ISSN 2234-3806 • eISSN 2234-3814

572 www.annlabmed.org https://doi.org/10.3343/alm.2019.39.6.572

Ann Lab Med 2019;39:572-576https://doi.org/10.3343/alm.2019.39.6.572

Brief CommunicationDiagnostic Immunology

EDTA Treatment for Overcoming the Prozone Effect and for Predicting C1q Binding in HLA Antibody TestingHoon Seok Kim , M.D., Ae-Ran Choi , M.T., Mina Yang , M.D., and Eun-Jee Oh , M.D., Ph.D.Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea

The Luminex-based single antigen bead (SAB) assay is widely used to detect HLA anti-body in transplant recipients. However, one limitation of the SAB assay is the prozone ef-fect, which occurs mostly as a result of complement interference. We investigated the effi-cacy of EDTA treatment for overcoming the prozone effect and predicting C1q binding of HLA antibody. We subjected 27 non-treated (naïve) and EDTA-treated serum samples from highly sensitized patients to IgG-SAB assays, and we confirmed the prozone effect in 53% and 31% of class I and class II antibody tests, respectively, after EDTA treatment. When we conducted additional assays after dithiothreitol treatment and serum dilution, EDTA was the most efficacious in eliminating the prozone effect. Reducing the prozone ef-fect by EDTA treatment strengthened the correlation between IgG mean fluorescence in-tensity (MFI) and C1q MFI values (ρ=0.825) as compared with the naïve sera (ρ=0.068). Although C1q positivity was dependent on the concentration of HLA antibody in EDTA-treated sera, the correlations varied individually. Overall, our results confirmed the efficacy of EDTA treatment for overcoming the prozone effect. EDTA treatment showed a positive effect on the correlation between IgG MFI and C1q MFI values.

Key Words: Prozone effect, EDTA, Single antigen bead assay, C1q binding, HLA antibody

Received: January 4, 2019Revision received: April 1, 2019Accepted: June 9, 2019

Corresponding author: Eun-Jee Oh, M.D., Ph.D.Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 06591, Korea Tel: +82-2-2258-1641Fax: +82-2-2258-1719E-mail: ejoh@catholic.ac.kr

© Korean Society for Laboratory MedicineThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecom-mons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Accurate assessment of HLA antibody is crucial for successful

transplant management [1]. The Luminex-based single antigen

bead (SAB) assay is widely used for sensitive detection of low

concentrations of antibody [2-5]. In the SAB assay, antibody

amount is determined semi-quantitatively and is expressed as

the mean fluorescence intensity (MFI) after reaction with anti-

human IgG antibody reagent labeled with a fluorescent dye.

MFI values correlate with transplant-related outcomes; thus,

monitoring MFI values may be useful for risk assessment after

transplantation [6, 7]. In addition, SAB assays for detecting C1q-

binding HLA antibodies (C1q SAB) have been introduced to bet-

ter discriminate clinically relevant antibodies [8].

However, clinical application of the SAB assay is limited be-

cause of technical issues, including assay variability, complement

interference, and the prozone effect [9]. Inhibition causing false-

negative or falsely decreased MFI values has been observed in

approximately 70% of highly sensitized patients [10]. Several

studies have attempted to eliminate the prozone effect by treat-

ment with EDTA or dithiothreitol (DTT), preheating, and/or dilu-

tion of serum [9, 11, 12]. There are multiple explanations for

the prozone effect, including bead saturation [13, 14], steric

hindrance from complement complex [11], and the presence of

IgM HLA-specific antibodies [15]. Complement interference in-

volves covalent binding and accumulation of C4 and C3 degra-

dation products on the immune complex, which prevent fluo-

rescent anti-IgG from binding to IgG. EDTA eliminates the pro-

zone effect because, as an iron-chelating reagent, it can inhibit

the formation of C1qrs complement complex and prevent com-

1 / 1CROSSMARK_logo_3_Test

2017-03-16https://crossmark-cdn.crossref.org/widget/v2.0/logos/CROSSMARK_Color_square.svg

