C4d - staining: Comparison of methodsC. Seemayer, A. Gaspert, M. Mihatsch

Introduction

Tx-Normal

Detection of C4d:

Frozen sections, mAb QuidelParaffin sections:C4dpAb, Biomedica

Comparison of results by 2 investigators

Renal allograft biopsies

Frozen sections

Formalin fixation

Paraffin sections

Indirect IF

Mouse monoclonal anti-C4d antibody

IHC with ABC method

Rabbit polyclonal anti-C4d antibody

Cryo-conservation

Aim of the study:

1.Comparison of frozen to frozen biopsies with respect to time-dependent stability and investigator dependent reproducibility of results

2.Comparison of F-IF and corresponding P-IHC staining patterns

3.Investigation of P-IHC staining patterns focusing on the inter- and intra-observer variability

Materials & Methods (1)

Patients and biopsies

– retrospective study

– 64 kidney allograft Bx (Basel 1996 - 2004)

– parallel Bx cores either frozen or formalin fixed

– 240 renal allografts Bx (50 different units)

– independent analysis by two renal pathologists

Materials & Methods (2)

Immunofluorescence

– snap frozen tissues

– indirect IF technique

– 1st ab: mouse anti-human anti-C4d antibody

– dilution 1:50

– Quidel (San Diego, CA, USA)

– 2nd ab: Alexa-Fluor 488-labeled goat anti-mouse IgG

– dilution 1:200

Materials & Methods (3)

Immunhistochemistry

– formalin fixed and paraffin embedded tissue sections

– rabbit polyclonal antibody

– anti-C4dpAb (Biomedica, Vienna, Austria)

– antigen: split product C4d (aa 1252-1256 of C4)

– dilution 1:10 and 1:20

– heat antigen retrieval

– ABC Elite complex method

Materials & Methods (4)

Evaluation / Scoring

– Diffuse expression: > 50% of PTC positive

– Focal expression: 10 PTC < 50% of PTC positive

– Focal minimal expression: 3 - 10 PTC positive

– Negative: < 3 PTC or completely negative

Results

Qualitative differences between frozen and paraffin sections

Staining patterns for C4d in frozen and paraffin sections

C4d in frozen sections C4d in paraffin sections

Specific staining in normal kidneys

Glomerular mesangium and less severe peripheral BM

Not present

Diagnostic staining in transplants

Linear staining of cortical PTC in unscared areas

Linear staining of cortical PTC in unscared areas

Special findings in cases with PTC positivity

Often stronger staining in PTC of the medulla than in the cortex

Strong staining in areas of fibrosis and tubular atrophy

Stronger staining of glomeruli

Special findings in diseased kidneys

Strong staining of glomerular BM in case of glomerular damage

Protein deposits in arteriolar hyalinosis (common)

Thickened tubular BM (rare)

Staining of unknown specificity Endothelium of arteries

Staining of plasma in capillaries

In some cases strongly staining granules along PTC making inrerpretation sometimes impossible

Protein droplets in tubular cells

Cellular interstitial infiltratesa

Results

1.Comparison of frozen to frozen biopsies with respect to time-dependent stability and investigator dependent

reproducibility of results

Results

1. Comparison of F-IF and correspondingP-IHC staining patterns

Overall comparison of C4d expression in frozen (IF) and formalin fixed (IHC) sections

No. of cases

Comparison of C4d expression in diffuses cases frozen (IF) versus paraffin embedded (IHC)

0

5

10

15

20

25

30

D (IF) D F FM Neg

Comparison of C4d expression in focal cases frozen (IF) versus paraffin embedded (IHC)

0

5

10

15

20

25

F (IF) D F FM Neg

No. of cases

Comparison of C4d expression in focal mini cases frozen (IF) versus paraffin embedded (IHC)

0

1

2

3

4

5

6

7

FM (IF) D F FM Neg

No. of cases

Comparison of C4d expression in negative cases frozen (IF) versus paraffin embedded (IHC)

0

2

4

6

8

10

Neg D F FM Neg

No. of cases

Number of PTC expressing C4d in diffuse cases: frozen (IF) versus paraffin embedded (IHC)

% of C4d PTC

IF IHC

Investigator dependent variability :Frozen (IF2) versus paraffin in investigator 1 / 2

Type of comparison Compared item Kappa-value

Frozen to paraffin Investigator 1

antibody-dilution 1:10 1:20

0.340.29

Investigator 2antibody-dilution 1:10 1:20

0.320.27

Inter- and intra-investigator dependent variability in paraffin: Investigator 1 / 2 and antibody

dilutions 1:10 / 1:20

Type of comparison Compared item Kappa-value

Paraffin to paraffin Investigator 1 / 21:101:20

0.570.63

Intra-observer (Inv.2)1:101:20

0.680.83

Conclusion

C4d staining results reported on frozen tissue samples

using IF with a monoclonal antibody appear to be better

suited for diagnostic as well as research purposes.

Future studies should correlate C4d staining patterns with

circulating donor specific antibodies.

Unanswered Questions

– Grading: negative, focal and diffuse

– Significance of C4d in glomeruli

– Co- staining of C4d and other complement factors

Take Home Message

1. C4d in PTC is a footprint of a humoral immune response:Search for donor specific Ab

2. Tx-glomerulitis, Tx endarteritis, leukocytes in PTC are the most important morphological indicators of humoral rejection:Search for C4d

3. Use -whenever possible- frozen sections for the detection of C4dFocal deposits in frozen sections may be lost in paraffin sections due to lower sensitivity of the method.

Open Questions

– Clinical significance of focal deposits of C4d in PTC

– Clinical and biological significance of late C4d deposits (after years)

– Is the „dose“ of antibodies relevant for the morphological sequelae?

– Why are donor specific antibodies not always associated with C4d deposits?

– Why is the morphology so variable in C4d positive cases?

– Why is only C4d present and no other complement components?

– Is the effect of antibodies against HLA Class I the same as against Class II?

– Pathogenesis of Tx-glomerulitis and Tx-endarteritis without C4d deposits.

Open Questions