Second Year Medical Student Fall Pathology Lab: Transfusion Medicine and Blood Banking October 21st and 22nd 2013 1 Outline, Presenters, Contact Info • Blood Donation, Component preparation, and Transfusion indications – Dr. Dan Waxman, M.D. • Executive Vice President and Chief Medical Officer of Indiana Blood Center • Clinical Professor of Pathology • [email protected] • Ordering a blood transfusion – Dr. Connie Danielson, M.D, PhD • Chief of Laboratory Service at Wishard Hospital • Clinical Associate Professor • [email protected] • Basic principles of transfusion – Dr. Julie Cruz, M.D. • Associate Medical Director of Indiana Blood Center • Volunteer Clinical Assistant Professor of Pathology • [email protected] 2 Outline, Presenters, Contact Info • Transfusion reactions – Dr. Nicole Hubbard, M.D. • Microbiology fellow • [email protected] • Case Presentation – Dr. Ted Kieffer, M.D., M.S. • PGY3 Pathology resident • [email protected] • Presentors – Dr. Morgan McCoy, M.D. • PGY3 Clinical Pathology resident – Dr. Stephanie Slemp, M.D. • PGY2 Pathology resident 3 Transfusion Medicine Lab Blood Donation, Component Preparation, and Transfusion Indications Transfusion therapy is a set of processes, not just a product Medical reason to TX Recruit donor Screen donor Pre-TX testing Collect unit Issue unit Prepare components Administer at bedside Monitor & evaluate Infectious diseases testing Product: Blood safety Entire process: Blood transfusion safety After S. Dzik, MD Blood Transfusion Service, MGH, Boston Whole Blood A. Description: 500+/- 50 ml mixed with 70 ml CPD B. Storage: 21 days stored at 1-6º C. Indications: Recently used in military hospitals in combat areas settings Proposed clinical trials to examine feasibility and efficacy in civilian setting Packed Red Blood Cells A. Description: 1. 200 ml of RBC with 111 ml of additive solution 2. Packed cell volume = 60% B. Storage = 42 days Packed Red Blood Cells C. Indications: 1. Acute blood loss exceeding 15-20% of blood volume (pediatric patients - 10-15 ml/Kg) and failure to obtain hemodynamic stability with reasonable volume of crystalloid and/or colloid solutions 2. Acute blood loss of any amount if there is clinical evidence of inadequate oxygen carrying capacity Packed Red Blood Cells C. Indications: (Cont’d) 3. Hemoglobin of ≤ 7 gm/dl (hematocrit ≤ 21%), if not due to a treatable cause (treatment of underlying case is preferable if patient is not symptomatic) 4. Symptomatic anemia regardless of hemoglobin level 5. Hemoglobin ≤ 8 gm/dl (hematocrit ≤ 24% ) and acute cardiac disease / or shock Packed Red Blood Cells D. Contraindications: 1. 2. 3. 4. For volume replacement In place of a hematinic To enhance wound healing To improve general “well-being” Leukocyte-Reduced Red Blood Cells A. Description: Packed red cells with leukocytes reduced (residual leukocyte count less than 5 X 106) Leukocyte-Reduced Red Blood Cells B. Processing of Product: 1. Product made during transfusion with filter attached to unit 2. Pre-storage leukocyte reduction at blood center Leukocyte-Reduced Red Blood Cells C. Indications: 1. Prevention of HLA/WBC alloimmunization 2. Prevention of recurrent non-hemolytic febrile reactions 3. Prevention of CMV transmission in select groups of patients Saline Washed Red Blood Cells A. Description: packed red cells washed with saline 1. 99% of plasma proteins are removed 2. 85% of leukocytes are removed 3. Post-wash K + is 0.2 meq/L Saline Washed Red Blood Cells B. Processing: manual and automatic methods C. Storage: once washed, 24-hour outdate Saline Washed Red Blood Cells D. Indications: 1. History of allergic or febrile reactions secondary to plasma proteins not prevented by pre-transfusion administration of antihistamines and leukocyte reduction 2. IgA deficiency with documented IgA antibodies 3. History of anaphylactic reaction to blood components Irradiated Blood Products A. Products irradiated: Whole blood, packed red cells, platelets and granulocyte concentrates Irradiated Blood Products B. Indications: preventing graft versus host disease 1. 2. 3. 4. 5. Immunocompromised patients Directed donations from blood relatives Premature infants ≤ 1200 gms Fetuses receiving intrauterine transfusions Neonatal exchange transfusions Irradiated Blood Products C. Processing and final product: 1. Irradiate with 2500cGy 2. Mitotic capacity of lymphocytes is reduced or eliminated without significant functional damage to other cellular elements Irradiated Blood Products D. Storage: Red cells outdate 28 days from irradiation (or original expiration if less than 28 days) Platelet Concentrates A. Description: 1. Random donor unit contains 5.5 X 1010 platelets suspended in 30-50 ml of plasma 2. Apheresis donor unit contains 3. 3.0 X 1011 platelets suspended in 300-400 ml of plasma 2-RBCs Plateletpheresis Plasmapheresis Platelet Concentrates B. Storage: 1. Stored at 20-24º C on a rotator 2. 