Immunohematology Success PDF

CHAPTER Immunohematology Outline 496 > Immunohematology Overview > Genetics > ABO and H Blood Group Systems and Secretor Status > Rh Blood Groups > Other Blood Group Systems > Bl...

CHAPTER Immunohematology Outline 496 > Immunohematology Overview > Genetics > ABO and H Blood Group Systems and Secretor Status > Rh Blood Groups > Other Blood Group Systems > Blood Bank Reagents and Methods > Direct Antiglobulin Testing X Identification of Unexpected Alloantibodies > Pretransfusion Testing >- Hemolytic Diseases of the Newborn >- Blood Collection > Blood Components: Preparation, Storage, and Shipment > Blood Component Therapy > Transfusion Therapy > Transfusion Reactions > Transfusion-Transmitted Diseases > Safety and Quality Assurance > Blood Usage Review Review Questions 546 Answers & Rationales 577 References 613 495 496 CHAPTERS: IMMUNOHEMATOLOGY I. IMMUNOHEMATOLOGY OVERVIEW A. Definition: Immunohematology is the study of blood group antigens and antibodies, HLA antigens and antibodies, pretransfusion testing, identification of unexpected alloantibodies, immune hemolysis, autoantibodies, drugs, blood collection, blood components, cryopreservation of blood, transfusion-transmitted viruses, tissue banking and organ transplantation, blood transfusion practice, safety, quality assessment, records, blood inventory management, and blood usage review. B. Immune System 1. Acquired immunity is a specific response of the immune system in which antibodies specific to a particular antigen are produced. Plasma cells produce antibodies. 2. Innate immunity is a nonspecific reaction of the immune system that attacks all invaders. It includes physical and biochemical barriers and cells such as leukocytes, including neutrophils, monocytes/macrophages, and natural killer cells. Physical barriers include intact skin, mucous membranes, etc. Bactericidal enzymes are biochemical barriers. C. Antigen Characteristics 1. Antigens are substances that combine with an antibody. An antigen that causes a specific immune response is an immunogen. Immunogens are made of protein, carbohydrates, and combinations of both. Antigens are found on the surface of platelets and WBCs as well as RBCs. Some immunogens produce a greater response than others. 2. There are 23 RBC antigen systems containing over 200 RBC antigens. RBC anti- gens are inherited and are composed of proteins, glycoproteins, and glycolipids. 3. Human leukocyte antigens (HLAs) a. Present on leukocytes and tissue cells b. Genes that encode the HLA antigens are part of the major histocompatibility complex (MHC). c. MHC is on chromosome 6 and is divided into Class I, II, and III. 1) Class I includes A, B, and C loci. 2) Class II includes DR, DP, and DQ. 3) Class III includes complement proteins. d. Immune response to transfused incompatible HLA antigens causes fever and chills. This is known as a febrile, non-hemolytic transfusion reaction. e. HLA must be matched for organ, tissue, bone marrow, and stem cell transplant donors and recipients. If the recipient is not matched correctly, a severe graft-versus-host disease results. f. HLA test applications include paternity testing, organ and tissue transplan- tation, bone marrow and stem cell transplantation, and platelet matching.  IMMUNOHEMATOLOGY OVERVIEW 497 4. Platelet antigens a. Membranes have protein antigens. b. Platelet antibodies occur less frequently in the general population because of less antigen variability. c. Antibodies reacting with platelets may be ABO-, HLA-, or platelet specific. d. Diseases: Neonatal alloimmune thrombocytopenia and posttransfusion purpura D. Antibody Characteristics 1. Molecular structure a. Each molecule has two heavy chains and two light chains. b. The heavy chain is responsible for the immunoglobulin group specificity. c. Antibody binding site is found in the variable region of the heavy and light chains. 2. IgM antibodies a. Composed of five basic immunoglobulin units (pentamer) b. Can directly bind with RBCs and produce agglutination c. Can activate complement d. Cannot cross the placenta because of large size of molecule e. React optimally at room temperature and below f. Usually clinically insignificant 3. IgG antibodies a. Single immunoglobulin unit b. Cannot visibly agglutinate RBCs c. Normally, cannot activate complement unless two molecules are present (i.e., IgG3) d. Can cross the placenta e. React optimally at 37°C f. Typically clinically significant; capable of causing transfusion reactions or hemolytic disease of the newborn (HDN) E. Antigen-Antibody Interactions 1. Follow the Law of Mass Action 2. Reversible 3. Antigen-antibody complex formed 4. Properties that influence antigen-antibody interactions: a. Fit of antigen into antibody binding site b. Size of antigen c. Shape of antigen d. Charge of antigen 5. Antigen-antibody complexes are held together by electrostatic charges, hydrogen bonding, hydrophobic bonding, and Van der Waals forces. 498 CHAPTERS: IMMUNOHEMATOLOGY F. Antigen-Antibody Reactions In Vivo 1. Transfusions can lead to antigen-antibody complex formation and complement activation in vivo, if wrong type of blood is transfused. 2. Transfusion of foreign antigens (RBC, HLA, and platelet) into a recipient can cause an immune response and antibody formation in the recipient (alloantibodies). 3. Antigen-antibody complexes are removed by the reticuloendothelial system: spleen, liver, and lymph nodes. G. Antigen-Antibody Reaction In Vitro 1. Reactions are detected by agglutination or hemolysis. 2. Some antigen-antibody complexes require two stages for detection: sensitization and lattice formation. a. Sensitization: Antibody attaches to antigen but does not produce visible agglutination or hemolysis. 1) Factors affecting first stage of agglutination a) Serum to cell ratio: This is the amount of antibody compared to the number of cells in solution. Increased amount of serum equals an increase in the number of antibodies in the solution. b) Reaction temperature: This is the temperature at which the antibody reacts best; most clinically important antibodies react best at 37°C. c) Incubation time: This is the time allowed for the antibody to attach to the antigen. This reaction occurs by chance. Times will vary according to the antibody and media used in vitro (i.e., albumin, LISS—low-ionic-strength saline). d) pH: The optimal pH for in vitro reactions is 7. b. Lattice formation: Random collisions of antibody-coated RBCs link antibodies together to form visual agglutination. 1) Factors affecting visual agglutination a) Reaction temperature b) Incubation time c) PH d) Repelling negative charges: In normal saline, RBCs have a net negative charge that repels other RBCs in solution. This charge inhibits agglutination. 3. Antigen and antibody agglutination a. Zone of equivalence: Antigen and antibody concentrations produce maximum agglutination. b. Prozone (antibody excess): Too much antibody compared with antigen concentration c. Antigen excess: Too much antigen compared with antibody concentration 4. Grading agglutination reactions a. To standardize the strength of agglutination reactions: 1 ) 4 + RBC button is solid with a clear supernatant.  GENETICS 499 2) 3 + RBC button breaks into several large clumps, with a clear supernatant. 3) 2 + RBC button breaks into many medium-sized clumps, with a clear supernatant. 4) 1 + RBC button breaks into many medium- and small-sized clumps, with background having many free RBCs (appears cloudy). 5) + w RBC button breaks into many clumps, barely or not visible macroscopically, with many RBCs in the background (use microscope to see clumps). 6) 0 = no agglutinated RBCs 5. Hemolysis is another indication of antibody-antigen reactions and is caused by complement activation. The supernatant appears clear red, with a smaller or nonexistent RBC button. GENETICS A. Definitions 1. Chromosomes: Structures that carry genetic information encoded on double- stranded DNA 2. Mitosis: Process of cell division that results in the same number of chromosomes in the new and old cells 3. Meiosis: Process of cell division that occurs in gametes resulting in one-half the chromosomes in each new cell 4. Blood group systems: Groups of related RBC antigens inherited according to Mendelian genetics 5. Phenotype: Physical, observable expression of inherited traits; detectable products 6. Genotype: Inherited genes; actual genetic makeup 7. Pedigree chart: Visual map that displays a family history and can display inheritance patterns for individual traits 8. Gene: Smallest unit of inheritance 9. Genetic locus: Site on chromosome where specific genes are located 10. Alleles: Alternative forms of a gene 11. Antithetical: Opposite form of a gene, different allele 12. Polymorphic: Having two or more possible alleles at a locus 13. Codominant: Equal expression of both alleles in phenotype 14. Recessive: Same allele must be inherited from both parents to be expressed, homozygous 15. Dominant: Only one allele must be inherited for it to be expressed; gene product always present 16. Autosomal: Genes expressed with equal frequency in males and females, on non-sex chromosome CHAPTER 5: IMMUNOHEMATOLOGY 17. Sex-linked dominant: Carried on the X chromosome; no father-to-son transmission; will be expressed if passed from father to daughter or from mother to son 18. Sex-linked recessive: It is carried on the X chromosome. Males inherit it from carrier mothers; traits are exhibited most commonly in males (e.g., hemophilia A). Females can exhibit the trait but must inherit it from both carrier mother and affected father. B. Mendelian Inheritance Principles 1. Law of Independent Segregation: Two members of a single gene pair passed from one generation to the next in separate gametes 2. Law of Independent Assortment: Traits inherited from different chromosomes expressed separately and discretely 3. Inheritance patterns: The inheritance of blood group antigens (A, B, O) can be predicted using a Punnett square. Punnett squares have the one person's genotype on the top and the other person's genotype on the side. See Table 5-lB. 4. Each square represents a possible genotype for an offspring. An offspring from these particular parents would have a 25% chance of inheriting any one of the four possible variants. Punnett squares are useful for understanding inheritance of blood groups and ramifications of heterozygosity or homozygosity. 