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Hemolytic Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Hemolytic and links to relevant pages within the course.

Learn more about laboratory continuing education for medical technologists to earn CE credit for AMT, ASCP, NCA, and state license renewal and recertification. Or get information about laboratory safety and compliance courses that deliver cost-effective OSHA safety training and continuing education to your laboratory's employees.



Alpha Thalassemia
Anemia in Alpha Thalassemia

When a patient has a type of thalassemia, there is often an excess production or accumulation of globin chains produced by genes that are not effected by the thalassemia deletion. This is a compensation mechanism that the body utilizes to maintain hemoglobin production (which requires globin chains).In alpha thalassemia, the body can produce excess gamma chains as a compensatory mechanism. This can lead to the production of gamma chain tetramers (hemoglobin Bart's) in the unborn child and as beta chain tetramers (hemoglobin H) in adults.This subsequent tetramer accumulation in response to thalassemia often leads to red blood cell damage and hemolytic anemia.

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Alpha Thalassemia Intermedia

Alpha thalassemia intermedia (Hemoglobin H Disease) results from a deletion of three out of four alpha chain gene loci. Infants born with alpha thalassemia intermedia appear normal at birth but often develop anemia and splenomegaly by the end of their first year. Development and life expectancy are usually normal, but some affected individuals may require splenectomy and transfusion therapy.Hepatomegaly is not a common finding and there may be some association with mental retardation. Due to the hemolytic nature of this anemia, there may be an increase in respiratory infections, leg ulcers and gallstones. Skeletal changes are not commonly seen in hemoglobin H disease.Any ethnic group can have occurrences of hemoglobin H disease; but it is most often seen in Southeast Asia, the Middle East and the Mediterranean islands.

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Serum Bilirubin

Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of bilirubin in the serum of adults is 0.2-1mg/dl. Bilirubin levels increase with liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dl.Persons with alpha thalassemia intermedia usually have an increased bilirubin level, because of ongoing hemolysis. This bilirubin is typically the unconjugated fraction of bilirubin.

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Serum Haptoglobin

Haptoglobin is the plasma protein responsible for binding free hemoglobin during episodes of hemolysis. Because of its role, haptoglobin would normally demonstrate decreased levels during a hemolytic crisis since free hemoglobin is spilled into the bloodstream from lysed red blood cells.The normal level of haptoglobin is 40-330mg/dL. Individuals who are in hemolytic crisis demonstrate greatly reduced levels to a complete absence of haptoglobin.In alpha thalassemia, however, haptoglobin levels remain normal or only slightly decreased, even during hemolytic events.The reason for this is that haptoglobin functions by binding the alpha chain portion of hemoglobin. With the absence of these chains in alpha thalassemia major and intermedia, haptoglobin cannot bind free hemoglobin. Therefore it is not consumed as it would be in other types of hemolytic anemia.

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Antibody Detection and Identification
Example of Clinically Significant Immune Antibody

The panel below shows reactions in the AHG phase only (clinically significant). Pattern reactivity of sample matches the pattern displayed by C on the panel. Anti-C is a clinically significant antibody that can cause both hemolytic disease of the newborn (HDN) and hemolytic transfusion reaction (HTR).ND= not done

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Course Introduction

Antibody screening and antibody identification are critical components in blood bank testing. Clinically significant antibodies must be identified so that appropriate blood products are selected for transfusion and the risk of adverse reaction is minimized. Clinically significant antibodies are capable of causing transfusion reactions, hemolytic disease of the newborn and in severe cases, death.This course will discuss the techniques that are used by blood bank technologists to detect and identify various types of antibodies.

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Significance of Reactions at Different Phases of Testing

Antibodies have optimum temperatures for reactivity. Reaction readings can be made at different phases: after immediate spin, after incubation at 37°C, and after the addition of antihuman globulin (AHG) and centrifugation. Reactivity in a certain phase will help to determine whether the antibody is cold reacting (IgM) or warm reacting (IgG). It will also help to distinguish between antibodies that are clinically significant and not significant. Clinically significant antibodies that are capable of causing acute and delayed hemolytic transfusion reactions (HTR) or hemolytic disease of the newborn (HDN) are usually IgG and react best in the AHG phase.Readings can be done at all three phases if a tube method is used. If a gel method is used, readings are done only at AHG. Immediate spin: Antibodies reacting in this phase tend to be cold reactive. They are usually IgM class and not clinically significant (with the exception of the A and B antibodies). 37°: Antibodies that react in this phase include strong IgM or IgG antibodies. After incubation, the tubes are examined for the presence of hemolysis. If complement was bound during incubation then hemolysis could be seen. NOTE: This reaction would only occur in serum samples. If EDTA plasma samples are used for testing, the complement cascade has been halted. Magnesium and calcium ions are not available for complement to be activated. AHG:Antibodies reacting in this phase are considered clinically significant. They are usually warm reactive and IgG.

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Authentic and Spurious Causes of Thrombocytopenia
Which of these conditions is associated with a pseudo-thrombocytopenia?View Page
References

Beavers C, Kern W, Blick K. Isolated acute thrombocytopenia in a 21-year-old caucasian male. Lab Med. June 2009;40(6):337-339.Bromberg MB. Immune thrombocytopenic purpura, the changing therapeutic landscape. N Engl J Med. 2006; 355:1643-1645. Glassy EF. ed. Color Atlas of Hematology. Northfield, IL: College of American Pathologists; 1998.Kwon JY, Shin JC, Lee JW. Predictor of idiopathic thrombocytopenic purpura in pregnant women presenting with thrombocytopenia. Int J Gynacol Obstet. 2007;85-88. Taghizadeh, M. An update on immune-mediated thrombocytopenia. Lab Med. 2008;39(1):51-54.Tarr PI, Gordon CA, Chandler WE. Shiga like toxin producing Escherichia coli and hemolytic uremic syndrome. Lancet. 2005;365:1073-86.Woelke C , Eichler P. Washington G, etal. Post transfusion purpura in a patient with HPA-1a and GP1a/11a antibodies. Transfus Med 2006;16:69-72. Wyrick-Glatzel J.Thrombotic thrombocytopenic purpura and ADAMTS-13: New insights into pathogenesis, diagnosis and therapy. Lab Med. 2004;35(12):733-737.

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Increased platelet destruction

The most common cause of thrombocytopenia is increased destruction of platelets. Platelets are eliminated from peripheral circulation faster than the bone marrow can produce new platelets.Increased platelet destruction may be the result of immune or nonimmune mechanisms. Immune platelet destruction begins when antibodies coat platelets. These sensitized platelets are then destroyed by macrophages, mostly from the spleen but also from the liver. Disorders that are associated with immune mechanisms of destruction include: Idiopathic (or immune) thrombocytopenic purpura (ITP) Heparin-induced thrombocytopenia (HIT) Neonatal alloimmune thrombocytopenia (NAIT)Increased destruction of platelets is not always caused by the immune system. Platelet destruction can occur as a result of abnormal platelet aggregation or endothelial cell injury. Both of these occurrences can cause fibrin to form in arterioles and capillaries. This leads to platelet activation and consumption. Conditions associated with nonimmune destruction and consumptive thrombocytopenia include: Thrombotic thrombocytopenic purpura (TTP) Hemolytic uremic syndrome (HUS) Disseminated intravascular coagulation (DIC) All of these conditions are associated with significantly decreased platelet counts that may become life threatening. Restoration of platelet numbers is essential to promote clotting and vascular patency.

