Results and Discussion

We used a standardized immune monitoring program based on recent advances in flow cytometry (exact quantification of surface marker expression) to investigate 54 patients with open heart surgery. All patients showed postoperative immunodepression, documented by decreased monocytic HLA-DR. During the postoperative follow-up, all markers investigated tended to recover.

One day after open heart surgery, monocytic HLA-DR expression revealed a marked attenuation compared with values determined preoperatively (9437 ± 854 ABC vs 30,644 ± 2868 ABC; Fig. 2A), as already described for patients after cardiopulmonary bypass surgery (10). Thereafter, expression of HLA-DR on monocytes slowly increased within 10 d to reach normal values in most of the patients. The time-course of the expression of both the membrane peptidases

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Fig. 1. Gating on the monocytes for determination of HLA-DR (A), and on the neutrophil granulocytes (B) for determination of neprilysin/CD10 and aminopeptidase N/CD13. (C) The estimation of the granulocytic expression of neprilysin/CD10 is shown in a FL2 histogram. The lines on the x-axis represent the geometric mean values of the beads measured that are related to molecules per cell. In this particular case, the patient's granulocytes expressed, on average, 2644 neprilysin/CD10 molecules per cell.

Fig. 1. Gating on the monocytes for determination of HLA-DR (A), and on the neutrophil granulocytes (B) for determination of neprilysin/CD10 and aminopeptidase N/CD13. (C) The estimation of the granulocytic expression of neprilysin/CD10 is shown in a FL2 histogram. The lines on the x-axis represent the geometric mean values of the beads measured that are related to molecules per cell. In this particular case, the patient's granulocytes expressed, on average, 2644 neprilysin/CD10 molecules per cell.

aminopeptidase N/CD13 and neprilysin/CD10 on granulocytes showed a similar behavior with a decrease, especially on day one after heart operation, and a return to values of healthy volunteers within 7 d after open heart surgery (Fig. 2B,C). Neutrophils are a key component of the innate immune system because they represent the first line of defense the body has upon invasion by a foreign micro-organism. These cells do not express major histocompatibility complex (MHC) class II molecules. However, quantification of Fcy receptor I/CD64 has been found to be a useful indicator of granulocytic activation in septic patients (11). Now, we report that the expression of membrane peptidases could be a useful marker for the immunomonitoring of patients undergoing open heart surgery. In future analyses, a possible relationship between an immunodeficiency of patients and the diminished expression of both membrane peptidases on granulocytes has to be investigated.

4. Notes

QuantiBRITE PE beads provide a useful tool for standardized analysis across labs. When used in conjunction with 1:1 conjugates of PE-to-MAb, the QuantiBRITE PE beads provide a simple yet robust means of quantitating

Time (days) Fig. 2. (Continued)

expression levels in terms of ABC (2). However, a number of variables are critical for standardized quantitative flow cytometry. Time of analysis, method of fixation, lysing method, the pH value of the buffers used, and other factors may all influence the quantitative values obtained with this procedure. As

Fig. 2. Changes in antigen expression of monocytes and granulocytes in 54 patients undergoing open heart surgery compared with healthy volunteers (hatched area). All results are expressed as antibodies bound per cell (ABC; mean ± SEM). Percentiles are indicated for healthy volunteers with P25 (25th percentile), P50 and P75. One day after surgery, all patients showed a diminished expression of the markers investigated compared to reference values. N = number of patients. (A) Monocytic HLA-DR of patients started preoperatively in the range of the 75th percentile of the control group and revealed a marked attenuation from day one to three after surgery. Thereafter, expression slowly increased within 10 d, reaching normal values in most of the patients. (B) Neprilysin/CD10 expression on granulocytes paralleled the decrease in monocytic HLA-DR expression with values less than those of the control group on days one to three after surgery and a normalization by day seven. (C) Similarly, expression of aminopeptidase N/CD13 on neutrophils revealed a sharp decrease 1 d after surgery. Recovery to normal values already started on day three.

Fig. 2. Changes in antigen expression of monocytes and granulocytes in 54 patients undergoing open heart surgery compared with healthy volunteers (hatched area). All results are expressed as antibodies bound per cell (ABC; mean ± SEM). Percentiles are indicated for healthy volunteers with P25 (25th percentile), P50 and P75. One day after surgery, all patients showed a diminished expression of the markers investigated compared to reference values. N = number of patients. (A) Monocytic HLA-DR of patients started preoperatively in the range of the 75th percentile of the control group and revealed a marked attenuation from day one to three after surgery. Thereafter, expression slowly increased within 10 d, reaching normal values in most of the patients. (B) Neprilysin/CD10 expression on granulocytes paralleled the decrease in monocytic HLA-DR expression with values less than those of the control group on days one to three after surgery and a normalization by day seven. (C) Similarly, expression of aminopeptidase N/CD13 on neutrophils revealed a sharp decrease 1 d after surgery. Recovery to normal values already started on day three.

such, the protocol must be precisely followed to assure comparability between laboratories.

1. Our own observations show that storage of blood at room temperature for longer then 4 h results in a time-dependent increase in the expression of HLA-DR on monocytes. One reason for this effect might be that the decrease in monocytic HLA-DR expression could be the result of an intracellular accumulation of HLA-DR molecules caused by an incomplete posttranslational processing (7). Keep blood specimen on ice after collection if you cannot finish staining procedure within 4 h.