5. Homozygous: Individual inherits identical alleles at the same gene locus from both parents. 6. Heterozygous: Individual inherits different alleles at the same gene locus from each parent. 7. Dosage effect: Agglutination reactions are generally stronger for homozygous cells and slightly weaker for heterozygous cells. 8. Cis: Genes are inherited on the same chromosome. 9. Trans: Genes are inherited on separate chromosomes. Genes inherited in transposition can weaken the trait's expression. 10. Linkage and haplotypes a. Linked genes: Genes that are close together on a chromosome and inherited as one unit. The Law of Independent Assortment does not hold with linked genes. TABLE 5-1 PUNNETT SQUARE Mother's Genotype Father's B AB BB Genotype AO BO  ABO AND H BLOOD GROUP SYSTEMS AND SECRETOR STATUS 501 b. Haplotype: Set of genes inherited via one of the two parental gametes c. Amorphs: Genes that do not produce a detectable product 1 1. Population genetics: Statistical calculation to determine the prevalence of antigens in specific populations a. Phenotype calculations: Determine the frequency of an antigen in a population b. If a person has multiple antibodies, determine the percentage of compatible units; the frequency for each antibody must be multiplied. For example, if the individual antigen probabilities in the population are 30% E-positive, then it is 70% E-negative = 0.70 78% M-positive, then it is 22% M-negative = 0.22 80% c-positive, then it is 20% c-negative = 0.20 Prediction of percentage of compatible units = the product of the individual probabilities or 0.70 X 0.22 X 0.20 = 0.03 or 3% if units are randomly chosen from inventory. 12. Parentage testing: HLA antigens follow Mendelian genetics principles and can be used to determine the parents of offspring. HLA genes are polymorphic with many alleles possible at each locus. The more alleles, the less likely it is to find two identical individuals. Parentage testing works on the principle of excluding falsely accused individuals using statistics. ABO AND H BLOOD GROUP SYSTEMS AND SECRETOR STATUS A. Landsteiner's Rule: If an individual has the antigen, that individual will not have the antibody. This is a universal law and has few exceptions. B. ABO Antigens 1. Found on RBCs, lymphocytes, platelets, tissue cells, bone marrow, and organs 2. These antigens can be secreted by tissue cells if the appropriate genes are present. 3. Glycolipid or glycoprotein 4. Developed in utero at 5-6 weeks of gestation 5. Full expression of ABO antigens occurs between 2 and 4 years of age. 6. Frequencies: See Table 5-2. C. Inheritance and Development of A, B, and H Antigens 1. The H antigen is the building block for the A and B antigens. There are only two alleles in the H gene: H and h. The H allele is found in 99.99% of the world's population, and h is a rare amorph allele. 502 CHAPTERS: IMMUNOHEMATOLOGY TABLE 5-2 FREQUENCIES OF ABO ANTIGENS Blood Type Whites, Frequency (%) Blacks, Frequency (%) 0 45 49 A 40 27 B 11 20 AB 4 4 2. The H antigen acts as the acceptor molecule for the two sugars that make up the A and B antigens. 3. The A blood type is the H antigen with A^-acetylgalactosamine attached. 4. The B blood type is the H antigen with o-galactose attached. 5. The O blood type is the H antigen with no additional sugar attached. D. ABO Subgroups 1. Subgroups differ in the amount of the antigen expressed on the RBCs. Subgroup Aj possesses both A and A t antigens on the RBC surface. Subgroup A2 only expresses A antigen. 2. Blood group A has two major subgroups, Aj and A2. 80% of group A people are A r and 20% of group A people are A2. 3. People with subgroups of the A antigen can produce antibodies against Al antigen. 4. Subgroups of A include A15 A2, A3, Ax, Am, Acl, and Abantu. 5. Subgroups of A can be detected by polyclonal Anti-A,B. This is produced by Group O individuals only. Anti-A,B will agglutinate A subgroups because it has specificity for both A and B antigens but cannot be separated into Anti-A and Anti-B. Anti-A, lectin is active against A,, but not the other A subgroups. 6. Subgroup A3 characteristically produces a mixed-field reaction with polyclonal Anti-A and polyclonal Anti-A,B. 7. If weak subgroups of A in recipients are not detected, there is no harm in a person with the subgroup receiving type O blood. However, if the person with the weak subgroup of A donates blood that is transfused to a group O patient, intravascular hemolysis may result. E. A and B Are Codominant Traits: If the allele is present, the antigen will be expressed. O is an amorph allele that produces no transferase to add sugars to the H determinant site.  Rh BLOOD GROUPS 503 F. Anti-A and Anti-B: These antibodies are produced by humans, who lack the corresponding antigen, as a result of exposure to naturally occurring substances that resemble A and B antigens. G. Anti-A and Anti-B Are IgM Antibodies: This means they activate complement and cause visible RBC agglutination. They may cause hemolysis at room temperature. H. Routine ABO Grouping 1. Forward type: Person's RBCs are mixed with reagent Anti-A and Anti-B. 2. Reverse type: Person's serum is mixed with reagent Aj and B RBCs. 3. ABO discrepancies occur when the forward and reverse groupings do not agree. a. Problems with forward grouping (extra antigen present, weak antigens) could be caused by acquired B phenotype, polyagglutination, rouleaux, ABO subgroups, transfusion of non-type specific blood, and bone marrow or stem cell transplants. b. Problems with reverse grouping (unexpected antibodies or weak/missing antibodies) could be seen in individuals with A subgroups with Anti-Ap cold alloantibodies, cold autoantibodies, and rouleaux, and in a newborn or elderly person. I. Bombay (Oh) Phenotype 1. Person inherits hh genotype. 2. Types as an O (forward and reverse); has alloanti-H capable of activating complement and causing a hemolytic transfusion reaction 3. These people can only be transfused with Bombay group blood. Blood may be collected and frozen as autologous or from siblings who are also Bombay. J. Secretor Status 1. Two alleles: Se and se 2. People who inherit Se are secretors and are capable of expressing ABO and H antigens in their secretions. 3. A, B, and H antigens, appropriate to the individual's ABO group, are found in saliva, urine, tears, bile, amniotic fluid, breast milk, exudate, and digestive fluids of secretors (Se). IV. Rh BLOOD GROUPS A. Rh Blood Group System 1. Controlled by two genes RED and RHCE. RHD controls D expression; no d allele. RHCE controls C, c, E, e expression. 2. Rh antigens are proteins. 504 CHAPTERS: IMMUNOHEMATOLOGY 3. Rh Terminology a. The most common individual antigens are named in the Fisher-Race terminology, D, C, c, E, e, Cw, G, etc. b. Haplotypes are often expressed in a modified Wiener terminology such as RjRj for CDe/CDe. 4. Phenotype: RBC antigens identified with specific antisera; Genotype: Genes present on person's chromosomes 5. Rh system antigens a. D antigen: Most immunogenic of Rh antigens b. WeakD 1) Weak D occurs when D is weakly expressed. Weak D must be detected by an IAT (indirect antiglobulin test). 2) Genetic cause: Weaker expression of the cDe haplotype may fail to react by direct agglutination testing, but it will react strongly by the IAT. 3) Position effect: Occurs when the C antigen is inherited trans to the D antigen. This weak D may be detected without carrying the test to the antiglobulin phase. 4) Partial D: Occurs when only part of the D antigen is inherited. There are multiple epitopes that make up the D antigen. A partial-D individual lacks one or more of these epitopes and is capable of making antibody to the epitopes that s(he) lacks. Partial-D individuals are usually detected because the antigen reacts strongly with monoclonal reagents. A partial D is suspected when a seemingly D-positive person makes anti-D after transfusion with D-positive blood. 5) Weakly reactive D means a person is D-positive. AABB Standards state that all Rh-negative donor units must be tested for weak D, and those units that test positive must be identified as D-positive. However, weak-D recipients are transfused with D-negative blood. c. Other Rh system antigens 1) f or ce: If c and e are present on the same haplotype, f antigen is expressed. 