Hematology - 08 Intro to RBC Disorders and Anemias

INTRODUCTION TO ERYTHROCYTE DISORDERS AND ANEMIA OUTLINE • Anemia o Clinical Findings of Anemia o Mechanisms of Anemia o Laboratory Diagnosis of Anemia • Classification of Anemia o Morphologic Classification o Pathophysiologic Classification o Anemia due to Blood Loss o Anemia due to Accelerated RBC Destruction o Intrinsic Hemolytic Anemia (Membrane Defect) o Intrinsic Hemolytic Anemia (Metabolic Defect) o Intrinsic Hemolytic Anemia (Hemoglobin Defect) o Extrinsic Hemolytic Anemia o Immune Hemolytic Anemia o Autoimmune Hemolytic Anemia o Iso/Alloimmune Hemolytic Anemia o Drug-induced Immune Hemolytic Anemia • • ERYTHROCYTE DISORDERS • 2 main type of RBC disorders o ↑ RBC ct. - Polycythemia (not beneficial) ▪ ↑ Hct >53% men; >51% women ▪ Present symptoms affecting oxygen transport • Hyperviscosity - thicker blood causing slower flow of blood in circulation decreasing oxygen flow to the tissues • Hypervolemia - increased blood volume causing increased pumping of blood o ↓ RBC ct. - Anemia (not a disorder but manifestation) • • PHYSIOLOGIC ADAPTATIONS • ANEMIA • • • • • • Greek word anaimia: “without blood”; “lack of blood” Deficiency of red blood cells or of hemoglobin in the blood Decrease in oxygen delivery to the tissue Manifestation of other underlying disease Functional Definition: o A decrease in the oxygen carrying capacity of the blood. It can arise if there is insufficient hemoglobin or the hemoglobin has impaired function. ▪ Insufficient Hgb - more common cause Operational Definition: o a reduction in the hemoglobin content of blood that can be caused by a decrease in RBCs, hemoglobin, and hematocrit below the reference interval. • CLINICAL FINDINGS OF ANEMIA • Pallor - paleness of skin; ↓ Hgb causing ↓ O2 leading redistribution of blood in essential organs (heart, brain, muscles) ▪ Skin only requires minimal oxygen o Low blood pressure o Hypotension Features to check on physical examination (for screening) o Pallor o Jaundice - occur in px w/ hemolytic anemia (liberated Hgb are degraded into bilirubin) o Petechiae - small red spots in skin o Hemorrhage o Bleeding of oral mucosa o Sternal tenderness o Lymphadenopathy o Cardiac murmurs/arrhythmia Severity of anemia depends on o Reduction of RBC mass ▪ Rapid loss - severe RBC ▪ Gradual loss - mild to moderate/less severe o Cardiopulmonary adaptation ▪ Tachycardia ▪ Higher heart rate = more severe anemia o Degree of progression of anemia Moderate anemia: Hgb 7-10 g/dL Severe anemia: Hgb <7 g/dL o HEMATOLOGY Classic Symptoms: Fatigue and shortness of breath o Low hemoglobin o Easy fatigability - inefficient O2 delivery due to ↓ Hgb o Dyspnea (fatigue and shortness of breath) ▪ inefficient O2 delivery o Vertigo - inefficient O2 delivery due to ↓ Hgb o Faintness - inefficient O2 delivery due to ↓ Hgb o Headache - inefficient O2 delivery due to ↓ Hgb o Tachycardia (due to rapid fall in Hgb) ▪ Increases heart rate and blood flow to improve O2 transport due to inefficient O2 delivery Response to Acute Loss of Blood (sudden blood loss) o Sudden blood loss - from trauma, injury ▪ Large amount of blood is loss in short time o Increased heart rate, cardiac output, respiratory rate ▪ Increase flow of oxygenated blood in body to satisfy O2 demand o Redistribution of blood flow to essential organs ▪ Causing pallor or skin paleness Response to Slowly Developing Anemia o Gradual decrease of Hgb, causing slower body