2. BD claims that it is not necessary to subtract the cell blank (negative cell population) from the positive cell population because in the first decade the fluorescence of the bead alone is very similar to that of the negative cell population. To subtract the isotype control value it would be necessary to order a PE-labeled isotype control antibody with the same 1:1 fluorochrome-to-protein ratio.

3. The specific number of PE molecules per bead varies from lot to lot, with low fluorescence intensity in the range of approx 800 and high fluorescence intensity in the range of approx 70,000 PE molecules per bead. Only within this range is the linearity of ABC values guaranteed.

4. The arithmetic mean is well suited to the analysis of data that is collected on a linear scale, whereas as a general rule, the geometric mean is better suited for use with data collected with a logarithmic amplifier. The geometric mean is calculated by multiplying the individual values of a group together, thus obtaining the nth root of the product.

5. The MAb for the labeling of blood cells have to be applied at saturating amounts. Therefore, a white blood cell count must be performed before processing, and the cell concentration adjusted accordingly. One should aim at a cell concentration of 1 X 106/test tube. Samples with a leukocytosis may need to be diluted.

6. The same reagent (single batch) from the same company and in the same dilution should be used throughout the experiment.

7. The mechanisms involved in the decrease of HLA-DR in sepsis include shedding of HLA-DR from the cell surface of monocytes (7). MHC molecules are known to be shedded very easily, e.g., after treatment of cell with interferon (9). To prevent shedding by antibody ligation, the staining procedure of MHC molecules should be carried out at 4°C. We observed no differences between results of labeling of membrane peptidases at 4°C or at room temperature. For reason of simplicity we used 4°C for all antibody incubation steps.

8. In an earlier study authors noted that erythrocyte-lysing reagents cause varying and sometimes substantial reduction in the fluorescence intensity of cells stained directly with CD34 MAb conjugates (1). Therefore, do not change the lysing solution within your study.

9. No-wash protocols and methods using washing steps can clearly differ in their results. We often observed that higher ABC values are obtained in no-wash procedures. Because most investigators in the field of quantitative flow cytometry use at least one washing step, we opted for this method.

10. Flow cytometers should be calibrated according to each laboratory's standard operating procedure. Compensation is a very important point that can affect inter-laboratory comparability and reproducibility. In your assay it is useful to avoid multicolor staining with fluorescent dyes that bleach into the PE channel.

11. Two thousand events are a 95% guarantee that the result is within 2% of the "true" value (binomial sampling). This sample mode assumes that the variability of determining replicates is <2%.

References

1 Serke, S., van Lessen, A., and Huhn, D. (1998) Quantitative fluorescence flow cytometry: a comparison of the three techniques for direct and indirect immunoflu-orescence. Cytometry 33, 179-187.

2 Pannu, K. K., Joe, E. T., and Iyer, S. B. (2001) Performance evaluation of QuantiBRITE phycoerythrin beads. Cytometry 45, 250-258.

3 Schmitz, J. L., Czerniewski, M. A., Edinger, M., et al. (2000) Multisite comparison of methods for the quantitation of the surface expression of CD38 on CD8(+) T lymphocytes. The ACTG Advanced Flow Cytometry Focus Group. Cytometry 42, 174-179.

4 Huschak, G., Zur Nieden, K., Stuttmann, R., and Riemann, D. (2003) Changes in monocytic expression of aminopeptidase N/CD13 after major trauma. Clin. Exp. Immunol. 134, 491-496.

5 Volk, H. D. (2002) Immunodepression in the surgical patient and increased susceptibility to infection. Crit. Care 6, 279-281.

6 Hershman, M. J., Cheadle, W. G., Wellhausen, S. R., Davidson, P. F., and Polk, H. C., Jr. (1990) Monocyte HLA-DR antigen expression characterizes clinical outcome in the trauma patient. Br. J. Surg. 77, 204-207.

7 Perry, S. E., Mostafa, S. M., Wenstone, R., Shenkin, A., and McLaughlin, P. J. (2004) HLA-DR regulation and the influence of GM-CSF on transcription, surface expression and shedding. Int. J. Med. Sci. 1, 126-136.

8 Riemann, D., Blosz, T., Wulfaenger J., Langner, J., and Navarrete Santos, A. (2002) Signal transduction via membrane peptidases. In: Ectopeptidases, (Langner, J. and Ansorge, S., eds.), Kluwer Academic/Plenum Publishers, New York, pp. 141-170.

9 Gershon, H. E., Kuang, Y. D., Scala, G., and Oppenheim, J. J. (1985) Effects of recombinant interferon-gamma on HLA-DR antigen shedding by human peripheral blood adherent mononuclear cells. J. Leukoc. Biol. 38, 279-291.

10 Strohmeyer, J. C., Blume, C., Meisel, C., et al. (2003) Standardized immune monitoring for the prediction of infections after cardiopulmonary bypass surgery in risk patients. Cytometry B Clin. Cytom. 53, 54-62.

11 Davis, B. H., Bigelow, N. C., Curnutte, J. T., and Ornvold, K. (1995) Neutrophil CD64 expression: Potential diagnostic indicator of acute inflammation and therapeutic monitor of interferon-y therapy. Lab. Hem. 1, 3-12

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