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An Overview: Polyethylene glycol (PEG) - Adsorption of Auto-abs & Detection of Allo-abs in WAIHA

An Overview: Polyethylene glycol (PEG) - Adsorption of Auto-abs & Detection of Allo-abs in WAIHA Cheng Chun Kwok. What is Polyethylene glycol (PEG)?. Linear, neutral, water-soluble, non-toxic, ethylene glycol polymer. Consistency (liquid to solid) depends on Mol. Wt.

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An Overview: Polyethylene glycol (PEG) - Adsorption of Auto-abs & Detection of Allo-abs in WAIHA

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  1. An Overview: Polyethylene glycol (PEG) - Adsorption of Auto-abs & Detection of Allo-abs in WAIHA Cheng Chun Kwok

  2. What is Polyethylene glycol (PEG)? • Linear, neutral, water-soluble, non-toxic, ethylene glycol polymer. • Consistency (liquid to solid) depends on Mol. Wt. • Surfactant in industry (food, cosmetics, pharmaceutics) • Biomedicine (dispersing agents, solvents, ointment, suppository bases, vehicles, tablet excipients).

  3. How Does PEG Work? • Macro-molecules  remove water  concentrate abs   abs uptake. • Test mixture cannot be centrifuged. • Ab detection depends on IAT phase. • Anti-IgG AHG is recommended.

  4. PEG in Blood Banking • First described by Nance & Garratty in 1987. • Mol. Wt. around 3,500 - 4,000. • Two types of PEG solutions. • 20% PEG (20 g PEG / 100 mL NISS) (4 drops 20% PEG + 2 drops serum) • PEG in LISS - commercial available (2 drops PEG + 2 drops serum) • Liew & Duncan proposed to use in auto-abs adsorption & allo-abs detection in 1995.

  5. Auto-immune Hemolytic Anemia (1) • 3 broad categories IHA: alloimmune, autoimmune, & drug-induced. • AI: auto-abs on patient rbc  in patient’s serum. • Patient  anemia ( Hb /  Hct) / compensated? • Anemia not present: DAT+ with free auto-abs. • Anemia compensated: compensated WAIHA. • Hemolytic anemia present: WAIHA. • Difficult to distinguish bet them in BB.

  6. Auto-immune Hemolytic Anemia (2) • Blood smear: spherocytes, reticulocytosis. • Biochem: unconjugated bilirubin , LDH , Hp. • Hemoglobinemia & hemoglobinuria. • Serological tests: DAT, AS / abs id on serum &/or eluate. • HA may due to structural membrane defect, erythrocytic enzyme deficient, abn Hb molecules.

  7. All Positive DAT  Free Auto-abs present HA? • No,  affected not clearly understood. • Positive DAT + free auto-abs - HA (not WAIHA). • Positive DAT + free auto-abs + HA (WAIHA). • Complicated. • Lots of factors may involved.

  8. Possible Factors Influence an Antibody to cause Hemolytic Anemia (1) • Thermal amplitude of abs reactivity. • Titer in serum. • Avidity for red cells antigen. • amount of abs bound to red cells. • Ability of abs to fix complement in vivo. • Activity of individual’s macrophages.

  9. Possible Factors Influence an Antibody to cause Hemolytic Anemia (2) • IgG subclass (IgG3 > IgG1 > IgG2 > IgG4) • Rh abs mostly IgG1 & IgG3. • Anti-K & anti-Fy usually IgG1. • Anti-Jk mainly IgG3. • Severe HDN mostly often associated with IgG1.

  10. Why Interested in WAIHA? • Create problems in BB  mask concomitant presence of clinically significant allo-abs. • Identify clinically significant allo-abs to avoid HTR. • Warm auto-abs adsorption procedures are tedious & time-consuming. • Auto-abs react with all donor red cells  compatible blood almost always impossible. • Question: If we give phenotype matched blood, should we border the tedious auto-abs adsorption & allo-abs detection?

