Sulen Sarioglu, Mehtat Unlu, Mustafa Sakar, Taner Camsari, Mehmet Turkmen, Hulya Ellidokuz

Departments of Pathology, Biomedical Technology, Nephrology, Pediatric Nephrology, Preventive OncologyFaculty of Medicine, Dokuz Eylül University

Izmir, Turkey

The staining patterns and intensity of immune deposits are valuable for the diagnosis of glomerular diseases

Semiquantitative measures have been applied pioneered by Pirani et al. for interstitial fibrosis, tubular atrophy and immune deposits

Lately quantitative methods are applied for interstitial inflammation, fibrosis and atrophic changes with clinical and prognostic correlations

Attempts for quantification of immune deposits are sparse. Previously Danielevicz et al. measured the electron dense deposit area in IgA nephropathy and mesangial proliferative glomerulonephritis.

In this series Semiquantitative scores (SS) for staining

were compared with mean optical density, intensity and total optical density measurements by image analysis software

The properties of staining in different diseases are described

Different methods of quantification is compared in a series of cases.

27 (54%) IgA nephropathy (IgAN) 8 (16%) membranous nephropathy (MN) 8 (16%) membranoproliferative glomerulonephritis (MPGN) 7 (14%) systemic lupus erithematosus nephritis (SLE-N) DIF: anti-IgG, IgA, IgM, C3, C1q, lambda and kappa (DAKO,

Carpinteria, CA) EM examination + Semiquantitative scores (SS) for DIF positive cases that

were previously given for reporting renal biopsies were used for comparison with measurements from these digital images.

The demographic characteristics, diagnosis and serum creatinin (Cr) values at the time of biopsy and the latest one during follow up were retrieved from the hospital files.

Digital images were used for the image analysis. Sparing the glomerular tuft, all the surrounding areas were

cleaned at the Windows Paint Brush Program Green colour were selected by the pathologist on visual

observation by multiple clicking on the image. Subsequently, the system selected the areas with the same configuration of staining, converted into pixel units.

By this application “intensity” was determined. The software also measured the optical density of the

selected pixels. The optical density of light was measured in a scale of 0 (black) to 255 (white) for each pixel.

The mean value of the optical densities of all selected pixels was “mean optical density” (MOD)

The “total optical density” (TOD) was equal to intensity x MOD.

All the slides were sectioned by the same technician, by the same frozen section technique at 4 micron meter.

The intensity (area) measurement of the programme was previously tested for inter and intraobserver reliability for histochemistry (*)

The measurement of optical density was performed by the software, always giving the same result for the selected area.

*Sis B, Sarioglu S, Sokmen S, et al. J Clin Pathol. 2005 Jan;58(1):32-8.

Correlation analysis, Kruskal Wallis Test with Bonferonni and linear regression analysis were performed by Statistical Package for the Social Sciences (SPSS) 11.00.)

The mean age of the patients was 35,14+20,34 (range 4-76).

The mean Cr values at biopsy was 1,43+1,32 (range 0,29-5,86).

The mean latest Cr values available was 1,25+1,24 (range 0,44-6,34) mg/dl.

The mean follow up was 8,35+6,5 (range 1-28) months.

A case of SLEN:

Semiquantitative score (SS) 2,

intensity: 12,

mean optical density (MOD):44,

total optical density (TOD):528 (anti C1q)

Original image

After paint brush process

Selected area with image analysis software

A case of MN;

SS:3

intensity:18

MOD:63

TOD:1134 (anti IgG)

Original image

After paint brush process

Selected area with image analysis software

MPGN;

SS 3

intensity:23

MOD:85

TOD:1636 (anti C3)

Original image

After paint brush process

Selected area with image analysis software

The highest mean values for IgG ◦ MN cases by SS, ◦ MPGN cases with MOD ◦ SLE-N cases with intensity and TOD.

The highest mean values for IgA ◦ IgAN cases by SS and MOD methods, ◦ SLE-N cases by intensity and TOD methods.

The highest mean values for C3◦ MPGN cases with SS, intensity,TOD and MOD

SS, intensity, MOD and TOD images with positive staining (199 cases).

