CLS_A_Bottomline_Approach_5e HEMA.pdf

53 HEMATOLOGY by Angela B. Foley...

53 HEMATOLOGY by Angela B. Foley Hematopoiesis Prenatal Hematopoiesis Sac 0 1 2 3 4 5 6 7 8 9 Conception ; Adult Hematopoiesis Months Birth Blood Cell Maturation HEMATOPOIETIC STEM CELL CFU- GEMM CLP COMMITTED CFU-E CFU-GM [EO, BASOJ CFU-MK CFU-TNK & CFU-B PROGENITORS GROWTH FACTORS GM-CSF, GM-CSF, IL-3,5 GM-CSF, IL-1 ,2,4,5,6,7, 12,15 IL-3 EPO IL-3 6,11 , TPO Bone Marrow (BM) Rubriblast Myeloblast Monoblast Megakaryoblast Lymphoblast t t t t t Prorubricyte Promyelocyte Promonocyte Prolymphocyte t t Pmmegl ryocyt, Rubricyte - Myelocyte - t t Metarubricyte Metamyelocyte Megakaryocyte t t Peripheral Blood Reticulocyte t Band Neutrophil t ! EPO CSF TPO = = = Erythropoietin Erythrocyte Colony Stimulating Factor Thrombopoietin Segmented Neutrophil Basophil GM = Monocyte Eosinophil Macrophage (tissue) Granulocyte, monocyte Platelet (thrombocyte) 7\\ NK Cell T Cell B Cell t GEMM = Granulocyte, Erythrocyte, CFU = Colony Forming Unit Plasma Megakaryocyte, Monocyte MK = Megakaryocyte CLP = Committed Lymphoid Progenitor L = Lymphocyte Cell IL ; Interleukin NK ; Nautural Killer 54 3. Protoporphyrin Synthesis a. Precursors (see memory tool below) b. Porphyrias - Enzyme deficiencies cause build-up of heme precursors - r ed or port wine colored urine Action of EPO and TPO ❖ Early precursors (Dl:!,""'LTA ala or PORphobilinogen)- neuropsycluatric symptoms, i.e. acute intermittent porphyria or ID Mature and Immature AIP Cells from Graphic Images ❖ Later precursors (UR, COP, PROTO) - cutaneous sysmptoms such as photosensitivity, facial Hemoglobin hair HEME SYNTHESIS 1. Must have iron and protoporphyrin 2. Iron transport and storage a. Transferrin - Fe transport protein b. Ferritin - major Fe storage form c. Hemosiderin - I-120 insoluble Fe ,;toragP. form (long-tP.rm) Conditiom Associated with lncreaaed Storage of lron d. Excess iron will be stored in tissues and body organs ~ can lead to hemosiderosis, hemochromatosis (organ damage) Fe TraIJBport Protein Fe Storage Forms REMEMBER! Y'ALL ARE HERE AL Heme Precursors ATJ30 R3~10 While in the DELTA, POUR YOUR COP, PRONTO, a cup of HEME. DELTA-aminolevulinic acid t PORphobitinogen t URoporphyrinogen t COProporphyrinogen t PROTOporphyrin t HEME + globin 4 hemoglobin..,_____,,,,, PORPHYRIAS: Exessive formation of porphyrins occurs iJ any enzymatic step in heme synthesis is blocked.  55 Sequence of Conditions Cau.mig- Heme Precursors Left/Bjght Sbifts GLOBIN SYNTHESIS Normal Fetal BD.d Adult HEMOGLOBIN TYPES IN NEWBORN ANO ADULT Hemoglobin Chains CHROMOSOME GLOBIN HGB NEWBORN ADULT CHAINS %.,,~ Basic Laboratory Procedures 16 11 s2 e2 Gower I ANTICOAGULANTS 0% 0% (l2 °2 Gower II (Embryonic) (Embryonic) 1. EDTA (ethylenediamine-tetra-acetate) - chelates Ca++.~: c t e s212 Portland 2. H eparin - anti-thrombin agent cxn2 Hgb F 60-90% 1% HEMOGLOBIN ai&i HgbA2 Anti-IF positive (Intrinsic Factor), ,f.MMA (methylmalonic acid), ,f.Homocysteine, ~ Malabsorption/ Dietary ~ t s12, Anti-IF negative Folate Deficiency tFolate levels, Anti-IF negative, t Retics, Oval Macrocytes, Hypersegmented Polys Liver Disease / Alcoholism ,¼.Liver Enzymes, Target Cells, Round Macrocytes NORMOCYTIC/N ORMOCHROMIC Antibody Mediated ,f.Bilirubin, t Haptoglobin, DAT+ ltiF PCH ,r;p Donath Landsteiner Ab (Anti P specificity) iW Cold Agglutinin Disease I& lgM Ab (Anti