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Thrombotic Thrombocytopenic Purpura and Hemolytic Uremic Syndrome

Thrombotic thrombocytopenic purpura (TTP) is an uncommon, but very serious consumptive platelet disorder. Its cause is unknown, but there are several possible precipitating factors including infection, carcinoma, and pregnancy. More women than men are affected by TTP. If left untreated, the mortality rate is in excess of 90% due to multiorgan failure. Hemolytic uremic syndrome (HUS) is also a platelet consumptive disorder. HUS is thought by some to be the same condition as TTP because both disorders have the same underlying pathology. However, HUS is more often associated with renal failure and TTP with neurological manifestations including visual impairment, weakness, headache, dizziness, disorientation. seizures, or coma. Microangiopathic hemolytic anemia, thrombocytopenia, and fever is associated with both TTP and HUS. The patient's condition can deteriorate rapidly while these symptoms are becoming evident. HUS is usually seen in children; it is the most common cause of acute renal failure in children. Patients may have bloody diarrhea and symptoms resembling colitis. Diarrhea-related HUS is usually associated with ingestion of undercooked beef contaminated with Ecoli O157:H7; it is the Shiga-like toxin from this serotype that causes the illness. Some patients may have long term kidney dysfunction as a result ofthis virulent infection. For patients who have experienced renal failure, dialysis may be required.

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Which of the following bacteria is often linked to diarrhea-associated hemolytic uremic syndrome?View Page
Which of the following laboratory results would you find with disseminated intravascular coagulation (DIC) but NOT with thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome (HUS)?View Page

Basics of Lean and Six Sigma for the Laboratory
Measuring Quality

Laboratory quality indicators are commonly measured as averages or percentages. For example:The turnaround time (TAT) for HIV Western Blot averages 3.5 days.Type O units are available 99.9% of the time in an emergency release.In the first example, it might appears that 3.5 days is a reasonable TAT for a sendout test at first glance, but an average of 3.5 days means the result can be available anytime between the same day to a week later. A patient may have to schedule an appointment at least one week later to ensure the result is available. This may potentially delay treatment. As for the physicians, they want to know when their patients' results will come back; they are not interested in knowing the average but exactly when the result will be available. In the second example, 99.9% might sound impressive but it would translated into 1 out of every 1000 patients requiring emergency transfusion being at risk of ABO hemolytic reaction. In fact if 99.9% is used to measured quality of the processes that happened in our everyday life, that would translate into:18 plane crashes daily17,660 mail mix-ups hourly3,700 medication errors daily10 dropped babies daily$24.8 million worth of incorrect charges hourly500 wrong procedures performed weekly.Other industries realized long ago that measuring quality based on percentage or average is not adequate to satisfy the customers. The need for a higher standard of quality led to the use of Six-Sigma.

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Beta Thalassemia
Anemia in Beta Thalassemia

In thalassemia, there is often an excess production or accumulation of globin chains whose genes are not affected by the deletion. In beta thalassemia, this may be seen as an increase in gamma chain and delta chain production, leading to increased levels of hemoglobin F and A2 respectively. These hemoglobins have a higher oxygen affinity, leading to decreased oxygenation of the tissues and clinical symptoms respective to this state.In addition, the excess free alpha chains produced in this condition form insoluble precipitates within the red blood cells, often lead to red cell membrane damage and decreased cellular deformability. This leads to a hemolytic anemia. Adding to the anemia is a decrease in the total amount of hemoglobin produced in spite of the erythroid hyperplasia of the bone marrow.

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Serum Bilirubin

Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of serum bilirubin for adults is 0.2-1mg/dL.Bilirubin levels increase with some liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dL.Persons with beta thalassemia major usually have an increased bilirubin level. This bilirubin is typically the unconjugated fraction of bilirubin.

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Blood Banking Question Bank - Review Mode (no CE)
The most common cause of severe life threatening hemolytic transfusion reactions is:View Page
A delayed hemolytic transfusion reaction is most likely to be the result of which of the following antibodies:View Page
Which of the following types of packed RBCs could be transfused to a group O patient:View Page
Fresh frozen plasma :View Page
Which of the following antibodies is the most common cause of hemolytic disease of the newborn:View Page
The use of the direct antiglobulin test is indicated in all the following except:View Page
IgM antibodies directed against red cells generally:View Page
Which of the following conditions is most frequently associated with anti-I:View Page
Which of the following set of conditions would preclude hemolytic disease of the newborn as a result of ABO incompatibility:View Page
To detect the presence of blocking antibodies fixed on the red cells of a newborn infant:View Page
Patients with which of the following conditions would benefit most from washed red cells:View Page
The most severe acute hemolytic transfusions reactions are the result of which of the following:View Page
Which of the following antigen groups is closely related to the ABO system:View Page
IgM antibodies produced against red blood cells generally:View Page

Case Studies in Clinical Microbiology
The patient was admitted to the hospital. The sputum specimen was inoculated to sheep blood agar. Based on the colony morphology and the alpha hemolysis seen in the image to the right, the most likely identification is:View Page
Middle ear damage in cases of S. pneumoniae infections are caused primarily by the: (Choose all that apply)View Page
The bacterial species shown growing on 5% sheep blood agar was recovered from the spun sediment of a midstream urine specimen after 24 hours incubation at 35C. Each of the following tests would be useful in supporting the presumptive identification of Enterococcus species except: (Choose all that apply)View Page
The spot test that is helpful in separating Enterococcus species (positive as shown in the image) from the viridans streptococci and S. pneumoniae (both negative) is:View Page
Most strains of S. anginosus (milleri) carry the F antigen (see image). Rare strains that carry the group A antigen can be differentiated from S. pyogenes by which of the following laboratory tests:View Page
Beta hemolytic colonies grew from the blood culture bottle after 18 hours incubation (see image). Which of following tests would be helpful in making a preliminary identification? (Choose all that apply)View Page
Thus, in follow-up to the previous discussion, the reaction shown in the image establishes the identification of a group A, beta hemolytic streptococcus.View Page
Shown in the image is a close-in view of the colony growth after 48 hours incubation. What are the possible presumptive identifications suggested by the colonies observed? (Choose all that apply)View Page

Chemical Screening of Urine by Reagent Strip
Match the following positive urine blood results with the possible conditions which cause them to occur.View Page
Clinical Significance of Urobilinogen in Urine

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction, as shown in the table below:ConditionUrine Bilirubin ResultUrine Urobilinogen ResultHemolytic diseaseNegativeIncreasedHepatitic diseasePositive or negativeIncreasedBiliary obstructionPositiveNormal* *Urine chemical reagent strip methods cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

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In hemolytic disease, the urine bilirubin test result is negative and the urine urobilinogen test result is:View Page

Chemical Screening of Urine by Reagent Strip (retired March 2012)
Clinical Significance

No blood is found in the urine of healthy individuals although samples from menstruating females, frequently, but not always, test positive for blood. Hematuria is associated with renal or genital urinary disorders in which the bleeding is the result of irritation to the involved organs or trauma. Examples include renal calculi, pyelonephritis, glomerulonephritis, tumors, trauma or exposure to toxic chemicals or drugs and/or strenuous exercise. Hemoglobinuria may be due to the lysis of red cells within the urinary tract. If it is caused by intravascular hemolysis, the hemoglobin is then filtered through the glomeruli. In the normal individual, the hemoglobin molecule attaches to haptoglobin and in this way bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, as in cases of hemolytic anemia, severe burns, transfusion reaction, infection or strenuous exercise, hemoglobin passes into the urine.

