ABO Discrepancies & ABO Discrepancies & other problemsother problems

Prepared ByPrepared ByAhmad Shihada Silmi Msc, FIBMSAhmad Shihada Silmi Msc, FIBMS

Medical Technology DeptMedical Technology DeptIslamic University of GazaIslamic University of Gaza

ImportanceImportance

It is important for students to recognize It is important for students to recognize discrepant results and how to (basically) discrepant results and how to (basically) resolve themresolve them

Remember, the ABO system is the most Remember, the ABO system is the most important blood group system in relation to important blood group system in relation to transfusionstransfusions

Misinterpreting ABO discrepancies could Misinterpreting ABO discrepancies could be life threatening to patientsbe life threatening to patients

DiscrepanciesDiscrepancies

A A discrepancydiscrepancy occurs when the red cell occurs when the red cell testing does NOT match the serum testing testing does NOT match the serum testing resultsresults

In other words, the In other words, the forward does NOT forward does NOT match the reversematch the reverse

WhyWhy??

Reaction strengths could be Reaction strengths could be weakerweaker than than expectedexpected

Some reactions may be Some reactions may be missingmissing in the in the reverse or forward typingreverse or forward typing

ExtraExtra reactions may occur reactions may occur

PatientPatientAnti-AAnti-AAnti-BAnti-BAA11 Cells CellsB CellsB Cells

1144++11++0044++

220044++11++00

3344++44++11++00

440033++0000

What do you doWhat do you do??

Identify the problemIdentify the problem Most of the time, the problem is technicalMost of the time, the problem is technical

Mislabeled tubeMislabeled tube Failure to add reagentFailure to add reagent Either Either repeat testrepeat test on on samesame sample, request a sample, request a

new samplenew sample, or , or wash cellswash cells Other times, there is a Other times, there is a real discrepancyreal discrepancy

due to problems with the patient’s red cells due to problems with the patient’s red cells or serumor serum

DiscrepancyDiscrepancy? ?

If a real discrepancy is encountered, the If a real discrepancy is encountered, the results must be recordedresults must be recorded

However, the interpretation is delayed until However, the interpretation is delayed until the discrepancy is RESOLVEDthe discrepancy is RESOLVED

ErrorsErrors

Technical ErrorsTechnical Errors Clerical errorsClerical errors

Mislabeled tubesMislabeled tubes Patient misidentificationPatient misidentification Inaccurate interpretations recordedInaccurate interpretations recorded Transcription errorTranscription error Computer entry errorComputer entry error

Reagent or equipment problemsReagent or equipment problems Using expired reagentsUsing expired reagents Using an uncalibrated centrifugeUsing an uncalibrated centrifuge Contaminated or hemolyzed reagentsContaminated or hemolyzed reagents Incorrect storage temperaturesIncorrect storage temperatures

Procedural errorsProcedural errors Reagents not addedReagents not added Manufacturer’s directions not followedManufacturer’s directions not followed RBC suspensions incorrect concentrationRBC suspensions incorrect concentration Cell buttons not resuspended before grading agglutinationCell buttons not resuspended before grading agglutination

Clotting deficienciesClotting deficiencies

Serum that does not clot may be due to:Serum that does not clot may be due to: Low platelet countsLow platelet counts Anticoagulant therapy (Heparin, Aspirin, etc)Anticoagulant therapy (Heparin, Aspirin, etc) Factor deficienciesFactor deficiencies

Serum that does not clot completely before Serum that does not clot completely before testing is prone to developing fibrin clots that testing is prone to developing fibrin clots that may mimic agglutinationmay mimic agglutination

ThrombinThrombin can be added to serum to activate can be added to serum to activate clot formationclot formation

OR, tubes containing EDTA can be usedOR, tubes containing EDTA can be used

Contaminated samples or reagentsContaminated samples or reagents

Sample contaminationSample contamination Microbial growth in tubeMicrobial growth in tube

Reagent contaminationReagent contamination Bacterial growth causes cloudy or discolored Bacterial growth causes cloudy or discolored

appearance…do not use if you see this!appearance…do not use if you see this! Reagents contaminated with other reagents Reagents contaminated with other reagents

(don’t touch side of tube when dispensing)(don’t touch side of tube when dispensing) Saline should be changed regularlySaline should be changed regularly

