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

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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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slide1

An Overview: Polyethylene glycol

(PEG) - Adsorption of Auto-abs &

Detection of Allo-abs in WAIHA

Cheng Chun Kwok

what is polyethylene glycol peg
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).
how does peg work
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.
peg in blood banking
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.
auto immune hemolytic anemia 1
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.
auto immune hemolytic anemia 2
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.
all positive dat free auto abs present ha
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.
possible factors influence an antibody to cause hemolytic anemia 1
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.
possible factors influence an antibody to cause hemolytic anemia 2
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.
why interested in waiha
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?
what is clinically significant ab
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.
all abs reactive at 37 o c or iat are clinically significant
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.
detection of allo abs in patients with auto abs 1
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.
detection of allo abs in patients with auto abs 2
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.
detection of allo abs in patients with auto abs 3
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 .

evaluations of peg in waiha abs adsorption detection
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.
barron brown immunohematoloty 1997 13 119 22
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).

barron brown immunohematoloty 1997 13 119 221
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).

barron brown result 1 immunohematoloty 1997 13 119 22
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.

barron brown result 2 immunohematoloty 1997 13 119 22
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).

barron brown result 3 immunohematoloty 1997 13 119 22
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.
cheng et al transfusion 2001 41 13 7
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).
cheng et al transfusion 2001 41 13 71
Cheng et alTransfusion 2001;41:13-7.

Conventional

PEG

serum

serum

PeG

untreated

red cells

red cells

cheng et al result 1 transfusion 2001 41 13 7
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.

cheng et al result 2 transfusion 2001 41 13 7
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.
cheng et al result 3 transfusion 2001 41 13 7
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.
judd dake immunohematology 2001 17 82 5
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).

judd dake immunohematology 2001 17 82 51
Judd & DakeImmunohematology 2001;17:82-5.

ZZAP

PEG

serum

serum

PeG

ZZAP (ficin)-

treated red cells

red cells

judd dake result 1 immunohematology 2001 17 82 5
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.

judd dake test result 1 immunohematology 2001 17 82 5
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.

judd dake test result 2 immunohematology 2001 17 82 5
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.

judd dake result 2 immunohematology 2001 17 82 5
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.
leger rm ciesielski dc garratty g 1 transfusion 1999 39 1272 3
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.
leger rm ciesielski dc garratty g 2 transfusion 1999 39 1272 3
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.
leger rm ciesielski dc garratty g 2 transfusion 1999 39 1272 31
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

leger rm ciesielski dc garratty g 3 transfusion 1999 39 1272 3
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.
what make the differences
What Make the Differences?
  • Method to separate serum-PEG mixture after adsorption.
  • ?? Centrifugation force ??
  • ??Duration ??
  • ??Temperature ??
how to perform peg incorporated one cell sample allogeneic adsorption
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)

advantages of peg
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.
disadvantages of peg
Disadvantages of PEG
  • Precipitates Ig.
  • Weak allo-abs may not be detected after PEG adsorption.
remedy
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.
conclusion
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.
slide44

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