Separation of red blood cells by field flow fractionation.

Field flow fractionation (FFF) is a new methodology described as being well-suited for the separation and characterization of biopolymers and particles. On theoretical grounds, cells may be separated with FFF if they differ in size, density or deformability. In the present study, we first tried to determine optimal separation conditions for red blood cells; thereafter we used FFF to examine red cell changes during a phenylhydrazine-induced hemolytic anemia. It has been shown that in less than 30 minutes, FFF is able to separate normal red blood cells from Heinz body-rich cells or reticulocytes that differ in size or density. The successive steps of hemolysis and regeneration appear clearly on the fractograms. Advantages and drawbacks of the method are discussed.

Increased bone marrow toxicity of doxorubicin bound to nanoparticles.

The in vivo myelosuppressive effects of free and polyalkylcyanoacrylate-bound doxorubicin were compared in a mouse model. After intravenous administration of 11 mg/kg body weight of doxorubicin either free or bound to polyisobutyl (doxo-PIBCA) or polyisohexylcyanoacrylate (doxo-PIHCA) nanoparticles, we studied the total and differential counts of blood, bone marrow and spleen cells; the number of granulocyte progenitors (CFU-GM) was determined by culture. Doxorubicin concentrations were measured with an HPLC method in the bone marrow and the spleen. Doxo-PIHCA nanoparticles showed the highest and longest myelosuppressive effects which correlated well with a high concentration of the drug in the bone marrow and the spleen. Moreover, it was found that PIHCA nanoparticles induced the release of colony stimulating factors, which might account for the observed increase of toxic effects of doxorubicin on bone marrow progenitors. These data also indicate that a more precise evaluation of the myelosuppressive effects of targeted formulations of anticancer drugs is needed, which may be attained by studies on bone marrow progenitors.

Elderly patients treated with tinzaparin (Innohep) administered once daily (175 anti-Xa IU/kg): anti-Xa and anti-IIa activities over 10 days.

Since low molecular weight heparins (LMWH) are partly eliminated by renal excretion, their pharmacodynamic profile may be modified in very elderly patients with age-related renal impairment. The aim of this prospective study was to determine whether tinzaparin (Innohep) 175 anti-Xa IU/kg administered subcutaneously once daily over 10 days does accumulate in hospital patients greater than 70 years of age. Plasma anti-Xa and anti-IIa amidolytic levels and APTT were determined prior to the first injection (day 0), and then, at peak level i.e. 5 h after the second injection (day 2) and subsequently on days 5, 7 and 10. Thirty consecutive inpatients (6 men, 24 women) requiring LMWHs at a curative dose for acute thromboembolic disease were included. Patients' characteristics (mean +/- SD) were: age 87.0+/-5.9 years (range 71-96), body weight 62.7+/-14.6 kg (range 38-90) and creatinine clearance 40.6+/-15.3 mL/min (range 20-72). The mean actual dose of tinzaparin delivered was 174.8 anti-Xa IU/kg. Since no patient had an anti-Xa activity above 1.5 IU/mL, the dose of tinzaparin remained fixed over 10 days. Anti-Xa and anti-IIa peak levels measured on day 2 were 0.66+/-0.20 IU/mL (range 0.26-1.04) and 0.33+/-0.10 IU/mL (range 0.18-0.55), respectively. Ex vivo anti-Xa/anti-IIa ratios were close to 2.1. APTT ratios (patient/control) were strongly correlated with anti-IIa activity (p <0.01). There was no progressive increase of the anti-Xa and anti-IIa activities after repeated administration of tinzaparin over the 10 day treatment period. No correlation was found between anti-Xa and anti-IIa activities and age, weight, or creatinine clearance. No major bleeding occurred during the study and only one minor haematoma at the injection site was reported. No thrombo-embolic complication or death occurred. Tinzaparin may thus be administered safely at a treatment dose (175 anti-Xa IU/kg) in older patients with age-related renal impairment. Neither dose adjustment, nor serial anti-Xa activity monitoring seems to be required in patients with creatinine clearance above 20 mL/min during the first ten day treatment.

PEGylated polycyanoacrylate nanoparticles as vector for drug delivery in prion diseases.

