*NCCT is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. Program, provider #122.

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1 COURSE DESCRIPTION Hemolysis is a recognized problem for both patients and medical laboratories. In patients, hemolytic anemias can cause serious medical concerns. In laboratories, hemolysis of blood specimens can affect the accuracy of test results, and it is reported to account for nearly 60% of rejected specimens. When the laboratory receives blood samples with hemolysis, it cannot automatically determine the cause, so recollection is the typical course of action. The best way to understand hemolysis is to review the big picture, including both in vivo (inside the body) and in vitro (outside the body) forms. *Valid for P.A.C.E. credit through 12/31/2019* * ASCLS P.A.C.E. is an approved continuing education agency by the California Department of Health Laboratory Field Services, Accrediting Agency #0001. *NCCT is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. Program, provider #122. Rev 6 January 2018 COPYRIGHT 2013 National Center for Competency Testing Reproduction or translation of any part of this work beyond that permitted by Sections 107 or 108 of the 1976 United States Copyright Act without the permission of the copyright owner is unlawful. No part of this work may be reproduced or used in any form or by any means-graphic, electronic, or mechanical, including photocopying, recording, taping, or information storage and retrieval systems without written permission of the publisher. 1

2 COURSE TITLE: Getting the Red Out: An Introduction to Hemolysis Author: Kay C. Bertrand, M.A. Ed, MT(ASCP) Vice President, Exam Development National Center for Competency Testing Number of Clock Hours Credit: 1.0 Course # Level of Instruction: Basic P.A.C.E. Approved: _X Yes _ No COURSE OBJECTIVES At the completion of this course, participants will be able to: 1. Differentiate between in vivo and in vitro hemolysis. 2. Describe the structure and function of the red blood cell. 3. Describe the function of hemoglobin. 4. Differentiate between hemolytic anemias that are inherited and acquired. 5. Identify causes for inherited and acquired hemolytic anemias. 6. Recognize symptoms for hemolytic anemias. 7. Recognize common laboratory tests used for diagnosis of hemolytic anemias. 8. Recognize common causes of in vitro hemolysis. 9. Understand how laboratory tests may be adversely affected by hemolysis. Disclaimer The writers for NCCT continuing education courses attempt to provide factual information based on literature review and current professional practice. However, NCCT does not guarantee that the information contained in the continuing education courses is free from all errors and omissions. 2

3 WHAT IS HEMOLYSIS? The simplest definition comes from examining the root word and suffix. Hemo refers to blood and lysis refers to breaking apart or disintegration. Thus, hemolysis occurs when the red blood cell membrane is compromised and its structure breaks down, causing the release of its cellular components. Since the largest component of red blood cells by weight is hemoglobin, and since hemoglobin gives red blood cells their characteristic red color, visible hemolysis can add a clear red tint to plasma or serum. Hemolysis may occur inside the human body (in vivo) or outside the body (in vitro). Hemolysis that originates inside the patient can cause problems for both the patient and the laboratory, depending upon the lab tests ordered. Hemolysis that originates outside the body, typically during specimen collection, can also affect the accuracy of laboratory tests. The color itself can cause problems, as can the release of the contents of the cells. This, in turn, may affect the accuracy of the diagnosis and treatment of patients. For these reasons, it is valuable for healthcare professionals to recognize and understand both types of hemolysis. IN VIVO HEMOLYSIS The red blood cell membrane is a barrier that keeps all the crucial cell components inside, allowing the cell to do its job throughout the human body during its typical lifespan of 120 days. If the membrane ruptures, the cell cannot do its job properly and its lifespan may be shortened considerably. RBC FUNCTION Red blood cells have many functions inside the body. Perhaps the most significant of them is to carry hemoglobin (Hgb), allowing the body to transport oxygen to the tissues. When people have normal amounts of normal hemoglobin, their red blood cells can do the job they were meant to do. Tissue is made up of cells; cells need oxygen to survive. To understand how red blood cells function, it is helpful to understand their composition. In humans, the hemoglobin molecule is composed of both heme and globin. Each hemoglobin molecule has four (4) globin subunits, each of which contains a protein chain and a heme group. The structure is very complex, with the globins arranged in folding patterns that are difficult to visualize in two dimensions. Heme is held within these globin folds. Each heme group contains iron. For oxygen to be properly carried by the molecule, iron must exist in the ferrous (Fe 2+ ) ion form. If there is a problem with any part of the structure of the hemoglobin molecule, or if iron exists in any other form, red blood cells may not be able to function properly. It is a delicate balance. Humans breathe to bring oxygen into their lungs, as all cells of the body need oxygen to function properly. In adults, normal adult hemoglobin molecules within red blood cells carry oxygen to cells throughout the body where it is released to provide energy for the body to carry out its daily functions. There, it also collects carbon dioxide (CO 2 ) for 3