Mild positive correlation between SS with intensity and TOD (r=0,433, p=0,000 and r=0,440, p=0,000 respectively),

No correlation between SS and MOD (r=0,085, p=0,236)

SS was correlated with intensity and TOD for IgG (r=0,491p=0,004 and, r=0,542, p=0,001 respectively), IgA (p=0,028 and r=0,344, p=0,016 and r=0,373 respectively) IgM (p=0,001 and

r=0,519, p=0,035 and r=0,387 respectively) and C3 (p=0,028 and r=0,344,

p=0,003 and r=0,492 respectively). Only intensity and TOD were included for

linear regression analysis for predicting SS, and only TOD was important for determining SS (p:0.000)

semiquantitative scores

3,002,001,00,00

mean of mean optical density measurements

80

60

40

20

0

semiquantitative scores

3,002,001,00,00

mean of total optical density measurements

1000

800

600

400

200

0

semiquantitative scores

3,002,001,00,00

mean of intensity measurements

14

12

10

8

6

4

2

0

INTENSITY MOD

TOD

SS and TOD (p=0,045 and r=0,717) for MN cases for IgG.

MPGN cases for SS with intensity and TOD (p=0,015 and r=0,850, p=0,015 and r=0,850 respectively) for IgG.

SLE-N cases for SS with intensity and TOD (p=0,002 and r=0,934, p=0,003 and r=0,926 respectively) for IgA.

SS with intensity and TOD for MPGN cases (p=0,049 and r=0,757, p=0,049 and r=0,757 respectively) for IgG.

There was difference between disease groups for SS of IgG and for IgA, (p=0,041 and

p=0,008 respectively) and for intensity of IgG (p=0,001),

SS of IgG for MN (mean= 2,36+0,74) was higher than IgAN (mean: 1,27+0,47)

SS for IgA (mean= 2,44+0,64 ) was higher for IgAN than MN (mean= 1,00+0,00) (p=0,004, p=0,005 respectively).

The intensity of IgG was higher for MN (mean=8,16+3,79) compared to IgAN cases (mean=

1,75+3,50) (p=0,000). The SS for IgA (mean= 6,60+7,56 ) was higher

than MPGN (mean= 1,75+3,49) for IgAN cases (p=0,003).

Only intensity of IgG was significantly more for SLE-N (mean=21,88+26,49) than IgAN cases (mean=1,75+3,50) (p=0,001).

There was no difference for MN and MPGN or SLE-N, and between MPGN and SLE when any of the methods was considered.

In one case the SS for IgA was equal to IgG (1 vs 1).

The digital measurements were both higher for IgA for intensity and TOD (0,40 vs 0,90 for

intensity and 23,60 versus 51,30 for TOD) For two IgAN cases MOD of IgA was higher

than IgG, but the intensity was slightly higher for IgG.

IgA nephropathy, both of the images SS were same but the intensity and TOD were higher for anti-IgA staining.

a: SS:1, intensity:0,40, MOD:59, TOD:23,6 (anti IgG)

b: SS:1, intensity: 0,90, MOD:57, TOD:51,3 (anti IgA)

a b

In one MPGN case the SS were same for IgG, IgA and IgM, (1,1 and 1 respectively) but intensity and TOD were highest for IgM and higher for IgG compared to IgA allowing easy differential diagnosis (For IgG, IgA and IgM; intensity: 0,15, 0,03 and 0,75 and TOD=15,75, 2,65 and 74,25 respectively).

For two of the SLE-N cases the SS and intensity for IgG, IgA were same, but TOD measurements could demonstrate that actually IgG was the predominant antibody.

For 4 (8%) cases the digital measurements and especially TOD were useful for determining the predominant immune deposit which could not be ascertained by SS.

When all the cases were considered creatinin at the time of biopsy was mildly correlated with intensity and TOD of IgM (p=0,004; r=0,44 and p=0,07; r=0,42 respectively).

For IgAN cases creatinin at the time of biopsy was moderately correlated with intensity and TOD of C3 (p=0,03; r=0,49 and p=0,03;

r=0,48 respectively). The SS were not correlated with first and

latest Cr values. There was no correlation between latest

Cr values and any immune deposit measurements.

SS given by the pathologists seems to be determined related to TOD.

SS presented results corraleted with TOD and intensity

For some cases only TOD allowed discrimination of the predominantly deposited antibody.

TOD might present better correlation with clinical features than SS but this should be evaluated in series with longer folow up.

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