For the online version of the manuscript: Crivellari et al. Human protein C zymogen concentrate in
patients with severe sepsis and multiple organ failure after adult cardiac surgery.
--Statistics and laboratory methods used to measure protein C and other parameters.
--Table 1: Microbiological findings.
--Table 2: laboratory variables
--Figure 3: fluid balance
Statistical Analysis
For repeated measurements Friedman’s two-way analysis of variance was used. Post hoc
testing of values versus baseline was performed by Wilcoxon-testing for paired samples; p < 0.05
(two-sided) was considered statistically significant. Data for continuous variables are given as
median and interquartile range. Statistical analyses were performed using Systat 7.0 for Windows
(SPSS Inc., Chicago, IL).
Laboratory methods
Serial venous samples (4.5 ml) were collected in siliconized Vacutainer tubes (Becton-Dickinson,
Plymouth, UK) containing (0.5 ml) tri-sodium citrate (0.129 M) and in tubes containing 0.5 ml of a
mixture of tri-sodium citrate and benzamidine-HCl (200mM) at the following times: before the
bolus dose, 6 hours after bolus and every 12 hours thereafter up to 78 hours. Within 1 hour from
collection, platelet poor plasma was obtained by centrifugation for 10 minutes at 2,000 x g at room
temperature. Prothrombin time (PT, Hemoliance Recombiplastin, Instrumentation Laboratory,
Lexington, MA), activated partial thromboplastin time (aPTT, STA aPTT Kaolin, Diagnostica
Stago, Asnier sur Seine, France ), fibrinogen (FG, clotting assay, STA Fibrinogen, Stago), and Ddimer (STA Liatest D-D, Stago) determinations were performed on fresh citrated plasma samples
with an automated coagulometer (STA, Stago). Plasma aliquots were snap-frozen with methanol
and dry ice and stored at – 70C for additional measurements in citrated plasma of protein C
anticoagulant activity (STA Protein C, Stago), and antithrombin (amidolytic activity, STA
Antithrombin, Stago). Blood samples collected in tri-sodium citrate and benzamidine-HCl were also
centrifuged as described above with plasma aliquots snap-frozen and stored at -70C. Within one
month, prothrombin fragment 1+2 (F1+2, Enzygnost F1+2, Dade-Behring, Marburg), thrombinantithrombin III complex (TAT Enzygnost TAT micro, Dade-Behring, Marburg), and interleukins
(IL) 6, 8 and 10 (R&D systems, Minneapolis, MN, USA) were measured with commercially
available ELISA kits. Quantification of activated protein C (APC) levels was by an home-made
ELISA as described by Liaw et al [18]. Briefly, 96-well vinyl microtiters plates (Costar) were
coated with the monoclonal antibody HAPC 1555 (5 µg/ml in coating buffer: 20mM HEPES,
pH7.5, 150mM NaCl, 5mM CaCl2). Benzamidine-HCl plasma samples were anticoagulated and
recalcified by addition of heparin 2 IU/ml, HEPES 20 mM, pH 7.5 and CaCl 2, 10 mM (final
concentrations) and incubated (100 µl) at room temperature for 30 min with the coated antibody.
Wells were washed thrice and the chromogenic activity of bound APC was measured by addition of
100 µl S2366 (0.5 mM) (Chromogenix, Mölndal, Sweden) in coating buffer. Substrate hydrolysis
was monitored at 405 nm. A standard curve was generated with increasing amounts of APC (Xigris,
Eli Lilly, Indianapolis, IN), from 0 to 50 ng/ml, spiked into coating buffer.
Table 1 online: Microbiological findings for the online version of the manuscript.
