Chapter 18: Immunohematology and Transfusion Medicine PDF

Chapter 18 IMMUNOHEMATOLOGY AND TRANSFUSION MEDICINE 1  Learning Outcomes 1. Define the terms immunohematology and transfusion medicine. 2. List the agencies involved in regulating the practice of transfusion medicine. 3. List the benefits and reasons for transfusing b...

Chapter 18 IMMUNOHEMATOLOGY AND TRANSFUSION MEDICINE 1  Learning Outcomes 1. Define the terms immunohematology and transfusion medicine. 2. List the agencies involved in regulating the practice of transfusion medicine. 3. List the benefits and reasons for transfusing blood products. 4. Correlate the various red blood cell components and derivatives used for transfusion and the reason for each. 5. List the factors necessary for donor selection, identification, criteria, donated blood testing, anticoagulants, labeling, and storage of blood products. 6. Define the terms autologous transfusion and directed transfusion, antigen, alloantibodies, isoantibodies and immune antibodies AND agglutination 7. Define the terms associated with the inheritance of red blood cell groups. 8. List the three means of detecting antigen-antibody reactions. 9. List the ABO phenotypes, genotypes and typing procedures used in blood banking. 2  Learning Outcomes 10. Understand which red blood cells may be given to a recipient based on their blood type. 11. Understand which plasma product may be given to a recipient based on their blood type. 12. Explain the difference between universal donor and universal recipient. 13. State the antigen involved in the Rh Group System that determines whether a patient is positive or negative. 14. Explain the difference between direct and indirect antihuman globulin testing. 15. Define transfusion reaction and list the signs and symptoms a patient may exhibit. 16. Explain the pathophysiology of HDFN and its occurrence in ABO and Rh antigens. 17. Define the acid elution stain and the assessment plan of fetomaternal hemorrhage. 3 Blood Transfusion: The Gift of Life A simple explanation of the steps taken for safe transfusion to patients as well as the explanation of the transfusion process.. https://youtu.be/zIPtekPhhjY?si=h9jeWrBYB54OXU6t 4  Overview of Blood Banking Learning Outcome 1: Immunohematology is the study of the immunologic responses to blood components. Transfusion medicine is the medical practice and techniques associated with the procurement, processing, and distribution of blood or blood components to patients. 5  Overview of Blood Banking Learning Outcome 2: Several government agencies in the United States regulate the practice of transfusion medicine. These include: ◦ National Center for Drugs and Biologics of the US Food and Drug Administration ◦ Centers for Medicare and Medicaid Services ◦ Occupational Safety and Health Administration (OSHA) ◦ State Departments of Health (inspections to ensure regulatory compliance) 6 Overview of Blood Banking Learning Outcome 2: Professional Leadership Organizations include: AABB( Association for the Advancement of Blood & Biotherapies)-an international professional association that provides leadership and mechanisms to deal with progress and change. College of American Pathologists (CAP) The Joint Commission (TJC) 7  Benefits and Reasons for Transfusion Learning Outcome 3: Restoring or maintaining oxygen-carrying capacity or hemoglobin (packed RBCs) Restoring or maintaining blood volume (acute blood loss- massive bleeding) Replacing coagulation factors (platelets or cryoprecipitate) Restoring or maintaining leukocyte functions (rare) 8 Whole Blood, Blood Components, and Derivatives for Transfusion –Learning Outcome 4 Whole blood contains formed elements (RBCs, WBCs, and platelets) and plasma. When it comes to red blood cell transfusions, the primary component used is "packed red blood cells" (PRBCs), which are essentially red blood cells with most of the plasma removed, allowing for efficient delivery of oxygen to tissues in situations like anemia or acute blood loss; Other derivatives like "washed red blood cells" might be used in cases of severe allergic reactions to plasma proteins, Irradiated red blood cells are given to immunocompromised patients to prevent graft-versus- host disease 9  Whole Blood, Blood Components, and Derivatives for Transfusion –Learning Outcome 4 Irradiated RBCs are red blood cells that have been treated