemolytic anemia in patients with G6PD deficiency. 58 9. Describe the pathophysiology and site of red blood cell destruction of immune-mediated hemlysis due to IgG, IgM, and complement. a. Antibodies to universal red cell antigens can cause hemolysis by either intravascular or extravascular destruction. Production of these antibodies can be in response to infection, can be associated with a malignancy or autoimmune disease, or can be drug-induced. b. Cold antibodies (referring to a 4°C temperature for maximal in vitro effect), typically of the IgM class, transiently bind red cell membrane in cooler areas of the body (fingers, toes, ears, skin). As they move back to central circulation, they avidly activate complement through the C5-9 attack complex which creates holes in the membrane. When they move more centrally, the antibody dissociates itself because of low affinity at higher temperatures, and complement is left to destroy the cell (intravascular hemolysis). c. Warm (maximal effect at 37°C) antibodies, usually IgG, bind the red cell with high affinity and have no or poor complement activating capacity, inciting the splenic macrophage to antibody-mediated phagocytosis through its Fc receptor. d. Occasionally, the small amount of C3 also induces phagocytosis through complement receptors. Clearance by phagocytosis results in extravascular hemolysis. 10. Describe the direct antiglobulin test (direct Coombs test) and the indirect antiglobulin test (indirect Coombs test). a. Antiglobulin or Coombs tests are used to detect IgG and/or complement on the surface of the cell. The direct antiglobulin test (DAT), also known as the direct Coombs test, evaluates the presence of either IgG or C3d or C4d on the surface of the red cell by the addition of Coombs reagent which has antibodies for IgG, C3d and C4d, causing agglutination. b. The indirect antiglobulin test (also known as the indirect Coombs test) detects the ability of patient’s serum to bind IgG and/or complement to test (normal) red blood cells. By definition, autoimmune hemolytic anemia should have a positive DAT. 59 11. Distinguish warm antibody-induced autoimmune hemolytic anemia from cold antibody-induced autoimmune hemolytic anemia on the basis of: immunoglobulin class of the antibody, direct antiglobulin test results, and clinical manifestations. a. + Patients with COLD AIHA have a positive DAT (complement only, no IgG), maximal reactivity at 4˚C and antigen specificity for I or i. + Patients with WARM AIHA exhibit a positive DAT (strong IgG +/- weak complement) with maximal reactivity at 4˚ C and panagglutin without antigen specificity. 12. Outline management options for inherited and acquired hemolytic anemia, including autoimmune hemolytic anemia. a. Hereditary Spherocytosis: supportive care for chronic anemia and intermittent complications and splenectomy, which usually resolves clinical manifestations. b. G6PD deficiency: avoiding oxidant drugs and foods for the most common variants seen in the U.S. For severe cases with chronic anemia, supportive care and folate are included. c. PK deficiency: supportive care, folate and transfusions if severe anemia continues. Splenectomy may partially ameliorate the disorder d. AIHA: identification and treatment of any underlying disorder (lupus, malignancy, etc.). 13. List some of the risks and benefits of splenectomy. Describe when prophylactic antibiotics are indicated post-splenectomy, what antibiotics are used, and the role of vaccination. a. Risks: The spleen is critical for clearance of intravascular microbes and in children, is critical to the development of the adaptive humoral response (and is the origin of IgM agglutinins, especially for encapsulated organisms). Most significant complication of splenectomy is overwhelming bacterial sepsis, particularly associated with S. pneumoniae. The risk is greatest in children < 5 years of age. In adults mortality from sepsis is 200 times that of the general population. b. Benefits: amelioration of symptoms associated with hemolytic anemia. c. Pre-surgery vaccination protocol: to avoid complication of splenectomy, vaccination against H. influenza b, S. pneumoniae, and Meningococcus takes place before surgery. d. Prophylactic antibiotics: should be given daily atleast during childhood; and the instruction to see a physician immediately for a febrile illness > 38.5˚ C is imperative. 60 WEEK 3 BL - Ontogeny of the Immune System 1. Define: a. stem cell: undifferentiated cells which when they divide give rise to another stem cell and daughter cell committed to differentiate. b. B cell: lymphocytes that play a large role in the humoral immune response. They make antibodies and become memory B cells c. T cell: T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They have t cell receptors and mature in the Thymus. d. pre-B cell: a B cell with cytoplasmic IgM but no surface IgM e. pre-T cell: in bone marrow, don’t have characteristic surface markers that distinguish them as T cells but are committed to expressing them in the right environment. They will go to the thymus. f. self-tolerance: the process by which the body does not mount an immune response to self antigens 2. Draw an outline diagram which shows bone marrow, thymus and lymph node. Indicate the development and movement of cells of the B and T lines, starting with the hematopoietic stem cell and ending with mature T and B cells. a. In the bone marrow, you find pre-T cells (no distinguishing surface markers, but committed to expressing them in the right environment). You will also find “pro-B cell”, pre-B cells, immature B cells and mature B cells . b. These cells go to the thymus, where they rearrange their receptor genes and are selected for their responsiveness to “self + antigen”. Cells in thymus display CD1 surface marker (not found on mature T cells in pheriphery). Most also display CD4 and CD8. Once cells have been selected in the thymus, those chosen are exported (1%) into the lymph nodes and the others die. 3. Define the Bursa of Fabricius, and discuss where its functions take place in mammals. a. Bursa of Fabricius: where precursors from the bone marrow go to finish their development in birds, burse is located in at the hind end of the gut. b. The mammalian equivalent is the bone marrow where B cells develop. 