  11. What is Clinically Significant Ab? • Known to cause HDN. • Known to cause HTR. • Unacceptably shorten survival of transfused red cells. • Examples: ABO, Rh, Duffy, Kidd, Kell, SsU, & MUR. • All of them are reactive at 37oC &/or IAT.

  12. All abs Reactive at 37oC &/orIAT are Clinically Significant? • No. • All clinically significant abs are reactive at 37oC &/or IAT. • Abs reactive at 37oC &/or IAT may not be clinically significant. • Can be distinguished in Blood Bank?  not easy. • When an allo-ab reactive at 37oC &/or IAT is identified  antigen negative cells are selected for transfusion.

  13. Detection of allo-absin patients with auto-abs (1) • 1-in-5 dilute auto-abs to detect allo-abs is unreliable & should be strongly discouraged. • “least incompatible” blood without allo-abs detection in urgent transfusion is unacceptable. • Auto-adsorption is ideal but procedures are tedious, labor intensive & time-consuming. • Urgent transfusion may be delay. • Limitation: patient severely anemia or recently transfused.

  14. Detection of allo-absin patients with auto-abs (2) • Allogeneic adsorption is an alternative. • Differential warm allogeneic adsorption. • One-cell sample allogeneic adsorption. • Differential warm allogeneic adsorption. • Patient phenotypes not known / uncertain. • Patient recently transfused. • Tedious, time-consuming & labor intensive. • Abs to high-incidence antigen may be removed. • Repeat the procedures in transfused patients.

  15. Detection of allo-absin patients with auto-abs (3) • One-cell sample allogeneic adsorption. • Patient not recently transfused. • Patient phenotypes known. • Abs adsorption with phenotype-matched red cells. • Serum insufficient. • Recently transfused patient: phenotype with reticulocyte-riched region red cells  gel(LISS-IAT). Young red cells: MCHC ; acetylcholinesterase activity .

  16. Evaluations of PEG in WAIHA Abs Adsorption & Detection • Barron & Brown • Immunohematoloty 1997;13:119-22. • Cheng et al • Transfusion 2001;41:13-7. • Judd & Dake • Immunohematology 2001;17:82-5.

  17. Barron & BrownImmunohematoloty 1997;13:119-22. • 19 patients with warm auto-abs were tested. • 14/19 contained allo-abs / + auto-abs specificities. • Adsorption: equal part papain-treated rbc & serum Vs equal part untreated rbc, serum, & 20% PEG in PBS. • Detection: LISS (2 drops serum + 2 drops LISS + 1 drop 5% reagent cells).Vs PEG-IAT(6 drops serum/PEG mixture + 1 drop 5% reagent cells). ?? Optimal reactivity of 20% PEG; serum:PEG:rbc = 2:4:1 (Nance & Garratty, Am J Clin Pathol 1987).

  18. Barron & BrownImmunohematoloty 1997;13:119-22. Ref PEG serum serum 20% PEG papain-treated red cells red cells ?? Optimal reactivity of 20% PEG; serum:PEG:rbc = 2:4:1 (Nance & Garratty, Am J Clin Pathol 1987).

  19. Barron & Brown (Result 1)Immunohematoloty 1997;13:119-22. RefPEG Total adsorption time 59.5 hrs 10 hrs Total number of adsorption 42x 30x Average time 161.5 mins 30 mins Abs reactivity 4 stronger 5 stronger anti-K, -Fya, 2 -E anti-E, -Jkb, 3 -C Remarks 1 auto- ND 1 auto- & 2 allo- ND ND - Not Detected. Ref: 10 mins enzyme + 10 mins wash + 60 mins incubation + 5 mins harvest. PEG: 15 mins inucbation + 5 mins harvest.

  20. Barron & Brown (Result 2)Immunohematoloty 1997;13:119-22. • 2 allo-abs not detected with PEG. • Anti-K weak reactive with ref but non-reactive with PEG. • Anti-Jkb microscopic positive but non-reactive with PEG. ?? Optimal reactivity of 20% PEG; serum:PEG:rbc = 2:4:1 (Nance & Garratty, Am J Clin Pathol 1987). ?? Macroscopic but not microscopic reading at all stages of LISS, + IAT reading, is recommended (Issit & Anstee, Applied blood group Serology).

