Deborah Baudler MS, MT(ASCP) SBB Assistant Professor University of Illinois-Springfield Patchwork Conference April 15, 2014 Investigative Techniques in.

Deborah Baudler MS, MT(ASCP) SBBAssistant Professor

University of Illinois-SpringfieldPatchwork Conference

April 15, 2014

Investigative Techniquesin Blood Banking

University of Illinois-Springfield

UIS CLS Students

• Identify common problems that occur in day to day blood banking

• Discuss various techniques for problem-solving

• Apply new knowledge to case studies for resolution

Objectives

“The science of deduction and analysis is one which can only be acquired by long and patient study...”

Sherlock Holmes

• ABO Discrepancies

• Weak Positive Antibody Screen……no antibody identified

• Miscellaneous Reactivity showing up on the antibody panel

• Incompatible Crossmatch when antibody screen is negative

Common Problems That Can Occur

• Cry a little• Start over, hoping the problem will just go

away• Shake the tubes harder• Pretend the weak reactions don’t exist• Call your blood bank supervisor at 2 am to

see if she/he is reading a good book• Leave it for the next shift to resolve!

Common Solutions Available

ABO Discrepancy

• A discrepancy occurs when the red cell testing does NOT match the serum test results

• In other words, the forward type does NOT match the reverse.• What is the discrepancy here?

Anti-A Anti-B Anti-D A1 cells B cells

2+ 4+ 4+ 1+ 0

• Recall: the production of ABO antigens is controlled by the genes we inherit

• ABO forward and reverse reactions are typically very strong: 3+ to 4+.

• Where do we start?

What we do know….

Anti-A Anti-B Anti-D A1 cells B cells

2+ 4+ 4+ 1+ 0

• Most of the time, the problem is technical• Failure to add patient plasma• Reversed the A1 and B cells in the rack• Reagent contamination• Incubation time too short• Clot in specimen• Interpretations not accurately recorded

Discuss the Possibilities

Forward vs Reverse

Courtesy of School of Health Related Professions University of Mississippi Medical Center

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

Rouleaux

• If you have extra reactivity:• Recent Bone Marrow/ Stem Cell Transplant: check

medical history• Excess protein coating red cells or Rouleaux: Wash red

cells and retest• Strong cold agglutinin coating cells: Treat cells with

0.01 M DTT• Antibody coated red cells causing autoagglutination:

can be seen in HDFN. Perform DAT and Incubate cells and wash several times with 37°C saline

Reasons for Red Cell Discrepancies

• Acquired B antigen: occurs in Group A individuals with gram neg sepsis. True group A cells will not agglutinate with patient’s own Anti-B in plasma

Reasons for Red Cell Discrepancies

Enzyme cleaves off acetyl group

D-Galactose

Acetyl group

• If you have missing or weak reactivity:• Subgroup of A: test cells with A1 Lectin, Anti-A,B and Anti-H• Massive red cell transfusion: check transfusion history• Cancer or Chemotherapy: require longer incubation period

Reasons for Red Cell Discrepancies

• If you have extra reactivity:• Rouleaux: Check for “stack of coins”

and perform Saline Replacement• Cold or RT alloantbody: Antibody ID

and repeat reverse cells with antigen negative cells

• Cold or RT autoantbody: Antibody ID and pre-warm plasma and reverse cells in separate tubes, combine and read

Reasons for Plasma Discrepancies

• If you have extra reactivity:• Issoagglutinins: Passive ABO

antibodies: check recent transfusion history

• Subgroup of A: A1 Lectin and antibody ID with A1 cells

Reasons for Plasma Discrepancies

• If you have missing or weak reactivity:• Check age of patient:

• Newborn: no antibody production until 4 mos• Elderly: extend incubation or increase serum/cell ratio

• Hypogammaglobulinemia: extend incubation or increase serum/cell ratio

• Chemotherapy or recent Bone Marrow Transplant: check medical history

Reasons for Plasma Discrepancies

• We have extra reactivity on the plasma side• Most frequent cause for ABO discrepancy