4. Describe the sequence of appearance of cytoplasmic and surface immunoglobulins in developing B cells. Using these data, derive a model that could explain self-tolerance at the B cell level (“clonal abortion”). a. “Pro B cell” progenitors: identifiable when they begin to make detectable cytoplasmic mu chains b. Pre B cell: a cell with cytoplasmic IgM but no surface IgM c. Immature B cell: a cell with surface IgM only and can interact with outside world. If immature B cell is exposed to antigen, the signal causes the cell to try receptor editing, if that fails it activates a suicide program and dies. This deletion is called “clonal abortion” and explains why we do not make antibody to self. In the bone marrow pre-B cells are differentitating into immature B cells and any cell whose receptors happen to be anti-self will almost surely encounter “self” in the bone marrow and will either make a new receptor or will be aborted. If it does not encounter antigen (not anti-self) then it will mature further so it expresses sIgM and sIgD. Then when it meets antigen, it will be stimulated. d. Mature B cell: a cell with both IgM and IgD (of the same specificity) are on cell surface 61 5. 6. Draw a graph showing the antibody response to a typical antigen in a primary and in a secondary response. Show both IgM and IgG antibody levels. a. During primary B cell responses to antigen, IgM is secreted first, then usually, helper T cells help B cells switch to IgG (or IgA or IgE). b. In response to secondary (booster) immunizations, IgM response is the same as in primary, but the IgG response (helped by T cells) is sooner, faster and higher and more prolonged because of immunological memory. Anamnestic Response. If a patient has NO functional T cells, there would be no IgG curve on the graph, only 2 similar IgM curves. 7. Draw a graph which shows relative IgG and IgM levels in a normal infant from conception to one year of age. Distinguish maternal from infant's antibodies. a. IgM is made by the fetus before birth (IgM cannot cross placenta) and the Mother’s IgG is actively transported across the placenta so at birth the baby has 100% of adult levels (much of the increase is seen in the last 2 weeks of pregnancy—therefore preemies have problems with IgG levels). After birth, the Mother’s IgG levels drop (half life=3 weeks). 3-6 months after birth, the baby begins to make its own IgG. The most vulnerable time for babies is at about 6 months, when the Mother’s IgG is low and the baby’s IgG is low. This is the reason breast feeding is so important, the milk contains antibodies to prevent infection/disease during this vulnerable period. IgA starts at 2-3 months. 62 8. Given a newborn's antibody titer, interpret its significance if the antibody is IgG or IgM. If IgG, calculate what the titer will be at 4 months of age, and state the assumptions that you made when you did the calculations. a. If the antibody is IgM, we know that it was produced by the baby (the baby was exposed to something) because mom’s IgM cannot cross the placenta b. If the antibody is IgG, it is from the mother (IgG can cross the placenta). The IgG has a half-life of 3 weeks, so in 4 months (16 weeks) the antibody will be 50% ( 3wks), 25% (6 wks), 12.5% (9 wks), 6.25% (12 wks), 3.125% (15 wks) of the original amount. 9. Discuss the decrease in diversity seen in the immune repertoire of older people. a. People can completely reconstitute their T cell numbers and diversity up to about 40 years old, then diversity becomes increasingly limited, and more and more cells show a “memory” phenotype (they have been exposed to a lot more), while fewer are naïve. Old people have fewer (new clone production slows), but larger, clones. Young people have smaller, but more, clones. Old people make good responses to antigens they saw in their youth, but fail to respond to new antigens (ie: SARS---fatal in many elderly patients). BL - T Cells - Part I 1. List the six main types of T cells, and define their functions. Discuss the positive and negative interactions between Th1, Th2, and Treg cells. a. Th1 cells: first called delayed hypersensitivity T cells, when circulating and they encounter antigen they secrete lymphokines, most importantly, interferon gamma, a pro-inflammatory and chemotactic for blood monocytes and macrophages. Monocytes and macrophages move into the area and are activated by interferon gamma becoming “classically activated M1”—ANGRY MACROPHAGES. These ingest and kill bacteria/invaders. The macrophages release their own cytokines that increase inflammation including tumor-necrosis factor alpha or IL-1. b. Th17 cells: makes pro-inflammatory lymphokine IL-17. Resembles Th1 in that its main job is inflammation. Implicated in several autoimmune diseases. Even angrier macrophages. c. Th2 cells: circulate through blood until they encounter antigen. Secrete IL-4 and IL-13 which attract and activate macrophages, but differently than IFNgamma. This is called “alternatively activated M2”. Involved in