Antibody Screening and Crossmatch Teting

2/16/2021

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Continuing Education

• PACE, Florida and California DHS • 1.0 Contact Hours • Each attendee must register to receive CE at: https://www.surveymonkey.com/r/AntibodyScreen • Registration deadline is 5 March 2021

• Certificates will be sent via email only to those who have registered 19 March 2021

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Antibody Detection & Crossmatch Testing: What? When? Why?

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Objectives

• Discuss the purpose antibody detection testing • Review techniques for antibody detection testing • Examine reagents used in antibody detection and crossmatch including potentiators and reagent red blood cells • Compare antibody detection testing to crossmatch testing

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Terminology

Antibody Detection Test VS Antibody Screen Test

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Why Antibody Detection Testing?

• After determining ABO/Rh, we want to know: • Is there already a defense mechanism in place to attack a foreign invader? • If there is a defense mechanism in place, should it end up in a foreign situation?

• To solve both questions, in the transfusion medicine arena, we need to perform an antibody detection test.

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Why Antibody Detection Testing?

• 1-2% of patients have unexpected alloantibodies1

• 20-50% of transfusion dependent patients have unexpected alloantibodies

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A History2…

• 1960s: Full antiglobulin crossmatch • In the event of incompatible crossmatch, • Continue crossmatching, rather than identifying the antibody

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A History2…

• Initial intent of antibody screen: • Detect anti-D in serum of pregnant women • And ‘other’ antibodies then either unknown or consider unimportant

• Source of screening red cells: • Collected fresh specimens from staff members. • Knickerbocker Biosales was the 1st to offer commercial screen cells.

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Screen VS Crossmatch

Full Full Antiglobulin + Antiglobulin Screen Crossmatch

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Publications

Publication Article Focus The Type and Screen: a Safe Alternative and Sensitivity of antibody screen test Supplement in Selected Surgical Procedures6

The Risk of Abbreviating the Major Crossmatch in Urgent or Massive Transfusion7 The Implementation of Antibody Screening in Frequency of ‘false negative’ crossmatches Pre-transfusion testing for patient safety in Jakarta Blood Transfusion Unit8 Time of Attention of Concentrate Red Blood Cells Turn-around-time for antibody screen test Through Type & Screen – Abbreviated Cross Test in a Blood Bank of a Hospital Emergency Center in Lima, Peru9

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Methods for Antibody Detection Testing

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Antibody Detection Methods

Most Frequent Reaction Phase for Commonly Encountered Antibodies1 Agglutinating Coating Anti-M, -N; -Lea, - Anti-D, -C, -c, -E, COLD Leb, -P1, -I, -I, -H, -e; -K; -Jka, -Jkb; WARM (Room -IH -Fya, -Fyb; -S, -s (37 C) Temperature) Usually naturally occurring Generally unwanted

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Antigen Antibody Reactions

• Ag/Ab reactions must be visualized to be interpreted properly

• Can be done by: • Agglutination (tube or column) • Adherence (solid phase) • Hemolysis • Precipitation • Color change (Absorbance, Florescence)

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Adherence

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Adherence

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Adherence

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Agglutination

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Agglutination

Phase I

Antibody uptake or “Sensitization”

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Agglutination

Factors Affecting Sensitization3 • Incubation Time • Temperature • pH • Ionic strength • Antigen distribution • Ratio of antigen to antibody

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Agglutination

Phase II

Can be either IgG or IgM

Visible agglutination as red blood cells bind into clumps

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Agglutination

Factors Affecting Binding4 • Positively charged molecules • Location/position of antigen • Zeta potential

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Agglutination

Zeta Potential

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Comparison of Agglutination & Solid Phase

Red Cell Surface Sample to Cell Sample Method Area Ratio Volume (µL) (mm2) (µL/mm2)

Solid Phase 25* 66.4 .38 Column Agglutination 25 598 .04 Tube Agglutination 100 2244 .04

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Potentiators5

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How do potentiators work?

• Protein/ Colloid Media • Increase dielectric constant which reduces zeta potential • Examples: albumin, PEG, polybrene

• Low Ionic Strength Media • Decreases ionic strength which reduces zeta potential • Examples: LISS

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Low Ionic Strength Solutions (LISS)

• 0.2% NaCl • Enhance antibody uptake by reducing the zeta potential • Reduce incubation time • Dependent on serum:cell ratio

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Polyethylene Glycol (PEG)

• H20-soluble polymer • Removes water & forces sensitized red blood cells closer together by concentration • Considered more sensitive than LISS for the detection of clinically significant antibodies • Can be used in adsorption procedures and with eluates.

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Reagent Red Blood Cells (red blood cells)

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Reagent red blood cells

• Genes can be either Homozygous or Heterozygous • ie. Fy*A / Fy*A or FY*A / FY*B

• Antigens show Dosage: • Double dose Fy(a+b-) or Single dose Fy(a+b+)

• Homozygous genes produces double dose antigens. • Heterozygous genes produce one dose of each antigen for each gene present.

