TABLE OF CONTENTS About the Author Hematicons & Altus Sheet MTLE Table of Specifications ‘SAMPLE COLLECTION Blood Sample Collection ‘Additives in Collection Tubes Bone Marrow Sample Collection 10 HEMATOPOIESIS 12 Bone Marrow, Stem Cells, Progenitors and Growth Factors 13 RED BLOOD CELLS 16 Erythropoiesis 16 Structure, Function and Metabolism 17 Hemoglobin Synthesis 18 Hemoglobinopathies 21 RBC Anomalies 23 Anemia 27 LABORATORY METHODS FOR RBCs and WBCs 39 Reference Values 39 Manual Cell Counts 40 Hemoglobin Determination 42 Reticulocyte Count 43 Erythrocyte Sedimentation Rate 44 Examination of Hemogram 45 Automated Blood Cell Analysis 46 Bone Marrow Examination 49 WHITE BLOOD CELLS 52) Non-Malignant WBC Disorders 55 Cytochemical Stains 58 Myelodysplastic Syndrome 59 Acute Leukemias 61 Myeloproliferative Neoplasms 65 Chronic Lymphoid Neoplasms 67 HEMOSTASIS AND THROMBOSIS 70 Megakaryopoiesis and Platelets 71 Primary Hemostasis. 73 Secondary Hemostasis. 74 Hemorrhagic and Thrombotic Disorders 81 Laboratory Tests of Platelets 85 Laboratory Tests of Secondary Hemostasis. 87 ‘Answers to Checkpoints 91 References 92 Na RONSAnna-Lee Baguio Bandoy obtained her Bachelor in Medical Laboratory Science from the University of the Immaculate Conception as Magna Cum Laude. She is a recipient of the PAMET-PASMETH Academic Excellence Award and Scholarship on Tertiary Education Program under the Educational Benefit System Unit (EBSU) of the office of the City Mayor of Davao. She went on to finish her Master of Science in Medical Technology from San Pedro College with High Academic Distinction Award (Benemeritus). She is also an active researcher and research mentor and received regional, national and international recognition for her works about antibiotic resistance profile of gram-negative bacilli from ants in selected hospitals. Currently, she is a registered Medical Technologist working as a Clinical Instructor and Asst. Professor Ill in undergraduate | courses namely Biochemistry, Endocrinology and Toxicology, Microbiology and Hematology | and graduate program course Advanced Clinical Chemistry at University of the Immaculate { Conception. 4 She also serves as a review lecturer in Medical | Technology Board Exam Courses and coach for the UIC National PAMET-PASMETH Quiz Show \ | team. \ \ \ The digital art shown is a visualization of her dream to become a physician-scientist. “Committ the Lord whatever you do, ane your plans willsucceed.” = Proverks 16:3 Widurus co. Page |1THE HEMATICONS ASSESSMENT rs sctencxtins stone oases icon ears ober MASTER! FUNDAMENTAL iN. : ICON i eee CRITICAL ICON _ Mese cons wl serve 3 your gue NOTICE: tye nay seen opter inane nic -@> _THE ALTUS SHEET ‘Alus Sheet is 2 review tool atached to each Altus Co. Review Notes. This sheet will help the user improve their memory skis. The review notes have Y£LL0¥ eolor fonts which indicate KEYNOTES that the author wants the USER to memorize by heart, Placing the Altus sheet on top of the designated page will make the yellow-colored font keynotes invisible, allowing the USER to assess and challenge himself to remember all the missing KEYNOTES from the topic. ont mason euuae Houten get 28a Sa ah Yo AOR Wa pagel naam wu cae yen pk Ley WA INTENSIFY your brainpower now with ALTUS SHEET! MiAurus co. Page |2ey MTLE Table of Specification — 1 Quality Assurance 5% 2 Blood Collection, anticoagulants, safety, etc. 5% 3° Hematology tests and procedures 30% 2.1 Routine Tests 15% 2.2 Automation 10% 2.4 Advance and special techniques 5% 4 Hematopoiesis 40% 3.1 Hematopoiesis (Overall process) 6% 3.2 Erythropoiesis and RBCs 12% 3.3 Leukopoiesis and WBCs 12% 3.4 Thrombopoiesis and platelets 10% Coagulation and Fibrinolysis 20% 5.1 Hemostasis 2% 5.2 Coagulation and Fibrinolysis tests and procedures 8% 5.3 Coagulation factors, diseases and reference values 10% TOTAL 100% Uidurus co. Page|3Ne Sample Collection if @ w Blood Sample Collection paolo Skin Puncture ~ technique of choice for newborn, infant, pediatric patients, geriatric patients, adults with bums or reserved vein for therapy or patients with thrombotic ‘tendencies and intense fear of needles. ~ sample obtained is a mixture of blood from venules, arterioles capillaries, and interstitial and intracellular fluids Equipment 41. Lancet or blade spring loaded in the puncture device 2. Container: a. Capillary tubes (red band = wiheparin; blue band = no anticoagulant) b. Microcollection tubes “bullets” a Minimum: filt 250pL Maximum fil: SOOpL Collection Site a, Lateral (outside) or medial (inside) plantar surface of the heel - INFANTS b. Third or fourth finger in the nondominant hand ->4 and adults Precautions: Puncture SHOULD NOT be mors than 2mm deep Wipe away the first drop to prevent contamination of tissue fluid and to facilitate free flow of blood. NOTE: * Puncture on the finger should be PERPENDICULAR to the fingerprint lines. * Fingers of infants should not be punctured due to risks for bone injury. © Warming of site increases the blood flow 7x (not greater than 42°C for 3- Smins) * DO NOT USE POVIDONE-IODINE: causes FALSE INCREASE in bilirubin, uric acid, potassium and phosphorus (BUrPP) Order of Draw for Skin Puncture 1. Tube for gas analysis 2. Slides, unless made from specimen in the EDTA microcollection tube, ; 3. EDTA microcollection tube G 4, Other microcoltection tubes with anticoagulants 5. Serum microcollection tubes Note: Wipe away the first drop of blood to remove residual alcohol and tissue fluid contamination. us co. Page|4Venipuncture Equipment a. Vein-locating devices - Use of portable transillur its walls) ~ Typically shine high-intensity LED or infrared red light to highlight veins Principle: Hemoglobin in blood within the veins absorbs light, causing veins to stand out as dark lines. Example: Venoscope Il, Neonatal Transilluminator tion (inspect an organ by passing fight through b. Toumiquet ~ used to provide a barrier against venous blood flow to help locate a vein ~ can be a blood pressure cuff (<40mm Hg), Velcro strap or elastic strap or latex free tourniquet ~ applied 3 - 4 inches above the venipuncture site no longer than 1 minute. c. Needles c-1, Multi-sample needles (Evacuated Tube System) ~ preferred by CLSI ~ allows numerous tubes to be collected in a single venipuncture ~ Components: (1) Muli-sample needle, (2) Tube holder “single use only (3) evacuated tubes *SESIP (Sharp with Engineered Sharps Injury Protection) ~a needle or tube with safety device £.2, Hypodermic needles (Syringe System) ~ used for patients with small or difficult veins ~ Length of needle: 4 to 1.5 inches = Bevel angle: <30 degrees GAUGES: a needle’s diameter in inversely proportional to the gauge number “the higher the gauge number, the smaller the actual diameter of the needle” Number [Color | Number Color 16 White 23 Blue 13 | Pink 24 | Lavender/Purple 9 | Cream 25 | Orange G Yellow, 26 Brown 1 Green 27 Grey Black ©.3. Winged Infusion (Butterfly) Needles = used for collecting small and difficult veins ~ allows more flexibility and precision than needle and syringe = components: (1) %to % inch stainless steel needle permanently connected to (2) § to 12 inches tubing with Luer attachment for syringe or multi- sample luer adapter Order of Draw for Venipuncture 1. Blood culture tube (yellow stopper) 2. Coagulation tube (light blue stopper) 3. Serum tube or without activator (red, gold stopper) : enone 6@ 5, EDTA tube (lavender or pink stopper) 6. Sodium fluoride with or without EDTA or oxalate (gray stopper) Tus co. Page|§Sites for Venipuncture (a) Median cubital vein (2) Cephalic vein (3) Basilic vein If not possible, alternate sites include: (1) Ventral forearm (8) Back of the hand (2) Wrist area (4) Ankle or foot Procedure: Not aeNe geenen 1". 12. 13. 14. 15. 16. 17. Reassure the pal Position the pi . Perform venipuncture by anchoring the vein with the thumb 1 to 2 inches below the site Greet and identify the patient (check patient identifiers) Sanitize hands, Verify and note patient isolation restrictions and diet restrictions ‘Assemble supplies and appropriate tubes for the requested tests. Verify paperwork and. tube selection. Reassure and position the patient Apply tourniquet and select appropriate venipuncture site (Priority: median vein) Put on gloves. Cleanse the puncture site Inspect the equipment and needle tip and inserting the needle, = Needle angle: 30 degrees; if donor bleeding 45 degrees then reduce to 10 ~ 20 degrees (skin to vein) Release tourniquet Position gauze over puncture site Remove needle and apply pressure If syringe is used, fil the tubes Discard the needles Label the specimen: Patient's full name, ID number, Date and time of collection, collector's initials Transport specimen promptly and properly nt: Crying causes increase in cell count i: Lying down - hemodilution; decrease in PCV by 8% and decrease in WBC. Up - Hemoconcentration increased PCV and WBC If site is with IV line: (1) Select the other arm; (2) Or STOP the IV for 2 mins, Draw blood below the line, Discard the first SmL. Notify the physician Reasons for Specimen Rejection PNOMRENs Test order requis ion and the tube identification do not match Tube is unlabeled, or the labeling, including the patient identifiers are incorrect. Specimen is hemolyzed. Specimen was collected at the wrong time. Specimen was collected in the wrong tube. Specimen is clotted, and the test requires whole blood or plasma. Specimen was contaminated with intravenous fluid. Specimen is lipemic, we Qaerus co. Page|6. 4 Sample Collection g fi Additives in Collection Tubes a A, Clot Activators ~ increases clotting process to occur. - blood specimen for serum testing must be allowed to clot for 30-60mins prior to centrifugation or removal of serum ~ example: glass or silica particles and thrombin B. Anti-glycolytic Agent ~ inhibits metabolism of glucose by blood cells . example: Sodium fluoride ees * Separator Gel ~ serves as a barrier between serumiplasma and cells eid Note: Tubes containing sodium fluoride ONLY yield SERUM while tubes containing sodium | 2 ee oan ne fluoride plus anticoagulant yield PLASMA bod ce (1.09 ina form of bai ter cerrtugation C. Anticoagulants - prevents blood from clotting tubes with anticoagulants must be gently inverted IMMEDIATELY after collection Most common Anticoagulants and their actions a. EDTA (Ethylenediaminetetraacetic acid) ~ 8x inversion: 1.8mg/mL of blood ~ optimum cane. - best preserves cell morphology: anticoagulant of choice for hematology cells counts and morphology Platelet Satellitism ~ Platelet adhere around the ~ action: chelates calcium neutrophil forming = forms: Powdered di-potassium, Ky (Versene) a Liquid ti potassium, Ko (Sequestrene) = May oecur using - blood smear the sample within 2hrs. of collection EDTA - Preferred anticoagulants for platelet a. If for platelet count: EDTA b. If for platelet aggregation: CITRATE - NOT FOR COAGULATION TESTS = it inhibits fibrinogen — thrombin interaction Correction: REPEAT THE COLLECTION using sodium citrate anticoagulant. Obtain. the platelet count by ‘multiplying the count With factor: 1.4 b. CITRATE - 3 - 4x inversion = binds calcium in SOLUBLE complex = most common form: sodium citrate (also used in ESR with black stoppers) - used for coagulation studies (light blue top) Concentration: 3.2% or 0.109 Ratio: 9:1 blood to