Immunohematology Review: Help, What Do I Need to Know to Pass the ASCP Exam?

Transcription

1 Immunohematology Review: Help, What Do I Need to Know to Pass the ASCP Exam? Dr. Phyllis Ingham EdD. MEd. MT(ASCP) Program Director/Chair Clinical Laboratory Technology West Georgia Technical College Phyllis.ingham@westgatech.edu American Society for Clinical Laboratory Science Annual State Meeting Georgia Chapter 2019

2 Take Off the Blindfold Today!

3 Blood Group Systems

4 ABO Blood Group System What We Know.. Inverse reciprocal relationship between the forward and reverse type Naturally occurring antibodies ABO antibodies are predominately IgM

5 ABO Antibodies Activate complement Room temperature or colder Produce strong direct agglutination reactions

6 ABO Blood Group Inheritance Inheritance by Mendelian genetics Codominant expression

7 How are ABO Red Cell Antigens Formed? ABH antigen formation results from: the interaction of genes at three separate loci (ABO, Hh, and Se) These genes produce specific glycosyltransferases that add sugars to a basic precursor substance.

8 ABO and Hh Gene Interaction Immunodominant sugars confer blood group specificity

9 H Antigen

10 ABO Antigens and Phenotypes

11 ABO Discrepancies

12 A SubGroups First described in 1911 by von Dungen A 1 and A 2 subgroups Differences between A 1 and A 2 are quantitative and qualitative A subgroups generally more common than B subgroups

13 A SubGroups Forward grouping reagent (Anti-A) strongly agglutinates both A 1 and A 2 phenotypes Anti-A 1 lectin reagent used in the differentiation of A 1 and A 2 phenotypes

14 A SubGroups Anti-A 1 lectin reagent agglutinates A 1 (or A 1 B) cells but does not agglutinate A 2 (or A 2 B cells) Lectins used in blood banking Dolichos biflorus: agglutinates A1 or A1B Bandeiraea simplicifolia: agglutinates B cells Ulex europaeus: agglutinates O cells (H specificity) and other ABO blood groups depending on the amount of H antigen available

15 Weak Subgroups of A Subgroups weaker than A 2 occur infrequently Most often recognized through an ABO discrepancy (unexpected reactions in the forward and reverse grouping ) Varying expression of four characteristics Weak A subgroups can be distinguished as A 3, A x, A end, A m, A y, and A el

16 Weak Subgroups of B Very rare and less frequent than A subgroups Usually recognized by variations in the strength of the reaction using anti-b and anti-a,b Result of alternate alleles at the B locus Serologic techniques characterize B subgroups in the following categories: B 3, B x, B m, and B el

17 Bombay Phenotype (Oh) First reported by Bhende in 1952 in Bombay, India Inheritance of a double dose of the h gene, producing the very rare genotype hh No H antigen made ABO genes cannot be expressed ABH antigens cannot be formed

18 Bombay Phenotypes RBCs are devoid of normal ABH antigens and fail to react with anti-a, anti-b, and anti-h. In RBC testing using anti-a and anti-b, the Bombay would phenotype as an O blood group.

19 Bombay Phenotype Unlike the anti-h found occasionally in the serum of A1 and A1B individuals, the Bombay anti-h can often be potent and reacts strongly at 37 C. It is an IgM antibody that can bind complement and cause RBC lysis Only blood from another Bombay individual will be compatible

20 ABO Discrepancies

21 ABO Discrepancies Unexpected reactions in the forward and reverse grouping due to Problems with the patient s serum (reverse grouping) Problems with the patient s red cells (forward grouping) Problems with both the serum and cells Can appear as extra positive or weak/missing reactions Must be resolved prior to reporting a patient or donor s ABO group

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23 ABO Discrepancy Categorization Group I discrepancies Group II discrepancies Group III discrepancies Group IV discrepancies

24 Common Group Two Discrepancies Enhancing weakly reacting antigens with room temperature incubation Pretreatment of RBCs with enzymes Acquired B antigen

25 Resolving ABO Discrepancies Enhancing weakly reacting antigens with room temperature incubation Pretreatment of RBCs with enzymes Acquired B antigen Consideration of cold autoantibodies The patient s RBCs can be tested with Dolichos biflorus to confirm the presence of an ABO subgroup Unexpected alloantibodies in the patient s serum other than ABO isoagglutinins may cause a discrepancy in the reverse grouping

26 Resolution of Group Three Discrepancies Effects of Rouleaux Effects of Wharton s jelly

27 Resolution of Common Group Four Discrepancies Consideration of cold autoantibodies The patient s RBCs can be tested with Dolichos biflorus to confirm the presence of an ABO subgroup Unexpected alloantibodies in the patient s serum other than ABO isoagglutinins may cause a discrepancy in the reverse grouping

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29 Chemistry/Antigens (Methodologies Traditional Tube/Microwell Testing) Fig. 2-5 Summary of ABO reagents. Blood banks are using monoclonal antibodies for ABO reagents in routine testing. Fig. 9-4 Results and interpretation of an ABO/Rh phenotype. In the hemagglutination test, agglutination is a positive result, and no agglutination is a negative result.