I specificity), Cold Agglutinin Titer+ e:w Warm Autoimmune l& lgGAb Hemolytic Anemia Membrane Defect llF Hereditary Spherocytosi~ 611" Spherocytes, ,f.MCHC, Abnormal Spectrin 1& Hereditary Elliptocytosis a- Elliptocytes (>15% to 100%), Abnormal Spectrin IIF PNH e:w CD55-, CD5~. FLAER Test positive Enzyme Deficiency 11F G6PD " t G6PD, Heinz Bodies _.., P vn 1v I(;""'"" /PK\ tri1'..PK II.In ~in7 R,-,,1;.,~ Decreased Production / Loss - Aplastic Anemia ,r;p "Dry Tap" Bone Marrow (BM), Hypocellular BM, t Retics, Pancytopenia - Acute Blood Loss 611" Normal BM, +Reties - Chronic Renal Disease - t EPO Hemoglobin Defects Definitive Poikylocytes on Smear (HbC crystals, Sickle Cells, SC crystals, etc.), Hb Electrophoresis REMEMBER! Trust in yourself. Your perceptions are often far more accurate than you are willing to believe - Claudia Black Correlate Lab Data to Determine Type ofAnemia 64 Special Tests TEST MEASURES INDICATIONS COMMENTS FLAER Test Absence of GPI Anchor Proteins PNH More specific and sensitive (Fluorescent Aerolysin) for CD 55 and CD59 Flow Cytometry Deficiency of CD 55 and CD 59 PNH Less sensitive than FLAER test on RBCs and granulocytes Heinz Body Prep Effect of Oxidizing Agent on G6PD Deficiency Formation Triggered by Oxidants (supravital stain) Hemoglobin Unstable Hemoglobins such as Anti-Malarial Drugs, (Precipitated globin chains) HbH Fava Beans & Sulphur Drugs Sickle Cell Screen Reduced Solubility of HbS Reducing Agent: Deoxygenated Hemoglobin S Na Dithionate Kleihauer-Betke Acid Resistance of Fetal Hemoglobin Fetal-Maternal Hemorrhage; Cells with +HbF Stain Pink; Elution to Acid Elution Hereditary Persistence of Fetal Normal Adult Cells ~ Ghost Cells Hemoglobin Hemo~obin Migration of Various Suspected Hemoglobinopathies May be Performed at Various pHs Electrop oresis Hemoglobins Cold Agglutinin Titer Presence of Cold Autoantibody Cold Autoimmune Hemolytic lgM Ab, Anti-I Specificity Anemia Donath Landsteiner Test Presence of Biphasic DL Antibody Paroxysmal Cold Hemoglobinuria lgG Ab, Anti-P specificity GPI- Glycosylphosphatidylinositol FLAER- Fluorescent Aerolysin REMEMBER! Kleiheur-Betke Fetal Hb is Resistant to Principles and Indications of Special Te8t8 Acid Elution (Adult Cells Appear as Ghost Cells.) (~~ ( "' 20% blasts) HEREDITARY CONDITIONS ❖ AML with recurrent chrom osomal CONDITION) CHARACTERISTICS COMMENTS abnormalities iw t(8;21) Alder-Reilly Large Azurophilic ,t.Mucopolysaccharides 1& Inv (16) or t(l6;16) Granules (Hunter, Hurler) i& t(l5 ;17) Chediak- Large Lysosomes (Fusion Albinism, i& llq23 Higashi of Primary Granules) + susceptibility to ❖ AML with dyspfasia Infection I& may follow MDS Does Not Affect ❖ AML & MDS therapy related May-Hegglin La~e Platelets, t Number, i:ih le Bodies in Segs, Leukocyte Function ❖ AML not otherwise classifi.ed - Monos, and Lymptis defaults to the FAB classification ❖ AML of ambiguous lineage Pelger-Huet Hyposegmented Polys Normal Function b. French , American, British (FAB) classi- fication (>30% blasts) MYELOPROUFERATIVE DISEASES 1. Myelodysplastic Syndromes- neoplastic, AML PREDOMINANT CELL SEEN clonal,stem cell disorder s characterized MO Myeloblast without differentiation by cytopenias and BM dyspoiesis Ml Myeloblast with minimal maturation a. Refractory Anemia (RA)- 20% plasm a cells i 11 p eripher al lmmunoglobulin lgG lgM circulation (Bence-Jones) (Heavy Chain: Special Cytochemical Stains/ Inclusions/ Markers STAIN/ INCLUSION/ MARKERS INDICATES: SIGNIFICANCE: Prussian Blue Iron Sideroblastic Anemia, Iron overload LAP Leukocyte Alkaline Phosphatase ,+. Leukemoid Reaction, P. vera; t CML Peroxidase/Sudan Black Myeloperoxidase/Lipid Myeloid Precursors Pos / Lymphoid Precursors Neg Specific Esterase Granulocyte Precursors Negative in Monocytic Leukemia Non-Specific Esterase Monocyte Precursors Positive in Monocytic Leukemias TRAP Tartrate-Resistant Acid Phosphatase Hairy Cell Leukemia Auer rods Coalition of 1° Granules Acute Myeloid Leukemia CD13, CD33 Myeloid Lineage Myeloid / Monocytoid Leukemias CD4 l , CD42, CD6l Megakaryocytes Megakaryocytic Leukemia CD14, CD64 Monocyte lineage Monocytoid Leukemias CO2, CD3, CDS, CD7 T-lineage T-cell Neoplasms CD10 (CALLA), CD19, C022 B-lineage B-cell Neoplasms CD34 Stem Cells Stem Cells for Transplantation, Acute Leukemia CD71 Transferrin receptor Erythroleukemia C045 Common Leukocyte Antigen Found on all Leukocytes CD103 , CDllc, CD25 HCL Also seen in other Lymphoproliferative Disorders JAK 2 Mutation PV Also seen in other MPN  67 Lysosome and Lipid storage Disorders DISEASE ACCUMULATED LIPID LAB DIAGNOSIS Gaucher Glucocerebroside BM Macrophages with wrinkled or striated cytoplasm Niemann-Pick Sphingomyelin BM Macrophages with globular or foamy cytoplasm, Sea-blue Histiocytes REMEMBER! Tartrate-Resistant Apid ,,, Phosphatase'for Hairy Cell Leukemi.a lR.A.P. the Hairy Beast REMEMBER! "!!)= REMEMBE c!] c::; (? JAK2 WHO mutation Classification and of Acute P. vera Leukemia JAcK loves PQly Yera ,, Be able to identifv mature, immature, and abnormal RBCs and WBCs from graphic images. Correlate abnormal findi.ngs with appropriate disease states. 68 HEMATOLOGY SAMPLE QUESTIONS 1. The major iron storage compound is 6. A patient with a negative dithionite solubility A. Hemosiderin test has a band in the A region and a band in B Ferritin the S region on cellulose acetate hemoglobin C. Siderotic granules electrophoresis at pH 8.6. On citrate agar D. Transferrin there is only a band in the A region. Which of the following is compatible with these results? 2. How would the following resul1ts on a 32 year A. HhAS old adult female be interpreted? B. HbAE Hemoglobin - 9.0 gm/dl C. HbAD MCV - 74 fl D. Hh ACHarlem MCH - 27 pg 7. The failure of gronulocytes to develop past the MCHC - 30.0 g/dl "band" or two-lobed stage is characteristic of RDW - 19.0 % A. Bernard-Soulier syndrome Serum ferritin - 4 ng/ml (N=20-250 ng/ml) B. Chediak-Higashi syndrome Serum iron - 29 g/dl (N=70-200 g/dl) C. May-Hegglin anomaly TIBC - 590 g/dl (N=250-435 g/dl) D. Pelger-Huet anomaly % Saturation - s 8. A patient with on elevated WBC count with A. Anemia of chronic inflammation neutrophilia, a left shift, toxic granulation, B. Iron deficiency anemia vacuoles, dohle bodies and an increased LAP C. Thalassemia minor probably has which of the following? D. Siderohlastic anemia A. Acute myelogenous leukemia B. Chronic myelogenous leukemia 3. How would the following results on a 72 year C. Bacterial sepsis old adult female be interpreted? D. Viral sepsis Hemoglobin - 6 g/dl 9. The following results were obtained on an MCV - 114 fl automated CBC. MCH - 39 pg MCHC - 34 g/dl Hemoglobin -11.2 g/dl RDW-18.S % Hct - 27% Oval macrocytes on Wright stain RBC - 2.1 X 106/ul Reticulocyte count - 1.2% MCV - 128 