Enclonar, Kimberly / MLS 3A

ABO Grouping & Discrepancies Techniques in ABO Grouping

Slide method
April 8, 2021
• Principle
Meshane Bato-on, RMT
o RBCs from the specimen are tested for the
presence of A antigen and/or B antigen using
known anti-sera (anti-A and anti-B)
Blood Grouping
o Agglutination indicates the presence of
• Identify the blood group of an individual
corresponding antigen
• Performed prior to blood transfusion (compatibility
• Procedure
testing)
o PROPER LABELING
• Determine the possibility of occurrence of HDN o Anti-sera (Anti-A and anti-B) is added
o Sample: Capillary blood, or venous blood
ABO Antigen and Antibody Relationship
• Prone to drying - causes false positive results
• Universal donor - Type O
• Report reaction immediately
• Universal Acceptor - Type AB
• Reporting: A, B, AB, O
• US: o (+) clumping
o O+
o (-) homogenous
o A+
o B+
Tube method
o AB+ - least frequent
• Used for both forward and reverse typing
• Gold standard and recommended method
• Incubation and centrifugation can be performed to
enhance the reaction
• Addresses the drying issue of the slide method
• Forward typing
o Reagents: anti-A & anti-B
o Sample: 4-5% RBC suspension
• Reverse typing
o Reagents: A cells & B cells
o Sample: Patient serum/plasma
• EDTA is used
• Plasma is collected from the middle
• Mix and centrifuge (60s)
• Conventional tube method
o Pipette, NaCl, antisera, test RBCs, tubes, rack,
ABO Grouping cell wash centrifuge
Forward Grouping (Front-type or Forward Typing) • Grading
• Identifies antigen using Anti-A (blue) and Anti-B o Negative to 4+
(yellow) reagent. o Hemolysis - troubleshoot
o If neg - check under microscope
Characteristics of Routine Reagents used Forward Grouping • Ratio of serum to RBC suspension: 2:1
Anti A Anti B
Gel Tech method
Type of Ab Monoclonal Monoclonal
• Standardized method
Specificity Highly specific Highly specific • Can be used for ABO & Rh grouping, compatibility, Ab
Ig Class IgM w/ 3-4+ reaction IgM w/ 3-4+ identification and DAT
reaction • Requires small sample volume
• Requires special centrifuge
Color (dye) Clear blue (Tryphan Clear yellow • Does not require washing of RBCs & microscopic
Blue) (acriflavin) examination
Lectin Dolichos biflorus Griffonia • Disadvantage: expensive
simplicifolia • Principle for Forward grouping
o Microtubes filled with dextran acrylamide gel
and antisera are used, the gel act as a reaction
Reverse Grouping (Back-type)
medium and as a size filter, red cells to be tested
• Identifies antibody using A cell and B cell reagent
are added on the top of the tube
• Tests patients serum/plasma
• Result
• Uses 4-5% suspension of A cell and B cell (cherry red)
o Positive = agglutinated cells trapped at the top
of the tube
Characteristics of Routine Reagents used in Reverse
o Negative = non-agglutinated cells = pass through
Grouping
the gel and form button at the bottom of the
A cells B cells tube
Source Human Human • MF = mix-field (bottom and top)
• A2 produces IgM against A1
Red cell 4-5% 4-5% o B is more potent than A2
suspension
Reaction IgM with 3-4+ IgM with 3-4+
reaction reaction
 Enclonar, Kimberly / MLS 3A
ABO Discrepancies Blood group specific soluble Wash with saline
• Occur when unexpected reaction occur in the forward substances (BGSS)
and reverse typing
Low incidence antigens Use new reagents
• Most common cause: Technical errors
• 4 classification: Group I, II, III, IV
• 1+ or 2+ results Anti-A Anti-B A1 cells B cells Cause
A +mf 0 0 +3 Leukemia
Common Sources of Technical Errors
• Incorrect identification of tubes A 4+ 2+ 0 4+ Acquired B
• Too heavy or too light cell suspension antigen
• Mix-up samples O 0+ 1+ 4+ 4+ Low
• Clerical error incidence
• Failure to add reagent/sample Ab in the
• Over-centrifugation/under-centrifugation reagent
• Contaminated reagents
• Uncalibrated centrifuge
Group III
• Problems in forward and reverse
Group I
• Elevated levels of plasma proteins, causes rouleaux
• Problems in reverse typing formation or pseudoagglutination
• Weak/missing antibody • Causes
• Causes: o Elevated levels of globulin from certain disease
o Newborns
▪ Multiple myeloma
o Elderly patients
▪ Waldenstrom maxroglobulinemia
o Patients with Leukemia
▪ Hodgkin's
(hypogammaglobulinemia) o Elevated levels of fibrinogen
o Immunosuppressed individuals o Plasma expanders (dextran,
o Patients taking immunosuppressive drugs
polyvinylpyrrolidone)
o Bone marrow and stem cell transplantation o Wharton's jelly in cord blood
• Resolution • Resolution
o Check age of patient o Microscopic examination
o Incubate patient serum with A and B cells at o Washing the cell with saline several times
room temp for 30min o Saline replacement technique
o Incubate patient serum with A and b cells at 4C o For cord blood sample:
for 15min, together with O cells and autocontrol ▪ Wash 6-8x
▪ Check for autoantibodies
Anti-A Anti- A1 B Cause
Forward Reverse Cause B cells cells
Typing Typing
AB 4+ 4+ 2+ 2+ Pseudoagglutination
Anti-A Anti- A1 cells B
B cells
Group IV
O 0 0 0 0 • Elderly/newborn • Forward or reverse
• Causes
• Group II o Spontaneous agglutination by cold reactive
• Forward typing autoantibody
• Weak/missing antigen o Unexpected ABO isoagglutinin
• Causes o Unexpected non-ABO alloantibody
o Subgroup of A or B may be present • Resolution
o Leukemia causing reduction in ABO antigen o Washing patient red cells with saline then retest
expression o Run DAT and auto-control
o Acquired B phenomenon o Run antibody screen
▪ Colon cancer patients
▪ Pathogens can traverse and modify A Anti-A Anti-B A1 cells B cells Cause
antigens and convert it to B antigen (E.coli
B 2+ 4+ 4+ 2+ Cold
serotype 086)
autoantibodies
▪ Intestinal obstruction
▪ Proteus vulgaris AB 4+ 4+ 1+ 0 Unexpected
▪ Can perform deacetylation - remove N- isoagglutinin
acetyl AB 4+ 4+ 1+ 1+ Non-ABO
• Blood group specific soluble substance alloantibody
• Low incidence antibodies in reagent anti-sera

Cause Resolution
Weak subgroup Incubation at room temp
for 30min
Acquired B phenomenon Acidification
Treatment of RBC with
acetic anhydride