HEALING (debris removal, scar formation, walling off pathogens). IL-4 is chemotactic for eosinophils what are specialized to kill worms and protozoans. Th2 give rise of Th2 –like follicular cells that go into lymphoid follicles and secrete IL-4 which pushes B cells to swtich from IgM/IgD to make IgE 63 (antibody of parasite resistance and allergy). Th2= 2 roles in parasites, 1: M2 macrophages and 2: stimulate B cells to make IgE. d. SIBLING RIVALRY: Th1 and Th2. IFNgamma (made by Th1) suppresses Th2 differentiation. IL-4 (made by Th2) suppresses Th1. BALANCE EACH OTHER. e. Follicular helper T cells (Tfh): DC arrives in lymph node, some activated follicular helper T cells migrate into follicles of cortex where B cell are abundant. They help B cells that have recognized antigen become activated and differentiate into antibody secreting plasma cells. Tfh secrete cytokines and direct B cells to switch from IgM to IgG, IgA or IgE. Tfh in the gut switch B cells to IgA, Tfh in the spleen switch B cells to IgG. Without Tfh, it would be hard to make any antibody class, especially those downstream from Ig. f. Regulatory T cells (Treg): 5% of T helper cells, suppresses the activation and function of other T helper cells. Most are CD4+/CD5+. They product TGFbeta and IL-10. Very potent (1 can suppress 1000 Th cells). g. Cytotoxic Killer T cells (CTL): signals the target cell to commit suicide by activating apoptosis that leads to rapid DNA fragmentation and nuclear collapse. 2 methods: 1. Activate Fas (CD95) “death receptor” on the target (CTLs bear the Fas ligand, CD95L). 2. Secrete contents of lytic granules which contain proteases called granzymes and perforins that allow penetration of granzymes into target cell— apoptosis. 2. Describe the surface markers that can be used to distinguish between T and B cells in humans. Describe markers that helper and killer T cell subpopulations in humans have on their surfaces. a. T cells have CD3 (virtually ALL T cells), CD4 (Th2, Th1 and Treg cells), CD8 (CTL cells) surface markers (CD= cluster designation). Th1 and Th2 cannot be distinguished via surface (must look at what lymphokines they make) b. B cells have antibody surface markers. B cells are readily distinguished using antibodies to immunoglobulins or their chains, or to the surface marker CD20. c. 3. Define a. Lymphokine: short range mediators made by lymphocyte that affects behavior of the same or another cell. A subset of cytokines. Examples: IL-2, IFNgamma, IL-4, IL-5, IL-10. b. Chemokine: small (6-14 kD) short range mediators made by any cell that causes inflammation. Examples: MIP-1 to -4, RANTES, CCL28, CXCL16 c. Cytokine: short range mediators made by any cell that affects behavior of the same or another cell. Examples: IL-1, TNFalpha, IL-12. 4. Describe an activity of interferon-gamma (IFNγ). a. Most important lymphokine secreted by Th1 cells. Pro-inflammatory, chemotactic for blood monocytes and tissue macrophages. Macrophages move into area where Th1 recognized antigen. IFNgamma activates them to be classically activated into M1 cells, “angry” macrophages. Ingest and kill bacteria. 5. Define mitogen, and name two T cell mitogens. Name a mitogen that stimulates both B and T cells in humans. a. Mitogen: protein that stimulates T cell division b. Examples of mitogens: phytohemagglutinin (PHA)—stimulates T cells to divide because it binds CD3, concanavalin A (Con A)—stimulates T cells to divide. c. Pokeweed mitogen (PWM) stimulates both B and T cells (non-specifically) to divide 64 6. Distinguish between the effects of a mitogen and an antigen, when added to normal blood lymphocytes. a. Antigens are specific, mitogens are nonspecific b. Mitogen: doesn't actually bind to antigen-binding site on T cell, like an antigen does. What it does bind to is the CD3 domain that controls signal transduction from the antigen-binding chains. This is like bypassing the light switch in your house- the lights are going to always stay on because there's no longer a mechanism for turning them off. 7. Compare and contrast the antigen receptors of T and B cells a. B cells: B-Cells: Bind antigen directly with surface antibodies. Because T cells see antigen only when it is complexed with cell-surface MHC molecules, T cells focus their attention on cell surfaces, and do not interact with free antigen; that is a job for the B cell and its antibodies. b. T cells: T-cell receptor for antigen (TCR) is made up of 2 chains, alpha and beta, and each has a common and variable portion. The T cell makes the receptor out of V, (D) and J regions recombined (just like B cells) and (like antibodies) each chain has 2 CDRs (complementarity-determining regions). This is taking place in the thymus. Both alpha and beta chains have trans-membrane domains (unlike surface Ig where ONLY the heavy chain is trans-membrane domain). c. CAREFUL CONTROL: 1. On the TCR is a complex of molecules called CD3. When T cell binds correct antigen + MHC with its TCR, the actual signal that turns the T cell on is transmitted by CD3. 2. When a Th0 binds to antigen-presenting cell, APC gives T cell boost by secreting IL-12. Needs these signals to be activated. 