30 Chemistry/Antigens (Methodologies Solid Phase Red Cell Adherence Assay - SPRCA) Fig. 9-6 SPRCA. Reactions and interpretation of

31 Chemistry/Antigens (Methodologies Gel Test) Fig ID-MTS Gel Test procedure for the detection of A, B, and D antigens.

32 Now You Give It a Try Group Activity

33 Rh Blood Group Rh specific antigens reside on proteins versus the carbohydrate antigens ABO and Hh. Rh antigens are very immunogenic. Rh antibodies are produced after exposure to foreign red blood cells.

34 Rh Blood Group Rh is the second most important blood group system after ABO. It is a complex blood group system composed of over 50 different RBC antigens. Individuals who lack RhD are Rh negative. Individuals who possess RhD are Rh positive. Determining the presence or absence of RhD is critical in pretransfusion testing.

35 Fisher Race-DCE Terminology Fisher and Race postulated that the antigens of the system were produced by three closely linked sets of alleles. Each gene was responsible for producing a product (or antigen) on the RBC surface.

36 Each antigen and corresponding gene were given the same letter designation (when referring to the gene, the letter is italicized). The phenotype (antigens expressed on the RBC detected by typing) of a given RBC is defined by the presence of D, C, c, E, and e expression.

37 Wiener: Rh-Hr Terminology Wiener believed there was one gene responsible for defining Rh that produced an agglutinogen containing a series of blood factors. This Rh gene produced at least three factors within an agglutinogen.

38 Comparison of Rh Genetic Theories

39 Rh Phenotypes and Conversions

40 Weak D: Variations of D expression Some individual s RBCs possess weaker expression of D antigen that requires an indirect antiglobulin test to detect the D antigen. Individuals with altered D antigen are categorized into different phenotypes defined as weakened D. C in trans to RHD Weak D Partial D

41 Weak D

42 Detection of Rh Antibodies and Antigens Most Rh antibodies are IgG and react optimally at 37 C or after AHG testing. Rh antibodies are usually produced following exposure to foreign RBCs. Rh antibodies may show dosage. Rh antibodies are enhanced when testing with enzyme-treated RBCs.

43 Weak D Procedure

44 Other Blood Group Systems

45 The Lewis System The Lewis blood group system is unique because the Lewis antigens are not intrinsic to RBCs but are on type 1 glycosphingolipids that are passively adsorbed onto the RBC membrane from the plasma. There are several Lewis antigens, but the two of primary concern are Le a and Le b. Two alleles at the Lewis locus, Le and the amorph le, and two alleles at the Secretor locus, Se and the amorph se.

46 Lewis Antigens

47 Lewis Antibodies Frequently naturally occurring antibodies made by Le(a b ) persons; that is, they occur without any known RBC stimulus. Generally IgM and do not cross the placenta. Not well developed on fetal RBCs. Anti-Le a is the most commonly encountered. Anti-Le b is not as common or generally as strong as anti-le a.

48 Synthesis of Lewis Antigens Lewis antigens produced in saliva and other secretions are glycoproteins, but Lewis cell-bound antigens absorbed from plasma onto the RBC membranes are glycolipids

49 Development of Lewis Antigens Depending upon the genes inherited, Le a and Le b glycoproteins will be present in the saliva of newborns, but Lewis glycolipids are not detectable in the plasma until about 10 days after birth.

50 All Other Blood Group Systems Must Know for each Group: Antigen frequency (high or low) Phase most likely to be detected at (IS: Cold reacting, AHG: 37 degrees) Reactivity with enzyme treated cells (are they enhanced or destroyed) Clinically significant capable of causing hemolytic transfusion reactions and/or HDFN

51 Antibody Specificities Anti-D, E, and K are most common in the US Anti-C, -c, -e, -Jka, -Jkb Fya, -S, -s sometimes seen Anti-Fya and anti-fyb rarely exist as single alloantibodies

52 Antibody Specificities Temperature (IgM = cold reactive, usually not clinically significant) Lemon Pie is best served cold (Lewis M, N, P1)

53 Knowledge of Antibody Specificities Antibodies to high incidence antigens are rarely seen (few people lack the antigen and can therefore form the antibody): k (Cellano), Kpb, Jsb, P, Pk, U, Lub, Vel, etc. Antibodies to low incidence antigens are rarely seen (although most people can form the antibody, the antigens are rarely found on donor blood antibodies usually formed through HDFN) Cw, Kpa, Jsa, Lua, etc.

