Clinical findings
A 43 year - old man, admitted to an hospital due to a severe ulcerative rectocolitis, submitted to total parenteral nutrition (TPN) for 5 days. He underwent abdominal surgery and remained on TPN which was administered without a multivitamin solution. After 3 weeks of receiving TPN he presented hypotension and tachycardia, confusional state and agitation. He complained fever, abdominal pain, nausea, and vomiting; abdominal local tenderness was present. The clinical course quickly worsening; on the fourth week of TNP the man suddenly experienced cardiac arrest and unexplained lactic acidosis: pH7.140; pCO2 31.1 mmHg; lactate 19 mmol/L; HCO3 11.1 mmol; SBE -18.7 mmol. He was immediately administered vasoactive drugs and bicarbonate. One hour later he died. The dosing of the thiamine resulted equal to 60 nmol/L (n.v. 66 - 200 nmol/L).
Immunohistochemical findings
Pathologic features were estimated using histologic sections stained by haematoxylin-eosin (H&E) and thrichrome stain. In addition, immunohistochemical investigation of samples was performed utilizing antibodies anti-CD 45, anti-CD15, anti-CD68 (DAKO, Copenhagen, Denmark) and TUNEL assay (Chemicon, Temecula, CA, USA). We used 4 mm thick paraffin sections mounted on slides covered with 3, amminopropyl-triethoxysilane (Fluka, Buchs, Switzerland). A pre-treatment was necessary to facilitate antigen retrieval and to increase membrane permeability to antibodies. The primary antibody was applied in a 1:600 ratio CD 45, in a 1:50 ratio CD15, in 1:200 ratio for CD68 and incubated for 120 min at 20°C. For TUNEL assay (Apotag Plus Peroxidase In Situ Apoptosis Detection Kit, Chemicon, Tamecula, CA, USA) sections were pre-treated with Proteinase K (Sigma-Aldrich, Buchs, Switzerland) (20 μg/ml) for 15 min, at 20°C; covered and incubated with the TdT enzyme, diluted in a ratio of 30% in reaction buffer for 60 min, at 38°C; put in a coupling jar containing working strength stop/wash buffer, agitated for 15 seconds, and incubated for 10 minutes at 20°C; covered and incubated with anti- digoxigenin conjugate for 30 minutes, at 20°C. The positive reaction was visualized by 3,3- diaminobenzidine peroxidation according to standard methods. The sections were counterstained with Mayer's haematoxylin, dehydrated, cover slipped and observed in a Leica DM4000B optical microscope (Leica, Cambridge, UK).
For semi-quantitative analysis, slides were scored in a blinded manner by two observers (MN, IR). Intensity of immunopositive expression was assessed semiquantitatively in the scale 0-4 as follows: 0 = no immunoreactivity, 1 = mild immunopositivity in scattered cells, 2 = immunopositivity in up to one third of cells, 3 = immunopositivity in up to half cells and 4 = strong immunopositivity in the majority or all cells. In cases of divergent scoring, a third observer (VF) decided the final category. The samples were also examined under a confocal microscope, and a three-dimensional reconstruction was performed (True Confocal Scanner, Leica TCS SPE).
In both cases, myocytes nuclei labelled by TUNEL assay showed an intense, wide, positive reaction. In order to obtain the determination of the fraction of myocytes nuclei labelled by TUNEL, the number of myocytes nuclei per unit area of tissue was determined by counting an average of 10 fields, 1.4 mm2 each, at a magnification of x10 in each area of myocardium sampled. The percentage of apoptotic myocytes nuclei was determined. In case 1, the immunohistochemical study revealed an intensive positive result to TUNEL assay: approximately 38 ± 18% apoptotic cells were observed. CD15 and CD45 positive reactions interesting the anterior left ventricular samples, were observed too.
In case 2, the immunohistochemical study revealed an intensive positive result to TUNEL assay: approximately 54 ± 16% apoptotic cells were observed. CD68 positive reaction was observed around the foci of myocardial necrosis.
In both cases the careful post-mortem examination led to acute cardiac failure due to beriberi as cause of death.