Reagent red blood cells, Pooled

Reagent red blood cells, 2-cell

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Reagent red blood cells, 3-cell

Reagent red blood cells

Pooled Cell 2 - Cell 3 - Cell Contains a D- cell no no yes Sensitivity by  Ag sites < sensitive >pooled, <3-cell Best Guaranteed JK & FY double dose no maybe yes

Publications

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Screen VS Crossmatch

The Type and Screen: a Safe Alternative and Supplement in Selected Surgical Procedures6 L I Boral, J B Henry, Transfusion. Mar-Apr 1977.

“The type and screen can be shown to be 99.99% effective in preventing the transfusion of incompatible blood.”

Screen VS Crossmatch

The Risk of Abbreviating the Major Crossmatch in Urgent or Massive Transfusion7 H A Oberman, B A Barnes, B A Friedman, Transfusion. Mar-Apr 1978.

“Issuance of blood in urgent situations after an “immediate spin” phase of the crossmatch, for patients whose red blood cells have been typed, and whose serums have been screened for unexpected antibodies, has a low level of risk.

Screen VS Crossmatch

The Implementation of Antibody Screening in Pre- transfusion testing for patient safety in Jakarta Blood Transfusion Unit8 M Maryunis, P Rini, NK Ritchie and S Salim, ISBT Abstract. 2015.

• 65,045 samples tested • 387 positive antibody screen test • 79 positive crossmatch • 308 false negative crossmatch

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Screen VS Crossmatch

Time of Attention of Concentrate Red Blood Cells Through Type & Screen – Abbreviated Cross Test in a Blood Bank of a Hospital Emergency Center in Lima, Peru9 L Fernandez Bolivar1, A Sagastegui Soto2, E Santiago3 and B Cordova Cervantes2, ISBT Abstract. 2020.

• 7,438 requests for blood • 29.1% available within 5 min • 60.2% available within 30 min • 85.9% available within 60 min

Summary

• Detection of unexpected antibodies is important for patient safety. • Reagent red blood cells are intentionally selected • Potentiators can be used to enhance antibody detection • The use of automation allows for fast-turn-around time for type, screen and crossmatch testing.

Boral LI, Henry JB. The type and screen: A safe alternative and supplement in selected surgical procedures. Transfusion 1977;17:163-8. Boral LI, Henry JB. The type and screen: A safe alternative and supplement in selected surgical procedures. Transfusion 1977;17:163-8.

References

1. Moulds, M. (2010). Guidelines for Antibody Identification. American Association of Blood Banks. 2. Reid, S. R. P. M. (2016). Bloody Brilliant! A History of Blood Groups and Blood Groupers. Bethesda, Maryland: AABB Press. 3. Coombs RRA, Race RR and Mourant AE. A new test for the detection of weak and “incomplete” Rh agglutinins. British Journal of Experimental Pathology 1945 Aug; 26(4): 255–266 4. N. C. Hughes-Jones, M.A. B.M. B.Ch. Ph.D., Nature of the Reaction between Antigen and Antibody, British Medical Bulletin, Volume 19, Issue 3, September 1963, Pages 171–177. 5. Harmening, D. M. (2018). Modern blood banking & transfusion practices (7th ed.). Philadelphia, PA: F.A. Davis Company. 6. Boral LI, Henry JB. The type and screen: A safe alternative and supplement in selected surgical procedures. Transfusion 1977;17:163-8 7. Oberman, H.A. , Barnes, B.A. , Friedman, B.A. The risk of abbreviating the major crossmatch in urgent or massive transfusion. Transfusion 1978; 18: 137–141. 8. M. Maryunis, P. Rini, N.K. Ritchie, and S Salim. (2015). The Implementation of Antibody Screening in Pre-transfusion testing for patient safety in Jakarta Blood Tranfusion Unit. Vox San, 109 S2, 72. 9. L. Fernandez Bolivar, A. Sagastegui Soto, E. Santiago, B. Cordova Cervantes. (2020). Time of Attention of Concentrate Red Blood Cells Through Type & Screen – Abbreviated Cross Test in a Blood Bank of a Hospital Emergency Center in Lima, Peru. Vox Sang, 115 S1, 101–102.

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Questions?

Continuing Education

• PACE, Florida and California DHS • 1.0 Contact Hours • Each attendee must register to receive CE at: https://www.surveymonkey.com/r/AntibodyScreen • Registration deadline is 5 March 2021

• Certificates will be sent via email only to those who have registered 19 March 2021

2021 Webinar Schedule Coming Soon!

Link to register: https://app.livestorm.co/immucor Text WEBINAR to 47177

17 2/16/2021

Thank you!