response o Increased 2-3-DPG/BPG ▪ Liberate Hgb in peripheral circulation ▪ Decrease O2 affinity with 2-3-DPG bound ▪ Increase distribution of oxygen in tissues ▪ Decreased 2-3-DPG allow Hgb to bind O2 but cannot dissociate causing inability to distribute O2 ▪ Shift to the right o Increased EPO ▪ EPO - produced in liver & kidney stimulating bone marrow erythropoiesis ▪ ↑erythropoietic rate = ↑ RBC = ↑ RBC in circulation MECHANISMS OF ANEMIA • Ineffective Erythropoiesis o Normal production o Production of defective erythroid progenitor cells ▪ ↑ RBC precursor cells in bone marrow ▪ ↓ circulating RBCs ▪ ↓ peripheral blood hemoglobin ▪ Defective erythroid progenitor cells - undergo apoptosis (programmed cell death) before maturity ▪ Cause lack of mature circulating RBCs causing decrease Hgb Decreased Hgb stimulate liver to produce ↑ EPO stimulating bone marrow to undergo erythropoiesis ▪ Produce progenitor cells and precursor cells ▪ Ineffective compensation by bone marrow o Ex. Megaloblastic anemia, sideroblastic anemia, thalassemia Insufficient Erythropoiesis o Decreased production of erythroid precursors cell in bone marrow (↓RBC production) o Factors causing a decreased RBC production: ▪ Iron deficiency • Malabsorption - iron in diet is not absorbed • Inadequate intake - insufficient iron in diet • Excessive loss - caused by bleeding ▪ EPO deficiency (renal disease - EPO production) ▪ Autoimmune reaction (aplastic anemia, acquired pure red cell aplasia) ▪ Infection (Parvovirus B19) ▪ Bone Marrow Infiltration (myelophthisic anemia) Acute Blood Loss and Hemolysis o Traumatic injury o Shortened RBC life span o Infection-related destruction o Defects in RBC membrane o Defects in enzyme system o Antibody-mediated o Mechanical fragmentation o • • LABORATORY DIAGNOSIS OF ANEMIA • • • • • Complete Blood Cell Count with Red Blood Cell Indices Reticulocyte Count Peripheral Blood Smear Examination Bone Marrow Examination Other Laboratory Tests (non-hematologic) PERIPHERAL BLOOD SMEAR EXAMINATION • • • • • BONE MARROW EXAMINATION • • COMPLETE BLOOD CELL COUNT WITH RED BLOOD CELL INDICES • • Can be performed manually or using an automated hematology analyzer (more common practice) Determine: o RBC count - important in diagnosis o Hgb concentration - important in diagnosis o Hematocrit - important in diagnosis o RBC indices (MCV, MCH, MCHC) - one of the most important values (esp. MCV & MCHC) ▪ MCV & MCHC - morphologically classify anemia o WBC count & differential count o Platelet count o RBC histogram o RDW • • • • • Young RBCs without nucleus Contains residual RNA Assess bone marrow’s ability to increase RBC production in response to anemia; o “Poor man’s bone marrow” - test bone marrow w/o bone marrow study Crucial for determining whether an anemia is caused by early RBC destruction or increased RBC production ↑ reticulocyte count = acute blood loss/↑ RBC production ↓ reticulocyte count= decreased RBC production Bone marrow aspiration and biopsy may help in establishing the cause of anemia o Bone marrow aspiration - preformed in posterior iliac crest, sternum, ribs Can point to the underlying cause of the anemia o Include abnormal cellularity (e.g., hypocellularity in aplastic anemia) o Cellularity - ratio between red marrow & yellow marrow o Hypocellular - higher yellow marrow than red marrow o Red marrow - only produces blood cells Evidence of ineffective erythropoiesis & megaloblastic changes o Eg. Folate/vitamin