5-day outdate Platelet Concentrates C. Indications: prevention and cessation of bleeding 1. Severely thrombocytopenic (less than 10,000 or 20,000 depending on institution) 2. Moderately thrombocytopenic and bleeding (less than 50,000) 3. Surgery or invasive procedure (less than 50,000) 4. Diffuse microvascular bleeding following cardiopulmonary bypass or with intra-aortic balloon pump (less than 100,000) 5. Bleeding with qualitative platelet defect 6. Massive Transfusion Protocols (MTP) Platelet Concentrates D. Contraindications: 1. Idiopathic Thrombotic Thrombocytopenic Purpura (ITP) 2. Thrombotic Thrombocytopenic Purpura (TTP) Platelet Concentrates E. Dosage: 1. 2. 3. 4. 4-6 platelet concentrates 1 apheresis unit Platelet count should increase 25,000 – 30,000/cc3 Each dose has equivalent of one unit of fresh plasma* * Unless using platelet additive solution (PAS) Frozen Plasma A. Description: 1. 225-275 ml of plasma and CPDA-1, including 25 meq of citric acid 2. Jumbo plasma 400 cc or greater 3. Frozen within 8 hrs = FFP 4. Frozen within 24 hrs = 24FP B. Storage: 1 year at -18º C Frozen Plasma C. Outdate once thawed (1-6º C) 1. 24 hours for FFP or 24FP 2. 72-120 hours if relabeled as Thawed Plasma 37 Frozen Plasma D. Indications: 1. Treatment of coagulopathy due to clotting factor deficiencies 2. Patient is bleeding actively with PT and/or PTT greater than 1.5 normal (INR > 1.8) and platelet count above 50,000 3. Coumadin overdose with major bleeding or impending surgery 4. Treatment of TTP 5. Massive Transfusion Protocol (MTP) Frozen Plasma E. Contraindications: 1. Volume expansion 2. Treatment of nutritional deficiencies Cryoprecipitate A. Description: each unit consists of 10-30 ml residual plasma than contains 80 units of factor VIII: C and 250 mg of fibrinogen B. Storage: 1 year at -18º C Cryoprecipitate C. Indications: 1. 2. 3. 4. Hypofibrinogenemia (≤ 100 mg/dl) Dysfibrinogenemia Factor XIII deficiency - rare MTP New/Future Products A. Growth Factors to stimulate cell production B. Sterilized products to prevent infections* C. Synthetic products to replace current transfusions* *Approval delayed due to regulatory reluctance. Questions ? TRANSFUSION MEDICINE LAB • ORDERING A BLOOD TRANSFUSION: • What does your patient need? – Why? How much? When? Alternatives? • Obtain Consent • Write Order • Obtain Specimen and send to Blood Bank Case Presentation The patient is a 64 year old man 2 days after a total hip arthroplasty. He has a history of coronary artery disease and had a MI 5 years ago. He complains that during physical therapy he becomes short of breath and develops chest pain which resolves with rest and sublingual nitroglycerine. Today his Hgb/HCT= 7.5 gms/dL/23%. You decide to order 1 unit of packed red blood cells to be transfused. Why? packed Red Blood Cells • Indication – patient is anemia AND symptomatic • Hgb < 8gm/dL • Patient complaining of shortness of breath *1 unit RBC/Adult increases Hgb 1 gm/dL* • Since the 1990’s it has become clear that “over-transfusion” can increase morbidity and mortality. Transfusion Guidelines: • In the USA currently accepted guidelines for transfusion of red cells is generally: – Hgb ≤7 g/dL many need transfusion – Hgb 7-9 g/dL variable need for transfusion – Hgb≥ 10 g/dL very few need transfusion Blood is a drug with a variable safety profile (discussed later) Normal levels of Hgb in adults • Men: 13.8 to 18.0 g/dL • Women: 12.1 to 15.1 g/dL • If the concentration is below normal, this is called anemia. • Hematocrit, the proportion of blood volume occupied by red blood cells, is typically about three times the hemoglobin concentration measured in g/dL. Blood volume of an Adult vs a Neonate Equations Hematocrit (HCT) Ex: 40% An adult has = = ~ 2L of RBC ~ 3L of plasma RBC volume Total blood volume ? RBC 2 liters volume 5 liters Obtain Consent: • • • • Written informed consent Expected benefits Possible risks Alternatives • If transfusion is refused -document Consent form 56 Obtain written informed consent: What are the patient’s concerns? What are the Blood Bank’s concerns? Complications of Transfusion Complications of Transfusion continued Write the order: Type and cross patient for 1 unit of red blood cells. Give when ready over 2-4 hours as tolerated Obtain Specimen: Identifiers on wrist band Identifiers on specimen label Identifiers on requisition All Identical A Patient’s Specimen must have: • • • • Patient identifiers Phlebotomist signature +/- witness signature Date and time collected* *A patient’s specimen can be used for 3 days Questions: A unit of red blood cells should raise the Hgb concentration in a nonbleeding adult by approximately: 1) 0.5 grams/dL 2) 1.0 grams/dL 3) 2.0 grams/dL 4) 5.0 grams/dL Questions: A unit of red blood cells should raise the Hgb concentration in a nonbleeding adult by approximately: 1) 0.5 grams/dL 2) 1.0 grams/dL 3) 2.0 grams/dL 4) 5.0 grams/dL Questions: A patient’s specimen sent to Blood Bank may be used for testing for : 1) 12 hours 2) 24 hours 3) 2 days 4) 3 days Questions: A patient’s specimen sent to Blood Bank may be used for testing for : 1) 12 hours 2) 24 hours 3) 2 days 4) 3 days Questions: A patient with a Hgb concentration of 10 g/dL would have a HCT of approximately: 1) 20% 2) 20 g/dL 3) 30% 4) 31 g/dL Questions: A patient with a Hgb concentration of 10 g/dL would have a HCT of approximately: 1) 20% 2) 20 g/dL 3) 30% 4) 31 g/dL Questions ? Basic Principles of Transfusion ABO, Rh, Other Blood Groups Pretransfusion Testing ABO Blood Groups • 4 Types: A, B, AB and O the name of the