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Match the following:View Page
Clinical Significance

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction. Urobilinogen is increased in hemolytic disease and urine bilirubin is negative. Urobilinogen is increased in hepatic disease, and urine bilirubin may be positive or negative. Urobilinogen is low with biliary obstruction, and urine bilirubin is positive. Reagent strips methods however, cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

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Urobilinogen is excreted in the urine in increased amounts in: (Choose ALL of the correct answers)View Page

Chemistry / Urinalysis Question Bank - Review Mode (no CE)
A spectrophotometric scan of amniotic fluid may be valuable in the determination of which of the following conditions:View Page
Which of the following conditions would be suggested by a marked rise in alkaline phosphatase, jaundice, and a moderate rise in ALT:View Page

Confirmatory and Secondary Urinalysis Screening Tests
Bilirubinuria

Bilirubin may be present in the urine when bile duct obstruction, liver disease, or liver damage is present. Bilirubinuria can be detected before other clinical symptoms, such as jaundice, are present or recognizable. The detection of small quantities of bilirubin is very important in early diagnosis of obstructive and hepatic jaundice. The urine bilirubin test is also useful in the differential diagnosis of obstructive jaundice (positive for bilirubinuria) vs. hemolytic jaundice (negative for bilirubinuria).

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Diabetes and the Current American Diabetes Association Guidelines
HbA1C versus Blood Glucose Measurement

Advantages of utilization of HbA1C over blood glucose measurement include: Fasting is not required Greater specimen stability Less fluctuations in day-to-day levels caused by stress and illness Disadvantages of utilization of HbA1C over blood glucose measurement include: Cost per test is higher than blood glucose. Conditions that shorten red blood cell (RBC) survival e.g., hemolytic anemia, homozygous sickle cell trait, pregnancy, or recent significant blood loss, will reduce exposure of RBCs to glucose, thereby lowering the HbA1C test value. Specimens with >10% fetal hemoglobin (HbF) may have a falsely decreased HbA1C test result. If onset of diabetes is rapid, blood glucose levels will more correctly reflect glycemia than HbA1C levels.

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Erythrocyte Inclusions
In which of the following disorders would you probably observe coarse basophilic stippling on a Wright-stained peripheral blood smear?View Page
Howell-Jolly Bodies

Howell-Jolly bodies are round, smooth, almost pyknotic, dark-purple bodies ranging in size from 0.5 to 1.0 micron in diameter. These RBC inclusions contain DNA. Howell-Jolly bodies are thought to be nuclear remnants or aggregates of chromosomes that have separated from the mitotic spindle and remain behind after the remainder of the RBC nucleus is expelled. The spleen normally removes these bodies from the RBCs. However, Howell-Jolly bodies can be observed on a Wright-stained peripheral blood smear post-splenectomy or when the spleen is not functioning properly. Howell-Jolly bodies may also be seen in hemolytic anemias such as sickle cell anemia.When Howell-Jolly bodies are present, they usually occur singly, as shown in the image on the right. It is possible, though, that two or more may be seen in a single RBC, usually occurring in the presence of megaloblastic anemia or abnormal erythropoiesis.

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Erythrocyte Inclusions (retired 7/10/2012)
Cabot rings may be seen in rare occasions in patients who have:View Page
More on Howell-Jolly Bodies

Under normal conditions, Howell-Jolly bodies are thought to be remnants of nuclear fragments due to incomplete expulsion of the nucleus. In pathological conditions, they are aggregates of chromosomes which have separated from the mitotic spindle during abnormal mitosis. Single or multiple Howell-Jolly bodies may be found in a red cell. A single HJ body in a red cell may be seen in megaloblastic anemia, hemolytic anemia such as sickle cell anemia and after splenectomy. Megaloblastic anemia or abnormal erythropoiesis is usually present when multiple Howell-Jolly bodies are observed in a single cell.

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In which of the following conditions would you expect to find Howell-Jolly bodies?View Page

Hematology / Hemostasis Question Bank - Review Mode (no CE)
Which of the following conditions might give rise to the red cell abnormality depicted here:View Page
Coarse basophilic stippling in all of the following EXCEPT:View Page
Identify the object contained in the cell in this illustration indicated by the arrow:View Page
The intracellular precipitates seen in the RBCs in this illustration is termed:View Page
Which of the following conditions is frequently associated with these cells?View Page
Which of the following is not primarily a hemolytic process?View Page
Spherocytes are associated with which two of the following conditions:View Page
Aplastic anemia may be caused by all expect the following:View Page

Hemoglobinopathies: Hemoglobin S Disorders
Clinical Manifestations

Sickle cell disease (SCD) manifests itself as a chronic hemolytic anemia. There is slowed growth and development in children with sickle cell anemia, who may present with dactylitis. In addition to the general symptoms of anemia (fatigue, weakness, pallor etc.) patients are prone to infection, cardiomegaly, usually due to iron deposits from frequent transfusions, and bone and organ infarcts. Male patients can experience priapism.Patients with SCD can experience vaso-occlusive, hemolytic, sequestration, and aplastic crises. The major symptom in SCD is pain. Pain is a warning sign that is related to vaso-occlusion and life-threatening complications.

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Hemolytic Crisis

Sickle cell anemia, in addition to being a hemoglobinopathy, is a hemolytic anemia. Hemolysis is both intravascular (about one-third) and extravascular (about two-thirds). Common markers of hemolysis include elevated LDH, bilirubin, and reticulocyte levels.The hemoglobin that is released into the plasma during intravascular hemolysis combines with nitric oxide (NO). The resulting decrease in NO availability contributes to the vaso-occlusive crisis by stimulating vasoconstriction, endothelial adhesiveness, and thrombosis.Hemolytic crisis also involves splenic sequestration, which occurs in an effort to remove damaged red blood cells. This can result in hypovolemia, which may lead to shock, especially in children. Children can also exhibit splenomegaly due to intrasplenic pooling of blood.Adults in hemolytic crisis may experience autosplenectomy. This occurs when the spleen has multiple infarctions, followed by fibrosis, which renders the spleen nonfunctional.

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RBC Morphology in Sickle Cell Disease (HbSS)

Sickle Cell Anemia (HbSS) is a hemolytic anemia, characterized by the presence of drepanocytes (sickle cells) and polychromasia (increased reticulocytes). Nucleated red blood cells (NRBCs) may be seen during episodes of severe hemolysis. The absence of polychromasia may indicate aplastic crisis. The homozygous state of hemoglobin SS causes RBCs to take on the characteristic sickle shape when hemoglobin is in a deoxygenated state. The name "sickle" comes from the tool (seen in the upper image) that is used to manually cut hay. When RBCs sickle they take on the same shape as the blade of the sickle, as seen in the bottom image.

