AB-ident_for-workshop-2019

Principles of antibody identification, enzyme treated red cells and other enhancing agents Nampeung Anukul Division of Transfusion Sciences AMS, CMU 24 July 2019 © Copyright Showeet.com

Outlines 1. What to know before antibody identification 2. How to do antibody identification - Panel interpretation 3. How to solve problems - Potentiators - Other agents 4. Case BB Workshop_2019 2

Blood transfusion  Donor processing Antibody screening  Crossmatching to detect red blood cell antibodies other than anti-A or anti-B.  Unexpected red cell alloantibodies Antibody Identification “determine Ab specificity and clinical significance” - for donor’s RBCs survival - prevent HTR Not present present BB Workshop_2019 3

Panel cells  8-11 panel cells Rh MNSs Lewis Lutheran Kell Duffy Kidd D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga RT 37ºC AHG CCC 1 0 + 0 + + + 0 0 0 +++0 0 + 0 + + 0 + 0 0 + + + + + 2 + + 0 0 + 0 0 0 + 0+0+ + + 0 0 + 0 + 0 0 + 0 + + + 3 + + 0 0 + 0 0 0 0 ++0+ + 0 + 0 + + + 0 0 0 + 0 + + 4 + 0 + + O 0 0 / 0 +00+ + 0 0 0 + 0 + 0 0 0 0 + 0 + 5 0 0 + + + + 0 / 0 +0++ + 0 + 0 + 0 + 0 0 + + + + + 6 0 0 0 + + + + + 0 0+0+ + 0 + 0 + 0 + 0 0 + 0 + 0 0 7 0 0 0 + + + 0 0 0 +00+W 0 + 0 + + 0 0 0 0 + + + 0 8 0 0 0 + + + 0 0 0 ++0+ + 0 0 0 + 0 + 0 + 0 0 + 0 + 9 0 0 0 + + + 0 / 0 ++++ 0 0 + 0 + 0 W + 0 + 0 + 0 + 10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + + refers to the presence of the antigen Auto Control BB Workshop_2019 0 refers to the absence of the antigen 4

What to know before antibody identification • Patient’s history – Transfusion history: within 3 months? – Sex: Woman (pregnancy) – Neonate: less than 6 months? – ABO types: A1B (anti-H) or A2B (anti-A1) – Rh types: D-negative (anti-D) • Diseases associate with red blood cell antibodies – Autoimmune hemolytic anemia (autoantibody) – Cold agglutinin syndrome, Infection with Mycoplasma pneumoniae (anti-I) • Null phenotype – O Bombay/Parabombay (anti-H) – Rhnull (anti-Rh29) – Pnull (anti-PP1Pk or anti-Tja) • IVIG, RhIG treatment or taking some medicines BB Workshop_2019 5

Indirect antiglobulin test • Label 11 tubes for panel cells and 1 tube for autocontrol 1 2 3 4 5 6 7 8 9 10 11   2 drops of the patients serum 12 AC +  1 drop of each panel cell 6 BB Workshop_2019

Rh MNSs Lewis Lutheran Kell Duffy Kidd D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga RT 37ºC AHG CCC 1 0 + 0 + + + 0 0 0 +++0 0 + 0 + + 0 + 0 0 + + + + + 2 + + 0 0 + 0 0 0 + 0+0+ + + 0 0 + 0 + 0 0 + 0 + + + 3 + + 0 0 + 0 0 0 0 ++0+ + 0 + 0 + + + 0 0 0 + 0 + + 4 + 0 + + O 0 0 / 0 +00+ + 0 0 0 + 0 + 0 0 0 0 + 0 + 5 0 0 + + + + 0 / 0 +0++ + 0 + 0 + 0 + 0 0 + + + + + 6 0 0 0 + + + + + 0 0+0+ + 0 + 0 + 0 + 0 0 + 0 + 0 0 7 0 0 0 + + + 0 0 0 +00+W 0 + 0 + + 0 0 0 0 + + + 0 8 0 0 0 + + + 0 0 0 ++0+ + 0 0 0 + 0 + 0 + 0 0 + 0 + 9 0 0 0 + + + 0 / 0 ++++ 0 0 + 0 + 0 W + 0 + 0 + 0 + 10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + RT = incubate 5 min (IgM) Auto Control 37°C = incubate 30 min (IgG) wash = removes unbound protein in serum AHG = antihuman globulin CCC = proves adding AHG, AHG works well, adequate washing BB Workshop_2019 7