fl Serum 812 - 55 pg/ml (N=200-1000 pg/ml) MCH - 53.3 pg Serum folate - 7 ng/ml (N=2-10 ng/ml) MCHC - 41.5 g/dL Anti-IF (intrinsic factor) antibodies - positive RDW - 19.0 % All results are flagged. The technologist found A. Folate deficiency B. Liver disease no evidence of clots in the sample. What C. Pernicious anemia should be done next? D. Reticulocytosis A. Ask for a redraw 4. Which of the following results from decreased B. Warm the sample to 37° C and synthesis of globin chains? rerun A. Beta-thalassemia C. report the results if controls are in B. Hemoglobin C disease range C. Hemoglobin M D. Rerun the sample and sign out if D. Sickle cell disease results match S. The normal M:E ratio for an adult is 10. Plasma cells evolve from which cell line? A. 1:1.5 A. Lymphocytic B. 3:1 B. Monocytic C. 5:1 C. Myelocytic D. 9:1 D. Megakaryocytic  69 11. In performing a manual white blood cell count, 14. A bone marrow differential performed on a 0.02 ml of blood was diluted with 1.98 ml of patient showed 20% blasts. Flow cytometry ammonium oxalate. An average of 50 cells studies demonstrated the blasts to be positive were counted using a Neubauer hemacytome- for CDlO, CDl9, CD22, and negative for CD13 and ter. What is the patient's white count? CD33. Which of the following diseases is most A. 5,000/µL compatible with these findings? B. 5,500/µL A. ALL C. 10,000/µL B. AML D. 11,000/µL C. CML D. CLL 12. A 4 ml EDTA tube was received in the laboratory containing approximately 1 ml of 15. Which of the following is diagnostic of acute whole blood. If performed on this sample, promyelocytic leukemia? which of the following manual laboratory tests A. t(9 ;22) is most likely to be affected? B. t(l5;17) A. Hemoglobin C. t(l6;16) B. Retie count D. t(8;21) C. Sed rate D. WBC count 16. Plasma cell (multiple) myeloma may be sus- pected if which of the following is seen on a 13. A peripheral blood smear stained with peripheral smear? Prussian blue demonstrates siderocytes. A. Basophilic stippling On a Wright stained smear, what would B. Bizarre blast cells be expected? C. Hyper segmented neutrophils A. Basophilic stippling D. Rouleaux B. Howell Jolly bodies C. Heinz bodies 17. Which parameter is most likely affected by D. Pappenheimer bodies lipemia? A. MCV B. WBC count C. Hemoglobin D. RBC count ANSWERS AND RATIONALE l. B 3. C Option A is a long-term water-insoluble iron storage compound hut not the major one. Oval macrocytes, decreased Bl2 and positive Hemosiderin can be found in found in IF antibodies are all indicators of pernicious macrophage lysosomal membranes and seen in anemia. Option A is incorrect because the folate bone marrow aspirates stained with Pmssian is normal. Option B is incorrect because the blue. Option C are iron inclusions found in red anti-IF antibodies would NOT be positive. Option D is incorrect because of the normal cells stained with Prussian Blue. Option D is the reticulocyte count and the additional abnormal transport protein specific for iron. data. 2. B 4. A An RDW greater than 14.5%, decreased All other options result from structural iron/increased TIBC and greatly decr eased abnormalities. ferritin (indicating no iron stores) support this diagnosis. Option A is incorrect because ferritin 5. B would NOT be decreased and the TIBC would be decreased. Option C is incorr ect because in The normal M:E ratio is between 3:1 and 4:1. thalassemia minor, the Fe and TIBC would 6. C probably be normal and the anemia would be less severe. Also, the RDW would proabably be Rb AS would give a positive solubility and in the normal range. Option D is incorrect would show a separate band in the S region on because the ferritin would be increased as would citrate agar. Rb E would give a negative the serum iron. solubility test but would migrate to the C position 70 on cellulose acetate. Hb C 1-farlem would give a 12. C positive solubihty test and would migrate to the C position on cellulose acetate. Underfilling results in excess EDTA causing the red cells to shrink. This would cause the sed 7. D rate to be falsely decreased since the smaller cells will settle out more slowly. The other values Option A is a platelet adhesion problem would probably not be affected. characterized by giant platelets. Option B is characterized by giant lysosomes in leukocytes. 13. D Option C is characterized by giant platelets and Doble bodies. Siderotic granules are composed of iron and on a Wright stained smear appear as 8. C Pappenheimer bodies within the red cell. They are frequently seen in sideroblastic anemia, Option A is incorrect because there would be alcoholism , thalassemia and some preleukemic a predominance of lymphocytes and an LAP states. Option A is composed of R A remnants would not be performed. Option B is incorrect and does not stain with prussian blue. It is because the LAP score would be decreased. associated with lead poisoning, thalassemia, and Option D would show a normal WBC count and hemolytic anemias. Option B is composed of LAP score with no dohle bodies, toxic DNA remnants and is associated with granulation or vacuoles seen. hyposplenism, pernicious anemia and thalassemia. Option C is denatured hemoglobin , 9. B is OT seen on a Wright stained smear and is These results violate the "Rule of Three" and associated with G6PD deficiency, exposure to are strongly suggestive of a cold agglutinin. oxidizing drugs, alpha thalassemia, and unstable Warming the sample to 37°C will usually cause hemoglobins. the agglutination to disperse. Option A would not 14. A be the first course of action. Options C and D are not acceptable because the results are flagged >20% blasts in the bone marrow is associated and indicate some type of interference or erro- with acute leukemias. CDlO, CD19 and CD22 neous result. are indicative of B- lineage ALL. Option B would be positive for CD13 and CD33. Options 10. A C and D would not have >20% blasts in bone Plasma cells evolve from B cells which are marrow. lymphocytes. 15. B 11. B The t(l5;17) translocation is diagnostic of The formula for calculating manual cell APL counts is as follows: 16. D # cells counted x _1_ x dilution factor tot vol Rouleaux due to increased plasma proteins (monoclonal immunoglobulin) may be seen in The dilution is 1:100. So the dilution factor is plasma cell myeloma. The serum viscosity is 100. volume factor can be eliminated by taking increased and the albumin:globulin ratio is 10% of the number of cells counted and adding decreased. Option A is seen in conditions this to the number of cells counted. associated with disturbed erythropoeisis. Option 50 X.1 = 5 Bis seen in leukemia states. Option C is seen in 50 + 5 = 55 pernicious anemia. So, 17. C 55 x 100 = 5,500/mm3 Options A,B and D are measured by the impedence principle and are not affected by lipemia. Hemoglobin is measured optically and lipemia will cause a false increased value.  