blood group corresponds to the presence of the antigen associated with the sugar residue (ie, a person whose red blood cells carry the A antigen has blood group type A. Type O red blood cells lack these terminal sugars, and the corresponding antigen is called the “H” antigen. Antigen Specificity Immunodominant sugar H L-fucose A N-acetylgalactosamine B D-galactose ABO Antigens and Antibodies Blood Group Antigen on Red Blood Cell Antibodies in serum Group O H (neither A nor B) Anti-A, Anti-B Group A A Anti-B Group B B Anti-A Group AB A and B Neither anti-A nor anti-B • ABO compatibility is the primary concern when selecting a unit of blood for transfusion • Other antigens are present on red blood cells, and antibodies to these antigens are also important. However, these antibodies occur only after the individual has been exposed (or sensitized) to the antigen through transfusion or pregnancy. This is also called alloimmunization. RED CELLS RECIPIENT CELLS TRANSFUSED (DONOR) CELLS RECIPIENT CELLS PLASMA TRANSFUSED (DONOR) CELLS ABO Frequencies Group O 45% Group A 40% Group B 11% Group AB 4% ABO Typing • forward type involves testing the patient’s red blood cells with commercial anti-sera containing anti-A and anti-sera containing anti-B. Reactivity with the specific anti-sera indicates that antigen is present on the patient’s red blood cells • reverse type combines the patient’s serum with reagent red blood cells that are either type A or type B. Reactivity indicates the patient has circulating antibody to the corresponding antigen on the reagent red blood cell. • Reactivity occurs in the form of agglutination – which is the clumping together of the red blood cells resulting from the interaction of the antibody and its corresponding antigen. This clumping can be observed visually or detected photometrically in automated equipment. Strength of agglutination is graded from 0 (no agglutination) to 4+ (very strong). ABO Typing sera and reagent red cells Agglutination 4+Agglutination (strong positive) 0 agglutination (negative) ABO Forward and Reverse Groupings Forward group: Patient’s cells with reagents Reverse Group: Patient’s serum with reagents Blood Group Anti-A Anti-B Antigen(s) on RBCs A1 cells B cells Antibody(ies) in serum O 0 0 No A or B + + A and B A + 0 A 0 + B B 0 + B + 0 A AB + + A and B 0 0 No A or B The Rh Blood Group System • RhD • The RhD gene is either present or absent – it’s absence is denoted by the placeholder “d”. There is no “d” gene, and no “d” antigen. • An individual who possesses no copies of the RhD gene (did not inherit RhD from either parent) is indicated by d, and said to be “Rh negative”. Eighty-five percent of Caucasians express the D antigen. • Some RBC’s exhibit a weaker than normal form of the D antigen, giving weaker or negative reactivity with standard anti-D reagent. This antigen expression is known as weak D. RhCE • The RhCE gene codes for either RhCe, RhcE, Rhce, or RhCE proteins. Again, these genes are inherited as a haplotype from each parent. The following table shows the possiblehaplotypes and their frequencies utilizing Fisher-Race and Weiner terminology. Prevalence (%) Haplotype Fisher-Race DCe dce DcE Dce dCe dcE DCE dCE Haplotype Weiner R1 r R2 R0 r’ r” Rz ry White Black Asian 42 37 14 4 2 1 <0.01 <0.01 17 26 11 44 2 <0.01 <0.01 <0.01 70 3 21 3 2 <0.01 1 <0.01 Calculating prevalence • Since one haplotype is inherited from each parent, the phenotype frequencies may be obtained by multiplying the haplotype frequencies for the population. For example, the frequency of the dce/dce (rr) phenotype in the Caucasian population is .37 X .37 = .14 or 14%. So, if an alloimmunized individual required red blood cells of this type for transfusion, approximately 14% of randomly collected donor units would be appropriate. • What frequency would you expect to find the dce/dce (rr) phenotype in the Black population? Other Blood Group Systems • Lewis – Antibodies to the antigens in the Lewis system are rarely clinically significant. The antigens are not synthesized on the red cells, but are absorbed from the plasma. The importance of the Lewis system is more its association with the Se gene and the determination of an individual’s secretor status. • Kell – After ABO and Rh, the K antigen is the most immunogenic blood group antigen causing both HDFN and delayed hemolytic transfusion reactions. About 10% of whites and 2% of blacks are positive for the K antigen. The antithetical antigen, k, (pronounced Cellano) is a high incidence antigen, meaning <1% of the population lacks this antigen. Other Blood Group Systems • Kidd – Two antigens, Jka and Jkb are responsible for the common phenotypes. The Kidd antigens are located on a membrane glycoprotein involved in urea transport in the red cell. They are often weakly reacting antibodies showing dosage. Dosage refers to stronger expression of the antigen when the individual inherits the gene homozygously, vs. heterozygously. It is important that antibodies to these antigens are ruled out on homozygous reagent cells, since a heterozygous cell may be nonreactive due to the low expression of the antigen. Kidd antibodies are notorious for weakening over time and even becoming undetectable. Despite