9234-qe best practices in the immunohematology reference

9234-QE Best Practices in the Immunohematology Reference Lab October 23, 2011 2:00 PM - 3:30 PM

Event Outline

Event Title: 9234-QE Best Practices in the Immunohematology Reference Lab Event Date: Sunday, October 23, 2011 Event Time: 2:00 PM to 3:30 PM

Presenters: Janis Hamilton, Regina Leger, Jennifer Schierts, Ann Steiner

Speaker Presentation

Janis Hamilton

Regina Leger Best Practices in the IRL: Considerations for Specialized Testing

Jennifer Schierts Best Practices in the Immunohematology Reference Lab

Ann Steiner

Event Faculty List

Event Title: 9234-QE Best Practices in the Immunohematology Reference Lab Event Date: Sunday, October 23, 2011 Event Time: 2:00 PM to 3:30 PM

Director/Speaker Janis Hamilton MS, MT(ASCP)SBB American Red Cross PO Box 33351 Detroit, MI 48232-5351 [email protected] Disclosures: Yes

Speaker Regina Leger MSQA, MT(ASCP)SBB, CMQ/OE(ASQ) American Red Cross Southern California Region Research Laboratory 100 Red Cross Circle Pomona, CA 91768 [email protected] Disclosures: Nothing to Disclose

Speaker Jennifer Schierts MT(ASCP)SBB Memorial Blood Centers 737 Pelham Boulevard St., Paul, MN 55114 [email protected] Disclosures: Nothing to Disclose

Speaker Ann Steiner MT(ASCP)SBB Ortho Clinical Diagnostics 1809 Fair Street Ann Arbor, MI 48103 [email protected] Disclosures: Not Provided

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AABB 2011 IRL Best Practices 1

Best Practices in the IRL:Considerations for Specialized Testing

Gina Leger, MSQA,MT(ASCP)SBB,CMQ/OESouthern California Region, Pomona

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Issues for Testing in IRLs

• Higher complexity of serological problems

• Non-routine tests required for problem solving, individual cases

• Knowledge of test background and principles required

• Lack of readily available control material for some tests

• Ensuring quality work performance

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Questions to Consider

What makes a laboratory test "good"?

How do we ensure we have interpreted a test correctly?

What controls are necessary?

Is what we are doing "good enough"?

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What makes a laboratory test "good"?

• Well-written procedure• Sufficient controls to ensure

correct interpretation• Method produces correct and

reproducible results• Results correlate with clinical

and/or other laboratory data

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Written Procedure

Elements:• Materials, equipment required• Steps to perform• Quality control

– Expected results– What to do if QC unsatisfactory

• Place / form to record results• How to interpret results

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Training of Staff

• Didactic• Not just "steps" to perform• Background, rationale for use• Significance of results• Problems that can occur

• Test performance and practice

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Competence of Staff

• Performance of test• Interpretation of results• Direct observation, monitoring

recording/reporting results, review test & QC results, discussion, problem solving, direct observation of equipment maintenance

• CLIA regulations 42 CFR 493.1451

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Laboratory Performance

Verify that routine procedures produce accurate and reliable results –provides confidence in lab's testing and reporting processes:

1. Proficiency testing for regulated analytes

2. Verification of accuracy of non-regulated analytes

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1. Proficiency testing (PT)• CLIA regulations 42 CFR 493.857• Immunohematology regulated

analytes– subscribe to CMS approved PT program: – ABO group /D type, antibody

detection, antibody identification, compatibility testing

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2. Verification of accuracy • CLIA regulations 42 CFR 493.1236(c)• Verify test accuracy twice annually:

– other tests or procedures not considered "regulated"

– e.g., blind samples, sample shared with another laboratory, other external assessment

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Quality Control

• Commercial reagent, method: follow instructions for use

• Non-commercial method: controls that validate test result and ensure accuracy and precision

• Qualitative tests: negative and positive controls

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Quality Control

CLIA 42 CFR 493.1256

Control procedures must

• Detect immediate errors and errors occurring over time due to– Test system failure – Changes in environmental

conditions– Operator performance

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Quality Control

CLIA 42 CFR 493.1256(h) If control materials not available: • alternative mechanism to detect

errors… • examples given in Interpretive

Guidelines: – test sample in duplicate– serial dilutions if positive to confirm positive reactions