4 return to the lungs and release from the body. All the cells of the body rely upon the red blood cells to provide them with a sufficient amount of normal hemoglobin to survive. CONDITIONS OF IN VIVO HEMOLYSIS The average span in the life of a red blood cell is 120 days. Problems may occur when red blood cells are destroyed before their normal lifespan is over. This may be a result of one of three types of hemolytic anemias: Inherited Acquired Unknown origin Inherited Hemolytic Anemias Inherited hemolytic anemias are caused by faulty genes inherited from one or both parents. Faulty genes may affect the cell membrane, cell enzymes, or hemoglobin. Some of these may be more familiar to healthcare professionals than others are. One of the most common in the United States is Sickle Cell Anemia. This and other examples are included below. Figure 1. Examples of inherited hemolytic anemias Types Sickle Cell Anemia G-6-PD Deficiency Pyruvate Kinase Deficiency Thalassemias Hereditary Spherocytosis Hereditary Elliptocytosis Causes Hemoglobin S (an abnormal hemoglobin) causes RBCs to live days instead of 120 days RBCs are missing the enzyme glucose-6-phosphate dehydrogenase (G-6-PD), making them more susceptible to rupture when in contact with trigger substances (e.g. sulfa drugs, infections) RBCs are missing the pyruvate kinase (PK) enzyme, necessary to fully metabolize glucose for energy Certain normal hemoglobins are not produced in sufficient quantities; there are fewer healthy RBCs RBC membrane defect causes cells to be spherical (rather than the normal flexible biconcave disc shape) resulting in a shorter cell lifespan RBC membrane defect causes cells to be oval shaped, resulting in a shorter cell lifespan Acquired Hemolytic Anemias Patients with acquired hemolytic anemia may have perfectly normal red blood cells, but something they acquire causes their bodies to destroy the cells inside the bloodstream or within the spleen. Acquired hemolytic anemias can be caused by immune disorders, infections, blood transfusions, medications, or mechanical problems in the body. 4

5 Immune Disorders Nearly half of all hemolytic anemias are autoimmune in origin. This means that a patient s own immune system produces antibodies that attack the patient s own red blood cells. Some of these antibodies are active at body temperature and others are active at cold temperatures (i.e. cold agglutinins). Patients with certain conditions, infections, or diseases may have a higher risk for these anemias. Following are some disease conditions that put people at risk of autoimmune hemolytic anemias. Human immunodeficiency virus (HIV) Hepatitis Systemic lupus erythematosus (SLE, lupus) Certain leukemias or lymphomas Epstein Barr virus (EBV) Cytomegalovirus (CMV) Stem cell transplants Blood Transfusions Some acquired hemolytic anemias are not autoimmune in origin. These are called alloimmune conditions. They may be caused by receiving a blood transfusion with blood that is incompatible with the patient s blood. For example, if an individual whose red blood cells are group A (i.e. cells contain the A antigen and serum contains anti-b antibodies) is given a unit of group B packed red blood cells, the anti-b antibodies will try to destroy the group B red blood cells from the transfused unit. The transfused red blood cells will be hemolyzed. Another example could be caused during pregnancy if a mother possesses antibodies against her baby s red blood cells because of an Rh incompatibility. (Note: Rh factor is just one of many antigens that can be found on red blood cells, but it is one that most people recognize.) Most typically, this occurs with Rh negative mothers that have Rh positive infants. Thankfully, the use of Rh immunoglobulin injections for Rh negative women just after miscarriage or delivery has been very effective in preventing this from happening to most women. Medications Certain medications, such as penicillin, may cause hemolytic anemias. This can occur when the medication binds to the surface of RBCs and the patient develops an antibody to the medication. This is one reason patients are always asked if they have any known drug allergies. Medication-induced hemolytic anemias can also occur in other ways and with other medications, such as anti-inflammatory drugs, antimalarial drugs, and some chemotherapeutic agents. 5