8. Discuss the structures recognized by T cell receptors (see also Immunogenetics). Distinguish between what is recognized by helper and cytotoxic T cells. Explain the special role of dendritic cells in this process. a. Antigen enters the body infects locally and causes innate response breakdown products are ingested by dendritic cell within DC endosome, proteins are broken down to peptides via lysosomethe endosomal peptides then fuse with vesicles containing Class II MHC molecules embedded in their membrane (facing IN) some of the peptide associates with the MHC molecule endosome goes to cell surface and fuses with plasma membrane and MHC + antigenic peptide are exposed to outside world the appropriate T cell (HELPER T’s) recognizes both the MHC + antigen and is turned on. b. Protein in made within the cell chopped up into peptides fuses with vesicles containing Class I MHC molecules embedded in their membrane some of the peptide associates with MHC molecules fuses with plasma membrane and MHC + peptide are exposed to outside world appropriate T cell (CYTOTOXIC) recognizes both the MHC + antigen and is turned on. 9. Discuss what is meant by “MHC-restriction”. Name the classes of MHC molecules by which CTL and helper T cells are restricted. 65 a. PART 1: T cells are restricted in their recognition of antigen, to antigen on the surface of cells (target cells) genetically identical to themselves. T cells do not “see” antigen alone, but nly antigen presented to them on the surface of a genetically identical cell. T cell and antigen-presenting cell must come from individuals who share alleles at the Major Histocompatability Complex (MHC)—codes for surface glycoproteins. Basically, T cells are MHC-restricted and my T cells won’t work in another person. b. PART 2: Two classes of MHC molecules, MHC Class I and MHC Class II. All nucleated cells have MHC Class I on their surfaces. Class II products are only on dendritic cells, macrophages and B cells (the cells involved with presenting antigenic peptides to T helper cells). Antigen is endocytosed and presented by dendritic cells via Class II MHC to the T cell. Proteins synthesized within a cell are presented via Class I MHC (Most are normal, but some can be viral, bacterial, etc). c. Th1, Th17, Tfh, Treg and Th2 (HELPERS) recognize peptides on CLASS II MOLECULES. d. Cytotoxic T cells (KILLERS) recognize peptides on CLASS I MOLECULES. 10. Describe the role of T cells in ridding the body of a viral infection. a. CTL cells will “see” an infected/foreign cell (because MHC Class I will have it bound) and then will activate the target cell to commit suicide because it has a Fas CD95L Ligand (binds to Fas CD95 receptor on target cell) OR secretes lytic granules that trigger apoptosis. b. HELPER cells will “see” antigen presented on a dendritic cell, B cell or macrophage via MHC Class II. They will activate the immune response and divide. 11. Describe the characteristics of T-independent antigens. a. Most antigens require T cell help to achieve a reasonable antibody response, but some don’t. These are T-independent antigens are usually have the same epitope repeated over and over (rare in proteins, but common in carbohydrates, ie: Streptococcus pneumoniae). Carbohydrate chain binds to the B cell antibodies cell is activated and begins to divide The response to T-independent antigens is almost all IgM (T cells needed to switch from IgM to IgG, IgA or IgE). So, if a person is very deficient in T cells, they still have make antibody to carbohydrates. With protein antigen—NO IgM and NO IgG is made without T cell help. 12. Outline an experiment that shows that an antibody response can be “T-dependent”. a. T-dependent response require T cell help to achieve a reasonable antibody response. To test this, we could expose cells to antigens and see what antibodies are produced. 13. Diagram the mechanism by which Tfh cells help B cells. a. B cell binds the epitope that the B cells receptor is specific for bound molecule is endocytosed and broken down in endocytic vesicle peptide fragments bind to MHC Class II moleciles brought in by other vesicles that fuse with the endosome and MHC-peptide complex moves to surface B cell displays antigen + Class II MHC correct Tfh comes along and sees its epitope + Class II MHC on B cell binds and focuses surface interactions and helper lymphokines on B cell. 66 b. NOTE: the epitope that the T cell “sees” is not the same as the one the B cell “saw”. 14. Describe the cellular and molecular events following intradermal injection of tuberculin antigen into a person who have cell-mediated immunity to it. Justify calling the process “delayed hypersensitivity”. Characterize the cells that would be seen in a 48-hour biopsy of the site with regard to whether T cells or macrophages predominate. a. TB antigen comes in; it gets picked up by dendritic cells and broken up, then displayed; circulating Th1 cells bind to dendritic cell surfaces, causing their activation; they begin to release IL-2, which activates any nearby Th1 or Th2 cells that can bind to TB as well, and also release IFN-gamma, that cause the capillaries to open up and macrophages to be attracted to the site (chemotactic) over 48 hours or so. Given that 1 activated T cell can attract 1000 macrophages with its chemotactic signals, I think it's a fair bet that the macrophages would outnumber the T cells. b. The process is called delayed - I think - because the first time you're exposed to the antigen, it doesn't happen. This has to do with T cell timing-- if you're exposed for the first time, your dendritic cells have to bring antigen fragments to the lymph nodes, you have to make antibodies, etc. If you're exposed the second time, you already have circulating T antibodies that recognize those antigen fragments at the site of infection, and can prompt a quick, local, and painful reaction. BL - Histology of the Thymus and Peripheral Lymphoid Organs 1. Describe the basic structure and general movement of lymph and lymphocytes through a lymph node. What differentiates activated nodules from non-activated? a. Afferent lymphatic vessels drains into node just beneath the capsule into the subcapsular space/sinus drains into trabecular sinuses drains into medullary sinuses converge at hilum and lymph leaves via efferent lymphatic vessels towards either a more central lymph node or ultimately for drainage into a central venous subclavian blood vessel. b. B cells migrate to the nodular cortex and medulla c. T cell migrate to the deep “paracortex” cortex d. When a lymphocyte recognizes an antigen, B cells become activated and migrate (within a follicle??) to germinal centers—regions of active cell proliferation and apoptosis (“secondary nodule” has germinal center, “primary” does NOT). Antibody producing plasma cells form and migrate to medullary cords. 