9/23/2018. Investigation of Hemolysis in the Clinical Laboratory. Objectives. What is hemolysis?

Transcription

1 Investigation of Hemolysis in the Clinical Laboratory Jason Anderson, MPH, MT(ASCP) Field Product Specialist Objectives 1. Define hemolysis. 2. Distinguish between intrinsic and extrinsic hemolysis 3. Explain where in the body hemolysis can take place. 4. Describe how the body physiologically reacts when hemolysis is present. 5. Discuss hematological and biochemical tests, used in the laboratory investigation of hemolysis. Copyright 2018 Sysmex America, Inc. All rights reserved. 2 What is hemolysis? Premature breakdown of red blood cells Where can hemolysis occur? Within the macrophages of the Mononuclear Phagocyte System (MPS) Within the blood vessels 1

2 What is the normal aging process of red blood cells? Average life span 120 days RBCs have no nucleus cannot synthesize new cellular components Old/damaged RBCS are removed by macrophages within the MPS, most notably the Spleen A small percentage break down within the circulation cellular fragments are engulfed by macrophages. What is the MPS? Mononuclear cells with pronounced phagocytic ability that are distributed extensively in lymphoid and other organs. Process of normal RBC breakdown Urobilinogen (urine) Macrophage RBC Protoporphyrin Globin Amino acids Iron Bilirubin Reabsorbed Amino acids Binds to transferrin Unconjugated bilirubin General protein synthesis Transported to the bone marrow for incorporation into erythroblasts (hgb formation Bilirubin glucoronides Stercobilinogen (feces) What causes hemolysis? Some diseases and disease processes cause RBCs to breakdown prematurely Normal response is for bone marrow to increase hematopoiesis Causes of hemolysis can be broadly classified as being either intrinsic or extrinsic to the RBC Intrinsic occurs as a result of RBC defect Most are hereditary PNH is an exception (acquired disorder) Extrinsic occurs as a result of extracorpuscular or environmental factors patient s own RBCs as well as any transfused RBCs will be affected as long as the causative factor remains in place 2

3 Intrinsic causes of hemolysis RBC defects Hereditary conditions RBC membrane defects 1. Hereditary Spherocytosis (HS) 2. Hereditary Elliptocytosis 3. Hereditary Stomatocytosis Enzyme defects 1. Glucose-6-phosphate dehydrogenase deficiency (G6PD) 2. Pyruvate Kinase deficiency (PKD) Hemoglobin defects 1. Sickle cell disease (HbS) 2. Hemoglobin C 3. Thalassemia Extrinsic causes of hemolysis Immune Idiopathic Autoimmune Leukemia/Lymphoma Drugs Infections Alloimmune Hemolytic transfusion rxn Hemolytic disease of the newborn Non-Immune Prosethetic heart valves TTP HUS DIC Preeclampsia/HELLP syndrome Infections Malaria Clostridia Extrinsic causes of hemolysis cont. Chemical & physical agents Certain drugs Burns Snake bites Secondary to other disease Liver and Renal disease Mechanical stress March hemoglobinuria 3

4 Where does hemolysis take place? Within the Mononuclear Phagocyte System (MPS) This is referred to as Extravascular Hemolysis Directly in the circulation This is referred to as Intravascular Hemolysis Generally speaking, intravascular hemolysis is more acute and more severe 10 Intravascular hemolysis RBCs broken down directly in the circulatory system. Free Hgb and RBC Enzymes (LDH) are released in circulation. Hgb tetramer broken into dimers and immediately bound by Haptoglobin which is saturated and immediately cleared by the liver A low Haptoglobin level is a hallmark of Intravascular hemolysis. After Haptoglobin is saturated, excess free hgb is filtered in the kidneys and reabsorbed in the proximal tubules Iron is recovered and converted into ferritin or hemosiderin If rate of hemolysis is > renal tubule absorptive capacity, free hgb will be excreted in the urine (hemoglobinuria). Can be detected with urine strip. Extravascular hemolysis Occurs when RBCs are phagocytosed by macrophages in the spleen, liver and bone marrow No free hgb is released into circulation No hemoglobinemia/hemoglobinuria with extravascular hemolysis alone The breakdown of hgb within macrophages into its constituent components, heme and globin, occurs in the same manner as the normal aging process. 4

