Multicentre evaluation of stable reference whole blood for enumeration of lymphocyte subsets by flow cytometry.

BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.

Long-term results of distal pancreatectomy for chronic pancreatitis in 90 patients.

OBJECTIVE: To determine the indications for distal pancreatectomy for chronic pancreatitis and to evaluate the risks, functional loss, and outcome of the procedure. SUMMARY BACKGROUND DATA: Chronic pancreatitis is generally associated with continued pain, parenchymal and ductal hypertension. and progressive pancreatic dysfunction, and it is a cause of premature death in patients who receive conservative treatment. Good results have recently been reported by the authors and others for resection of the pancreatic head in this disease, but distal pancreatectomy is a less popular option attended by variable success rates. It remains a logical approach for patients with predominantly left-sided pancreatic disease, however. METHODS: A personal series of 90 patients undergoing distal pancreatectomy for chronic pancreatitis over the last 20 years has been reviewed, with a mean postoperative follow-up of 34 months (range 1-247). Pancreatic function was measured before and after operation in many patients. RESULTS: Forty-eight of 84 patients available for follow-up had a successful outcome in terms of zero or minimal, intermittent pain. There was one perioperative death, but complications developed in 29 patients, with six early reexplorations. Morbidity was unaffected by associated splenectomy or right-to-left dissection. Late mortality rate over the follow-up period was 10%; most of these late deaths occurred because of failure to abstain from alcohol. Preoperative exocrine function was abnormal in two thirds of those tested and was unchanged at follow-up. Diabetic curves were seen in 10% of patients preoperatively, while there was an additional diabetic morbidity rate of 23% related to the procedure and late onset of diabetes (median duration 27 months) in another 23%. Diabetic onset was related to percentage parenchymal resection as well as splenectomy. Outcome was not clearly dependent on the etiology of pancreatitis or on disease characteristics as assessed by preoperative imaging. However, patients with pseudocyst disease alone did better than other groups. Twenty-one of 36 patients who failed to respond to distal pancreatectomy required further intervention, including completion pancreatectomy, neurolysis, and sphincteroplasty. Thirteen of these 21 patients achieved long-term pain relief after their second procedure. CONCLUSIONS: Distal pancreatectomy for chronic pancreatitis from any etiology can be performed with low mortality and a good outcome in terms of pain relief and return to work in approximately 60% of patients. Little effect is seen on exocrine function of the pancreas, but there is a diabetic risk of 46% over 2 years. Pseudocyst disease is associated with the best outcome, but other manifestations of this disease, including strictures, calcification, and limited concomitant disease in the head of the pancreas, can still be associated with a good outcome.

Affordable CD4(+)-T-cell counting by flow cytometry: CD45 gating for volumetric analysis.

The flow cytometers that are currently supported by industry provide accurate CD4(+)-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between -29 and +46 cells/mm(3) with very little bias for CD4 counts (in favor of the Trio method: +8 CD4(+) lymphocytes/mm(3); 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between -118 and +98 cells/mm(3), again with a negligible bias (-10 CD4(+) lymphocytes/mm(3)). In the volumetric method using CD45/CD8, the strongly CD8(+) cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8(+) cells/mm(3); 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.