2) Ce or rh.: C and e are inherited as a haplotype made by D-positive individuals who make anti-C. 3) Cw: Low-frequency antigen 4) V or ces: 30% prevalence in African-Americans 5) G: In test tube appears to be anti-D and anti-C 6) Rh:29: Antibody to Rh:29 is the antibody to the high-frequency Rh antigen made by Rhnull people. d. Unusual phenotypes 1) D deletion: No reaction occurs when tested with anti-E, anti-e, anti-C, and anti-c. Written as D —. 2) Rhnull phenotype: This appears to have no Rh antigens. The membranes of their RBCs are abnormal and the RBCs have a shortened  OTHER BLOOD GROUP SYSTEMS 505 life span. This can result from inheriting two nonfunctional RHCE alleles along with the dual deletion of the RHD alleles. Rhnull phenotype can also result from inheriting two recessive regulator alleles at the RHAG locus. The latter individuals pass on normal RHD and RHCE alleles to their children. 6. Rh antibodies a. Produced in humans through pregnancy or transfusions b. IgG antibody; Rh antibodies generally do not activate complement c. Optimal reaction temperature: 37°C d. Reaction phase: AHG (antihuman globulin) e. Agglutination enhancement occurs with LISS, enzymes, and PEG (polyethylene glycol). f. Stronger reactivity of antibody with cells from homozygous individuals is shown with anti-C, anti-c, anti-E, and anti-e (dosage). g. C and e and E and c are usually found together. h. These antibodies produce hemolytic transfusion reactions (HTRs). Antibodies may not be currently detectable, but the person should always receive antigen negative blood if they have a history of Rh antibodies. i. Rh antibodies can cause hemolytic disease of the newborn (HDN), because they can cross the placenta. Rh immune globulin (RhIG) administered after delivery (within 72 hours) can protect a woman from making anti-D. V. OTHER BLOOD GROUP SYSTEMS A. Kell Blood Group System 1. Abbreviation: K 2. Antibody class: IgG 3. Optimal reaction temperature: 37°C 4. Reaction phase: AHG 5. Enzyme treatment: No effect 6. Antigens: K (Kell), k (Cellano), Kpa, Kpb, Kpc, Jsa, Jsb, and Ku; common Kell system antigens k, Kpb, and Jsb 7. Allelic pairs: Include K and k, Kpa and Kpb, Jsa and Jsb 8. K is very immunogenic. Although the K antigen is found in only about 9% of the population, anti-K is encountered quite frequently and can cause HTR and HDN. 9. Kellnu,,: This is also known as KQ. It occurs when RBCs lack the Kell antigens but have the Kx antigen. 10. The Kx antigen is produced by a gene located on a different chromosome than the Kell system genes. This antigen is inherited independently from the Kell antigens; the Kx antigen structure appears to be required for the expression of the Kell system antigens. Knull individuals have increased amounts of Kx. 508 CHAPTERS: IMMUNOHEMATOLOGY 1 1. McLeod phenotype a. Individuals who have an alteration of the allele-producing Kx on the X chromosome lack Kx on the red blood cells and have greatly decreased expression of Kell antigens. b. These individuals have decreased RBC survival as well as RBC morphologic and functional abnormalities. B. Duffy Blood Group System 1. Abbreviation: Fy 2. Antibody class: IgG 3. Optimal reaction temperature: 37°C 4. Reaction phase: AHG 5. Enzyme treatment: Destroys Fya and Fyb 6. Clinically significant: a. Anti-Fya and anti-Fyb can cause HTR and HDN. b. The Fy(a-b-) phenotype is more resistant to malarial infection by Plasmodium vivax. 1. Antigens: Fya, Fyb 8. Four phenotypes: Fy(a+b—); Fy(«—£>+); Fy(a+£>+); Fy(a-b-) 9. Alleles: Pya, Fyb, and Fy (silent allele) 10. Commonly show dosage effect: Weak antibodies react more strongly with homozygous cells. C. Kidd Blood Group System 1. Abbreviation: Jk 2. Antibody class: IgG 3. Optimal reaction temperature: 37°C 4. Reaction phase: AHG 5. Enzyme treatment: Enhances agglutination 6. Clinically significant: Associated with HTR and mild HDN 7. Antigens: Jka, Jkb, Jk3 8. Four phenotypes: Jk(a+b -); Jk(a - b+); Jk(a+b+); Jk(a-b-) 9. Alleles: Jk* codes for Jka and Jk3; Jkb codes for Jkb and Jk3. 10. Show dosage effect: Weak antibodies agglutinate homozygous cells more strongly than heterozygous cells. 11. These antibodies bind complement. 12. These antibodies deteriorate in storage, declining quickly to below the detectable level in human serum, and commonly cause delayed HTR (DHTR). D. Lutheran Blood Group System 1. Abbreviation: Lu 2. Antibody class: Lua IgM; Lub IgG 3. Optimal reaction temperature: Lua 4°C; Lub 37°C  OTHER BLOOD GROUP SYSTEMS 507 4. Reaction phase: Lua room temperature; Lub AHG 5. Enzyme treatment: Variable effect 6. Clinically significant: a. No clinical significance. Anti-Lua can be present without prior transfusion or pregnancy. b. Anti-Lub is rare and associated with HTR and HDN. 7. Antigens: 18 total, including Aua and Aub 8. Alleles: Lua, Lub E. Lewis Blood Group System 1. Abbreviation: Le 2. Antibody class: IgM 3. Optimal reaction temperature: Most often 4°C, sometimes 37°C 4. Reaction phase: Room temperature, 37°C, and AHG 5. Enzyme treatment: Enhanced agglutination 6. Clinically significant: No 7. Produced by tissue cells and secreted into fluids. The antigens are adsorbed onto the RBC membranes. 8. May take 6 years to fully develop these antigens. 9. Genetics: IfLe gene inherited, Lea is adsorbed onto RBCs—Le(a+b-). Lea is the only antigen that can be secreted by a nonsecretor. 10. If Se gene is also inherited, Leb is adsorbed onto the RBC—Le(a - b +). 11. Bombay phenotypes are Lea positive if they inherit the Le gene. 12. Cells type as Le(a+i>+) (transiently during first years of life), Le(—), Le(a-b+), Le(a-b-). 13. Lewis antibodies are sometimes formed during pregnancy but weaken and disappear after delivery. F. I Blood Group System 1. Abbreviation: I 2. Antibody class: IgM 3. Optimal reaction temperature: 4°C 4. Reaction phase: Immediate spin (IS) and occasionally 37°C 5. Enzyme treatment: Enhanced agglutination 6. Clinically significant: No 7. It can be a bothersome antibody, masking the reactions of a clinically significant alloantibody. May need to prewarm cell suspension and reagent or do cold autoabsorption to find clinically significant alloantibodies. 8. Strong anti-I is associated with Mycoplasma pneumoniae infection. G. P Blood Group System 1. Abbreviation: Pt 2. Antibody class: IgM (anti-Pj) 508 CHAPTERS: IMMUNOHEMATOLOGY 3. Optimal reaction temperature: 4°C 4. Reaction phase: IS, 37°C and AHG 5. Enzyme treatment: Enhanced agglutination 6. Clinically significant: a. Anti-Pj is not clinically significant. b. Anti-Pi + P + Pk is an IgG clinically significant antibody. 7. Phenotypes: P p P2, p, Pf, P2k, and Luke 8. Alleles: Pp P, Pk, andp 9. Anti-Pj can be neutralized by soluble P1 reagent. 10. Autoanti-P is Donath-Landsteiner antibody. Naturally occurring biphasic antibody associated with paroxysmal cold hemoglobinuria. It binds to the antigen on the patient's RBCs in the cold and fixes complement. The RBCs are hemolyzed when the temperature reaches 37°C. 11. Patients with autoanti-P may require a blood warmer for transfusion. 12. Anti-PPjPk is found in individuals of the p phenotype. It is clinically significant and associated with spontaneous abortions. Need compatible blood from other p phenotype individuals. H. MNS Blood Group System 1. M and N antigens a. Abbreviation: MN b. Antibody class: IgM c. Optimal reaction temperature: 4°C or 37°C d. Reaction phase: IS, 37°C, or AHG e. Enzyme treatment: Destroys antigens f. Clinically significant: No g. Antigens: M and N associated with glycophorin A 2. S and s antigens a. Abbreviation: Ss b. Antibody class: IgG c. Optimal reaction temperature: 37°C d. Reaction phase: AHG e. Enzyme treatment: Variable effect f. Clinically significant: Yes g. Antigens: S, s, and U associated with glycophorin B 3. Anti-M a. It is clinically significant if IgG; IgM antibody is not clinically significant. b. Demonstrates dosage effect 4. Anti-N is very rare. 5. Anti-S, Anti-s, and Anti-U a. Clinically significant, causing HTR and HDN b. Anti-U is rare and occurs in S-s-U- people.  BLOOD BANK REAGENTS AND METHODS 509 I. Miscellaneous Blood Group Systems 1. Diego: Dia, Dib, Wra, Wrb; Dib and Wrb are high-incidence antigens. 2. Cartwright: Yta and Ytb; Yta is a high-incidence antigen. 3. XG: Xga antigen has a higher incidence in females than in males. 4. Scianna: Scl, Sc2, and Sc3; Scl and Sc3 are high-incidence antigens. 5. Dombrock: Doa, Dob, Gya, Hy, and Joa; Gya, Hy, and Joa are high-incidence antigens. 6. Colton: Coa, Cob, and Co3; Coa is a high-incidence antigen. 7. Chido/Rodgers: Cha and Rga are both high-incidence antigens. 8. Gerbich: Ge2, Ge3, and Ge4 are high-incidence antigens. 9. Cromer: Cra and several others are high-incidence antigens. 10. Knops: Kna, McCa, Sll, andYk a are high-incidence antigens. 1 1. Cost: Csa and Csb; Csa is a high-incidence antigen. 12. Vel: Vel is a high-incidence antigen. Anti-Vel is a hemolytic, clinically significant antibody. 13. John Milton Hagen: JMH is a high-incidence antigen. 