– additional review prior to release

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Method or Procedural Controls

• Help to verify a constituent of test is not causing a false positive or false negative result

• Help to minimize incorrect interpretation

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Examples of Method Controls

• Endpoint of titration determined (at least one nonreactive dilution), especially if testing treated RBCs

• Dilution control, e.g. for inhibitions

• 37C control for biphasic hemolysin in Donath-Landsteiner Test

• Nonreactive diluent control, e.g. PBS substituting for drug solution

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Controls: DTT/2-ME-Treated Plasma

Ensure DTT or 2-ME is causing desired effect:

• Positive control = known IgM antibody

• Negative control = known IgG antibody

Additional method control

• Saline dilution control

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Differentiation of IgM vs. IgG

Interpretation of agglutination:Plasma + DTT/2-ME = negPlasma + saline = pos → IgMPlasma + DTT/2-ME = posPlasma + saline = pos → IgG or

IgM + IgG (may need titration)Potential Problems:Gelling of plasma/serum + DTT→ invalid;Reaction of plasma + saline needs to be sufficient to differentiate negative result

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Example: Differentiation of IgM vs. IgGTesting for both agglutination and AGT

30 ' RT

30'37C

anti-IgG

Interp

Plasma + DTT 0 2+ IgM + IgGPlasma + saline 2+ 2+

IgM ct + DTT 0

IgM ct + saline 4+

IgG ct + DTT 1+ 3+

IgG ct + saline 0 3+

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Controls: Enzyme-Treated RBCs

Ensure enzyme causing desired effect:

• Antibody to denatured antigen, e.g. anti-Fya

• Antibody showing reactivity enhancement, e.g. diluted anti-D

• Inert plasma (detect overtreatment)

Other control:

• Glycine max – reactivity only indicates RBCs were treated

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RBCsAnti-Fya Anti-D*

Inert plasma

Glyc. max

37C AHG 37C AHG 37C AHG IS

Untreated 0 3+ 0 4+ 0 0 0

Enzyme-treated

0 0 4+ 4+ 0 0 4+

*Anti-D diluted to react only at the AGT

Antigen denaturation

Reactivity enhancement

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Which control is best?

Depends…

What is desired effect?

Antigen denaturation or

antigen-antibody enhancement?

Additional control when used as part of antibody identification:

Results consistent with specificity

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Tests Without Positive Controls:

Donath-Landsteiner (D-L) Test

• Diagnostic test for paroxysmal cold hemoglobinuria (PCH)

• Rare autoimmune hemolytic anemia historically associated with syphilis

• Now, more commonly presents as acute, transient hemolysis in children post-viral infection

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Donath-Landsteiner Test

Expected serology in these cases:• Direct antiglobulin test (DAT)

positive for C3 only • Eluate nonreactive • Antibody screen negative • Normal 4C agglutinin titer (<64)

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• Test not performed very often• Small sample volume may be

submitted if from a child• Rare antibody so positive samples

are often not available

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Testing for:• Cold-reactive IgG complement-

binding antibody that reacts in vitro as biphasic hemolysin

– Binding of antibody to RBCs occurs at cold temp

– Hemolysis occurs as coated RBCs are warmed to 37C

• Specificity is often anti-P (D-L test tested with P– RBCs)

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Test: Pt. serum + RBCs

0C→ 37C

Control: Pt. serum + RBCs

37C

Test: Pt. serum + C' + RBCs

0C→ 37C

Control: Pt. serum + C' + RBCs

37C

C' Control: C' + RBCs

0C→ 37C

C' = complement source

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A2 = serum + C' + RBCs

A1 =serum + RBCs

A3 (C' control) = C' + RBCs

Positive Donath-Landsteiner TestTubes 0C → 37C; 37C control not shown

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What makes test invalid?• Hemolysis in 37C control and test• Hemolysis in C' control and test + C'