  21. Barron & Brown (Result 3)Immunohematoloty 1997;13:119-22. • Decreases in adsorption time. • Efficient & cost-effective. • Very weak allo-abs may not be detected. • Option to reduce time & cost in adsorptions. • When non-detection is suspected, use standard procedures.

  22. Cheng et alTransfusion 2001;41:13-7. • 16 patients with warm auto-abs were tested. • 8/16 contained allo-abs / + auto-abs specificities. • Adsorption: equal part untreated rbc & serum Vs equal part untreated rbc, serum, & PEG (PeG). • Detection: gel (LISS-IAT)Vs PEG-IAT(4 drops serum/PEG mixture + 1 drop 5% reagent cells).

  23. Cheng et alTransfusion 2001;41:13-7. Conventional PEG serum serum PeG untreated red cells red cells

  24. Cheng et al (Result 1) Transfusion 2001;41:13-7. ConventionalPEG Total adsorption time 2,400 hrs 360 hrs Mean time 150 mins 22.5 mins Mean fold 2.5x 1.5x Auto-abs adsorption (3x) 2 Not adsorbed All adsorbed Allo-abs detection All are demonstrated Con: 60 mins incubation. PEG: 15 mins incubation.

  25. Cheng et al (Result 2) Transfusion 2001;41:13-7. • 3 allo-anti-E, 1 allo-anti-e, & 3 allo-anti-Mur were able to be demonstrated by both methods. • Reactivity strength not mentioned & compared between the two methods. • 40% efficiency in number of adsorptions. • 85% decreases in adsorption time. • Effective in allogeneic adsorption.

  26. Cheng et al (Result 3) Transfusion 2001;41:13-7. • Method awaits standardization. • Fully replace conventional method not recommended. • Further studies on weak allo-abs loss during adsorption or IAT. • Other techniques incorporated to enhance abs detection - gel (LISS-IAT). • Safe to male has no recent transfusion history / female has not been pregnant or no recent transfusion history.

  27. Judd & DakeImmunohematology 2001;17:82-5. • 11 warm reactive auto-abs selected to compare ZZAP & PEG adsorption. • 12 allo-abs were selected to compare abs detection after ZZAP- & PEG-adsorption. • Adsorption: ZZAP adsorption (ficin) Vs equal part untreated rbc, serum, & PEG (PeG). • Detection: NISS-IAT (3 drops serum + 1 drop 3-4 % reagent cells, 60 mins 37oC, PS-AHG)Vs PEG-IAT(4 drops serum/PEG mixture + 1 drop 5% reagent cells). ?? NISS-IAT: serum to cell = 2 to 1 (Technical Manual).

  28. Judd & DakeImmunohematology 2001;17:82-5. ZZAP PEG serum serum PeG ZZAP (ficin)- treated red cells red cells

  29. Judd & Dake (Result 1)Immunohematology 2001;17:82-5. ZZAPPEG Auto-abs removal power Comparable Allo-abs studies 1 rxn grade or more weaker anti-Fya 1+S ND anti-c 1+S weak anti-Jka 1+ - 2+S w - 1+w ND - Not Detected.

  30. Judd & Dake (Test & Result 1)Immunohematology 2001;17:82-5. • Two fold adsorption of 7 allo-abs (2 anti-D, 1 anti-E, 1 anti-K, 1 anti-Jka, & 1 anti-Jkb) with antigen negative red cells. • PEG-serum parallel run with saline-serum. • Titration studies on adsorbed sera with saline: 60 mins at 37oC with anti-IgG. • 5/7 were 2 folds lower with PEG. • 2/7 were 1 fold lower with PEG. Titers of PEG-serum Vs saline-serum : 2-8 Vs 4-32. ?? Serially dilute PEG-serum mixture with saline.