Column agglutination technology BB Workshop_2019 8

http://www.chemoquip.com/matrixgel.php 9 BB Workshop_2019

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NaCl, Enzyme Test and Cold Agglutinins http://www.bio-rad.com/en-sg/product/nacl-enzyme-test-cold-agglutinins?ID=LO2QF3JSZ https://diahem.com/ BB Workshop_2019 11

The automated platforms • BIO-RAD (Column agglutination technique, CAT) • Ortho-Clinical Diagnostics (CAT) • Grifols (CAT) • IMMUCOR (Solid Phase Red cell Adherence Assay) • DIAGAST (Erythro-Magnetic Technology) BB Workshop_2019 12

Panel Interpretation Look at Result Interpretation 1. Autocontrol Negative Alloantibody Positive Autoantibody 2. Ruling out Negative Rule out antigen that is positive on Positive that panel. Never rule out using positive reactions. BB Workshop_2019 13

Panel 1 Rh MNSs Lewis Lutheran Kell Duffy Kidd D C E c e f V VS Cw M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga IS RT 37 AHG CC 1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 1+ 2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 1+ 3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+ 4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 0 0 + 0 + 0 0 0 0 2+ 5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 0 2+ 6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 0 2+ 7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 0 2+ 8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 2+ 9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 0 2+ 10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 0 2+ 11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 1+ Auto 00 0 0 2+ Control “Ruling out” means crossing out antigens that did not react 1. Rule out by drawing 1 line through the antigen specificity at the top of the sheet 14 2. Repeat the process for each cell in the panel BB Workshop_2019

Panel Write-up 1. List all of the circled specificities C, Cw, Lea, Lua 2. List of rare antigens Cw , Lua 3. Look at the phase of reaction MN, ABO, P1, Lewis, Lua IS = room temp. = cold reacting antibodies (IgM) 37ºC/AHG = warm reacting antibodies (IgG) 4. Look at the pattern of the agglutination reactions of 15 antibodies that cannot be ruled out Anti-C BB Workshop_2019

Panel Write-up 1. List all of the circled specificities C, Cw, Lea, Lua C 2. List of rare antigens Cw , Lua 3. Look at the phase of reaction IS = room temp. = cold reacting antibodies (IgM) 37ºC/AHG = warm reacting antibodies (IgG) Look at Result Interpretation 3. Phases of reaction IS or RT Cold or IgM antibody (MN, ABO, P1, Lewis, Lua) Mia 37°C - May be cold (IgM) if Rx starts at RT AHG - May be warm (IgG) if Rx are not seen at Anti-C RT but noticed at AHG Warm or IgG antibody; clinically significant BB Workshop_2019 16

Panel Write-up 4. Look at the pattern of the agglutination reactions of antibodies that cannot be ruled out Look at Result Interpretation 4. Reaction strength Single strength 5. Matching pattern Varying strength One antibody specific - More than one antibody 6. Rule of three Single Ab - One antibody showing dosage The reaction pattern matches one of the BB Workshop_2019 Multiple Ab antigen column. 3 positives Difficult to match 3 negatives Suspected Ab reacts with at least 3 panel cells that are antigen positive. Suspected Ab is not react with at least 3 panel cells that do not posses the antigen. 17

