Josep M. Jou

Preanalytical treatment of EDTA-anticoagulated blood to ensure stabilization of the mean platelet volume and component measured with the ADVIA counters

The mean platelet component (MPC) parameter calculated by the ADVIA blood cell analyzers provides direct information on density, or granularity, of platelets and could become a useful biomarker to detect in vivo platelet activation. Unfortunately, it is largely affected by time and storage conditions in standard anticoagulants based on EDTA. The present study was designed to improve the stability of the MPC in blood specimens to facilitate a more standardized use in different laboratories. Blood from healthy controls was collected into EDTA plus additives, and stored at different conditions. MPC and the mean platelet volume (MPV) were assessed at 30 min and at 1, 3, 6 and 24hours after blood drawing on the ADVIA 2120 system. Flow cytometry was used to evaluate platelet-activation proteins. Ultrastructural morphology of platelets was assessed using electron microscopy. Storage in EDTA increased MPV, decreased MPC, reduced the number of alpha-granules, and induced changes in the phosphorylation patterns of platelet proteins. A solution based on EDTA containing wortmanin and tyrphostin (ED-WORTY), both inhibitors of signaling pathways, provided good stability for most of the parameters tested up to 6 hours at room temperature. Storage at lower temperatures produced more favorable results. ED-WORTY solutions preserved adequate morphology and had minimal influence on other parameters provided by the ADVIA 2120 system. Thus, the additives included in ED-WORTY may be useful for maintaining the stability of MPC for prolonged periods and to facilitate the transport and exchange of samples among institutions and laboratories.

mTOR inhibition and erythropoiesis: microcytosis or anaemia?

BACKGROUND Anaemia and microcytosis are common post kidney transplantation. The aim of this study was to evaluate the potential role of mammalian target of rapamycin (mTOR) inhibition in the development of anaemia and microcytosis in healthy animals and in human erythroid cultures in vitro. METHODS Rats with normal kidney function were treated with sirolimus (n = 7) or vehicle (n = 8) for 15 weeks. Hemograms were determined thereafter. In the sirolimus withdrawal part of the study, rats received sirolimus (SRL) for 67 days (n = 4) 1 mg/kg three times per week or for 30 days (n = 4) and were observed until Day 120. Hemograms were performed regularly. Peripheral blood mononuclear cells from healthy controls (HC; n = 8), kidney transplant patients with sirolimus treatment with (SRL + MC; n = 8) or without microcytosis (SRL - MC; n = 8) were isolated and cultured in the absence or presence of SRL (5 ng/mL). RESULTS SRL-treated animals had a reduced mean corpuscular volume (MCV) and elevated erythrocyte count compared with control animals after 15 weeks of treatment. This effect was evident as early as 4 weeks (MCV: 61.5 ± 1.8 versus 57 ± 1.7 fL; P = 0.0156; Red blood count 7.4 ± 0.3 × 10(9)/L versus 8.6 ± 0.5 × 10(9)/L; P = 0.0156) and was reversible 90 days after SRL withdrawal. SRL in the culture medium of erythroid cultures led to fewer colonies in cultures from HC as well as from kidney transplant patients (without SRL: 34.2 ± 11.4 versus with SRL: 27.5 ± 9.9 BFU-E-derived colonies P = 0.03), regardless if the cultures were derived from recipients with normocytic or with microcytic erythrocytes. The presence of tacrolimus in the culture medium had no influence on the number and size of colonies. CONCLUSION mTOR inhibition induces microcytosis and polyglobulia, but not anaemia in healthy rats. This might be caused by growth inhibition of erythroid precursor cells.

Automated assessment of the neutrophil and platelet activation status in patients with essential thrombocythemia

Neutrophil and platelet activation are consistently found in essential thrombocythemia (ET), but the techniques employed to demonstrate such abnormalities are complex. To ascertain whether the ADVIA 120 analyzer can be employed to assess neutrophil and platelet activation status in ET, 55 such patients and the same number of matched healthy individuals were studied and the results correlated with neutrophil CD11b and platelet P-selectin expressions measured by flow cytometry. Compared with controls, ET patients had significantly higher values of neutrophil myeloperoxidase index (MPXI), mean platelet volume (MPV), platelet distribution width (PDW), and platelet component distribution width, and significantly lower values of neutrophil lobularity index and mean platelet component (MPC). Patients with the JAK2 mutation had significantly lower values of MPC and higher values of MPV and PDW than those with wild-type allele. A positive correlation was observed between MPXI and neutrophil CD11b expression and a negative correlation between MPC and platelet P-selectin expression. The intensity of the agreement between the variables obtained by the two methods was moderate. These results support the possible value of MPC as surrogate parameter of platelet activation in ET.

Effects of chronic normovolemic anemia on gastric microcirculation and ethanol-induced gastric damage in rats

The effects of chronic normovolemic anemia on gastric microcirculation and gastric mucosal susceptibility to ethanol-induced gastric damage were investigated in anesthetized rats. Blood exchange by a plasma expander during four consecutive days rendered the animals anemic with a 34% decrease in the baseline hematocrit but without affecting blood volume. Chronic anemia induced a decrease in whole blood viscosity, an increase in gastric mucosal blood flow measured by hydrogen gas clearance, a decrease in gastric vascular resistance, and a decrease in gastric hemoglobin content without changes in the gastric oxygen content, the latter two parameters being measured by reflectance spectrophotometry. Gastric mucosal blood flow was lowered by intragastric administration of 100% ethanol in both anemic and control rats, but the final blood flow was significantly higher in anemic than in control animals. Macroscopic gastric damage induced by ethanol administration was significantly lower in anemic than in control rats. We conclude that chronic normovolemic anemia increases gastric mucosal blood flow and leads a protecting mechanism against gastric mucosal damage induced by absolute ethanol.