anticoagulant ratio (coagulation studies) 4:1 blood to anticoagulant ratio (Standard Westergren: black top) ©. OXALATE ~ 8 - 10x inversion ~ binds calcium to form INSOLUBLE calcium oxalate. Potassium oxalate (Paul-Hellers): shrinks cell- most widely used ~ Ammonium oxalate (Wintrobe's): swells cell crus co. Page|?d, HEPARIN ~ 8X inversion Formulation: Ammonium, Lithium and Sodium Heparin Optimum concentration: 15 (0 20 Uiml of blood ~ inhibits THROMBIN; natural anticoagulant acid mucopolysaccharide that inhibits coagulation by inactivation of thrombin ~ used for minimal hemolysis of red blood cells ~ used in chemistry STAT tests (e.9. electrolyte tests) + anticoagulant of choice for osmotic fragility tests or samples for defibrinated blood - NOT FOR BLOOD FILM PREPARATION. a. distorts WBC and platelet morphology b. produces Bluish background - NOT FOR COAGULATION ~ inhibits all stages of coagulation Order of Draw, Stopper Colors and Rationale for Collection Order Order of Draw Tube Stopper Color Rationale for Collection Order Blood cultures Yellow SPS Minimizes chance of microbial contamination (sterile collections) Sterile media bottles Coagulation tubes Light blue The first additive in the tube in the order because all other additive tubes affect coagulation tests Glass nonadditive tubes Red Prevents contamination by other additives in other tubes Plastic clot activator ‘Red tubes Filled after coagulation tests because silica particles, activate clotting and affect coagulation tests (carryover of silica into subsequent tubes can be overridden by Serum separator tubes Red and gray rubber anticoagulant in them). (SsTs) Gold plastic Plasma-separator tubes Green and gray Heparin affects coagulation tests and interferes in (PSTs) rubber collection of serum specimens; it causes the least Heparin tubes Light-green plastic interference in tests other than coagulation tests Green EDTA tubes Lavender, pink, or Responsible for more carryover problems than any other Plasma preparation purple additive: elevates Na and K levels, chelates and decreases tubes calcium and iron levels, elevates PT and PTT results Oxalateffiuoride tubes Gray Sodium fluoride and potassium oxalate affect sodium and potassium levels, respectively. Filled after hematology tubes because oxalate damages cell membranes and Causes abnormal RBC morphology. Also, oxalate interferes in enzyme reactions, Qidurus co. Page| 8Anticoagulant Tube Guide @ BD Vecutziner Venous Blood Collection gg @ ‘Stopper Additive Tnversions Laboratory Use Mechanism of Color Action GOLD ‘Clot activator 3 a forserum determination in chemistry Sica clot activator ‘and ge! for serum separations used for routine blood danar screening and agnostic testing of serum for ifectous ciseases Noe: ube inversions ensues mixing of clot acivator blood eltng time: 29 mins LIGHT Lithium Heparin = 8 «for plasma determination in chemisty Inhibits formation of GREEN and Gel thrombin RED 3 Siicone Coated (Glss} Da for serum determination in chemistry ‘Silica clot activator Clot activator, scone 5 bse for routine blood donor screening and coated (plastic) diagnostic testing of serum for infectious diseases Note: tube inversions ensures mixing of clat activator blood eating time: 60 mins ‘ORANGE Thrombn-basoa 7 Forse sum dtominaion w oars Git aivaor cbt atvator ood cloting ie: Eins ROYAL BLUE. Cit acvalor (Sse seu) oa fortace-iomens, toxcoony Teparin inhibits ‘b.K2 EDTA (plastic) 8 and nutritional chemistry detreminations thrombin Fmulion a2 EDTALines calcium _ GREEN» Sodio Hepar a fr plana determination nchonisry inhib formation of Lio Heparn a thrombin GRAY 2. Potassium Oe] © for gucose deamination Te Oslo and sodium fuorde eWTAmieoaguas b. Sodium tuordets2 EDTA. wil plasma ¢. Sodium thor (rum : sanples, oo Sodium odes the antigheote agent TAN REEDTA sy Ta forked detminaions Tis ube i cei to contin ss than tpg (oom) ea YELLOW 2. Sodium pohanaal Ta SPS for Bod eure specimen colesion na A-conplement sulfonate (SPS) miroblony and an-phagooyic 8. Ao Gil Detrose (ACD) g_—_— ACD for usin blood bank ties, HLA RBC preserve pheneyrng and DNA an pater sing. TAVENDER @.Ligid KUEDTA oss) 3 a KREDTA and EDTA Tor whole Dood Chel calc hematology determinaion b, Sray-coted K2 EDTA 8. K2EDTA‘or tine immunchematlogy and (oat) iao@ donor steering WHITE K2EDTAand 7 Formaleulr dagnosi tea meio gel or plasma separation 4 z — PINK Spraycnted KZ EDTA(psie) 0 For oaine mmanohenalalogy Cheba cam and blod donor screening TIGHT BLUE — Bufered Sou Ciate Ttod For ecaquaion determination 2.01064 (3.24) glass os 8.01004 (2.2%) past CLEAR BLUE Gira, theophyine Sioa Forpldee incon ess . Chelaes ac adenosine and cpydamcle and routine coagulation determination (cTAD) (CLEAR RED NONE (pay 0 Forvse as discard tbe or secondary specimen tube Sates co. pagela_s Sample Collection Bone Marrow Sample Collection a Bone marrow — produces 6 billion cells per kg per day : ~ at early stage BM is primarily composed of red marrow (active cell production) ~at 7 year, adipocytes (yellow marrow replaces the red marrow in the long bones) Indication of Bone marrow examination: 1. Neoplasia diagnosis and staging 4, Infections 2. Marrow failure and cytopenia 5, Monitoring of treatment 3. Metabolic disorders CORE BIOPSY (Trephine Biopsy) io aie eee = demonstrates bone marrow architecture (spatial relationship of uaa hematologic cells to fat, connective issue and bone stroma) a ‘ta coor = used to estimate cellularity ~~ = used to evaluate diseases that produce focal lesions e.g. Hodgkins lymphoma, multiple myeloma, metastatic tumor ore iopsy = needle: Jamshidi biopsy needle, Westerman-Jensen Needle sete wi or Sara a ‘ay ducing mses ASPIRATION needle: gauge 14 to 18 (University of Ilinois Aspiration Needle) * volume: 1 mL to 1.5 mL (collection of > 1.5 mL may indicate peripheral blood dilution) BONE MARROW COLLECTION SITES 1. Posterior superar lise crest (spine) of the pelvis ~ safest ***(for core and aspiration) 2. Anterior superior iliac crest of the pelvis 3. Sternum — for aspiration only 4, Anterior medial surface of the tibia - for children <2 yrs. old: aspiration only 5. Spinous process of the vertebrae, ribs, etc. BONE MARROW SPECIMENS. a. Direct aspiration smears = use 6 ~ Bx ethanol washed slides ~ perform wedge smear (similar to peripheral blood smear) b, Anticoagulated Aspirate Smears transfer BM aspirate to vials with Ks EDTA . Crush smears ~ gently press to crush spicules = use ethanol washed slides 4. Imprints (Touch Preparations) -valvable when specimen has clotted or DRY TAP ~ core biopsy specimen and clotted marrow ma washed glass or coverslip ©. Concentrate (Bufty Coat) Smears ~ useful when there is decreased nucleated cells in direct marrow cells ~ place 1.5mL of KxEDTA anticoagulated marrow s + centrifuge at 2500g for 10 mins. 1. Histologic sections (Cell black) yybe hel in forceps and repeatedly touched to a (eg. Aplastic Anemia) specimen to a narrow-bored glass or plastic tube LTUS co. Page| 10Checkpoint Line 1 Topic: Sample Collection Part. Multiple Choice. Encirce the best answer. 1. What blood collection technique is indicated for burn unit patients? a Venipuncture ©. Ear puncture b, Skin puncture «blood collection is prohibited 2, The tourniquet should be placed _ above the venipuncture site a.3-4om c..3- 4 inches b.75- 100m 4. 75 10inches 3. Wha isthe preferred needle to be used for venipunctura according to CLSI? 2, syringe system . multiple-samples needle system b. winged infusion needles 4. hypodermic needles 4, What is the stopper color indicated for molecular diagnostics test? a. pink ©. royal blue b. orange white 5. What is the molar concentration of crate used for coagulation studies? 332M 32M . 109M 4. 1.09M 6. What methad is also known as core biopsy? a Touch preparation method ¢. Trephine Biopsy ». Crush biopsy method 6. Cel block 7. The physician tries to rule-n leukemia for his 40-yr.old patient, the best sample for this is bone marrow aspirate, Whereis the best site for sample coletion? a. Slemum c, Anterior superior iliac crest of the pelvis 'b, Posterior superior iliac crest of the pelvis d, vertebrae 8, Platelet salellitism may occur in peripheral blood smears prepared with EDTA, what additive should be used instead? a. Heparin cc. Lithium oxalate b, Sodium citrate d. Versene 8. What anticoagulant causes the formation of bluish background when used for peripheral blood smear preparation? a. Heparin 6. Lithium oxalate b. Sodium citrate d. Versene 410, A blood sample was collected from a patient from 10-hour fasting. Foreseeing delay in processing, the medical technologist immediately placed the blood in a tube with sodium fluoride additive, What type of sample was yield from the preparation? a, Plasma , Serum ', Whole blood 4, Both A and B Part Il Enumeration 1, List the order of draw for skin puncture technique. 2. List the order of draw for venipuncture technique. 3, Enumerate § bone marrow specimen preparation techniques. actus co. Page| 11» -4 Hematopoiesis Ras Hematopoietic Stages = Hematopoiesis ~2 regulated process of blood cell production includes cellular renewal, formation, proliferation, differentiation and maturation ~ a HEMATOPOIETIC STEM CELL is capable of self-renewal and directed differentiation into all required cell ineage, its system consists of bone marrow, liver, spleen lymph nodes and thymus. | Locations: Yok sac to (AGM) region [ mesoblestic phase] >> fetal ver [hepatic phase] >> bone marrow [medullary phase] vei-gnabomsare Stages: (1) hiesoblastic stage - embryo (2) Hepatic stage - fetal 3) Medullary (Wyeloid) Phase ~birth through adult 1, MESOBLASTIC STAGE = Begin at the 19" day of embryonic development after fertilization; ~ blood islands remain active for 8 to 12 weeks = hematopoietic activity is limited to RBC production - Products produced: HSCs, primitive erythroblasts and embryonic hemoglobin: (Gower 1, Gower 2 and Portland) 2. HEPATIC PHASE ~ begins at 5 to 7 gestational weeks ~ characterized by recognizable clusters of developing erythroblasts, granulocytes and monocytes ~ 3" month, yolk sac discontinues its role, fetal liver becomes active (RBC and WBC production) by the end of 4" month, primitive cells disappear with an increase in more definitive erythroblasts, granulocytes and megakaryocytes ~ other active sites: spleen, thymus and lymph nodes = hemoglobin production: Hemoglobin F, Hemoglobin Ay and Hemoglobin Az ~ bone marrow becomes active around the fifth month of gestation 3, MEDULLARY (MYELOID) PHASE ~ Bone marrow is the primary site = hematopoiesis occurs in most bones but primarily in the flat bones of sternum, ribs, vertebrae, skull and pelvis ~ In adults, the principal source of production is STERNUM, and other flat bones. - Thymus activity decreases after childhood ~ babies and children have more red marrow activity, whereas adults have equal composition of red and yellow marrow LTUs co. Page| 124 Hematopoiesis ~~ Bone Marrow, Stem Cells, Progenitors and Growth Factors Basic Terms and Cell Functions Stem Cell ~ capable of self-renewal and differentiation into mult-lineage cell + Embryonic Stem Cell ~ pluripotent; they can generate all tissues in the body Hematopoietic Stem Cells (1'SC) — renew and differentiate into committed hematopoietic progenitors Retrogression — replacement of red marrow (active marrow) by adipocytes to