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

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Presentation on theme: "An Overview: Polyethylene glycol (PEG) - Adsorption of Auto-abs & Detection of Allo-abs in WAIHA Cheng Chun Kwok."— Presentation transcript:

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)? zLinear, neutral, water-soluble, non-toxic, ethylene glycol polymer. zConsistency (liquid to solid) depends on Mol. Wt. zSurfactant in industry (food, cosmetics, pharmaceutics) zBiomedicine (dispersing agents, solvents, ointment, suppository bases, vehicles, tablet excipients).

3 How Does PEG Work? zMacro-molecules remove water concentrate abs abs uptake. zTest mixture cannot be centrifuged. zAb detection depends on IAT phase. zAnti-IgG AHG is recommended.

4 PEG in Blood Banking zFirst described by Nance & Garratty in zMol. Wt. around 3, ,000. zTwo types of PEG solutions. y20% PEG (20 g PEG / 100 mL NISS) (4 drops 20% PEG + 2 drops serum) yPEG in LISS - commercial available (2 drops PEG + 2 drops serum) zLiew & Duncan proposed to use in auto-abs adsorption & allo-abs detection in 1995.

5 Auto-immune Hemolytic Anemia (1) z3 broad categories IHA: alloimmune, autoimmune, & drug-induced. zAI: auto-abs on patient rbc in patients serum. zPatient anemia ( Hb / Hct) / compensated? zAnemia not present: DAT + with free auto-abs. zAnemia compensated: compensated WAIHA. zHemolytic anemia present: WAIHA. zDifficult to distinguish bet them in BB.

6 Auto-immune Hemolytic Anemia (2) zBlood smear: spherocytes, reticulocytosis. zBiochem: unconjugated bilirubin, LDH, Hp. zHemoglobinemia & hemoglobinuria. zSerological tests: DAT, AS / abs id on serum &/or eluate. zHA may due to structural membrane defect, erythrocytic enzyme deficient, abn Hb molecules.

7 All Positive DAT Free Auto-abs present HA? zNo, affected not clearly understood. zPositive DAT + free auto-abs - HA (not WAIHA). zPositive DAT + free auto-abs + HA (WAIHA). zComplicated. zLots of factors may involved.

8 Possible Factors Influence an Antibody to cause Hemolytic Anemia (1) zThermal amplitude of abs reactivity. zTiter in serum. zAvidity for red cells antigen. zamount of abs bound to red cells. zAbility of abs to fix complement in vivo. zActivity of individuals macrophages.

9 Possible Factors Influence an Antibody to cause Hemolytic Anemia (2) zIgG subclass (IgG 3 > IgG 1 > IgG 2 > IgG 4 ) yRh abs mostly IgG 1 & IgG 3. yAnti-K & anti-Fy usually IgG 1. yAnti-Jk mainly IgG 3. ySevere HDN mostly often associated with IgG 1.

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

11 What is Clinically Significant Ab? zKnown to cause HDN. zKnown to cause HTR. zUnacceptably shorten survival of transfused red cells. zExamples: ABO, Rh, Duffy, Kidd, Kell, SsU, & MUR. zAll of them are reactive at 37 o C &/or IAT.

12 All abs Reactive at 37 o C &/or IAT are Clinically Significant? zNo. zAll clinically significant abs are reactive at 37 o C &/or IAT. zAbs reactive at 37 o C &/or IAT may not be clinically significant. zCan be distinguished in Blood Bank? not easy. zWhen an allo-ab reactive at 37 o C &/or IAT is identified antigen negative cells are selected for transfusion.

13 Detection of allo-abs in patients with auto-abs (1) z1-in-5 dilute auto-abs to detect allo-abs is unreliable & should be strongly discouraged. zleast incompatible blood without allo-abs detection in urgent transfusion is unacceptable. zAuto-adsorption is ideal but procedures are tedious, labor intensive & time-consuming. yUrgent transfusion may be delay. yLimitation: patient severely anemia or recently transfused.

14 Detection of allo-abs in patients with auto-abs (2) zAllogeneic adsorption is an alternative. yDifferential warm allogeneic adsorption. yOne-cell sample allogeneic adsorption. zDifferential warm allogeneic adsorption. yPatient phenotypes not known / uncertain. yPatient recently transfused. yTedious, time-consuming & labor intensive. yAbs to high-incidence antigen may be removed. yRepeat the procedures in transfused patients.

15 Detection of allo-abs in patients with auto-abs (3) zOne-cell sample allogeneic adsorption. yPatient not recently transfused. yPatient phenotypes known. yAbs adsorption with phenotype-matched red cells. ySerum insufficient. yRecently 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 zBarron & Brown yImmunohematoloty 1997;13: zCheng et al yTransfusion 2001;41:13-7. zJudd & Dake Immunohematology 2001;17:82-5.

17 Barron & Brown Immunohematoloty 1997;13: z19 patients with warm auto-abs were tested. z14/19 contained allo-abs / + auto-abs specificities. zAdsorption: equal part papain-treated rbc & serum Vs equal part untreated rbc, serum, & 20% PEG in PBS. zDetection: 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 & Brown Immunohematoloty 1997;13: PEGRef serum 20% PEG red cells serum papain-treated red cells ?? Optimal reactivity of 20% PEG; serum:PEG:rbc = 2:4:1 (Nance & Garratty, Am J Clin Pathol 1987).

