ABO Discrepancies Disclosure Objectives Importance Recognition ...

4/12/2011 

ABO Discrepancies 

Developed by: Kelly Kezeor, MT(ASCP) 

Farai Tsimba-Chitsva, MT(ASCP)SBB 

Kerry Burright-Hittner, MT(ASCP)SBB 

Disclosure 

I have no real or apparent conflict of interest or other 

relationships related to the content of this presentation. 

There is no off-label and/or investigational use of products 

discussed in this presentation. I have no relevant financial 

relationship to disclose. 

The need is constant. 

The gratification is instant. 

Give blood ṬM 

Objectives 

Correlate ABO/Rh testing with 

the expected result and define 

various ways to resolve an ABO 

discrepancy. 

Identify various clerical l and 

technical errors that can affect 

ABO/Rh interpretation. 

Discuss the effects of disease 

on the expression of ABH 

antigens and antibodies. 

Importance 

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 severe, 

if not fatal, transfusion complications in 

patients. 

What IS an ABO Discrepancy? 

Definition: 

When the results of the forward grouping 

(patient’s cells) does not correspond to the 

reverse grouping (patient’s plasma/serum). 

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 

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4/12/2011 

ABO Discrepancies MUST be Resolved 

In PATIENTS, an ABO discrepancy must be 

resolved before ANY blood component is 

transfused. 

– If an ABO discrepancy cannot be resolved before 

blood product is needed, transfuse with Group O red 

blood cell units 

In DONORS, the discrepancy must be resolved 

before any blood is labeled with a blood type. 

Identify the Discrepancy 

Front Type 

Reverse Type 

Patient Anti-A Anti-B Anti-A,B A 1 cells B cells 

JK 0 3+ 3+ 0 0 

Front type: B 

Reverse Type: AB 

Theory: Weak reverse type 



Front Type 

Reverse Type 


KK 4+ 4+ 4+ 3+ 3+ 

Front Type 

Reverse Type 


CK 4+ 0 4+ 0 4+ 

Front Type: AB 

Reverse Type: O 

Front Type: A 

Back Type: A 

Theory: Additional antibodies in the reverse type 

Theory: NO DISCREPANCY NOTED 


Front Type 

Reverse Type 


AK 0 w+ 1+ 4+ 0 

Front Type: B?? 

Reverse Type: B 

Steps for Resolution 

Recheck specimen for identification 

Rewash EDTA cell suspension 

Repeat testing 

Confirm patient’s t’ diagnosis, i age, medication, 

and pregnancy history 

Theory: Weak reacting antigen in the front type. 

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Types of ERRORS 

Clerical Errors 

Mislabeled tubes 

Patient misidentification 

Inaccurate interpretations recorded 

Transcription error 

Computer entry error 


Reagent or equipment problems 

Using expired reagents 

Using an un-calibrated centrifuge 

Contaminated or hemolyzed reagents 

Incorrect storage temperatures 


ABO Discrepancy Categories 

Procedural errors 

Reagents not added 

Manufacturer’s directions not followed 

RBC suspensions incorrect concentration 

Cell buttons not re-suspended before grading 

agglutination 

Problems with 

Red Blood Cells 

Weak-reacting/Missing 

antigens 

Extra antigens 

Mixed field reactions 

Problems with 

Plasma/Serum 

Weak-reacting/Missing 

antibodies 

Extra antibodies 

Grouping 

Forward 

Reverse 

Missing/Weak Extra Mixed Field Missing/Weak Extra 

Subgroup A/B Acquired B O Transfusion 

Young 

Elderly 

Immunocompromised 

Cold 

Autoantibody 

tib Forward Grouping Problems 

Disease 

(cancer) 

B(A) Phenotype 

Bone Marrow 

Transplant 

Cold 

Alloantibody 

Rouleaux 

True Chimera 

Rouleaux 

Anti-A 1 

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Forward Grouping Problems 

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 A 3 , A x and B 3 and 

B can traditionally present a mixed field reaction. 

Chimerism due to the intrauterine exchange of red cells, fraternal 

twins, and mosaicism arising from dispermy presents mixed field even 

though it rarely occurs. 


Resolving Mixed Field (mf) Reactions 

Determine the CAUSE of the mixed field reaction 

Checking the patient’s 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, saliva, and transferase studies 

Molecular testing 


Weak or Missing Antigen 

Result of: 

Inheritance of a weak ABO subgroup 

Malignancies may result in the loss of ABH transferases 

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. 