6 Mechanical Problems If blood cell membranes are physically damaged, they may have shorter lifespans. There can be many causes for mechanical RBC membrane damage; these include, but are not limited to the following. Implantation of an artificial heart valve Hemodialysis machines Heart-lung bypass during open heart surgery Serious problems with blood pressure (e.g. eclampsia) Snake venom Tick-borne diseases SYMPTOMS OF HEMOLYTIC ANEMIAS Most patients with any kind of anemia complain of being tired, a result of the lack of oxygen in their tissues. Other signs or symptoms may include the following. Pale skin Shortness of breath Cold extremities Headache Dizziness Heart problems When the anemia is hemolytic in origin, additional signs may include: Jaundice Pain from gallstones Pain from an enlarged spleen Leg pain, particularly in sickle cell anemia patients With a severe hemolytic transfusion reaction, signs may also include: Fever and chills Low blood pressure Shock DIAGNOSTIC TESTS FOR HEMOLYTIC ANEMIAS A wide variety of clinical laboratory tests may be ordered when trying to determine the cause of any anemia, including hemolytic anemia. Some tests will be ordered to rule out common causes of anemia, and other tests will be ordered to zero in on the precise cause(s). Figure 2 provides a list of tests with explanatory information. 6

7 Figure 2. Diagnostic Tests for Hemolytic Anemia Complete Blood Count (CBC) Differential (Diff): Automated or manual smear (blood on a slide) Reticulocyte (Retic) Count Direct Coombs (also called direct antihumanglobulin test or DAT) Haptoglobin Bilirubin (and other liver function tests) Hemoglobin Electrophoresis Osmotic Fragility G-6-PD Deficiency, test or screen Sickle Cell screen Bone Marrow Iron or Vitamin-related tests (i.e. B12) Includes: RBC count WBC count Platelet count Hemoglobin (g/dl) Hematocrit (% RBC in total volume) MCV (average RBC volume) MCH (average RBC hemoglobin) MCHC (average cell Hgb concentration) RDW (RBC distribution width) Provides an overview of morphologic characteristics of the cellular components in blood (i.e. shapes, sizes, inclusions, and more) These are RBCs that are newly released from the bone marrow, and are increased in many anemias Tests for antibodies that are already attached to RBCs Plasma haptoglobin levels decrease if RBCs are lysed in the bloodstream, as haptoglobin will combine with hemoglobin Increased levels indicate RBC breakdown, as bilirubin is a breakdown product Identifies abnormal hemoglobin types (e.g., Hemoglobin S in sickle cell anemia) Tests the fragility of the red blood cells (i.e. spherocytes are very fragile) Tests for the presence or amount of the enzyme; sometimes performed by state mandate on newborns Sometimes performed by state mandate on newborns Determines whether enough blood cells and platelets are being produced by the marrow These may be ordered to rule out other causes for anemia IN VITRO HEMOLYSIS When hemolysis occurs outside the body it is called in vitro hemolysis. If the hemolysis is sufficient, the plasma or serum of a whole blood sample may be tinged with a pink to red discoloration when this occurs. When the laboratory receives a blood sample with obvious hemolysis, the sample must often be recollected as hemolysis can interfere with a significant number of laboratory tests. If the test results cannot be interpreted 7