with gamma or X-rays to inactivate donor lymphocytes. This process is used to prevent transfusion- associated graft-vs-host disease (TA-GVHD) in patients who are at risk. Irradiated RBCs are safe for all patients, but they have a shorter shelf life. 10 Whole Blood, Blood Components, and Derivatives for Transfusion –Learning Outcome 4 Plasma transfusions are used to treat a variety of conditions and deficiencies by replacing missing proteins in the blood. ◦ 1. Fresh frozen plasma-good source of labile clotting factors used to correct deficiency in coagulation factors or to treat shock due to plasma loss from burns or massive bleeding. ◦ 2. Plasma-derived products or plasma derivatives (PDMPs) are pharmaceuticals made from human plasma, the liquid part of blood. These products are used to treat various conditions, including bleeding disorders, immune deficiencies, and autoimmune diseases. 11 Whole Blood, Blood Components, and Derivatives for Transfusion –Learning Outcome 4 Some examples include: Factor VIII concentrate: replaces the missing factor VIII to help prevent or stop bleeding Cryoprecipitate: used to treat fibrinogen deficiencies 3. Plasma substitutes: include albumin and plasma protein fraction. Products heat treated to eliminate the risk of infectious diseases. Are used to treat patients who need replacement of blood volume. 12  Whole Blood, Blood Components, and Derivatives for Transfusion-Learning Outcome 4 1. Platelets are used in transfusions to treat and prevent bleeding in patients who have low platelet counts or platelet disorders. Platelets are tiny cell fragments that help form clots and stop bleeding. Plateletpheresis is a high-yield method for obtaining platelets for transfusion. Because all the components, except platelets, are returned to the donor in plateletpheresis, it is possible to obtain three single donor units from one donor during a single apheresis session. 2. Random Donor Platelet concentrates are platelets prepared by centrifugation and removal of plasma from a fresh unit of donor blood and subsequent separation of platelets from platelet-poor plasma. Units of platelets are stored at room temperature (20–24°C) with continuous, gentle agitation for up to 5 to 7 days. Bacterial contamination can be a problem encountered with the platelet blood component. 13  Whole Blood, Blood Components, and Derivatives for Transfusion Key points about platelet storage and bacteria: Short shelf life: Due to bacterial growth concerns, platelets are typically only stored at room temperature for up to 5 days. Reason for concern: Room temperature is ideal for bacterial multiplication, making platelet units susceptible to contamination if not handled properly. Sepsis risk: Bacterial contamination of platelets can lead to severe transfusion reactions, including sepsis, which is why careful screening and storage practices are crucial. Potential solutions: Some research explores methods to extend platelet shelf life by storing them at colder temperatures, which can slow bacterial growth, but this may also affect platelet function. 14 How Apheresis Gave Emma Her Life Back https://youtu.be/8mnOUoI3bDk?si=KhUW-BnEemb152hy 15 Blood Donation: Donors, Collection, Storage, and Processing- Learning Outcome #5 Collection and Processing of Red Blood Cells involves: 1. Donor selection, identification and criteria 2. Donated blood testing 3. Anticoagulants used to preserve donated blood products 4. Labeling of the donor blood product bag 5. Storage of donated Blood 16  Collection, Storage, and Processing-LEARNING OUTCOME 5 The FDA safety system includes regulating and monitoring the functions of: 1. Donor selection and identification: ◦ Donor screening is essential to ensuring the safety of the U.S. blood supply. ◦ FDA regulations require that a donor is free of any disease transmissible by blood transfusion as determined by a health history and physical examination. 2. Donor Criteria include: Age, weight, hemoglobin, temperature, travel history, blood pressure, tattoos/piercings, and medications 17 Blood Donation: Donors Screening, Collection, Storage, and Processing- #5 Learning Outcome Donated blood must be quarantined until it is tested and shown to be free of infectious agents. ◦ In addition to federal regulations, the FDA periodically issues guidance documents providing recommendations to decrease the potential for transmission of infectious diseases when new information or testing methodologies are available. 