Evaluation of Hemolysis

Transcription

1 Evaluation of Hemolysis Matthew Elkins, MD PhD Upstate University Hospital Hemolysis Definition: Disruption of the cell membrane of RBCs resulting in the release of free hemoglobin Intravascular hemolysis disruption within the blood stream with release of RBC contents into the circulation Extravascular hemolysis disruption outside of blood stream (in reticuloendothelial system) Hemolysis Sequelae Anemia decrease in hemoglobin below the patient s usual baseline Acute anemia loss of RBC, usually without loss of intravascular volume Chronic anemia long term decrease in RBC mass, compensation Pallor, lethargy, diaphoresis, nausea/vomiting, tachycardia, headache, dizziness, depression, shortness of breath, high output cardiac failure, death 1

2 Hemolysis Sequelae Schaer et al Blood Hemolysis Sequelae Free hemoglobin binds to nitric oxide to form methemoglobin Methemoglobin induces release of proinflammatory cytokines from endothelial cells (e.g. IL 6, IL 8) Fever, chills, SIRS Decrease in NO results in increased vascular tone and hypercoagulability resulting in hypertension and stroke Hemolysis Sequelae Schaer et al Blood 2

3 Hemolysis Sequelae Damage to renal tubule epithelium Acute tubular necrosis Acute, severe back/flank pain Can result in permanent kidney damage Hemolysis Sequelae Schaer et al Blood Causes of Hemolysis Non immune hemolysis Mechanical shearing (artificial heart valve, fibrosis) Trauma, toxins, infections Abnormal RBC or hemoglobin Microangiopathic hemolytic anemia (MAHA) Immune hemolysis Immune destruction of transfused RBCs due to alloantibodies produced by recipient Immune destruction of patient RBCs due to antibodies in transfused plasma/antibody concentrate (e.g. RhoGam) Immune destruction of patient RBCs due to autoantibody 3

4 Hemolysis workup History, physical exam Symptoms and duration, PMHx Hematology Lab tests CBC, Reticulocyte counts, Peripheral smears, Hemoglobin electrophoresis, Osmotic fragility testing, Bone marrow evaluation Chemistry Lab tests Haptoglobin, Bilirubin, LDH, Serum free hemoglobin, Urinalysis (hemoglobinuria) Blood Bank Evaluation Type and screen, DAT, Eluate, RBC antibody identification Hemolysis Work up Is the patient undergoing hemolysis? Haptoglobin Lactate dehydrogenase Bilirubin Serum free hemoglobin Urinalysis Chemistry Evaluation Haptoglobin Measured by immuno turbidometric measurement Mix patient s serum with anti human h t l bi Ab haptoglobin Abs Resultant aggregate blocks light transmittance on a spectrophotometer Hpt is also an acute phase reactant, so may see increase with inflammation Hpt is produced from the liver, so low levels can be seen due to liver failure 4

5 Patient Evaluation Hemolysis Anemic? Hemolysis may occur without anemia due to the capacity of bone marrow to replenish destroyed RBCs Anemia occurs if hemolysis exceeds erythropoiesis capacity of the bone marrow High rate of hemolysis Decreased erythropoiesis capacity Hematology Lab Tests Complete Blood Count (CBC) Automated counting of RBCs Platelets WBCs Determined by size of cellular component Measured using either impedance or light scatter Hematology Lab Tests Complete Blood Count (CBC) Measured by impedance method: Set up two chambers with maintained electrical current at set voltage + 5

6 Hematology Lab Tests Complete Blood Count (CBC) As cells pass through aperture between chambers, resistance to electrical flow increases in relation to cell size RBC and platelets tested in one reaction Second sample tested after hydrolysis of RBCs + Hematology Lab Tests Complete Blood Count (CBC) Light scatter measured using a flow cytometer Cellular constituents pass through a laser beam one at a time The light is refracted as it passes through the cell The amount of refraction (spread) correlates to the size of the cell Laser Detector Side scatter used to quantitate WBC Patient Evaluation Hemolysis Anemic What is the RBC morphology? RBC morphology can provide insight i htinto the underlying cause of anemia/hemolysis 6

7 Peripheral Blood Smear Sample of whole blood is spread on a glass slide Thin layer of blood is examined microscopically to evaluate morphology of platelets, RBCs, and WBCs Case 1: Patient Presentation 52 year old woman Recent upper respiratory infection Left frontal headache Gingival bleeding Gross hematuria New ecchymoses bilateral extremities and petechiae on trunk Case 1: Laboratories LDH 1698, Bilirubin 3.1 Haptoglobin <10 Blood Type: B+ Antibody Screen: Negative DAT: Negative 7