Equipment problemsEquipment problems

Routine maintenance should be performed Routine maintenance should be performed on a regular basis (daily, weekly, etc)on a regular basis (daily, weekly, etc)

Keep instruments like centrifuges, Keep instruments like centrifuges, thermometers, and timers calibratedthermometers, and timers calibrated Uncalibrated serofuges can cause false resultsUncalibrated serofuges can cause false results

HemolysisHemolysis

Detected in serum after centrifugation (red)Detected in serum after centrifugation (red) Important if not documentedImportant if not documented Can result from:Can result from:

Complement bindingComplement binding• Anti-A, anti-B, anti-H, and anti-LeAnti-A, anti-B, anti-H, and anti-Leaa

Bacterial contaminationBacterial contamination

Red supernatant

ABO discrepanciesABO discrepancies

ABO DiscrepanciesABO Discrepancies

Problems with Problems with RBCsRBCs Weak-reacting/Missing antigensWeak-reacting/Missing antigens Extra antigensExtra antigens Mixed field reactionsMixed field reactions

Problems with Problems with serumserum Weak-reacting/Missing antibodiesWeak-reacting/Missing antibodies Extra antibodiesExtra antibodies

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

YoungElderly

Immunocompromised

Cold Autoantibody

Anti-A1

Rouleaux

Cold Alloantibody

RouleauxMay cause all + reactions

Forward Grouping Forward Grouping ProblemsProblems

Red Cell ProblemsRed Cell Problems

Affect the Affect the forward groupingforward grouping results results Missing or weak antigensMissing or weak antigens Extra antigensExtra antigens Mixed field reactionsMixed field reactions

Forward Grouping:Forward Grouping:Missing or Weak antigensMissing or Weak antigens

ABO SubgroupsABO Subgroups Disease (leukemia, Hodgkin’s disease)Disease (leukemia, Hodgkin’s disease)

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

00000044++

• Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)

Group O Group A

Subgroups of A (or B)Subgroups of A (or B)

Subgroups of A account for a small portion Subgroups of A account for a small portion of the A population (B subgroups rarer)of the A population (B subgroups rarer)

These subgroups have These subgroups have less antigen sitesless antigen sites on the surface of the red blood cellon the surface of the red blood cell

As a result, they show weakened (or As a result, they show weakened (or missing) reactions when tested with missing) reactions when tested with commercial antiseracommercial antisera

Resolution:Resolution: test with Anti-A test with Anti-A11, Anti-H, and , Anti-H, and anti-A,B for A subgroups anti-A,B for A subgroups

Forward Grouping:Forward Grouping:Extra AntigensExtra Antigens

Acquired B Acquired B B(A) phenotypeB(A) phenotype RouleauxRouleaux PolyagglutinationPolyagglutination Wharton’s JellyWharton’s Jelly

Anti-AAnti-AAnti-BAnti-BA1 A1 CellsCells

B B CellsCells

44++11++0044++

EXAMPLE

Acquired B PhenomenonAcquired B Phenomenon CauseCause: : there are two causes of acquired B phenomenon:there are two causes of acquired B phenomenon:

In vivoIn vivo, patients with bacterial infections and often cancer of the , patients with bacterial infections and often cancer of the

colon or rectum may developcolon or rectum may develop a a false B-like antigenfalse B-like antigen..

The mechanism: The bacterial produce a deacetylase (enzyme) The mechanism: The bacterial produce a deacetylase (enzyme) which chemically alters the terminal sugar of A antigens (N-which chemically alters the terminal sugar of A antigens (N-acetyl-D-galactosamine) into D-galactosamine. acetyl-D-galactosamine) into D-galactosamine.

Because the terminal sugar of the B antigen is galactose, anti-Because the terminal sugar of the B antigen is galactose, anti-B antisera will cross react with the B-like D-galactosamine B antisera will cross react with the B-like D-galactosamine antigen. Because of this, antigen. Because of this, in vivoin vivo, only group , only group AA people can people can develop an acquired B-like antigen. The condition is transient develop an acquired B-like antigen. The condition is transient and disappears when the infection is cured. and disappears when the infection is cured.