18  Blood Donation: Donors, Collection, Storage, and Processing- #5 Learning Outcome FDA monitors donated blood and is tested for a variety of things, including: Blood type The donor's blood type is determined and compared to their previous donations. Infections Blood is tested for infections like HIV, hepatitis B and C, syphilis, HTLV, West Nile virus, cytomegalovirus, and Zika virus. Viral genetic material Nucleic Acid Amplification Testing (NAT) looks for viral DNA or RNA in the blood sample. Blood is tested to ensure that it is safe for patients to receive a transfusion. After a first donation, blood is also tested for STDs to determine if the donor is allowed to donate again. 19 Blood Donation: Donors, Collection, Storage, and Processing –Learning Outcome 5 Various anticoagulant-preservative solutions are used to prolong the shelf life of blood cells. CPDA-1 is FDA - approved for storage of up to 35 days at 1⸰C to 6⸰C. ADSOL extends the shelf life of stored blood to 42 days. The ABO blood group and Rh type are shown on the label, once these tests are completed. Proper labeling of each unit of blood or blood component must include additional information like 1. Donation type 2. Any divisions 3. Attributes like: Washed or irradiated 4. Unique donation number 5. Blood Center information where the unit was collected and processed like the American Red Cross 20 ISBT LABEL FOR RED BLOOD CELL UNIT 21 Blood Donation: Donors, Collection, Storage, and Processing –Learning Outcome 5 Storage of Blood: Preserved RBCs must be stored in a refrigerator with a constant temperature of 1 C to 6 C. Some type of alarm must be available that will go off when the temperature is not within limits. A thermometer for recording the temperature must be installed. Stored packed RBCs are inspected daily for color, turbidity, appearance of clots, and presence of hemolysis. RBCs units are removed when they do not meet the appearance criteria established by the transfusion service. 22  Other Types of Blood Donations-learning outcome 6 Autologous transfusions ◦ The safest blood a recipient can receive is his or her own blood. ◦ Prevents transfusion-transmitted infectious diseases and eliminates the formation of antibodies to antigens in transfused RBCs from others and the possibility of GVHD (Graft versus Host disease) ◦ Blood donation also stimulates erythropoiesis by repeated preoperative phlebotomy. Directed transfusions ◦ The patient directly solicits blood for transfusion from family or friends. 23 Antigens and Antibodies in Immunohematology Transfusion medicine is based on antigens and antibodies. An antigen is defined as a foreign substance or a nonself antigen. Antibodies that react with antigens from a genetically different individual of the same species are referred to as alloantibodies. 24  Antigens and Antibodies in Immunohematology- Learning Outcome 7 Inheritance of red blood cell groups involves: ABO PHENOTYPES AND GENOTYPES ◦ Genes - antigens present on RBCs, WBCs, and platelet membranes controlled by a gene. ◦ Chromosomes- a complete set of genetic material (23 chromosomes) is inherited from each parent. ◦ Gene location (linkage)-genes located on the same chromosome and are normally inherited together. ◦ Alleles-variants of a gene for a particular trait ◦ A person who has identical alleles for a trait = homozygous ◦ A person who has two different alleles for a trait = heterozygous ◦ Phenotypes (what is seen by tests made directly on the RBCs, WHAT YOUR BLOOD TYPE IS) and genotypes (actual total genetic makeup of the individual, WHAT YOU GOT FROM YOUR PARENTS.) ◦ https://www.youtube.com/watch?v=9O5JQqlngFY 25  Isoantibodies and Immune Antibodies Isoantibodies ◦Isoantibodies result from internal (such as bacterial) or external antigenic stimuli. Immune antibodies ◦Immune antibodies result from stimulation by specific blood group antigens. Immune antibodies are also referred to as unexpected antibodies. ◦ These antibodies are the result of immunization caused by pregnancy or prior blood transfusion. 