Correlate ABO/Rh testing with the expected result and define various ways to resolve an ABO discrepancy. Identify various clerical and technical errors that can occur inR affect ABO/Rh interpretation. Discuss the effects of disease on the expression of ABH antigens and antibodies 2 Recognition and resolution are two very important skills that blood bank technologists must possess. Of all the blood group systems, ABO is THE MOST IMPORTANT. ABO misinterpretation can lead to serious transfusion complications in patients. 3 ABO Discrepancies MUST be Resolved
In PATIENTS, an ABO discrepancy must be resolved before any blood component is transfused. AABB policy 5.12.1 & 5.12.2 says blood shall not be released for a patient until any discrepancy in patient ABO grouping is resolved.
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In DONORS, the discrepancy must be resolved before any blood is labeled . 5 Landsteiners Rule 6 What IS an ABO Discrepancy? Definition: When the results of the forward grouping (patients cells) does not correspond to the reverse grouping (patients plasma/serum 7 What CAUSES an ABO Discrepancy? Weak or Missing antigens in the FRONT type Weak or Missing antibodies in the
REVERSE type Additional antigens in the FRONT type Additional antibodies in the REVERSE type 8 Reasons for ABO discrepancy Types of ERRORS Clerical Errors Reagent or equipment problems Procedural errors TRUE DISCREPANCY SITUATION Problems with Red Blood Cells Problems with Plasma/Serum 9 Clerical Errors Mislabeled tubes Patient misidentification errors
Inaccurate interpretations recorded 10 Reagent or equipment problems Using expired reagents Using an un-calibrated centrifuge Contaminated or hemolyzed reagents Incorrect storage temperatures Types of Errors Procedural errors Reagents not added Manufacturers directions not followed RBC suspensions incorrect concentration Cell buttons not re- suspended before grading agglutination
11 Grouping Forward Reverse Missing/Weak Extra Mixed Field Missing/Weak Extra A/B Subgroup Disease (cancer) Acquired B B(A) Phenotype O Transfusion Bone Marrow Transplant Young Elderly Immunocompromised Cold Autoantibody Anti-A 1
Rouleaux Cold Alloantibody Rouleaux May cause all + reactions 12 patient red cells (type A) FORWARD GROUPING PROBLEMS anti-A antibodies AHG 13
Mixed field (mf) agglutination Weak or missing antigens Additional or unexpected antigens Polyagglutinable cells Mixed Field (mf) Reactions Small agglutinates with many un-agglutinated cells Result of: Mixed cell population from a massive transfusion of another blood group. non-O individual receiving O red blood cells)
Bone marrow transplants having both the original type and donor marrow cells. The inheritance of weak ABO subgroups such as A3, Ax and B3 and B can traditionally present a mixed field reaction.
Chimerism 14 Resolving Mixed Field (mf) Reactions Determine the CAUSE of the mixed field reaction Checking the patients transfusion history and clinical history e.g. HPC transplant
If it is determined that it is a weak subgroup, perform specialized tests e.g. adsorption and elution.
Molecular testing 15 Weak or Missing Antigen Result of: Inheritance of a weak ABO subgroup Malignancies may result in the loss of ABH transferases H d ki di Forward Grouping Problems Hodgkins disease Lymphomas Leukemias Massive transfusion with group O blood to a non-group O person e.g. a group A person receiving lots of group O blood. Bone marrow transplant and chemotherapy 16 Forward Grouping Problems Resolving Weak or Missing Antigens
Check the patients transfusion and clinical history Read the forward group microscopically
Use anti-A,B and monoclonal antisera that is known to react with Ax and A3 weak subgroups.
Perform adsorption and elution studies
17 Forward Grouping Problems Additional Antigens Result of: Bacterial enzymes deacetylate the A antigen to a B antigen and the patient front types as an AB and reverses as an A. Acquired B antigens are observed in patients with recurring GI or colon infections with GI bacteria 18 Acquired B Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar. Group A individual N-acetyl galactosamine Acquired B Phenotype Bacterial enzyme removes acetyl group Galactosamine now resembles D- galactose (found in Group B) 19 This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but still technically it is extra). Patients should receive Group A units. Acquired B usually goes away when the condition resolves Forward Grouping Problems Resolving Additional Antigens
Check clinical history for evidence of colon infections with Gram negative sepsis Auto control is negative- that proves the blood group as A.
As the patients own anti-B will not react with their own AB cells
Acidify the anti-B to a p.H. of 6 and retest Acquired B antigens will not react in acidified antiserum, whereas as normal B antigens will react.