this, they cause severe delayed hemolytic transfusion reactions. They are also implicated in HDN, but it is usually a milder form. Other Blood Group Systems • MNS – The most important antigens that affect transfusion are M, N, S, s and U. The M and N antigens are carried on glycophorin A, and S,s, and U are carried on glycophorin B. Anti-M and anti-N can occur as naturally occurring agglutinins (IgM) and as such usually react at room temperature or below. They do not cause HDFN. These antibodies can usually be ignored for transfusion, unless they are also reactive at 37 C due to the presence of an IgG component. In contrast, S and s are IgG reactive at 37 C, are clinically significant and cause HDFN. Anti-U is a rare antibody which is associated with HDFN and hemolytic transfusion reactions, usually seen in the African American population. It occurs in individuals who are S-s-U-, and individuals who have anti-U are incompatible with greater than 99% of the donor population. Other Blood Group Systems • Duffy – The Duffy system is made up of six antigens that reside on an acidic glycoprotein . The glycoprotein carrier molecule is also known as the Duffy antigen receptor for chemokines (DARC) and functions as a cytokine receptor. It is also a receptor for the malarial parasite Plasmodium vivax. The null phenotype Fy(a-b-) is rare in Caucasians but common in blacks. It is thought that this developed as a survival advantage in malarial regions of Africa, since such individuals are naturally resistant to infection by P. vivax. Anti-Fya is a relatively common antibody, while anti-Fyb is less common. Both are clinically significant and associated with hemolytic transfusion reactions and HDFN. Antibody Screen The goal of antibody screening is to detect unexpected clinically significant red cell antibodies. In general, clinically significant antibodies are antibodies known to have caused hemolytic transfusion reactions, hemolytic disease of the fetus and newborn (HDFN), or shortened survival of transfused red blood cells. The method used must be capable of detecting clinically significant antibodies, which requires that the antibody screen method include a 37 C incubation with reagent red cells followed by an indirect antiglobulin test (IAT). The Indirect Antiglobulin Test (IAT): The IAT is used to detect in-vitro sensitization and detects antired cell antibodies in patient’s serum or plasma. Procedural steps are as follows: 1. Patient’s plasma or serum is incubated at 37 C with red cells (screen or panel cells of known antigenic composition, ie. reagent red cells, or donor cells as in the crossmatch). 2. A potentiator (LISS, PEG) may or may not be added 3. During incubation, if an antibody is present in the plasma or serum and the corresponding antigen is present on the red cells, the cells become sensitized by the antibody adsorbing to antigens on the red cell surface 4. After incubation, the red cells are washed with saline to remove unbound antibody 5. Antihuman globulin serum (anti-IgG or polyspecifc AHG, usually the former) is added and forms RBC agglutinates if the antibody has attached to the antigen sites during incubation Indirect antiglobulin test Crossmatch Methods: • Immediate spin crossmatch: The immediate spin (IS) crossmatch is performed only after an antibody screen is done and found to be negative on a current specimen. It is performed at room temperature without enhancement. The patient should have no history of clinically significant antibodies. The immediate spin crossmatch is meant to detect ABO incompatibility. It can also detect cold reactive (generally clinically insignificant) antibodies that react at room temperature. • Antihuman Globulin (AHG) crossmatch The AHG is performed as described for the IAT above, and tests the patient’s serum or plasma against the donor RBCs intended for transfusion to the patient. It will detect ABO incompatibility as well as other clinically significant antibodies, such as antibodies in the Rh, Kell, Kidd, and Duffy systems. Computer-Assisted (Electronic) Crossmatch Principle: • Using a validated computer system, patients with no history of clinically significant antibodies and a negative antibody screen on the current specimen are issued ABO specific or compatible donor units. Specific conditions must be met: – – – – – Two independent determinations of the ABO group of the patient No history of clinically significant antibodies A negative antibody screen on a current specimen Donor units are ABO confirmed The computer system is validated on site, results are entered directly into the computer, and there is logic in the system to recognize incompatibility In the last section: • You ordered type and cross for 1 unit of red blood cells for your patient. • Now, you are the blood bank technician: • You receive an appropriately labeled and collected specimen with an order to type and cross for one unit of red blood cells. First, you perform the ABO and Rh Typing: Forward type: tube on left contains anti-B, tube on right contains anti-A. Reverse type: Tube on left contains A1 Cells, Tube on right contains B cells Forward Anti-A Reverse Anti-B Type? A Cell B Cell Type? Patient RBC with anti-D Rh Typing Anti-D • What is