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Hemolytic Disease of the Fetus and Newborn
Foreword

This course is a refresher on current concepts and practices in hemolytic disease of the fetus and newborn (HDFN). As such it is a survey course that provides a broad overview of the field and presents an opportunity to review significant aspects of HDFN and its laboratory investigation and prevention. Because it is a survey course with many topics, not all will be covered in depth. However, Rh immune globulin (RhIg) will be reviewed extensively since it prevents the most severe form of HDFN and is one of the biggest success stories of modern medicine. The course assumes that participants have a basic background knowledge of immunohematology theory and practice. Reading the resources in Further Reading for more information on any topic is encouraged. In brief, the course will: Recap relevant background information on HDFN and its treatment Review the characteristics and uses of Rh immune globulin (RhIg) Discuss typical laboratory findings and their interpretations Examine current best practices in perinatal testing programsThe course is a companion to "Rh negative female with anti-D at delivery: A case study on dealing with the issues" and complements its content.

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Prenatal Treatment

Prenatal treatment of severe HDFN due to anti-D consists of in utero transfusions. Because of significant risks, transfusion is indicated only if fetal monitoring suggests significant hemolytic disease. 1. Intrauterine Transfusion (IUT)IUTs are done when fetal monitoring indicates severe HDFN and the fetus is too premature for early delivery. IUTs involve the intraperitoneal infusion of packed red cells. The success of the procedure depends on absorption of the red cells through the subdiaphragmatic lymphatic vessels of the fetus. 2. Intravenous transfusion (IVT)Because there may be erratic and inconsistent absorption of intrauterine transfusions in severely hydropic fetuses, IVTs were developed. IVTs involve transfusing donor RBC directly into the umbilical vein.

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Literature and Online Resources

The following published literature and online resources, while useful, should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.References indicated by * provide a broad overview of HDFN and are highly recommended.LITERATUREAvent ND, Reid ME. The Rh blood group system: a review. Blood 2000 Jan 15;95 (2):375-87.Bowman J. Thirty-five years of Rh prophylaxis. Transfusion 2003 Dec;43(12):1661-6.* Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematology 2006;22(4):188–195. (scroll to article)Eder, AF, Manno, C.S. Alloimmune hemolytic disease of the fetus and newborn. In Wintrobe's Clinical Hematology, 11th ed. (Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader BE, (eds). Philadelphia, PA: Lippincott, Williams & Wilkins, 2004.Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol. 2006 Mar-Apr;13(1-2):4-12. Kennedy MS, McNanie J, Waheed A. Detection of anti-D following antepartum injections of Rh immune globulin. Immunohematology 1998;14(4):138-40.Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis. BJOG. 2009 Sep;116 (10): 1307-14. Epub 2009 Jun 17.* Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ. 2005 May 28;330(7502):1255-8. (UK perspective but much valuable information relevant to all)* Murray NA, Roberts IAG. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007 Mar; 92(2): F83–F88. Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G; DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006 Jul 13;355(2):156-64.Ramsey G. Inaccurate doses of Rh immune globulin after Rh-incompatible fetomaternal hemorrhage: survey of laboratory practice. Arch Pathol Lab Med 2009 Mar; 133(3):465-9. Reid ME. The Rh antigen D: a review for clinicians. Blood Bulletin 2008 Apr; 10(1).Sandler SG. Effectiveness of the RhIg dose calculator. Arch Pathol Lab Med 2010 Jul;134(7): 967-8.Shulman IA, Calderon C, Nelson JM, Nakayama R. The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies. Transfusion 1994 Aug;34(8):666-70.Tamul KR. Determining fetal-maternal hemorrhage with flow cytometry. Advance 2000. Posted online June 5, 2000.Westhoff CM, Sloan SR. Molecular genotyping in transfusion medicine. Clin Chem 2008;54(12): 1948-50.ONLINE RESOURCESPaxton A. Bringing new rigor to RhIg calculations. CAP TODAY. May 2008. Accessed January 18, 2011.*Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010. Accessed January 18, 2011.

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Introduction to the ABO Blood Group System
Immunoglobulin

The predominant immunoglobulin class for the B antibodies produced by individuals with group A phenotype and the A antibodies produced by individuals with group B phenotype is IgM. Small quantities of IgG and IgA may also be present.The ABO antibodies found in the serum of group O individuals include anti-A and anti-B. An antibody designated anti-A,B is also present. Anti-A,B in group O individuals tends to be predominantly IgG, although IgM and IgA components are also present.Infants of group O mothers are at higher risk for hemolytic disease of the fetus and newborn (HDFN) than those born to mothers with group A or B because IgG immunoglobulins readily cross the placenta. IgM molecules do not cross the placenta because of their larger size. However, the HDFN that results is usually mild and often subclinical. Infants generally survive with little or no intervention.It is important to note that immune antibodies are usually IgG. Both naturally occurring and immune ABO antibodies are critically important in transfusion since both sensitize, and usually hemolyze, red cells with the corresponding antigen.

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Medical Error Prevention (retired)
Sentinel Event Categories

Sentinel Events are sentinels--they function as guards or watchkeepers. They indicate serious situations that require immediate attention: Patient deathParalysisComaPermanent loss of functionAny procedure on the wrong patient, the wrong side of the body, or the wrong organ Hemolytic transfusion reaction involving major blood group incompatibility

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Postanalytic Medical Errors

Errors also occur after analyses are completed and reported. Postanalytic errors begin with the medical professionals who receive test results, and they include interpretation of the results. These errors can occur at--the bedside, chair-side, hospital, clinic-- wherever the patient and the medical professional are located. The possibility for postanalytic medical error continues through diagnosis and treatment procedures and processes. These medical errors occur during the time after the laboratory reports test results. Examples: Wrong test value associated with patient Wrong test interpretation Wrong diagnosis Wrong treatmentLaboratory professionals might believe they are not associated with postanalytic medical errors, but they can be. One deadly example is fatal hemolytic transfusion reaction involving laboratory error.

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Sources of Laboratory-Related ErrorsView Page

Microbiology / Serology Question Bank - Review Mode (no CE)
Which of the following substances produced by Group A Streptococci is responsible for producing type specific immunity:View Page
On sheep blood agar Haemophilus influenzae may exhibit satellite formation around all but which of the following organisms:View Page

Multi-drug Resistant Organisms: MRSA, VRE, and Clostridium difficile
Identification of Enterococcus species from clinical cultures

Gram stain: gram-positive cocci in singles, pairs, or chains; cells can be ovoid to coccobacillaryColony morphology: on blood agar after 24 hours of incubation, colonies are nonhemolytic or alpha hemolytic (rare strains may be beta hemolytic), and approximately 1 to 2 mm in diameter.Catalase: negativePresumptive identification: Growth on bile esculin agar and in 6.5% salt broth are two characteristics that have commonly been used to identify Enterococcus to the genus level. A positive esculin in combination with a positive PYR reaction is another approach to presumptive identification.Species identification: E. faecalis and E. faecium are usually easily identified by most commercial systems. Successful identification of the other species on these systems may vary. With respect to vancomycin intermediate or resistant strains, two key characteristics are motility and pigment. E. casseliflavus is both motile and possesses a yellow pigment; E. gallinarum is also motile but non-pigmented. E. faecalis and E. faecium demonstrate neither characteristic.