8 Case 2: Peripheral Blood Smear Case 2: TTP Thrombotic thrombocytopenic purpura Anti ADAMTS13 antibody or congenital defect Directly inactivates ADAMTS13 and causes increased clearance ADAMTS13 cleavesvwfextravwf extra large largemultimers Absence of functional ADAMSTS13 results in abundance of HMW vwf multimers Larger multimers activate platelets multiple thromboses Platelets used up thrombocytopenia Case 2: TTP Thrombotic thrombocytopenic purpura ADAMTS13 assay: Normal pooled plasma Patient Sample Dilution (same final conc. as 1:1 mix) Result = 53% 1:1 Mix Result=23% Corrected 23/53=43.4% Percent Inhibition=56.6% Normal >67% activity or <33% inhibition Patient result <5% activity or >95% inhibition 8

9 Peripheral Blood Smear Schistocytes Remnants of RBCs from intravascular shearing Small platelet fibrin clots transiently lodge in vessels, narrowing vascular channel and causing activation of complement cascade on vessel wall Peripheral Blood Smear Fibrin strands attach to endothelial walls RBCs passing through the vessels are transiently bound by these fibrin strands RBCs torn apart Resultant fragments cleared by the spleen Microangiopathic Hemolytic Anemias Pathophysiology Activation of platelets/coagulation cascade in small vessels resulting in fibrin stranding and microthrombi Vascular occlusion, hemolysis, consumptive coagulopathy (platelets and/or coagulation factors) 9

10 Microangiopathic Hemolytic Anemias Causes Thrombotic Thrombocytopenic Purpura Systemic malignancy Malignant hypertension Autoimmune diseases (SLE, PAN) Severe infection (sepsis, DIC) Drugs Quinine best example, but many causes Schistocytes TTP Schistocytes = mechanical shearing of RBCs Non MAHA Schistocytes Marching hemolysis / drummer hemolysis Stenotic heart valves Artificial heart valves Peri valvular leak Valvular stenosis Mechanical valves LVADs, ECMO, bypass Fibrosis of liver, bone marrow Peripheral Smear Spherocytes RBC with smaller diameter and no central clearing Caused by either: Lack of functional cytoskeletal components Antibodies specifically recognizing i self RBC antigens 10

11 Peripheral Smear Spherocytes Abs recognized by macrophages in the splenic sinusoids which phagocytize RBC membrane Both pathways result in loss of cell membrane RBC cytoplasm and membrane equilibrates Anemia results due to clearing of the spherocytes Peripheral Smear Other RBC morphologies Stomatocytes hereditary, Rh null syndrome Acanthocytes (spur cells) liver disease, dyslipidemias, McLeod phenotype Echinocyte (burr cells) renal failure, burns phosphate deficiency Degmacyte (bite cells/blister cells) oxidative hemolysis (ex. G6PD deficiency) resulting in condensation of hemoglobin which gets removed by macrophages Peripheral Smear Sickle cells, HgbC crystals Condensation of abnormal hemoglobin resulting in insoluble crystals Distorts cell membrane Intravascular and extravascular hemolysis Chronic inflammatory state 11

12 Patient Evaluation Hemolysis Anemic RBC morphology Immune hemolysis? What blood is compatible with patient? Blood Bank Testing ABO and Rh Typing Forward typing: Patient s RBCs with anti A, anti B and anti D Blood Bank Testing ABO and Rh Typing Reverse typing: Patient s plasma with A RBCs and B RBCs 12

13 Blood Bank Testing Antibody Screen non ABO antibodies: Patient s plasma with screening O cells Case 2: Patient Presentation 45 yo woman G2P2 Admitted with a subarachnoid hemorrhage due to right carotid bifurcated aneurysm. Four days after surgical repair, she was transfused two units RBC for Hgb=7.0 mg/dl. Two weeks later she was transfused two additional units (Hgb=7.1 mg/dl). Case 2: Patient Presentation Early in the 2nd unit, the patient became febrile ( C), tachycardic (99 106), hypertensive (128/72 153/76). The nurse also noted new cranberry colored urine in her catheter bag. Her past transfusion history was significant for RBC approx. 15 yrs earlier. 13

14 Case 2: Laboratory Workup LDH= 821 Bilirubin (T/D)= 1.9 / 0.5 Haptoglobin= <6 Serum Free Hemoglobin= 87 UA= Hemoglobinuria Visual hemolysis check: positive Case 2: Laboratory Workup Patient ABO/Rh type: O+ (pre, post samples) ABO/Rh RBC units 1+2: O+ Direct Coombs: negative (C3, IgG: pre, post) Repeat p Antibody screen: anti Le a, C, S (using enhanced methods) Repeat Full Crossmatch: Unit 2, Incompatible (C+, S+) + Blood Bank Testing Direct Agglutination Test (DAT) Direct Coombs test Test for antibodies or complement bound to patient RBCs Use AHG for IgG Anti C3 for IgM 14

15 Immune mediated Hemolysis with Negative DAT Antibody present, but all Ag+ cells hemolyzed IgA, IgD, or IgE autoantibody (only IgG and C3 detected) Low affinity binding antibody dislodged during wash steps in DAT Blood Bank Testing Indirect Coombs Test Tests patient serum for anti RBC antibodies Combine serum with test RBCs Add AHG to agglutinate if antibodies bound Eluate Evaluation Determine specificity of antibodies bound to the patient s RBCs Elute off antibodies from patient s RBCs Test against screen RBCs with AHG 15

