Antibody Screening and Crossmatch Teting Webinar SLIDES

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All Content © Immucor 8 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

All Content © Immucor 9 Antibody Detection & Crossmatch Testing: What? When? Why?

All Content © Immucor 10 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

All Content © Immucor 11 Terminology

Antibody Detection Test VS Antibody Screen Test

All Content © Immucor 12 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.

All Content © Immucor 13 Why Antibody Detection Testing?

• 1-2% of patients have unexpected alloantibodies1

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

All Content © Immucor 14 A History2…

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

All Content © Immucor 15 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.

All Content © Immucor 16 Screen VS Crossmatch

Full Full Antiglobulin + Antiglobulin Screen Crossmatch

All Content © Immucor 17 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

All Content © Immucor 18 Methods for Antibody Detection Testing

All Content © Immucor 19 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

All Content © Immucor 20 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)

All Content © Immucor 21 Adherence

All Content © Immucor 22 Adherence

All Content © Immucor 23 Adherence

All Content © Immucor 24 Agglutination

All Content © Immucor 25 Agglutination

Phase I

Antibody uptake or “Sensitization”

All Content © Immucor 26 Agglutination

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

All Content © Immucor 27 Agglutination

Phase II

Can be either IgG or IgM

Visible agglutination as red blood cells bind into clumps

All Content © Immucor 28 Agglutination

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

All Content © Immucor 29 Agglutination

Zeta Potential

All Content © Immucor 30 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

All Content © Immucor 31 Potentiators5

All Content © Immucor 32 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

All Content © Immucor 33 Low Ionic Strength Solutions (LISS)

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

All Content © Immucor 34 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.

All Content © Immucor 35 Reagent Red Blood Cells (red blood cells)

All Content © Immucor 36 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.

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

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.”

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.

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

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

• 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.

All Content © Immucor 47 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.

• 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

Link to register: https://app.livestorm.co/immucor Text WEBINAR to 47177 All Content © Immucor 52 All Content © Immucor 53 Thank you!