Pathogens
Source of sepsis
negative culture
Enterobacter Cloacae
Pneumonia
Acinetobacter Baumanii, Klebsiella Pneumofila
Pneumonia
Acinetobacter Baumanii, Citrobacter Braakii PNEUMONIA
Pneumonia
negative culture
MRSA Staph. epidermidis sepsis
Blood
MSSA Staph. Aureus sepsis in bronchial sample, E.Coli in urine
sample
Pneumonia + blood + urine
Pseudomonas Aeruginosa, Serratia Marcescens PNEUMONIA
Pneumonia
negative culture
Table 2 online. Medians and interquartile range of laboratory variables for the online version of the manuscript. P values are according to
Friedman’s two-way analysis of variance. The asterisks (*) indicate the first time point showing a statistically significant difference over baseline
levels (p ≤ 0.03)
PT (Ratio)
Normal
range
0.90-1.18
aPTT (Ratio)
0.80-1.24
AT (% of normal)
70-130
PC:C (% of normal)
65-140
D-dimer (mg/L)
0.22-0.77
APC (g/L)
0.1-2.4
Fibrinogen (g/L)
1.5-4.00
F1.2 (pmol/L)
69-229
TAT (g/L)
1.0-4.1
Platelet count
(x 109/L)
IL-6 (ng/L)
130-400
IL-8 (ng/L)
<3.12
<31.2
Baseline
1.58
1.32-1.67
1.19
1.17-1.24
55
44-63
40
38-49
4.50
2.24-5.97
0.3
0.1-0.4
6.05
5.66-8.31
370
205-430
9.60
6.65-18.7
78
66-138
474
367-574
121
53-135
6
1.25
1.16-1.41
1.11
1.09-1.16
66
54-68
73*
68-82
3.50
2.52-4.03
0.2
0.1-1.1
6.44
5.87-8.68
334
171-523
9.40
5.95-15.5
75
65-128
195*
121-280
161
79-163
Hours after protein C concentrate bolus infusion
18
30
42
54
66
1.18
1.20*
1.17
1.18
1.20
1.08-1.47 1.11-1.23 1.13-1.21 1.16-1.28 1.18-1.23
1.03
1.06*
0.96
1.12
1.08
1.01-1.25 0.99-1.13 0.94-1.02 0.99-1.18 0.99-1.20
69
73
81*
80
77
60-77
56-85
65-88
58-88
69-87
88
92
86
82
89
80-109
65-104
66-112
75-98
87-106
2.61
3.35
4.03
6.13
4.97
1.68-3.80 2.61-5.54 2.35-5.52 3.63-7.02 3.75-6.48
0.2
0.2
0.3
0.2
0.2
0.0-0.7
0.0-0.8
0.1-0.5
0.0-0.4
0.2-0.7
6.71
6.61
7.06
6.47
7.00
5.75-9.07 6.07-7.79 5.55-8.13 5.40-7.52 5.24-7.12
211
191
212
234
250
141-326
90-444
148-295
132-319
157-315
6.90
6.90
5.60
5.10*
5.60
5.65-10.2 5.75-8.35 4.90-7.40 4.15-5.45 4.75-7.15
104
120
119
122
128
90-171
106-160
108-152
105-148
106-156
123
97
33
65
59
87-211
62-110
26-72
16-86
13-91
96
66
46*
62
48
60-151
40-94
43-90
40-73
37-51
p*
78
1.18
1.17-1.21
1.06
0.92-1.12
72
63-86
90
75-97
5.96
3.56-8.53
0.3
0.1-0.4
6.09
5.31-7.20
234
111-342
10.0
5.55-10.2
133
106-165
55
30-64
59
46-81
0.04
0.01
0.002
0.001
0.09
0.83
0.91
0.38
0.03
0.46
<0.0001
0.04
IL-10 (ng/L)
<7.8
174
116-347
Abbreviations:
PT : prothrombin time;
aPTT: acitvated partial thromboplastin time;
AT: antithrombin;
PC:C: protein C anticoagulant activity;
APC: circulating activated protein C;
F1.2: prothrombin fragment 1.2;
IL: interleukin.
60
48-111
38*
31-90
54
24-65
38
17-71
35
26-72
32
21-46
21
15-36
0.002
Fig 3 (for the online version of the manuscript)
Daily fluid balance immediately and for four days after protein C
bolus administration
1000
500
0
-500
-1000
-1500
-2000
-2500
-3000
-3500
day -1
bolus day
day 1
day 2
day 3
day 4