b12 deficiency or myelodysplastic syndrome o Ineffective erythropoiesis = defective precursor cells o Defective precursor cells - seen only in bone marrow; do not mature in circulation due to apoptosis before maturity Gold standard for diagnosis of iron deficiency anemia OTHER LABORATORY TESTS • RETICULOCYTE COUNT • • • Important in evaluation of anemia Note on RBC diameter, shape, color and inclusions o Anisocytosis - variability in size o Poikilocytosis - variability in shape o Anisochromia - variability in color/Hgb Wright-stained blood films Serves as quality control on results from automated analyzers Normal RBC: 6-8um • • • Routine Urinalysis o Hemoglobinuria - evident in hemolytic anemia ▪ Liberation of Hgb due to intravascular hemolysis causing high Hgb excreted through urine Fecal Occult Blood (FOBT) o Detect blood in stool - Indicate GI bleeding Parasitic infection o T. trichiura, hookworms, D. latum - can cause anemia Chemistry o Serum Haptoglobin, Lactate Dehydrogenase, Unconjugated bilirubin ▪ Haptoglobin - bind free Hgb, increases in hemolytic anemia to bind liberated Hgb ▪ • Lactate Dehydrogenase - enzyme where high concentration is in RBC; in hemolytic anemia, it is increase as it is liberated with Hgb during intravascular hemolysis ▪ Unconjugated bilirubin - increased degradation of Hgb from hemolysis produce increased bilirubin • Indirect bilirubin/B2 o Iron Studies ( TIBC, Transferrin, Serum Ferritin) ▪ Important in differentiating microcytic, hypochromic anemia Direct Antiglobulin Test (DAT) o Serologic test determining if the cause of intravascular hemolysis is caused by antibodies ANEMIA DUE TO BLOOD LOSS • CLASSIFICATION OF ANEMIA • • Morphologic Classification o Based on MCV and MCHC values 1. Normocytic, Normochromic anemia 2. Microcytic, Hypochromic anemia 3. Macrocytic, Normochromic anemia Pathophysiologic Classification o Based on mechanism of anemia 1. Erythrocyte destruction 2. Blood loss 3. Impaired RBC production • MORPHOLOGIC CLASSIFICATION RBC MCV MCHC ANEMIAS Morphology (80-100 fL) (32-36%) Normocytic, Normal Normal No/↓ Reticulocytosis Normochromic • Aplastic Anemia • Renal Disease w/ Reticulocytosis • PNH • Sickle Cell Anemia • Enzyme Deficiencies • Hemolytic anemia Microcytic, Low Low Thalassemia Hypochromic <80fL <32% Anemia of Chronic Disease (TAILS) Iron-Deficiency Anemia Lead Poisoning Sideroblastic Anemia Macrocytic, High Normal Megaloblastic Anemia (Vit Normochromic >100fL B12, Folate def, pernicious anemia, D. latum infection) Non-megalobastic Anemia (Liver Disease, Alcoholism, Bone Marrow Failure) • • Subtypes of Normocytic, Normochromic - Normocytic, Normochromic with & without Reticulocytosis More commonly used in classifying anemia ANEMIA DUE TO ACCELERATED RBC DESTRUCTION • • • • • Blood Loss (Hemorrhage) o Acute o Chronic Hemolytic (Erythrocyte Destruction) o Inherited Defects/Intrinsic Hemolytic Anemia (RBC) o Acquired Disorders (not RBC caused) Impaired or Defective Production o Aplastic o Iron Deficiency o Sideroblastic o Anemia of Chronic Disease o Megaloblastic Anemias due to Henolysis Intrinsic Hemolytic Anemia o Defect of RBC itself (membrane, metabolic, hemoglobin defects) o Normocytic, normochromic with reticulocytosis Extrinsic Hemolytic Anemia o Factors outside RBC and acting upon it. INTRINSIC HEMOLYTIC ANEMIA (MEMBRANE DEFECT) HEREDITARY SPHEROCYTOSIS (HS) • • • • • • PATHOPHYSIOLOGIC CLASSIFICATION • Acute Post-Hemorrhagic Anemia/Blood