14. Sid: Sda is a high-incidence antigen. VI. BLOOD BANK REAGENTS AND METHODS A. Principle of Blood Bank Tests Ag + Ab 15 mL RBCs, more than one dose is required to neutralize the RBCs. 4. Fetal screen (Rosette test): A suspension of maternal RBCs is incubated with anti-D. Anti-D binds to Rh-positive fetal RBCs, if present in the maternal circulation. D-positive indicator cells are added that bind to the anti-D, forming a rosette around the sensitized Rh-positive fetal RBCs. This is a screening method to detect fetomaternal bleeds >15 mL. If the fetal screen is positive, a Kleihauer-Betke test is required to quantify the amount of bleed that has occurred. 5. Kleihauer-Betke (KB) acid elution is used to determine the amount of a fetomaternal hemorrhage. Principle: Fetal hemoglobin is resistant to acid elution. A blood smear from the mother is made, then dipped in an acid buffer and stained with a counterstain. The buffer lyses the mother's cells (ghost cells) and does nothing to the fetal cells. Pink fetal cells are counted. Results are reported as percent of fetal cells (# fetal cells -H total cells counted). The amount, in milliliters (mL), of fetal blood in maternal circulation equals the % fetal cells X50. Divide the mL of cells by 30 to determine the number of Rh immune globulin doses needed. Note: Flow cytometry assays have been developed that can replace the traditional Kleihauer-Betke test.  BLOOD COLLECTION 523 H. Exchange Transfusions 1. Selection of blood for exchange transfusion a. Infant cells must be tested for ABO and D. ABO group of RBCs chosen for transfusion must be compatible with mother's ABO group. Group O blood is typically used. b. Mother's blood is used for antibody screen. c. Units must be antigen negative for all antibodies in mother's blood. 2. FFP is used to reconstitute packed RBCs to a hematocrit of approximately 40-50%. Group AB FFP is typically used. 3. Any blood products to be transfused must be hemoglobin S negative, CMV negative, and irradiated. XI. BLOOD COLLECTION A. Donor Selection 1. Registration questions include full name, address, home and work phone numbers, date of birth, gender, date of last donation, written consent, photo identification, race (optional), and intended use of donation. 2. Educational material is distributed to the donor. The donor must read material, and if the prospective donor shows symptoms of an infectious disease, the donor is excluded from donation. 3. Donor history questions include: a. Have you ever donated or attempted to donate blood using a different (or another) name here or anywhere else? b. In the past 8 weeks, have you given blood, plasma, or platelets here or anywhere else? c. Have you for any reason been deferred or refused as a blood donor or told not to donate blood? d. Are you feeling well and healthy today? e. In the past 12 months, have you been under a doctor's care or had a major illness or surgery? f. Have you ever had chest pain, heart disease, recent or severe respiratory disease? g. Have you ever had cancer, a blood disease, or a bleeding problem? h. Have you ever had yellow jaundice, liver disease, viral hepatitis, or a positive test for hepatitis? i. Have you ever had malaria, Chagas disease, or babesiosis? j. Have you ever taken etretinate (Tegison) for psoriasis? k. In the past 3 years, have you taken acetretin (Soriatane)? 1. In the past 3 days, have you taken piroxicam (Feldene), aspirin, or anything that has aspirin in it? m. In the past month, have you taken isotretinoin (Accutane) or fmasteride (Proscar) (Propecia)? 524 CHAPTERS: IMMUNOHEMATOLOGY n. In the past 4 weeks, have you taken any pills or medications? o. In the past 12 months, have you been given rabies shots? p. Female donors: In the past 6 weeks, have you been pregnant or are you pregnant now? q. In the past 3 years, have you been outside the United States or Canada? r. Have you ever received human pituitary-derived growth hormone? s. Have you received a dura mater (or brain covering) graft? t. Have you or any of your blood relatives ever had Creutzfeldt-Jakob disease or have you ever been told that your family is at an increased risk for Creutzfeldt-Jakob disease? u. In the past 12 months, have you had close contact with a person with yellow jaundice or viral hepatitis, or have you been given hepatitis B immune globulin (HBIG)? v. In the past 12 months, have you taken (snorted) cocaine through your nose? w. In the past 12 months, have you received blood or had an organ or a tissue transplant or graft? x. In the past 12 months, have you had a tattoo applied, ear or skin piercing, acupuncture, accidental needlestick, or come in contact with someone else's blood? y. In the past 12 months, have you had a positive test for syphilis? z. In the past 12 months, have you had or been treated for syphilis or gonorrhea? aa. In the past 12 months, have you given money or drugs to anyone to have sex with you? bb. At any time since 1977, have you taken money or drugs for sex? cc. In the past 12 months, have you had sex, even once with anyone who has taken money or drugs for sex? dd. Have you ever used a needle, even once, to take drugs that were not prescribed for you by a doctor? ee. In the past 12 months, have you had sex, even once, with anyone who has used a needle to take drugs not prescribed by a doctor? ff. Male donors: Have you had sex with another male, even once, since 1977? gg. Female donors: In the past 12 months, have you had sex with a male who has had sex with another male, even once, since 1977? hh. Have you ever taken clotting factor concentrates for a bleeding problem such as hemophilia? ii. In the past 12 months, have you had sex, even once, with anyone who has taken clotting factor concentrates for a bleeding problem such as hemophilia? jj. Do you have AIDS or have you had a positive test for the HIV virus? kk. In the past 12 months, have you had sex, even once, with anyone who has AIDS or has had a positive test for the HIV virus?  BLOOD COLLECTION 52S 11. Are you giving blood because you want to be tested for HIV or the AIDS virus? mm. Do you understand that if you have the AIDS virus, you can give it to someone else even though you may feel well and have a negative AIDS test? nn. Were you born in, or have you lived in, or have you traveled to any African country since 1977? oo. When you traveled there, did you receive a blood transfusion or any other medical treatment with a product made from blood? pp. Have you had sexual contact with anyone who was born in or lived in any African country since 1977? qq. In the past 12 months, have you been in jail or prison? IT. Have you read and understood all the donor information presented to you, and have all your questions been answered? 4. Examples of donor deferrals: See Table 5-5. 5. All donors must pass a physical exam with the following criteria: a. Appear to be in good health b. 38% hematocrit (minimum) c. 12.5 g/dL hemoglobin (minimum) d. Body temperature must be below 99.5°F (37.5°C). e. Blood pressure must be below or equal to 180/100 mm Hg. f. Pulse must be between 50 and 100 bpm and regular. g. Weight must be a minimum of 110 pounds. 6. Confidential unit exclusion (optional) a. This is used to give donors a way to indicate if this unit should be used for transfusion or discarded. The most common way to accomplish this is to give the donor two bar-coded labels: One states that the blood is OK to use and the other states that the blood should not be used. The donor chooses the label and applies it to his/her records. Once the label is pulled from the backing, the only way of knowing which label is on the records is to scan the bar code. 7. Informed consent: The donor must sign a form that allows blood to be collected and used for transfusion. B. Phlebotomy 1. Identification is a crucial step. The donor must be identified before phlebotomy can be done. 2. Bag labeling: The bag, attached satellite bags, sample tubes, and donor registration must have the same unique identification number. The labels consist of letters and bar codes. 3. Postdonation care: After donating, donors are urged to avoid alcohol and smoking immediately, drink lots of fluid for the next 3 days, and be aware that dizziness and fainting can occur a few hours after donation. 