the patient’s ABO/RH? Antibody screen • A 3 cell antibody screen is performed and two of the three cells are positive. What does this mean? You test the patient’s serum against a panel of reagent red blood cells with the following results: • Antibody is present to what antigen? • What does this imply for transfusion? • A unit of RBCs that is Type A, Rh positive and confirmed negative for the Jkb antigen is selected for the patient. The AHG crossmatch is performed and the unit is compatible. Is this unit appropriate for the patient? Questions ? Transfusion Reactions Nicole Hubbard M.D. Microbiology fellow Outline • Definition • Types of transfusion reactions – Acute febrile transfusion reactions – Acute non-febrile transfusion reactions – Delayed febrile transfusion reactions – Delayed non-febrile transfusion reactions • Risk of Transfusion reactions • Approach and follow-up to suspected transfusion reactions 105 Definition • Unfavorable events that occur during or after transfusion • Can happen with any blood component • PRBC, Plasma, Platelets, Cryoprecipitate • Rho (D) immune globulin, IVIG, Factor concentrate • Stem cells, Human albumin, Granulocytes Categorization of transfusion reactions Febrile Afebrile Acute Hemolytic (HTR) TRALI Bacterial contamination Non-hemolytic (FNHTR) TACO Urticarial/allergic Premedicated febrile Delayed Hemolytic (DHTR) TA-GvHD PTP Iron overload Acute hemolytic transfusion reactions (AHTR) • Most severe reaction (may be fatal!) • All reactions are hemolytic until proven otherwise • ABO incompatibility most commonly from clerical error • IgM antibodies bind to the RBCs → complement activation → formation of MAC → RBC lysis • Coagulation cascade activation from Ag-Ab complexes → • Intravascular thrombi → schistocytes • Coagulation cascade and complement → cytokine production → additional symptoms • Treatment is supportive Signs and Symptoms • Fever/Chills 1°C or 2° F (most common) • Tachycardia • Hypotension • Bleeding • Dyspnea • Impending doom • Nausea/Vomiting • Pain: flank, back, chest, head, infusion site • Dark urine (hemoglobinuria) • Death Laboratory Findings • Hemoglobinemia (pink or red serum/plasma) • Hemoglobinuria (NOT hematuria) • Usually positive direct antiglobulin test (DAT) but can be negative (all Ab coated cells already lysed) • Elevated indirect bilirubin and LDH • Decreased haptoglobin • Hyperkalemia • Peripheral smear: Schistocytes Transfusion Related Acute Lung Injury (TRALI) • #1 cause of fatal transfusion reactions in the US • Usually donor anti-HLA or anti-neutrophil antibodies attach to neutrophils in lung → neutrophil activation → vascular damage → edema and impaired gas exchange • Begins during or within 6 hours of transfusion • No pre-existing acute lung injury • Prevented by use of male plasma – Why??? Symptoms and Other Findings • • • • • Dyspnea Fever Tachycardia Hypotension Frothy endotracheal aspirate • Death • CXR shows whiteout/edema Suspected TRALI • Donor testing for anti-HLA and anti-neutrophil antibodies • Permanent deferral for donor if positive Bacterial Contamination • Contaminated blood product → sepsis • Most common in platelet products – Why??? • Symptoms: – Rapid high fever, hypotension, tachycardia, and shock – May also have violent rigors, nausea, vomiting, diarrhea – Shock and death Febrile non-hemolytic transfusion reactions (FNHTR) • Most frequently reported • Temperature increase of >1°C or >2°F with no other explanation • Cytokines from WBC in product during storage or antibody activation of WBC • Usually mild symptoms – Fever – Chills (may be only symptom if pt is premedicated) • Treatment: – Antipyretics (acetaminophen) – Leukoreduced products (pre-storage) Categorization of transfusion reactions Febrile Afebrile Acute Hemolytic (HTR) TRALI Bacterial contamination Non-hemolytic (FNHTR) TACO Urticarial/allergic Premedicated febrile Delayed Hemolytic (DHTR) TA-GvHD PTP Iron overload Transfusion associated circulatory overload (TACO) • Fluid overload precipitated by blood product • Risk factors: Renal impairment and heart failure • Signs and symptoms: Dyspnea, tachycardia, hypertension, jugular venous distension • Treatment: Support respiratory function and diuretics TRALI v. TACO TRALI • Dyspnea • Tachypnea • Tachycardia • Hypotension • Fever TACO • Dyspnea • Tacypnea • Tachycardia • Acute Hypertension • Jugular venous distension • Rales • BNP levels Allergic Reaction • Second most common type of transfusion reaction • Only reaction that allows the restart of procedure if symptoms resolve • Caused by type I hypersensitivity to donor plasma proteins • Symptoms: Pruritis, urticaria, erythema • Treatment/Prevention – Benadryl – Could give plasma deficient products (washing) Anaphylactic Reaction • Most severe allergic reaction • Symptoms: Rapid onset of laryngeal edema leading to dyspnea, tachypnea, hypotension, tachycardia, afebrile • Often associated with recipients that lack IgA AND have anti-IgA • Can give washed or IgA deficient products Categorization of transfusion reactions Febrile Afebrile Acute Hemolytic (HTR) TRALI Bacterial contamination Non-hemolytic (FNHTR) TACO Urticarial/allergic Premedicated febrile Delayed Hemolytic (DHTR) TA-GvHD PTP Iron overload Delayed Hemolytic