20 Forward Grouping Problems Spontaneous Agglutination Result of: Strong potent cold auto antibodies Forward Grouping Problems Would appear as AB in the front type and O in the reverse type Strong positive DAT with IgG, C3d and saline control Whartons jelly in cord blood 21 Resolving Spontaneous Agglutination Incubate plasma and cells (separately) at 37C for 5 to 15 minutes Wash cells 5 to 6 times with warm saline Forward Grouping Problems Retest warm washed cells with warm plasma Retest the DAT and saline control Treat cells with 0.01M DTT Wash cord blood a minimum of 6 times with saline 22 Forward Grouping Problems Resolving Spontaneous Agglutination Incubate plasma and cells (separately) at 37C for 5 to 15 minutes Wash cells 5 to 6 times with warm saline Retest warm washed cells with warm plasma Retest the DAT and saline control Treat cells with 0.01M DTT
Wash cord blood a minimum of 6 times with saline 23 Polyagglutination Result of: Inheriting acquired abnormalities of the red cell membranes with exposure to crypt antigens e g T activation Bacterially contaminated sample
Resolve by: Avoid testing with human antisera; use monoclonal antisera.
Collection of a new sample 24 patient serum-plasma (with anti-B Ab) AHG reagent red cells (type B) Reverse Grouping Problems 25 Plasma or serum ABO discrepancies are more common than red cell discrepancies.
Newborns Antibodies are not present at birth and only develop after 3 to 6 months of age.
Elderly Weakened antibody activity
Hypogammaglobulinemia
Little or no antibody production (immune-compromised patient)
NO agglutination on reverse grouping 26 Reverse Grouping Problems Resolving Weak/Missing Antibodies Determine patients age and diagnosis Incubate serum testing for 15 minutes at room temperature or 18 C to enhance antibody reactions If negative, incubate serum testing at 4C for 15 minute 27 Extra Antibodies Result of: Cold antibodies (allo- or auto-) Cold antibodies may include anti-I, H, M, N, P, Lewis Rouleaux Anti-A1 in an A2 or A2B individual 28 Extra Antibodies: Rouleaux Result of: Abnormal concentrations of serum proteins Altered serum/protein ratios High-molecular-weight volume expanders Associated with: Multiple myeloma Waldenstroms macroglobulinemia (WM) 29 30 Resolution of Extra Antibodies: Rouleaux
REMOVE PROTEINS!! If the forward grouping is affected, wash cells to remove protein and repeat test.
Reverse Grouping Problems The reverse grouping is affected, perform salinereplacement technique (more common)
Reagent cells and patient serum are centrifuged to allow antibody attachment (if present)
Serum is removed and replaced by an equal volume of saline which disperses cells Tube is mixed, centrifuged, and re-examined for agglutination 31 Extra Antibodies: Anti-A1 Result of: A2 (or A2B) individuals development of anti-A1 antibody A2 (or A2B) individuals have less antigen sites than A1 individuals.
anti-A1 is a naturally occurring IgM antibody Antibody reacts with A1 cells, but not A2 cells 32 A sub-groups 33 Reverse Grouping Problems Resolution Extra Antibodies: Anti-A1 Type patient red blood cells with Anti-A1 lectin Test patient serum with A1 , A2 and O cells 34 CASE 1 35 Case Study #1: Patient History 88 year old male Diagnosis: Immunocompromised Medications: Corticosteroids Transfusion History: Massive plasma infusion Lab: Hemoglobin: 6.5 g/dL 36 What is the problem? Both a front AND reverse typing issue Investigation Patient age: 88 years old Diagnosis: Hypogammaglobulinemia Medications: Immunosuppressive drugs Resolution: weak front type, weak reverse type A B AB AntiD D CONTROL A1cells A2cells Bcells Patient 0 0 0 3+ 0 W+ W+ 0 37 A B AB AntiD D C A1cells A2 cells Bcells patient 0 0 0 3+ 0 W+ W+ 0 30 RT 0 1+ 2+ 1+ 1+ 0 Weak front type and weak reverse type resolution:
Enhance forward type Incubate patient cells with antisera (per manufacturers directions)
Enhance reverse type Incubate reverse type cells with patient serum for 15 to 30 minutes Room temperature or 18C (per manufacturers directions) 4C for 15 minutes test concurrently with autologous cells and group O screening cells
Enzyme treat reverse type cells with FICIN.