  31. Judd & Dake (Test & Result 2)Immunohematology 2001;17:82-5. • To demonstrate abs activity loss in PEG-adsorption procedure but not on post-adsorption storage. • Incubate PEG-serum & saline-serum at 37oC, 15 mins, centrifuge & harvest the supernatants. • Measure the IgG levels with nephelometer. • PEG-serum mixture: 128-243 mg/dL. • Saline-serum mixture: 265-505 mg/dL. • IgG level of PEG-serum mixture was 50% lower. ?? Compare IgG levels of ZZAP & PEG adsorbed serum.

  32. Judd & Dake (Result 2)Immunohematology 2001;17:82-5. • PEG adsorption effective in removing auto-abs. • Fail to detect allo-abs due to Ig precipitation.

  33. PEG Does precipitate Immunoglobulin ??

  34. Leger RM, Ciesielski DC, & Garratty G (1)Transfusion 1999;39:1272-3. • Investigate possible loss of ab reactivity of PEG-adsorbed sera upon storage. • 7 sera contain single ab specificities • Anti-E, -K, -Fya, & -Jka. • 2 sham PEG-adsorptions with ag neg red cells. • Fresh PEG-adsorbed serum reactivity: 1+ - 3+. • PEG-sera mixture left at 4oC for 1 - 4 days.

  35. Leger RM, Ciesielski DC, & Garratty G (2)Transfusion 1999;39:1272-3. • Stored sera mixture divided into 2 aliquots. • ‘Mixed’ & ‘Settled’ • ‘Mixed’ was mixed before sampling. • ‘Settled’ was allowed ppt to form, settle, & sample the clear supernatant. • PEG-adsorbed sera mixture were tested and compared to the adsorbed sera at the time of adsorption.

  36. Leger RM, Ciesielski DC, & Garratty G (2)Transfusion 1999;39:1272-3. PEG-adsorbed Sera 4oC Storage PEG-unadsorbedMix Before Centrifuge & SeraSamplingSample Supernatant 1 x anti-E 1+S 1+ 1/2 + 1 x anti-Fya 2+ 2+ 1+ 5 x abs (day 0) - Same Degree 5 x abs (day 4 ) - Same Degree 4 abs Same degree 1 anti-Fya weaken

  37. Leger RM, Ciesielski DC, & Garratty G (3)Transfusion 1999;39:1272-3. • Precipitate formed in stored PEG-serum mixture. • Once precipitate formed, DO NOT centrifuge. • Remix the mixture before sampling. • Test PEG-adsorbed serum on the day of preparation.

  38. What Make the Differences? • Method to separate serum-PEG mixture after adsorption. • ?? Centrifugation force ?? • ??Duration ?? • ??Temperature ??

  39. How to Perform PEG-incorporated One-cell Sample Allogeneic Adsorption Mix equal parts of patient serum + PEG + phenotyped packed red cells  incubate at 37oC, 15-30 minutes  centrifuge & harvest adsorbed serum/PEG mixture  perform PEG-IAT with SC / PC 4 drops serum/PEG mixture to 1 drop 5% red cells 37oC, 15-30 minutes, IAT(anti-IgG)

  40. Advantages of PEG • Enhance auto-abs adsorption. • Prior red cells treatment not necessary. • Direct benefit: time saving, & minimize the delay of urgent transfusion in WAIHA patients. • Indirect benefit: labor & cost effectiveness.

  41. Disadvantages of PEG • Precipitates Ig. • Weak allo-abs may not be detected after PEG adsorption.

  42. Remedy?? • Avoid overnight storage PEG-serum mixture at 4oC - precipitate formed. • Adsorbed PEG-serum mixture should be tested as soon as possible. • precipitate formed, mix thoroughly before sampling & DO NOT centrifuge. • Other techniques may be incorporated to enhance abs detection after PEG adsorption  gel (LISS-IAT)?? • Cells to PEG/serum mixture ratio. • AHG in gel: MS/PS.

  43. Conclusion • Potential technique in WAIHA auto-abs adsorption & allo-abs detection. • Safe to male has no recent transfusion history / female has not been pregnant or no recent transfusion history. • ?Method to separate serum-PEG mixture after adsorption.

  44. End Questions & Comments are welcome

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