Transfusion Reactions (DHTR) • Anamnestic response = resurgence of antibody faster and more severe after second exposure • IgG antibodies bind to the RBCs(sensitized cells) → processed by reticuloendothelial system→ RBCs removed by spleen • Incompletely removed cells result in spherocytes • Main causes Kidd (Jk) and Duffy (Fy) systems Signs and Symptoms • • • • • Fever/chills (mild) Mild jaundice Anemia/pallor Splenomegaly Rarely death Laboratory Findings • • • • • DAT positive Hyperbilirubinemia Usually no free hemoglobin (versus AHTR) “New” antibody Spherocytes on peripheral smear Transfusion-Associated Graft versus Host Disease • Highly (> 90%) fatal • Donor WBC recognize pt’s HLA antigens → WBC activation → destruction of host tissues • Signs and symptoms: Fever, diarrhea, skin rash, bone marrow suppression • Treatment/Prevention – Supportive care – Irradiation of blood products • Why??? Categorization of transfusion reactions Febrile Afebrile Acute Hemolytic (HTR) TRALI Bacterial contamination Non-hemolytic (FNHTR) TACO Urticarial/allergic Premedicated febrile Delayed Hemolytic (DHTR) TA-GvHD PTP Iron overload Post-transfusion Purpura • Patient abs against donor platelet antigens (usually PLA1) • PL-A1 negative patients (RARE) • Signs/symptoms: Profound thrombocytopenia, purpura, bleeding • Patient’s own platelets undergo destruction as well (unknown mechanism) • Treatment: supportive/plasmapheresis Iron Overload • Transfusion dependent anemia (i.e. sickle cell and thalassemia patients) • Iron accumulates with multiple transfusions • Results in end-organ damage • Treatment/prevention • Chelation therapy • Exchange transfusions Risks of Blood Product Transfusion •Hives: 1 in 30 to 100 •Febrile reactions: unknown quantity, but common •Transfusion Associated Circulatory Overload (TACO): 1 in 3,000 to 12,000 •Transfusion-Related Acute Lung Injury (TRALI): 1 in 5,000 •Acute Hemolytic Reactions: 1 in 15,600 to 35,700 •Bacterial Infection: 1 in 20,000 •Hepatitis B: 1 in 200,000 •HTLV-1: 1 in 641,000 •Hepatitis C: 1 in 1.2 million •HIV: 1 in 1.4 million •Other infectious diseases such as West Nile virus: less than 1 in million •Other infections such as Babesiosis or Chagas: very rare •Anaphylaxis: 1 in 20,000 Indianapolis Coalition for Patient Safety, Transfusion consent form. What To Do For Suspected Transfusion Reaction? • • • • • • • Stop Transfusion Keep line open with saline Report to physician Order transfusion work up Clerical check at bedside Unit and tubing need to be returned to blood bank Patient blood sample sent to lab Laboratory Work-Up • • • • Examine blood for hemolysis Perform DAT Repeat type and cross If greater then >2°C, send retention segment and bag for culture • Also, blood cultures on patient • If suspect HTR • Collect first voided urine • Check bilirubin 6 hours post transfusion • Check haptoglobin • Check CBC References • References available upon request. • Thank you: • Judi Seidel, MT (ASCP) SBB • Dr. Steven Gregurek M.D. Questions ? Case Presentation MS2 Pathology Lab Blood Bank Lab 10/21 & 10/22 Ted Kieffer PGY3 Case Presentation • A 25 year old pregnant Chinese female is brought to the emergency department following a motor vehicle accident. She does not speak English but appears to be in significant pain and grasping her lower abdomen. What do you do? Case Presentation • History and review of systems cannot be obtained due to language barrier and no translator readily available. • Vitals – HR 110 beats per minute – Resp 20 breaths per minute – BP 135/90 – Sats 97% on room air Case Presentation • Physical exam – Relatively unremarkable aside from • Abdominal exam which reveals a band-like contusion across the lower abdomen and a firm, tender uterus with a fundal height of 26 cm • Vaginal exam remarkable for blood What else might you want to know? (i.e. who else might you want to evaluate?) Case Presentation • Fetal heart rate by doppler – 160 bpm Reasurring? Current Differential Diagnosis? Placental abruption Preterm Labor Placenta previa Uterine rupture Subchorionic hematoma Case Presentation Current Differential Diagnosis: Placental abruption – acute onset, tender & firm abdomen/uterus, maternal hypotension Preterm Labor – gradual onset, progressive, Mucus plug may be mistaken for vaginal bleeding Placenta previa – characteristically painless vaginal bleeding at >20 weeks gestation Uterine rupture – sudden onset FHR abnormalities, vaginal bleeding, abdominal pain, and maternal hypotension Subchorionic hematoma – light vaginal bleeding, generally no abdominal pain, <20 weeks gestation Case Presentation • Lab tests? – – – – CBC BMP + Ca, Mg, Phos Type and cross Coag studies • Including: Fibrinogen, fibrinogen degradation products (FDP), D-dimer – *other tests • Alpha fetal protein (AFP), human chorionic gonadotropin (hCG) • **Kleihauer-Betke test** • Imaging? – Ultrasound • Anything else? Orders? – Fetal heart rate monitor – Pain medication for mom Case Presentation Abdominal US/FAST scan Diagnosis!!! Placental Abruption Radiologist eventually reads sonogram and interprets findings as small abruption Case Presentation • What do you do now? – Vitals still stable – Lab results • CBC – 10.2 8.9>------<347 31 • BMP – 142/105/10 ---------------<95 4.0/24/1.1 Ca – 4.9 mg/dL (wnl) Mg – 2.0 mg/dL (wnl) Phos – 3.5 mg/dL (wnl) Anything else? Treat Patient – Tocolysis Magnesium Sulfate Case Presentation • What do you do now? – Lab results • Coag tests – PT – 9 seconds (normal 11-13.5 sec) – aPTT – 21seconds (normal 25-35 sec) • • • • • Fibrinogen – 350 mg/dL (wnl) FDP – 5 mcg/mL (wnl) D-dimer - <5μg/mL FEU (wnl) AFP & hCG wnl Kleinhauer–Betke test – 0.2% Hypercoag? Why did you order these? Case Presentation B-cells Anti-D Anti-A A-cells Anti-B • What do you do now? – More lab results • Type and cross – – – – ABO type A Rh negative Screen positive DAT positive with mixed field DAT Antibody screen 4+ 4+ Case Presentation Antibody Panel 0 4+ 4+ 4+ 0 0 0 0 0 0 0 M Anti-D with a mixed field on patient sample Case Presentation • At that moment the translator arrives and informs you the patient is feeling much better, abdominal pain has decreased, and that her contractions appear to have ceased. • Any other questions you would like to ask? Prior birth history? Case Presentation • The translator informs you that the patient has had two children prior to this pregnancy, all with the same father. The first was born without complications but the second was jaundiced for several weeks. All prenatal and postnatal care was received in rural China. Case Presentation • So what is happening? What must you be concerned about? What are your next steps? • Lets bring it all together – Diagnosis of placental abruption – Blood tests indicate two different RBC in mom’s blood – Mother is Rh negative – Anti-D antibody identified on RBC panel – History of complicated pregnancy FMH HDFN Case Presentation Causes of Fetal Maternal Hemorrhage (FMH) Delivery Indications for administering RhIG if mother is Rh- and fetus Rh+/unk At 28 weeks of gestation Induced abortion Spontaneous abortion Spontaneous abortion, threatened abortion, induced abortion Ectopic pregnancy Ectopic pregnancy Partial molar pregnancy Chorionic villus sampling Cordocentesis Invasive procedures: genetic amniocentesis; chorionic villus sampling; multi-fetal reduction; fetal blood sampling Percutaneous fetal procedures (eg, fetoscopy) Amniocentesis External cephalic version Abruptio placenta Hydatidiform mole Fetal death in the second or third trimester Blunt trauma to the abdomen Antenatal hemorrhage Maternal abdominal trauma Antepartum hemorrhage in the second or third trimester (eg, placenta previa or abruption) Spontaneous Manual removal of the placenta External cephalic version Case Presentation • Management of FMH – RhIG – Kleihauer-Betke test • standard method of quantitating fetal-maternal hemorrhage for calculating RhIG dose • Maternal blood smear exposed to acid pH which dissolves adult hemoglobin and leaves fetal hemoglobin intact – special stain applied (Shepard’s) which stains hemoglobin and percentage of fetal cells is calculated out of 2000 Case Presentation • Management of FMH – RhIG – Using Kleihauer-Betke test • Calculate maternal blood volume: – prepregnant wt kg x 70ml/kg x [1.0 + (0.5 x wks gestation/36)] - est blood loss – Or estimate 5L • Calculate fetal blood volume in maternal circulation: – (% from KBT) x maternal blood volume • Calculate dose of RhIG – 1 vial (300μg RhIG)/30mL fetal blood – fudge factor – round up one vial for values of n.0 to n.4; round up two vials for values of n.5 to n.9 Case Presentation • Management of FMH – RhIG – Using Kleihauer-Betke test in our patient • Calculate maternal blood volume: – No info given – using 5L or 5000mL • Calculate fetal blood volume in maternal circulation: – (0.2%) x 5000mL = 0.002 x 5000mL = 10mL • Calculate dose of RhIG – 1 vial/30mL fetal blood x 10mL = 0.33 vials = 1 vial (rounded up) – You administer 1 vial of RhIG to the patient – Why did you go through all of that effort? Hemolytic Disease of the Fetus/Newborn (HDFN) Defined as the destruction of fetal and neonatal red blood cells by maternal antibodies (termed alloantibodies) – Historically IgG Rh/anti-D antibodies Pathophysiology of HDN • Maternal IgG antibodies cross the placenta and attach to fetal red blood cells • Red blood cells hemolyzed or removed via reticulo-endothelial system • Resultant anemia causes accelerated production of RBCs by bone marrow, termed erythroblastosis fetalis • In severe disease, bone morrow inevitably fall short of necessary RBC production • Body responds with extramedullary hematopoesis in the spleen and liver • Hepato-splenomegaly causes portal hypertension and hepatocellular damage • Anemia coupled with hypoproteinemia leads to massive, diffuse edema and high cardiac output heart failure Erythroblastosis and Hydrops Fetalis Sequelae of HDN • Hyperbilirubinemia – RBC destruction does not cease with delivery • IgG due to its small size as a monomer distributes throughout tissues (intravascular and extravascular) • IgG has a half life of 25 days – Prior to delivery bilirubin is transported across placenta, conjugated by maternal liver – Bilirubin conjugation system in a neonate is immature – Without therapy unconjugated/indirect bilirubin can reach toxic levels (18-20 mg/dL) and diffuse into the brain causing kernicterus and acute bilirubin encephalopathy Kernicterus and Acute Bilirubin