yellow marrow ‘Mesenchymal cells ~ embryonic tissue that differentiates into structural elements, e.g. stromal cells Stromal cells ~ originate from mesenchymal cells; they are connective tissue cells of any organ @.g. fibroblasts endothelial cells, adipocytes, osteoblasts, etc. Roticular Adventitial Cells (Fibroblast) ~ forms a support lattice for developing hematopoietic cells Adipocytes ~ regulates the volume of the marrow; also secretes cytokines for hematopoietic regulation Osteoblast ~ bone-forming cells Osteoclast ~ bone-resorbing cells pies BONE MARROW CELLULARITY - Bone marrow can produce 2.5 billion RBC, 2.5 billion platelets and 1 billion WBC /Kg/Body Weight/day = Hematopoietic Stem Cell (HSC) in BM ratio: 1:1000 - percentage of marrow space occupied by hematopoietic cells compared with fat = normocellular marrow for adult: a. Fat (yellow marrow): 10 ~ 50% - composed of adipocytes (fat cells), undifferentiated mesenchymal cells, and macrophages b. hematopoietic cells (red marrow): 40 ~ 60% (average: 50%) ~ consist of developing blood cells and their progenitors child <2yrs old has 100% red marrow Types of Human Stem Celts a. Totipotential Stem Cell = most versatile type of stem cell and can develop into any human cell ype b. Pluripotential Stem Cell ~ can develop to any cell type except into fetus c. Multipotential Stem Cell ~ derived from pluripotent stem cells = mature into limited specific cell types to form tissues Theories of Hematopoietic Progenitor Origin a, Nonophyletic Theory (widely accepted) all blood cells are derived from a single progenitor stem cell called pluripotent hematopoietic stem cell b, Polyphyletic Theory ~ suggests that each blood cell ineages is derived from its own unique stem cell SiAurus co. Page| 13Diagram of Hematopoiesis The diagram of homatopoiesis shows derivation of calls from the multipotent stem cells. Based rom: Keotan, EM, Sh, L J, & Walega, JM. 2018), Rod's Hemaloy Cina Prneple and Appsons. lever Sunder. @ Did you know Nuiton in CDRS and CxCRE hive een sorted to HW Lineage Specific Markers, Myeloid 33, cbith, con Engthoid O71, Glyeophorin A (GPA) Megakaryooje cai, C05 e-Lym coi0, cove Lymohoid C03. 607 (CFU.GEMM (undifferentiated) Cos, com ‘Committed Myebid Progenitor C053, co3E ‘Committed Lymphoid Progenitor | co10, cose Early-Acting Multilineage Growth Factors KIT Ligand (Stom Cell Factor) binds to KIT receptor in HSC; | restate. CORS 2 XC ae Ge SEM ew oh BE rato ten, HW cane inet jecteases in level during differentiation binding triggers cel proliferation; stimulates growth viability and adhesion FLT-3 Ligand (FL) OXCL2 (SDF-1) Other Growth Factors ‘Stimulates primitive progenitor cali; often synergistic with SCF | @ chemokine expressed by BM stromal celis and microvascular endothelial colls ‘CXCL12-CXCR4 chemokine signaling is involve in HSC engraftment and maintenance "Noe: Hamstopoitc Stam Cals (HSC) expresses CXCRA, the receptor of CXCL12 Cytokine | Primary Cell Source Action Target Po Kidney (pentubular | stimulates prolferaion of erythroid progenitor Bone marrow enhroid progenitor interstitial els) (BFU-E and CFU-E) G-CSF Endothelial cls, stimulate granuloye colonies ‘Neutrophil precursors placenta, monocjtes | stimulate progenitors to neutrophil ineage Fibroblasts GM-CSF | Firobests, Tels, — | promotes antigen presenation and T cel homesslass | BM progenior call, macrophages, macrophages Hematopoietic ell growth faclor dendric cls, NKT calls IL-3 ‘Actveled T cells proliferation of hematopoietic progenitors Hemalopoiatc stem cells and NK cols progenitors 110 (C04, Th2T calls, | Inhibits cytokine production Teel 08" cals, Inhibits macrophages Macrophages macrophages TFN-a Dendtic cells, NK | Aniiral Wacrophages cells, Tand B cells | anhances MHC expression NK cells ay, WG (Lrus co. Page| 14Checkpoint Line 2 Topic: Hematopoiesis ‘Muttiple Choice. Encirle the best answer, 1. Allo the following are true for hepatic phase hematopoiesis, except: 2. begins al 5®— 7" week of gestation ', spleen and thymus are ative sites for hematopoiesis ©. products produced are the embryonic hemoglobin 4. by he end of 4" month, primitive cells disappear with an increase in more definitive cell stages 2. What organ is responsible forthe production of erythropoietin? a. Bone marrow «Kidney ». Spleen d. Liver 3. Which ofthe following are the lineage specific markers ofthe erythroid progenitors? a. CD10, CD38 ©, CDIT, GPA b. GPA, CD71 d. C033, CD11 4. Which of the following are the lineage specific markers ofthe megakaryocyte progenitors? a. CD47, C038 . CD17, GPA b. GPA, CD71 4. CD61, C41 Which of the following statements fis the biological action of I-10? a. promotes antigen presentation and T cell homeostasis b. proliferation of hematopoietic progenitors ¢. Inhibits eytokine production d, enhances MHC expression 6. Which of the following statements fis the biological ation of interferon-alpha? a. promotes antigen presentation and T cell homeostasis b. proliferation of hematopoietic progenitors 6. Inhibits cytokine production d. enhances MHC expression 7. This chemokine binds to CXCRA to create a signal for hematopoietic engraftment and maintenance, a. SOFA ¢. FLT-3 Ligand b. KT-Ligand 4. CORS Ligand 8. Whats the Hematopoietic Stem Cell (HSC) in BM ratio? 21:10 ©. 11000 b. 10001 ¢. 410000, 9. The mutation of is associated with the occurrence of undifferentiated cells inthe peripheral blood, a, SDF ©. FLT-3 Ligand b. KiT-Ligand 4. Allof the above 10. The hematopoietic diagram in page #15 is derived from what hematopoietic origin theory? 2. Monophiyletc Theory ¢. Totpotential Stem Cell Theory ». Polyphiyetic Theory None of the above ttus co. Page| 15mJ Red Blood Calls “ Erythropoiesis Erythropoiesis = process by which erythroid precursor cells differentiate to become mature RBC = typically occurs in erythroid islands (macrophages surrounded by RBC precursors) = the primary regulator is &YTHROP OIFTIW (produced by the kidney cells) ~ takes approximately 3-5:ioys (pronormoblast to reticulocyte) ~ the reticulocytes remain in the BM for # (o 2 days before its release in the circulation. in the peripheral blood, the reticulocyte matures after 4 day. ~1 Rubriblast = 16 RBC Erythrokinetics Fe: es toe? ~ describes the dynamics of red blood cell production and destruction Eth iands ae macoshoes Hypoxia that te surauded by RBC Precusors. Macrophages were efeved to prove ion fr the RBC precursors, “sstbng i ‘honomeren However, developing RBC cian ign wa Vnsfern, Ths, no need (or rest conc Toy, macephages ae known to emacs elke stimusion and seme as car anchor for serves as the stimulus for red blood cell production its sensing system is located at the peritubular fibroblasts of the kidney. Functions of Erythropoietin a. Promotes early release of reticulocyte from the bone marrow b. Prevents apoptotic cell death (reduces Fas receptor “death receptor") ©. Reduces maturation time (via reduction of cell cycle) elon Rs. ‘SUMMARY OF STAGE MORPHOLOGY Cell or Stage Thnator | WS mocked Remarks Wormoblastie Tubrbacte Bnfroblaste Pronormoblast _Rubriblast__Proertirablast | 12200m | 81 | 1-2 —| Deeply basophile ofoplasm No granules; Fine chromatin Capabie of mitosis; increase iron uplake and protoporphyrin synthesis Entiest recognizable precursor Basophili Prorubrieyte Basophilic Tsim [G1 [C1 [intensely basophilic eoplsm ‘normoblast ‘engtroblast Chromatin sightly coarse Nuceal are usually not visible Capable of mitosis With detectable level of hemoglobin Polychromatephilic ioe Pokehromaophiic | T0-Tam [ET | —o Fue ray to pink cytoplasm Normoblast enythrobast Remarkable lave of hemoglobin present Chromatin is increasingly clumped Las slage capable of mitosis “Onthochromie TWetarubrieyie— Orhochromic Boum | 420 | Salmon pink cytoplasm (acidophii Normablast enfhroblat Small pyknoie nucleus Hote: Pyrenocyte— enveloped extruded nucleus ofthe RBC Polychrommtophilic Polychromato Polyehromatophilic | @10ym | No 0 | ** called reticulocyte anftrrocya philic ‘rythrocyte sucess Bink to stightly pinkish gray erythrocyte sytoplasm contains basophilic reticulum of RNA, visualized via SUPRAVITAL STAIN Eyiirooye Tnhrocye Enfthrocyte aim Salmon pink in color ‘on-nisclealed, round, biconcave \idurus co. Page | 16__4_Red Blood Calls Structure, Function and Metabolism . RBC MEMBRANE ~ the biconcave shape of the RBC is crucial to its function ~ allows close to maximum surface-to-volume ratio and optimal gaseous exchange Main physiologic functions: a, Maintain cell structure and deformability b. Maintain osmotic balance between plasma and cell cytoplasm ©. Acts as supporting skeletal system for surface antigens and receptors d. Aid in the transportation of essential cellular ions and gases Components: ‘A. Carbohydrate (8%) B. Protein (52%) © Integral protein: Glycophorin A and Component A © Peripheral protein: Spectrin and actin ©. Lipid (40%) © Externat: phospholipid, phosphatidylcholine, phosphatidylcholine, glycolipid and sphingomyelin ‘* Internal phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine ‘Note: the cholesterol content ofthe membrane depends ‘upon the concentration of plasma cholesterol, bile acids and the actly of lecithin cholesterol acyltransferase (car Metabolism of Red Blood Cells - mature RBCs have no mitochondria. Thus, they rely on different metabolic pathways for functional maintenance 1. Embden-Meyerhof Pathway (MAJOR PATHWAY) ~ requires energy source (from glucose: 90% of glycolysis: ANAEROBIC) = maintains cation gradients (potassium IN: sodium OUT) = maintain the RBC membrane flexibility - ** affected with Pyruvate kinase deficiency 2. Hexose Monophosphate Shunt (Pentose Phosphate Pathway) = 10% glycolysis (AEROBIC) - gives protection to RBC from oxidant damage by the production of REDUCED GLUTATHIONE - prevents denaturation of hemoglobin =" affected with Glucose-6-phosphate dehydragenase (GBPD) deficiency: SOLE SOURCE OF NADPH 3. Rapoport - Luebering Pathway = regulates oxygen delivery to tissues by the production of 2,3 bisphosphoglycerate (2,3 BPG) = Increased 2,3 BPG, decreased 02 affinity - decreased 2,3 BPG, increased 02 affinity affected with Pliosphoructokinase 4, Methemoglobin Reductase Pathway = maintains IRON in FERROUS FORM (Functional form) for hemoglobin - to limit the production of Methemoglobin = *** affected with NADH cytochrome BS reductase Q@urus co. Page| 17»s_-4_Red Blood Cells Y Hemoglobin Synthesis HEMOGLOBIN STRUCTURE B® itis composed of 4 subunits, each containing heme and globin its main fun is to transport oxygen from the lungs to tissue and carbon dioxide from tissue to lungs ~ hemoglobin in RBC: 34g/dL; molecular weight: 64,000 daltons; 1.34 mL oxygen! gram of hemoglobin ~ contributes in acid-base balance by binding and releasing H+ and transports nitric oxide (a vasodilator) HEME SYNTHESIS = consists of a ting of carbon, hydrogen and nitrogen atoms called PROTOPORMYRIN IX, with a central Fe2 ~ the synthesis begins in MITOCHONDRIA with the formation of D-ALA from glycine and succinyl coenzyme A Ghycine + Succinycon “SAZMHHE eS Protoporphyrinogen IX Protporetyrinogen | Protoporptyrin 1x Ferocneatase «Fed Heme The diagram of hempobin synthesis. GLOBIN CHAINS AND ITS SYNTHESIS - synthesis occurs in the RIBOSOMES ®° 0. Amiooievtini ai Oytosot I ALraenydenase Porphobitinogen (P8G) 86 desinase Hydroxymetnyibilane | Uroporpyrinagen