71 COAGULATION by Daniel Haun Platelets The bottom line.. ? PRODUCTION After adhesion and aggregation a platelet plug 1. Produced from megakaryocytes is built at the injury site. The PFA test asks 2. Distribution the important question: a. 30% spleen Do the platelets properly adhere and b. 70% peripheral blood aggregate at the injury site? c. Reference range - 150,000- 400 ,000/mm3 2. Localization of the platelet plug d. Life span - 9-12 days a. Secreting platelets release arachidonic acid which converts to prostaglandin, (becomes Thrornhoxane A2) in the platelet b. Arachidonic acid is processed by adjacent endothelial cells to form platelet-inhibiting prostacyclin Platelet Precursor Cell The bottom line.. ? The platelet plug is limited to the injury site. Platelet Reference Ranges FUNCTIONS 3. Assembly and localization of the fibrin clot 1. Initial arrest of bleeding and formation a. Platelet release components include of the platelet plug fihrinogen, Factor V and Factor a. Adhesion VIII ❖ Glycoprotein lb binds to b. Fihrinogen is bound on the platelet exposed collagen surface during aggregation ❖ Requires von Willebrands factor c. Factor VIII is bound to the platelet ❖ Results in release (secretion) of surface with von Willebrand factor ADP and other granule d. Shape change exposes platelet components (including Factor V membrane phospholipid (PL); the and fibrinogen) template for the assembly of the b. Aggregation factor complexes ❖ Other platelets are stimulated b y ❖ Historically called Platelet Factor 3 ADP to undergo shape change ❖ Binds the Factor VIII and IXa (disc to sphere to pseudopods) complex (requires Ca++J - no exposing the glycoprotein Ilb I wonder Hemophilia A and Illa complex which binds Hemophilia B ( Factor VIll and fibrinogen (this is the complex IX deficiencies) are clinically that is blocked by a number of identical GP Ilb/IIIa inhibitor drugs) ❖ Binds the Factor V and Xa ❖ Fibrinogen binding links the complex; also requires Ca++ platelets; the first (and reversible) phase of aggregation ❖ With weak stimuli, the The bottom line.. ? aggregates can disassociate but strong stimuli cause the The platelet plug is a wonderful place to aggregating platelets to release produce a fibrin clot and without the platelet (secrete); with release, the presence, the fibrin won't form. It is clot aggregation is irreversible promotion and clot localization all in one. 72 3. Fibrinogen proteins a. Factors I, V , VIII , XIII b. Consumed during clotting (therefore not in serum) c. ,t- in acute phase (pregnancy and Steps in Formation of inflammation) Platelet Plug Plasma Coagulation Factors FUNCTIONS 1. Substrate - Factor I (fibrinogen) Characteristics of Coagulation Proteins 2. Cofactors - accelerate enzymatic reactions a. Factors III, V and VIII FACTOR NAMES b. Factor HMWK (high molecular weight kininogen ) 1. Noted by Roman numerals 3. Enzymes 2. Exceptions: a. Serine protea ses - cleave peptide a. Prekallik:rein bonds (Factors II, VII, IX, X , XI b. High molecular weight kininogen and XII) STABILIZED CROSSLINKED FIBRIN b. Transamidase - XIII only 1. Turn it on CHARACTERISTICS OF COAGULATION PROTEINS a. Activated intrinsically by the collagen ( via Factor XII) contact Coagulation Groups