Resolving Weak or Missing Antigens 

Check the patient’s transfusion and clinical history 

Read the forward group microscopically 

Use anti-A,B and monoclonal antisera that is known to react with 

A x and dA 3 weak ABO subgroups 

Perform adsorption and elution studies 


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 Gram negative 

bacteria 

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Resolving Additional Antigens 

Check clinical history for evidence of colon infections with 

Gram negative sepsis 

Test an auto control 

The patient’s 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. 


Spontaneous Agglutination 

Result of: 

Strong potent cold auto antibodies 

Would appear as AB in the front type and O in the reverse type 

Strong positive DAT with IgG, C3d and saline control 

Wharton’s jelly in cord blood 


Resolving Spontaneous Agglutination 

Incubate plasma and cells (separately) at 37°C 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 


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 

Reverse Grouping Problems 

▪Plasma or serum ABO discrepancies are 

more common than red cell discrepancies. 


Weak/Missing 

Additional Antibodies 

Rouleaux 

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4/12/2011 


Weak/Missing Antibodies 

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 

(immuno-compromised patient) 

NO agglutination on reverse grouping 


Resolving Weak/Missing Antibodies 

Determine patient’s 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 4°C for 15 

minutes 


Extra Antibodies 

Result of: 

Cold antibodies (allo- or auto-) 

Cold antibodies may include anti-I, H, M, N, P, Lewis 

Rouleaux 

Anti-A 1 in an A 2 or A 2 B individual 


Resolving Extra Antibodies: Cold Alloantibodies 

Perform antibody identification at IS and or RT 

Use the pre-warm technique 

Incubate the serum and red cells separately at 37°C before 

testing 

Perform a cold adsorption 

Incubating equal amounts of red cells and serum at 4C for 30 to 

60 minutes and test adsorbed serum against reverse type cells 

Antigen type reverse cells for the offending antigen 

If reverse type cells are antigen positive find reverse type cells 

that are antigen negative and incubate with serum to resolve 

discrepancy. 


Extra Antibodies: Rouleaux 

Result of: 

Abnormal concentrations of serum proteins 

Altered serum/protein ratios 

High-molecular-weight volume expanders 

Associated with: 

‣ Multiple myeloma 

‣ Waldenstrom’s macroglobulinemia (WM) 

‣ Hydroxyethyl starch (HES), dextran, etc 


Resolution of Extra Antibodies: Rouleaux 

REMOVE PROTEINS!! 

If the forward grouping is affected, wash cells to 

remove protein and repeat test 

If the reverse grouping is affected, perform saline 

replacement 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 salinewhich 

disperses cells 

Tube is mixed, centrifuged, and re-examined for agglutination 

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Result of: 

Extra Antibodies: Anti-A 1 

A 2 (or A 2 B) individuals development of anti-A 1 antibody 

A 2 (or A 2 B) individuals have less antigen sites than A 1 

individuals 

anti-A 1 is a naturally occurring IgM antibody 

Antibody reacts with A 1 cells, but not A 2 cells 


Resolution Extra Antibodies: Anti-A 1 

Type patient red blood cells with Anti-A 1 lectin 

Test patient serum with A 1 , A 2 and O cells 

Case Study #1 



Give blood. TM 

Case Study #1: Patient History 

•88 year old male 

•Diagnosis: Immunocompromised 

•Medications: Corticosteroids 

•Transfusion T f i History: Massive plasma infusion 

i 

•Lab: Hemoglobin: 6.5 g/dL 

Anti-A Anti-B Anti-A,B Anti-D 

D 

Control 

A 1 cells A 2 cells B cells 

Patient 0 0 0 3+ 0 w+ w+ 0 

What is the problem? 

Both a front AND reverse typing issue 

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 

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4/12/2011 

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 18°C (per manufacturers directions) 

4°C for 15 minutes 

test concurrently with autologous cells and group O screening cells 

Enzyme treat reverse type cells with FICIN 

Case Study #2 


D 

Control 


Patient 0 0 0 3+ 0 w+ w+ 0 

30’ RT 0 1+ 2+ 1+ 1+ 0 






D 

Control 


• 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 

Patient 0 4+ 4+ 3+ 0 3+ 3+ 1+ 

Screening 

Cells 

DAT 

IS 

PEG/IAT 

I 1+ 0 

II 1+ 0 

III 1+ 0 

Anti-IgG 

Anti- 

C3dC3b 

Saline 

Control 

Patient 0 2+ 0 


Reverse typing issue 

Additional antibodies: cold?? 