Msyetiace Uroporpiyrinagen I Ussporyhynogen ‘secamayace Copropoepinyinonen Mi HEME + Globin Chains 1 HEMOGLOBIN - consist of 4 polypeptide chains; globin chains determine the type of hemoglobin present DEVELOPMENTAL EXPRESSION OF HEMOGLOBIN ‘Gower | (ta and epson) z= Gover (apa ad esos) Hg Porn (ea and pi) =a ‘Swiching zac alpha cle Genetic coding for globin chains * Chromosome 16: alpha gene and zeta gene * Chromosome 11: beta gene, delta gene, gamma gene and epsilon gene he Pode ee} | Switching epsilon to gamma @C!! | Normal Human Hemoglobins Formation of Hemoglobin F Hemoglobin Molecular Structure Stage of Life Gower 1 2 zela, 2 epsilon ‘embryonic a et te toe eee eres inet Portland 22eta, 2 gamma ‘embryonic DOWNeplatonolgarmacan] Gower 2 2 alpha, 2 epsilon embryonic AL alpha, 2 beta newbom and adult sin EEE [eer | 2 teeta yy Nacres co. Page| 18Effects of various factors on Oxygen Dissociation Curve (DC) Globin Chains in Hemoglobin ‘Shift caused by: Greek Greek No. of Factors Factor Increased Factor Decrease designation Name amino acids Temperature R L a Alpha 141 a DPG 7 i 8 sa ied - R + 46 Carbon dioxide R L 7 sens ue Note: The shape of the ODC curve of hemoglobin is SIGMOIDAL, il Verse 0c of myoglobin s nei ee HEMOGLOBIN DERIVATIVES 4. Methemogtobin (Hi) ~ derivative of hemoglobin in which of ferrous iron is oxidized to ferric state, resulting to the inability of Methemoglobin to combine reversibly to oxygen ~ associated with exposure to exogenous oxidants such as nitrite, primaquine, dapsone and benzocaine. ~ hereditary causes linked to mutation in NADH-cytochrome bS reductase 3 (CYB5R3) ~ causes chiocolate brown discoloration of blood; cyanotic (1.Sgr/ng/éL) ~ measured via spectrophotometric techniques: wavelength: 525nm Note: An abnormal hemoglobin (Hb M) may also be responsible for methemoglobinemia noted at birth or inthe first few months of life, 2, Sulfhemaglobin ~ formed by irreversible oxidation of hemoglobin by drugs to treat bacteremia caused by Clostridium Wwelchit (sulfonamides and phenacetin) and other oxidants (eq nitrites and phenylhydrazine) ~ formed by the addition of SULFUR atom to the pyrrole ring of heme ~ produces GREEWISH PIGMENT = can result to formation of Heinz bodies; can combine with carbon monoxide to form carboxysulfhemoglobin = Blood is mavy-lavender in sulfhemoglobinernia = CANNOT be converted to normal hemoglobin A, persist in life until breakdown = quantitated via spectrophotometry, has similar peak with Hi - However, sulfhemoglobin curve does not shift when CYANIDE is added. 3. Carboxyhemoglobin (HbCO) ~ results from the combination of carbon monoxide (CO) with heme iron, = light sensitive and has a typical color civecry-reé its affinity to hemoglobin is 240x that of oxygen; SHIFT TO THE LEFT ~ Carbon monoxide is termed a5 91. £11T KILLER, (colorless and odorless and victims quickly becomes hypoxic) + detected by spectral absorption instruments as 540am, = TREATMENT: removal of patient from CO source and administration of 100% oxygen. Nomenclature and Absorption Maxima of Hemoglobins Absorption peak 1 | Absorption peak 2 | Absorption peak 3 Term Symbol | e x t x t Hemoglobin Ho 431 (140) ‘555 (13.04) lOxyhernoglobin IHoo2 415 (131) 542 (14.37) 877 (18.37) |Carboxyhemoglobin lHbco 420 (192) 539 (14.36) 568.5 (14.31) Hemiglobin (methemoglobin) Hi 406 (162) 500 (9.04) 630 (3.70) Hemiglobincyanide (cyanmet Hb) _|HiCN 421 (122.5) 540 (10.99) ‘Note: The wavelength (A) in nanometers fro each maximum Is followed by the extinction coeficient (e) placed lh parenthesis, The wavelength in nanometers is followed by the extinction coeficient placed in parenthesis Mkurus co. Page [19Checkpoint Line 3 Erythropoiesis, RBC Structure, Function and Metabolism Hemoglobin Synthesis and derivatives Multiple Choice. Encircle the best answer. 1. For the genetic coding of globin chains, chromosome 16 is responsible forthe synthesis of a. zela and epsilon gene 6. alpha and zeta gene b, zea and beta gene 4, alpha and gamma gene 2. Hemoglobin For Fetal hemoglobin stats to occur onthe 10" week of gestation, the formation of such hemoglobin is due to the combination of which globin chains? 2. alpha and beta globin chains ©. alpha and epsilon globin chains . alpha and zeta globin chains 4. alpha and gamma globin chains 3. What RBC metabolic pathways is responsible forthe maintenance of reduced from of iron in the hemoglobin? a, Pentose phosphate pathways «, Rapoport-Luebering pathway ', Methemoglobin reductase pathway 4, Hexose Monophosphate shunt 4, What stage in the erythropoiesis isthe last stage capable of miosis? 2, Polychromatophilic nrmoblast . Orthochromic enthroblast b. Metaruoricyte 6, Polychromatophilic erythrocyte 5 Which ofthe following sequences follows the correct order of erythropoiesis? a, Rubriblst, basophilic enthroblst,rubriy, orthochromic normoblast, reticulocyte, erythrocyte ». Basophili normobiast, proerythroblst, rubriyte, metarubriyt, orthrechromic normablast, erythrocyte 6. Pronormoblst,prorubriyt, orthachromic erthroblas,polychromatopilicnormoblast, erythrocyte 4. Pronormoblas, basonhilc erythroblas,polychromatophikc erythrocyte, orthachromic normoblat, erythrocyte 6. Which ofthe following is NOT a function of EPO? 4. promotes early release of reticulocyte ©. reduction of call cycle b. reduces Fas receptor 4, increases rate of cel cycle 7. The exposure of which phospholipid, hints a signal to the macrophages to perform phagocytosis as means of cell death? a phosphatidyicholine ©. phosphatidyethanolamine b. phosphatdylserine «. phosphatidylinastl 8. A mutation in the gene synthesizing uroporphyrinogen decarboxylase will ead to the accumulation of whal substance? a. Uroporphyrinogen ill ©. Porphobilinogen b. Coproporphyrinogen tt ¢. Hydroxymethybilane 8. A mulation in cytochrome BS reductase 3 is associated withthe accumulation of ati ¢. Sulfhemogblobin b. Hbco 6. Cyanmethemoglobin 10. An increase to the level of 2,3 biphosphoglycerae is observed with in the ODC curve, a. hyperbolic graph 6. shift tothe left b. sigmoidal granh 4. shift tothe right LTUs co. Page| 20HEMOGLOBINOPATHY Red Blood Cells Hemoglobinopathies a = most common genetic disease ~ the hemoglobin molecule has an altered amino acid sequence within the globin chains which changes the Structure impairing its function — QUALITATIVE DEFECT HEMOGLOBIN S "Sickle Cell Disease Sickle Cell Trait Hemoglobin C Hemoglobin E Hemoglobin SC = mutation at the 6" position of beta globin; substitution of glutamine fo valine = polymerizes into long, thin polymers to form sickles when 02 saturation decreases = most common hemoglobinopathy = may be reversible (assoc wvasoocclusion) or irreversible Homozygous Hgb S Normocytic, normochromic anemia; Poikilocytes: Drepanocytes, target cells Increased reticulocyte count “*Dithionite Solubility (Sickle screen) - POSITIVE Hemoglobin electrophoresis - POSITIVE for Hgb S and NO Hgb A is present Observed to be resistant against Plasmodium falciparum infection Major threat: 8 ACTER|AL |NFECTIONS (septic shock) from Staphylococcus aureus, ‘Streptococcus pneumoniae and Haemophilus influenzae Hallmark: VASOOCCLUSIVE CRISIS Heterozygous for Hgb S: majority of gb present is hemoglobin A Patients are usually asymptomatic, Sickle Cell Screen Positive _| Hgb A and Hgb S are present Mutation at the 6° position of beta globin, substitution of Glutamine to Lysine Polymerizes into thick crystals when oxygen saturation decreases Crystals resembles "ars of gold” or “Washington Monument” Mutation at the 20° position of beta globin substitution of Glutamine i Lysine Usually affects patients from Southeast Asia, Thailand Solubilty testing NEGATIVE; confirm via HPLC or electrophoresis Combination disorder with inheritance of mutations for both Hab S and Higb C MOST COMMON COMPOUND DISORDER Similar picture to Sickle Cell Disease, however, does not manifest till teenage years Solubilily testing POSITIVE Hemoglobin M Involves a mutation in alpha, beta and gamma globin genes via substitution of a tyrosine amino acid for either proximal or distal histidine - it causes auto-oxidation of heme iran (iron in ferric state) forming methemoglobin ‘THALASSEMIAS = disorders caused by genetic mutations that lead to QUANTITATIVE DEFECTS in the amount of globin chains produced, = types and severity of thalassemia depends on the globin gene mutated (a arf) and the number of genes affected A. a-Thalassemia ~ caused by mutation or deletion in the a-globin genes - leads to the formation of excess gamma chains: omoolobin Bort (4 gamma chains) = After 6 months gamma switches to beta chain: Hemoglobin Wi (4 beta chains) = Hemoglobin Bart and Hemoglobin H ~ INCREASE AFFINITY to oxygen ~ Causes marked hypoxia leading to cardiac failure and hydrops fetalis ‘Avrus co. Page| 21GENOTYPE DISORDER REMARKS, ae Bart's Hydrops Fetalis Not compatible with life as the blood is unable to ‘Alpha Thalassemia Major —_| oxygenate tissue because of high 02 affinity of Hb Bart =-l-a Hemoglobin H Disease Unpaired B chain will form tetramers of Hgb H Decreased RBC and Hgb Microcytic, hypochromic RBCS, target cells, poikilocytes Hgb H inclusions - stained via supravital stain (NMB) _____=-/aa____ | Alpha Thalassemia Minor | Two gene deletion a-/a- Asymptomatic presentation aala- Silent Carrier ‘One gene deletion, no clinical symptom aa/aa None Normal hemoglobin B, B- Thalassemia ~ caused by mutation or deletion in the B-globin gene ~ Asymptomatic till 6 months due to increase Hemoglobin F ~ Symptoms appear between 6-24 months after the completion of gamma to beta switch INCREASED EPO but still exhibits ineffective erythropoiesis; promotes BM EXPANSION = Inctease Extramedullary hematopoiesis | = Increase Iron accumulation: Increase erythropoiesis = decrease hepeidin | ~ Progresses to reduction of bone mineral density and thinning | = Increase risk to pathologic fractures ~ Exhibits FRONTAL BOSSING GENOTYPE DISORDER REMARKS : 7B), (8B), (BR) Cooley's Anemia Severe hemolytic anemia diagnosed at 6* month Beta thalassemia Major —_| With hepatosplenomegaly and distinct bone changes (87 B*"), (6878), | Beta Thalassemia intermedia | Clinical signs and symptoms vary because of (@"/38°) multiple genotypic presentation Usually not transfusion dependent 78), 878) Beta Thalassemia Minor | Usually with mild anemia Normal to elevated RBC wih decreased Hgb and ct : Poikilocytes (target cells) vp Silent Carrier no aiinical symptom diagnosis via genetic analysis no therapy needed Vidurus co. Page| 22i» -4_ Red Blood Cells “Rae Anomalies a Classification of Variation: a. Size d. Inclusions in erythrocytes b, Shape e. Alteration in the erythrocyte distribution in peripheral blood smear c. Color (hemoglobin content) A. SIZE: ANISOCYTOSIS: Variation in size = Red cell distribution Width (ROW) ~ numerical expression that correlate with degree of anisocytosis NORMOCYTIC CELL: MCV: 80-100 fL RBC Abnormality Cell Description ‘Commonly Associated Disease Anisocytosis ‘Abnormal variation of RBC volume or diameter Hemolytic, Megaloblastic, IDA Microcytes Decrease in cell size: 8um), MCV >100fL Megaloblastic anemia Myelodysplastic syndrome Non-megaloblastic anemia Chronic liver disease B. CELL SHAPE: POIKILOCYTOSIS: Variation in SHAPE a, Poikilocytosis secondary to developmental macrocytosis ~ Macrocytes/ oval macrocytes b. Poikilocytosis