8 with certainty because the sample shows obvious hemolysis, the test is of no value in making decisions about patient diagnosis or treatment. Healthcare practitioners need to understand how important it is to try to prevent hemolysis when they collect blood samples. As the GIGO saying goes, Garbage in, garbage out. Good, clean samples help to produce good results. CAUSES OF IN VITRO HEMOLYSIS In vitro hemolysis may be a result of cellular aging, temperature extremes, and/or mechanical, chemical, or osmotic causes. These may be due to improper specimen collection, transfer of blood, storage, processing, or transport. Since blood samples can be collected from patients in several ways, collection/transfer problems vary with the apparatus used. See Figure 3. Figure 3. Hemolysis Caused by Specimen Collection Specimen Collection Issues that May Increase Chance of Hemolysis Improper site selection Drawing from a site distal to antecubital region of the arm Tourniquet on too long Interstitial fluid leaks into tissue Too small needle bore Large vacuum force causes RBC stress Too large needle bore Faster and more forceful flow through needle Wet alcohol from prep Alcohol may enter tube with blood, causing lysis of red cells Poor technique If blood flows too slowly, lumen of needle may be too close to inner vein wall, causing occlusion and rupture Syringe draws Pulling plunger back too far or too fast Blood drawn from a peripheral IV catheter Higher chances of hemolysis; loosely connected blood collection assemblies can result in frothing Hemolysis may also occur when blood samples are being processed or transported for testing. Most healthcare professionals are aware that shaking or mixing blood samples too vigorously can cause the red blood cells to rupture, which is the reason that care must be used to gently invert tubes with anticoagulant additives after blood collection. There are other processing warnings that are less commonly known. When whole blood samples are not allowed enough time to fully clot before being processed, fibrin strands can form in the serum. When processors use applicator sticks to dislodge the fibrin, red blood cells can rupture, causing hemolysis. Exposing blood to temperatures that are either too high or too low can cause hemolysis, as will the prolonged contact of serum or plasma with cells. Refrigeration of whole blood inhibits the sodium/potassium ATPase pump in the RBCs, resulting in a loss of potassium (K+) from the RBC into the plasma and diffusion of sodium (Na+) into the red blood cell. Separating serum/plasma from cells as soon as possible will reduce these effects. 8

9 Use of a pneumatic tube system in transporting blood samples may also cause mechanical trauma to blood cells resulting in hemolysis. The amount of trauma can vary with system configuration, length, and speed. EFFECTS ON LABORATORY TESTS The effects of hemolysis upon laboratory tests may vary by the amount of hemolysis and by the actual test method used, which often differs from laboratory to laboratory. Some of the most significant problems occur when there are very large differences in the concentration of a substance inside and outside the red blood cell. If there is a high concentration of any chemical inside the RBC, and a low concentration of the same chemical in the plasma, it will take very little hemolysis to release a significant amount of that chemical into a plasma or serum. Serum levels could be easily artificially elevated. As examples, in vitro hemolysis would have a proportionately larger effect on tests like potassium (K), lactate dehydrogenase (LD), or folate. Sometimes, the actual amount of hemolysis makes a difference. While there are some tests that cannot tolerate any hemolysis at all (e.g., insulin, protein electrophoresis, and triglycerides), there are other tests that may not be adversely affected by a very small amount. Thus, the best advice is to prevent hemolysis whenever possible. WHAT CAN BE DONE? To prevent hemolysis, healthcare professionals performing venipuncture should use the median cubital, basilic, or cephalic veins when possible. They should choose needle sizes with care. Tourniquets should never be left on a patient s arm for more than one minute at a time, and fists should not be pumped. If syringes are used, excessive force should be avoided when pulling back on the plunger. Alcohol cleansing should always be allowed to air dry before drawing blood. Manufacturer s directions should be followed when inverting tubes with clot activators or other additives, and when allowing whole blood (serum) samples to clot. Instructions may vary by tube manufacturer. One final word of advice is to read the manufacturer inserts prior to initial use and whenever changes are flagged. These are all simple actions that can make a big difference in the quality of the sample collected and the quality of the laboratory information derived from testing. SUMMARY Decisions made using a compromised sample (e.g. hemolyzed) may adversely affect the patient s diagnosis or treatment. Who would want to take that chance? The best course of action is to watch for it, to prevent it from happening whenever possible, and to recollect the sample if it does. Individual laboratory guidelines will dictate the follow up actions, which may include the immediate transmission of identified lab results (such as potassium levels in emergencies) in case immediate intervention by the primary care provider is required. 9

