Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research.

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (≈transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies. EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 °C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for <24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes. Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population.

Leukemia in a trisomy 21 mosaic: specific involvement of the trisomic cells.

Acute monoblastic leukemia was diagnosed in a 32-month-old boy with mild signs of Down's syndrome. Chromosome analysis of cultured skin fibroblasts and peripheral blood lymphocytes disclosed a constitutional mosaicism (46,XY/47,XY,+21). At initial diagnosis of acute leukemia, additional chromosomal changes were found in bone marrow blasts that were consistent with the M5 subtype. The cytogenetic markers of the acute leukemia were restricted to the trisomic subset and disappeared during remission. These findings add further weight to the suggestion that the propensity of Down's syndrome patients to develop leukemia is directly related to their karyotype abnormality and that leukemia might be clonal in origin.

CSF cells in tuberculous meningitis. Humoral and cellular immune response.

In 10 patients with tuberculous meningitis both T and B lymphocyte activation was observed in the CSF from clinical onset of disease. During the course of the illness, the humoral response remained most marked, disappearing by 2 months, while the cellular immune response continued for about another 3 months. This contrasts with acute viral meningitis, in which both responses cease within 1-2 weeks of disease.

Burkitt's lymphoma--a correlated light and electron microscopic study on the malignancy in the CSF.

In a sporadic case of primary maxillar Burkitt's lymphoma and secondary spinal epidural dissemination, the massive malignancy in the CSF was investigated by light and electron microscopy. At both levels the CSF tumor cells were strikingly similar, with the undifferentiated lymphoblasts that characterize Burkitt's lymphoma in tissue. However, the starry-sky appearance in the CSF was produced more often by degenerating malignant cells than by histiocytes as classically described in tissue with Burkitt's lymphoma.

CSF cytology of acute viral meningitis and meningoencephalitis.

The CSF of 12 patients with acute meningitis and meningoencephalitis of viral nature (proven in 10 and probably in 2) was investigated by light microscopy. In early CSF, 1--31% reactive lymphocytes and 1--6% plasma cells were found consistently. In addition, various lymphoid cell features of marked immune activation were disclosed in association: cell polymorphism, normal-appearing mitosis and binucleation, abnormal nuclear lobulation and even partition, as well as Russell bodies, morular appearance and clasmatosis in plasma cells. The CSF findings are discussed with respect to those described in tuberculous meningitis and multiple sclerosis and distinguishing features are suggested to occur in early CSF of viral meningitis.

Platelet survival in myelodysplastic syndromes.

A platelet survival study with indium-111 oxine was performed in 6 patients with a myelodysplastic syndrome. A markedly shortened platelet life-span (range 0.50-2.65 days; normal 6.5-12 days) was obtained in each case. Platelet production ranged from 53,100 to 430,800 platelets/mm3/day (normal 66,200 +/- 14,600). A second isotopic study was performed in 1 case with platelets from a normal compatible donor. The nearly complete normalisation of the platelet life-span (from 0.85 to 5.50 days) indicates an intracorpuscular origin of the defect.

Neutrophil dysfunctions in thalassaemia major: the role of cell iron overload.

The susceptibility to infections was recorded in 13 patients with beta thalassaemia major (T.P.). The following parameters were also investigated in their polymorphonuclear neutrophils (PMN): nitro blue tetrazolium (NBT) reduction, heated yeast and Escherichia coli phagocytosis, Escherichia coli killing and myeloperoxydase activity. These results were compared to those obtained in healthy controls (H.C.). The Perls's reaction was performed on PMN and graded according to a scoring system with the aim of quantifying the iron intoxication of PMN. Phagocytosis and Perls's reaction of PMN from H.C. were also studied after 20 h of incubation with thalassaemic serum. 6 T.P. out of 13 developed septicaemia during their lifetime and in all 9 septicaemic episodes were noted. Phagocytosis was greatly impaired, disclosing both cellular and serum abnormalities. The mean percentage of Perls's positive PMN was 13% in T.P., contrasting with the constant negative reaction in H.C. The incubation of PMN from H.C. with serum from T.P. induced the simultaneous appearance of a phagocytosis defect and of a positive Perl's reaction. It was concluded that in beta thalassaemia major the phagocytosis of PMN was altered due to a combination of serum and cellular abnormalities and that both may be related to the iron overload.