26 Means of Detecting Antigen–Antibody Reactions Antisera= type of substance must be available to show what antigens are present on the red cell. A reaction is agglutination of RBCs. When antiserum is mixed with RBCs, an antigen–antibody reaction may or may not absent, and the result is a negative reaction. occur. If agglutination occurs, the result is a positive reaction. If no agglutination occurs, the antigen is negative. Agglutination- the clumping of RBCs caused by the reaction of a specific antibody and antigen on the cells. 27 Means of Detecting Antigen–Antibody Reactions Factors that affect an antigen-antibody reaction: ◦ Use of adequate serum and RBCs ◦ The correct concentration of cell suspensions ◦ The testing medium ◦ Proper temperature and duration of incubation ◦ Proper use of centrifugation ◦ The condition and correct use of reagents ◦ Accurate reading and interpretation of agglutination reactions 28 Means of Detecting Antigen–Antibody Reactions Hemolysis is the result of lysis, or destruction, of a RBC by a specific antibody. The antigen–antibody reaction causes the activation of complement, which results in the rupture of the cell membrane and the subsequent release of hemoglobin. It is important that blood bank testing be performed on serum or plasma that is free of hemolysis and that whenever hemolysis occurs, it is interpreted as a positive reaction. 29  Blood-Banking Techniques- Learning outcome 8 Traditionally, RBC group tests are performed in test tubes with the specimen of choice for all blood bank testing being plasma. 30 Blood-Banking Techniques Recent advances in technology have led to other methods besides test tube reactions to detect antigen- antibody reactions. These methods include: Other methods of detecting antigen-antibody reactions (learning outcome 8): ◦ Gel technology ◦ Microplate testing methods ◦ Solid-phase red blood cell adherence methods 31  Blood-Banking Techniques Fig. 17.5. Appearance of reaction patterns and grading for gel or column agglutination technology. (From Cooling L, Downs T: Immunohematology. In McPherson RA, Pincus MR, editors: Henry’s clinical diagnosis and management by laboratory methods, ed 22, Philadelphia, 2011, Elsevier/Saunders.) To read an Ortho gel card, after centrifugation, visually inspect each microtube on the card, observing both the front and back, to see if red blood cells have agglutinated (clumped together) within the gel, indicating a positive reaction; if no agglutination is present, with the red blood cells forming a compact button at the bottom, the result is negative; the strength of a positive reaction can be graded based on how high the agglutinated cells are distributed within the gel column. 32 Blood Banking Techniques Factors that affect the reactions used to detect an antigen–antibody reaction include : ◦ Use of adequate serum and RBCs ◦ Correct concentration of cell suspensions ◦ The testing medium ◦ Proper temperature and duration of incubation ◦ Proper use of centrifugation ◦ The condition and correct use of reagents ◦ Accurate reading and interpretation of agglutination reactions 33  ABO Red Blood Cell Group System-learning Outcome 9 ABO phenotypes: A, B, AB, O ABO genotypes: AA, AO, BB, BO, AB, OO ABO typing procedures ◦Front typing ◦Reverse grouping ◦Red cell typing for antigen ◦Serum typing for antibody 34 ABO Red Blood Cell Group System Results may be grouped as follows: Group A blood (RBCs): positive reaction of cells with anti- A antiserum Group B blood (RBCs): positive reaction of cells with anti- B antiserum Group O blood (RBCs): negative reaction of cells with both anti-A and anti-B antiserum Group AB blood (RBCs): positive reaction of cells with both anti-A and anti-B antiserum 35 ABO Red Blood Cell Group System Fig. 17.8. Interpreting ABO blood typing results. Type A, Cells containing A antigen agglutinate with the blue anti-A serum. Agglutination causes the cells to fall our of the blue solution. Type B, Cells containing B antigen agglutinate with the anti-B serum. Type AB, Cells with both A and B antigens agglutinate with both antisera. Type O, Cells with no antigens do not react with either antiserum. The blue and yellow background colors of the typing sera are not visible because of the suspended cells that have not agglutinated. (From Garrels M, Oatis CS: Laboratory testing for ambulatory settings: a guide for health care professionals, ed 2. Procedure 6-4 E p. 231, 2010, Elsevier.) 