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Preliminary Identification of the Primary Select Agents of Bioterrorism
Bacillus anthracis

Culture Characteristics:Growth may be noted as soon as eight hours after inoculation and occurs on most routine media, including sheep blood agar (SBA), chocolate agar (CHOC), and routine blood culture media Does not grow on MacConkey (MAC) agarColony Morphology on SBA at 35°C, 18-24 hours:Flat or slightly raised, gray to white with a "ground glass" appearance Described as "tenacious" or "sticky" like petroleum jelly, shown in the top image B. anthracis is NOT hemolytic, while B. cereus is hemolyticCharacteristic Features:After 18 hours of incubation on SBA at 35°C, the slightly undulate margin may show curling, displaying a so-called "Medusa head" or described as comma-shaped protrusions, shown in the lower image

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
The peripheral blood smear represented by the image on the right was submitted for hematologic review. The identification of the RBC inclusions shown are most likely identified as:View Page
Which of the following conditions is associated with the defective erythrocytes that are indicated by the arrows in this image?View Page
A 5-year-old girl was brought to the emergency department with bloody diarrhea and severe abdominal pain. A complete blood count produced these results:CBC ParameterPatient ResultReference IntervalWBC9.6 x 109/L4.3 - 10.8 x 109/LHemoglobin9.1 g/dL11.5 - 13.5 g/dLHCT28%37 - 48%MCV80 fL86 - 98 fLRDW13.111 - 15Platelets90.1 x 109/L150 - 450 x 109/LThe peripheral blood smear is represented in the image to the right. Which of the following condition(s) could be present in this patient when considering the information above and the cells indicated by the arrows on the peripheral smear?View Page
The arrangement of the erythrocytes in this peripheral smear should be reported out as rouleaux formation.View Page
A blood smear represented by the photograph was submitted for hematologic review. Based on the erythrocyte morphology and the accompanying histogram, which of the following choices is the most likely situation or condition?View Page
The complete blood count was obtained from a patient recently admitted to the emergency room. The red blood cell indices obtained revealed an MCV of 115 femtoliters (fL) (normal range 80 - 90 fL). The patient met the criteria for a peripheral blood smear examination. A representative field is shown on the right.Which of the following conditions may be indicated by the results seen on this peripheral blood smear?View Page
Case continued:Which hematologic condition could be associated with the findings shown in these images?View Page
A 49-year-old male with pneumonia was treated with high-dose intravenous penicillin. He became jaundiced with yellow sclera. The image on the right is typical of other fields that were observed on his peripheral blood smear.Since penicillin may, in some individuals, cause autoimmune hemolytic anemia, the clinician requested a direct antiglobulin test (DAT) be performed. The DAT was positive, indicating that antibodies to the drug were produced, which then attached to the drug on the surface of the red cells. Hemolysis occured due to the drug-induced antibody attachment, leaving the patient with various abnormal red blood cell morphologies. Which of the following cell types would you report for this patient?View Page
Poikilocytosis Review Table

Cell TypeImageCellular DescriptionAssociated Diseases and ConditionsTeardrop cellRed blood cells (RBCs) are shaped like a teardrop with a projection extending from one end.Myelofibrosis with myeloid metaplasia (MMM)SpherocyteRBCs smaller than normalNo central pallorRound rather than disc-shapedHereditary spherocytosisCertain hemolytic anemiasSevere burnsTarget cellRBCs with characteristic bull's-eye morphology due to hemoglobin distribution.Hemoglobinopathies (e.g., sickle cell disease)Certain thalassemiasIron deficiency anemiaSplenectomySevere liver diseaseSickle cellRBCs contain hemoglobin S.Thorn or crescent-shapedSickle cell anemiaStomatocyteRBCs with thin, elongated area of central pallor (slit-like, or coffee-bean-shaped on peripheral blood smears).Three-dimensionally, RBCs are cup-shaped.Hereditary stomatocytosisAlcohol-related diseaseLiver diseaseRh null phenotypeArtifactSchistocyte (fragmented red cells)RBC blood cell fragments or piecesVary widely in size and shapeSevere burnsHemolytic uremic syndrome (HUS)Microangiopathic hemolytic anemia (MAHA)Disseminated intravascular coagulation (DIC)Thrombotic thrombocytopenic purpura (TTP)Ovalocyte (elliptocyte)RBCs are elongated-oval, cigar, or pencil-shapedHereditary elliptocytosisMegaloblastic anemiaMyelophthisic anemiaCertain thalassemiasSevere iron deficiency Acanthocyte (Spur cell)RBCs demonstrating irregularly-spaced, spiny projections that vary in size and numberNo central pallor.AbetalipoproteinemiaSevere hepatic diseaseMyeloproliferative disordersMAHANeuroacanthocytosissyndromesEchinocyte (Burr cell)RBCs have short and evenly-spaced, rounded projections surrounding the cellCentral pallor presentUremiaHeart diseasePyruvate kinase deficiencyStomach cancersBleeding peptic ulcersBite cellRed cells that appear to have bites taken out of them (Image A)Supravital stain reveals the presence of Heinz bodies--precipitated denatured masses of hemoglobin (Image B) Disorders associated with Heinz body formation:Unstable hemoglobinsChemical poisoningG-6PDHemolytic anemia associated with severe alcoholic liver disease

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A known case of hemolytic disease of the newborn (HDN) is presented in the image on the right. Many different cellular morophologies are present. Apart from the obvious anisocytosis (microcytes and macrocytes), which additional red blood cell morphologies are worth reporting?View Page
Dimorphic (Double Cell) Population

Dimorphic is a term used to describe two circulating red cell populations. One is the patient's basic red cell population while the other is a second population with distinct morphological features. The distinct populations can be observed in the top image on the right. The bottom image on the right illustrates the two distinct peaks that are observed on the RBC histogram from the automated hematology analyzer.Dimorphic red blood cell populations can be found in conditions/situations such as: red blood cell transfusions, myelodysplasia, refractory anemia with ringed sideroblasts, hemolytic processes involving a reticulocyte response, and when patients are given erythropoietin therapy.It is important to recognize when a population of cells in the peripheral smear is not in context with anticipated laboratory findings and the clinical situation.

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Red Cell Morphology
Microcyte with Normal Hemoglobin Content

The arrow points to a microcyte with normal hemoglobin content (one-third of central pallor). Since many of the other cells in this field are normal or larger than normal, the mean corpuscular volume (MCV) would be within the normal range although the diameter and volume of this individual cell would be lower than normal. This type of microcyte can be seen in some hemolytic anemias and the rare enzyme deficiency, pyruvate kinase deficiency anemia.

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Which of the following characteristics can be seen in the microcytes present in some hemolytic disorders?View Page
Another Example of Microcytes

Another example of microcytes seen in a slide from a patient with hemolytic anemia. Compare the two microcytes in the center of the field with the lymphocyte to the right. Notice the larger red cell just below the microcytes is about the same size as the lymphocyte. Several other microcytes can also be seen in this field.

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Another Knizocyte

Another example of a knizocyte is seen in this slide. These forms are seen in conditions in which spherocytes are visible and in some types of hemolytic anemia.

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Conditions Associated with Spherocytes

Examples of conditions in which spherocytes can be seen include hereditary spherocytosis and immune hemolytic anemias (i.e., ABO incompatibility). Spherocytes can also form in conditions where there has been a direct physical or chemical injury to the cells. An example would be a smear from an individual who has suffered severe burns. In hereditary spherocytosis, a condition where spherocytes are numerous, the MCHC value will be at the upper limits of normal, or about 36. The identification of spherocytes on the smear of a patient with hereditary spherocytosis can aid significantly in the diagnosis of the disorder. Artifactual spherocytes can appear when blood is stored for a prolonged period of time.

