Контроль гіпотетичного прояву хронічного синдрому втоми: попередні спостереження

Immunophenotyping

Anticoagulated blood (EDTA) was collected between 9 and 11 a. m. and used for white blood cell enumeration, differential counts (Celldyn 4000, Abbott Laboratories, Abbott Park, IL 60064, USA) and flow cytometric studies. Lymphocyte populations were analysed with dual colour direct immunofluorescence on a EPCS® XL flow cytometer (Carlter, Miami, FL, USA), with aid of the «System I™» computer software. One hundred pi of whole blood was incubated with the appropriate combination of monoclonal antibodies for 25 minutes at 4°C. Then red cells were lysed using lysis buffer (Becton Dickinson) for 7 minutes, spun down and washed once with 2 ml phosphate buffered saline (PBS). Resuspension was immediately followed by cell analysis. Commercially available (Becton-Dickinson) phycoerythrin (PE) or fluorescein isothiocyanate (FITC) monoclonal antibodies were used and are listed in Table 3. Estimates of absolute numbers of lymphocyte subsets were determined by multiplying peripheral lymphocyte counts by the percentage of each surface marker.

Statistical Analysis

All the data were administered into Excel 98. 0. The data were coded and transferred to the University of Newcastle, Callaghan, Australia where the statistical analysis was done. Data distributions were evaluated for normality and linearity and those data not showing normality were transformed for multivariate analysis. Subject characteristics were assessed using chi-square probability and student t-tests. Uni-variant group differences were assessed on un-transformed data using the student t-test. Immunophenotyping profiles were assessed by forward stepwise discriminant function analysis as both percentage distribution and cell counts. The patient classification capacity of the discriminant function module was used to assess the patient compliance within each model. This allowed an evaluation of the predictive capacity of the different immunophenotypes in determining a potential diagnosis of CFS.

 

TABLE 3

Monoclonal Antibodies Used for Immunophenotyping

Complementarity DeterminationMonoclonal

AntibodiesSubset

CD2+Leu5b+FITCE-rosette receptor

CD3+Leu4+FITCT-cells

CD3+HLADR+Leu4+HLADR+PEActivated T-cells

CD25+IL2R1-PEActivated cells

CD4+Leu3a+FITCHelper/inducer T-cells

CD4+CD45RA-Leu3aLeu18-PEMemory CD4-cells

CD4+CD45RA+Leu3al_eu18+Virgin CD4-cells

CD8+Leu2a+FITCCytotoxic/Suppressor T-cells

CD8+CD11b+Leu2aLeu15+PESuppressor cells/NK-subset

CD8+CD11fa-Leu2aLeu15-Cytotoxic T-cells

CD-I 9+Leu12+PEB-cells

CD19+CD5+Leu1+Leu12+Mature B-cells

CD3- CD16+CD56+Leu4-Leu16+PE Leu19+PENK-cells

CD3+CD16+CD56+Leu4+Leu16+Leu19+Subset cytotoxic T-cells

 

These data were processed using Access97™ (Microsoft, Redmond, WA, USA), Excel97™ (Microsoft) and Statistica™ (Ver. 5. 1, Statsoft, Tulsa, OK, USA).

 

 

Twenty-seven CFS patients were recruited who complied with the Fukuda criteria. Of the 27 Fukuda defined CFS patients, 16 patients (59. 3%) also fulfilling the Holmes criteria. No difference in age or sex distribution was found between patients and healthy volunteers (C = controls)  (mean age ± SD: CFS = 41. 1 ± 8. 6, C = 38. 6 ± 10. 4 years; age range: CFS = 19-66, C = 21 -58 years; N and percent (%) females: CFS = 25 or 92. 6%, C = 18 or 90%).

Table 4 shows the standard biochemical measures, ranges and prevalence of subjects outside the reference ranges. Six patients had elevated c-reactive protein (CRP) levels and 10 elevated erythrocyte sedimentation rates (ESR). The patients with an elevated c-reactive protein levels were more likely to have a raised ESR (raised CRP = 5 of 6; normal CRP 5 of 2-P < 0. 008) consistent with an acute phase reaction. Patients with raised CRP levels had higher serum calcium levels than the remaining CFS patients (CRP > 4 = 9. 2 ± 0. 4; CRP < 4 = 8. 9 ± 0. 2-P <0. 05). Patients with raised ESR levels had lower serum potassium levels than the remaining CFS patients (ESR >10 = 3. 7 ± 0. 1; ESR < 10 = 3. 9 ± 0. 2-P < 0. 02). Four patients had low serum calcium levels and these four CFS patients also had lower whole body potassium levels (low Ca = 31. 4 ± 4. 3; normal Ca = 38. 4 ±7. 7-P< 0. 05). Thus CFS patients do have an alteration in electrolyte levels which appear partially associated with alterations in measures of acute phase reactions.

 

TABLE 4

Standard Measures and Prevalence Outside Reference Ranges fo the CFS Patients

ParameterMean (95% CL) Prevalence

N (%) Reference Range

RNase-L19, 8 (10, 1-29, 6) 23 (85, 2) High<2, 0LMW/HMW x 10

Neutrophil count4, 0 (3, 4-4, 6) 01, 48 -7, 10 x 103/mm3

Lymphocyte count2, 3 (1, 9-2, 7) 00, 68 -4, 22 x lo3/mm3

C-reactive protein3, 4 (2, 0-4, 8) 6 (22, 2) High< 4 mg/L

Erythrocyte sedimentation rate10, 7 (7, 0-14, 3) 10 (37, 0) High0-10