19 Ref PEG Total adsorption time 59.5 hrs 10 hrs Total number of adsorption 42x 30x Average time mins 30 mins Abs reactivity 4 stronger 5 stronger anti-K, -Fy a, 2 -E anti-E, -Jk b, 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. Barron & Brown (Result 1) Immunohematoloty 1997;13:

20 Barron & Brown (Result 2) Immunohematoloty 1997;13: z2 allo-abs not detected with PEG. yAnti-K weak reactive with ref but non-reactive with PEG. yAnti-Jk b 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: zDecreases in adsorption time. zEfficient & cost-effective. zVery weak allo-abs may not be detected. zOption to reduce time & cost in adsorptions. zWhen non-detection is suspected, use standard procedures.

22 Cheng et al Transfusion 2001;41:13-7. z16 patients with warm auto-abs were tested. z8/16 contained allo-abs / + auto-abs specificities. zAdsorption: equal part untreated rbc & serum Vs equal part untreated rbc, serum, & PEG (PeG ). zDetection: gel (LISS-IAT) Vs PEG-IAT (4 drops serum/PEG mixture + 1 drop 5% reagent cells).

23 Cheng et al Transfusion 2001;41:13-7. PEGConventional serum PeG red cells serum untreated red cells

24 Cheng et al (Result 1) Transfusion 2001;41:13-7. Conventional PEG 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. z3 allo-anti-E, 1 allo-anti-e, & 3 allo-anti-Mur were able to be demonstrated by both methods. zReactivity strength not mentioned & compared between the two methods. z40% efficiency in number of adsorptions. z85% decreases in adsorption time. zEffective in allogeneic adsorption.

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

27 Judd & Dake Immunohematology 2001;17:82-5. z11 warm reactive auto-abs selected to compare ZZAP & PEG adsorption. z12 allo-abs were selected to compare abs detection after ZZAP- & PEG-adsorption. zAdsorption: ZZAP adsorption (ficin) Vs equal part untreated rbc, serum, & PEG (PeG ). zDetection: NISS-IAT (3 drops serum + 1 drop 3-4 % reagent cells, 60 mins 37 o C, 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 & Dake Immunohematology 2001;17:82-5. PEGZZAP serum PeG red cells serum ZZAP (ficin)- treated red cells

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

30 Judd & Dake (Test & Result 1) Immunohematology 2001;17:82-5. zTwo fold adsorption of 7 allo-abs (2 anti-D, 1 anti-E, 1 anti-K, 1 anti-Jk a, & 1 anti-Jk b ) with antigen negative red cells. zPEG-serum parallel run with saline-serum. zTitration studies on adsorbed sera with saline: 60 mins at 37 o C with anti-IgG. z5/7 were 2 folds lower with PEG. z2/7 were 1 fold lower with PEG. Titers of PEG-serum Vs saline-serum : 2-8 Vs ?? Serially dilute PEG-serum mixture with saline.

31 Judd & Dake (Test & Result 2) Immunohematology 2001;17:82-5. zTo demonstrate abs activity loss in PEG-adsorption procedure but not on post-adsorption storage. zIncubate PEG-serum & saline-serum at 37 o C, 15 mins, centrifuge & harvest the supernatants. zMeasure the IgG levels with nephelometer. zPEG-serum mixture: mg/dL. zSaline-serum mixture: mg/dL. zIgG 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. zPEG adsorption effective in removing auto-abs. zFail to detect allo-abs due to Ig precipitation.

33 PEG Does precipitate Immunoglobulin ??

34 Leger RM, Ciesielski DC, & Garratty G (1) Transfusion 1999;39: zInvestigate possible loss of ab reactivity of PEG-adsorbed sera upon storage. z7 sera contain single ab specificities zAnti-E, -K, -Fy a, & -Jk a. z2 sham PEG-adsorptions with ag neg red cells. zFresh PEG-adsorbed serum reactivity: zPEG-sera mixture left at 4 o C for days.

35 Leger RM, Ciesielski DC, & Garratty G (2) Transfusion 1999;39: zStored sera mixture divided into 2 aliquots. zMixed & Settled zMixed was mixed before sampling. zSettled was allowed ppt to form, settle, & sample the clear supernatant. zPEG-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: PEG-adsorbed Sera 4 o C Storage PEG-unadsorbed Mix Before Centrifuge & Sera Sampling Sample Supernatant 1 x anti-E 1+S 1+ 1/2 + 1 x anti-Fy a x abs (day 0) - Same Degree 5 x abs (day 4 ) - Same Degree 4 abs Same degree 1 anti-Fy a weaken

37 Leger RM, Ciesielski DC, & Garratty G (3) Transfusion 1999;39: zPrecipitate formed in stored PEG-serum mixture. zOnce precipitate formed, DO NOT centrifuge. zRemix the mixture before sampling. zTest PEG-adsorbed serum on the day of preparation.

38 What Make the Differences? zMethod to separate serum-PEG mixture after adsorption. z?? Centrifugation force ?? z?? Duration ?? z?? 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 37 o C, minutes centrifuge & harvest adsorbed serum/PEG mixture perform PEG-IAT with SC / PC 4 drops serum/PEG mixture to 1 drop 5% red cells 37 o C, minutes, IAT(anti-IgG)

40 Advantages of PEG zEnhance auto-abs adsorption. zPrior red cells treatment not necessary. zDirect benefit: time saving, & minimize the delay of urgent transfusion in WAIHA patients. zIndirect benefit: labor & cost effectiveness.

41 Disadvantages of PEG zPrecipitates Ig. zWeak allo-abs may not be detected after PEG adsorption.

42 Remedy?? zAvoid overnight storage PEG-serum mixture at 4 o C - precipitate formed. zAdsorbed PEG-serum mixture should be tested as soon as possible. zprecipitate formed, mix thoroughly before sampling & DO NOT centrifuge. zOther techniques may be incorporated to enhance abs detection after PEG adsorption gel (LISS-IAT)?? yCells to PEG/serum mixture ratio. yAHG in gel: MS/PS.

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

44 End Questions & Comments are welcome


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