secondary to membrane abnormalities ~ Acanthocytes/ Spur cell Thorn cell ~ Echinocytes/ Burr cell - Codocyte/target celliMe» - Spherocyte - Stomatocyte - Eliptocytes Ovalocytes ¢. Poikilocytosis secondary to trauma - Schiistocytes/Schizooyte/Keratocyte/helmet cell - Dacrocytefteardrop cell - Microspherocyte/pyropoikilooytes - Semi-lunar bodies/crescent cell 4d, Poikilocytosis secondary to abnormal hemoglobin content Drepanocyte / Sickle cell / Menisocyte mn hat cell \&urus co. Page| 23‘Summary of Poikilocytes Red Cell Type ‘Morphologic Appearance Defect or Change Disease Association Ticanthocyte — Spheroid with 3-12 ivegular spikes Increased ration of cholesterol ‘Abetalipoproteinemia to lecithin Pyruvate kinase deficiency Eng-stage liver disease Fchinocyles Regular 10-30 scalloped Deplalion of ATP Uremia (wir cells) short projections Exposure to hypertonic solution Pyruvate kinase deficiency Axia drying Chronic renal disease Codoeyies Peripheral rim of hemoglobin Excess surface membrane Hemogiobinapathies ((erget cetl) surrounded by clear area and (0 volume ratio Thalassemia central hemaglobinized area Post splenectomy (@uls eye) Liver disease Decroyocyie Teardrop or pear shaped with ‘Squeezing and fragmentation ‘Myelopthisic Anemia {coardrop cell) single elongated point or tail uring spleenic passage Agnogenic Myeloid Metaplasia Primary Myelofibrosis Dropanceyie — Cresent shape cel that lacks Polymerization of deoxygenated Sickle cel anemia (cichle cell) zone of central pallor hemoglobin “Filiptocyie Rod or cigar-shaped Polymerization of hemoglobin HerediaryEliptooyosis ‘Ovalayte Egglike or oval shaped cell Hemoglobin has bipolar Megaloblastc anemia wider than eliptocyles arrangement, reduction Myelodysplasia in membrane cholesterol Sickle cell anemia Schistocyte Fragments of RBCs varying in Exireme fragmentation Disseminated Intravascular size and shape produced by damage of RBC Coagulation (OIC) fibrin, altered vessel walls and Microangiopathic hemolytic prosthetic heart valves ‘Anemia (MAHA) Spherscyie Smaller diameter than normal Lowest surface area to volume Herediiary Spheroojosis RBC with concentrated hemoglobin ratio with defective membrane Immune hemolytic anemia content; no visible central pallor defect in spectrin Tlomatoeyte Normal sized cell with sli-ike —__-Knawn to have increased Hereditary Slomalocyjtosis area in the center permeability to sodium RH Null Disease RED CELL MORPHOLOGY GRADING MORPHOLOGY GRADE AS, MORPHOLOGY (GRADE AS Polychromatophilia += 110 S/field — [Rouleux 7+ = aggregates of 3 to 4 RBC Helmet cell 2+ = 6 to 10lfield 2+ = aggregates of 5 to 10 RBC Teardrop cell 3+ = >t0rfield 3+ = numerous aggregates with Acathocytes only few free RBC Schistocytes Spherocytes Poikilocytosis F#= 310 forield [Sickle cols “GRADE AS POSITIVE ONLY Ovalocytes 24= 11 to 20,field|Basophilic stippling Elliptocytes 3+ = >20field |Pappenheimer bodies Burr calls Howall-Jolly bodies Bizarre-shaped RBC Target colls Stomatocytes \idurus co. Page |24€. HEMOGLOBIN CONTENT: ANISOCHROMIA: Variation in hemoglobin content Normochromic — normal hemoglobin content: 1/3 cell diameter Hypochromic © Central pallor exceeds 1/3 of the diameter of the cell; Decreased MCHC: e.g. IDA and thalassemia Hyperchromic ~ should be called spherocyte © No central pallor; Increased MCHC: SPHEROCYTOSIS Polychromasia © Indicates reticulocytosis; Blue-gray coloration ‘ ‘© Indicates young RBC; increased in erythropoietic activity e.g. hemorrhage, hemolysis Oe Hypochromia Grading Polychromasia Grading Area of central pallor is 1/2 of cell "Percentage of RBC that are Polychromatophilic 1+__| diameter ‘Slight 7 2+ | Area of pallor is 2/3 of cell diameter 1+ 3% 3+__ | Area of pallor is 3/4 of cell diameter a 5% ‘4+ | THIN RIM of hemoglobin 3a 10% a 211% _ : RED CELL INCLUSIONS Type Morphologic Appearance Defect or Change Disease Association Howell-Jolly Bodies Coarse round densely staned Tier remnants confining Magalablastic Anemia purple 2pm granules eccenticaly NA Accelerated erythropoiesis located on periphery of membrane __Maybe single or double Cabot Ring Rings, loops or figures of eight Rernnants of micolubules of Dyseryihropoiesis red to purple mitt spindles Heinz Bodies ‘Deep purple iragulr shaped Represent precited, Defect in HS incusions 2 to 3m denatured EPO deficiency Found on RAC inner surface of membrane hermaglobn due to oxidative Unstable hemoglobin injuy Dasophilic Stipplings — Round dark blue granules Represents impaved Lead poisoning uniformly distbuted exatropciesis Pyrimidine-5-nucleoidase RNA remnants deficiency Pappenheimer ‘Small 2to Sym ireguar Unused ion deposi Siderobasic anemia bedi basophiic inclusions that defective entropoiesis aggregate in small ustrs near periphery with Wrights stain Finged Siderobiest WNudleated REC that contains excessive Ton overoad in Siderobasic anemia non-heme iron paticles arranged in mitachondria of nomablats fing form ue to delecive heme synthesis Siderocyte TNOW-nucleted RBC conning iron ‘entassive ron overoad in Siderobiestc anemia ritochondia of rarmoblsts Aue ta detective heme synthesis = (MISCELLANEOUS RBC ABNORMALITIES Autoaggiutination ‘Clumping of RBCs Presence of antibody Cold agglutinin Autoimmune hemolgic anemia “Vouleaur ‘Alignment of RBCs linear appearing ‘caused by increased Muliple Myeloma 3 sack of coins | ‘concentration Waldenstrom of globuin Macrogiobulinemia Vidurus co. Page| 25Checkpoint Line 4 Topic: Hemoglobinopathies and RBC anomalies Part |, Multiple Choice. Encircie the best answer. 1. tis defined as the variation in red blood cell stucture/formation, a, anisooylosis _. poikilooytosis, . Polychromesia 4, Metachromasia 2. What isthe hypochromia grading of the red blood cell given the description: rea of pallor is 75% ofthe cell diameter? att bd 0.3 04 3. An 8month-old baby was brought tothe ER due to dificult in breathing, pallor and fracture. The physician also noted “frontal bossing”. Based on the history, patient signs and symptoms. What isthe most key clinical impression? a. Cooleys anemia . Bats hydrops foals b. Sickle cell anemia 1, Hemoglobin H disease 4. What abnormal hemoglobin is formed in patients with alpha-thalassemia major during the 7 month of gestation? a, Hemoglobin Barts «. Hemoglobin Mi b, Hemoglobin H d, Hemoglobin S ‘2 mutation characterized by he substituion of Glutamine to Lysine atthe 6* postion of beta globin chain? a. Hemoglobin C ¢. Hemoglobin SC b. Hemoglobin € 4. Hemoglobin S 6. Lis 2 mutation characterized by the substtution of Glutamine to Lysine atthe 26° positon of beta globin chain? 2. Hemoglobin C c, Hemoglabin SC b. Hemoglobin E 4. Hemoglobin § 7. Patients with Sickle Cell Disease were observed to have cll sicking allow oxygen tension. This phenomenon renders the patient to be resistant ta infections against 3, Babesia spp. «6, Pesmodium falioarum b. Staphylococcus aureus a. Haemophilus influenzae Part I. Matching Type. Match column A to column B. ‘COLUMN A 4. Nuclear remnants containing DNA 2 Denatured hemoglobin due to oxidative injury '3. Remnants of microtubules of mitotic spindles 4. Seen in abetlipoproteinemia 5. Seen in RH Null Disease 6. Seen in Myelopthsic anemia 7. Composed of 4 gamma chains 8. Resembles bars of gold 8. Seen in GOPO deficiency 16. Seen in nemolytc transfusion reactions ‘COLUMN B a, Spherooytes b. Port hemagiobin «. Heinz bodies 4. Cabot Rings «. Barts hemoglobin {. Hemoglobin H 4. Stomatonytes h Elipiocytes i. Basophilic stipplings j. Dacryocytes . Acanthocytes |. Howell-lolty bodies rm, Hemoglobin C 1. Hemoglobin € \idurus co. Page |26Red Blood Cells ™ Anemia ANEMIA ~ defined as the decrease in the ability of blood to carry oxygen ~ decrease in the amount of hemoglobin in the blood that may be caused by decrease in number of RBCs, ‘hemoglobin and hematocrit below the reference range for healthy individuals Common signs and symptoms: fatigue ~ shortness of breath = pallor + cardiac issues Common tests for initial anemia evaluation: ~ complete blood count (CBC) with peripheral smear view: Hgb, Hct and RBC indices Note: smear examination will reveal the appearance of RBCS. ~ Reticulocyte count: shows the bone marrow response to decreases in RBCS ‘Anemias may be classified by combinations of different criteria: 2. Morphology - RBC indices are used to gauge size and hemoglobinization (1) NormocyticiNormochromic (2) Microcytic/Hiypochromic (3) MacrocyticiNormochromic b, Function: defects leading to RBC decreases: (1) Proliferation: RBCs are not produced at normal rates (2) Maturation: RBCs are produced in the marrow but may not mature appropriately (8) Survival: RBCs are produced appropriately but are lost/destroyed prematurely J ANEMIA OF IRON AND HEME DISORDERS a Iron Deficiency Anemia (IDA) lack of iron to form adequate heme . Anemia of Chronic Inflammation ‘adequate on slres thal have impaired release for incorporaion . Sideroblastic Anemia ‘adequate/excess iron that is not able to be effectively incorporated into heme ¢. Hemochromatosis ‘won disorder thats NOT anemia; with excess iron absorption end stores Overview of Iron Cycle and Regulation in the body. =a t + Pepayis [om [tds [temo Uustinmae abate emo om [seen] ise + teats totes ae ins se ec cng ae hrapst sion Ron sch 1+ Medan pac HEFCE mse rovasonin ed whoo sere ‘Wis tgue is aed a: Rehan, M, Sith. & Wg, MOV), Roda Henalgy Cia Pris ad Appears. Clini Sunde \durus co, Page |27‘A. IRON DEFICIENCY ANEMIA (IDA) 5:5 anemio” Causes: - Inadequate intake (approx. 1mg daly replacement from et) ~ Increased requirement (e.g. pregnancy, nursing, infants) ~ absorption issues (e, g. decreased acidity, celiac dse, mutation in regulatory proteins (matriptase 2)) ~ chronic RBC loss (chronic hemorrhage or hemolysis, prolonged menorshagia, chronic Gl bleeding) ~ parasitic infection (#7, americanus, A. duodenale, T. trichiura, S. mansoni, S. haematobium) Development of Iron deficiency anemia _ < #¢§ G Normal Iron Status Stage 1 Stage 2 Stage. = ee Fern Depletion Tansferin Depletion Furoal Yon Depeton etn Depa poise Tin (ron Defcieney Anema) “rank Anemia" ren pep ein. ——————- tron Storage Chamber (Ferritin level) | + Functional Iron Chamber (Hemaglabin level) L TST Be ROR Kaa HST, BW WE ToS TS STI Sunde Laboratory Tests Values Stage 4 Stage 2 Stage 3 Hemoglobin Normal Normal — Decreased Serum Iron Normal Decreas Decreased TIBC Normal Increased Increased Ferritin Decreased Decreased Decreased ‘Slage 1 (Storage Iron Depletion) ‘Stage 2 (Transport Iron Depletion) ‘Stage 3 (Functional Iron Depletion) progressive loss of iron stores | - exhaustion ofthe storage iron poo! | - depletion of storage and transport iron 'o evidence of iron deficiency | - increase in transferin receptor in iron | ~ blood pictur: iicrocyiic¥ypechramte = latent or subetinical stage starved cells Severe signs: ~ increase in soluble transferrin receptor | ~ °° (Sore tongue) ~ decrease in ion avilable for = sugulay chovlosis lame cracks at enytropoiess the comer ofthe mouth) ~ hoilonyels (spooning of nats) = pica (raving af non-food items e.g sol paper, ie-papophani Laboratory Diagnosis f Screening Tests Diagnostic Tests Specialized Tests Complete blood count (CBC) Perform Iron Studies = increased fee erythrocyte = increase anisocyosis ~ decreased serum iron Protoporphyrin (FEP) detected as zinc blood picture (microyicfhypochromie)_ | ineeased