526 CHAPTERS: IMMUNOHEMATOLOGY TABLE 5-5 POSSIBLE REASONS FOR DONOR DEFERRALS a. Hepatitis B IgG 12 months b. Tattoo/piercing 12 months c. Exposure to blood 12 months d. Sexual contact with a person at high risk for HIV 12 months e. Imprisonment (>72 hours) 1 2 months f. Postblood transfusion 12 months g- Rape victim 12 months h. Aspirin and aspirin-containing drugs 72 hours i. Human pituitary growth hormone injection indefinite j- Sexual contact with anyone who used a needle to take illegal drugs indefinite k. Taken clotting factors indefinite 1. AIDS or HIV positive indefinite m. Males having sex with other males indefinite n. Had viral hepatitis indefinite 0. Positive HBsAg indefinite P- Positive HBc indefinite q- Positive HTLV-I or HTLV-II indefinite r. History of Creutzfeldt-Jakob disease indefinite s. History of Chagas disease or babesiosis indefinite C. Special Blood Collection 1. Autologous donation: A donation of blood given by a person to be used for transfusions on themselves at a later date. There are four types—preoperative, intraoperative hemodilution, intraoperative collection, and postoperative collection. a. Advantages: No diseases transmitted, no alloantibodies formed, no transfusion reactions possible  BLOOD COLLECTION 527 b. Disadvantages: High waste amount (unused if surgery postponed), adverse donor reactions, and increased cost 2. Preoperative collection a. Blood is drawn and stored before surgery. b. Used for stable patients having a surgery that may require a transfusion c. Especially good for patients with existing alloantibodies for whom it may be difficult to find compatible units d. Process begins with a physician's order. e. Patients must sign informed consent. f. Not asked detailed questions about high-risk behavior g. Facility makes policy regarding patient's health, age, weight, etc. Hemoglobin should not be below 11 g/dL or hematocrit below 33%. h. Blood not drawn sooner than every 72 hours and not drawn within 72 hours of surgery, i. Patient's name, transfusion facility, unique patient identification number, expiration date, and "For Autologous Use Only" or "Autologous Donor" tag is on the bag. j. ABO and D must be performed at the collecting facility. These tests must be repeated if the transfusing facility is different from the collecting facility, k. If transfused outside of the facility, HBsAg, anti-HBc, hepatitis C antibody, HIV 1/2 antigen and antibody, and serologic testing for syphilis must be performed before shipping. 1. If donor is positive for any of the above, physician's permission is required to use the unit, and a biohazard sticker is attached to the unit before shipping, m. An autologous unit cannot be used for allogeneic transfusion; if it is not used by the donor, it must be discarded. 3. Intraoperative hemodilution (acute normovolemic hemodilution) a. One to two units of the patient's blood are removed at the beginning of surgery and replaced by volume expanders. b. Units must be labeled with patient's name, unique identification number, date and time of phlebotomy, and "For Autologous Use Only." c. This blood can be stored at room temperature for up to 8 hours or at 1-6°C for 24 hours. 4. Intraoperative collection (intraoperative salvage) a. Blood lost into the abdominal cavity is collected by a machine. It is washed with saline and transferred back into patient. Blood should not be used if blood will be contaminated with bacteria, as in peritonitis. 5. Postoperative collection a. Collect blood from surgical drains and deliver into sterile containers. b. Collected blood must be transfused within 6 hours. 6. Directed donations a. Patients choose their own donors. b. All AABB Standards for donation apply to directed donations. CHAPTERS: IMMUNOHEMATOLOGY c. Policies about switching units from directed donation to general donor pool vary among institutions. 7. Hemapheresis a. Leukopheresis: Only WBCs removed from donor blood b. Plateletpheresis: Only platelets removed from donor blood c. Plasmapheresis: Only plasma removed from donor blood d. Red cell pheresis: Only red cells removed from donor e. Apheresis instrument: An electronic instrument that takes blood from a donor, separates the desired component, and returns the remaining components to the donor. (Process takes from 20 minutes to 2 hours.) f. All AABB Standards for donation apply to apheresis donors also. However, frequency of donation and additional testing are different for the three types of apheresis: 1) Plateletpheresis: Platelet count of 150,000/jxL; 48 hours required between donations, up to 24 times/year 2) Leukopheresis: Not more than twice a week, 24 times/year 3) Plasmapheresis: Every 4 weeks; total protein, IgG, and IgM monitored 4) Red cell pheresis: Every 16 weeks 8. Therapeutic phlebotomy a. One unit of blood is removed from a patient in a specified time interval. b. This is done to treat patient symptoms in polycythemia, hemochromatosis, and porphyria. XII. BLOOD COMPONENTS: PREPARATION, STORAGE, AND SHIPMENT A. Definitions 1. Whole blood: Blood collected from donors contains all cellular and liquid elements. 2. Components: Parts of blood used for treating patients, including RBCs, plasma, platelets, and cryoprecipitated antihemophiliac factor 3. Hemotherapy: Use blood or blood components to treat a disease in a patient B. Blood Collection Bag 1. It is a closed system consisting of main bag with needle, tubing, and up to four satellite bags attached. The entire system is sterile. 2. Standard phlebotomy = 450 mL ± 45 mL or 500 mL ± 50 mL C. Anticoagulant Preservative Solutions 1. Standard volume: 63 mL for 450 mL collections or 70 mL for 500 mL collections 2. If an autologous unit is drawn on a patient weighing less than 110 pounds, the anticoagulant must be reduced. a. Reduced Volume Factor (A) = weight of patient 4- HOlb A X 70 mL = amount of anticoagulant needed (B)  BLOOD COMPONENTS: PREPARATION, STORAGE, AND SHIPMENT 529 70 — B = amount of anticoagulant to remove A X 500 mL = amount of blood to collect b. Example: 90-lb donor 90 Ib -i- 110 Ib = 0.81 = A 0.81 X 70 mL - 56.7 mL = B 70 mL — 56.7 mL = 13.3 mL of anticoagulant to be removed from bag 0.81 X 500 mL = 405 mL of blood to be collected Types of anticoagulants and preservatives a. Adenine: Used in ATP synthesis b. Citrate: Chelates calcium to prevent coagulation c. CPD: Citrate-phosphate-dextrose d. CP2D: Citrate-phosphate-2-dextrose e. CPDA-1: Citrate-phosphate dextrose adenine-1 f. Dextrose: Sugar to support RBC life g. Sodium biphosphate: Buffer to prevent decreased pH Storage a. Shelf life: This is the amount of storage that blood can take that yields at least 75% of original RBCs still in recipient's circulation 24 hours after transfusion. Remember, blood is still "alive" when it is in a blood bag. b. Glucose, ATP, 2,3-BPG, and pH decrease as RBCs are stored. After cells are transfused, ATP and 2,3-BPG levels are restored in about 24 hours. c. Substances that increase during storage are all metabolic end products such as potassium, hydrogen ions, etc. Additive solutions a. AS-1 contains mannitol. b. AS-3 contains citrate and phosphate. c. AS-5 contains mannitol. d. These must be added within 72 hours of collection. e. Usually, an additive solution is added to RBCs after plasma is separated off. f. Additives extend the shelf life to 42 days and reduce RBC viscosity during transfusion. Rejuvenation solution a. Contains phosphate, inosine, pyruvate, and adenine b. Its purpose is to restore 2,3-BPG and ATP levels before freezing or transfusing a unit. c. May be necessary for autologous or rare units d. RBCs can be rejuvenated up to 3 days past the expiration date and can then be frozen for future use. e. RBCs can be rejuvenated, stored up to 24 hours at 1-6°C, and transfused. The cells must be washed before transfusion to remove the inosine. Blood component preparation a. Whole blood is centrifuged and can be separated into RBCs, platelets, fresh-frozen plasma (FFP), and cryoprecipitated antihemophiliac factor. 530 CHAPTERS: IMMUNOHEMATOLOGY b. Process: Whole blood bag is centrifuged; plasma is separated off into a satellite bag. If platelets are to be prepared from whole blood, two spins are required. The first centrifugation will be a "soft" spin, leaving platelets sus- pended in the plasma layer. If platelets will not be produced, a single "hard" spin (increased time and rotations per minute [rpm]) will be performed. c. AS-1 is put into RBC bag (if additive solution is used). d. RBC bag is sealed and removed from system. e. Plasma bag is centrifuged to sediment platelets ("hard" spin). f. Plasma is separated into FFP bag, leaving platelets with 40 to 70 mL of plasma in platelet bag. g. Platelet bag is sealed off and cut. h. Plasma is either frozen to make FFP within 8 hours of collection or frozen and later thawed in refrigerated conditions to make cryoprecipitate and cryo-poor plasma. 8. Storage temperature and expiration dates for components a. Whole blood: Storage 1-6°C; expires with CPD, CP2D anticoagulants in 21 days, with CPDA-1 anticoagulant in 35 days, with Adsol (AS-1, AS-3, orAS-5)in42days b. RBCs: Storage 1-6°C; expires with CPD, CP2D anticoagulants in 21 days, with CPDA-1 in 35 days, with AS-1, AS-3, and AS-5 in 42 days c. Platelets: Storage 20-24°C with rotation, expires in 5 days d. FFP: Storage -18°C, expires in 1 year; storage -65°C, expires in 7 years e. Cryoprecipitate: Storage-18°C, expires in 1 year f. RBCs (frozen): Storage -65°C, expires in 10 years g. RBCs (deglycerolized, washed): Storage 1-6°C, expires in 24 hours after thawing (deglycerolization) h. RBCs (irradiated): Storage 1-6°C, expires in 28 days or on originally assigned outdate, whichever comes first i. Platelets (pooled): Storage 20-24°C, expires in 4 hours after pooling j. Cryoprecipitate (pooled): Storage 20-24°C, expires 4 hours after pooling k. FFP (thawed): Storage 1-6°C, expires in 24 hours 1. Plateletpheresis: Storage 20-24°C, expires in 5 days m. Granulocyte pheresis: Storage 20-24°C, expires in 24 hours D. Storage and Transportation 1. FDA requirements and AABB Standards define calibration and maintenance procedures, storage temperature limits, and monitoring parameters for equipment used to store blood products. 