Back to Our Case

Anti-A Anti-B Anti-D A1 cells B cells

2+ 4+ 4+ 1+ 0

In This Case

Anti-A Anti-B Anti-D A1 cells B cells

2+ 4+ 4+ 1+ 0

1. Patient has never been transfused and is not pregnant

2. Patient is here for elective surgery

3. Antibody Screen is negative, Auto control = 0

4. Checked under the scope, no Rouleaux

5. What’s left to do?

1. Recall: 20% of group AB individuals are actually A2B. 25% of A2B will make an alloantibody called Anti-A1

2. Test patient’s red cells with A1Lectin. A2B will not agglutinate with A1Lectin

3. Test patient’s plasma with several lots of A1 cells to confirm that the antibody is Anti-A1

Solution

Agglutination

No Agglutination

A1 cells

A2 cells

• Patient is an A2B Pos with Anti-A1

• Solution: Use A2 reverse cells to eliminate extra reactivity and resolve discrepancy

In This Case

Anti-A Anti-B Anti-D A2 cells B cells

2+ 4+ 4+ 0 0

• 2. Weak Positive Screen: Negative Antibody ID

• Get the Patient’s Medical History• Possible Solutions:

• Repeat antibody screen and ID by a second method• Check expiration dates of reagents• Increase serum/cell ratio• Increase incubation time• Contact the manufacturer

• How should this be reported?

Weak Antibody Activity

• 3. Positive Screen: No specific antibody identified• All alloantibodies have been ruled out!

Miscellaneous Antibody Activity

D C E c e K Fya Fyb

+ + 0 + + 0 + 0

+ 0 0 + + 0 0 +

+ + 0 0 + + + +

0 + + + + 0 + +

0 + 0 0 + 0 + 0

0 0 + + 0 + 0 0

0 0 0 + + 0 + +

Jka Jkb M N S s Lua

0 + + + + + 0

+ 0 + 0 + 0 0

0 + 0 + 0 + +

+ 0 0 + 0 + 0

+ 0 0 + 0 + 0

0 + + 0 + 0 0

+ + + + + + 0

Lub IS AHG cc

+ 0 + nt

+ 0 0 √

+ 0 0 √

+ 0 + nt

+ 0 0 √

+ 0 0 √

+ 0 + nt

What Should You Do?

• 3. Positive Screen: No specific antibody identified

• Possible Solutions• Check lot number of antigrams!• Check expiration dates of reagents • Repeat antibody screen and ID by a second method • Increase serum/cell ratio• Increase incubation time

Miscellaneous Antibody Activity

• Highlight positive reactions• Check for Dosage

Miscellaneous Antibody Activity

D C E c e K Fya Fyb

+ + 0 + + 0 + 0

+ 0 0 + + 0 0 +

+ + 0 0 + + + +

0 + + + + 0 + +

0 + 0 0 + 0 + 0

0 0 + + 0 + 0 0

0 0 0 + + 0 + +

Jka Jkb M N S s Lua

0 + + + + + 0

+ 0 + 0 + 0 0

0 + 0 + 0 + +

+ 0 0 + 0 + 0

+ 0 0 + 0 + 0

0 + + 0 + 0 0

+ + + + + + 0

Lub IS AHG cc

+ 0 + nt

+ 0 0 √

+ 0 0 √

+ 0 + nt

+ 0 0 √

+ 0 0 √

+ 0 + nt

• Additional Suggestions: • Get the Patient’s Medical History• Enzyme panel• Check Direct Coombs• Perform an Eluate

Miscellaneous Antibody Activity

• For a DAT to become positive: > 200 molecules of IgG on red cell

• Purpose of an eluate:• Removes an antibody that’s coating the red cell• Concentrates antibody• Allows identification of newly forming or weak antibodies• Can be positive even when DAT is negative

Benefits of an Eluate

Miscellaneous Antibody Activity

• What antibody is detected?