54 Knowledge of Antibody Specificities Usually clinically significant: ABO, Rh, Kidd, Duffy, S, s, U, P Rarely (if ever) cause clinically obvious symptoms: Bg(HLA), Ch/Rg(C4), Leb, JMH, Xga Sometimes: Cartwright (Yt), Lutheran (Lu), Gerbich(Ge), Dombrock(Do), M,N, Lea, Vel, LW, Ii, H, Ata, Inb, Mia, Csa

55 Blood Group Disease Associations Hemolytic anemias (Rh null, McLeod phenotype) McLeod Syndrome (Chronic Granulomatous Disease) Malaria (Duffy) Cold Hemagglutinin Disease (I, IH) Paroxysmal Cold Hemoglobinuria (P IgG biphasic hemolysin; Donath Landsteiner antibody test)

56 Antibody Screening and Identification

57 Immune Response what really happens?

58 Immune Response (Primary vs Secondary) Fig. 1-6 Primary and secondary immune responses. The initial exposure to an antigen elicits the formation of IgM, followed by IgG antibodies and memory B cells. The second response to the same antigen causes much greater production of IgG antibodies and less IgM antibody secretion.

59 Immune Response (Macrophages) Fig. 1-4 Antibody attaches to the Fc receptor on a macrophage to signal clearance. The variable portion of the immunoglobulin attaches to the antigen on the red cell, while the macrophage attaches to the Fc portion. The red cell is transported to the spleen and liver for clearance.

60 Immunoglobulins TABLE 1-2 Comparison of IgM and IgG

61 Immunoglobulin Structure

62 Complement Serum proteins that assist with the clearance of antibody-coated red cells. Biologic functions include: Opsonization enhancing phagocytosis of antigens Chemotaxis attracting macrophages and neutrophils Cell Lysis rupturing membranes of foreign cells Agglutination clustering and binding of pathogens together (sticking)

63 Stages- Agglutination Reactions TABLE 1-5 Factors Affecting Agglutination

64 Potentiators TABLE 2-14 Summary of Antibody Potentiators

65 Direct and Indirect Antiglobulin Test Comparison

66 Sources of Error in Antiglobulin Testing

67 Clinical Examples Causing a Positive Direct Antiglobulin Test

68 Applications of Indirect Antiglobulin Test in the Immunohematology Laboratory

69 Antibody Screening Fig. 7-1 Screening cell antigram. Methodology (Tube, Gel, Solid Phase)

70 Antibody Screening Fig. 7-2 Screen interpretations. Tentative interpretations that can be made after testing of the antibody screen and direct antiglobulin test. IS, Immediate-spin; 37 C, 37 C incubation; AHG, antihuman globulin; CC, check cells;, check cells agglutinate; NT, not tested; Poly, polyspecific antiglobulin reagent; C3, anticomplement reagent.

71 Guidelines for Interpretation of a Panel

72 Antibody Panel

73 Multiple Antibodies

74 Select Cell Panel

75 Ficin Treated Panel

76 Strong Cold Autoantibody

77 Mini Cold Panel

78 Warm Autoantibody

79 Elution Technique

80 Special Procedures for Antibody ID Neutralization good for Lewis (Lewis Substance); P 1 antibodies (hydatid cyst fluid), anti-sd a (fresh urine) Ch and Rg (plasma). Adsorptions: Autologous can only be performed if patient has not been recently transfused or unless cell separation procedure is performed. If DAT is positive, antibody must be removed before procedure can be performed. Allogeneic use R 1 R 1, R 2 R 2 and rr cells. Must be careful during interpretation as clinically significant antibodies can be excluded. Commercially available W.A.R.M and RESt kits are available. RBC Phenotyping DAT must be negative. If DAT is positive and patient has not been recently transfused, Chlorquin Diphosphate can be used to remove IgG from patient s red cells. Recent transfusion requires cell separation procedure to be performed prior to phenotyping.

81 Special Reagents to help in Antibody ID 2-ME (2-Mercaptoethanol) and DTT (Dithiothreitol) cleaves disulfide bonds of IgM antibody molecules. Helps distinguish between IgM and IgG. AET (2-aminoethylisothiouronium bromide) creates red cells that lack Kell antigens ZZAP (DTT + papain) used to remove immunoglobulins and complement from the surface of red blood cells, commonly when evaluating a potential autoantibody. ZZAP also deactivates a multitude of red cell antigens on the red cell surface.