Acquired BAcquired B

Bacteria (Bacteria (E. coliE. coli) have a ) have a dedeacetylating acetylating enzyme that effects the A sugar….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)

Another mechanismAnother mechanism

In vitroIn vitro, blood specimens can get an acquired B-like , blood specimens can get an acquired B-like antigen if they are bacterially contaminated. This is antigen if they are bacterially contaminated. This is because the membranes of some bacteria (e.g., because the membranes of some bacteria (e.g., E. E. colicoli and and P. vulgarisP. vulgaris ) have determinants which are ) have determinants which are chemically similar to the B antigen. chemically similar to the B antigen.

In this case, anti-B antisera is actually reacting with In this case, anti-B antisera is actually reacting with the bacterial antigens which have attached to the red the bacterial antigens which have attached to the red cells. cells.

In vitroIn vitro, both group O and group A cells can acquire , both group O and group A cells can acquire the B-like antigen. Note: most examples of acquired the B-like antigen. Note: most examples of acquired B phenomenon detected in the blood bank happen B phenomenon detected in the blood bank happen in in vivovivo to group A people only. to group A people only.

ES4 Anti-B antiseraES4 Anti-B antisera

The use of monoclonal ABO typing antisera The use of monoclonal ABO typing antisera (specifically an anti-B clone designated "ES4") (specifically an anti-B clone designated "ES4") initially caused an increase in acquired B initially caused an increase in acquired B phenomenon. phenomenon.

because because

The ES4 monoclonals can detect even a small The ES4 monoclonals can detect even a small number of galactosamine molecules on red cells. number of galactosamine molecules on red cells. However, the reactions are particularly sensitive to However, the reactions are particularly sensitive to pH and can be reduced (not eliminated totally) if the pH and can be reduced (not eliminated totally) if the pH is lowered, something that the manufacturers pH is lowered, something that the manufacturers have done. have done.

Typical reaction patternTypical reaction pattern

The reactions with antiThe reactions with anti--B are weaker than B are weaker than expected expected ((ee..gg.., 1+ or 2+, 1+ or 2+). ). The patient's The patient's autocontrol is negative even though antiautocontrol is negative even though anti--B B is present is present ((patient is group Apatient is group A).).

The patient's own antiThe patient's own anti--B will not recognize B will not recognize and agglutinate the Band agglutinate the B--like antigen, but like antigen, but everyone else's antieveryone else's anti--B B ((including the including the typing seratyping sera) ) willwill. .

Resolution of acquired BResolution of acquired B Check the past records in case the patient is a known group Check the past records in case the patient is a known group

AA. . Check the diagnosis for bacterial infection (with or without Check the diagnosis for bacterial infection (with or without

Cancer of the colon or rectum).Cancer of the colon or rectum). Test the red cells with anti-B reagent acidified to pH 6.0 If using human polyclonal reagents, redo the ABO group If using human polyclonal reagents, redo the ABO group

using monoclonal anti-A and anti-B typing sera, which may using monoclonal anti-A and anti-B typing sera, which may resolve the problem. resolve the problem.

If using monoclonal reagents, redo the ABO group using If using monoclonal reagents, redo the ABO group using human polyclonal anti-A and anti-B typing sera.human polyclonal anti-A and anti-B typing sera.

Do autologous control it should give negative result. Do autologous control it should give negative result. Try secretor status studies (usually not necessary). If the Try secretor status studies (usually not necessary). If the

patient is group A and a secretor, he will secrete A and H patient is group A and a secretor, he will secrete A and H antigens only. antigens only.

Implication in blood transfusionImplication in blood transfusion

Group A people Group A people ((especially children with a small especially children with a small blood volumeblood volume) ) who have acquired B who have acquired B phenomenon should receive group A washed phenomenon should receive group A washed red cells red cells ((or group O washed red cellsor group O washed red cells). ).

The red cells should be washed to remove all The red cells should be washed to remove all traces of donor antitraces of donor anti--B which can react with the B which can react with the patient's Bpatient's B--like antigenslike antigens..

