Hemolytic Information and Courses from MediaLab, Inc.

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.

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.

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

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.