82 Prewarm Technique

83 Strategies for the weak antibody or one that does not fit a pattern

84 Saline Replacement for Rouleaux

85 Antibody ID You Give it a Try! Group Activity

86 Crossmatching, Here we come!

87 Compatability Process Pre-Analytical: Label the Tube Correctly!

88 Comparison of immediate-spin, computer, and antiglobulin crossmatch requirements

89 Unexpected Incompatibilities in Immediate-Spin Crossmatch

90 Antibodies can be missed in compatibility testing if: The corresponding antigen is absent from screening cells The antibody is so weak that it detects only homozygous expressions of the antigen (dosage effect) The antibody is detectable only by a method not routinely employed (e.g., in the presence of a particular enhancement medium) Antibody history is unknown

91 ABO Product Compatibility

92 Compatibility Testing - Incorrect ABO Grouping Resolution: Check all tube labeling against positive ID of donor and patient Repeat ABO/Rh of patient and donor Request new sample if necessary Patient plasma + Donor cells IS 37 AHG CC 4+

93 Serological Steps to Resolution of Incompatibility Verify integrity of specimen (patient identification, labeling) Check patient history (diagnosis, medications) Perform antibody ID If panel results are inconclusive (or cannot rule out all possibilities) must run selected cells, special techniques (neutralizations, absorption, elution, enzymes, etc.) Type antibody-producer for corresponding antigen (if not transfused within last 3 months) Screen for antigen negative (ABO/Rh) compatible units

94 Blood Donation, Transfusion Therapy Transfusion Reactions

95 Donor Selection Process Registration (positive identification) Educational Materials Screening Physical Examination Informed consent Self-exclusion

96 Donor Collection 0.7% aqueous scrub solution of iodophor compound to remove surface dirt and bacteria and begin germicidal action 10% povidone-iodine is applied beginning at the intended venipuncture site and continuing outward in a concentric spiral. The area is allowed to air dry for 30 seconds before being covered with sterile gauze For donors sensitive to these solutions, another method should be designated by the blood bank physician, such as chlorhexidine (ChloraPrep 2%) and 70% isopropyl alcohol ABBB standards require the use of collection containers that divert the first 10 to 20 ml of blood into a diversion pouch when platelet products are to be prepared from whole blood donation Primary bag used for blood collection, all attached satellite bags, sample tubes, and the donor registration form must be labeled with a unique identification number.

97 Physical Exam

98 Donor Referrals Must know: Temporary Permanent Indefinite

99 Adverse Donor Reactions

100 Special Donations Directed Donations Autologous Donations Doctor s prescription No age limit Lower hemoglobin (11.0 g/dl) Cannot donate within 72 hours of surgery Abbreviated testing per AABB standards A patient must give consent and have his/her physician submit a written request for the Red Cross to collect blood from the selected donors. No evidence that patients can select safer donors than a volunteer blood system provides. All donated blood products are tested with the same tests for HIV and other infectious diseases, which further enhances the safety of the blood supply. Social pressure associated with directed donations may compromise the reliability of the donor s answers to health-history questions.

101 FDA Regulations Blood is classified as a drug under the Federal Food, Drug and Cosmetic Act and is therefore subject to strict regulatory requirements during the manufacturing process as well as subsequent handling by transfusion facilities including labeling. It may only be dispensed with a prescription from a physician. Any equipment used in the manufacturing of blood products is also regulated under Medical Devices by the FDA (e.g., blood collection scales, apheresis machines, computer hardware/software). Any adverse reactions during donation or during subsequent transfusion must be reported to FDA (Biological Product Deviation Reporting) Fatalities attributable to blood component transfusion must be reported verbally to FDA within 24 hours and a written report filed within 7 days.

102 Donor Processing Must Knows! AABB Standards Testing: ABO/Rh Antibody Screening Infectious Disease Testing Component preparation: Storage Quality control

103 Donor Testing

104 Component Preparation and Storage

105 Blood Component Storage: What happens inside the blood bag?

106 FFP and Platelet Component Preparation FFP: Plasma frozen within 8 hours of collection and stored at or below 18 C for up to 1 year or stored at or below 65 C for 7 years. FFP contains the labile coagulation factors (factors V and VII) and stable factors. Plasma frozen within 24 hours of phlebotomy (PF24): Contains similar coagulation factors as FFP, although factor VIII levels are reduced and factor V may be variable compared with FFP. Recovered plasma for further manufacture, which is usually shipped to a fractionator for processing into derivatives such as albumin, immune globulin, and coagulation factor concentrates.