16 Antibody mediated Hemolysis Alloantibody to antigen present on transfused RBCs Transfused alloantibodies into patient with RBCs expressing antigen Transfused antibody concentrate (IVIG, RhoGam) Transfused non ABO compatible plasma All reported cases are type O plasma into type A or type AB patients Antibody mediated Hemolysis Autoantibody recognizing RBC antigen Abnormal antibody recognizing normal antigen Warm autoantibody : IgG, reacts at 37⁰, extravascular hemolysis Cold autoantibody : IgM, reacts at 24 ⁰, intravascular hemolysis Case 3: Patient presentation 5 year old girl 2 week history of progressive dry cough Afebrile 37.4 (99.4 F) Vomiting, diarrhea Tired, lethargic, shortness of breath Chest pain No significant PMHx Multiple family members with cold symptoms 16

17 Case 3: Patient presentation V/S= T 37.6, HR 135, BP 98/55, RR Imaging shows bilateral pneumonia with left empyema Pleural fluid culture showed Streptococcus pneumoniae Over 3 days, Hgb 6.3 Case 3 : Strep pneumo ahus Neuraminidase produced by S. pneumo cleaves N acetylneuraminic acid on glycoproteins of cell membrane proteins Exposure of Thomsen Fi Friedenreich ihantigen (Tantigen) on glycophorins A and B on RBC and renal endothelial cell membranes Oliver et al 2010 High titers of antibodies against T antigens are normally found in all patients Case 3 : Strep pneumo ahus Binds to RBC ag polyagglutination, hemolysis Binds to renal endothelium HUS Lectins T Th Tn HEMPAS Cad Arachis hypogea Glycine max + (soja) Salvia sclarea Salvia horminum Dolichos biflorus DAT + in 90% Lectin assay most sensitive/specific Treatment may require minimization of exposure to donor plasma 17

18 Antibody mediated Hemolysis Autoantibody recognizing RBC antigen Abnormal antibody recognizing normal antigen Warm autoantibody : IgG, reacts at 37⁰, extravascular hemolysis Cold autoantibody : IgM, reacts at 24 ⁰, intravascular hemolysis Normal antibody recognizing abnormal antigen Bacterial toxins resulting in exposure of cryptic antigens or creation of foreign antigens Case 4: Patient presentation 54 year old man History of coronary artery disease, GERD Transferred to UMHS for treatment of hemolytic anemia Case 4: Patient presentation 6/25 Prostate biopsy 6/28 Sent home on antibiotics for UTI 6/28 Presents at OSH w/ fever, chills, headache, urinary frequency 7/6 For presumed AIHA, given IVIG, steroids, and 2 units RBCs 7/5 Re presents to OSH with same sx, and now hematuria 7/7 Transferred to UMHS for treatment of hemolytic anemia 18

19 Case 4: Clinical Presentation On admission: Hemoglobin 7.4, Hematocrit 21.8 LDH 1955 Total bilirubin 4.7 Haptoglobin <10 DAT UMHS Blood Bank work up Polyspecific= 3+ Anti IgG= 3+ Anti C3= 3+ IVIG given at OSH UMHS Blood Bank work up Serum: Negative Eluate: Negative 19

20 Antibiotics 6/25 Prostate biopsy 6/28 Sent home on antibiotics for UTI 6/28 Presents at OSH w/ fever, chills, headache, urinary frequency 7/6 For presumed AIHA, given IVIG, steroids, and 2 units RBCs 7/5 Re presents to OSH with same sx, and now hematuria 7/7 Transferred to UMHS for treatment of hemolytic anemia Drug induced hemolysis Drug dependent Autoantibody 20

21 Antibiotics 6/25 Prostate biopsy 6/28 Sent home on antibiotics for UTI 6/28 Presents at OSH w/ fever, chills, headache, urinary frequency 7/6 For presumed AIHA, given IVIG, steroids, and 2 units RBCs 7/5 Re presents to OSH with same sx, and now hematuria 7/7 Transferred to UMHS for treatment of hemolytic anemia Cefotetan and hemolysis Cephamycin antibiotic with coverage similar to 2 nd generation cephalosporins Most common cause of drug dependent drug induced hemolysis Commonly given prior to surgery as prophylactic Detectable drug persists on RBCs up to 92 days after last dose (Davenport et al. 2004) Treatment is supportive until drug is eliminated DAT+ with negative eluate Drug dependent antibody Most commonly beta lactam antibiotics Antibody with rare antigen specificity False positive DAT (hyperviscosity, contamination by Wharton s jelly) Dialyzed patients exposure to formaldehyde from tubing alters RBC antigens, eluate will react with formalin treated test RBCs 21

22 Questions? 22