Total lon Binding Capacity | protoporphyrin (Fucrometry) ~ decrease MCV, MCH, MCHC (mie) ~ increased soluble transfetin receptor decrease RBC count and hematocrit | - decreased transferrin saturation (STF) ~ (lnmmunoassay) decrease RPI (ineective erythropoiesis) | - decreased serum ferritin Treatment: (1) Treat the underlying causes (2) dietary supplement (oral ferrous sulfate) \idurus co. Page| 28B. ANEMIA OF CHRONIC INFLAMMATION (act) ~ acquired anemia characterized by abundant iron stores, yet iron cannot be readily incorporated into the serum or RBCs for use ~ oocurS as a result of increases in various acute phase reactants present with inflammation which slows down iron release that is needed by the developing cells ~@ common complication in patients with disorders such as inflammation (cheumatoid arthritis), infection (luberculosis, HIVIAIDS) and malignancy (SLE, neoplasms) ~ central feature: sideropenia in the face of abundant iron stores —- due to impaired iron kinetics, OG Mechanism of Anemia of Chronic Inflammation Chronic infection (TB, HIV) ‘Malignancy (neoplasms) Autoimmune dysregulation (SLE) ‘Aclvated| macrophages and T cells >—— Deen bao ‘rate mare TNF-a, IL-1 INF-y ts — [ven —- “nates L__. decaiset stesso ton 1 ‘he macropoges ens mptootes Decent Dees yop inthe predion a EPO tae meow inthe drew ‘Wis que istases ro: Kahan, Mt, Sih LJ, & Wega, JM. (206) Rass Hemslogy Cll Principles and Aplatos, eer Saunders. HEPCIOIN ~ a hormone that controls the absorption and recycling of ron targets ferroporin| ~ decreases iron release from macrophages and hepatocytes and absorption of iron in the intestine ~ an acute phase reactant TACTOFERRIN | - competes with transfer for plasma iron, (RBCs has NO Lacloferrin receptors) ~ an ion-binding protein in the granules of neutrophils = an acute phase reactant FERRITIN ~ binds iron ((RBCs and erythroid precursors has NO ferritin receptors) ~ an acute phase reactant Other inflammatory o7tokines: tissue necrosis-o (TNF- o) and inlerleukin-1 from macrophages: interferon-y from T-cells Note: Hepatocyte production of transferrin is regulated by intracellular iron levels. Thus, the low level of toll iron binding capacity (TIBC) in = ‘ACI teflets abundant iron stores in the bod. Laboratory Diagnosis Screening Tests Diagnostic Tests Specialized Texts Complete blood count (OBC) Perform Iron Studies NORMAL soluble Wenfein ecopior ~ leukocytosis and thrombocytosis ~ decreased serum iron (STR) — (mmmuncassay) + blood picture: ~ desea ul kon in Capac (18) | - positive inion sian (Prussian blue) 4. normocytic, normachromic decreased transferrin saturation “perform specie tests for nflarmatign b. microcytieMrypochramic- ton defeney | ~ increased serum fertin (04. CRP) or test for cancer ~ decrease RPI (neflectve erylliropoiess) Treatment: (1) Effective control of underlying condition (2) Therapeutic administration of EPO and iron \idtrus co. Page| 29C. SIDEROBLASTIC ANEMIA ~ characterized by the presence of normal or increased iron that is not effectively incorporated into heme due to interference in the production of adequate amount of protoporphyrin T HALLMARK; owes Siceroblasts (aomobiastswiiron depois inthe ichanasurouning the nucleus in Prussian ie sn) ~ Hereditary: X-linked or autosomal Treatment: Some respond to pyridoxine (a cofactor) ~ Acquired: refractory anemia, anttubercular drugs, chloramphenicol, alcohol, LEAD and chemotherapeutics = Lead Poisoning (acquired porphyria) - interferes the following enzymes: Affected enzymes Accumulating by-product Analyte of interest and sample 2. PRG synthase (ALA dehydratase) | Aminolevulinic acid (ALA) | - measure ALA in urine b. Ferrochelatase Protoporphyrin IX | = measure FEP as ZPP in blood Note: Lea also inhibits pyrimidine S'-mucieotidase (in RNA breakdown of rliculocyles) “FE (rex eto protoporphyrin ZF ie olga PG smbase (Porphobinogen sass) Clinical presentation: ~ abdominal pain, constipation, vomiting and muscle weakness ~ LEAD LINE: linear blue-black deposit of lead sulfide in the gums near the teeth BASOPHILIC STIPPLINGS. Treatment: (1) removal of the offending agent (2) salts of ethylenediaminetetraacetic acid (EDTA)- chelates lead = Porphyrias hereditary conditions that impair production of protoporphyrin cog Type of Poriyria ‘Afected enzyme | Accumulating by-product Clinical features a. Congenital Erythropoieic | Uroporpliyrinogen IN | Uroporphyrinogen! | - photosenstivly Porphyria (CEP) synthase hemolytic anemia “Gunther's disease” Gene defect: UROS = Fink o deep burgundy urine teeth wl uoreseence red under UY light 8. Porphyria Cutanea Tarda Uroporphyrinogen Uroporphyrint [= photosensitivity (PCr) decarboxylase = reddish or brownish urine 6. Erythropoetic. Ferrochelatase Protoporphyrin 0X | = photosensitivity Protoporphyria (EPP) Gene defect: FEGH a Ylinked Erythropoietc. ALAsyntiase 2 | Aminolevulinic acd (ALA) [= photosensiiiy Protoporphyria (XLEPP) (gain function) ~ mild microcytic, Gone deiect: ALAS 2 hypochromic anemia Ace nlermitent Hepa | Uroporplirinogen | ALA and porphabiinogen | ~ psychologic disturbances Porphyria (AIHP) synthase Porphyrin precursors | - neurologic dysfunctions ‘Hereditary Coproporphyia_| Coproporphyrinagen | Coproporphyrinagen I| ~resembles “AHP oxidase 4. Varegate Porphiyia Protoporphyinogen | Proloporphyrinogen | ~ resembles *POT oxidase Coproparphyrin | - with neurologic fe : dysfunctions WAurus co. Page [30D. IRON OVERLOAD = HEMOCHROMATOSIS (0)(0117¢ pineryes) CJ - iron problem that does not involve anemia * prora Dsbts is chrcterted fromthe ~ increased iron stores (absorption is greater than loss) | Sensi ol hesiern in te st | gesting cbr nde darage * Stored as feritin and hemosiderin (heart, liver, pancreas) wns cas ne or 1. Hereditary Hemochromatosis Phenotypes Phenotype Gene defect Protein defect Normal funcion of afected protein a, Hemochromatosis Type 1 WE Hereditary inhibits TfR1-mediated iron hemochromatosis | intake; regulates hepcidin prot expression ’b, Hemochromatosis Type ‘HE2 (HM) Hemojuvelin regulates hepeidin expression 2A, Juvenile 6, Hemochromatosis Type AMP Hepcidin ‘downregulate ferroporin 28, Juvenile ‘mediated iron transport in = macrophages and enterocytes Hemochromatosis Type 8 TRE Transferrin receptor | provides hepatocyte iran protein 2 uptake; regulates hepcidin expression e. Hemochromatosis Type 4 (SLC40A1 Solute carrier family 40 | transports iron out of member 1 (Jerroportin-t) | enterocytes and macrophages {, Hemochromatosis Type 5 FTHt Forritin heavy chain iron storage |, Acquired: transfusion related, chronic liver disease, alcoholism, supplemental or dietary iron overload Treatment: Iron chelators and phlebotomy SUMMARY OF IRON STUDIES FOR ANEMIA OF IRON DISORDERS Se: © Diagnostic Use Tron Deftiency | Anemia of Chronic | Sideroblasio Anemia Infammation ‘Anemia Pasaing 2, Serum feritin Indicator ofiron stows - [Decreased | increasea Inoreased Normal . Serum ron | tndicatr of available anspor mn | Decreased | _Decreasod Increased Variable c.TIBC Indies indicator of iron stores | ~ ncreased Decreased Decreased Normal 4. Transferrin | Indirect indicator of ion stores | Decreased | Detreased/Normal | ncreasea ~~} lnoveasea ‘Saturation ve transport ton . FEPIZPP Taciator of fancinal ron increased Thoreased tnereased | Markedly availabe in red blood cells increased BM iron Visual qualtalve assessment of | Vo sainable | icreased/Normal | increased Normal (Prussian blue tissue ion sores iron ‘ringed singed stain) = sideroblasts | _sideroote Note: Transferrin Saturation % = serurn iron (uq/iL) x 100 TIBC (pf) Lrus co. Page| 31{get ANEMIA OF DNA METABOLISM DEFECTS MACROCYTIC ANEMIAS (1) Megaloblastic Anemia: Vitamin 812 and folic acid deficiencies ‘ (2) Non-megatoblastic Anemia: Liver disease, alcoholism, hypothyroidism and reticulocytosis Functions of Vitamin B12 and Folic Acid = Vitamin B12 (Cobalamin) 1. Isomerization of methyimalonyl CoA to succinyl CoA 2. Transfer of methyl group from S-methyltetrahydrofolate (S-methyl THF) to homocysteine to generate methionine * Folic Acid (Folate) a amino acids and nucleotides ~ circulates in the blood as 5-methy! THF (inactive \ransfer carbon unit forms of methyl group necessary for the metabolism of 2@ form) SUMMARY OF ABSORPTION OF VIT. B12 (COBALAMIN) 1. Protein bound dietary Vit. B12 is released inthe stomach via the scion of pepsin and hydrochloric ai, 2. Haptocorrin(R prcein) binds Vit. 812 and remains bound unt intestinal proteases (e.g trypsin) catalyes ts release 3. In the duodenum, intrinsic factor (F) binds Vit B12, 4. Cubitn-amniontess (Cubam) and’ megetin receptors inthe leal enterocyte binds Vi.812- intrinsic factor complex, then releases vi B12, 5. Transcobalamin from the enterozytes binds Vit. B12 and transports ito the circulation, 6. The melaboialy active form Vit 812 Mt B72-tanscobalamin complex ar Holotanscobalamia) binds to specticrecepior of cells for is uilizaton. ‘SUMMARY OF THE ROLE OF VIT. B12 AND FOLIC ACID IN DNA SYNTHESIS. Inactive folate (S-methy THF) enters the cel 2. The methyt group from S-methyiTHF is transfered to homocysteine, ‘Methionine synthase with Vit. 812 as a cofactor ‘converts homocysteine to methionine and forming tetrahydroolate (THR) the active form as by-product. By the donation of metyl group from serine, THF is converted to 5,10 methyl THF. The metnyl group of 5,10 methyl THF is transferred to \deoxyundinemonophosphate (UMP) to form 40 yrs. old patients without HLA identical sibling MiAurus co. Page |34B. PURE RED CELL APLASIA = bone marrow exhibits decreased Production of RBCs and RBC precursors, whereas other cell ines are present and produced normally a. Acquired * Primary is idiopathic or autoimmune * Secondary is usually associated with tumors, infection (Parvo & Virus), drugs (Chloramphenicol), Chemicals (enzene) b, Diamond-BlacKfan Anemia (Congenital Pure Red Cell Aplasia) ~ congenital erythroid hypoplasia = mutation at RPS19 (25%), RPS7, RPS10, RPS17, RPS24 and RPS26 in the 40s subunit ~ Mutation at RPLS, RPL11 and RPL35A in the 60s subunit €. CONGENITAL DYSERYTHROPOIETIC ANEMIA (CDA) ~ heterogeneous group of rare disorder characterized by refractory anemia, reticulocytopenia, hypercellula bone marrow with markedly ineffective erythropoiesis and distinctive dysplastic, changes in BM erythroblasts Classification: Mutation Remarks 2ODAT ‘GDAMTT gene on chromosome 1% ~ malformation of fingers or toes, brawn ~ encodes codanin-t for cel cycle regulated | skin pigmentation & neurologic defects ‘uctear protein erythroblasts are megaoblastid with internuclear chromatin bridges ~ characteristic spongy heterochromatin with “Swiss cheese” appearance b.CDAT ‘SECZ3B yene on chromosome 20 ~ circulating RBCs hemolye with Hams ‘aka HEMPAS ~ encodes a component ofthe coat protein | acidified serum test but not with sucrose {ered tay enyroblasic complex (COPI) that forms vesicle for hemolysis test ‘mukinucearty wth positve | transport of secretory poten from aided serum) endoplasmic reticulum to golg apparatus «CDA ‘AiFZ3 gene ~ bone marrow has megaloblstic changes codes for protein involved wth eyfoknesis | - giant erythroblasts with up to 12 nucle D. MYELOPHTHISIC ANEMIA - infitration of abnormal cells (metastatic sold tumors e.g. lung, breast and prostate) into the bone marrow and subsequent destruction and replacement of normal hematopoietic