Acquired B PhenotypeAcquired B Phenotype

Limited mainly to Group Limited mainly to Group AA11 individuals with: individuals with: Lower GI tract diseaseLower GI tract disease Cancer of colon/rectumCancer of colon/rectum Intestinal obstructionIntestinal obstruction Gram negativeGram negative septicemia septicemia

(i.e. (i.e. E. coliE. coli))

Resolving Acquired BResolving Acquired B

Check patient diagnosis: Infection?Check patient diagnosis: Infection? Some manufacturers produce anti-B Some manufacturers produce anti-B

reagent that does not react with acquired Breagent that does not react with acquired B Test patients serum with their own RBCsTest patients serum with their own RBCs

The patients own anti-B will not react with the The patients own anti-B will not react with the acquired B antigen on their red cell acquired B antigen on their red cell ((autologousautologous testing) testing)

B(A) phenotypeB(A) phenotype

Similar to acquired BSimilar to acquired B Patient is Group B with an apparent extra Patient is Group B with an apparent extra

A antigenA antigen The B gene transfers small amounts of the The B gene transfers small amounts of the

A sugar to the H antigenA sugar to the H antigen Sometimes certain anti-A reagents will Sometimes certain anti-A reagents will

detect these trace amount of A antigendetect these trace amount of A antigen ResolutionResolution: test with another anti-A : test with another anti-A

reagent from another manufacturerreagent from another manufacturer

Other reasons for “extra” antigensOther reasons for “extra” antigens

PolyagglutinationPolyagglutination – agglutination of RBCs with – agglutination of RBCs with human antisera no matter what blood typehuman antisera no matter what blood type Due to bacterial infectionsDue to bacterial infections Expression of hidden Expression of hidden T antigensT antigens react with antisera react with antisera

RouleauxRouleaux – extra serum proteins – extra serum proteins Wharton’s JellyWharton’s Jelly – gelatinous substance derived – gelatinous substance derived

from connective tissue that is found in cord blood from connective tissue that is found in cord blood and may cause false agglutination (Remember: and may cause false agglutination (Remember: only forward typing is performed on cord blood)only forward typing is performed on cord blood) Wash red cells or request new sample from heel, etcWash red cells or request new sample from heel, etc

Forward Grouping:Forward Grouping: Mixed Field AgglutinationMixed Field Agglutination

Results from two different cell populationsResults from two different cell populations Agglutinates are seen with a background Agglutinates are seen with a background

of unagglutinated cellsof unagglutinated cells All groups transfused with Group O cellsAll groups transfused with Group O cells Bone marrow/stem cell recipientsBone marrow/stem cell recipients AA33 phenotype phenotype

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

0022++44++00

Mixed Field AgglutinationMixed Field Agglutination

Reverse Grouping Reverse Grouping ProblemsProblems

Reverse GroupingReverse Grouping

Affect the reverse grouping resultsAffect the reverse grouping results Missing or weak antibodiesMissing or weak antibodies Extra antibodiesExtra antibodies

Reverse Grouping:Reverse Grouping:Missing or Weak antibodiesMissing or Weak antibodies

NewbornsNewborns Do not form antibodies until laterDo not form antibodies until later

ElderlyElderly Weakened antibody activityWeakened antibody activity

Hypogammaglobulinemia Hypogammaglobulinemia Little or no antibody production (i.e. Little or no antibody production (i.e.

immunocompromised)immunocompromised) Often shows Often shows NONO agglutination on reverse agglutination on reverse

groupingsgroupings

Missing or Weak antibodiesMissing or Weak antibodiesExampleExampleAnti-AAnti-AAnti-BAnti-BA1 cellsA1 cellsB cellsB cellsTentative groupTentative group

#1#1++44------AA

#2#2--++44----B or ABB or AB

#3#3--------OO

#1: Patient is a newborn: Anti-A and anti-B are not present at birth and develop about 3-6 months of age. (Usually the reverse group is not done when grouping newborns.)

#2: Patient is very elderly: Anti-A and anti-B levels decrease in old age because levels of immunoglobulins decrease. Because the levels may only be decreased and not totally missing, further investigation can be done. (Note: It would be unusual for an elderly person to totally lack ABO antibodies in the absence of an immune disorder.)

#3: Patient has a- or hypogammaglobulinemia: Anti-A and anti-B will be weak or missing in patients with a gammaglobulinemia or hypogammaglobulinemia.

ResolutionResolution

Check the age of the patient Check the age of the patient Repeat the ABO group at 4°C [anti-A and anti-B Repeat the ABO group at 4°C [anti-A and anti-B

react best at 4°C]. react best at 4°C]. QC required: because all persons have a QC required: because all persons have a

harmless auto-anti-I reactive at 4°C, include an harmless auto-anti-I reactive at 4°C, include an autocontrolautocontrol. (Auto-anti-I may agglutinate the A1 . (Auto-anti-I may agglutinate the A1 cells, the B cells, and the patient's own cells at cells, the B cells, and the patient's own cells at 4°C.) 4°C.)

Check the diagnosisCheck the diagnosis. . If undiagnosed, have gammaglobulin levels If undiagnosed, have gammaglobulin levels

tested tested

Resolving Weak or Missing Resolving Weak or Missing antibodiesantibodies

Determine patients age, diagnosisDetermine patients age, diagnosis Incubate serum testing for 15 minutes (RT) Incubate serum testing for 15 minutes (RT)

to enhance antibody reactionsto enhance antibody reactions If negative, place serum testing at 4°C for 5 If negative, place serum testing at 4°C for 5

minutes with autologous control (a.k.a. minutes with autologous control (a.k.a. Autocontrol, AC)Autocontrol, AC)

This is called a “This is called a “mini-coldmini-cold” panel and ” panel and should enhance the reactivity of the should enhance the reactivity of the antibodiesantibodies

Reverse Grouping:Reverse Grouping:Extra AntibodiesExtra Antibodies

Cold antibodies (allo- or auto-)Cold antibodies (allo- or auto-) Cold antibodies may include anti-I, H, M, N, P, Cold antibodies may include anti-I, H, M, N, P,

LewisLewis RouleauxRouleaux Anti-AAnti-A11 in an A in an A22 or A or A22B individualB individual

Cold antibodiesCold antibodies

Sometimes a patient will develop cold-reacting Sometimes a patient will develop cold-reacting allo- or auto-antibodies that appear as “extra” allo- or auto-antibodies that appear as “extra” antibodies on reverse typingantibodies on reverse typing

AlloantibodiesAlloantibodies are made against foreign red cells are made against foreign red cells AutoantibodiesAutoantibodies are made against ones own red are made against ones own red

cells. cells. Cold Cold reacting antibodies cause reacting antibodies cause agglutination with red cells at room temperature agglutination with red cells at room temperature and below. The autocontrol will be positive.and below. The autocontrol will be positive. Resolution:Resolution: warming tube to 37° and washing red warming tube to 37° and washing red

cells can disperse agglutination; breaking the IgM cells can disperse agglutination; breaking the IgM bonds.bonds.

RouleauxRouleaux Can cause both extra antigens and extra Can cause both extra antigens and extra

antibodiesantibodies ““stack of coins” appearancestack of coins” appearance May falsely appear as agglutination due to the May falsely appear as agglutination due to the

increase of serum proteins (globulins)increase of serum proteins (globulins) Stronger at IS and weak reaction at 37°C and no Stronger at IS and weak reaction at 37°C and no

agglutination at AHG phaseagglutination at AHG phase Associated with:Associated with:

Multiple melomaMultiple meloma Waldenstrom’s macroglobulinemia (WM)Waldenstrom’s macroglobulinemia (WM) Hydroxyethyl starch (HES), dextran, etcHydroxyethyl starch (HES), dextran, etc

Resolving RouleauxResolving Rouleaux Remove proteins!Remove proteins! If the If the forward groupingforward grouping is affected, is affected, wash cellswash cells

to remove protein and repeat testto remove protein and repeat test If the If the reverse groupingreverse grouping is affected, perform is affected, perform

saline replacementsaline replacement technique (more common) technique (more common) Cells (reagent) and serum (patient) centrifuged to Cells (reagent) and serum (patient) centrifuged to

allow antigen and antibody to react (if present)allow antigen and antibody to react (if present) Serum is removed and replaced by an equal volume Serum is removed and replaced by an equal volume

of saline (saline disperses cells)*of saline (saline disperses cells)* Tube is mixed, centrifuged, and reexamined for Tube is mixed, centrifuged, and reexamined for

agglutination (macro and micro)agglutination (macro and micro)

*some procedures suggest only 2 drops of saline.*some procedures suggest only 2 drops of saline.

ExampleExampleAnti-AAnti-AAnti-BAnti-BA1 cellsA1 cellsB cellsB cellsTentative groupTentative group

#1#1++44--++11++44AA

#2#2++44++44++22--ABAB

#3#3--++44++44++11BB

Anti-A1 in A2 or A2B people: examples #1 and #2 illustrate the presence of anti-A1. The autocontrol (not shown) would be negative.

Irregular IgM Alloantibodies: All three examples could represent the presence of irregular IgM alloantibodies such as anti-M, -N, -Lea, -Leb, or -P1. The A1 cells (or B cells) may be agglutinating because they are positive for the corresponding antigen. The autocontrol (not shown) would be negative.

Extra AntibodiesExtra Antibodies

Rouleaux: providing both cells in the reverse grouping show agglutination (examples #1 and #3), the discrepancy could be due to Rouleaux. The autocontrol (not shown) would be positive.Causes: Rouleaux is a type of false agglutination caused by an increase in serum globulins. This can occur in diseases such as multiple myeloma or macroglobulinemia or can be caused by infusion of macromolecular substances such as dextran or polyvinyl pyrollidone (PVP), which are used as blood volume expanders.Autoanti-I: Many people have a harmless autoanti-I that is IgM and reacts best at 4°C. The harmless autoanti-I of most people will not react above 10°-15°C, but some people have an autoanti-I that can react at RT and cause unexpected agglutination in both cells of the reverse serum group (examples #1 and #3).

ExampleExampleAnti-AAnti-AAnti-BAnti-BA1 cellsA1 cellsB cellsB cellsTentative groupTentative group

#1#1++44--++11++44AA

#2#2++44++44++22--ABAB

#3#3--++44++44++11BB

Anti-AAnti-A11

Sometimes ASometimes A22 (or A (or A22B) individuals will B) individuals will

develop an anti-Adevelop an anti-A11 antibody antibody

AA22 (or A (or A22B) individuals have less antigen B) individuals have less antigen

sites than Asites than A11 individuals individuals

The antibody is a naturally occurring IgMThe antibody is a naturally occurring IgM Reacts with AReacts with A11 Cells, but not A Cells, but not A22 Cells Cells

Anti-A1 from patient

+ A1 cells

+ A2 cells

AGGLUTINATION

NO AGGLUTINATION

Resolving anti-AResolving anti-A11 discrepancy discrepancy

2 steps:2 steps: Typing patient RBCs with Anti-ATyping patient RBCs with Anti-A11 lectin lectin

Repeat reverse grouping with ARepeat reverse grouping with A22 Cells instead Cells instead

of Aof A11 Cells Cells Both results should yield NO agglutinationBoth results should yield NO agglutination

Anti-AAnti-AAnti-BAnti-BA1 A1 CellsCells

B B CellsCells

44++0022++44++

Resolution of discrepancies caused Resolution of discrepancies caused by antiby anti--A1A1

First stepFirst step::

We must determine if the person is group A1 or group We must determine if the person is group A1 or group A2. (If group A1, the discrepancy with the A1 cells is A2. (If group A1, the discrepancy with the A1 cells is NOTNOT due to anti-A1). due to anti-A1).

To do this, we antigen type the person's red cells with the To do this, we antigen type the person's red cells with the anti-A1 lectin which is anti-A1 lectin which is Dolichos biflorusDolichos biflorus . If the red cells . If the red cells agglutinate, the person is group A1. If the red cells do not agglutinate, the person is group A1. If the red cells do not agglutinate, the person is not group A1, and probably is agglutinate, the person is not group A1, and probably is group A2 assuming the red cells reacted strongly (3+ or group A2 assuming the red cells reacted strongly (3+ or 4+ with anti-A).4+ with anti-A).

Resolution of discrepancies caused by Resolution of discrepancies caused by antianti--A1A1

Second stepSecond step::

If the person appears to be group A2, we must If the person appears to be group A2, we must prove that the extra antibody is anti-A1, and not prove that the extra antibody is anti-A1, and not some other IgM irregular antibody. some other IgM irregular antibody.

To do this we test the person's serum against a panel To do this we test the person's serum against a panel of 3 A1 cells and 3 A2 cells. If anti-A1 is present, of 3 A1 cells and 3 A2 cells. If anti-A1 is present, only the A1 cells should agglutinate.only the A1 cells should agglutinate.