2. All refrigerators, freezers, and platelet incubators must have a. Recording devices that monitor the temperature at least every 4 hours b. Audible alarms that ensure response 24 hours a day c. Regular alarm checks d. Power failure and alarm activation emergency procedures  BLOOD COMPONENT THERAPY 531 e. Emergency power backups (continuous power source for alarms) f. Calibrated thermometers that are checked against referenced thermometers g. Written procedures for all the above 3. Transportation a. Temperature for RBCs of 1-10°C is required during transport. A predetermined amount of wet ice in plastic bags is placed on top of the blood units to maintain the temperature for 24 hours. b. RBCs are packed in cardboard boxes with a styrofoam box inside. The ice is double-bagged and weighs approximately nine pounds. c. Frozen components are shipped on dry ice. These should be well wrapped because dry ice evaporates, and space in the box for movement should be allowed. d. Platelets are shipped at room temperature. Platelets can survive without agitation for a maximum of 24 hours. e. When component shipments are received, observe and record the temperature and appearance of units. If temperature is out of range, units must be evaluated before transfusion. Institutions have policies for determination of the disposition of the units. All problems and dispositions must be documented and stored with blood bank records. E. Administration of Blood Components 1. Positive identification of patient, sample, and crossmatched unit 2. Only normal saline should be infused with blood components. 3. A standard 170-micron filter must be used with all blood components. Leukoreduction filters may be used to reduce the number of leukocytes transfused with RBCs. 4. The maximum transfusion time allowed for one unit to be transfused is 2-4 hours. If the unit cannot be completely infused within 4 hours, the unit should be divided into two satellite bags and transfused as two separate units. 5. Documentation and accurate recordkeeping are vital. XIII. BLOOD COMPONENT THERAPY A. Whole Blood 1. Used in actively bleeding patients, patients who have lost at least 25% of their blood volume, or patients requiring exchange transfusions 2. When whole blood is not available, reconstituted whole blood (RBCs mixed with thawed type AB FFP from a different donor) may be used. B. RBCs 1. Used in oncology patients undergoing chemotherapy or radiation therapy, trauma patients, surgery patients, dialysis patients, premature infants, and patients with sickle cell anemia 532 CHAPTERS: IMMUNOHEMATOLOGY 2. Transfusing one unit usually increases the patient's hemoglobin approximately 1 g/dL and the hematocrit by 3%. C. Leukocyte-Reduced RBCs 1. Used in chronically transfused patients or patients having known febrile transfusion reactions 2. The standard 170-micron filter does not remove leukocytes. A special filter is required for bedside filtration. Leukoreduction (filtration) can also occur in the manufacturing process, which typically occurs within 72 hours from the time of collection. 3. AABB Standards for leukocyte reduction states that 85% of RBCs must remain and leukocytes must be reduced to less than 5 X 106 WBC/unit. D. Frozen RBCs 1. Method: RBCs are frozen by adding glycerol to prevent cell hydration and the formation of ice crystals that can cause cell lysis (40% weight per volume). 2. The unit is transferred to a polyolefm or polyvinyl chloride bag, and then the bag is placed in a metal or cardboard canister. 3. Initial freezing temp is -80°C, then for long-term storage at -65°C for 10 years. E. Deglycerolized RBCs 1. Frozen RBCs are thawed, and then the glycerol must be removed. 2. Deglycerolization: Glycerol is drawn out of the RBCs by washing the RBCs with a series of saline solutions of decreasing osmolality. 3. Deglycerolization involves entering the bag, so the deglycerolized RBCs expire in 24 hours. F. Washed RBCs 1. Used for patients who have a reaction to plasma proteins (allergic, febrile, and/or anaphylactic) 2. Used in infant or intrauterine transfusions 3. 10-20% of RBCs are lost in the process of washing the RBC unit with normal saline. G. Irradiated RBCs 1. T cells can cause graft-versus-host disease, with 90% of cases being fatal. 2. Gamma irradiation prevents T cell proliferation. 3. AABB Standards require irradiation of cellular components (RBCs and platelets), if a donor is a blood relative of the intended recipient or donor unit is HLA matched for recipient. Recommended minimum dose of gamma irradiation is 25 Gy (2500 rads).  BLOOD COMPONENT THERAPY : 533 4. Used for intrauterine transfusions, immunodeficient recipient, premature infants, chemotherapy and radiation patients, and bone marrow or progenitor cells transplant patients H. Platelets 1. Purpose: Used to control or prevent bleeding 2. Not indicated in patients with ITP (idiopathic thrombocytopenia) 3. Indicated in patients with chemotherapy, post-bone marrow transplant patients, or patients experiencing postoperative bleeding 4. Transfused platelets have a life span of 3 to 4 days. 5. No crossmatch necessary, but ABO type-specific preferred. 6. Platelet concentrates a. Prepared from whole blood unit b. Contain approximately 5.5 X 1010 platelets/unit c. Raise platelet count by 5000 juL/unit after transfusion 7. Pooled platelets a. Procedure is to choose one platelet bag of those to be pooled and empty content of other bags into it. b. Usual platelet order is 6-10 units. c. Opening the unit reduces the shelf life of the bag to 4 hours. Platelets should be pooled immediately before transfusion. 8. Plateletpheresis a. HLA-matched patients who receive numerous platelet transfusions can develop antibodies to the class IHLA antigens on platelets. These patients require HLA matching before transfusion. If platelets to be transfused are not HLA matched, the platelets will not last for 5 days in the patient's circulation. b. Plateletpheresis packs contain approximately 3 X 1011 platelets per unit. 9. Leukocyte-reduced platelets a. Filters can reduce the number of leukocytes in a bag while being transfused. b. Specific apheresis instruments can reduce leukocyte numbers during collection. I. Fresh-Frozen Plasma 1. Purpose: To replace coagulation factors in the patient 2. Indicated in: a. Bleeding patients who require factors II, V, VII, IX, and X b. Abnormal coagulation due to massive transfusion c. Patients on anticoagulants who are bleeding or require surgery d. Treatment of TTP and hemolytic uremic syndrome e. Patients with liver disease to prevent or correct bleeding 534 CHAPTERS: IMMUNOHEMATOLOGY f. Antithrombin III deficiencies g. DIG when fibrinogen is > 100 mg/dL 3. Thawing a. Thawed in water bath at 30-37°C for 30-45 minutes before transfusion b. Unit should be placed in watertight container before immersing in water bath to keep ports clean and prevent contamination. c. Water baths with agitators are preferred because the unit thaws faster. d. FDA-approved microwaves can also be used. J. Cryoprecipitated Antihemophilic Factor (Cryoprecipitate) 1. Insoluble precipitate is formed when FFP is thawed between 1 and 6°C. It contains factor VIII, fibrinogen, factor XIII, and von Willebrand factor. 2. It is used for patients with factor XIII deficiency, von Willebrand disease, and fibrinogen deficiency, and as a fibrin sealant. Note: Patients with Factor VIII deficiency are routinely treated with Factor VIII concentrates. 3. Each unit must contain at least 150 mg/dL of fibrinogen and 80IU of factor VIII. 4. Pooled cryoprecipitate a. Like platelets, cryoprecipitate is pooled into one bag before transfusion. b. Units are thawed in a similar fashion to FFP before pooling. c. Cryoprecipitate must be given within 4 hours after pooling. d. Formula for figuring factor VIII in cryoprecipitate: plasma volume X (desired level % — initial level %) # of units = 80 lU/bag e. Fibrin glue from cryoprecipitate: 1-2 units of cryoprecipitate are mixed with thrombin and applied topically to the bleeding area. K. Granulocyte Pheresis 1. Granulocyte transfusions are rare and limited to septic infants. 2. The pheresis bag contains > 1.0 X 10'° granulocytes, platelets, and 20-50 mL ofRBCs. 3. The cells deteriorate rapidly and must be transfused within 24 hours of collection. 4. Store at 20-24°C with no agitation until transfused. 5. Crossmatching is required because of RBC contamination. L. Labeling 1. Must conform with Title 21 of the Code of Federal Regulations (CFR), specifically 21 CFR 606.120 and 606.121, as well as FDA current thinking as described in "Guidance for Industry: Recognition and Use of a Standard for Uniform Blood and Blood Component Container Labels" (9/22/2006). In  TRANSFUSION THERAPY 535 addition, facilities accredited by AABB must have implemented ISBT 128 labeling systems by May 1, 2008, in accordance with the "United States Industry Consensus Standard for the Uniform Labeling of Blood and Blood Components Using ISBT 128" (November 2005). 2. Current labeling requirements include proper name, unique number, amount of blood collected, amount and type of anticoagulant, volume of component, expiration date, storage temperature, ABO/D type, reference to the "Circular of Information for the Use of Human Blood and Blood Components," warning regarding infectious agents, prescription requirements, donor classification, and FDA license number if applicable. 