Unit 2 : ABO Discrepancies

What is an ABO discrepancy?

• exist when interpretation of the forward typing and reverse serum / plasma grouping for a sample differ

• or when current type does not match historical record

• ex: missing or “extra” reactions from expected reactions

What is ABO Forward Typing?

• you test the patients RBC with known antisera

  • Anti-A

  • Anti-B

  • Anti-A,B

• discovers what antigens are on the patients RBC

What is ABO Reverse typing?

• you test the patients plasma/serum against known RBCs

  • A1 cells

  • B cells

• discovers what antibodies are in patients plasma

Where do ABO discrepancies come from?

• preanalytical errors

• analytical errors

• other causes

What are examples of preanalytical errors that cause ABO discrepancies?

• incorrect identification of patient

• incorrect labeling of specimen

• sample mix-up (joint commission)

What clerical errors are associated with pre/post analytical errors that cause ABO discrepancies?

• Labeling of tubes

• transcription error

• incorrect recording of results

What technical errors are associated with analytical errors that cause ABO discrepancies?

• cell suspension too heavy or too light

• mix up of samples

• failure to add reagents or patient sample

• failure to follow manufacterer’s instructions or SOP

• improper resuspension of cell button

• failure to note hemolysis

• uncalibrated centrifuge / over or under centrifugation

• contaminated reagents

• warming during centrifugation

What are the different classifications of discrepancies?

• Weak or missing antibodies

  • problem in REVERSE typing

• Weak or missing antigens

  • problem in FORWARD typing

• Abnormal plasma proteins

  • causes rouleaux

• Miscellaneous causes

What unexpected reactions can occur in Reverse grouping?

• weak/negative/missing reverse typing

• extra reactions in reverse typing with POS O ccells/ POS AUTO

• Extra reactions in the reverse typing

What unexpected reactions can occur in Forward Grouping?

• due to weak or missing antigens

• due to extra antigens

What is the most common discrepancy?

• unexpected reactions in reverse grouping due to weakly reacting or missing antibodies

What is this an example of?

• ABO discrepancy due to unexpected reaction in reverse grouping

  • due to weak reacting/ missing antibodies

  • This person forward types as B blood

  • reverse type does not show result

What people are more likely to have an ABO discrepancy in reverse grouping due to weakly reacting / missing antibodies?

• newborns

  • do not produce antibodies till 4-6 months age

• elderly patients

• people w’ leukemias, lymphomas, hypo/agammaglobulinemia

• people on immunosuppressive drug therapy

• congenital hypo/agammaglobulopathies

• bone marrow transplant recipients

How do you resolve an ABO discrepancy in reverse grouping?

• try to enhance weak / missing reaction

  • room temp incubation

  • 4 C incubation

Why would a 4 C incubation help resolve ABO discrepancy for reverse grouping?

• ABO antibodies are mainly IgM and react better at cold temps

  • also have IgG component

If you are trying to resolve an ABO discrepancy due to issues in reverse typing, what must also be run at the same temperature?

• autocontrol (pt cells / plasma)

• O cells (screening cells)

  • if pt plasma react with O cells, something other than anti-A or anti-B may be responsible

Describe Auto Anti-I.

• cold reactive (IgM immunoglobulin)

• I antigen is present on mostly all adult red cells

What does this mean?

• ABO discrepancy in reverse grouping due to extra reaction in reverse groups typing with pos O cells

  • this means pt may have antibody reacting with something OTHER THAN A or B antigens

  • could be cold autoantibody or other cold-reactive antibody

What does this mean?

• extra unexpected reaction in reverse grouping

  • could mean there are ABO subgroups

What does this mean?

• extra reaction in reverse grouping with ABO subgroups

• A₂B with anti-A₁

What is A₂B with anti-A₁?

• person has

  • A2 antigen

  • B antigen

• they do not have A1 antigen

• some A2 or A2B individuals can make anti-A1

  • therefore their plasma may react with A1 reagent cells

• use Anti-A1 lectin to investigate

  • Anti-A = +

  • ANti-B = +

  • AntiA1 lectin = 0

  • these support A2B phenotype

What can cause unexpected reactions in forward grouping due to weak / missing antigens?

