Page 84 - Cardiac abnormalities after aneurysmal subarachnoid hemorrhage

Chapter 6
of the local medical ethics committee, and in accordance with the Declaration of Helsinki. Use of material for research after completion of a pathological examination is part of the patient contract in the participating hospitals.
Immunohistochemistry
The heart tissue samples of both the SAH patients and the control subjects were fixed in 4% buffered formaldehyde solution and embedded in paraffin for the preparation of 4 μm sections. Sections were then dewaxed, dehydrated, and antigen retrieval was performed by boiling in 10 mM sodium citrate buffer, pH 6.0, for 10 minutes in a microwave oven. Sections were pre-incubated with normal serum for 10 minutes. Rabbit serum was used for monoclonal antibodies; swine serum was for polyclonal antibodies. Pre-incubation was followed by incubation with primary antibodies for 1 hour.
The primary antibodies that we used are: Myeloperoxidase (MPO), mouse anti- human CD68, mouse anti-human CD45, rabbit anti-human Complement C3d and mouse anti-human CD31, all from Dako Cytomation, Denmark.
Sections were subsequently rinsed in Phosphate Buffered Saline (PBS), and incubated with a biotin-labeled secondary antibody (Rat-anti-Mouse-biotin (RαM-biotin) or Swine-anti-Rabbit-biotin (SαR-biotin)) for 30 minutes. After washing in PBS, sections were incubated with streptavidin-biotin complex/HRP (sABC/HRP) for 1 hour. After the sABC/HRP incubation, sections were rinsed with PBS, followed by visualization with 3,3’-diaminobenzidine (DAB 0.1 mg/mL, 0.02% H202). Sections were subsequently counterstained with hematoxylin, dehydrated and covered. As a control, the same staining procedure was used, but instead of primary monoclonal or polyclonal antibody, PBS was used.
Morphometric analysis
Two observers (I.B., W.L) scored the number of extravascular neutrophil granulocytes (MPO positive), lymphocytes (CD45 positive) and macrophages (CD68 positive). This was done by counting the number of positive cells within a fixed grid drawn on the microscopic slide with the specimen. To minimize inter-observer variability, some of the slides were scored on a two person multi-viewing microscope, some were done separately. Additionally a third observer, an experienced pathologist, checked samples at random. Both observers were blinded with respect to the origin of the slides (SAH versus controls). In case of significant differences in scoring results between the 2 observers, both observers examined the same myocardial slides simultaneously.
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