Example of dosage effect: Duffy Rh MNSs P Lewis Lutheran Kell Duffy Kidd D C E c e f V VS CW M N S s P1 Lea Leb Lua Lub K k Kpa Jsa Fya Fyb Jka Jkb Xga RT 37 AHG CC 1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 W+ 2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 2+ 3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 2+ 4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 + 0 + 0 + 0 0 2+ 5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 1+ 6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 2+ 7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 2+ 8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + + + + 0 + 0 0 W+ 9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 2+ 10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 W+ 11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 W+ Auto 0 0 0 2+ Control BB Workshop_2019 18

Rule of three  to confirm the presence of the antibody  This gives a 95% confidence (p-value ≤ 0.05)  Patient serum MUST be:  Positive with 3 cells with the antigen  Negative with 3 cells without the antigen Look back your result on the panel! How many cells for C+ and C- ? BB Workshop_2019 19

What if the “rule of three” is not fulfilled? • additional cells from another panel • different lot numbers of panel cells BB Workshop_2019 www.beckmancoulter.com 20

To confirm Ab identification result,….. RBC Phenotyping  Only perform this if the patient has NOT been recently transfused (donor cells could react)  Patient who has anti-C and anti-Lua should have C-/Lua- phenotype. Selected cells e.g. suspect anti-C and anti-Lua in serum C-/Lua+ pos. with pat. serum = serum contains anti-Lua neg. with pat. serum = serum not contains anti-Lua C+/Lua- pos. with pat. serum = serum contains anti-C neg. with pat. serum = serum not contains anti-C BB Workshop_2019 21

Enhancement of Ag-Ab reaction Sometimes observe weak reaction!  Homozygous cells/antigen expression  Specific antigens are not in the list of panel cells  Serum: Cell ratio  Incubation temperature  pH  Length of incubation time  Potentiators BB Workshop_2019 http://www.medical-labs.net/precipitation-curve-with-prozone-postzone-and-zone-of-equivalence-2481/ 22

Potentiators 23 1. Polybrene 2. Polyethylene glycol (PEG) 3. Low ionic strength solution (LISS) 4. Albumin BB Workshop_2019

Polybrene PEG Albumin LISS structure Positively charged Soluble polymer 22-30% albumin Hypotonic solution reaction polymer indication neutralize –ve charge of removes water, decreases zeta reduced Na+ and of use RBCs concentrating potential by Cl- surrounding antibody buffering with +ve RBCs charge - tested at RT for 1 minute wash immediately - enhance reaction - lower ionic - must use monospecific after 37ºC and test strength (0.03M) anti-IgG in AHG with monospecific of Rh and anti-P1 than normal saline anti-IgG in AHG antibodies (0.17M) - Incubation time - 30 min incubate of 10 minute preferred limitations - cannot be used for the - inhibit reaction of - does not enhance - enhances cold detection of antibody to ABO and Lewis warm autoantibodies Kell antibodies autoantibodies - anti-K may not - anti-Jka may not react well in LISS http://www.sigmaaldrich.com/ be detected - enhances warm autoantibodies www.thermofisher.com BB Workshop_2019 24

Techniques to Isolate, Remove or Depress Antibody Reactivity - Inhibition tests : to eliminate reactivity of antibodies with certain specificities. - Inactivation of blood group antigens : – Use of proteolytic enzymes – Use of sulfhydryl reagents – Use of chloroquine - Adsorption : to remove autoAb to detect/rule out alloAb - Elution : to recover Ab in a usable form or recover intact RBCs free of Ab BB Workshop_2019 25

Inactivation of blood group antigens • Inactivated RBC will lose their activities with antibody. • Applications: – Use to confirm multiple antibodies – Used to destroy high incidence antigens in case of cannot find antigen negative cells.  Use of proteolytic enzymes  Use of sulfhydryl reagents  Use of chloroquine BB Workshop_2019 26