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108 Expected Changes in Patient with Component Therapy In a 70-kg adult (per unit): Whole Blood and RBCs: Hgb 1 g/dl, Hematocrit 3% Random Donor Platelets: 5-10K Apheresis Platelets: 30-60K Fresh Frozen Plasma (FFP)* coagulation factors 20% (3-6 units) Cryoprecipitate (CRYO)*: fibrinogen 5-10 mg/dl *FFP and CRYO should only be given in the event that there are no suitable coagulation factor concentrates that are available.

109 Neonatal Transfusion Therapy <4 months of age do not have to repeat compatibility testing if initial screen and DAT are negative Fresh blood < 7 days CMV seronegative Leukoreduced Irradiated Hgb S negative

110 Emergency Transfusion RBCs - Group O (Rh positive or Rh negative selection may be sex dependent in some facilities) Plasma Group AB Uncrossmatched no sample or not enough time to complete testing; donor blood must be conspicuously labeled and segments pulled for later compatibility testing (when sample received and testing completed)

111 Massive Transfusion replacement of one or more blood volumes within a 24 hour period. Adult EBV 5000 ml (about 10 units whole blood) Will also develop deficiencies of clotting factors and platelets so these will need to be replaced as well Switching blood types may be necessary to avoid depleting blood supply of a particular type (e.g. A+ to O+). Care must be taken when switching back to original type. Why?

112 Sickle Cell Patients Transfusion Therapy Sickle cell patients tend to make antibodies more readily than do other patients. This is due to the fact that Sickle Cell disease is a disease that occurs in the black population who share antigenic similarities. The majority of the donor population is white with antigenic dissimilarities. This fact alone contributes to the increased immunogenicity of donor blood antigens in Sickle Cell patients. Sickle cell patients should be given blood that is ABO/Rh compatible and negative for C, E, and K antigens. Blood must also be Hgb S negative.

113 Component Indications

114 Patient Disease and what product should be given

115 What to do for Adverse Reactions

116 Adverse Reaction Workup Additional testing Initial Investigation to determine if a reaction is occurring

117 Immune Mediated and Non Immune Mediated Reactions Summary

118 Hemolytic Disease of the Fetus and Newborn

119 Hemolytic Disease of Fetus and Newborn (HDFN) Fig Metabolism of bilirubin. A, Before delivery, fetal bilirubin produced by the breakdown of sensitized red cells in the fetal spleen is safely metabolized by the maternal liver. B, After delivery, the newborn's liver does not produce glucuronyl transferase and cannot convert bilirubin to an excretable form. As a result, it collects in tissues and causes brain damage.

120 HDFN Testing TABLE 11-2 Prenatal Testing: Tests to Identify Women at Risk of Hemolytic Disease of the Fetus and Newborn Fig Twofold serial dilutions of the serum containing the antibody are prepared with saline as the diluent. Saline is first added to tubes 1:2 1:256. Serum is then added to tube 1 and 1:2. The serum is transferred from 1 to 1:2 and then to 1:4 continuing to the last tube, changing pipette tips to prevent carryover. The red cell selected for testing is usually homozygous and tested by the antiglobulin technique using anti-igg. The titer is reported as the reciprocal of the highest dilution that gives a 1+ reaction.

121 Post Partum Testing

122 RhIG is it required?

123 Fetal Maternal Screen and Kleihauer Betke Acid Stain Rosette test for detection of fetomaternal hemorrhage. RhIG, Rh immune globulin; lpf, low power field. Acid elution test for determination of hemoglobin F. After staining, fetal red cells appear dark pink, and adult cells appear as pale ghost cells. Fetal hemoglobin resists acid elution and remains intact, whereas the adult cells lose the hemoglobin and do not take up the stain.

124 HDFN and RhIG Rule of Thumb: 1 vial of Rhogam will cover a packed red blood cell bleed of 15 ml or a whole blood bleed of 30 ml.

125 Almost Done! Make a specific study schedule that works for you! Don t procrastinate! Utilize the BOC Study Book Extensively! Get a great nights rest before the test Eat a good breakfast! Rule out the answers you are certain it is not, then pick the best answer between the last two possibilities Take deep breaths and remain calm!

126 References Harmening, D. (2019) Modern Blood Banking and Transfusion Practices 7th ed Publisher: F. A. Davis Co. Wise, Scott MS, MT(ASCP)SBB, Associate Professor, Georgia Regents University, ASCLS-GA 2017