Investigation 


Diagnosis: Anemia 

Medications: None 

Resolution: 

Additional antibody in reverse 

type 

Additional antibody in reverse type resolution: 

Prewarm Technique 

Incubate the serum and red cells separately at 37°C before testing 

Cold Adsorption 

Incubate equal amounts of red cells (adsorbing cells) and patient 

serum at 4°C for 30 to 60 minutes 

Test adsorbed serum against reverse typing cells 

Case Study #3 

Screening 

Cells 

IS 

4°C Adsorbed 

Serum 

I 0 

II 0 

III 0 


Patient 3+ 3+ 1+ 

4°C 

Adsorbed 

Serum 

3+ 3+ 0 




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4/12/2011 



D 

Control 


• 45 year old man 

• Diagnosis: Recurrent GI bleed 

• Medications: None 

• Transfusion History: 10 in past year; none in last 

3 months 


Patient 4+ w+ 4+ 4+ 0 w+ 2+ 3+ 

Screening 

Cells 

DAT 

IS 

PEG/IAT 

I 1+ 0 

II 1+ 0 

III 1+ 0 

Anti-IgG 

Anti- 

C3dC3b 

Saline 

Control 

Patient 0 0 0 


Forward and Reverse typing 

issue 

Investigation 


Diagnosis: Recurrent GI bleed 

Medications: None 

Resolution: 

Additional antibody in front 

type and reverse type 

Additional antibody in the front and reverse type resolution 

Forward type: 

Wash red cells extensively and repeat testing 

Removes proteins 

Reverse type 

Saline replacement technique; 

Patient cells and serum combined and centrifuged 

Allows for antigen/antibody reaction (if present) 

Remove serum and replace with equal volume of saline 

Mix, centrifuge, and reexamine for agglutination 

Both macroscopically and microscopically 

Front Type: 

Anti-A Anti-B Anti-A,B 

Patient 4+ w+ 4+ 

Patient Cells 

washed X6 

with Saline 

4+ 0 4+ 

Reverse Type: 


Patient w+ 2+ 3+ 

Saline 

Replacement 0 1+ 3+ 

Screening IS 

PEG/IAT 

Cells 

(saline 

replacement) 

I w+ 0 

II 0 0 

III w+ 0 

Forward type resolved: 

Patient forward type: A 

Reverse type NOT resolved: 

cold alloantibody?? 

Anti-M identified at IS 

53 

54 

Reverse grouping cells need to be antigen typed for the M antigen. 

A 1 cells 

(original source) 

A 2 cells 


B cells 


A 2 cells 

(second source) 

B cells 

(second source) 

Anti-M 

0 

2+ 

1+ 

0 

0 

•The original source of A 2 and B 

cells are M antigen positive. Since 

the patient has anti-M identified at 

IS, this antigen is interfering with 

the reverse grouping (causing a 

discrepancy). 

•A second source of A 2 and B cells 

were located that are M antigen 

negative. These cells will be used 

to perform reverse grouping on this 

patient. 


D 

Control 


Patient 4+ w+ 4+ 4+ 0 w+ 2+ 3+ 

Washed X6 with normal saline 

(second source, 

M antigen negative) 

Saline 

replacement 

4+ 0 4+ 0 0 3+ 

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4/12/2011 


Case Study #4 


• Diagnosis: Pregnancy #3 

• Medications: Pre-natal vitamins 

• Transfusion History: No transfusions 




57 

58 


D 

Control 


Patient 4+ 0 4+ 4+ 0 2+ 0 3+ 

Screening 

Cells 

IS 

PEG/IAT 

I 0 0 

II 0 0 

III 0 0 


Reverse typing issue 

Investigation 


Diagnosis: Pregnant 

Medications: vitamins 

Resolution: 

Additional antibody in reverse type 

•Test the patient’s serum 

against different A 1 and A 2 

cells 

Patient Serum 

A 1 cells 3+ 

A 1 cells 3+ 

A 1 cells 3+ 

A 2 cells 0 

A 2 cells 0 

A 2 cells 0 

•Test the patient’s red blood 

cells against anti-A 1 lectin 

(Dolichos biflorus) 

Anti-A 1 Lectin 

(Dolichous biflorus) 

Patient 0 

Resolution: Patient is (probable) 

type A 2 with anti-A 1 in her serum 


Case Study #5 


• Diagnosis: COPD 

• Medications: Morphine 

• Transfusion History: No known transfusion per 

blood bank records 





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61 


D 

Control 


Patient 4+ mf 0 4+ mf 4+ 0 1+ w+ 4+ 

Screening 

Cells 

IS 

PEG/IAT 

Investigation 

I 1+ 2+ 

II 0 0 

III 1+ 2+ 


Forward and Reverse typing issue 


Diagnosis: COPD 

Medications: Morphine 

Resolution: 

Mixed field in front type and extra 

antibody(ies) in reverse type 

Additional antibody in the front and reverse type resolution 

Forward type: 

Mixed field 

What is causing mixed field—has this patient been transfused? 