Loss o Blood is lost over a short period of time in amounts sufficient to cause anemia o Rapid loss of >20% blood volume o Occur due to trauma or severe injuries o Laboratory Findings: ▪ RBC: normocytic, normochromic ▪ Decreased RBC count, Hemoglobin, Hematocrit • Initially, Hct & Hgb not decreased; blood cell and plasma loss is balanced • Compensation causes hypovolemia making extracellular fluid move into the intravascular space diluting RBC in blood ▪ Increased Reticulocyte Count (3-5 days) Chronic Post-Hemorrhagic Anemia/Blood Loss o Loss small amount of blood over extended period o Common cause: GI bleeding o Laboratory Findings: ▪ RBC: Microcytic, hypochromic • Initially, normocytic, normochromic • As more blood loss, more iron is lost • GI bleeding - leading cause of iron deficiency ▪ Retics: Normal or slightly increase ▪ Hgb/Hct: Low ▪ low serum iron and ferritin • aka Congenital Hemolytic Jaundice/Anemia Most common membrane defect; common in northern European ancestry Autosomal Dominant trait (75%), Nondominant (25%) Protein mutation that disrupts vertical linkage between transmembrane proteins and underlying cytoskeleton o Cause increased sodium permeability of membrane Hallmark: Spherocytes on PBS Characterized by: o Splenomegaly, jaundice, anemia o Increased permeability to Na o Decreased RBC lifespan ▪ Due to spherocytes trapped in splenic sieve which are phagocytized by splenic macrophage Laboratory Findings: o Osmotic Fragility Test - increased ▪ Due to decreased surface-to-volume ratio of cell o Reticulocyte count – increased (5-20%) o Direct Coombs Test (DAT) – Negative o MCV – normal to slightly decreased o MCHC – increased o WBC & platelet cts. – increased during period of hemolysis o Total bilirubin - elevated • Peripheral Smear: o Microspherocytes - cells smaller than normal RBC and with no central pallor o Polychromasia - indicate reticulocytosis HEREDITARY ELLIPTOCYTOSIS (HE) • • • • • • PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) • • • aka Marchiafava-Micheli Syndrome Rare chronic acquired defect of the RBC membrane which renders it sensitive to lysis by complement causing chronic intravascular hemolysis (acidic pH at nighttime) o Patient lack CRP (complement-regulatory protein) RBC lacks glycosylphosphatidylinositol (GPI) - anchored protein: o Decay Accelerating Factor (DAF) – CD55 o Membrane Inhibitor of Reactive Lysis (MIRL) – CD59 o Deficiency allow complement binding in RBC membrane producing and end product complement of MAC (membrane attack complex) resulting to lysis HEREDITARY PYROPOIKILOCYTOSIS (HPP) • • • • • • • • Characterized by: o Anemia o Pancytopenia o Thrombosis (Budd-Chiarri Syndrome) ▪ Budd-Chiarri Syndrome - hepatic vein thrombosis o Hemoglobinuria, Hemosiderinuria o Jaundice Laboratory Findings: o Sucrose Hemolysis Test - Positive ▪ Aka Sugar Water Test o Ham’s Acidifed Serum Test – Positive (hemolysis) ▪ RBC mixed w/ complement-containing serum in low pH allowing binding of complement in RBC membrane activating complement and producing MAC causing lysis o Hemosiderinuria o RBC: Normocytic, normochromic o Reticulocytes - increased aka Ovalocytosis Autosomal Dominant inheritance Involves defect association of spectrin, resulting in free, unconnected dimers o Spectrin - RBC membrane in pairs; RBCs have normal lifespan; px are often asymptomatic (90%) o 10% - develop moderate to severe anemia Laboratory Findings: o OFT - increased Peripheral Smear: o ≥25% elliptocytes or ovalocytes (only RBC) ▪ ↓ surface:volume ▪ Elliptocytes - cigar-shaped RBC; elongated form ▪ Ovalocyte - oval-shaped o Retics and nRBC – normal shape Autosomal recessive disorder Severe hemolytic anemia with poikilocytosis among infants Rare subtype of HE (also involves defective spectrin) Seen in blacks Laboratory Findings: o MCV – low (50-65 fL) o RBC fragments after 1 hr incubation at 41-45C (heat sensitivity); normal RBC do not fragment at 49C Peripheral Smear o Microspherocytes, elliptocytes o RBC fragments initial observation after incubation OVERHYDRATED HEREDITARY STOMATOCYTOSIS (OHS) • • • • • Aka Hereditary Hydrocytosis RBCs become overhydrated due to membrane defect o Cause moderate--severe anemia due to RBC lysis RBC membrane is excessively permeable to sodium and potassium Laboratory Findings: o OFT – increased o Retics count – increased o Serum potassium – decreased Peripheral Smear: o Stomatocytes – 5-50% of RBC (slit/mouth-like RBC) HIGH PHOSPHATIDYLCHOLINE HEMOLYTIC ANEMIA • • • Inherited condition and represents an imbalance in the membrane phospholipid content of the RBC. The anemia may increase in the presence of infection or under condition of stress Laboratory Findings: o Causes mild anemia with morphologically normal cells INTRINSIC HEMOLYTIC ANEMIA (METABOLIC DEFECT) • DEHYDRATED HEREDITARY STOMATOCYTOSIS (DHS) • • • • • Aka Hereditary Xerocytosis RBC membrane has defect in cation permeability o Also presents defect in water entry in RBC causing dehydrated RBC (xerocytes) Most common form of stomatocytosis Laboratory Findings: o OFT – decreased o Retics count – increased o Jaundice o Splenomegaly Peripheral Smear: o Stomatocytes – up to 10% of RBC G6PD (glucose-6-phosphate dehydrogenase) DEFICIENCY • • • Rhnull DISEASE • • • • • It represents an absence of all Rh-Hr antigens on the red cells which results in membrane abnormalities inherited due to gene suppression or presence of silent Rh gene (Xo - encodes for D antigen for Rh blood group) o Deficiency of all antigens for Rh blood group system Mild to moderate hemolytic anemia Laboratory Findings: o Retics count – moderately increased o OFT - increased Peripheral Smear: o Stomatocytes and spherocytes RBC enzymopathies - defect in enzymes in RBC or with enzymes responsible in RNC metabolic processes • • • Sex-linked enzyme defect Most common among African and Mediterranean cultures o African - mild cases o Mediterranean - most severe cases Most common enzyme deficiency in the hexose monophosphate shunt (relies in G6PD) o G6PD - important enzyme o RBC become susceptible to oxidation by oxidants (H2O2) o Normal RBCs produce reduced glutathione which reduce oxidants to water and O2 o Absence of G6PD allow oxidation of Hgb to Methemoglobin (cannot bind oxygen) Most common RBC enzyme defect Oxidation of hemoglobin to methemoglobin (Fe3+); denatures to form Heinz bodies (Hgb precipitates) o Heinz bodies - removed from the body by hemolysis causing hemolytic anemia Anemia is unusual in G6PD px o Anemia occur when exposed to oxidants (sulfa drugs, primaquine, soy, fava bonds) HEREDITARY ACANTHOCYTOSIS • • • • • • Aka abetalipoproteinemia autosomal recessive inheritance MTP gene mutation o MTP - needed to transfer & assemble lipids into apo-B lipoproteins (LDL, VLDL, CM) Mild anemia associated with steatorrhea (fat in stool sample), neurological and retinal abnormalities Laboratory Findings: o Absent in Plasma: CM, LDL, VLDL o Decreased: Cholesterol (LDL), Triglyceride (CM, VLDL) o Retics: Normal to slightly increased Peripheral Smear: o Acanthocytes: 50-100% of RBCs Laboratory Test Methyl violet or crystal violet stains Dye reduction test Principle Heinz bodies stain G6P, NADP, and brilliant Cresyl blue dye mixture is incubated; if G6PD is present in hemolysate, NADP will be oxidized, and blue dye will change color Ascorbate Hemoglobin is oxidized to HbM by ascorbate cyanide test more rapidly in G6PD-deficient patients because they lack GSH Fluorescence A drop of G6P and NADP is placed on filter spot test paper with patient’s hemolysate; if G6PD is present, NADP is oxidized to NADPH, which fluoresces with UV light Quantitative Rate of reduction of NADP to NADPH can be Assay of G6PD measured spectrophotometrically at 340nm PYRUVATE KINASE (PK) DEFICIENCY OTHER RBC ENZYMOPATHIES • • • • • • • Autosomal recessive 2nd most common RBC enzymopathy Most common RBC enzyme deficiency in EmbdenMeyerhoff pathway o Pyruvate kinase - convert phosphoenolpyruvate to pyruvate; ATP is also a product o ↓ pyruvate kinase = ↓ ATP product Decreased RBC deformability due to decreased ATP Mild to moderate hemolytic anemia with splenomegaly Peripheral Smear: o Usually normal RBC in PBS o Post-splenectomy ▪ Echinocytes/burr cells (3-10%) ▪ Target cells ▪ Howell-Jolly bodies, Pappenheimer bodies ▪ Polychromasia (↑ reticulocytes) • INTRINSIC HEMOLYTIC ANEMIA (HEMOGLOBIN DEFECT) • • • • Laboratory Findings: o Reticulocytes – increased (5-fold post-splenectomy) o Fluorescent Spot Test – positive o Quantitative Assay for PK o WBC & Platelet counts – normal to slightly increased o Indirect bilirubin – increased o LDH – increased o Serum haptoglobin – decreased o Urinary Urobilinogen - Increased • • • 3rd most common RBC enzyme deficiency P5’NT-1 – dephosphorylates pyrimidine-5’-ribonucleoside monophosphate to form ribonucleoside & inorganic phosphate o P5’NT-1 deficiency accumulate pyrimidine-5’ribonucleoside monophosphate causing coarse basophilic stippling in RBCs Occurs in lead poisoning and probably responsible for the basophilic stippling in that condition. Mild to moderate hemolytic anemia with reticulocytosis, jaundice and splenomegaly Diagnostic procedure: o Marked basophilic stippling in red cells o Retics count increased o Decreased nucleotidase activities This are disorders that can occur from genetic mutation giving rise to either hemoglobinopathy or thalassemia. Hemoglobinopathies o Qualitative defect o They represent majority of abnormal Hb disorders. o Results from a single amino-acid distribution in one of the polypeptide-chains alpha, beta, gamma, delta ▪ Replacement of at least one amino acid which can change the function of hemoglobin ▪ Can cause defective Hgb Thalassemias o Quantitative (abnormally long/short polypeptide chain) o They are caused by abnormal long or short polypeptide resulting from gene coding termination, errors, frame-shift mutation, cross over in phase, deletion of codons of fused hybrid chains. EXTRINSIC HEMOLYTIC ANEMIA • • PYRIMIDINE-5’-NUCLEOTIDASE DEFICIENCY (P5’NT-1) • • Glucosephosphate isomerase, Triosephosphate isomerase, hexokinase and diphosphoglycerate mutase deficiencies o Causes an abnormality in anaerobic glycolysis/EMP Glutathione synthetase, glutathione peroxidase, glutathione reductase deficiencies. o Enzyme required in HMP o Hemolysis increases w/ oxidant drug exposure or infection Causes