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Keratocytes, continued

Another example of a keratocyte (helmet or horn cell) is seen in the center of this field. Examples of conditions in which keratocytes can be seen include disseminated intravascular coagulation (DIC), microangiopathic hemolytic anemia, glomerulonephritis, and rejection of renal transplants. The diagnosis of these disorders is not based on the presence of keratocytes.

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Rh negative female with anti-D at delivery: A case study
Introduction

This case concerns a common scenario in the transfusion service (TS) laboratory, the detection of anti-D at delivery in a female who has received Rh immune globulin (RhIg) during pregnancy.Distinguishing between passive and immune anti-D is important clinically: If passive anti-D is misinterpreted as immune, RhIg prophylaxis may be omitted leading to D sensitization. If immune anti-D is misinterpreted as passive, appropriate follow-up of the antibody may be curtailed putting the fetus at risk.Unfortunately, differentiating between immune and passive anti-D is often impossible. This case study presents an opportunity to review perinatal testing programs and the crucial role of RhIg in preventing hemolytic disease of the fetus and newborn (HDFN) due to anti-D. The case also examines practical aspects of routine serologic testing involving neonates and women who have received RhIg during pregnancy. The case is a companion to "Hemolytic Disease of the Fetus and Newborn" and complements its content.In brief, the case will: Guide participants through laboratory findings that need to be interpreted and resolved; Examine current best practices in perinatal testing programs; Review the characteristics of RhIg and its use in pregnancy; Review and investigate key issues associated with detection of anti-D in women who have received antenatal RhIg; Discuss crossmatch and LIS policies related to RhIg-derived passive anti-D.

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The positive DAT on the newborn means that the infant probably has clinically significant hemolysis.View Page
Routine Serologic Tests - Mother

Tests done routinely as part of perinatal testing programs vary from country to country and within countries. Below is one example of serologic tests typically done when pregnant females lack clinically significant antibodies. Other test protocols exist.Mother ABO, Rh, and antibody screen at first prenatal visit; Optional (not mandated by blood safety standards): Test for weak D, if initial Rh typing appears to be D-negative; D-negative females: Tested again (ABO, Rh, and antibody screen) at ~ 28 weeks weeks gestation prior to administration of RhIg (depending on the country) and again at delivery. Note: The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. When available, the mother can be typed for D using molecular methods, but this is usually not done unless she is weak D. The purpose is to determine using molecular methods which D variant the mother has, weak D or partial D, since the latter can produce anti-D. (see Further Reading) Molecular typing is reviewed more fully in Refresher on Hemolytic Disease of the Fetus and Newborn and Its Prevention, a companion course that complements this one.

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Literature and Online Resources

The following published literature and online resources, while useful, should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.References indicated by * provide a broad overview of HDFN and are highly recommended.LITERATUREAvent ND, Reid ME. The Rh blood group system: a review. Blood. 2000 Jan 15;95 (2):375-87.Bowman J. Thirty-five years of Rh prophylaxis. Transfusion 2003 Dec;43(12):1661-6.* Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematology. 2006;22(4):188–195. (scroll to article).Eder, AF, Manno, C.S. Alloimmune hemolytic disease of the fetus and newborn. In Wintrobe's Clinical Hematology, 11th ed. (Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader BE, (eds). Philadelphia, PA: Lippincott, Williams & Wilkins, 2004.Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol. 2006 Mar-Apr;13(1-2):4-12. Kennedy MS, McNanie J, Waheed A. Detection of anti-D following antepartum injections of Rh immune globulin. Immunohematology 1998;14(4):138-40.Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis.BJOG. 2009 Sep;116 (10): 1307-14. Epub 2009 Jun 17.* Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ. 2005 May 28;330(7502):1255-8. (UK perspective but much valuable information relevant to all)* Murray NA, Roberts IAG. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007 Mar; 92(2): F83–F88. Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G; DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006 Jul 13;355(2):156-64.Ramsey G. Inaccurate doses of Rh immune globulin after Rh-incompatible fetomaternal hemorrhage: survey of laboratory practice.Arch Pathol Lab Med 2009 Mar; 133(3):465-9. Reid ME. The Rh antigen D: a review for clinicians. Blood Bulletin 2008 Apr; 10(1).Sandler SG. Effectiveness of the RhIg dose calculator. Arch Pathol Lab Med 2010 Jul;134(7): 967-8.Shulman IA, Calderon C, Nelson JM, Nakayama R. The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies. Transfusion 1994 Aug;34(8):666-70.Tamul KR. Determining fetal-maternal hemorrhage with flow cytometry. Advance 2000. Posted online June 5, 2000.Westhoff CM, Sloan SR. Molecular genotyping in transfusion medicine. Clin Chem 2008;54(12): 1948-50.ONLINE RESOURCESPaxton A. Bringing new rigor to RhIg calculations. CAP Today May 2008. *Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010.

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The Disappearing Antibody: A Case Study
Risks of transfusing unmatched RBC

We often "get away" with transfusing unmatched RBC because the incidence of unexpected antibodies in patients experiencing medical emergencies is thought to be relatively low ( ~3-5% is sometimes cited, but with little solid evidence).Antibody incidence may vary according to several factors: Genetic disposition Patient's underlying disease Number of prior transfusions Gender (females may get exposed to foreign antigens via fetomaternal bleeds as well as transfusion) Concordance of antigen phenotypes of patients vs blood donors in a given locale.In general, antibody incidence increases with the number of transfusions that are given, although most antibody producers will respond within the first 3 - 4 transfusions. Antibody incidence in transfusion-dependent patients, such as those with sickle cell anemia or thalassemia, is very high. Regardless of likelihood, transfusing uncrossmatched blood to a patient with unexpected antibodies can result in a serious hemolytic transfusion reaction.

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Balancing the risks

Life-Threatening HemorrhageDespite potential risk, sometimes immediate transfusion is necessary, even for patients with red cell antibodies. In such cases transfusion service staff should alert the medical director, who can discuss options with clinical staff.The medical director will generally talk to the staff attending the patient and indicate that, if possible, they should hold off transfusion. But if it is a case of massive bleeding where exsanguinating hemorrhage is likely, it is better to give some blood and monitor for a delayed hemolytic transfusion reaction than to let the patient bleed to death.Transfusing when bleeding is brisk will result in much of the autologous and incompatible blood bleeding out, with the possibility of a delayed hemolytic reaction once the patient's antibody rebounds and destroys still present antigen-positive donor red cells.Some transfusion services also try to minimize the risk of unmatched blood by typing their emergency supply of O Rh negative RBCs for the K antigen, since anti-K is a relatively common clinically significant antibody. See Resources for two papers that discuss the risks of transfusing un-crossmatched emergency blood.

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Think about your responses to each of the following questions, then click on the questions.View Page
The antibody screen is positive but the transfusion of the O Rh-negative RBCs is already in progress. What are the transfusion service (TS) laboratory's priorities in this case?Place the following procedures that will be followed by the TS in the appropriate order of priority.View Page
Follow-up with clinical staff

The patient's physician was notified that compatible blood was unavailable and that the patient's antibody was still being investigated.When asked whether or not the patient was experiencing a transfusion reaction due to the transfusion of the two unmatched and incompatible O Rh negative RBC, the nurse in the OR stated that the patient was undergoing surgery and completely sedated. A transfusion reaction was not apparent but they would investigate and closely monitor.Hemolytic Transfusion Reactions (HTR)Before proceeding to the next page, make a short list of signs and symptoms associated with immediate hemolytic transfusions reaction and another list associated with delayed hemolytic transfusion reactions.

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Signs and symptoms - Precaution

Signs and symptoms are used only as a general guide to the type of transfusion reaction that may be occurring.Lower back pain, for example, would suggest an acute hemolytic reaction, whereas fever is associated with several types of reactions: Hemolytic (immediate and delayed) Febrile Bacteriogenic

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Which of the following signs and symptoms is most likely to indicate a severe immediate hemolytic transfusion reaction?View Page

Transfusion Reactions
Categories of Transfusion Reactions

Adverse complications of transfusions can be classified into several categories: Immune-mediated transfusion reactions are those that trigger a response from the patient's immune system. Many transfusion reactions are mediated by the recipient's immune system. These reactions occur as a result of antigen-antibody interactions. Antibodies involved include those with specificity towards antigens on red cells, white cells, or platelets. In general, the immune responses occur in three stages: the immune system detects foreign material (antigen) the immune system processes the antigen the immune system mounts a response to remove the antigen from the body Non-immune mediated hemolytic transfusion reactions are caused by the physical or chemical destruction of transfused RBCs, bacterial contamination, circulatory overload, or citrate toxicity. Acute reactions are those that occur during or within 24 hours after the transfusion. There is usually a rapid onset of symptoms and these reactions may be fatal. Delayed reactions occur weeks or months after the transfusion of blood or blood components.

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In Vivo Red Cell Destruction

Important events that occur in an immune-mediated hemolytic transfusion reaction (HTR) include: Antibody Binding to Red Blood Cells Antibodies may be either IgM or IgG class. IgM antibodies activate complement and lead to intravascular hemolysis where free hemoglobin is released into the plasma. IgG antibodies rarely activate complement but they are often involved in effecting phagocytosis. The concentration of the antibody is directly related to the severity of the HTR. Activation of Complement The end result of complement activation is red cell lysis. Activation of Mononuclear Phagocytes and Cytokines Sensitized red cells are removed from circulation by mononuclear phagocytes. Macrophages in the spleen and Kupffner cells in the liver are active in this process. Activation of Coagulation Antibody-antigen complexes may initiate coagulation and cause disseminated intravascular coagulation (DIC). Shock and Renal Failure Hemolysis can be intravascular or extravascular. In intravascular hemolysis, free hemoglobin, RBC stroma, and intracellular enzymes are released into the blood stream. This results in hemoglobulinemia and hemglobinuria which can lead to kidney damage. In extravascular hemolysis, there is no release of free hemoglobin. Sensitized red cells are removed from the circulation by the monocytes and macrophages in the reticuloendothelial system.

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Preliminary Laboratory Investigation

When the laboratory receives notification of a transfusion reaction, the first step is a clerical check. The clerical check should be performed as soon as possible to identify any possible ABO incompatibility. The technologist will compare the component bag, label, paperwork, and patient sample and look for errors. If an error is found, the physician must be notified. Once the post-transfusion sample is received, the sample should be examined for the presence of hemolysis. Both the pre-transfusion sample and post-transfusion sample can be compared. Destruction of red cells and release of free hemoglobin will result in a pink to red supernatant. Pink or red colored serum may indicate intravascular hemolysis. The patient's serum may appear icteric if the hemolytic process is extravascular. The ABO testing must be repeated on the post-transfusion specimen as well. Examination of a post-reaction urine sample made aid in the diagnosis of acute hemolysis. Free hemoglobin in the urine indicates intravascular hemolysis. A direct antiglobulin test (DAT) must be performed on the post-transfusion sample. An EDTA lavender top tube is the required specimen type. If the DAT is positive on the post-transfusion sample, then one should be performed on the pre-transfusion sample. If the pre-transfusion DAT is negative and the post-transfusion is positive, the presence of incompatible red cells should be suspected. All findings must be reported to the supervisor or medical director, who may request additional tests.

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Additional Testing

If preliminary testing suggests hemolysis or if the results are misleading, additional testing may be required. If human error has been ruled out during the clerical check, repeat ABO/Rh testing should be performed on the unit of blood or its segment and the pre-transfusion sample to detect any sample mix ups and clerical errors. Antibody detection studies should be performed on the pre- and post-transfusion samples to look for any unidentified antibodies. If an antibody is identified, the donor cells should be tested for the corresponding antigen. The crossmatch should be repeated with pre-and post-tranfusion specimens using the indirect antiglobulin test (IAT). An incompatible crossmatch with the pre-transfusion sample indicates an original error, either clerical or technical. Incompatibility with only the post-transfusion sample indicates a possible anamnestic response, as in a delayed hemolytic transfusion reaction (DHTR), or sample misidentification. The patient's first voided urine specimen should be examined for the presence of free hemoglobin. The patient's bilirubin levels may also be evaluated. A change from normal pale yellow serum to a post-transfusion bright or deep yellow serum should prompt an investigation for hemolysis. The maximum concentration of bilirubin following hemolysis is not usually detectable until 3 to 6 hours after transfusion. The hemoglobin and hematocrit can be tested to detect a drop in hemoglobin or failure of the hemoglobin to rise after transfusion. Important information about physical or chemical hemolysis may be gained from examining the returned unit bag. If hemolysis is present in the bag or tubing, a process which affected the blood, such as inappropriate warming or faulty infusion pump, should be suspected. If bacterial contamination is suspected, the unit can be cultured. A positive culture indicates a reaction due to bacterial contamination.

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Causes

Acute hemolytic transfusion reactions (AHTR) are caused when red cells are transfused to a patient with a pre-existing antibody that destroys the transfused incompatible red cells through intravascular or extravascular hemolysis. Life threatening acute hemolytic reactions most commonly occur from the transfusion of ABO incompatible blood. Naturally occurring ABO antibodies bind complement on the red cell surface and have efficient lytic properties which cause intravascular hemolysis. Extravascular hemolysis is characterized by antigen-antibody complexes which do not activate complement. AHTRs feature rapid destruction immediately after transfusion. Rapid hemolysis of as little as 10 mL of incompatible red cells can produce symptoms of an AHTR. Signs and symptoms can occur within minutes after starting the transfusion. Fever is the most initial symptom followed by the chills. These reactions are mostly associated with the transfusion of ABO-incompatible red cells. Causes include clerical errors, such as mislabeled patient samples and mislabeled blood products. Although acute hemolytic reactions are rare with an incidence of 1 to 9 in 100,000 transfusions, they are the most dangerous and are severely life threatening.

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Clinical Signs and Symptoms

Although there is no consistent clinical picture of an acute hemolytic transfussion reaction (AHTR), common symptoms include chills, hypotension, and fever. Some patients have experienced pain at the infusion site, flank pain, and anxiety with a feeling of doom. Red or dark urine may be the first sign of intravascular hemolysis. If patients are unconscious or in surgery, changes in vital signs, unexplained bleeding, or hemoglobinuria may be the only signs. Additional signs and symptoms include, but are not limited to: rigors, facial flushing, chest and abdominal pain, nausea and vomiting, dyspnea, oliguria/anuria, diffuse bleeding, shock, and renal failure. The severity of symptoms is related to the amount of incompatible blood transfused. Patients with underlying diseases that involve intravascular hemolysis can make diagnosis extremely difficult.

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Management and Prevention

The first component of therapy is to stop the transfusion immediately. Vital signs must be closely monitored. Management involves treatment of hypotension and disseminated intravascular coagulation (DIC). It is essential to maintain blood volume and adequate renal blood flow. Diuretics, substances that increase urine output, may be administered. If the patient enters renal failure, dialysis must be initiated rapidly. It is impossible to prevent all hemolytic transfusion reactions. The purpose of pre-transfusion compatibility testing is to decrease the probability of a hemolytic transfusion reaction by performing ABO/Rh testing, detecting and identifying alloantibodies, and crossmatching compatible blood. Human error, the most common cause of hemolytic transfusion reactions, cannot be completely eliminated. Steps must be taken to reduce the possibility of human error in identification of patient samples, donor units, and recipients. Each person involved in the transfusion process, from collection of the blood sample to administration of the donor unit, must carefully adhere to each step outlined in the standard operating procedures. All appropriate protocols must be followed. Some examples are: Technologist checks blood sample to ensure proper labeling. Patient's previous transfusion records are examined and all transfusion testing is performed correctly and accurately. Technologist ensures correct unit is released from the blood bank. Transfusionist ensures the recipient is correctly identified.There must be a mechanism in place to train and assess all personnel involved in the transfusion process.

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An acute hemolytic reaction may be caused by which of the following? (Choose all that apply)View Page
Definition and Epidemiology

Transfusion-associated acute lung injury (TRALI) is a complication of blood transfusion that results in shortness of breath due to pulmonary edema, fever, and hypotension. The pulmonary edema is noncardiogenic which means it does not originate from the heart. TRALI is a severely life-threatening adverse reaction. Symptoms manifest within 6 hours of transfusion. Products typically implicated in TRALI are Whole Blood, Red Blood Cells, Fresh Frozen Plasma, Cryoprecipitate, and Platelets, with Fresh Frozen Plasma being the most often implicated product. In combined fiscal years 2005 through 2009, transfusion-related acute lung injury (TRALI) caused the higest number of reported fatalities (48%), followed by hemolytic transfusion reactions (26%) due to non-ABO (16%) and ABO (10%) incompatibilities. Complications of microbial infection, transfusion-associated circulatory overload (TACO), and anaphylactic reactions each accounted for a smaller number of reported fatalities. TRALI has accounted for the highest number of reported transfusion-related fatalities throughout the first decade of 2000.Cases occur in all age groups and genders. Most patients that develop TRALI have no history of adverse reactions. TRALI is generally under-diagnosed and under-reported and the true incidence may be higher than stated estimates. Under-diagnosing is due to lack of recognition of the condition and that it can be easily confused with other diseases. Also, TRALI may be attributed to the underlying condition of the patient.Reference: U.S. Food and Drug Administration Website. Fatalities reported to FDA following blood collection and transfusion: Annual summary for fiscal year 2009. Available at: http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/TransfusionDonationFatalities/ucm204763.htm. Accessed April 26, 2011.

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Definition and Incidence

Delayed hemolytic transfusion reactions (DHTR) are reactions that occurs 3 to 10 days after the transfusion. Usually, the blood appears serologically compatible at initial testing. Delayed reactions are common in patients who have been immunized to a foreign antigen from a previous transfusion or pregnancy, but the antibody titers decrease over time and the antibody is not detectable during pre-transfusion testing. The transfusion leads to a secondary (anamnestic) response, causing increased antibody production that sensitizes antigen-positive donor red cells. Hemolysis is extravascular. Sensitized cells are removed from circulation by the reticuloendothelial system, also called the monocyte-macrophage system. Because there is a delay in the presentation of symptoms, DHTR is not usually considered as a cause of the clinical presentation. The transfusion service usually initiates investigation of a DHTR because of serologic findings in a post-transfusion specimen. DHTRs occur more frequently than acute hemolytic reactions. Approximately 1:2500 transfusions result in a DHTR.

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Diagnosis

The symptom most commonly associated with a delayed hemolytic transfusion reaction (DHTR) is unexplained decrease in hemoglobin and hematocrit. Patients may also present with fever and jaundice. Hemolysis occurs slowly and is primarily extravascular. Unlike an acute hemolytic transfusion reaction (AHTR), hemoglobinuria, acute renal failure, and disseminated intravascular coagulation (DIC) are not generally seen. On some occasions, patient's may not present with any symptoms. Serologic findings include a positive direct antiglobulin test (DAT) and/or a positive antibody screen in post-transfusion testing. In many cases, the physician will send a request for an additional transfusion because of the decreased hemoglobin levels, and not suspect a DHTR. The positive antibody screen will trigger an investigation including antibody identification. The DAT may have a mixed field appearance because of the antibody-sensitized transfused red cells and the non-sensitized patient red cells. Segments from the donor unit can be tested for the offending antigen once the antibody has been identified.Antibodies that are most often reported as the cause of DHTR are anti-Jka and anti- Jkb. Other antibodies that are also commonly implicated in a DHTR include Kell, Rh, and Duffy system antibodies.The patient's physician should be notified so that additional clinical and laboratory evidence can be evaluated.

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Severe Delayed Hemolytic Transfusion Reactions (DHTR)

Generally, the clinical symptoms of a delayed hemolytic transfusion reaction (DHTR) resolve within 2-3 weeks without medical intervention other than transfusion support. On the other hand, severe DHTRs can occur with a life-threatening anemia. Severe delayed reactions occur most often in patients with sickle cell anemia. Sickle cell anemia patients have a high alloimmunization rate which puts them at greater risk for developing a DHTR. Diagnosis of a DHTR can be difficult in sickle cell patients because symptoms can be misdiagnosed as sickle cell crisis pain. Delays in medical treatment may lead to death. It is important for the transfusion service to obtain an accurate transfusion history. It is unclear what causes such severe reactions in sickle cell patients. Several explanations include bystander hemolysis, sickle cell hemolytic transfusion syndrome, and hyperhemolysis. In any case, it is important to recognize that severe DHTR in sickle cell patients is not uncommon. Treatment requires rapid diagnosis and transfusion support with antigen-negative red cells.

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Delayed hemolytic transfusion reactions (DHTR) typically occur 3 hours after transfusion.View Page

White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
The upper photograph of a bone marrow section reveals distinct hyperplasia with total replacement of marrow fat. A bone marrow smear stained with Wright/Giemsa is displayed in the lower photograph. Calculate the M:E ratio between myeloid and erythroid cells found in the lower photograph. The total peripheral blood white blood cell count was 5,400/cumm. This bone marrow architecture may be found in each of the following conditions except:View Page
The upper photograph of this bone marrow section also reveals distinct hyperplasia with total replacement of the fat. The lower photograph is a Wright/Giemsa stain. Calculate the M:E ratio of the distribution of myeloid and erythroid cells in the lower photograph. The peripheral white blood count was 18,500/cumm. The most likely associated condition is:View Page
The peripheral blood smear shown in this image was held for review because of an increase in platelets. Conditions in which platelets are increased as noted in this image include:View Page
Erythrophagocytosis

Illustrated in the image is a phagocyte devouring several erythrocytes. This uncommon phenomenon occurs in the bone marrow and in the spleen as part of the process of erythrocyte destruction. Erythrophagocytosis is found in histological sections of the spleen in cases of hemolytic anemia. This phenomenon appears also in splenic sections in lupus erythematosis, and in rheumatoid arthritis. Our example is from a patient with a myeloproliferative disorder and is a rare example of a circulating erythrophagocytic cell in the peripheral blood.

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