36 ABO Typing Procedures Red blood cells are mixed with antibodies against type A and type B blood in a test tube. If the red blood cells clump together, the blood type is A or B, depending on which antibody caused the clumping. Reverse typing The liquid part of the blood, called serum, is mixed with type A and type B red blood cells. Red cell typing for antigens, also known as red blood cell antigen typing, is a test that determines the presence or absence of specific blood group antigens on red blood cells: How it's performed Red blood cells are tested with specific antisera to see if they agglutinate, or clump together. Agglutination can occur at different temperatures depending on the antisera used. 37 ABO TYPING PROCEDURES "Serum typing for antibody" refers to a laboratory test that analyzes the liquid portion of a blood sample (serum) to identify which specific antibodies are present, typically used to determine a person's blood type by identifying the naturally occurring antibodies against the ABO blood group antigens not present on their red blood cells; for example, someone with type A blood will have anti-B antibodies in their serum, while type B blood will have anti-A antibodies https://www.youtube.com/watch?v=L06TJTMVkBo 38  ABO Red Blood Cell Group System Variation in isoantibodies is not seen in newborns who do not normally begin to produce antibodies until 3 to 6 months of age. The titer of isoantibodies normally increases gradually through adolescence and then decreases. Subgroups of group A or group B antigen exist with the most important subdivision being group A into A1 and A2. Subgroups occur when someone inherits less effective forms of the enzymes that create A and B antigens. This can result in a weaker form of the A antigen, which is known as an "A subgroup" H substance is a precursor of A and B blood group antigens. Key points about H substance: Function: It acts as a building block for the A and B antigens on red blood cells. Blood group association: Individuals with O blood type primarily express the H substance because their genes do not code for the enzymes needed to convert it into A or B antigens. 39 ABO RBC GROUP SYSTEM Immune antibodies of ABO system arise from the stimulation by ABH substances ( which are Sugar molecules attached to proteins or lipids on cell surfaces, defining the ABO blood group)that are widely distributed in nature. Physical and chemical properties of immune antibodies and isoantibodies differ in physical and chemical properties and their serologic behavior. IgM antibodies are unable to cross the placental barrier, but IgG antibodies can cross the barrier which is important in the cause of HDFN. (is a complex and potentially life-threatening condition arising from maternal-fetal blood group incompatibility.) 40  Other Blood Group Systems Human blood groups were discovered in 1900. In addition to the antigens of the ABO and Rh systems, the International Society of Blood Transfusion (ISBT) has defined 30 blood group systems, with numerous associated antigens in these systems. Some of these antigens are common (high frequency); others are uncommon or rare (low frequency). 41 Other Blood Group Systems Beyond the well-known ABO and Rh blood group systems, several other blood group systems exist, including the Duffy, Kidd, Kell, MNS, and Lutheran systems, each with their own unique antigens that can trigger antibody production if exposed to incompatible blood, which is important to consider during antibody identification for blood transfusion purposes 42 Learning Outcome 10- Practice Blood Bank Game posted on Blackboard 43  Universal Donors and Recipients Learning Outcomes 12 The terms universal donor and universal recipient pertain to the transfusion of packed RBC products in emergencies. The universal donor is the person with group O RBC. Group O red cells can be transfused into a person WITH ANY ABO blood type. The universal recipient is the patient with group AB RBCs. Their serum does not contain either Anti-A or Anti-B and therefore these patients could receive RBC transfusion of any ABO blood type. Universal donor-Group O neg Universal recipient-Group AB pos 44 Rh RBC Group System The Rh system is based on work by Landsteiner and Wiener (1940) & Levine and Stetson (1939.). 45  Rh Red Blood Cell Group System-Learning Outcome 13 Historical background ◦Rh-positive and Rh-negative status ◦ Persons whose RBCs contain D antigen either in the homozygous or heterozygous are known as Rh positive– 85% of the population. ◦ Persons whose RBCs lack the D antigen are termed Rh negative—about 15% of the population Typing blood for transfusion ◦When RBCs are to be transfused, the patient must be tested for the presence or absence of the D (Rho) antigen. 