10 REFERENCES Accessed 10/19/2012: Accessed 10/21/2012: Accessed 10/21/2012: Accessed 10/29/2012: Accessed 10/29/2012: Accessed 10/29/2012: Accessed 10/29/2012: Accessed 10/29/2012: Arzoumanian, Lean. Tech Talk, Volume 2, Number 2. October Becton, Dickinson, and Company (BD). Howanitz PJ. Errors in laboratory medicine: practical lessons to improve patient safety. Arch Pathol Lab Med 2005 Oct;129(10): Journal of Emergency Nursing: Official Publication of the Emergency Department Nurses Association [2003, 29(2): ] QUESTIONS Getting the Red Out # Directions: Answer sheets: Read the instructions to assure you correctly complete the answer sheets. Online: Log in to your User Account on the NCCT website o NOTE: If the online test questions differ from the course test that follows the reading material, the CE course you are using is outdated or the question has been revised since you downloaded it. The online question is the most current and it should be answered accordingly. Select the response that best completes each sentence or answers each question from the information presented in the course. If you are having difficulty answering a question, go to and select Forms/Documents. Then select CE Updates and Revisions to see if course content and/or a test questions have been revised. If you do not have access to the internet, call Customer Service at

11 1. What is the difference between in vivo and in vitro hemolysis? a. In vivo occurs more rapidly b. In vivo occurs only in test tubes c. In vitro is caused by disease d. In vitro occurs outside the body 2. What is the typical lifespan of a normal red blood cell? a. 120 minutes b. 120 hours c. 120 days d. 120 months 3. Which of these is perhaps the most significant function of red blood cells? a. Carrying hematocrit b. Carrying hemoglobin c. Transporting chemicals d. Transporting fluids 4. Why is it important for iron to exist in the ferrous (Fe 2+ ) state inside the hemoglobin molecule? a. For red blood cell membrane protection b. For longevity of the red blood cell membrane c. For oxygen to be properly carried to tissue d. For the porphyrin ring to remain intact 5. Which of these is a hemolytic anemia that contains Hemoglobin S, causing the red blood cell lifespan to be greatly decreased? a. Hereditary Spherocytosis b. Sickle Cell Anemia c. Thalassemia d. G-6-PD Deficiency 6. Which of these might cause an acquired hemolytic anemia? a. G-6-PD deficiencies b. Autoimmune conditions c. Thalassemias d. Pyruvate kinase deficiencies 11

12 7. Which of the following conditions does not typically put patients at higher risk for acquired hemolytic anemias? a. Influenza type A b. Epstein Barr virus c. Systemic lupus erythematosus d. Hepatitis 8. Which of the following is not a sign or symptom of hemolytic anemia? a. Jaundice b. Pain from an enlarged spleen c. Leg pain d. Blurry vision 9. Which of these is not a typical sign or symptom of a hemolytic transfusion reaction? a. Fever and chills b. High blood pressure c. Shock d. Hypotension 10. Which of these groups of laboratory tests is most likely to have been ordered specifically for a patient suspected of hemolytic anemia? a. CBC, Diff, Bilirubin, Osmotic Fragility b. CBC, Diff, Lipid Profile, Chemistry Panel c. Type and Crossmatch, Indirect Coombs d. Renal function tests with Electrolytes Panel 11. Which of these is not a common cause of in vitro hemolysis? a. Using a small gauge needle b. Allowing blood to fully clot before separation c. Collection through an IV catheter d. Shaking blood in tubes with anticoagulants 12. For which of these laboratory tests performed on serum would hemolysis be expected to have a significant impact on the accuracy of results? a. Glucose b. Chloride c. Potassium d. Magnesium *End of Test* 12

13 P.A.C.E. Course Evaluation NCCT 7007 College Boulevard Suite 385 Overland Park, KS Directions: Please let us know whether this CE Course met your expectations by answering the following questions. Your feedback helps us to make our products better for you! Course Title: Getting the Red Out Course Number: OBJECTIVES Yes No Yes No 1. Did you meet the objectives while reading this CE course? 2. Did the test measure what you learned? COURSE CONTENT Yes No Yes No Yes No 3. Were you satisfied with this course? 4. Was the CE course organized and useful for learning? 5. Was this CE course written at the right level for the practicing professional? VALUE Yes No Yes No Maybe 6. Did you learn anything new? 7. Did you learn anything you might use at work? What can NCCT do to make the CE courses better for you? What would you like to learn about in the future? Please list specific topics! *Please include this evaluation with your answer sheet.* 13