Harvest reticulocytes (that aren’t coated with antibody) and re-test front type 

Upon further investigation, patient was transfused with 4 group O 

Positive units last week at a different facility (explaining the mixed field 

reactivity identified in the front type). The antibody screen was negative 

at that time. 

Anti-A Anti-B Anti-A,B 

Reticulocyte Separation Cells 

Patient 4+ 0 4+ 

A 2 cells 

2+ 

64 

63 

Reverse type 

Antibody identification 

What antibody is in the screen? 

Could it be interfering with the reverse type? 

An elution of the patient’s red blood cells should be performed to 

Reverse type resolution: 

Antigen type the reverse group RBCs for Jk a to determine if the 

antibody is interfering with ABO resolution 

Absorb the anti-Jk a out of the patient sample and re-test the 

serum with the reverse type cells to resolve the ABO. 

identify the antibody that is coating the red blood cells (positive auto- 

control) 

Anti-Jk a 

Elution of the patient’s red blood cells identifies anti-Jk a is coating 

the cells. 

Antibody identification in the serum identifies anti-Jk a A 1 cells 


2+ 


B cells 

2+ 


65 

Since the patient has been recently transfused, differential adsorptions 

are performed at a cold temperature to fully remove the anti-Jk a from 

the patient serum. 

A 1 

cells 

A 2 

cells 

B 

cells 

Patient 1+ w+ 4+ 

Adsorbed serum 

0 0 4+ 

Case Study #6 


D 

Control 


Patient 4+ mf 0 4+ mf 4+ 0 1+ w+ 4+ 

Reticulocyte Separation Cells 

Adsorbed serum 

4+ 0 4+ 0 0 4+ 




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68 



D 

Control 


• 73 year old male 

• Diagnosis: Lymphoma and severe anemia 

• Medications: Aspirin 

• Transfusion History: 3 units 6 months ago 

• Laboratory: Hemoglobin: 4.5 g/dL 

• 4 units requested STAT!! 

Patient w+ 4+ 4+ 4+ w+ 4+ 2+ 0 

Screening 

Cells 

IS 

PEG/IAT 

I 0 0 

II 0 0 

III 0 0 

Auto 1+ 3+ 

DAT 

Anti-IgG 

Anti- 

C3dC3b 

Saline 

Control 

Patient 3+ 2+ w+ 


Forward typing issue 

Investigation 


Diagnosis: Lymphoma 

Medications: Aspirin 

Resolution: 

Weak reactive front type, 

positive D control, positive 

saline control 

69 

70 

Forward type: 

Weak Reactivity 

Weak ABO subgroup? Malignancy? Spontaneous agglutination? 


D 

Control 

Patient w+ 4+ 4+ 4+ w+ 

Warm washed cells 

w+ 4+ 4+ 4+ w+ 

DAT 

Anti-IgG 

Anti- 

C3dC3b 

Saline 

Control 



1+ w+ w+ 

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. 


D 

Control 


Patient w+ 4+ 4+ 4+ w+ 4+ 2+ 0 


w+ 4+ 4+ 4+ w+ 

0.01 M DTT Treated Cells 

0 4+ 4+ 4+ 0 

71 

72 

DAT 

Anti-IgG 

Anti- 

C3dC3b 

Saline 

Control 



1+ w+ w+ 

0.01 M DTT treated Cells 

1+ w+ 0 

Resolution: 

•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. 

ABO discrepancy recognition AND 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. 

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73 

References 

• Harmening, D.M. (2005). Modern Blood Banking and Transfusion 

Practices (5 th Ed.) 

• Roback, John et.al. Technical Manual, 16th Edition. City: American 

Association of Blood Banks (AABB), 2008. 

• American Red Cross: ABO Discrepancy Standard Operating 

Procedure 

• Community Blood Bank: University of Texas at Galveston Specialist 

in Blood Bank: ABO Discrepancies. 

13

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