Kim HS, et al.EDTA overcomes prozone effect in HLA antibody test

https://doi.org/10.3343/alm.2019.39.6.572 www.annlabmed.org 573

plement cascade activation [16]. DTT disrupts the pentameric

structure of IgM and the disulfide bonds in complement mole-

cules, thus reducing the prozone effect [15]. In this respect,

SAB assays as a standard should include serum treatment to

correct the prozone effect because MFI values are often used

as a surrogate marker of antibody strength. Although a recent

meeting report recommended EDTA and/or titration [1], there is

currently no verified standard method, and different treatment

methods are used across different laboratories.

We compared MFI values of SAB assays in serum samples

treated with or without EDTA, DTT, or dilution. In addition, we

evaluated the correlation between IgG-MFI and C1q-MFI values

after EDTA treatment. To our knowledge, this is the first study to

investigate the effects of EDTA on specific HLA loci in combina-

tion with C1q-MFI.

We collected leftover sera from 27 highly sensitized patients

(median age=49 years, M:F=14:13). All samples contained

strong HLA antibodies (MFI≥10,000), and the median panel-

reactive antibody (%) was 95%. The patients had become sen-

sitized by previous transplantation with or without transfusion or

pregnancy (81.5%, N=22), previous pregnancy and transfu-

sion (7.4%, N=2), previous pregnancy alone (7.4%, N=2), or

previous transfusion alone (3.7%, N=1). The Institutional Re-

view Board of Seoul St. Mary’s Hospital, Seoul, Korea, approved

this study (KC18SESI0323). LABScreen Single Antigen kits and

the LABScan 3D system (One Lambda, A Thermo Fisher Scien-

tific Brand, Canoga Park, CA, USA) were used to detect HLA

antibodies (11 samples for class I only, eight samples for class II

only, and eight samples for both class I and class II). For EDTA

treatment, a 0.5 M EDTA solution was diluted 1:20 (final con-

centration of 25 mM), added to the serum, vortexed, and centri-

fuged at room temperature for 10 minutes before testing [10].

We defined the prozone effect as doubled MFI values, with an

MFI cutoff of 1,000, or 5,000 MFI increment after serum treat-

ment compared with the results from naïve serum, considering

recent recommendations and reports [1, 12, 17].

In a comparison of naïve and EDTA-treated samples, we in-

vestigated MFI data from 3,363 beads (1,843 class I and 1,520

class II) from 35 tests (19 class I and 16 class II) (Fig. 1). The

prozone effect was identified in 53% (10 out of 19) of class I

antibody tests and in 31% (5 out of 16) of class II antibody tests

after EDTA treatment. Of all beads, 8.3% (26.5% of positive

beads) were affected by complement inhibition, with the me-

dian MFI increasing from 2,921 (range, 0–13,890) to 21,043

(1,024–27,874) after EDTA treatment. Regarding HLA specific-

ity, prozone effect incidences were as follows: HLA-A, 28.1%;

HLA-B, 43.6%; HLA-C, 3.6%; HLA-DR, 2.7%; HLA-DQ, 18.0%;

and HLA-DP, <0.1% of positive beads. These findings were

consistent with a previous report wherein HLA-C beads were the

least affected among class I beads and HLA-DQ beads were the

Fig. 1. Comparison of MFI values in naïve and EDTA-treated sera. (A) Class I IgG-SAB results in 19 sera (1,843 beads); (B) Class II IgG-SAB results in 16 sera (1,520 beads). Abbreviations: MFI, mean fluorescence intensity; SAB, single antigen bead.