Normocytic, normochromic anemia w/ reticulocytosis Causes: o Chemical agents – drugs, chemicals o Physical agents – Heat, trauma o Vegetable and animal poisoning o Infectious agents – Malarial parasite, bacteria o Presence of autoantibodies, isoantibodies or drug-related antibody causes IMMUNE HEMOLYTIC ANEMIA • • • • • Result of an antibody-mediated mechanism assoc. with: o Autoantibodies - Ab directed against self-antigen (Ag) o Isoantibodies (alloantibodies) - Ab against foreign Ag o Drug-related antibodies - Ab against drugs/metabolite Antibodies bind to RBC which can activate complement which can bind to RBC RBCs bound with antibodies/complement are removed from the circulation intravascularly/extravascularly o Intravascular removal - cause hemolysis Anemia will only occur if hemolysis are no longer compensated by bone marrow Two main class of Antibody involved: o IgG - reacts at 37C; warm acting Ab o IgM - reacts at - -394C; cold acting Ab • Direct Antiglobulin Test - aka Direct Coomb’s Test o Detect in vivo sensitization of RBCs ▪ Tests if RBC are sensitized by Ab o Reagent - polyclonal antihuman globulin (AHG) ▪ Has FC receptor specificity in Ab ▪ Binds to Ab ▪ Cause agglutination (+ rxn) • • • • Indirect Antiglobulin Test o In vitro sensitization of RBC o Detect Ab that can sensitize RBC o RBC has no Ab coating but serum has Ab o Reagent RBC have antigen in cell surface ▪ May react w/ Ab in serum causing binding of Ab w/ Ag o After binding, AHG is added which may cause agglutination (+ rxn) o Present Ag in reagent RBC react with specific Ab PAROXYSMAL COLD HEMOGLOBINURIA (PCH) • • AUTOIMMUNE HEMOLYTIC ANEMIA • Involves autoantibodies WARM AUTOIMMUNE HEMOLYTIC ANEMIA (WAIHA) • • • • • Most common autoimmune hemolytic anemia RBC coated with IgG and/or complement Causes: o Idiopathic - unknown cause o Secondary: Lymphoproliferative disease, nonlymphoid neoplasm, autoimmune disorders, immunodeficiency, viral infections Laboratory Findings: o Reticulocytosis o Autohemolysis Test – Positive ▪ Presence of intravascular hemolysis o OFT – increased o DAT – positive ▪ IgG only ▪ IgG + C3b ▪ C3b only - rare finding o MCHC – increased Peripheral Smear: o Spherocytosis o Anisocytosis o Polychromatophilia - due to reticulocytosis o Macrocytosis o nRBC present Causes: o Idiopathic o Secondary: Mycoplasma pneumoniae infection, infectious mononucleosis, other viral infections Laboratory Findings: o MCHC: Increased (>37%) o DAT – Positive o Cold Agglutinin Titer – Increased o Bilirubin – increased o Reticulocytosis o Prewarm blood specimens before testing (maintain at 37C) Peripheral Smear: Polychromatophilia, Spherocytosis, Agglutination of RBC (from IgM) o Prewarm the slide at 37C to dissociate Ab and prevent agglutination of RBC • • • Acute form of cold-reactive hemolytic anemia Associated antibody: Donath-Landsteiner Antibody (Anti-P) o Anti-P binds to P antigen in RBC promoting complement binding (<20C) ▪ Antigen-antibody complex activate complement o Complement coated RBC lyse when warmed to 37C ▪ Complement can cause RBC lysis o At 20C - sensitization of Ag of Rb w/ Anti-P o At 37 - lysis Hemolysis will only occur of blood is warmed after exposure to cold temperature Causes: o Idiopathic o Secondary: late-stage syphilis, viral respiratory infection, non-Hodgkin lymphoma Laboratory Findings: o Retics count – Increased o Donath-Landsteiner Test (Rosenbach/Ehrlich/Sanford Test) – Positive ▪ Use two patient