Hemolysis in 37C control can be due to alloantibody, e.g. anti-Lea, or to a higher thermal amplitude D-L antibody• If hemolytic alloantibody excluded,

set up the 37C control prewarmed

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Causes of false negative test:• Autoadsorption of cold autoantibody allow blood to clot at 37C prior to separating serum

• Low complement levels in patient who is hemolyzing add C' source

• Biphasic hemolysin in children can be transient (no longer detectable) obtain sample close to time of hemolysis


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Causes of false positive test:• A monophasic lysin, e.g. in cold

agglutinin syndrome, can cause false positive test as test warms from 0C → 37C if D-L test is positive and 37C

control is negative, gently mix the 37C control and place at RT for 60 min., centrifuge; a positive result invalidates positive D-L


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Tests Without Positive Controls:

Drug antibodies• Rare, drug-induced immune

hemolytic anemia (DIIHA) est. 1 in 1 million

Testing for drug antibodies• Drug-treated RBCs• Untreated (and enzyme-treated)

RBCs tested in the presence of a solution of drug

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When To Test For Drug Antibodies

• Evidence of unexplained immune hemolytic anemia: hb/hct, indirect bilirubin, LDH, retics, haptoglobin

• Positive DAT, nonreactive eluate (usually)

• Drug previously implicated in DIIHA• Temporal relationship between drug

administration and hemolysis

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Testing Drug-Treated RBCs

• Test plasma/serum, eluate and last wash against drug-treated and untreated RBCs in parallel

• If drug causes nonimmunologic protein adsorption (NIPA), also dilute plasma 1 in 20

Controls• Pool of normal sera• Saline• Positive control if available

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• Positive test = hemolysis, agglutination or sensitization (AGT)

• If no positive control and tests with drug-treated RBCs are negative

Report:

Antibody to ________ not detected. No positive control available to ensure that drug-treated RBCs were coated with drug.

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Typical Results (Penicillin-Type Antibody)

Drug-tt’d RBCs Untt’d RBCs

37C AGT 37C AGT Pt's serum +/0 + 0 0 Pt's eluate +/0 + 0 0 Controls:Normal sera 0 0 0 0Saline 0 0 0 0Positive +/0 + 0 0Last wash 0 0 0 0

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• Be aware of reactivity of normal sera/plasma with RBCs treated with:

cefotetan, piperacillin, oxaliplatin, cephalothin and others…

• Testing of normal sera as a negative control is essential to appropriately interpret results

• Expect eluate to react with drug-treated RBCs (recover drug antibody from patient's RBCs)

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Testing in the Presence of Drug

Test untreated & enzyme-treated RBCs

• Serum + drug solution

• Serum + C' + drug solution

Controls in parallel

• Serum + PBS

• Serum + C' + PBS

• C' + drug solution

• C' + PBS

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Positive test:

• Agglutination, hemolysis or sensitization in serum ± C' + drug and no reactivity in PBS control

Negative test results could be due to:

• Drug antibody not present

• Antibody may be to a metabolite of the drug

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Typical Results When Testing In Presence Of Drug

UT RBCs ET RBCs

37C AGT 37C AGTSerum + drug +/0 + + +Serum + PBS 0 0 0 0Serum + C' + drug +/0 + +/H +Serum + C' + PBS 0 0 0 0

C' + drug 0 0 0 0

C' + PBS 0 0 0 0

(H = hemolysis)

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Controls should be negative:

• Complement controls ensure hemolysis is due to drug antibody and not reactivity from the complement source

• Reactivity in any of the PBS controls (no drug added) should be investigated

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If the serum + PBS control is positive:

• Patient may have alloantibody

• Patient may have autoantibody

• Drug and/or drug-anti-drug complexes may be present in patient's sample (drug still circulating)

• Drug present in RBC diluent, eg, diluent for gel test

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Minimize Potential to Misinterpret Results

• Be familiar with literature on method, e.g., reported problems

• Unexpected or unusual result?• Consult expert colleague• Consult manufacturer of reagent, if

applicable

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Summary

What makes a "good" test?• One that works!What controls are needed?• Validate the test resultsAre we doing enough?• Staff demonstrate competence• Lab performance demonstrated• Test results correlate with clinical

data

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WWW.MBC.ORG • ©2011 MEMORIAL BLOOD CENTERS • CONFIDENTIAL & PROPRIETARY