3. Other products must be labeled as follows: a. Irradiated components must have name of the facility performing the irradiation. b. Pooled components must include final volume, unique number assigned to the pool, time of expiration, and name of facility preparing the pooled component. c. Autologous units must be labeled: "For Autologous Use Only." 4. "Circular of Information for the Use of Human Blood and Blood Components": Guidelines that provide a description of each component, indications and contraindications for use, and information of dosage, administration, storage, side effects, and hazards XIV. TRANSFUSION THERAPY A. Emergency Transfusions 1. Rapid loss of blood can result in hemorrhagic shock. a. Symptoms: Hypotension, tachycardia, pallor, cyanosis, cold clammy skin, oliguria, decreased hematocrit, decreased central venous pressure (CVP), CNS depression, and metabolic shock 2. Priorities in acute blood loss a. Replace and maintain blood volume. b. Make sure oxygen-carrying capacity is adequate. c. Maintain coagulation system integrity. d. Correct metabolic imbalances. e. Maintain colloid osmotic pressure. 3. Massive transfusion: Replacement of a person's entire blood volume (approximately 10 units) within 24 hours 4. Emergency transfusions result from trauma (gunshot wounds, stabbings, vehicular accidents, etc.) and surgical needs. 5. Emergency release of blood: It is preferable to transfuse type-specific blood. If time is not available to type the patient, type O, D-negative blood is transfused into women of childbearing age. Type O, D-positive blood is transfused into men. Physician must request emergency release indicating that 536 CHAPTERS: IMMUNOHEMATOLOGY no crossmatch is performed before the blood is transfused. The crossmatch is performed during or following the transfusion. B. Neonatal and Pediatric Transfusions 1. Smaller blood volume than adults 2. Premature infants may need transfusion to offset the effect of hemoglobin F in their system. Hemoglobin F does not give up oxygen readily. 3. latrogenic blood loss (blood taken from the neonate or infant for laboratory tests) causes the neonate or infant to develop an anemia that may be severe enough to transfuse. 4. Neonates and infants do not tolerate hypothermia well, so blood warmers may be used. 5. Washed or fresh blood is preferred for neonates or infants because of the liver's inability to metabolize citrate anticoagulants and potassium, which leaks from RBCs in donor units over time. 6. Transfusions are given in small volumes in multiple packs taken from a normal size blood unit. 7. Infants do not form antibodies for the first 4 months, so no crossmatch is necessary. 8. Transfuse CMV-negative and/or leukoreduced blood. C. Transplantation 1. Liver transplant patients require large amounts of blood products (on average 20 units of RBCs, 25 units of FFP, 17 units of platelets, and 5 units of cryoprecipitate) because the liver produces many coagulation factors and cholesterol for RBC membranes. 2. ABO compatibility is important in kidney, liver, and heart transplants. It is not important in bone, heart valves, skin, and cornea transplants. 3. Progenitor cell transplants a. Allogeneic or autologous b. Derived from bone marrow or umbilical cord blood c. Transfusion support with leukocyte-reduced products to prevent alloimmunization and a greater chance of rejection d. Conditions treated: Severe combined immunodeficiency disease, Wiskott-Aldrich syndrome, aplastic anemia, Fanconi anemia, thalassemia, sickle cell disease, acute leukemia, CML, lymphoma, myelodysplastic/myeloproliferative disorders, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, and testicular cancer D. Therapeutic Hemapheresis 1. Replacement of blood from a patient to improve a patient's health 2. Conditions indicated for therapeutic exchanges: Multiple myeloma, Waldenstrom macroglobulinemia, hyperleukocytosis, TTP/HUS, sickle cell, myasthenia gravis, acute Guillain-Barre syndrome  TRANSFUSION REACTIONS 537 E. Oncology 1. Chemotherapy drugs kill all cells that are undergoing mitosis: Stem cells, gastrointestinal epithelial cells, and hair follicles. 2. Action of chemotherapy drugs: a. Stopping DNA replication b. Interfering with mRNA production F. Chronic Renal Disease 1. Dialysis patients have an increased uremic (blood urea nitrogen or BUN) content in blood that alters the RBC shape and causes the cells to be removed from circulation by the spleen. 2. Dialysis itself mechanically destroys RBCs. 3. Nonfunctioning kidneys do not produce erythropoietin to stimulate RBC production. 4. The use of transfusions in dialysis patients has been dramatically reduced since erythropoietin therapy was initiated. G. Sickle Cell Anemia 1. An abnormal hemoglobin (e.g., Hgb S) causes cells to be removed from circulation, resulting in a lowered hematocrit. 2. Because these patients require many transfusions, phenotypically matched units are preferred. 3. Severe cases may be treated by bone marrow transplants. H. Thalassemia 1. Decreased synthesis of the a- and (3-globin chains 2. Hemolytic anemia results 3. Transfusion support necessary I. Aplastic Anemia 1. Blood transfusion support is usually needed until bone marrow transplant can occur. XV. TRANSFUSION REACTIONS A. Types of Transfusion Reactions 1. Transfusion reactions are an adverse physiological reaction to the infusion of blood. a. Hemolytic: This is a reaction that destroys the transfused blood cells in vivo. Large amounts of free hemoglobin are released into the blood and can cause systemic damage. b. Nonhemolytic: Febrile and allergic 2. Acute reactions occur rapidly, within hours of transfusion. 3. Delayed reactions occur days or weeks after transfusion. 538 CHAPTERS: IMMUNOHEMATOLOGY 4. Immune-mediated transfusion reactions are due to RBC or HLA antigens and antigen-antibody reactions. 5. Transfusion reactions can also be caused by bacteria, viruses, or parasitic organisms. B. Hemolytic Transfusion Reactions 1. May be either acute or delayed a. Intravascular reactions are usually acute, whereas extravascular reactions are usually delayed. b. Symptoms are variable; they may not be correlated with type of hemolysis. 2. Mechanism a. Antibody binding to RBCs 1) Intravascular hemolysis: IgM antibodies activate the classical pathway of complement that lyses RBCs intravascularly. The lysis releases hemoglobin and RBC remnants into the blood. The excess hemoglobin binds to haptoglobin. Haptoglobin can only bind so much hemoglobin, so the excess hemoglobin is found in the blood and urine. 2) Extravascular hemolysis: Antibody-coated RBCs are removed from circulation by the liver and spleen. The cells lyse when sequestered and, subsequently, bilirubin is released into the blood. Antibodies responsible for this type of hemolysis do not activate the complement cascade or only partially activate it. b. Anaphylatoxins cause hypotension by triggering serotonin and histamine release. c. Cytokine activation: Sensitized RBCs are cleared from the blood by phagocytes. The phagocytes release cytokines that cause fever, hypotension, and activation of T- and B cells. d. Coagulation activation: Antigen-antibody-complement complexes activate the clotting system and cause DIG. e. Renal failure is caused by systemic hypotension, reactive renal vasoconstriction, and intravascular thrombi. C. Acute and Delayed Hemolytic Transfusion Reactions 1. Acute hemolytic transfusion reactions a. Clinical signs/symptoms: Severe, rapid onset, fever, chills, flushing, pain at site of infusion, tachycardia, hemoglobinemia, hemoglobinuria, hypotension b. Major sequelae: DIG, renal failure, irreversible shock, death c. Mechanisms: Antigen-antibody reaction activates complement or coats RBCs (i.e., ABO incompatible blood and antibodies to Vel or PP{Pk antigens) d. Occurrence: 1:25,000 transfusions e. Most common cause: Identification error in patient, unit, and/or specimen f. Diagnostic laboratory tests: Elevated plasma free hemoglobin, elevated bilirubin (6 hours posttransfusion), decreased haptoglobin, and positive DAT  TRANSFUSION REACTIONS 539 2. Delayed hemolytic transfusion reactions a. Usually less severe than acute hemolytic transfusion reaction, and dependent on the concentration of antibody in the blood rather than the type of antibody b. Clinical signs: 5-7 days posttransfusion, fever, mild jaundice c. Major sequelae: Usually none. However, antibodies in the Kidd system can cause major delayed hemolysis. d. Causes: Alloantibodies to Rh, Duffy, and Kidd antigens; patient with low concentration of alloantibody experiences anamnestic response when reexposed to RBC antigen e. Occurrence: 1:2,500 transfusions f. Diagnostic laboratory tests: Positive DAT, positive posttransfusion antibody screen, and decreased hemoglobin and hematocrit D. Causes of Non-Immune-Mediated Mechanisms of RBC Destruction 1. Transfusion of hemolyzed units 2. Malfunctioning or unregulated blood warming units 3. Improper thawing and deglycerolization of a frozen RBC unit 4. Physical destruction by needles, valves, or equipment 5. RBC defects 6. Administration of drugs and/or non-isotonic solutions with blood unit E. Immune-Mediated Nonhemolytic Transfusion Reaction 1. Clinical signs a. Fever with temperature increase 1 °C over baseline temperature 8-24 hours posttransfusion b. Nausea, vomiting, headache, and back pain 2. Causes: HLA antibody in recipient to donor antigens; cytokines in blood products containing WBCs and platelets 3. Occurrence a. Common in patients with multiple pregnancies and transfusions b. Multiple exposures to HLA antigens c. Common in women d. 1:200 donor units transfused F. Allergic Transfusion Reactions 1. Urticarial reactions a. Clinical signs: Wheals, hives, itching b. Sequelae: None c. Causes: Recipient forms antibodies to foreign proteins in donor plasma d. Occurs in 1-3% of recipients 2. Anaphylactic reactions a. Clinical signs: Rapid onset, severe wheezing and cough, and bronchospasms 540 CHAPTERS: IMMUNOHEMATOLOGY b. Sequelae: Syncope, shock, death c. Cause: Genetic IgA deficiency d. Occurs very rarely G. Transfusion-Associated Graft-versus-Host Disease 1. Clinical signs: 3-30 days posttransfusion, fever, erythematous maculopapular rash, abnormal liver function 2. Sequelae: Sepsis, hemorrhage, 90% mortality rate 3. Cause: Transfused T cells react against recipients 4. Occurs rarely H. Bacterial Contamination of Blood Products 1. Bacterial contamination usually occurs during phlebotomy or during thawing of frozen blood components. 