2. Outline the vasculature of lymph nodes. Know the importance of the high endothelial venule. a. Each lymph node has a blood supply that enters through a small artery at he hilus and leaves via a small vein. Artery branches repeatedly to supply the entire node with O2 and nutrients. In node, vessels are lined by special endothelium, HIGH ENDOTHELIAL VENULE (round and protrube into lumen of vessel). These are sites for recognition and diapedesis of lymphocytes from the blood into lymphatic space of the node. Cells of these venules have receptors that function in initiating passage through the endothelium. 67 3. Describe the blood flow through the thymus. Be able to recognize the differences between a lymph node, thymic lymphoid tissue, and that of the spleen. a. Small arteries enter thymus through outer capsule and penetrate the thymus and bifurcate within the connective tissue septa between lobules. Endothelial cells of vessels have tight junctions and surrounded by connective tissue ensheathed by endothelioreticular cells. These combined layers (but functionally primarily the endothelioreticular layer) form the Blood-Thymus Barrier. Maturing thymocytes are NOT exposed to molecules circulating in blood. Stromal cells protect them from exposure to antigens and provide conditions for maturation and selection. 4. Be able to recognize the nuclei and cell bodies of reticuloendothelial cells in the thymus, Hassall’s corpuscles, and know how they are relevant to lymphocyte selection. a. Reticuloendothelial cells: forms blood-thymus barrier no matureing thymocytes aren’t exposed to stuff in the blood. Involved in selection process (+ and -) for thymocytes as they progress towards medulla. b. Hassall’s corpuscles: in medullary region, make thymic stromal lymphoprotein to suppress autoimmune events. Produce lymphokines that promote thymocyte maturation into adult T cells. 68 5. Describe the blood flow through the spleen. How does it differ markedly from that of the thymus and lymph node? a. Different because it has an open blood circulation through porous splenic sinuses. Recieves blood through splenic artery that branches into central arterioles that run into pulp of spleen and become lined with discontinuous endothelial cells, the discontinuities are so large that platelets, RBCs are laukocytes leave the vessels and enter sinuses that contain loosely packed arrangements of cells. Bulk of lymphoid tissue is arranged around central arterioles called periarteriolar lymphoid sheath (PALS). Germinal centers are in this sheath. The spleen is drained by splenic vein. Red pulp: where blood flows through loosely arranged channels/sinuses. White pulp: more organized lymphoid tissue. Like lymp nodes, spleen has reticular fibers that are in red pulp. Also contains macrophages (in white and red pulp) to remove senescent RBCs and platelets and recycle iron and remove debris they encounter. 6. Be able to recognize the cellular components of white pulp and red pulp. a. White pulp: lymphocytes (T-cells), The purpose of the white pulp is to mount an immunological response to antigens within the blood. The white pulp is present in the form of a periarteriolar lymphoid sheath. This sheath contains B cell follicles and T cells. b. Red pulp: 75% of spleen, connective tissue and splenic sinuses that are engorged with blood—thus RED color. Filters blood of antigens, microorganisms and old RBCs. Has platelets, granulocytes, RBCs and plasma. 7. Be able to recognize and describe the regions of mucosal-associated lymphoid tissue. a. Notable collections include the tonsils (palatine, lingual and pharyngeal (adenoids), esophageal nodules, the appendix, bronchial nodules, and a large number of aggregations of lymphocytes in the intestine, usually increasing in size and abundance along its length until in the colon, there are very abundant multiple groups of nodules both in the mucosa and submucosa known as Peyer’s patches. 8. Delineate the differences between primary and secondary lymph organs. a. Primary: bone marrow and thymus gland, major sites of development of B lymphocytes and T lymphocytes, respectively. Lymphopoiesis (differentiation of lymph cells from pluripotent progenitors) b. Secondary: aggregates of lymphocytes found in close proximity to antigen presenting cells and can also furnish an adaptive immune response, lymph nodes, tonsils, adenoids, Peyer’s patch and spleen. Seeded with cells from primary organs. 9. Be able to describe the structure of all major lymph organs. Explain the function of the main lymph organ cell types and/or parts. 