• ABO subgroups

• Hodgkin’s disease / leukemia

• Acquired B - associated with cancers of digestive tract

  • excess blood group specific soluble substance (BGSS)

    • carcinoma of stomach / pancreas

  • neutralizes reagent antisera

What is the resolution for Weak / missing antigens in forward typing?

• room temp incubation (if weak or missing reactions)

• 4 C incubation ( if weak or missing reactions)

  • requires autocontrol and O cell control

• Enzyme enhancement (if weak or missing reactions)

What can cause an ABO discrepancy in forward grouping due to Extra Antigens?

• Chimerism

• non-type specific bone marrow transplants and stem cell therapy

• exchange transfusions

• fetal-maternal bleeding

• use of non-ABO type specific RBC components for transfusions

What is Chimerism?

• blood group chimera can occur when more than one zygote/blastocyst contributes to one individual

• results in two genetically distinct cell populations

• pts blood may contain RBC populations with different ABO types

• see mixed field agglutination

How do you resolve ABO discrepancies with extra reactions in the forward grouping?

• wash red cell suspension (if unexpected)

• repeat with a different lot # (to rule out low frequency Ab in reagent)

• Acquired B ( if HX is suggestive of mixed field reactivity)

How do you test for Acquired B?

• Test with monoclonal Anti B (clone ES4) - agglutination is stronger

• if the pt is a sector, perform secretor studies

• treat with acetic anhydride - reference lab processes

What can cause unexpected reactions due to abnormal plasma proteins?

• multiple myeloma

• Waldenstroms macroglobulinemia

• Hodgkin’s lymphoma

• elevated fibrinogen

• plasma expanders (received albumin / crystalloids)

• Whartons Jelly (cord blood samples only)

What is roulaous?

• occurs when RBCs stack on top of one another (like a stack of coins)

• can look like agglutination

• is NOT true antigen - antibody agglutination

• can create a false-positive looking reaction during ABO testing

What can cause Roulox?

• abnormal proteins in plasma can decrease the normal repulsive forces between RBCs

• cells become arranged in stacks

How do you distinguish rouleaux from true agglutination?

• Saline Replacement (SSR)

What are the steps in Saline replacement (SSR)

• look at reaction microscopically - stacked coin appearance

• respin the affected tubes (often entire set so other tubes can serve as controls)

• gently remove pt plasma carefully (do not disturb RBC button)

• add 2 drops normal saline to replace removed plasma

• resuspend / read macroscopically

How do you interpret the results of Saline Replacement (SSR)?

• if “agglutination” disappears → rouleaux

  • you removed abnormal plasma proteins and replaced plasma with saline

• if agglutination remains → true agglutination

What miscellaneous problems can cause ABO discrepancies?

• cold reactive autoantibodies

• cold reactive alloantibodies

• bone marrow transplant

• ABO isoagglutinins

What is the most important troubleshooting approach?

• always start with a clerical check and repeat with a washed cell suspension

What does “suspect the weaker reaction” mean?

• Forward

  • Anti-A= 4+

  • Anti-B = 0

• Reverse

  • A1 cells = 0

  • B cells = 1 +

• forward typing strongly says A

• reverse anti-B reaction is weak

• you should ask “ why is the reverse reaction weak”

What does the Anti-A1 lectin reagent do?

• helpful for investigating A subgroups (A2/A2B situations)

What does the Anti-A,B reagent do ?

• can help detect weak A or B antigen expression

What do A2 cells do?

• helpful when investigating subgroup-related reverse reactions

What do O cells do?

• useful for determining whether an unexpected reaction is due to something other than anti-A / anti-B

How do you know which one it is in ABO discrepancies?

• suspect the weaker reaction

• review patients case history

• suspect the serum

How do you know what to do first in ABO discrepancies?

• always start with a clerical check and repeat with a washed cell suspension

• don’t suspect the most unusual fist - work through the easy checks first

What additional reagents might be needed for ABO discrepancies?

• Anti-A₁ and -H lectin (H lectiv rarely available in TSLs)

• Anti-A,B

• A_2, O, and autocells