2. Bacteria (Yersinia enterocolitica, most common) live and multiply in bag during storage. 3. Bacterial endotoxins can be present in the unit of blood and cause symptoms similar to hemolytic transfusion reactions. 4. 2% of units are contaminated. 5. Workup: Blood cultures drawn from patient; gram stain and culture of the unit 6. Person issuing unit needs to check for discoloration, clots, cloudiness, or hemolysis before unit is released. I. Circulatory Overload 1. Too much blood in a patient's vascular system caused by transfusing a unit too fast; most often occurs in children and elderly patients 2. Symptoms: Dyspnea, severe headache, peripheral edema, and signs of congestive heart failure occurring after transfusion; can be fatal J. Other Complications 1. Hemosiderosis: This condition, which is characterized by the deposition of the iron-containing pigment hemosiderin in organs such as the liver and spleen, occurs in chronically transfused patients, especially those with hemolytic anemias. 2. Citrate overload: Massive transfusions introduce large amounts of citrate into the body. Citrate binds ionized calcium, but it can be alleviated by calcium chloride or calcium gluconate injections. K. Transfusion Protocol and Suspected Transfusion Reaction Workup 1. Transfusionist checks and rechecks all paperwork, requisition, and blood bag tag before beginning the transfusion to ensure there were no clerical errors made. 2. Vital signs (blood pressure, temperature, respiration, and pulse) are taken before beginning and every 15 minutes for the first hour and then hourly until the transfusion is completed.  TRANSFUSION-TRANSMITTED DISEASES 541 3. If a reaction is suspected: a. Stop the transfusion. b. Notify the physician and the laboratory. c. Physician evaluates the patient. d. Draw EDTA and red top tubes, and collect first voided urine for laboratory testing according to institutional policy. 4. Laboratory responsibilities a. Check all samples, requisition, histories, and bags for identical patient identification. Clerical errors are responsible for most transfusion reactions. b. Examine pretransfusion and posttransfusion patient samples for hemolysis. c. Perform DAT on posttransfusion patient sample. If the posttransfusion sample is positive, the DAT is then performed on the pretransfusion sample. d. If clerical errors are eliminated and pre- and post-transfusion patient samples show no hemolysis and have negative DAT, the workup is considered to be not indicative of a hemolytic transfusion reaction. e. If any positive DAT or hemolysis is found in posttransfusion samples that was not present in pretransfusion samples, further testing is required. Repeat ABO and D on pretransfusion patient sample, posttransfusion patient sample, and segments from the bag; repeat antibody screen and crossmatch on old and new patient samples. Other tests may include hemoglobin, hematocrit, haptoglobin, urine hemoglobin, and bilirubin. 5. Transfusion reaction workup records a. Must be retained in the blood bank indefinitely b. Bacterial contamination and transmitted diseases are reported to blood collection facility. c. Fatalities are reported to FDA's Office of Compliance, Center for Biologies Evaluation and Research, within 24 hours. XVI. TRANSFUSION-TRANSMITTED DISEASES A. Donor Infectious Disease Testing (Test and Date Testing Started) 1. HBsAg (before 1980) 2. HBc antibody (1986) 3. HCV antibody (1990); HCV NAT testing (1999 under IND/licensed in 2002) 4. HIV-1/2 antibody (HIV-1: 1985;HIV-2: 1992) 5. HIV-1 p24 antigen (1996, discontinued 2002); HIV-1 NAT testing (1999 under IND/licensed in 2002) 6. HTLV-I/II antibody (1997) 7. Syphilis (before 1980) 542 CHAPTERS: IMMUNOHEMATOLOGY 8. CMV (only performed on small portion of inventory; CMV negative blood needed for premature infants, intrauterine transfusion, and immunocompromised recipients) 9. T. cruzi antibody/Chagas disease (2007/currently not mandated) 10. West Nile Virus NAT testing (2003 under IND/license 2007) B. Look-Back Studies 1. FDA requires notification of patients who received units from donors that subsequently tested positive for HIV-1/2 or HCV. a. Identify any blood products previously donated by a donor currently testing positive. b. Identify all blood products donated by that donor 12 months before the last negative screening test. c. Notify facilities that received units involved in the look-back investigation. d. Trace to patients and notify patients of potential exposure. XVII. SAFETY AND QUALITY ASSURANCE A. FDA Regulations 1. Mandate adherence to Current Good Manufacturing Practice (cGMP) a. Write standard operating procedures. b. Follow standard operating procedures. c. Record and document all work. d. Qualify personnel by training and education. e. Design and build proper facilities and equipment. f. Clean by following a housekeeping schedule. g. Validate equipment, personnel, processes, etc. h. Perform preventive maintenance on facilities and equipment, i. Control for quality, j. Audit for compliance with all the above. B. Records 1. Good recordkeeping a. Use permanent ink on documents. b. Record data on proper form. c. No white-out correction fluid is permitted; cross out mistake and have person making correction date and initial it. d. No ditto marks used. e. Record "broken, closed, or not in use" when appropriate. 2. Retention (indefinite) a. Donor's identification information, medical history, physical exam, consent, and interpretations for disease markers b. Information on blood and components from an outside source, including numeric or alphanumeric identification on old unit and identification of the  SAFETY AND QUALITY ASSURANCE 543 collecting facility, needs to be retained. However, the information from an intermediate facility may be used, if the intermediate facility retains the unit number and identification number of the collecting facility. c. Identification of facilities that carry out any part of the preparation of blood components, and the functions they perform d. Final disposition of each unit of blood or blood component e. Notification to donors of permanent deferral f. Records of prospective donors who have been placed on surveillance or indefinitely deferred for the protection of the potential recipient g. Notification to transfusing facilities of previous receipt of units from donors subsequently found to be confirmed positive for HIV and human T cell lymphotropic vims type 1 (HTLV) h. Difficulty in blood typing, clinically significant antibodies, and adverse reactions to transfusions i. Notification to recipients of potential exposure to disease transmissible by blood j. Names, signatures, initials, or identification codes and inclusive dates of employment of those authorized to sign or review reports and records 3. Retention (minimum of 10 years) a. Donor's ABO, D, difficulty in blood typing, severe adverse reactions to donation, and apheresis procedure clinical record b. Records of blood component inspection before issue c. Patient's ABO and D type, interpretation of compatibility testing, and therapeutic procedures, including phlebotomy, apheresis, and transfusion d. All superseded procedures, manuals, and publications e. Control testing of components, reagents, and equipment f. Proficiency testing surveys, including dates, performed tests, observed results, interpretations, identification of personnel carrying out the tests, and any appropriate corrective actions taken g. Documentation of staff qualifications, training, and competency testing h. Quality systems audits and internal assessment records C. Document Control 1. Must be complete, organized, appropriately stored, retrievable, and secure D. Personnel Qualifications 1. Job descriptions written with specific job duties are required. 2. Selection criteria for an employee must be developed. 3. Training must be provided during new employee orientation and whenever procedures change or the employee performs poorly. 4. Competency asses