69 a. Lymph nodes: small organs all over the body, found individually or in chain/clusters. Non-specific filters of debris/microorganisms and are a site for antigen presentation in adaptive immunity. Lymphocyte in interstitial space enters small lymphatic vessel connects to afferent lymphatic vessel enters node into subcapsular space. b. Thymus gland: bilobed thymus with connective tissue capsule from which connective tissue speta (tabeculae) divide the organ into pseudolobules, where all thymocytic cells undergo processes (proliferation, differentiation and selection) that results in release of immunocompetent T-lymphocytes. Provides environment for negative and positive selection of thymocytes occurs. Total lymphocyte mass of thymus decreases thru childhood and around puberty fills with fat cells and connective tissue. Densely packed set of developing thymocytes: CORTEX. More mature thymocyte precursors are less dense: MEDULLA. Epithelioreticular cells (or stromal cells)—includes epithelial-like cells, dendritic cells, macrophages. These cells provide a matrix and envelop developing thymocutes in large infolds as they matures and move from cortex to medulla, important for positive and negative selection events. Thymus has NO reticular fibers (no bulk fluid flow), stromal cells provide support. Hassal’s corpuscles: concentric circular layers of reticular cells, in the medullary regions, produce thymic stromal lymphoprotein that suppresses autoimmune events. At the end of selection, thymocytes leave thymus via the lymphatics and blood vessels. 70 c. Spleen: Multi-purpose lymphoid organ, role in adaptive immunity. See above for more info + picture. d. Mucosal-Associated Lymphoid Tissue (MALT): unencapsulated collections of lymphoid cells and their associated support cells and macrophages, includes: tonsils, esophageal nodules, appendix, bronchial nodules, aggregations of lymphocytes in intestine (Peyer’s patches). each of which is well situated to encounter antigens passing through the mucosal epithelium. Within intestinal MALT, special surface epithelial cells, M cells, deliver antigen to underlying lymphoid tissue so that foreign antigens in lumen of gut or in airways can elicit adaptive immune response. MALT lymphocytes enter lymph and blood circulation. 10. Describe the types of lymphoid cells. Understand the functions and interactions between these cells. a. Helper T cells b. B cells c. Cytotoxic T cells d. See Cohen’s lectures. 71 WEEK 4 Immunohematology 1. For persons of the A, B, AB and O blood groups, give the following data: most and least common groups; red cell antigens; specificities of the ABO antibodies in their plasma; safe donors to that type; safe recipients of blood from that type; possible genotypes. Blood Possible Red cell ABO Can donate Can receive Most and least Group genotypes antigens antibodies in to? blood of? common groups plasma A AA, AO A Anti-B A and AB A and O 42% white, 27% black, 100% certain Peruvian Indians B BB, O B Anti-A B and AB B and O 9% white, 21% black, very common in Vietnam and central Asia AB AB A and B NONE AB only A, B, AB 3% white, 4% black and O O OO NONE Anti-A and A, B, AB and O only 46% white, 48% black, (Bombay Anti-B O rare in China appears as O also) 2. Name the class of most ABO isohemagglutinins. a. Because blood group antigens are glycolipids (lipid backbone spans plasma membrane and terminal sugars confer the antigenic specificity) you will inevitably come in contact with these carbohydrates in the environment (during infancy, appear in blood 3-6 months of age) and become immunized to them. This is how a person with Type A blood has anti-B antibodies in their blood, but no antibody to A. These are called “naturally occurring” antibodies: Isohemagglutinins. They are of the IgM class (10 heavy, 10 light, 1 joining chain). b. Occasionally people do make IgG isohemagglutinins (especially Group O people). A or B fetuses of these women are at some risk of ABO hemolytic disease. 3. Explain the ABO antigen situation in a person of Bombay blood type, and the consequences of a transfusion of non-Bombay blood into such a patient. a. Bombay phenotype occurs when a person LACKS the transferase gene that puts the final sugar on the “core” and thus do not express even the H antigen (that O people express). It is rare. If you have Bombay blood, you cannot receive, blood from A, B or O groups, only from other Bombay blood donors. During routine typing, Bombay blood appears as “O”, but will have anti-O, anti-A and anti-B in plasma. 72 4. Define the cross match, and explain why it is important. Explain how red cells are destroyed following a mismatched transfusion, and why this may be devastating to the recipient. a. During blood banking, the blood is Typed and Screened for ABO and Rh, “unexpected antibodies”, syphilis, HepB, HepC, HIV, West Nile Virus. Reverse typing is then done to make sure the isohemagglutinins in plasma reflect the determined red cell type (ie: Group A blood has anti-B). b. Prior to transfusion, the recipients is typed for ABO and Rh and plasma screened for antibodies. The donor units must be compatible (identical) with recipient in terms of ABO and Rh. Cross match question: Are there antibodies in recipients plasma that can react with antigens on this donors RBCs?? If yes, and you give blood, the red cells will die earlier and patient may not survive. c. Cross match: plasma from recipient is mixed with RBCs of donor. If there is agglutination, it means there are lots of high-avidity antibodies in the serum (probably IgM). There are DANGEROUS because of potential complement-activation. If no agglutination, there may still be an antibody, not enough to agglutinate, but enough to opsonize the cells. So you enhance changes of agglutination by doing test in low ionic strength diluent. Then to maximize detection of IgG, you do indirect anti-globulin test. If no agglutination, then they are compatible. 