Use of Proteolytic enzymes Efficiency with IgG antibody: ficin > papain > bromelin > trypsin asclsga.org/wp-content/uploads/.../Immunohematology-Review-ASCLSGA-2015.pdf Antigens destroyed: MNS (s: uncertain), Duffy, Mia, Chido, Rodgers, Lutheran (with trypsin & chymotrypsin but papain and ficin have little effect), Antigens enhanced: ABO, Rh, Kidd, Lewis, I, and P Unaffectted: Kell, Diego, Colton BB Workshop_2019 27

One-stage enzyme Two-stage enzyme • Serum 2 drops + 2-5% red • Washed packed red cells + enzyme cells 1 drop + enzyme 1. solution = 1:1 1. solution 2 drops • Mix, incubate at 37°C, optimum 2. time • Mix, incubate at 37°C • 3X wash red cells with saline, dilute 2. 15 min 3. to 2-5% • Centrifuge, observe • Enzyme treated cells 1 drop + agglutination 4. serum 2 drops 3. • 3X wash with saline, add AHG 5. • Mix, incubate at 37°C 15 min • Centrifuge, observe agglutination 6. • 3X wash with saline, add AHG BB Workshop_2019 28

Panel 2 Multiple antibodies? Anti-Fya and anti-E? Panel from Blaney KD, Howard PR, editor: Basic & applied concepts of blood banking and transfusion practices, ed 3, Mosby, Inc., St. Louis, Missouri, 2013. BB Workshop_2019 29

Use of sulfhydryl reagents DTT: treat red blood cells to eliminate Kell antigen www.thermofisher.com (Kell antigens are held together by disulfide bonds of Cysteines) • Disrupt tertiary structure of proteins, unable to bind Ab • Blood group-bearing proteins that are sensitive to reducing agents include Kell, Dombrock, Knops, Lutheran, Indian, Cromer, JMH, AnWj, Yt, LW, and Scianna. http://www.redcross.org/cgi-bin/pubs/16.3sm.pdf Kell positive cell + DTT = no agglutination with anti-Kell BB Workshop_2019 30

Use of sulfhydryl reagents • Cleave the (Inter) disulfide bonds of IgM molecules http://www.labome.com/method/ • Abolish both agglutinating and complement-binding Mouse-Antibody.html activities • IgG antibodies are generally unaffected with optimal concentration. (0.0025-0.005 M, pH 7-8) • Applications: – help differentiate between IgM and IgG antibodies – Good to use when you have both IgG and IgM antibodies (warm/cold)  Dithiothreitol (DTT)  2-mercaptoethanol (2-ME)  2-aminoethylisothiouronium bromide (AET) Serum (IgM + IgG) + DTT = IgG BB Workshop_2019 31

ZZAP reagent (marketed as W.A.R.M.; Immucor, Inc.,Norcross, GA) • A combination of enzyme (e.g. papain) and DTT • Denatures Kell, MNS, Duffy, Lutheran and other less frequent blood group antigens • Does not denature the Kx antigen • Used as a one-step treatment of RBCs aiding in Ab iden • Treated cells are incubated at 37°C • Used to remove IgM, IgG and complement from DAT- positive RBCs prior to autoadsorption – “frees” autoantibody off patient’s cells BB Workshop_2019 32

Use of chloroquine  Chloroquine diphosphate (CDP) www.sigmaaldrich.com/catalog/product/sigma/c6628 • Cleave or inhibit noncovalent antigen-antibody binding • Used to remove IgG from DAT positive cells before performing RBC phenotyping by IAT • RBCs of patients with autoimmune hemolytic anemia • Partial removal may be enough to antigen type the cells or to be used for autoadsorption of warm autoantibodies • Can remove HLA class I (Bg antigens on RBCs, HLA on plt.) • removes β-2-microglobulin • Can weaken Rh antigens • No effect on RBC membrane and C3 • Very useful if the RBC needs to be antigen typed • After treatment, should use monospecific anti-IgG (AHG) • May cause RBC hemolysis if incubate longer than 2 hours • add CDP solution, mix and incubate at RT for 30 min  Glycine acid EDTA reagents : destroy Kell and Bg 33 BB Workshop_2019