46  Reaction (Coombs Test) –Learning Outcome 14 AHG test procedures ◦ Direct antihuman globulin test (DAT) ◦ Performed on RBCs suspected of being coated with antibody ◦ Used to demonstrate antibody that has coated or reacted with the RBCs in the patient’s body (in vivo) ◦ Indirect antihuman globulin test ◦ Used to detect antigen–antibody reaction that occurs in the test tube (in vitro) ◦ It tests for antibodies that are freely circulating in the plasma or antisera and reacts with specific antigens on RBCs in vitro. https://www.youtube.com/watch?v=JWXC8yXe9dE 47 Compatibility Testing and Crossmatching Definition and general considerations ◦ Whenever RBCs are to be transfused, two considerations are foremost: ◦ RBCs must be selected that will not be harmful to the patient or result in a transfusion reaction. ◦ RBCs must be selected that will be of maximum benefit to the patient. 48  Compatibility Testing and Crossmatching Whenever blood is to be transfused, it must be tested for compatibility between the donor and the recipient. Compatibility is much more than crossmatching, is just one part of the testing procedures. Compatibility testing involves a series of tests. For example, ABO and RH typing of donor and recipient, unexpected antibody screening and identification and crossmatching, etc. 49  Compatibility Testing and Crossmatching Crossmatching ◦ Crossmatch procedure of the donor’s RBCs with the patient’s serum ◦ Antihuman globulin crossmatches major crossmatch mixes the donor’s RBCs with a patient's serum to detect antibodies in the patient's serum that react with donor RBCs. ◦ Abbreviated crossmatch-(immediate spin) crossmatch can detect ABO incompatibility. ◦ Other crossmatching techniques ◦ Electronic crossmatching- -refers to blood issued without direct serologic crossmatching. 50 Adverse Effects of Transfusion- Learning Outcome 15 Clinically, the result of RBC destruction is a transfusion reaction. The signs and symptoms of a transfusion reaction vary from patient to patient. Generally, chills, high temperature, pain in the lower back, nausea, vomiting, and shock, as indicated by decreased blood pressure and rapid pulse, characterize an immediate reaction. Adverse effects of transfusion can generally be characterized as immune or nonimmune. Both immune and nonimmune types can occur as immediate adverse effects of transfusion. 51 Immediate Immunologic Adverse Reactions- #15 Examples OF adverse effects of transfusion include hemolytic, febrile, allergic, anaphylactic, and TRALI (transfusion-related acute lung injury), alloimmunization https://www.youtube.com/watch?v=DU3hhciAKWA 52 Immediate Non- Immunologic Adverse Reactions-#15 Examples OF adverse effects of transfusion include: Bacterial contamination, nonimmune hemolysis, circulatory overload 53 Hemolytic Disease of the Fetus and Newborn (HDFN)#16 HDFN occurs when a baby inherits an antigen for which the mother is negative. For this occur, the fetus must be positive for an antigen and the mother must be negative for that specific antigen. This disease most often involves antigens of the ABO or RH blood group systems. The D antigen of the RH system is one of the most potent antibody stimulator in a pregnant female if the fetus is D + and the mother is D-. 54  Hemolytic Disease of the Fetus and Newborn (HDFN)- #16 Pathophysiology- Begins in utero ◦ The mechanism involves sensitization or immunization of the mother to a foreign antigen present on her child’s RBCs. ◦ If any incompatible fetal RBCs enter the mother’s circulatory system, she can become sensitized to the antigen and potentially develop an antibody to the antigen on the fetal RBCs. If immunization occurs, it is permanent. ◦ The antibody formed by the mother is of the IgG type. IgG antibodies can cross the placenta into the circulatory system of the fetus, react with the corresponding antigen on the RBCs of the fetus, and destroy the fetus or newborn infant’s RBCs. https://www.youtube.com/watch?v=4hCzGhQPrzk 55 Hemolytic Disease of the Fetus and Newborn (HDFN)-#16 ABO antigens ◦ Most often, HDFN occurs as a result of antigens in the ABO system. The mother is usually group O, and the child inherits