Encephalopathy Development of alloantibodies • Mother must be antigen negative – Antigen positive individuals will not form antibodies • Mother must be exposed to antigen – – – – • Feto-maternal hemorrhage Transfusion with ABO compatible, minor group incompatible blood Injection with needles contaminated with minor group antigen positive blood Minor blood group mismatch allogeneic stem cell transplant Antigen exposure induces antibody formation – Exact volume unknown (varies between individuals) but as little as 0.1 mL of Rh+ RBC have been shown to stimulate antibody production – Larger volume of exposure tends to produce a more robust response • Following antibody production mother must become pregnant with an antigen positive fetus – The initial response will be IgM which will not cross the placenta • In most cases, the first minor group incompatibility between mother and fetus will not be affected, with exceptions – Subsequent exposure will induce memory cells to produce IgG antibodies which will in turn cross the placenta and cause hemolysis or clearance Development of fetal alloantibodies • Mendelian genetics – Genes code for an enzyme (ABO system), surface protein (Rh system), or nothing (silent/amorph) – Blood group genes major (ABO)and minor (all others) are inherited in a Co-dominant pattern • Each parent contributes half of the inheritance • Individual traits are inherited independent of each other Development of fetal alloantibodies Rh+ Rh Rh- Dd dd Dd Rh + Dd Rh + dd Rh - dd Rh - Development of fetal alloantibodies Rh + Rh Rh- Dd dd Unlike A and B antibodies, many minor antibodies are not naturally occurring First Rh + child sensitizes Mother Mother develops Anti-D antibodies Dd Dd First Child IgG Alloantibodies cross placenta Rh + Second Child Second child conceived Rh + dd Rh - dd Rh - Why only IgG antibodies? • Only IgG type antibodies can cross the placenta • Actively transported via a receptor specific to IgG Fc region • Starts in the second trimester and continues until birth Antigens associated with HDFN • Major incompatibility • ABO antigens – usually mild HDFN, A and B antigens less well developed in neonate (actually protects from minor incompatibility) • Minor incompatibility – Frequently associated with severe HDFN • Rh (D, c) and Kell (K) – Infrequently associated with severe HDFN • See next slide Infrequently associated with severe disease Colton Coa MNS Mta Rhesus HOFM Co3 MUT LOCR ELO Mur Riv Dia Mv Rh29 Dib s Rh32 Wra sD Rh42 Wrb S Rh46 Duffy Fya U STEM Kell Jsa Vw Tar Diego Antigens infrequently associated with severe HDFN Jsb Rhesus Bea Other antigens HJK k (K2) C JFV Kpa Ce JONES Kpb Cw Kg K11 Cx MAM K22 ce REIT Ku Dw Rd Ula E Kidd Jka Ew MNS Ena Evans Far e Hil G Hut Goa M Hr Mia Hr0 Mit JAL Rh Alloimmunization: diagnosis • Rh(D) typing and screen – Performed at first prenatal visit – If Rh(D)-, screen negative, and prenatal course uncomplicated • Repeat at 28 weeks • Repeat at delivery – If Rh(D)-, screen positive diagnostic tests • Indirect antiglobulin test on maternal serum with titer • Direct antiglobulin test on fetal red blood cells with titer • Positive agglutination in saline or albumin indicate Anti-D IgM production Prevention of Rh Alloimmunization • Anti-D immune globulin/RhIG – IgG anti-D manufactured from human plasma (primarily male who undergo repeat injections of Rh+ blood) – Preparation methods • HyperRho S/D®, RhoGAM® – Cohn cold ethanol fractionation followed by viral-clearance ultrafiltration – intramuscular only as IgA and other plasma proteins have the potential to produce anaphlyaxis • Rhophylac®, WinRho SDF® – Ion-exchange chromatography isolation – intramuscular or IV Prevention of Rh Alloimmunization • Anti-D immune globulin – Mechanism of action: epitope masking?, rapid clearance? – Guidelines • Weak D positive managed as Rh• Mother Rh-, fetus confirmed or suspected Rh+ – 300 micrograms early in third trimester – 300 micrograms if there is increased risk of feto-maternal hemorrhage » Repeat doses if risk is ongoing, guided by titers – 300 micrograms within 72 hrs after delivery of a Rh+ infant » If inadvertently not administered, give ASAP – partial protection has been seen up to 13 days after birth Diagnosis and Treatment of Intrauterine fetal anemia • Diagnostic techniques: – – – – Ultrasound Doppler assessment of MCA peak velocity Percutaneous umbilical cord sampling Allele-specific PCR on fetal cells in amniotic fluid • Itrauterine fetal transfusion – Transfuse when fetal hematocrit falls below 2 standard deviations of mean hematocrit for gestational age – Can be performed between 18 and 35 weeks – Intraperitoneal transfusions not as effective as intravascular transfusions in hydropic fetus due to congested lymphatics – ABO type O, Rh- packed RBC utilized – Fetal loss 1-2% with overall survival of 85% after transfusion Case Presentation • Finalizing the case – What do you order? • Maternal Anti-D titer – 1:64 • US of fetus and MCA peak velocity – Slightly hydroptic fetus – MCA peak velocity elevated • Fetal CBC – Moderate anemia • Intrauterine fetal transfusion performed by IR – Mom transferred to high risk OB service where last you heard she was doing very well and expecting to be discharged Questions ?