D C E c e K Fya Fyb

+ + 0 + + 0 + 0

+ 0 0 + + 0 0 +

+ + 0 0 + + + +

0 + + + + 0 + +

0 + 0 0 + 0 + 0

0 0 + + 0 + 0 0

0 0 0 + + 0 + +

Jka Jkb M N S s Lua

0 + + + + + 0

+ 0 + 0 + 0 0

0 + 0 + 0 + +

+ + + + + + 0

+ 0 0 + 0 + 0

0 + + 0 + 0 0

+ + + + + + 0

Lub AHG ELU cc

+ + + nt

+ 0 0 √

+ 0 + nt

+ + + nt

+ 0 + nt

+ 0 0 √

+ + + nt

• Possibilities:• Perform clerical check on specimen• Check agglutination under scope if <2+• Specimen at room temp or out of

refrigerator• Age of specimen: protein precipitation

• If Reactivity is 3-4+• Repeat patient’s blood type • Strong Cold Agglutinin

Incompatible Crossmatch When Antibody Screen is Negative

• Other Possibilities:• Patient has an antibody to a Low

Incidence antigen on unit• Unit has positive DAT

• Most likely• Return unit to blood center

• Solution: Try another unit

Incompatible Crossmatch When Antibody Screen is Negative

Let’s do some Investigating

Case 1

• 71 yr. old woman comes through the ER on a Friday night with a 6 g Hb. While her antibody screen is incubating, you get the following blood type:

Anti-A Anti-B Anti-D A1 cells B cells

0 4+ 0 4+ 2+

• ABO discrepancy present• Most probably blood type?• What should we do next?

Thoughts?

Anti-A Anti-B Anti-D A1 cells B cells

0 4+ 0 4+ 2+

Oh No!

• Next thing you know, her antibody screen comes up positive, YIKES!

D C c E e K Fya Fyb Jka Jkb M N S s IS AHG cc

1 + 0 + 0 + 0 + + + 0 0 + + 0 0 0 √

2 + 0 + + 0 + 0 + 0 + + + 0 + 0 0 √

3 0 + + 0 + 0 + 0 + + + 0 + + + + nt

Let’s Do the Cross-out Technique

Which Antibodies Could Possibly be Present?

D C c E e K Fya

Fyb Jka

Jkb

M N S s IS AHG cc

1 + 0 + 0 + 0 + + + 0 0 + + 0 0 0 √

2 + 0 + + 0 + 0 + 0 + + + 0 + 0 0 √

3 0 + + 0 + 0 + 0 + + + 0 + + + + nt

Which Antibody is Present?

Antibody ID Results

D C E c e K Fya Fyb

+ + 0 0 + 0 + +

+ 0 0 + + 0 0 0

+ + 0 0 + + + 0

+ 0 + + 0 0 0 +

0 + 0 0 + + + +

0 0 + + 0 0 0 +

0 0 0 + + 0 + 0

Jka Jkb M N S s Lua

0 + + 0 + 0 0

+ 0 + 0 + + 0

0 + 0 + 0 + +

0 + + + + 0 0

+ 0 0 + 0 + 0

0 + + 0 + + 0

+ 0 + + 0 + 0

Lub IS AHG cc

+ + + nt

+ + + nt

+ 0 0 √

+ 0 0 √

+ 0 0 √

+ + + nt

+ 0 0 √

•Antibody identified as Anti-M

•Anti-M can possess both IgM and IgG components

•Phenotype patient for M if not recently transfused

•Test B Neg, M Neg cells with patient plasma

Are We Done?

Anti-A Anti-B Anti-D A1 cells B cells

0 4+ 0 4+ 0

• 62 yr. old man comes through the ER on a Saturday night with abdominal pain. He is rushed to surgery for a possible bowel obstruction

• While his antibody screen is incubating, you get the following blood type:

Case 2

Anti-A Anti-B Anti-D A1 cells B cells

3+ 0 4+ 0 4+

• Patient’s medical history indicates he had cardiac by-pass surgery 4 weeks ago and received 3 units of prbcs

• Perform cross-out technique• Which antibodies can not be ruled out?

The Antibody Screen Results

D C c E e K Fya Fyb Jka Jkb M N S s IS AHG cc

1 + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + nt

2 + 0 + + 0 + 0 + 0 + + 0 0 + 0 0 √

3 0 + 0 0 + 0 + + + + + + + + 0 + √

The Antibody Screen Results

• Anti-C, e, Fya, Jka, N and Anti-S are not ruled out

D C c E e K Fya Fyb Jka Jkb M N S s IS AHG cc

1 + 0 + 0 + 0 + 0 + 0 0 + + 0 0 + nt

2 + 0 + + 0 + 0 + 0 + + 0 0 + 0 0 √

3 0 + 0 0 + 0 + + + + + + + + 0 + √

Conclusion? What is the next step?

Antibody ID Results

D C E c e K Fya Fyb

+ + 0 0 + 0 + +

+ 0 0 + + 0 0 0

+ + 0 0 + + + 0

+ 0 + + 0 0 0 +

0 0 + + + + + +

0 0 + + 0 0 0 +

0 0 0 + + 0 + 0

Jka Jkb M N S s Lua

0 + + 0 0 + 0

+ 0 + 0 + 0 0

+ + 0 + 0 + +

+ 0 + + + + 0

+ + 0 + 0 + 0

+ 0 + 0 + + 0

+ + 0 + 0 + 0

Lub IS AHG cc

+ 0 + nt

+ 0 0 √

+ 0 + nt

+ 0 + nt

+ 0 0 √

+ 0 + nt

+ 0 0 √0 + + + 0 0 0 +

0 0 0 + + 0 + 0

0 + + 0 + + 0

0 + 0 + 0 + 0

+ 0 + nt

+ 0 0 √

Repeated panel with PeG. Who is the culprit?

Antibody ID Results

D C E c e K Fya Fyb

+ + 0 0 + 0 + +

+ 0 0 + + 0 0 0

+ + 0 0 + + + 0

+ 0 + + 0 0 0 +

0 0 + + + + + +

0 0 + + 0 0 0 +

0 0 0 + + 0 + 0

Jka Jkb M N S s Lua

0 + + 0 0 + 0

+ 0 + 0 + 0 0

+ + 0 + 0 + +

+ 0 + + + + 0

+ + 0 + 0 + 0

+ 0 + 0 + + 0

+ + 0 + 0 + 0

Lub IS AHG cc

+ 0 + nt

+ 0 + √

+ 0 + nt

+ 0 + nt

+ 0 + √

+ 0 + nt

+ 0 + √

0 + + + 0 0 0 +

0 0 0 + + 0 + 0

0 + + 0 + + 0

0 + 0 + 0 + 0

+ 0 + nt+ 0 0 √

• 27 yr. old man is medevac to your facility from a small community hospital. The patient has been in a motor vehicle accident and is bleeding internally. He is being prepped for the OR.

Case 3

• So you perform a STAT Type and Screen• While the antibody screen is incubating, you record the following

results for the blood type:• Any problems?

Case 3

Anti-A Anti-B Anti-D A1 cells B cells

2+mf 2+mf 2+mf 4+ 4+

• The patient’s antibody screen results are Negative!• Now what?

The Antibody Screen Results

D C c E e K Fya Fyb Jka Jkb M N S s IS AHG cc

1 + 0 + 0 + 0 + 0 + 0 0 + + 0 0 0 √

2 + 0 + + 0 + 0 + 0 + + 0 0 + 0 0 √

3 0 + 0 0 + 0 + + + + + + + + 0 0 √

• Patient received:• 10 units Group O Negative rbcs• 4 Group O Single Donor Platelets

Check Medical History

• Patient’s ABO discrepancy was due to massive transfusion of out of group blood products.

• Important Clue: Mixed-field agglutination• Information from transferring hospital confirmed that patient

was AB Positive

• What blood type of rbcs should be transfused?

Resolution

You did it!