the A or B antigen from the father. Rh antigens ◦ In the case of Rh incompatibility, the mother is negative for D (d/d), and the father is positive for D. ◦ The child inherits this antigen from the father and is D positive (D/d). If any D-positive RBCs of the fetus cross into the mother’s circulation, she may develop an immune anti-D antibody. This IgG crosses the placenta and reacts with the RBCs of the fetus. ◦ Rhogam https://www.youtube.com/watch?v=VsMw7DwwSPU 56  Hemolytic Disease of the Fetus and Newborn (HDFN) Fig. 17.13. Postpartum effects of hemolytic disease of the fetus and newborn (HDFN). After delivery, the accumulation of bilirubin can severely harm a newborn infant. The liver of the newborn does not produce the enzyme glucuronyl transferase, which is necessary for converting bilirubin to an excretable form. Consequently, bilirubin accumulates, and if not removed will be deposited in lipid-rich tissues such as the brain. Excessive bilirubin in the circulation also produces jaundice. (Redrawn from Turgeon ML: Fundamentals of immunohematology, ed 2, Baltimore, 1995, Williams & Wilkins.) 57  Hemolytic Disease of the Fetus and Newborn (HDFN) Routine laboratory prenatal and postnatal testing ◦ Type the mother for ABO and Rh; screen serum for antibody; if antibody found, recheck titer throughout pregnancy ◦ After birth, infant’s blood is typed and tested. Treatment ◦ Variability in severity of HDFN: phototherapy, transfusion for the anemia 58 Hemolytic Disease of the Fetus and Newborn (HDFN) Qualitative screening of Prevention of Rh immunization ◦ Mechanism of action- To prevent Rh immunization, an Rh-negative pregnant woman is given an injection of "RhoGAM" (Rh immune globulin), which works by essentially "masking" any fetal Rh-positive red blood cells that may enter her bloodstream, preventing her immune system from recognizing them as foreign and triggering antibody production, thus stopping her from developing Rh antibodies that could harm future Rh- positive babies. 59 Rosette Screen ◦ Rosette screen is a highly sensitive method to qualitatively detect 10 ML or more of fetal whole blood in the maternal circulation. ◦ If negative, one vial of Rhogam is enough, if positive, it indicates an FMH exceeding 10 ML or more and requires quantification of the FMH by the Kleihauer- Betke acid elution test or flows to determine how many more vials of Rhogam are needed. 60  Hemolytic Disease of the Fetus and Newborn (HDFN) The rosette test is a blood test that screens for fetal-maternal hemorrhage (FMH) in pregnant women with Rh-negative blood: Purpose: Determines if there is fetal blood in the mother's circulation When it's performed: When it's known that the mother is Rh-negative How it's performed: A maternal blood sample is incubated with anti-Rho(D) immune globulin, then indicator cells are added What it shows: If the test is positive, the blood will clump together in a pattern that resembles a rose, which is how the test gets its name Follow-up testing: If the rosette test is positive, a quantitative test like the Kleihauer-Betke test is performed to confirm and measure the amount of FMH When it might be inaccurate: The rosette test can be falsely positive if the mother is weak-D positive, or falsely negative if the baby is weak-D positive 61 Hemolytic Disease of the Fetus and Newborn (HDFN)#17 Quantitative tests for measuring fetal RBCs in maternal circulation: Fetal hemoglobin or HgbF can be measured by various techniques such as the acid-elution technique (modified Kleihauer-Betke Test) and flow cytometry - based on antibodies against HgB F. Acid Elution test is a widely used confirmatory test for quantifying FMH. The purpose is that Hgb F is resistant to acid elution, but adult hgb is not. If a thin blood smear is exposed to an acid buffer, the adult RBC loses its hgb into the buffer, leaving only the rbc stroma, but the fetal rbc is unaffected and retains its hgb. 62 Assessment Plan For FMH The concentration of postpartum prophylaxis is determined for each patient by a 4-step procedure: 1. A rosette fetal RBC screening for FMH 2. Quantitation of the amt of FMH by Kleihauer Betke acid elution assay or flow cytometry assay 3. Estimation of the volume of FMH 4. Calculation of the required number of vials of Rhogam The amount of fetal rbc present in the maternal circulation is important for the rhogam dose. 63