Final diagnosis---B +ve 38 Case Study #2: Patient History 33 year old female Diagnosis: Anemia Medications: None Transfusion History: 2 units of packed red blood cells 5 years ago Lab: Hemoglobin: 9.1 g/dL Case 2 39 What is the problem? Reverse typing issue Additional antibodies: cold
Investigation Patient age: 33 years old Diagnosis: Anemia Medications: None Resolution: Additional antibody in reverse Anti-A Anti-B AntiD
D C A1 Cells A2 cells B cells patient 0 4+ 4+ 0 3+ 3+ 1+ Screening cells IS IAT 1 1+ 0 2 1+ 0 3 1+ 0 DAT Anit- IgG Anti C3d c3b cont Patient 0 2+ 0 40 Additional antibody in reverse type resolution:
Prewarm Technique Incubate the serum and red cells separately at 37C before testing
Cold Adsorption Incubate equal amounts of red cells (adsorbing cells) and patient serum at 4C for 30 to 60 minutes Test adsorbed serum against reverse typing cells. FINAL DIAGNOSIS B+ve Screening cells IS 4c adsorbed serum 1 0 2 0 3 0 A1 Cells
A2 cells
B cells Patient 3+ 3+ 1+ 4C adsorbed serum 3+ 3+ 0 41 36 YEAR OLD FEMALE DIAGNOSIS: PREGNANCY #3 MEDICATIONS: PRE-NATAL VITAMINS TRANSFUSION HISTORY: NO TRANSFUSIONS Case Study 3 42 Anti-A Anti- B Anti- AB Anti- D D contro l A1 cells A2 cells B cells Patient 4+ 0 4+ 4+ 0 2+ 0 3+ Screening cells IS IAT 1 0 0 2 0 0 3 0 0 43 What is the problem? Reverse typing issue
DAT -NEGATIVE
Patient Investigation Patient age: 36 years old Diagnosis: PREGNANT Medications: VITAMINS Resolution: Additional antibody in reverse type Patient serum A1 cells 3+ AI cells 3+ A2 cells 0 Anti A1 Lecitin(Dolic hous biflorus Patient 0 44 Resolution: Patient is (probable) type A2 with anti-A1 in her serum 73 YEAR OLD MALE DIAGNOSIS: LYMPHOMA AND SEVERE ANEMIA MEDICATIONS: ASPIRIN TRANSFUSION HISTORY: 3 UNITS 6 MONTHS AGO LABORATORY: HEMOGLOBIN: 4.5 G/DL Case Study #6 45 Anti A Ant B Anti AB Anti D DC A1 cells B cells patient W+ve 4+ 4+ 4+ W+ 4+ 0 Screeni ng cells positive IS IAT 1 0 0 2 0 0 3 0 0 Auto control 1+ 3+ DAT Anti -IgG Anti-c3d Ptaient 3+ 2+ What is the problem?
Patient Patient age: 73 years old Diagnosis: Lymphoma Medications: Aspirin Resolution: Weak reactive front type, positive D control, positive saline control 46 Forward type: Weak Reactivity Weak ABO subgroup? Malignancy? Spontaneous agglutination?.
47 Spontaneous agglutination was not resolved with warm washed cells. DTT (0.01M) treatment of the red blood cells should be performed to resolve the ABO/Rh discrepancy and disperse the spontaneous agglutination.
: Patient is B Positive Positive DAT and autocontrol was further investigated by a reference laboratory, and it was discovered the patient had a warm autoantibody in his plasma Example 7 Anti-A Anti-B A1 Cells B Cells 3+ 1+ 0 4+ Problem: Causes: Resolution: Take Home Message
ABO discrepancy recognition & resolution is imperative in the blood bank
ABO discrepancies can present themselves as a front type problem, reverse type problem, or combination of both.
If ABO discrepancy resolution cannot be performed, and blood is needed immediately, transfusion of type O blood may be necessary . 49 Relation of H-substance (antigen) and ABO groups 50 O h Phenotype (Bombay) Occurs when two hh genes are inherited at the Hh locus. Possess normal A or B genes (if they were inherited) but unable to express. Must have H on red cell membrane Can transmit A or B gene to offspring Term Bombay used since first discovered in Bombay, India 51 O h Phenotype (Bombay) Symbol O h denotes this phenotype RBCs not agglutinated by anti-A, -B or A,B Serum/plasma agglutinates A and B cells Not recognized until serum tested against group O cells and causes strong agglutination. Have anti-A, -B, -A,B and H Can only be transfused with Bombay blood <0.01% 52 Grading of Reactions 53 Blood group 54 O h Phenotype (Bombay) Confirmatory testing Anti-H lectin (Ulex europaeus) negative Agglutination of A, B, AB and O cells Serum/plasma will not agglutinate Oh cells. 55 Results in Tube method 56 57 Antigen / Antibody 58 Reading in tube method 59 Principle of Gel Technology J The sephadex gel matrix acts as a sieve.
J Large agglutinates remain on or near the top of the gel interface. J Smaller agglutinates pass partway through the gel, depending on size. J Unagglutinated cells pass to the base of the microtube 60
61 Extra Antibodies: Rouleaux Result of: Abnormal concentrations of serum proteins Altered serum/protein ratios Reverse Grouping Problems High-molecular-weight volume expanders Associated with: Multiple myeloma Waldenstroms macroglobulinemia (WM) Hydroxyethyl starch (HES), dextran, etc 62 Extra Antibodies: Rouleaux Result of: Abnormal concentrations of serum proteins Altered serum/protein ratios Reverse Grouping Problems High-molecular-weight volume expanders Associated with: Multiple myeloma 63 Extra Antibodies: Rouleaux Result of: Abnormal concentrations of serum proteins Altered serum/protein ratios Reverse Grouping Problems High-molecular-weight volume expanders Associated with: Multiple myeloma 64 Waldenstroms macroglobulinemia (WM) Hydroxyethyl starch (HES), dextran, etc