5 The body s reaction to hemolysis Kidneys sensitive to changes in oxygenation of hgb. Drop can occur due to a reduction in RBC mass (hemolysis or blood loss) Release hormone Erythropoietin (EPO) EPO is transported through plasma to BM where it accelerates erythropoiesis EPO mechanism acts like a thermostat, increasing or decreasing erythropoiesis based on need When hemolysis is taking place, the bone marrow will increase the production of RBCs relative to the amount of EPO produced If rate of RBC destruction is greater than ability for BM to compensate the individual will become anemic. Detecting hemolysis in the lab The approach in the dx of a hemolytic state involves: Establishing that RBC destruction is accelerated Establishing that erythropoiesis is increased The cause of hemolysis must be determined as well. If hemolytic anemia is suspected the following should be performed Complete blood count Reticulocyte count Peripheral blood smear examination Tests reflecting increased RBC destruction Test Intravascular hemolysis Extravascular hemolysis 1. Haptoglobin Decreased/depleted Normal 2. Serum bilirubin Increased unconjugated bilirubin Increased unconjugated bilirubin 3. Urine test strip for hgb Positive Negative 4. Urine Hemosiderin Positive Negative 5. LDH Increased Normal 6. Hemopexin test Decreased/depleted Normal 7. Urine test strip for urobilinogen Positive Positive 8. Schumm s test for methemalbumin Positive Negative 5

6 Test reflecting increased RBC production Reticulocyte Count Reticulocytes remain in the BM for ~2 days before being released into the peripheral blood # of Reticulocytes in the peripheral blood provides information about BM activity (erythropoiesis) In the event of hemolysis, the BM will try to compensate by upregulating erythropoetic activity. Reticulocyte population can be counted via manual or automated methods. Copyright 2018 Sysmex America, Inc. All rights reserved. 16 Impact of artefactual hemolysis The following practices can result in RBC lysis inside the collection tube (in-vitro hemolysis) Poor venipuncture technique Exposure to excessively hot or cold temperatures (freezing) Prolonged storage prior to analysis It is important to be aware of this, as artefactual hemolysis may be very difficult to distinguish from intravascular hemolysis. In both cases on visual inspection, the plasma will have a reddish-brown color. Copyright 2018 Sysmex America, Inc. All rights reserved. 17 Laboratory findings in artefactual hemolysis The following findings suggest that in-vitro hemolysis has taken place: A low RBC count and low HCT value with a normal HGB value. As a result, MCHC and MCH will appear raised. No reticulocytosis even in the presence of RBC fragments. Biochemical tests for intravascular hemolysis would be negative. Copyright 2018 Sysmex America, Inc. All rights reserved. 18 6

7 RBC Morphology in Hemolytic Anemias Copyright 2018 Sysmex America, Inc. All rights reserved. 19 RBC morphology assoc. with hemolytic conditions Basophilic Stippling Punctate basophilic inclusions Precipitated ribosomes Thalassemia and other anemias Copyright 2018 Sysmex America, Inc. All rights reserved. 20 Bite Cells Smooth semicircle removed from the margin of the cell Heinz bodies G6PD and drug-induced oxidant hemolysis Copyright 2018 Sysmex America, Inc. All rights reserved. 21 7

8 Howell-Jolly bodies Small, discrete basophilic dense inclusions; usually singular Nuclear remnant Hemolytic anemias Copyright 2018 Sysmex America, Inc. All rights reserved. 22 Microcytes Cells smaller than normal (<7um) Abnormal hgb production Thalassemia Copyright 2018 Sysmex America, Inc. All rights reserved. 23 Polychromatophilia Grey or blue hue frequently seen in reticulocytes Ribosome material Reticulocytosis, premature release of RBC from BM Copyright 2018 Sysmex America, Inc. All rights reserved. 24 8

9 Schistocytes Distorted, fragemented cell, 2 or 3 pointed edges Mechanical destruction Microangiopathic HA s, prosthetic valves, burns Copyright 2018 Sysmex America, Inc. All rights reserved. 25 Stomatocytes Mouth- or cuplike deformity Membrane defect with abnormal cation permeability Hereditary stomatocytosis, Immune-hemolytic anemia Copyright 2018 Sysmex America, Inc. All rights reserved. 26 Target Cells Target-like appearance, hypochromic with central hgb Relative membrane excess due to decreased hgb inside the cell Thalassemia, Hb C disease Copyright 2018 Sysmex America, Inc. All rights reserved. 27 9

10 Sickle Cells Sickle-shaped, pointed at both ends Molecular aggregation of hemoglobin S Sickle cell disorders Copyright 2018 Sysmex America, Inc. All rights reserved. 28 Spherocytes Spherical cell with dense hemoglobin and absent central pallor; usually decreased in diameter Loss of surface membrane Hereditary Spherocytosis, Immune-hemolytic anemia, incompatible blood transfusion Copyright 2018 Sysmex America, Inc. All rights reserved. 29 Copyright 2018 Sysmex America, Inc. All rights reserved