5. Compare and contrast the techniques of the direct and indirect antiglobulin tests and the question they are designed to answer. a. Direct (DAT): is there antibody already on the red cells I am interested in? Take red blood cells, rinse them and add antiglobulin. Detects cells that were coated with antibody in-vivo. Good for determining is there is autoimmune hemolytic anemia. b. Indirect: is there unexpected antibody to red cell antigens in the PLASMA of recipient? Take red cells from donor, add plasma from recipient, rinse cells (assuming no agglutination), add antiglobulin. If cells now agluttinate, there must have been any antibody to them in the plasma because the antiglobulin alone won’t react with the cells. Good for transfusion compatability. 6. Define heterophile antibody, and identify a common disease in which one type is increased enough to be useful diagnostically. a. Antibodies to 1 antigen which bind to another (cross-reactive). Example: infectious mononucleosis— antibody appears in serum, as response to viral antigen, but reacts with sheep red blood cells (leads to a cheap, quick test for mono-Monospot). Syphilis antibody that people make reacts with phospholipids extracted from beef heart simple test. 7. In Hemolytic Disease of the Newborn, explain: a. The consequences of severe hemolysis in the newborn i. The fetus will be born jaundiced. High levels of bilirubin (from break down of hemoglobin) can cross BBB and damage basal ganglia cerebral palsy +/- death. b. The way in which the mother becomes sensitized. i. AKA: erythroblastosis fetalis, occurs in Rh(D)+ children of Rh(D)- mothers. If the last trimester and during delivery, some red cels from baby enter mom’s circulation. If she is Rh(D)-, she may make anti-Rh(D) after exposure to babies Rh(D)+ blood. For first baby, it is no problem because baby is delivered. c. The class of antibody to Rh(D) the mother makes i. Because the antibodies can cross the placenta, the class is IgG. d. The consequences of sensitization to subsequent fetuses i. In subsequent pregnancies with another Rh(D)+ fetus, the mom’s antibodies formed after frist pregnancy can cross placenta and destroy the fetus’ red blood cells. Each pregnancy with Rh(D)+ fetus boosts her antibody responses. e. The role of Rh-immune globulin. i. When mom delivers first Rh(D)+ baby, she is given IgG antibody to Rh(D) (Rh-immune globulin), RhoGAM. These antibodies combine with the left over fetal red blood cells, opsonize them and 73 destroy them BEFORE they can immunize the mom. She must receive Rh immune globulin each time there is a chance of being immunized by Rh(D)+, deliveries, abortions, amniocentesis. Now, women are given the RhoGAM shot at 28 weeks gestation, to prevent immunization via transplacental bleeds in 3rd trimester. Then after delivery, baby will be tested an is Rh(D)+, then another shot will be given. 8. Explain the situation in which ABO hemolytic disease of the newborn can occur. a. ABO hemolytic disease of newborn occurs when women make IgG isohemaggluinins (ie: antibodies against B blood that are IgG and can cross the placenta). Common is Group O blood people. There is no RhoGAM shot for this. BL - Immunogenetics and Transplantation 1. Define Major Histocompatibility Complex. Distinguish between HLA-A and HLA-B antigens on the one hand, and the HLA-D group on the other, in terms of: which associate with foreign antigens for recognition by helper T cells; which, in association with foreign antigens, are the targets for killer T cells. a. Major Histocompatibility Complex: a family of genes, coded for on a single chromosome, that code for histocompatibility antigens, some of which are expressed on the surfaces of all nucleated cells. MHC in humans is HLA (Human Leukocyte Antigen). The most important loci within the HLA are HLA-A, HLA-B and HLA-D, of which HLA-DR is the most interesting for transplanters. b. HLA-A and HLA-B code for Class I MHC and so they are on all nucleated cells and are recognized by cytotoxic c. HLA-D (DP, DQ and DR) code for Class II MHC and are on antigen presenting cells only (macrophages, dendritic cells, etc), they are recognized by T helper cells. 2. Define: a. Alloantigen: antigen that is a part of an animal's self-recognition system. e.g., Major histocompatibility complex molecules. When injected into another animal, they trigger an immune response aimed at eliminating them. Therefore, it can be thought of as an antigen that is present in some members of the same species, but is not common to all members of that species. If an alloantigen is presented to a member of the same species that does not have the alloantigen, it will be recognized as foreign. They are the products of polymorphic genes b. Haplotype: combination of alleles (DNA sequences) at adjacent locations (loci) on the chromosome that are transmitted together. The MHC gene set that you inherited from your parents is called a haplotype. 