specimen: ▪ Sample 1 (test sample): Incubated at 4C (30 minutes), then at 37C (30 minutes) • 4C - complement binding/activation • 37C - MAC causing hemolysis • Sample should be hemolyzed ▪ Sample 2 (Px control): Incubated at 37C for 1 hour • Sample should no have hemolyzed o Increased indirect bilirubin o Hemoglobinuria COLD AUTOIMMUNE HEMOLYTIC ANEMIA (CAIHA) • • aka Cold Agglutinin/hemagglutinin Disease RBC coated with IgM that reacts optimally at 4C - >30C o >30C - no reactivity w/ IgM; dissociation of Ag from IgM o Assoc. antibodies: Anti-I, anti-i, or anti-Pr ▪ Anti-I - majority; anti-Pr - rare finding ▪ Ii - blood group system; most RBC have I antigen • Can virtually agglutinate almost all RBC ▪ I Ag - virtually present in all adult RBC surfaces • Anti-I can agglutinate all RBC • Peripheral Blood Smear: o Polychromasia o Spherocytes, schistocytes o nRBC o Anisocytosis, Poikilocytosis o Erythrophagocytosis - phagocytosis of RBC by phagocytes due to opsonins surrounding RBC ISO/ALLOIMMUNE HEMOLYTIC ANEMIA • • • • • Immune response to foreign antigen Occurs in newborns following transplacental passage of maternal anti-fetal red cell antibody. 2 types are basically due to blood group incompatibility Acute/Delayed Hemolytic Transfusion Reaction o ABO incompatibility o Rh incompatibility Hemolytic Disease of the Newborn/Fetus HEMOLYTIC TRANSFUSION REACTION • • • • Recipients’ plasma has antibodies against antigens present on donor RBC causing hemolysis o Patients are transfused with incompatible blood May be due to ABO and/or Rh incompatibility (and other blood group) o Eg. Recipient - “A”; has Anti-B (naturally occurring) ▪ Donor - “ B”; has Anti-A ▪ B Ag in Donor’s blood react w/ anti-B in recipient’s plasma causing hemolysis Acute HTR – occurs within minutes to hours of transfusion o Most common cause is transfusion of ABO incompatible blood ▪ Due to naturally occurring antibodies Delayed HTR – occurs days to week after transfusion o Associated antibody: IgG (Ab against Duffy antigens, Kidd antigens - blood groups) ▪ Associated Ab of Duffy & Kidd - immune Ab (Ab only produced when exposed to certain antigens) o Eg. Recipient - RBC Duffy (-); plasma; anti-Duffy (-) ▪ Donor - RBC Duffy (+) ▪ Recipient becomes exposed to Duffy (+) stimulating production of Anti-Duffy ▪ Production of Ab occur for days to weeks ▪ 2nd exposure to Duffy (+) - reaction of Anti-Duffy with Duffy (+); becomes Acute HTR HEMOLYTIC DISEASE OF THE NEWBORN/FETUS • • • • Due to Rh incompatibility (Erythroblastosis fetalis) o Only occurs if the mother is Rh (-) and father is Rh (+) Eg. Mother w/ Rh (-) is impregnated with Rh (+) father o Fetus is Rh (+) causing mother to form Anti-D (against Rh) o First baby may be safe - due to minimal Anti-D that reacts with Rh (+) o Second pregnancy - Ab synthesized during 1st pregnancy can act with RH Ag with 2nd baby ▪ 2nd baby may die Laboratory findings: o Severe anemia, nRBCs, Positive DAT o Very high bilirubin (kernicterus - bilirubin accumulation in brain) Treatment/Prevention: o Exchange transfusion in utero (baby’s blood exchanged) o RhoGam (Rh immunoglobulin) ▪ Bind with Anti-D present preventing HDN DRUG-INDUCED IMMUNE HEMOLYTIC ANEMIA • • • May occur following administration of drugs such as penicillin, stibophen and alpha methyldopa Antibody Characteristic: o Drug-Dependent Antibodies ▪ Requires presence of drug to activate o Drug-Independent Antibodies ▪ After initial exposure 3 Mechanisms: