Interesting Case Studies from The Mayo Clinic Reference

Laboratory

Georgette Benidt, MT(ASCP)

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Case 1

• 81 year old with B-cell lymphoproliferative disorder

• Clinician ordered the Donath Landsteiner Test

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DONATH-LANDSTEINER (DL)

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Case 1 – objectives

• Significance of the test

• Incidence of positive tests

• Testing challenges

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DL Significance

• Paroxysymal Cold Hemoglobinuria is an ideopathic disorder occurring in <1% of hemolytic anemias

• IgG biphasic autoantibody (usually anti-P) • Fixes complement at 4 C• Activates complement at 37 C

• Patient needs to avoid cold exposures (MN winters, air conditioners)

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Donath-LandsteinerTesting Challenges

• Need to maintain the specimens and controls at 37°C.

• Length of time from start to finish is minimum of 2 ½ hours

• Need fresh donor samples for complement and RBC’s

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Case 2

• 68 Y.O. male

• O Rh negative

• Myelodyplasia Syndrome

• Transfusion Dependent

• Previous Anti-K, Anti-E, Warm Autoantibody

• Presents now with the following results:

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  D C Cw E c e f v K k Fya Fyb Jka Jkb M N S s IS PEG AHGCC

R1R1 + + + 0 0 + 0 0 0 + + 0 0 + + 0 + + 0 2+  

R2R2 + 0 0 + + 0 0 0 0 + 0 + 0 + + 0 + + 0 4+  

rr 0 0 0 0 + + + 0 + + 0 + 0 + + + + + 0 2+  

rr 0 0 0 0 + + + + 0 + 0 0 + 0 + + + + 0 1+  

rr 0 0 0 0 + + + 0 0 + + + + + + 0 + 0 0 w+  

rr 0 0 0 0 + + + 0 0 + + 0 0 + + 0 + + 0 W+

rr 0 0 0 0 + + + 0 0 + 0 + + + + 0 + 0 0 w+  

rr 0 0 0 0 + + + 0 0 + 0 + 0 + + 0 + 0 0 w+  

Ror + 0 0 0 + + + 0 0 + 0 0 + 0 0 + 0 + 0 2+  

Auto w+  

Case 2: Initial Panel

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Case 2

• Do you see a pattern?

• Is there varying reactivity?

• We know that the patient has a warm autoantibody, what next?

• At Mayo, we absorb onto 3 different cells: R1R1, R2R2, and rr

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  D C Cw E c e f v K k Fya Fyb Jka Jkb M N S sIS PEG AHG CC

rr 0 0 0 0 + + + 0 + + 0 + 0 + + + + +   2+  

rr 0 0 0 0 + + + + 0 + 0 0 + 0 + + + +   2+

R1R1 + + 0 0   +      0 + + + + + + + 0 +   0 2+

r"r 0 0 0 + + + + 0 0 + 0 + + + 0 + 0 +   4+

rr 0 0 0 0 + + + 0 0 + 0 + + + + 0 + 0   0 2+

rr 0 0 0 0 + + + 0 0 + 0 0 + 0 0 + 0 +   0 2+

rr 0 0 0 0 + + + 0 0 + + 0 + + 0 + 0 +   0 2+

                                           

                                           

R1R1 + + 0 0 0 +     0    0 0 0 + 0 0 0 0  Absorbing Cell   

Case 2: R1R1 Absorbate

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  D C Cw E c e f v K k Fya Fyb Jka Jkb M N S sIS PEG AHG CC

r'r 0 + 0 0 + + + 0 0 + 0 + + + + + 0 +   1+  

rr 0 0 0 0 + + + 0 + + 0 + 0 + + + + +   2+  

rr 0 0 0 0 + + + + 0 + 0 0 + 0 + + + +   2+

rr 0 0   0         0 + + + + + + + 0 +   0 2+ 

rr 0 0 0 0 + + + 0 0 + 0 + 0 + + 0 + 0   0 2+

R2R2 + 0 0 + + 0 + 0 0 + 0 + + + + 0 + 0   0 2+

rr 0 0 0 0 + + + 0 0 + + 0 + + 0 + 0 +   0 2+

                                           

R2R2 + 0 0 + + 0     0   0 0 + 0 0 0 0 0  Absorbing Cell   

Case 2: R2R2 Absorbate

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  D C Cw E c e f v K k Fya Fyb Jka Jkb M N S sIS PEG AHG CC

r'r 0 + 0 0 + + + 0 0 + 0 + + + + + 0 +   1+  

Ror + 0 0 0 + + + 0 0 + 0 0 + 0 + + + 0   1+  

r"r 0 0 0 + + + + 0 0 + 0 + + + 0 + 0 +   4+

rr 0 0 0 0 + + + + 0 + 0 0 + 0 + + + +   2+

rr 0 0 0 0 + + + 0 0 + + + + + + 0 + 0   3+  

rr 0 0   0         + + + + + + + + 0 +   0 2+

rr 0 0 0 0 + + + 0 0 + 0 0 + 0 0 + 0 +   0 2+

rr 0 0 0 0 + + + 0 0 + + 0 0 + + 0 + +   0 2+

R1R1 + + 0 0 0 + 0 0 0 + 0 + + + + 0 + 0   1+

rr  0 0 0 0 + + + + 0 + 0 + 0 + + + + +   2+  

rr 0 0 0 0 + +     +   0 0 + 0 0 0 0 0  Absorbing Cell   

Case 2: rr Absorbate

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Case 2

• What antibodies were identified:• Anti-G, Anti-C, Anti-E, Anti-K, Anti-

Mur, Anti-V, and Warm Auto

• Why do we care about underlying antibodies:• Possible DHTR• Difficulty of finding antigen

negative blood

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Case 2

• What is significant about Anti-G?• Belongs to the Rh family• G antigen is present on all D+ and

or C+ RBCs• IgG and does not fix complement• Stimulus from the transfusion of

C+ RBCs following trauma

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Case 2

• More on anti-G• For Transfusion:• Provide D-, C- crossmatch

compatible RBCs• For OB Patients• Adsorption/elution studies may be

necessary to determine if anti-D is also present• RhIG administration??

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Case 2

• Antigen Incidence• Blacks• 92%

• Caucasians• 84%

• Asians• 100%

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Case 2: Conclusion

• Anti-G has been shown to be present years after the exposure of D+ or C+ RBC’s

• Why did we care in this case?• The patient had a previous Anti-C• The patient has only received Rh

negative blood that we know of• Do we have a rr, G+ donor?

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Case 3

• 20 YO female

• A Rh negative

• 38 week gestation in 2nd pregnancy

• No other information available

• Initial panel results are:

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  D C c E e K k Fya Fyb Jka Jkb M N S s ISPEG AHG CC

R1R1 + + 0 0 + 0 + 0 + 0 + 0 + 0 + 0 W+  

R1R1 + + 0 0 + + + + + + 0 0 + 0 + 0 1+  

R1R1 + + 0 0 + 0 + + 0 0 + + 0 + 0 0 1+  

R2R2 + 0 + + 0 0 + + 0 + + + 0 0 + 0 1+  

R2R2 + 0 + + 0 + + 0 + 0 + + 0 + + 0 1+  

R2R2 + 0 + + 0 0 + 0 + + 0 0 + 0 + 0 1+  

R2r + 0 + + + + 0 + + + + 0 + 0 + 0 1+  

r'r 0 + + 0 + 0 + 0 + 0 + + + + + 0 1+  

r"r 0 0 + + + 0 + + W + + + 0 + + 0 W+  

rr 0 0 + 0 + + + 0 + + + + 0 + 0 0 W+  

rr 0 0 + 0 + 0 + 0 0 + 0 0 + 0 + 0 1+  

rr 0 0 + 0 + 0 + + 0 + 0 0 + + + 0 1+  

rr 0 0 + 0 + 0 + 0 0 + 0 0 + 0 + 0 W+  

AUTO                               0 0 +

Case 3: Initial Panel

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Case 3

• Do you see a pattern?

• What should be done next?• Why?

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Case 3

• Possible antibody to high incidence antigen• Perform phenotype• Test serum against phenotypically

similar cell• If negative, look for multiple

common antibodies• If positive, consider high

incidence

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Case 3

• Our results• Phenotypically similar cell reacted

1+ with patient serum• Antibody was titered to determine

if it exhibited HTLA characteristics• Antibody did not have a high

titer

• Now what?

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Case 3

• DTT and papain treated cells were tested

• The antibody did not react with the treated cells. Antigen is assumed to be sensitive to treatments

• A list of high incidence antigens was compiled

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Case 3

• Based on the sensitivity of papain and DTT, a Yt(a-) cell was thawed and tested

• This cell was negative at AHG, and 2 more Yt(a-) cells were thawed and tested

• We now have our 3 negative cells to confirm the presence of an Anti-Yta

• The patient’s antigen status was Yta-

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Case 3

• In most populations, Yta has an antigen incidence of >99.8%

• Yta can bind complement

• Yta has been shown to cause anywhere from no transfusion reactions to moderate/delayed reactions

• Yta has not been shown to cause HDN

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Case 4

• 26 Y.O. female

• A Rh negative

• Presented during pregnancy

• No known antibody history

• Patient presents now with the following results:

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  D C c E e K k Fya Fyb Jka Jkb M N S s ISPEG AHG CC

RZR1 + + 0 + + 0 + + 0 + + + 0 + + 0 1+  

R2R2 + 0 + + 0 0 + 0 + 0 + 0 + + + 0 1+  

r'r 0 + + 0 + 0 + 0 + + 0 + 0 0 + 0 1+  

r"r 0 0 + + + 0 + + 0 + + + 0 0 + 0 1+  

rr 0 0 + 0 + 0 + + 0 + 0 + + 0 + 0 1+  

RZR1 + + 0 + + 0 + 0 + + 0 + 0 0 + 0 1+  

R1R1 + + 0 0 + + + + + + 0 0 + 0 + 0 1+  

rr 0 0 + 0 + 0 + 0 0 + 0 + + + 0 0 1+  

rr 0 0 + 0 + + + + 0 0 + + 0 + 0 0 1+  

AUTO                               0 0 +

Case 4: Initial Panel

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Case 4

• Possible Suspects• Multiple allo-antibodies• High-Titer-Low-Avidity• High Incidence

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Case 4

• Phenotype was performed

• Phenotypically similar cell was tested against serum and reacted 1+ AHG.• Ruled out the common multiple

alloantibodies.

• What would you do next?

• HTLA titers were done x2 with possible HTLA identified

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Case 4

• I was not convinced of the HTLA

• HTLA negative cells (Ch,Rg,Kn,Mc) were run with similar results

• We papain and DTT treated the same panel cell to see if we could rule out antigens• Papain cell still reacted• DTT cell did not react, and upon

repeating, reacted at micro positive.

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Case 4

• Based on the Papain and DTT results, high incidence negative cells were tested

• Lu(a-b-); Sc:-1,2; K null; Yt(a-); Ge:-2,-3; Lu:-8; Lu:-6 cells were all W+

• At this point, we decided to send it to New York Blood Centers to see if they could identify the antibody

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Case 4

• NYBC identified an Anti-Jra

• We picked ourselves up, dusted off and confirmed these results with our own reagents.

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Case 4

• A little about anti-Jra (Junior)

• Anti-Jra can bind complement

• Can cause transfusion reactions but no cases of HDN have been identified

• This antigen has an incidence of >99% in most of the population

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Case 4

• What went wrong?• We forgot that antibodies do not read

textbooks!• Jra antigen should be resistant to

DTT• Anti-Jra antibodies shouldn’t look

like HTLAs• Our patient wasn’t Japanese

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Case 4

• Outcome of patient:• Patient was urged to donate units

while she was still pregnant in case she needed them• Baby was antigen positive, but

there were no complications• Patient remains an allogeneic

blood donor

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Conclusions

• HTLA’s and High Incidence antibodies can mimic each other

• High Incidence antibodies can titer out to HTLA levels

• It is important to differentiate between HTLA and High Incidence antibodies

• Certain patient populations will continue to form antibodies

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Conclusions

• It is helpful to perform phenotypes, especially on patients you expect to have multiple transfusions

• Tests that seem like a waste of time can sometimes surprise you!

• Remember to take a picture of a positive DL…you may never see another one.

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References

• The Blood Group Antigen Facts Book, M.E. Reid, C.L. Francis

• Applied Blood Group Serology, P.D. Issitt, D.J. Anstee

• Technical Manual, 15th edition

• Mayo Clinic Transfusion Medicine SOP’s

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Thanks

• Craig Tauscher for helping me prepare this presentation

• Sheila Muenster for reviewing my presentation

• The MT students who had to sit through my rough draft

• Bob Stowers for having the DL

• The rest of my coworkers for their help

Any Questions??