35,000

30,000

25,000

20,000

15,000

10,000

5,000

0

0 5,000 10,000 15,000 20,000 25,000 30,000 35,000

MFI values for naïve samples

MFI

valu

es fo

r EDT

A-tre

ated

sam

ples

Prozone+

HLA-AHLA-BHLA-C

A

35,000

30,000

25,000

20,000

15,000

10,000

5,000

0

0 5,000 10,000 15,000 20,000 25,000 30,000 35,000

MFI values for naïve samples

MFI

valu

es fo

r EDT

A-tre

ated

sam

ples

Prozone+

HLA-DRHLA-DQHLA-DP

B

Kim HS, et al.EDTA overcomes prozone effect in HLA antibody test

574 www.annlabmed.org https://doi.org/10.3343/alm.2019.39.6.572

most affected among class II beads [18].

We selected five sera and additionally tested class I IgG after

1:8 dilution in phosphate-buffered saline and treatment with 5

mM DTT at 37°C for 30 minutes [19]. When we compared the

three serum treatment methods, EDTA treatment was the most

efficacious for overcoming prozone effect (Fig. 2). This finding

was consistent with previous studies that showed the superior

efficacy of EDTA for overcoming the prozone effect [12, 16].

DTT treatment and dilution resulted in small increases in MFI

values in some of the beads, indicating a residual prozone ef-

fect, presumably because only IgM antibodies were removed

(Supplemental Data Table S1).

We subjected 10 samples with the prozone effect (N=5 for

class I and N=5 for class II HLA antibodies) to C1qScreen as-

says (One Lambda). We selected beads that were positive in

EDTA-treated sera to calculate Spearman’s correlation coeffi-

Fig. 2. MFI values for naïve samples and samples treated with 1:8 dilution, DTT, and EDTA, and C1q assay using sera from five sensitized patients. (A) MFI values for all samples with various treatments. (B) Correlations between MFI values for naïve, diluted, DTT-treated, and EDTA-treated samples.Abbreviations: MFI, mean fluorescence intensity; DTT, dithiothreitol.

A

B

Kim HS, et al.EDTA overcomes prozone effect in HLA antibody test

https://doi.org/10.3343/alm.2019.39.6.572 www.annlabmed.org 575

Fig. 3. Correlation of MFI values from C1q- and IgG-SAB assays using (A) naïve sera and (B) EDTA-treated sera. Abbreviations: MFI, mean fluorescence intensity; SAB, single antigen bead.

70,000

60,000

50,000

40,000

30,000

20,000

10,000

0

0 5,000 10,000 15,000 20,000 25,000 30,000

MFI values for naïve samples

MFI

valu

es fo

r C1q

sam

ples

Class IClass II

ρ=-0.0684Class I: ρ=-0.358Class Il: ρ=0.391

A

70,000

60,000

50,000

40,000

30,000

20,000

10,000

0

0 5,000 10,000 15,000 20,000 25,000 30,000

MFI values for EDTA-treated samplesM

FI va

lues

for C

1q sa

mpl

es

B

Class IClass II

ρ=0.825Class I: ρ=0.886Class Il: ρ=0.749

cient (ρ). MFI values were summarized as median (range). For

statistical analysis, we used MedCalc Statistical Software version

18.9 (MedCalc Software, Ostend, Belgium).

When naïve sera were used, the correlation between IgG-MFI

values and C1q-MFI values was weak (ρ=-0.0684; class I: -0.358,

class II: 0.391). After EDTA treatment, the correlation between

IgG-MFI values and C1q-MFI values became stronger (ρ=0.825;

class I: 0.886, class II: 0.749) (Fig. 3). Beads that displayed low

IgG-MFI values in naïve sera, but high C1q-MFI values, showed

high IgG-MFI values after EDTA treatment. In naïve sera, C1q-

SAB beads with MFI≥1,000 (19.7% of all beads) had a median

IgG-MFI value of 2,842 (range, 0–19,256). In EDTA-treated

sera, C1q-positive beads had a median IgG-MFI value of 20,900

(1,539–27,874). These results supported previous findings that

C1q binding ability depends on the concentration of HLA anti-

body [12]. Tambur and Wiebe [9] reported that MFI cutoffs that

predicted C1q positivity varied from 6,237 to 14,154 using non-

treated samples. However, not all beads that had high IgG-MFI

values in EDTA-treated sera showed C1q positivity, and some

high-level IgG antibodies did not bind to C1q. When we plotted

IgG-MFI against C1q-MFI values from the data in 10 patients

separately, we found a wide range of correlation coefficients

(0.283–0.977). Therefore, it remains to be elucidated whether

there is an interdependence between C1q positivity and IgG-

MFI values.