3. Distinguish Class I and Class II histocompatibility antigens. a. Class I histocompatibility antigens: present on all nucleated cells (and platelets) b. Class II histocompatibility antigens: present on antigen presenting cells (B cells, macrophages and DCs) 4. Identify the chromosome on which the MHC is found in humans a. The human HLA is found on chromosome 6, short arm. 5. Discuss HLA-A and B typing in terms of how many antigens a person has at each locus. Given two unrelated parents' haplotypes, predict their children's phenotypes. a. Like most genes, each of these loci are expressed codominantly, so that at the HLA-A locus you have both paternal and maternal alleles expressed. b. Father HLA phenotype: A1, A3, B5, B7, DR9, DR11. Mother HLA phenotype: A2, A4, B6, B8, DR10, DR12. Child: A1, A4, B6, B7, DR11, DR12. The haplotypes are as follows: i. Child: A1, B7, DR11 and A4, B6, DR12 ii. Mother: A4, B6, DR12 and A2, B8, DR10 iii. Father: A1, B7, DR11 and A3, B5, DR9 74 6. Name the best probable donors of tissues or bone marrow to an individual, and discuss the reasons for your choices. a. HLA-DR (Class II) matching is the most important thing T helper cells won’t be stimulated. In terms of Class I matching, HLA-A and HLA-B are the most importantkiller T cells won’t be stimulated but Th1 cells will be. b. The best probably donor will be an identical twin, followed by a sibling, there is a 25% chance that two siblings will have identical haplotypes and will therefore be accepted as tissue/bone marrow donations. c. Parents will always be ½ different from their child so they are not good donors. Likewise, children are ½ different from their parents, so they are not good donors. d. You can also use volunteer donor if they match via HLA. e. For bone marrow, you look for matches at A, B, C, DR and DQ. A 10/10 match is desired. 7. Describe the one-way mixed leukocyte reaction (MLR) and discuss its use. a. To answer the question: How strongly do the recipient’s T cells recognize the Class II of this potential donor versus that one? b. Cells from the donor are treated with DNA synthesis inhibitors or radiation (kills lymphocytes, but not APCs like macrophages) to prevent their division. You want to know if the recipient can recognize the donor’s MHC. Then the cells from donor and recipient are mixed together to see if recipient’s Th cells divide in response to the donor’s HLA-D (mostly DR, on monocytes). A strong reaction (burst of cell division) may preclude the transplant. 8. Distinguish between “HLA-D” and HLA-DR, -DP, -DQ. a. HLA-D are all Class II MHC, but HLA-DR has to do with transplantation, which HLA-DQ and HLA-DP have to do more with autoimmune diseases. 9. Describe the cellular and molecular events which go on during graft rejection, both of the usual type and hyperacute rejection. Include: killer T cells, Th1 cells, angry macrophages, antibody + complement a. Th 1 cell recognizes foreign HLA-DR (not on all cells, just the APC’s like macrophages and DCs) on graft cells. Th1 proliferates (this is what the MLR measures) and secrete lymphokines like IFNgamma to attract a macrophage inflammatory response. The macrophages product pro-inflammatory cytokines like TNF-alpha and the graft is destroyed. Meanwhile, CTL’s recognize foreign HLA-A and HLA-B which are on all cells (including macrophages and DCs). This recognition is insufficient to activate them; they also require Th1-derived interleukins as a 2nd signal. Once activated the CTL’s become highly cytotoxic. Exactly parallels what happens in normal immune response (like a virus) except that in a normal response a peptide + self-MHC is recognized, in rejection its foreign MHC. b. In hyperacute rejection graft tissue is rejected almost immediately because there was circulating antibody against the graft’s tissue (from a previous failed graft) or against the graft’s residual blood in the tissues endothelium. The antibodies attached to endothelium and activate lots and lots of complementanaphylatoxin (C3a, C4a, C5a) from mast cells vasospasm, constricting vessels and tissue ischemia. COMPLEMENT MEDIATED IS FAST REJECTION. 75 10. Explain the interaction of T cells recognizing HLA-D and T cells recognizing HLA-A or B in the generation of killer T cells. Include the roles of cytokines in your discussion. a. If the donor and recipient are identical at HLA-A and HLA-B, but different at HLA-DR: you will activate Th1, but no CTL will be generated. You will still reject the graft but since only Th1 and not CTL are involved, rejection will be slower. b. If the donor and recipient are different at HLA-A and HLA-B, but identical at HLA-DR, you will get no MLR/no Th1 activation/no IL-2 generation and few CTL will be activated. Therefore, a good HLA-DR match is the most important. 11. Give an example of a disease whose incidence is tightly linked to a particular HLA allele. Speculate on the mechanism which might explain the linkage. a. Ankylosing spondylitis: arthritic condition in which there is inflammation of the insertions of tendons into bonescalcification of joints and they become inflexible (ankylosed). 92% of people with this are HLA-B27. Your risk of getting this disease is 90x greater if you are B27 than if you are not. Unknown association but some ideas are: unknown pathogen with a surface antigen that so closely resembles the B27 molecule that B27+ people can’t recognize it as foreign OR antigen may cross react with B27 so that a response to the foreign antigen might lead to autoimmunity OR cross-reaction between B27 and Klebsiella OR unknown ankylosing spondylitis gene to the left of HLA-B that is in strong linkage disequilibrium with B27. b. Association between HLA-DR3 and –DR4 and juvenile diabetes. 12. Discuss how T cells selected to recognize "self + X" also recognize foreign MHC (allorecognition). a. The recognition of foreign MHC is a chance cross-reaction; the receptors are actually selected to recognize self-MHC and antigen. b. T cells are selected to see something that is not quite “you”: you + foreign peptide. 5-10% of T cells also will response in a one-way MLR against foreign cells (it is like they were immunized, but they have never seen it before). c. 5% of T cells will bind a foreign MHC strong enough to cause activation. But none of the T cells that see MHC + foreign peptide will also see foreign MHC + peptide, since they were selected for a specific MHC (not the foreign MHC). This is why we can’t give T cells to other people. 76