cells E. ANEMIA OF CHRONIC KIDNEY DISEASE ~ individuals suffer anemia due to inadequate renal production of erythropoietin (Aurus co. Page| 35Oy! HEMOLYTIC ANEMIA 2@ HEMOLYTIC ANEMIA - disorders of premature RBC destruction, leading to anemia Classifications: (1) Intrinsic VS Extrinsic defects (2) Acquired VS Hereditary defects (8) Intravascular VS Extravascular hemolysis General features: = general symptoms of anemia = Splenomegaly ~ jaundice - Gallstones (chronic hemolysis) Laboratory findings: ~ INCREASED BILIRUBIN (62) + DECREASED HAPTOGLOBIN ~ INCREASED RETICULUCYTE COUNT, RPI HEMOLYTIC ANEMIAS: INTRINSIC DEFECTS ‘A. ABNORMALITIES IN RBC MEMBRANE a. Hereditary Spherocyto: - Autosomal Dominant inheritance; possible mutation in ANK7, SLC4A1, SPTA1, SPTB or EPB42 ~ Mutations produces defective proteins that disrupt the vertical linkages between the lipid bilayer and cytoskeletal networks - Presence of spherocytes; decreased area-to-volume ratio, = NEGATIVE DIRECT ANTIGOBULIN TEST (DAT) = INCREASED Osmotic Fragility (specimen: fresh heparinized blood) = AUTOHEMOLYSIS: Correction with ATP and Glucose b. Hereditary Elliptocytosis = Mutations in genes coding for a-SPECTRIN (SPTA1), 8-SPECTRIN (SPTB) or BAND 4.1 (EPB41) ~ defect in proteins thal disrupt the horizontal linkages in the protein cytoskeleton ¢. Hereditary Acanthocytosis - Defect in RBC membrane LIPID BALANCE, often result from liver issues - In severe liver disease, excess free plasma cholesterol that accumulates in the membrane = Neuroacanthocytosis ~ characterized by neurologic impairment and presence of acanthocytes in PBS Example disorders: Abetalipoproteinemia, McLeod syndrome and chorea acanthacytosis 4, PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) ~ RARE ACQUIRED INTRINSIC DEFECT resulting from mutation in 2/6: ene (detects aso in platelets and WBC) PIG gene cates for phosphtiylosiol N-aceyllucosaminytransferase suburitA necessary fr GP synthesis Cell lacks Gi. Y/COSYLPHOSPHATIOYLINOSITOL (GP), an anchor protein to COS (DAF) and C039 (RIRL) = RBCs are susceptible to lysis because CDSS and CD59 which inhibit complement are ABSENT + Screening Test: Sucrose hemolysis test ~Confirmatory Test (old): flaws Acilfied Serum Test = Confirmatory Test (new): MMultiparamter Flow Cylometsy Note * Diagnosis of PNH is accomplished by the detection of the absence of GPl-anchored proteins on WBCs using multiparameter flow cytometry Methods: a, Fluorescent monoclonal antibodies to GPl-anchored protein (CD59, CDSS, CD24, CD16, CDséb, (CD14) with ineage specific antibodies to non-GPI anchored proteins (CD15, CD33 or CD64)” b. Alternative Flow Cytometry using fluorescein-labeled proaerolysin variant (FLAER) Treatment: Classic PNH — FCULIZUMAD MAtrus co. Page| 36B, ABNORMALITIES IN ENZYMES (1) Glucose-6-phosphate dehydrogenase deficiency {RBCs are unable to reduce glutathione, leading to oxidation of hemoglobin to Heinz bodies Presence of bite cells and blister cells ~ Auto-hemolysis test: Correction with glucose (2) Pyruvate Kinase deficiency ~ leads to adenosine triphosphate (ATP) depletion and increase in 2,3 BPG ~ Auto-hemolysis test: Correction with ATP/ADP HEMOLYTIC ANEMIAS: EXTRINSIC DEFECTS a, Microangiopathic Hemolytic Anemia (MAHA) ~ group of disorders characterized by intravascular fragmentation of RBCs as they move through blood vessels obstructed by micro clots or endothelial damage ~ Presence of Schistocytes, increased B1, decreased haptoglobin Disseminated Intravascular coagulation (DIC) ~ activation of all parts of hemostatic system leading to the production of fibrin clots, consumption of platelets and coagulation proteins and degradation of fibrin - BOTH CLOTTING AND BLEEDING OCCUR - Coagulation tests are ABNORMAL ~ POSITIVE D-Dimer and Fibrin Degradation Products €. Thrombotic Thrombocytopenic Purpura (TTP) ~ NMOSCHOVIT2 SYNDROME ~ Patients have long von Willebrand factor (WWF) multimers that bind vascular endothelium and platelets ~ Platelets are used up and micro clots block small blood vessels ~ Mutation at ADAN/S7/3— cleaves long VWF multimers ~ Upshaw-Schulman Syndrome (Inherited TTP) 4, Hemolytic Uremic Syndrome (HUS) ~ MAHA with thrombocytopenia and renal involvements as a result of clot forming in the kidney vasculature ~ Has 2 general types: (1) Typical HUS: associated with Hemonhagic co!/(01S7:H7) and Shigella dysenteriae (Shiga toxin asso. HUS) (2) Atypical HUS: caused by unregulaled activation of alternative comploment pathway Other causes of Extrinsic Hemolytic Anemia a. HELLP Syndrome (Hemolysis, elevated liver enzymes and low platelet count) Hypertensive Crisis and Malignant hypertension Mechanical damage Infectious Agents: Malaria, Babesia, Clostridium pertringens alpha toxin Additional: Drugs, Chemicals, Venoms, Thermal Injury IMMUNE-MEDIATED HEMOLYTIC ANEMIA RBC lifespan is shortened because of presence of antibodies - DAT POSITIVE = Increased bilirubin and lactate dehydrogenase and decreased haptoglobin 4. Autoimmune Hemolytic Anemia i, Warm Autoimmune Hemolytic Anemia ii. Cold Agglutinin Disease ~ associated with Aiycoplasina pnevmonise i. Paroxysmal Gold Hemoglobinuria ~ associated with Donath-Landsteiner Ab (anti-® autoantibody) . Drug-induced Hemolytic Anemia b. Alloimmune Hemolytic Anemia i. Acute Hemolytic Reactions |i, Hemolytic Disease of the Fetus and Newborn (HOFN) Vidurus co. Page| 57Checkpoint Line 5 Topic: Anemia Part. Multiple Choice. Encicle the best answer. ‘1. Which of the folowing is not an example of hemolytic anemia with intrinsic defects? a, Paroxysmal Nocturnal Hemoglobinuria o. Pyruvate kinase deficiency ». Paroxysmal Cold Hemoglobinuria 4d, GEPD deficiency 2. This condition is characterized as MAHA with thrombocytopenia wth renal involvement due to unregulated activation of aterative complement pathway. 2. Alypical HUS c. HELLP Syndrome b. Typical HUS o.T1P 3. Which of the following test results difereniates non-immune mediated hemolytic anemia from immune mediated ‘hemolytic anemia? 3, Positive antinuman globulin test «. Increased haptoglobin . Negative antihuman globulin test 4, Decreased haptoglobin 4, What isthe soreening test for paroxysmal noctumal hemoglobinuria? a, Osmotic Fragility Test «. Hams Aciifed Serum Test b. Aulo-hemolysis Test 4. Sucrose Hemoiysis Tast 5, This type of rare disorder is characterized with refractory anemia with swiss-cheese appearance of heterochromatin. 2.CDAI «. COA It b.CDA I 4. HEMPAS 6, What disease is also known as the congenital pure red cell aplasia? ‘a, Fanconi Anemia c. Shwachman-Bodian-Diamond Syndrome b. Diamond-Blackfan Anemia 4, Upshaw-Schulman Syndrome 7. What isthe diagnostic tes! for congenital aplastic anemia? a, Mulliparameter Flow Cytometry ¢. Inlerphase fluorescence in situ hybridization b. Chromosomal Breakage Test d. SDS-PAGE 8. Which of the following parasites are associated with Vit. B12 deficiency? a. Necalor americanus ©. Dibothriocephalus latus ». Shistosoma haematobium 4. None of the above 9. What test wil best identify underying iron deficiency anemia in a patent with systemic lupus erythematosus? a. Zine protoporphyrin Test c. Total Iron Binding Capacity Test b. Transferrin Saturation Test 6, Soluble Transferrin Receptor Test 410. Wha isthe expected result of serum methylmalone acid in a patient with folate deficiency? a. Normal . Decreased b. Increased d, Wrong sample \idurus co. Page | 384 Laboratory Methods for RBCs and WEEs B@e Reference Values, ‘COMPLETE BLOOD COUNT (ADULT) ___ Units] Reference aes | Assay [Ut 10% pL (07H) | 4.20 6.00 | WBC x10%/ pL (x17) 36 - 10.6 RBC, female X1OuL (AOA) [3.50 — 5.70 | Neutrophil % | HGB, male yal 135-180 [Neutrophil (ANC) | x10%pL (x10%/L) 17-75 (g/t) (135 ~ 180) WCB, female g/dl T20- 7150 | Lymphocyte % a-a2 Bs (gh) (120 - 150) HCT, male % 40-54 Lymphocyte x07 pL (1071) 10-32 wy (40-054) | HCT, female % 35-49 | Monooyte % 2-11 {aly (0.35 - 0.49) Mc fL 80-100 | Monooge xT PAO) | 04-13 CH ml 2534 | Eosinophil % 1-3 MCHC g/d 32—35 | Eosinophil x07 (107) 0-03 ROW % 115145 — | Basophil % 0-2 RETIC x1O%L GO | 20-115 | Basophil x17 (HOV) 0-02 RETIC % 05-25 | Phtelet xIO%pl (0%) [150 - 450 NRBC per 100 WBC 0 MPV i 70-120 OTHER COMMON HEMATOLOGIC TESTS Assay, Units [Reference Vaties [Assay __Units | Reference Vatues] ESR, male mm Thour | 0 15 (0- §0y.0) | ESR, female mm Vhour | 020 (0 — 80y.0) festergren) 0-20 (-50y.0) | (Westeraren) 0-30 (250.0) ‘Serum Iron wok 50—160__| Serum Vit. B12 palm 200-900 TIBC g/dl. 250 400 | Haptoglobin ‘mg/m 30-200 Transferrin % 20-85 | Free serum mg/mL 0-10 saturation % hemoglobin Serum fein, male ng 40 400 | Serum folate g/m 340 Serum fern, female ng/ml. 12— 160 [RBC folate ng/mL 3120 BONE MARROW ASPIRATE REFERENCE VALUES (ADULT) Wao Diferental Reference Values WAC Differential Reference Valves Blasts 0-3 Eosinophils 0-3 Promyelocytes 1-5 Basophils 0-1 N.myelocytes 6-17 Lymphocytes 5-18 N. metamyelocytes 3-20 Plasma cells o-1 ands 9-32 Monocytes o-t N. segmental 7-30 Histiocytes (macrophages) 0-1 Enfhrocytes Series Reference Values Others Reference Values | Pronormoblasts a) MEE ratio 15 = 3:31 Basophilic Normoblasts (NB) 14 ‘Megakaryocytes 2-10 /ipt | Polychromatophilic NB E 10-20 Onthochromic NB §—10 "eon aca vs aH Mh, Bain MOTE, a Hanan ics Pps an ples Eee Sage a \durus co. Page| 39Manual Cell Counts Laboratory Methods for R df ry Methods for RBCs and WBCs u® @ ee — a saa The” counting rule Reger gm eS 6 iL a ay mene ce C8 ——_ops ecard erent! en enon FA Phin hin henge Ces Caren loot soa wee Thence andthe eo Seu we of ingroved Netaver ang ening The rs fr sncard WC ae ole AB. Co 0, The aranj quotes led 2:34 and Sar ued ler veel cout. Manual Cell Counts, = performed using a counting chamber or hemocytometer ~ manual dilutions are made with calibrated, automated pipettes and diluents Hemacytometer - Levy Chamber with improved Neubauer Ruling (MOST COMMON) Composition Part 2. 