Some limitations of the present study were that we could not

test serial dilutions of samples, and we did not confirm the HLA-

specific antibodies against intact HLA antigen on the lympho-

cyte. Because high-level antibodies against denatured HLA pro-

tein might also bind to C1q [20], future studies on interference

by denatured HLA are needed. In addition, we selected samples

with high panel-reactive antibody and strong HLA antibodies.

Thus, our results may not represent the frequency of the pro-

zone effect in SAB assays in general.

In conclusion, we confirmed the efficacy of EDTA treatment

for eliminating the prozone effect. Further, EDTA treatment had

a positive effect on the correlation between IgG MFI and C1q

MFI values. Our study highlights the importance of method stan-

dardization to correct the prozone effect.

Authors’ Disclosures of Potential Conflicts of Interest

No potential conflicts of interest relevant to this article are re-

ported.

Acknowledgments

This work was supported by the National Research Foundation

of Korea (NRF) grant funded by the Korea government (MSIP)

(NRF-2017R1A2B4011181), Korea.

Kim HS, et al.EDTA overcomes prozone effect in HLA antibody test

576 www.annlabmed.org https://doi.org/10.3343/alm.2019.39.6.572

ORCID

Hoon Seok Kim https://orcid.org/0000-0001-8980-8177

Ae-Ran Choi https://orcid.org/0000-0001-6150-1800

Mina Yang https://orcid.org/0000-0001-7431-6380

Eun-Jee Oh https://orcid.org/0000-0001-5870-915X

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A*69

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711

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210

647

479

058

870

250

370

188

6112

837

3

(Con

tinue

d to

the

next

pag

e)

Kim HS, et al.EDTA overcomes prozone effect in HLA antibody test

www.annlabmed.org https://doi.org/10.3343/alm.2019.39.6.572

A llel

e sp

ecifi

city

Sam

ple 1

Sam

ple 2

Sam

ple 3

Sam

ple 4

Sam

ple 5

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

A*80

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B*07

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B*08

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B*13

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B*13

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B*14

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B*14

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B*15

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B*15

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B*15

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B*15

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B*15

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B*15

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B*15

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B*18

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B*27

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B*27

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B*38

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B*39

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B*40

:01

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B*40

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510

,091

(Con

tinue

d to

the

next

pag

e)

Supp

lem

enta

l Dat

a Ta

ble

S1. C

ontin

ued

Kim HS, et al.EDTA overcomes prozone effect in HLA antibody test

https://doi.org/10.3343/alm.2019.39.6.572 www.annlabmed.org

A llel

e sp

ecifi

city

Sam

ple 1

Sam

ple 2

Sam

ple 3

Sam

ple 4

Sam

ple 5

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

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00

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Supp

lem

enta

l Dat

a Ta

ble

S1. C

ontin

ued

(Con

tinue

d to

the

next

pag

e)

Kim HS, et al.EDTA overcomes prozone effect in HLA antibody test

www.annlabmed.org https://doi.org/10.3343/alm.2019.39.6.572

A llel

e sp

ecifi

city

Sam

ple 1

Sam

ple 2

Sam

ple 3

Sam

ple 4

Sam

ple 5

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

Naïve

Dilu

tion

DTT

EDTA

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Bea

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the

proz

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effe

ct a

re b

olde

d an

d un

ders

core

d.*M

FI v

alue

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ead

was

≥1,

000

in th

e na

ïve

sam

ple

and

decr

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<1,

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tens

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, sin

gle

antig

en b

ead;

DTT

, dith

ioth

reito

l.

Supp

lem

enta

l Dat

a Ta

ble

S1. C

ontin

ued