system b. Activated extrinsically by disrupted GROUP CONTACT PROTHROMBIN FIBRINOGEN endothelial cell membrane ( tissue fa ctor or tissue thromboplasti.n) Factors XI, XII, PK, 11, VII, IX, X i, V, VIII, XIII complex with Factor VII to directly HMWK activate F actor X Vitamin K No Yes No 2. Cofactors Dependent a. Factor VIII is bound with a ctivated Consumed in No No (except for II) Yes F actor IX b y calcium to the platelet Clotting phospholipid membrane (PL) b. Together these factors activate Factor X (in the comJnon pathway 1. Contact proteins Factor Vis the cofactor to Factor a. Factors XII , XI, P K (prekallikrein) and HMWK (high molecular weight X a in a similar arrangement with calcium and PL) kininogen) c. This prothrombinase complex b. Participate in the initial phase of converts prothrombin to the active the intrinsic system thrombin c. NOT consumed during clotting (found in both serum and plasma) 3. Thrombin d. NOT Vitamin K dependent a. Cleaves peptides off of the fibrinogen molecule to form fibrin 2. Prothrombin proteins which polymerizes to form insoluble a. Factors II, VII, IX, X fibrin strands b. Vitamin K dependent b. Thrombin also activates Factor XIII c. NOT con sumed during clotting which crosslinks the fibrin strands (ex cept II) at the " D" region (birth of the D- d. Present in fresh and stored plasma dimer) and serum  73 Coagulation Cascade Intrinsic Extrinsic (APTT) (PT) Collagen Tissue Factor HMWK Kallikrein t ca++ X11t ----- Xlla VII xr- Xia ~ FIBRINOLYTIC SYSTEM 1. Turn it on Monitors IX ~ ~ / Monitors Oral a. Activated intrinsically by collagen Heparin Therapy C ++ a j Anticoagulants (Coumarin) via the Factor XII / contact pathway that initiates intrinsic clotting or extrinsically by tissue plasm.inogen activator ( TPA)........... _ ':_ft P_"°_'_P,;,f; ______ pcmm~ f iliw Y... b. Activators convert the precursor x--► Xa plasminogen to plasmin ca++ V 2. What does it do? ca++ a. Plasmin cleaves fibrin strands to Pit. Phospholipid soluble fragments of fibrin (fibrin Prothrombin ca++ Thrombin - I XIII ca++ degradation products are X, Y, D orE) b. Can come from fibrin clot (fibrinolysis) or from unclotted Fibrinogen - - - ~Fibrin - --- -- - --Xllla fibrinogen (fibrinogenolysis) ! Stabilized Cross/inked Fibrin c. D-dimer comes from crosslinked clot (clo t specific) 3. Turn it off Other functions of thrombin a. TPA inactivated by tissue plasminogen activator inhibitor Feeds back to "potentiate" factors (TPAI) - stops activation V and VII I b. Active plasmin inhihited by Alpha-2 Recruits and aggregates platelets Plasmin Inhibitor if it escapes the area of the clot. This prevents Turns on endothelial cell thrombomoduliA fibrinogenolysis (receptor/activator for Protein C and Protein S system) to inactivate Factors V and VIII Excessive and inappropriate fibrinolysis 4. Turn it off Major feature of disseminated intravascular a. Heparan sulfate on the endothelial coagulation {DIC); response to excessive cell binds antithrombin (AT) which clotting inactivates the activated serine proteases (heparin works this way, Also seen in liver disease (activators too!) are not cleared and the inhibitors are b. Activated Protein C and its cofactor diminished) Protein S ( when bound to its receptur/1:1(.:Livutur tl1rumhuL11udulin) Complications of cancer or surgery inactivates Factors VIII and V of the prostate or urinary tract where