11 Case study #1: history A 59 y.o. woman reports to the ED with complaints of fatigue, shortness of breath and swelling in her right ankle. She had just returned from an out of town trip and due to mechanical issues before takeoff, she had spent 11 hours on the plane. Upon examination she was found to be hypoxemic with a saturation of 88%. Her d-dimer was elevated and CT angiogram of the thorax revealed a left segmental pulmonary emboli. A CBC was also performed and results revealed a hemoglobin of 7.2 g/dl. Patient was referred to local hematology/oncology center for a hematology consultation. Case study #1: hematology results Copyright 2018 Sysmex America, Inc. All rights reserved. 32 Case study #1: hematology results - cont. RETICULOCYTE Final Value Unit Reference Range Retic % 17.9 High % Retic Absolute High x10^6ul Immature Retic Fraction High % Reticulated Hgb 37.1 High pg Questions 1. Based on what we know so far, can we conclude that this is a hemolytic anemia? 2. What can we gather from the test results thus far? Copyright 2018 Sysmex America, Inc. All rights reserved

12 Case study #1: hematology results - cont. Manual differential/smear review was performed What do you see? Spherocytes Copyright 2018 Sysmex America, Inc. All rights reserved. 34 Case study #1: biochemistry results CMP, LDH, and Haptoglobin was ordered All chemistry results were within the normal reference range except for the following Result Value Units Range Glucose 117 High Mg/dL Total Bilirubin 2.4 High Mg/dL LDH 771 High U/L Haptoglobin < 10 Low Mg/dL Copyright 2018 Sysmex America, Inc. All rights reserved. 35 Case study #1: diagnosis Additional medical history reveals patient has Rheumatoid Arthritis (RA) and is taking Methotrexate as part of their treatment plan Diagnosis? Autoimmune Hemolytic Anemia People with one type of autoimmune disease, such as RA, are prone to develop another type of autoimmune disease. Drugs such as Methotrexate can also trigger an AIHA Copyright 2018 Sysmex America, Inc. All rights reserved

13 Case study #1: Treatment Patient was treated with Prednisone and blood transfusions Weekly lab tests were performed to monitor progress Patient did not tolerate the prednisone very well, so her treatment was switched to Rituximab which resulted in a remission state of the patient s AIHA Copyright 2018 Sysmex America, Inc. All rights reserved. 37 Case study #2: history 39 y.o male admitted to the hospital with complaints of epigastric pain, nausea and vomiting. CT scan revealed mass in the pancreatic head plus liver lesions Biopsy positive for adenocarcinoma of the pancreas Patient started on Folfirinox as palliative chemotherapy After 2 cycles of chemo, bilirubin increased and CT scan showed disease progression Chemotherapy switched to Gemzar and Abraxane Copyright 2018 Sysmex America, Inc. All rights reserved. 38 Case study #2: history cont. After the 1 st cycle of the new treatment regimen, pt s hemoglobin dropped significantly. Lab tests were drawn including a CBC with manual diff, retic panel, CMP, LDH, Haptoglobin and Iron studies Copyright 2018 Sysmex America, Inc. All rights reserved

14 Case study #2: hematology results Copyright 2018 Sysmex America, Inc. All rights reserved. 40 Case study #2: hematology results cont. Result Units Ref. Range Copyright 2018 Sysmex America, Inc. All rights reserved. 41 Case study #2: hematology results - cont. Manual differential/smear review was performed What do you see? Schistocytes Copyright 2018 Sysmex America, Inc. All rights reserved

15 Case study #2: hematology results - cont. Copyright 2018 Sysmex America, Inc. All rights reserved. 43 Case study #2: biochemistry results CMP and Iron study results were unremarkable aside from a slightly elevated BUN, AST and moderately elevated ALP What is the Haptoglobin and LDH telling us? Copyright 2018 Sysmex America, Inc. All rights reserved. 44 Case study #2: diagnosis Diagnosis? Microangiopathic Hemolytic Anemia (MAHA) Additional testing done to try to find the cause of the MAHA including: Enteric Pathogen screen, ADAMTS13 and G6PD. All were ruled out. Conclusion was MAHA was drug related, most likely the Gemzar as there is documentation that hemolytic anemia can be a side effect of this drug. Copyright 2018 Sysmex America, Inc. All rights reserved

16 Lab investigation of hemolysis - summary Artefactual hemolysis can mimic intravascular hemolysis. The laboratory must make every effort to identify its presence An elevated reticulocyte count is essential for the diagnosis of hemolysis as this signals increased RBC production. RBC morphology is informative in determining the cause of hemolysis. Biochemical tests of hemolysis confirm the presence of RBC breakdown and are useful in distinguishing between intravascular and extravascular hemolysis. Copyright 2018 Sysmex America, Inc. All rights reserved. 46 References Sysmex Europe (2015, October 22). SEED: Laboratory Investigation of Haemolysis. Retrieved 9/4/18 from ysis.pdf U.S National Library of Medicine (2016, November 23). Mononuclear Phagocyte System MeSH Descriptor Data Retrieved 9/4/18 from Bain, B.J. (2005, August 4). Diagnosis from the Blood Smear. Retrieved 9/4/18 from 16