ประวตั ิผ้ปู ่ วย Case - ผ้ปู ่ วยเพศชาย อายุ 74 ปี ป่ วยเป็ น multiple myeloma และกาลงั ได้รับยา Daratumumab (DARA) - ปัจจบุ นั มารพ. ด้วยอาการซีด แพทย์ขอเมด็ เลือดแดงเข้มข้น 3 units ประวตั กิ ารรับเลือด เม่ือปีที่แล้ว IAT AHG - Antibody screen : neg Screening cell w++ O1 - DAT : neg ผลการตรวจเลือดปัจจบุ นั - ABO/Rh : O, D+ C+ c+ E- e+ O2 w++ - DAT : neg w++ - Antibody screen : weak O3 w++ - Antibody identification (IAT) w+ O4 - With untreated cells : weakly positive w+ - With papain treated cells : very weakly in few cells O5 papain- - Autologous control : w++ treated - DAT : neg O6 papain- - Elution : neg eluate treated BB Workshop_2019 Sofia Lejon Crottet, Swiss National Immunology Reference Laboratory 34 Case from: ISBT website, 2016

Case What could be the cause of panreactivity seen in IAT? Daratumumab (DARA) – human monoclonal IgG1kappa targeting CD38 - anti-CD38 therapy for multiple myeloma - CD38 expressed on B, T, NK, plasma cells and RBCs Daratumumab (DARA) – interfere IAT : seen up to 6 months after last dose - Positive IAT: may occur in all methods, usually weak but stronger in solid phase (up to 4+) - doesn’t interfere IS - cause panreactivity and mask clinically significant alloantibody BB Workshop_2019 Ref: Oostendorp M, et al. Transfusion 2015;55:1555-1562. 35

Management of DARA in BB DTT - denature CD38, prevents anti-CD38 binding - CD38 consists of 5 disulfide bonds, sensitive to reducing agent - after treat, exclusion of antibodies to common RBC antigens except anti-K IAT DTT treated IAT O1 2+ neg O2 2+ neg To treat: - 0.2M DTT: Dissolve 1 g DTT in 32 ml PBS, pH 8.0 - mix 1 vol washed packed RBC : 4 vol 0.2M DTT - incubate at 37°C for 30-45 min - wash 4 times, discard if excessive hemolysis - resuspend to 2-5% for testing - QC: test treated and untreated cell for an antigen expected to be destroyed (anti-k) BB Workshop_2019 Ref: PLoS ONE 7(4): e34918. doi:10.1371/journal.pone.0034918 36 http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0034918

Recommendation Expiration of DTT-treated cells? No published data, at least 1 wk Can ZZAP be used? Less efficient, Ficin/papain-sensitive antigens will be destroyed Any other management strategies? - use cord red blood cells - have negligible CD38 : non-reactive with DARA plasma - need phenotype known - not for crossmatch - DTT-treated donor red cells may be performed in AHG crossmatch - pheno- or genotype the patient (if possible before treatment with DARA) - perform baseline type & screen (if possible before treatment with DARA) - transfusion of ABO/RhD and K compatible blood is recommended BB Workshop_2019 Ref: Schmidt AE, et al. Transfusion,55:2292–2293. doi:10.1111/trf.13174 37

References 1. Blaney KD, Howard PR, editor: Basic & applied concepts of blood banking and transfusion practices, ed 3, Mosby, Inc., St. Louis, Missouri, 2013. 2. Fung, editor: Technical manual, ed19, Bethesda, Md, 2018, AABB. 3. วทิ ยาศาสตรก์ ารบรกิ ารโลหติ เลม่ 1 และ 2, 2558 4. คูม่ อื ปฏบิ ตั กิ ารธนาคารเลอื ด, 2561 BB Workshop_2019 38