Best Practices in the Immunohematology Reference Lab

Jennifer Schierts, MT(ASCP)SBBManager, Transfusion Support ServicesImmunohematology Reference LabMemorial Blood CentersOctober 23, 2011

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Ficin/Papain

• Proteolytic enzymes– Break down negatively charged protein molecules

• Ficin – derived from the sap of fig trees• Papain – derived from the unripe fruit of the

papaya • Enhances reactivity to:

– Rh, Kidd, P1, Lewis, and I antigens• Destroys reactivity to:

– Duffy, MNS, Ch, Rg, Yta, and JMH


Trypsin

• Proteolytic enzymes– Cleaves proteins at the carboxyl terminus of amino

acids arginine and lysine– Cleaves glycophorin A but NOT glycophorin B

• Enhances reactivity to:– Rh, Kidd, P1, Lewis, and I antigens

• Destroys reactivity to:– MN, Ch, Rg, Lutheran, Dombrock, Knops, and JMH

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AET/DTT

• AET (2-aminoethylisothiouronium)• DTT (Dithiothreitol)• Sulfhydryl reagent

– Reduce the disulfide bonds in highly folded protein structures

• Inactivates Kell, Lutheran, Dombrock, and Cromer blood group system antigens

• Destroys:– JMH, Kna, McCa, Yka, Yta, and LW


EDTA-Glycine-Acid

• Weakens antigens of the Kidd blood group system

• Denatures Kell blood group system, the Er collection, and Bg antigens

• Dissociating IgG from RBCs – Allows for phenotyping using IAT reagents


Chloroquine Diphosphate

• Inactivates Bg antigen

• Dissociating IgG from RBCs – Allows for phenotyping using IAT reagents

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DTT and 2-ME

• Inactivation of red cell antibodies using thiol reagents

• Cleave the interchain disulfide bonds that join the subunits of the IgM pentamer while leaving the IgG antibodies intact

• Most often used when investigating the potential of an alloantibody to cause HDFN– Titer post treatment


Neutralization

– Anti-P1• Hydatid cyst fluid, pigeon droppings, turtle doves’ egg whites• Commercial substances available

– Anti-Lewis• Plasma or serum• Commercial substances available

– Anti-Cha and Rga

• Plasma or serum– Anti-Sda

• Urine– Anti-I

• Human breast milk


Serum Acidification

• pH sensitive antibodies such as anti-M and anti-Pr react strongly at decreased pH (around pH 6.0)

• Carboxyl groups become charged on the sialoglycoproteins

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Other Miscellaneous Techniques

• Saline Replacement– Rouleaux

• ‘stacks of coins’

• Antibody titration• Prewarm

– Determine clinical significance of anti-A1, -M, -N, -Lea, -Leb, -P1

• Adsorption of HLA antibodies using platelet concentrates• Anti-Ch/Rg Rapid Procedure using C4d coated red cells• Adsorption for Anti-Ch/Rg using C4d coated red cells


Adsorption/Elution Studies

• Separate antibodies in serum samples:– containing more than one specificity such as

an antibody to a high incidence antigen– concentrate weakly-reactive antibodies

• Detect weakly-expressed antigens by using combined adsorption and elution tests– ABO subgroup resolution


Separation of Transfused Cells From Autologous Cells

• Reticulocyte separation using microhematocrit centrifugation

• Hypotonic saline to lyse transfused cells in a sickle cell patient

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Drug studies

• Drug-independent autoantibody

• RBC membrane binding drugs

• RBC membrane non-binding drugs

• Adsorption of protein

• Process for recognizing the presence of a drug antibody and refer if necessary

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GENOTYPING!


References

• Blaney, KD, Howard, PR. Basic & Applied Concepts of Immunohematology. St. Louis, Missouri: Mosby, Inc., 2000.

• AABB Technical Manual, current ed. Bethesda, Maryland: AABB Press.

• Judd, WJ, Johnson, ST, Storry, JR. Judd’s Methods in Immunohematology, 3rd Ed.Bethesda, Maryland: AABB Press, 2008.

9234-qe best practices in the immunohematology reference

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