2 raised surface b. H= shaped moat c. Counting area or Grid d.Depth Remarks Total area: 9mm? = 1 grid is composed of 9 squares, each measures mm x 1mm = Each comer squares (WBC squares) is subdivided to 16 squares ‘Area per square: .0625mm? (1/16) Each square is 0.2mm x 0.2mm the cover sip: 0.1mm Each surface: 3mm x 3mm counting area or grid = Center Square is subdivided to 25 squares Area per square: 04mm? (1/25) ; 7 ~ the distance between the counting surface and insane oc Csntig belek no es odes re ‘ote Contig te bt at ph pen ek a8 roamed | GENERAL FORMULA j,T8scout= cols cout aon car ‘Area (mm?) x depth (0.1) oR Teal coun'= cls cure x on fadors 10° i = caer Example: The NT perfomed 2 manual WBC count ‘using 1:100 dilution of acetic acid. An | separates the 2 raised surface = average of 45 calls weve eoured inthe ce counting area on both sides of the chamber. Calculate the WBC count. WAC count= sels counted dluton factor 10° ea (mm) = _s8colisx 100 x om | WaCcount = “so00%mm*orsoont oF 50x 10%pLor50x 10" Manual Cell Counts with Mé mmon Dilutions and its Counting Areas Celi Counted _ Dilution | Objective rea Coan White Blood Cells | 1% anunoniuin «ila | 10x ‘4mm "2-86 acatie acid 10x omn 1% tydrchlonic acid : Red Blood Gals Tsoluntc Saline F200 | 40x | O26 sal squares of the conta squaey Shortcut factor= 10,000 Plats Teammennm ontae | 1100 | 40 Truss pase conta rpg Midurus co. Page| 40= White Blood Cell Count ELEVATED LEVELS + Bacterial infections, leukemia, pregnancy, HDN, ulcers Viral diseases (measles), brucellosis, typhoid fever, infectious hepatitis, rheumatoid arthritis, lupus WBC Diluting fluids = 1-3 % acetic acid = 1% hydrochloric acid | NOTE: = Turks fluid: aqueous yentian violet and glacial acetic acid | Anytime there are 8 or more | nucleated red blood eels per 100 PRESENCE OF NUCLEATED RBC (NRBC) white blood cels in a diferent = NOT LYSED by WBC diluents, : | the WBC count SHOULD BE lents, caused FALSE increase in WBC CURED ADULT: >5 NRBCI100 WBC differential count NEWBORN : >10 NRBC/00 WAC differential count Formuta: — | Corrected WBC Uncorrected WBC x 100 100+ # of NRBC per 100 WBC counted Example: = Given @leukoryte count of 180 xio¥t and | “ORS WBC = ___Uncorected W8C___x 100 an NRBC count of 15/100 WAC. What isthe ee econ corrected WBC count? © Botox 100 100-15 [ConectesWaC = 1565x101 = Red Blood Cell Count = highest in the morning and lowest in the evening ~ elevated levels are observed in Polycythemia Vera and secondary polycythemia (due to dehydration and increased altitude) - Decreased level in anemia RBC Diluting Fluids - Normal Saline Solution (NSS) - Toisson's ~ 3.8% sodium citrate ~ Bethell’s - Dacies or Formol citrate ~ Gowers ~ Hayern’s = Platelet Count Platelets are difficult count manually because of the following: - Platelets adhere to foreign objects and to each other = They are small and can be confused easily with dirt or debris, = REFERENCE METHOD: (149i CONTRAST MICROSCOPY HALO APPEARANCE by: [iveciier and Cronkile = Diluting fluid: 1% Ammonium oxalate (1:100 dilution) - They are counted in the 25 small squares in the large center square * Disposable Blood Cell Count Dilution Systems (Unopette System) For WBC and Platelet Counts: = Capillary Pipette (calibrated up to 20uL) = Diluent Reservoir System ~ contains 1.98mL of 1% buffered ammonium oxalate ay \durus co. Page| 41<< g HEMOGLOBIN DETERMINATION 2@ Hemoglobin Determination IREE : 3 x RBC = Hemoglobin 4, COLORIMETRIC 3x Hgb = Hematocrit (+ 3%) a. Direct/Visual ' = Acid Hematin s ~ Alkali Hematin | Microhematocrit b. IndirecUPhotoelectric 1 -Hematocrit is the volume of packed RBC ‘occupying a specific volume of whole blood (or LA) ~ Measured by placing whole blood in capilary tubes and centrifuging to read the packed cell ~ Cyanmethemoglobin/Hemiglobincyanide (HiCN) 2, GASOMETRIC (Van Slyke Oxygen Capacity Method) ‘gram Hgb = 1.34 mL oxygen 2 volume 3, SPECIFIC GRAVITY METHOD (Copper Sulfate Method) : Adam Method: = 30mL per container! 25 tests 1 Capillary tube: length. 7-7.5 cm! 70-75mm - the solution is changed DAILY 2 Bore: {mm (1.2 mm) - Distance: drop of blood: ‘cm | Anticoagulant: Biue (none) or Red (heparin) NOTE: Acceptable drop will sink in the solution within 15 seconds: Hgb > 12.5g/dL Speed: 10,000 to 15,000g for 5 mins MACROHEMATOCRIT: Wintrobe and Landsberg ‘= Anticoagulant: Double oxalate, EDTA i Length ofthe tube: 11.5em : Bore size: 3mm i 4. CHEMICAL (Kennedy's, Wong's) i Centrifugation: 2,000 to 3,000g for 30mins ‘Note: Calibration for let sda is for ESR (top is zero) CYANMETHEMOGLOBIN METHOD 3 Calibration for rah side is for hematocrit (tn is 100) Reference method approved by the CLS! Principle: In the cyanmethemoglabin method, blood is diluted in an alkaline Drabkins solution of potassium | ferricyanide, potassium cyanide, sodium bicarbonate and a surfactant. The hemoglobin is oxidized to methemoglobin (Fe3+) by the potassium ferricyanide, KsFe (CN)s. The potassium cyanide then converts the ‘methemoglobin to cyanmethemoglobin. Hemoglobin (Fe2+) + KsFe (CN)s-> Methemoglobin (Fe) + KCN > cyanmethemoglobin Cyanmethemoglobin Reagents: Reagent | Remarks : “Potassium ferricyanide | converts ferrous hgb toferrichgb [converts HitoHiCN ___ Anticoagulant EDTA or Heparin orionic detergent | improves cell lysis; decreases turbidity _! Dilution: 4:281 (.02mL of bload to Dihydrogen potassium | - allows test reading after 3 mins compared to; ‘Sml Drabkins reagent) phosphate sodium bicarbonate (15mins) + Drains reagent is PHOTOSENSITIVE measure the color intensity at 40nm ____* “Wote: a. Al forms of hemoglobin are measured EXCEPT: Sulfiemogiotn b. Over-anticoagulant does not affect the results ' Turbidity will cause FALSE ELEVATED results to: | AUTOMATED METHODS use = High WBC count: remedy is to re-centrifuge the sample, { SODIUM LAURYL SULFATE (SLS) to convert then, read the supernatant + hemoglodinto SS gb = Hemoglobin and C: remedy: dilute 1:1 with water, This method does not generate toxic waste, results should be multiplied by 2 i Vikurus co. Page | 42i | RETICULOCYTE COUNT Ww Reticulocyte Count - INDEX of BM production of RBC -Reticulocytes are the final stage before an RBC ~ Reliculocytes may be counted manually to determine the RBC production and release from the bone marrow - INCREASED IN: hemolytic anemias, acute blood loss = DECREASED IN: Aplastic Anemia, Severe IDA reaches maturity. PRINCIPLE: Whole blood (with EDTA) is stained with SUPRAVITAL STAIN such as New Methylene Blue. Any non-nucieated RBC that contains two or more particles of blue stained granulofilamentous material after new ‘methylene blue staining is defined as a reticulocyte. The number of reticulocytes in 1000 RBCs is determined. Other SUPRAVITAL STAINS: Now Methylene Blue, Brilliant Cresyl Blue (for Heinz bodies), Nile Blue Sulfate ‘the number of rai i i 2 Ratculocyte Count] he muntr of ebaaRYes MOOT Tao) = nurberof rules x 100 1000 (RBCs counted) 1 Absolute Ratculooye | rele th aaa ramberf = : Ga refclogtesin tLofvbole boot, | ARS = srs x red cell count (10°2/L)x 1000 ‘. Corrected Reticulocyte | correcis the observed reticulonje Count court to anormal hematocnt of 45 |CRC = reticulocyte (%6) x patient hematocrit (L/L) UL to allow corecton forthe degree normal hematocrit (0.45 L/L) of patient anemia “4 Reculoojte Production | gereralindeator ofthe rate of FBC = Index production RPI = crc maturation time of rates Pale Hentort Comet Factor |__Mauraion Tine, Days) w-8 = fl Sesser a is eas 2 ae a ee aeennnreIE=a $s <5 3 “MILLER DISC = designed to reduce the labor-intensive process. = composed of 2 squares, with the area of the smaller square 1/9 the area of larger square. = the disc is inserted in the EYEPIECE of the microscope | Reticulocyte (94) = _number of reticulocytes in square A (large square) x100 ] } _fumbar of eticulocytes in square A (2 umber of RBCS in square B (small square) x 9 | Miller Outar Disc Counting Grid 4 Square Bis 1/9 the area of square A, Qa crus co. Page| 43a ane ERYTHROCYTE SEDIMENTATION RATE (ESR) 7 Erythrocyte Sedimentation Rate - screening test used to screen or monitor for various inflammatory states = The ESR looks at how much RBC setting will occur in a well-mixed whole blood sample over 60 mins. NOTE: 2, RBCs normally have a net negative charge, causing them to repel each other in a whole blood sample, leading to slow setting of the RBC over time, When a change to the charge occurs, usually resulting from increases in plasma proteins, the cells become attracted to each other, leading to increased setting speed of the RBCs. ELEVATED ESR: Pregnancy (after fist trimester), acute and chronic infections, rheumatic fever, rheumatic arthritis, ‘macroglobulinemia, tuberculosis, myocardial infarction, menstruation : DECREASED ESR : Polyeythomia, congestive heart failure, hypofibrinogenermia, and presence of RBC abnormalities Stages of ES! NORMAL VALUE (1) Initial routeaux formation (LAG PHASE: 10mins) ‘Westergren aie ‘< 50 yrs. old: O - 15¢ 1 (2) Rapid Setting of RBCs (DECANTATION: 40mins) venom ok: Dam ) Final Sedimentation of RBCs (Final Packing: 10mins) Female =< 50 fs. olf:0— 20mm > 50s, old: 0-30mm | FACTORS THAT AFFECTS THE ESR Children: 0~ 10mm 4.Erythrocytes | Level | 2. Plasma composition | Level | 3. Technical error__| Level ee ae 1 Macrocytes toc _| Fibrinogen toc _| Tilting <34eq 30% amor |! Female :0—20mm i Microcyes ‘Dec | Alpha 1 oloutin ‘nc | increased temp wwe | children :0- 13mm Poikoeytes | Dec | Alpha 2 globutn Tne | Length and diameter ae of tne ube Se” wm Potyeyhemia | Dec | Abbumin Dec _| Over antenagulant [Deo ow [neni ine_| Cholesterol Ine ft 4 NOTE: Seting te rack of ESR tubes ontop ofthe refrigerator willed othe following Witte Tn a. Falsely elevated ESR attributed to vibralions from opening and closing of the refrigerator and freezer doors a b. Falsely decreased ESR because of lower temperatures from air rushing out upon a ‘opening the refrigerator pigskin ¢. Falsely elevated ESR cause of mechanical heat tenon o Wintrbes tube is used for bath ematocn and ESR. The let sie, top mating 20 fo ESR we he igh Methods : sie op ig 0 for hentaet Wintrobe and Landsberg ‘Standard/Original Westergren Modified Westergren (Most common) a, Length: 11.5em a. Length: 200mm Length: 200mm b. Bore: Seam b. Bore: 2.5mm (internal) Bore: 2.55mm © Anticoagulant: Double Oxalate, | c. Anticoagulant: Citrate (Black) with | Anticoagulant: EDTA, EDTA 4:4 anticoagulant to blood ratio d. Calibration/graduation: up to 200 ‘+ Wiesiagrenmalod i the mos sentive math because of loger ue engi and increased sample ‘Wiebe method requires 2 smaller amount of ood and imelves ro dion ZETASEDIMENTATION RATIO. performed using Zetafuge and special capillary tubes ane =the result is dependent on the concentration of fibrinogen and y-globulins Penn A) x 100 = requires small amount of specimen and is NOT AFFECTED by anemia lacrit (%) ie Mikurus co. Page | 44 pees nentDoxo((J EXAMINATION OF THE PERIPHERAL BLOOD 4 FILM AND SO" CORRELATION WITH COMPLETE BLOOD COUNT (HEMOGRAM) Hemogram - the peripheral film evaluation is the capstone of C8C regardless the method, the numerical values should be consistent with the examination derived by examining the cells microscopically. Preparation and Staining Procedures for Blood Smear (1) Cover glass smear (Ehriich): 22mm x 22mm (2) Cover glass and slide (Beacom) (3) Wedge Smear *** ~ angle between 2 slides: 25°/30 - 40°30 ~ 45 * a. Thinner film ~ smaller blood drop - low angle ~ slow speed spread - less pressure b. Thick film - larger blood drop —_- increase angle fast speed spread - increase pressure (4) Spun Smear! Automated’ Hemaspinner = Buffy Coat Smear 1. Pxt WBC ot < 110% 2. Demostration of LE cell "Thick Blood Smear: for blood parasites FIXATIVE: METHANOL ‘STAINING: ROMANOWSKY STAIN = Wright's, Giemsa (preferred stain for parasites), Modified Wright's- Giemsa, Leishman, Jenner and May-Grunwald = Contains: Methylene blue (or Azure B) and Eosin = blood smears should be stained 2 to 3 hours of specimen collection = pH 6.8 for blood and BM staining; pH 7.2 for malarial parasite Staining Problems: Excessively Blue Stain: ‘= Thick films, prolonged staining time, inadequate washing, or too high alkalinity of stain or diluent Excessively Pink Stain "= Insufficient staining, prolonged washing time, mounting the coverslip Before they are dry, or too high acidity or stain or buffer COUNTING METHODS 8. Cross-sectional or Crenellation \ re MBCe are counted in consecutive fields asthe blood fim is moved form to side 6. Longitudinal Method - WBCs are counted in consecut head of the smear attlement Method ~ uses pattern of consecutive fields tive fields from the tail toward the BOG Fal ee P-Pressure A- Angle S - Speed S- Sample | Eat CA ok Ca ein oe fatal Ciel Rn ss ws Battlement Pattern Nikurus co. Page [45