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.
 Surfactant in industry (food, cosmetics,
pharmaceutics)
 Biomedicine (dispersing agents, solvents,
ointment, suppository bases, vehicles, tablet
excipients).
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
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)
 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)
 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?
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)
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)
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?
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?
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 37oC &/or
IAT 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)
 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)
 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)
 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
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.
 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;13:119-22.
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.
Total adsorption time
Total number of adsorption
Ref
PEG
59.5 hrs
10 hrs
42x
30x
Average time
161.5 mins
30 mins
Abs reactivity
4 stronger
5 stronger
Remarks
ND - Not Detected.
anti-K, -Fya, 2 -E
1 auto- ND
anti-E, -Jkb, 3 -C
1 auto- & 2 allo- ND
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.
 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.
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.
 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;41:13-7.
Conventional
PEG
serum
serum
PeG
untreated
red cells
red cells
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
2 Not adsorbed
All adsorbed
Auto-abs adsorption (3x)
Allo-abs detection
Con: 60 mins incubation.
PEG: 15 mins incubation.
All are demonstrated
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.
 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.
 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;17:82-5.
ZZAP
PEG
serum
serum
PeG
ZZAP (ficin)treated red cells
red cells
Judd & Dake (Result 1)
Immunohematology 2001;17:82-5.
ZZAP
Auto-abs removal power
Comparable
Allo-abs studies
anti-Fya
anti-c
anti-Jka
ND - Not Detected.
PEG
1 rxn grade or more weaker
1+S
1+S
1+ - 2+S
ND
weak
w - 1+w
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.
 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.
PEG adsorption effective in removing
auto-abs.
Fail to detect allo-abs due to Ig
precipitation.
PEG Does precipitate Immunoglobulin
??
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.
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;39:1272-3.
PEG-adsorbed Sera
4oC Storage
PEG-unadsorbed
Sera
1 x anti-E
Mix Before
Sampling
Centrifuge &
Sample Supernatant
1+S
1+
1/2 +
1 x anti-Fya
2+
2+
1+
5 x abs (day 0)
-
5 x abs (day 4 )
-
Same Degree
Same Degree
4 abs Same degree
1 anti-Fya weaken
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?
Method to separate serum-PEG
mixture after adsorption.
?? Centrifugation force ??
?? Duration ??
?? Temperature ??
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
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
Precipitates Ig.
Weak allo-abs may not be detected
after PEG adsorption.
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
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.
End
Questions & Comments are welcome