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

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 &/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-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.


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.


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 & 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 & BrownImmunohematoloty 1997;13:119-22.

PEPEGG

RefRef

serum

20% PEG

red cells

serum

papain-treatedred cells


Ref PEG

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 1) 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.


?? Macroscopic but not microscopic reading at all stages of LISS, + IAT reading, is recommended (Issit & Anstee, Applied blood group Serology).


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 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 alTransfusion 2001;41:13-7.

PEPEGG

ConventionConventionalal

serum

PeG

red cells

serum

untreatedred 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

Auto-abs adsorption (3x) 2 Not adsorbed All adsorbed

Allo-abs detection All are demonstrated

Con: 60 mins incubation.PEG: 15 mins incubation.


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.


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 & 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 & DakeImmunohematology 2001;17:82-5.

PEPEGG

ZZAPZZAP

serum

PeG

red cells

serum

ZZAP (ficin)-treated red cells

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-Fya 1+S NDanti-c 1+S weakanti-Jka 1+ - 2+S w - 1+w

ND - Not Detected.

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 specificitiesAnti-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.


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.


PEG-adsorbed Sera 4oC Storage

PEG-unadsorbed Mix Before Centrifuge & Sera Sampling Sample 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


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 ofpatient 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 cells37oC, 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.

ConclusionPotential 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.

EndQuestions & Comments are

welcome

Documents

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