urokinase can leak into the circulation 74 "Clotbusters" Routine Tests of ~ Streptokinase (commercial) Hemostatic Function 1_ Urokinase (commercial) PROTHROMBIN TIME (PT) 1. Screen for extrinsic & common ~ Tissue plasminogen activator (commercial pathways and in vivo) 2. Measures factors I , II, V, VII and X 3. Monitors oral anticoagulants (warfarin, coumarin, dicoumarol) 4. Reagent - tissue thromboplastin & Role of Thrombin, Pll18lDia CaC12 and Fibrin in Hemostasis- 5. Sen sitive to Vitamin K factors 6. International normalized ratio: INR Specimen Collection/Handling l. Sodium citrate 3.2% (Ca++ is necessary INR = (. patient result }ISi for both coagulation and platelet aggregation studies) \ mean of reference range (ISi = International Sensitivity Index from manufacturer) 2. Whole blood - anticoagulant ratio = 9: 1 7. Reference range = < 13 seconds 3. Use plastic tubes or siliconized a. Therapeutic goal: INR 2.0 - 3.5 glassware (glass activates factor XII ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) and platelets will adhere to glass). 1. Screen for intrinsi c & common 4. Coagulation samples can now be drawn pathways first when u sing evacuated tubes ( vacutainers). Historical r eports of 2. Measures all factors except VII and contamination with tissue XIII thromboplastin during collection a r e unfounded. 3. Monitors heparin therapy 5. Hemolyzed samples should NOT be 4. Reagents - activator (kaolin, celite or u sed for platelet aggregation studies ellagic acid), platelet phospholipid (PL) (red cells contain ADP). & CaC12 6. Lipemic samples may cause problems with coagulation and aggregation studies (may obscure changes in optical density). Anticoagulant Used for REMEMBER! Coagulation Studies Monitoring by PT and APTT APTT=2 T's Together Remind You of an "H"=Heparin Ratio of Anticoasu]ant : Blood PT=Coumarin· Vitamin KFactors  75 5. Reference range = 20 - 40 seconds a. Therapeutic goal: 1.5-2.5 times "normal" or u se Heparin Response Curve FIBRINOGEN ASSAY Correlate Conditions with 1. Quantitative measure of Factor I Bleeding Time Results 2. Reference range= 200-400 mg/ell 3. Anemia and hypofibrinogenemia t clot THROMBIN TIME (71) r etraction 1. Does NOT measure defects in intrinsic/ extrinsic pathways 4. Rapid dissolution of clot indicates ,t. fibrinolytic activity (example= DIC) 5. Glanzmann Thromhasthenia - no clot r etraction Special Tests of Hemostatic Function PLATELET AGGREGATION 2. Affected b y t fihrinogen levels and 1. Necessar y for platelets to stick to each presence of heparin and other anti- other thromhins 2. In vivo aggregating agents 3. Reference range =< 20 seconds a. ADP b. Collagen BLEEDING TIME c. Epinephrine 1. Historically measured platelet function d. Thromhin and numbers hut generally replaced by e. Serotonin PFA f. Arachidonic acid g. Ristocetin PLATELET FUNCTION ASSAY (PFA TEST) h. Snake venom 1. Measures platelet function with 1. Antigen-antibody complexes collagen, ADP and epinepherine J· Fibrin degradation products (FDPs) 2. Sensitive to aspirin, vWD , and ADP 3. In vitro aggregating agents receptor problems a. ADP b. Collagen 3. Replaces the bleeding time as a scr een c. Ristocetin d. Epinephrine CLOT RETRACTION e. Thromhin 1. Evaluates platelet function, fibrinogen , f. Arachidonic acid red cell volume and fibrinolytic activity 4. Measured with photo-optics 2. Abnormal if Elatelet count