The study material consisted of 102 astrocytomas, of which 9 were grade I pilocytic astrocytomas and 93 were grade II-IV diffusely infiltrating astrocytomas [grade II (n = 21), grade III (n = 16) and grade IV (n = 56)]. Of the 93 diffusely infiltrating astrocytomas (grades II-IV), 67 were primary tumors and 26 recurrent. First, the astrocytoma specimens were fixed in 4% phosphate-buffered formaldehyde and processed into paraffin blocks. On the basis of H&E-stained slides, one neuropathologist (H.H.) evaluated the tumors according to the WHO 2007 criteria . One histologically representative tumor region was selected from each sample specimen. Thus collected samples were mounted into multitissue blocks, which were constructed with a custom made instrument (Beecher Instruments, Silver Spring, MD, USA). The sample diameter of the tissue cores was 1000 μm. Also samples representing normal, non-neoplastic brain were included into blocks to serve as controls.
The samples were obtained from surgically operated patients at the Tampere University Hospital, Tampere, Finland, during 1983 to 2001. The tumors were radically resected if possible and most patients with grade III-IV astrocytomas also received radiotherapy. Adjuvant radiochemotherapy was not used in the period of the study. Mean patient age was 59 years, the youngest patient being 12 years old and the oldest 85 years old. Overall survival was known for 62 patients. Patient survival was examined in a five year follow-up study. The follow-up time started after primary resection of the astrocytoma and ended after five years of follow-up or if the patient died. The study protocol was approved by the Ethical Committee of Tampere University Hospital and the National Authority for Medicolegal Affairs of Finland.
Two slides were produced from each multitissue paraffin block, cut at 5 μm thickness. Fully automated immunostaining was performed by a Ventana BenchMark LT Automated IHC Stainer (The BenchMark Series automated slide preparation system by Ventana Medical Systems, Tucson, AZ, USA). Ventana EZ Prep solution (catalogue No 950–100, Ventana) was used for deparaffiniztion. For epitope retrieval CC1: Tris -EDTA buffer pH 8.0 (catalogue No 950–124,Ventana) was used at 95°C to 100°C for 30 minutes. The slides were rinsed between steps with Ventana Tris-based Reaction buffer (catalogue No. 950–300, Ventana). Slides were incubated at 37°C for 32 minutes with a C4d specific rabbit polyclonal anti human C4d antibody (Biomedica Medizinprodukte GmbH & Co KG, Wien, Austria) using a 1:10 dilution. The staining kit used was the Ventana Ultraview DAB Detection Kit. A sample of a rejected kidney allograft served as a positive control for C4d immunostaining.
To compare the C4d endothelial staining extent with the overall tumor vascularity, the second slide from each multitissue block was stained with CD34 antibody. Lyophilized mouse monoclonal antibody, specific to the human CD34 molecule ( Novocastra Laboratories Ltd, Newcastle Upon Tyne, UK) was used as an endothelial cell marker. The dilution used was 1: 500.
The extent of the staining reaction was determined by area fractions of the positively stained tumor samples, using a point counting grid . In this method, the sample is viewed with a microscope at magnification x400, and the point counting grid is placed over the tumor sample and the points covering the sample are counted. A value was given to each sample using the formula ((Σ C4d-P)/(Σ Pt)) x 100, where “Σ C4d-P” is the number of C4d positive points of a tumor sample and “Σ Pt” is the number of all points of the grid covering the entire tumor sample. C4d positive tumor area fractions and C4d positive endothelium area fractions were counted separately. Necrotic areas were omitted from the analysis.
The CD34 positive area fraction of the tumor sample was counted using the same morphometric method as for the C4d fractions. CD34 was assumed to stain specifically the endothelial cell cytoplasm and therefore the value acquired from the morphometric count was considered to represent the percentage of the total microvessel area of the tumor sample.
For further analyses, the tumor samples were scored for the intensity of C4d positivity with scores from zero to three (C4d INT). Zero was given if the sample was negative, one if slightly positive, two if clearly positive and three if strongly positive. Both endothelial positivity and tumor cell positivity were taken into account.
The tumor samples were also divided into three groups according to the extent of the staining reaction on the grounds of the C4d positive tumor area fraction (C4d EXT). 1 = no positivity, 2 = 2% or less positive tissue, 3 = more than 2% positive tissue. The tissue samples were also grouped according to the C4d positive endothelial area fraction (C4d END). 1 = no positively stained endothelium was seen, 2 = 0.5% or less of the tissue area was positively stained endothelium, 3 = more than 0.5% of the tissue area was positively stained endothelium.
To statistically compare the C4d endothelial positivity to overall vascularity of the tumor, a variable was created in which the percentage of the C4d positive endothelial area fraction was divided by the CD34 tissue area fraction percentage.
The statistical analyses were performed using SPSS for Windows (Chicago, IL, USA). The significance of associations were defined using Kruskal-Wallis test, Mann–Whitney test and chi-square test. The significance of correlations was determined utilizing Pearson correlation. Kaplan-Meier curves and log-rank test were used in the survival analyses.