Resolution of discrepancies caused by Resolution of discrepancies caused by antianti--A1A1

NOTE:NOTE: 3 A1 and 3 A2 cells are required in order to ensure that 3 A1 and 3 A2 cells are required in order to ensure that

the antibody reacting is anti-A1 and not some other the antibody reacting is anti-A1 and not some other antibody.antibody.

With 3 cells of each group we can achieve a statistical With 3 cells of each group we can achieve a statistical probability of 95% that the right antibody has been probability of 95% that the right antibody has been identified. identified.

For example, if only one A1 cell and one A2 cell were For example, if only one A1 cell and one A2 cell were tested, by chance, another antibody like anti-M or anti-tested, by chance, another antibody like anti-M or anti-P1 could react with the A1 cells (if they were M+ or P1+), P1 could react with the A1 cells (if they were M+ or P1+), but not the A2 cells (if they were M- or P1-).but not the A2 cells (if they were M- or P1-).

OthersOthers … …

The Bombay phenotype (extremely RARE) results when The Bombay phenotype (extremely RARE) results when hhhh is inherited is inherited

These individuals do not have any antigens and naturally These individuals do not have any antigens and naturally produce, anti-A, anti-B, anti-A,B, and anti-Hproduce, anti-A, anti-B, anti-A,B, and anti-H

Basically, NO forward reaction and POSITIVE reverseBasically, NO forward reaction and POSITIVE reverse

ResolutionResolution: test with anti-H lectin (Bombay’s don’t have : test with anti-H lectin (Bombay’s don’t have H and will not react)H and will not react)

Finding the problemFinding the problem……

Forward type tests for the Forward type tests for the antigen (red cell)antigen (red cell)

Reverse type tests for the Reverse type tests for the antibody (serum)antibody (serum)

Identify what the patient Identify what the patient types as in both Forward types as in both Forward & Reverse Groupings& Reverse Groupings

Is there a weaker than Is there a weaker than usual reaction?usual reaction?

Is it a missing, weak, or Is it a missing, weak, or extra reaction??extra reaction??

Resolving ABO DiscrepanciesResolving ABO Discrepancies

Get the patient’s history:Get the patient’s history: ageage Recent transplantRecent transplant Recent transfusionRecent transfusion Patient medicationsPatient medications The list goes on….The list goes on….

Let’s practice !Let’s practice !

Example 1Example 1

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

33++000011++

Problem:

Causes:

Resolution:

Example 2Example 2

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

33++11++0044++

Problem:

Causes:

Resolution:

Example 3Example 3

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

22++00++11++44++

Problem:

Causes:

Resolution:

Example 4Example 4

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

00000033++

Problem:

Causes:

Resolution:

Example 4Example 4

Anti-A,BAnti-A,B

Patient RBCPatient RBC11++

• Probably a subgroup of A (Ax)

• if the result was negative (0), adsorption or elution studies with anti-A could be performed (these will help determine what A antigens)

Example 5Example 5

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

0022++mfmf33++00

Problem:

Causes:

Resolution:

Example 6Example 6

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

44++44++0011++

Problem:

Causes:

Resolution:

Example 7Example 7

Anti-AAnti-AAnti-BAnti-BA1 CellsA1 CellsB CellsB Cells

00000000

Problem:

Causes:

Resolution:

Example 8Example 8

Screening Screening Cells Cells (I and II)(I and II)

Autocontrol Autocontrol (AC)(AC)

ConclusionConclusion

Patient Patient Serum 1Serum 1

PosPosNegNegCold Cold alloantibodyalloantibody

Patient Patient Serum 2Serum 2

PosPosPosPosCold Cold autoantibodyautoantibody

• if alloantibody – antibody ID techniques

• if autoantibody – special procedures (minicold panel, prewarming techniques

ReferencesReferences Rudmann, S. V. (2005). Rudmann, S. V. (2005). Textbook of Blood Banking and Textbook of Blood Banking and

Transfusion Medicine (2Transfusion Medicine (2ndnd Ed.). Ed.). Philadelphia, PA: Elsevier Philadelphia, PA: Elsevier Saunders.Saunders.

Blaney, K. D. and Howard, P. R. (2000). Blaney, K. D. and Howard, P. R. (2000). Basic & Applied Concepts Basic & Applied Concepts of Immunohematology. of Immunohematology. St. Louis, MO: Mosby, Inc.St. Louis, MO: Mosby, Inc.