Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

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Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are obtained in 5-20% of samples. We evaluated whether 6-color FCM, including additional markers and new marker combinations, improved the results. Bone marrow samples were obtained from 363 ALL patients at day 15, 33 and 78 and MRD was analyzed using 6-color (218 patients) or 4-color (145 patients) FCM in parallel to routine PCR-based MRD diagnostics. Compared with 4-color FCM, 6-color FCM significantly improved the concordance with PCR-based MRD data (88% versus 96%); particularly the specificity of the MRD analysis improved. However, PCR remained more sensitive at levels <0.01%. MRD-based risk groups were similar between 6-color FCM and PCR in 68% of patients, most discrepancies being medium risk by PCR and standard risk by FCM. Alternative interpretation of the PCR data, aimed at prevention of false-positive MRD results, changed the risk group to standard risk in half (52%) of these discordant cases. In conclusion, 6-color FCM significantly improves MRD analysis in ALL but remains less sensitive than PCR-based MRD-diagnostics.

Stored platelets release nucleotides as inhibitors of platelet function.

It is well known that the function of platelets decreases progressively during storage of platelet concentrates at room temperature. To investigate this phenomenon in more detail, we have resuspended platelets that had been stored for 24 h or 72 h in fresh plasma, and we have measured the aggregation response and the ATP secretion. Conversely, the effect of plasma in which platelet concentrates (PC) had been stored for 24 h or 72 h, was tested on fresh platelets. Both the aggregation response to collagen and ADP and the collagen-induced ATP secretion of stored platelets partially recovered after incubation with fresh plasma (p < 0.05). The same parameters measured with fresh platelets incubated in stored PC-plasma were found to be significantly reduced in comparison with the response of fresh platelets in fresh plasma (p < 0.05). Finally, platelets were stored in a plasma-free medium, suitable for platelet storage and the supernatant was tested. This supernatant inhibited the function of fresh platelets in a storage time-dependent fashion. Boiling of these supernatants did not change the inhibiting capacities, whereas filtration over active charcoal did. Analysis of this supernatant revealed AMP and diadenosine tetraphosphate, which both inhibit platelet function. These data show that stored platelets release nucleotides that inhibit platelet function in a reversible manner. This phenomenon may contribute to the decrease of platelet function during storage and the recovery of platelet function after transfusion.

In vitro evaluation of buffy-coat-derived platelet concentrates stored in a synthetic medium.

Human blood platelets, prepared by the buffy-coat method, were prepared and stored in different synthetic media. In a synthetic medium based on gluconate, acetate and citrate (GAC), the pH was 6.8 on day 6. This medium was chosen for further evaluation. The total platelet count and the leukocyte contamination were significantly lower in the platelet concentrates (PCs) prepared in GAC compared to PCs prepared in plasma. Platelets stored in plasma or in GAC were equally functional when tested for aggregation and adenosine triphosphate (ATP) secretion. Only stimuli that act through the arachidonic-acid pathway induced a lower platelet response in GAC. Platelet morphology was quantified by measuring the difference in light transmission during stirring at different rates in an aggregometer; no significant differences for platelets stored in GAC as compared to plasma were observed. Activation of platelets was measured by binding of monoclonal antibodies (McAb) against the Gp IIb/IIIa complex and against activation-dependent antigens (GMP 140 from the alpha-granules and a 53-kD glycoprotein from the lysosomal granules). There was no difference in binding of these McAb between platelets prepared and stored in plasma or GAC. We conclude that platelets prepared by the buffy-coat method and stored in GAC have the same in vitro qualities as platelets stored in plasma, except for the lower aggregation response by the arachidonic-acid pathway. This is probably due to an acetate-induced decrease in intracellular pH.

Selective effects of trypsinization on established and tumour-derived mouse 3T3 cells.

Explanted tumour cells are much more sensitive to the deleterious effects of routine trypsinization than are the parent 3T3 or SV40 transformed established cell lines. This differential sensitivity causes the disappearance of tumour-derived cells when grown in co-culture with untransformed 3T3 cells and accounts in some tumor explants for the emergence of trypsin-resistant varient cells which have lost tumour-specific properties.

Comparison of cell-surface glycoproteins of rat hepatomas and embryonic rat liver.

Cell-surface glycoprotein of 3 rat hepatoma strains and late-embryonic liver was metabolically labelled in vivo with [3H]- or [14C]-fucose. Trypsinization of the cells and exhaustive pronase digestion of combined hepatoma-liver trypsinates followed by gel filtration over Sephadex-Biogel mixtures, yielded elution profiles that contained more early-eluting (high-mol.-wt.) glycopeptides for hepatomas than for liver. At least 3 factors were identified which acted to augment the fraction of early-eluting tumour glycopeptides: (a) increase of neuraminidase-sensitive sialic acid, (b) increase of neuraminidase-insensitive sialic acid that was sensitive to mild HCl hydrolysis, and (c) presence of sugar sulphate groups contributing to a restricted extent, relative to possible unknown factor(s). Whether (a), (b) or (c) operated depended on the hepatoma strain or its mode of growth. Notwithstanding these differences in the nature of the increase in early-eluting glycopeptides, the increase itself appears not to be due to growth per se, nor to an embryonic expression, but rather may serve as a marker of tumourigenicity.