Laboratory methods

In the retrospective part of the study (I-II) serum concentrations of C-reactive protein (CRP) were collected from the individual patient charts. CRP was routinely determined three times per week during neutropenia. During clinically significant infection, CRP measurements were performed at 1-2 day intervals.

Table 7. Chemotherapy protocol in the AML-92 study of the Finnish Leukaemia Group (2) _________________________________________________________________________

Therapy Dosage Administration Days administered

_________________________________________________________________________ First course (IAT)

Idarubicin 12 mg/m2 _{i.v., 10–15 min 1, 3, 5}

Cytarabine 50 mg/m2 i.v.,bolus 1

100mg/m2 i.v., continuously 1(–7)–9 Thioguanine 75 mg/m2 _{p.o., twice daily 1(–7)–9} Second course (HDAraC-Ida)

Cytarabine 1500 mg (1.0 g)/m2 i.v., 3 h, twice daily 1–5 Idarubicin 8 mg/m2 _{i.v., 30 min 6–8} Third course (MEA)

Etoposide 100 mg/m2 i.v.,1h 1–4

Cytarabine 1000 mg (0.5 g)/m2 i.v., 2 h, twice daily 1–4 Mitoxantrone 12 mg (8 mg)/m2 _{i.v., 30 min 2–5} Fourth course (Amsa-HDAraC)

Amsacrine 115 mg/m2 _{i.v.,2h 1–5}

Cytarabine 3000 mg/m2 i.v., 3 h, twice daily 1–2 Fifth course (HDAraC-Ida)

Cytarabine 1500 mg (1.0 g)/m2 _{i.v., 3 h, twice daily 1–5} Idarubicin 8 mg/m2 _{i.v., 10–15 min 6–8}

_________________________________________________________________________ Dosage and days administered in parenthesis indicate patients 56 years old.

Abbreviations: i.v, intravenously; p.o, orally.

For the prospective part of the study (III-IV), the first blood samples for the measurement of CRP, vascular endothelial growth factor (VEGF) and amino-terminal pro-brain natriuretic peptide (NT-proBNP) serum concentrations were drawn at the beginning of neutropenic fever concomitantly with samples taken for blood cultures. Further samples were collected next morning and then every 24 hours up to three or five days (Figure 3). Platelet and leukocyte counts were routinely examined daily throughout the neutropenic period.

Blood samples for VEGF and NT-proBNP were centrifuged (2200 G 10 minutes) by laboratory technicians. VEGF samples were then stored at -20°C for later analysis and NT- proBNP samples were analysed within 12 hours.

4.2.3.1. Blood cultures

In case of neutropenic fever (neutropenia and a single oral measurement of 38.3°C or >38°C for one hour) 2-3 blood cultures were drawn from cubital veins at 1 hour interval by laboratory technicians. Blood cultures were processed using the automated

blood culture system Bactec 9240 (Becton Dickinson, Sparks, USA). Incubation period for both aerobic and anaerobic bottles were 7 days and for MYCO F / Lytic bottles 42 days.

A single positive blood culture was considered significant if the microbe was a clinically relevant cause of infection. Common skin contaminants (e.g. coagulase-negative staphylococci) were considered significant only if they were found in two consecutive blood cultures or if there was a concurrent skin or catheter infection.

4.2.3.2. Serum C-reactive protein

The concentration of serum CRP was measured with an immunoturbidometric method (Otsuji, 1982, Clin Chem) using a multichannel Hitachi 717 Automatic Analyzer (Hitachi Ltd, Tokyo, Japan) until the year 2000 and then with a Konelab60i Clinical Chemistry Analyzer (Lab systems CLD, Konelab, Helsinki, Finland). The reference value for serum CRP in healthy persons was <10 mg/l.

In the retrospective studies (I-II) also CRP slope velocity was analysed. For all febrile neutropenic periods following intensive chemotherapy, baseline CRP (< 48 h prior to rise of fever, CRP0) and the CRP level 2-3 days after the onset of fever (CRP2-3) was registered. Peak CRP and the time from the rise of fever to the highest CRP value of a given neutropenic period was also registered. CRP slope velocity was defined as the difference between CRP2-3 and CRP0, divided by the number of days and expressed as mg/l/d.

CRP2-3 – CRP0 CRP slope velocity = --- Number of days (2 or 3)

In the prospective studies (III-IV), CRP was measured at the start of neutropenic fever and then every morning during the study period (Figure 3).

Figure 3. Prospective study protocol.

Written informed consent from all patients •with AML < 70 years

•with HDT supported by ASCT

On the first morning after the beginning of neutropenic fever

study blood samples (d1) Neutropenic fever:

neurophil count < 0.5 x 109_{/l and}

fever (single oral temperature 38.3° or a temperature 38.3° for 1 h)

No neutropenic fever

On the second morning from the beginning of neutropenic fever

study blood samples (d2)

On the third morning from the beginning of neutropenic fever

study blood samples (d3)

On the fourth morning from the beginning of neutropenic fever

study blood samples (d4)

Study blood samples were

not collected At the beginning of neutropenic fever

at the same time when blood cultures

and other clinically important samples were obtained study blood samples (d0)

4.2.3.3. Serum vascular endothelial growth factor

Serum VEGF concentrations were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Quantikine ®, R&D Systems; Minneapolis, MN), which recognises the soluble isoforms (VEGF121 and VEGF165). The same VEGF ELISA method was used and described in detail in a recent Finnish study called FINNSEPSIS (15). In brief, the optical density at 450 nm was measured with wavelength correction at 540 nm (Multiscan RC plate reader; Thermolabsystems, Helsinki, Finland) and serum VEGF concentration was determined with Genesis (Life Sciences, UK) computer software capable of generating a four parameter logistic curve fit.

The intra-assay coefficient variation (CV) for a control sample (mean concentration 345 pg/ml) was 5.7% (n=10) and for pooled serum (mean concentration 96 pg/ml) 6.5% (n=10). The inter-assay CV for two control samples (mean concentration 167 pg/ml and 1002 pg/ml) was 8.9% and 4.3% (n=12). For pooled serum the inter-assay CV was 8.5% (n=12). In the FINNSEPSIS study, the samples of 30 healthy adult controls were collected. The mean age of these controls was 36±7 years. The number of males and females was equal (M/F 15/15). The median concentration of VEGF in the control group was 260 (IQR 126-459, range 63-809) pg/ml, which was used as the control value in the FINNSEPSIS study (15).

4.2.3.4. Plasma amino-terminal pro-brain natriuretic peptide

Samples for the measurement of serum NT-proBNP were drawn by venipuncture into Li heparin-containing tubes from patients lying in the supine position. Plasma was separated and NT-proBNP was determined within 24 hours using an electrochemiluminescence immunoassay (ECLIA, Roche Diagnostics) on a Cobas e 601 analyzer (Hitachi High Technology Co, Tokyo, Japan). The measuring range, defined by the lower detection limit and the maximum of the master curve provided by the manufacturer was 0.6-35 000 ng/l. The reference values were given according to age and gender of the patients: for males under 50 years of age 0-84 ng/l, 50-65 years of age 0-194 ng/l and over 65 years of age 0-229 ng/l. For females under 50 years of age 0-155 ng/l, 50-65 years of age 0-222 ng/l and over 65 years of age 0-352 ng/l.