PEGylated polymeric nanoparticles are hereby presented as a potential efficient drug carrier for the delivery of active therapeutic molecules in prion experimental diseases. Based on their blood long-circulating characteristics, these PEGylated particles made by the amphiphilic copolymer poly [methoxy poly(ethylene glycol) cyanoacrylate-co-hexadecyl cyanoacrylate] (PEG-PHDCA), showed comparatively conventional non-PEGylated nanoparticles, a higher uptake by the spleen and the brain which are both the target tissues of PrPres accumulation in scrapie infected animals.

Polyalkylcyanoacrylate nanoparticles as carriers for granulocyte-colony stimulating factor (G-CSF).

The human recombinant granulocyte colony-stimulating factor (rhG-CSF) is largely used in the treatment of neutropenia occurring during chemotherapy. After injection, this glycoprotein distributes through the whole body. Thus, to obtain high and durable bone marrow concentrations, targeting with polyalkylcyanoacrylate nanoparticles was considered. Two methods of preparation were investigated: anionic polymerization and precipitation of the preformed polymer. By anionic polymerization, it was possible to associate more than 66% of rhG-CSF with nanoparticles (polyisobutyl- or polyisohexylcyanoacrylate nanoparticles) when the glycoprotein was added at the end of the polymerization process. It has been shown that the rhG-CSF was mainly adsorbed on the surface of the nanoparticles and most of the colony stimulating activity was conserved. Using precipitation of performed polyisohexylcyanoacrylate, 90% of rhG-CSF was associated with nanoparticles, the protein being mainly adsorbed onto the nanoparticle surface. In this case, a decrease of the colony stimulating activity was however observed. Whatever the method used, the in vitro release of rhG-CSF from the polyisohexylcyanoacrylate nanoparticles, was progressive during 8 h in seric conditions. Nevertheless, using mice as an animal model, it has been shown that the short-term effects of intravenously injected rhG-CSF were not increased by its association with polyisohexylcyanoacrylate nanoparticles.

In vitro evaluation of nanoparticles spleen capture.

After intravenous injection, the main part of nanoparticles trapped by the spleen are concentrated in the marginal zone. The first step of this capture is the adhesion of the particles to the marginal zone macrophages. As classical techniques of cell suspension preparation did not allow to isolate without damage these actively capturing cells, tightly bound to a well-developed reticular meshwork, we designed a tissue slice incubation method, in order to study in vitro the interaction of nanoparticles with these particular macrophages, in conditions close to in vivo. In a serum supplemented medium, this in vitro model was able to give similar uptake profile than after intravenous injection of nanoparticles thus proving its validity. Surprisingly, no significant decrease of nanoparticles capture was observed when the medium was depleted from complement, immunoglobulins or proteins affine for heparin, while substitution of serum by purified albumin allowed a near optimal uptake. Addition of competitive ligands for lectin-like receptors did not show any clear inhibition of spleen capture. On the other hand, the scavenger receptor blocking agents, such as maleylated albumin or polyinosinic acid, induced a strong reduction of the spleen nanoparticles uptake. Thus, this paper proposes an in vitro binding assay as a reliable method to investigate the spleen capture of a large variety of nanoparticulate drug carriers. It is also a useful methodology to highlight the interactions between spleen cells and nanoparticles. The data obtained suggest that capture of nanoparticles depends on a multifactorial and complex phenomenon involving for a part albumin and the scavenger receptor.

Cells involved in the capture of nanoparticles in hematopoietic organs.

The affinity of nanoparticles for hematopoietic organs could be valuable for the targeting of certain stimulating factors to those tissues, but this affinity should also be taken into account in the toxicological evaluation of those carriers, especially when they are loaded with antimitotic compounds such as doxorubicin. However, the cells responsible for the capture of the nanoparticles and their localization in these organs is an important point to know before trying to modulate the nanoparticle's tissue distribution. Thus, we have studied, in this paper, the capture, the localization, and the retention in the bone marrow and in the spleen of biodegradable poly(isohexyl cyanoacrylate) nanoparticles as well as of nonbiodegradable polystyrene nanoparticles. The histological localization of these nanoparticles has been completed by cytological localization with a method used in cytochemistry for the evaluation of intracellular accumulation of various substances, such as iron deposits in bone marrow sideroblasts. These data indicate that, in the bone marrow, after a quick passage through the endothelium, nanoparticles were dispersed throughout in the tissue and captured by all types of phagocytizing cells. In the spleen, nanoparticles were mainly localized in large angular capturing cells in the marginal zone of the lymphoid follicles.

Ruling out acute deep vein thrombosis by ELISA plasma D-dimer assay versus ultrasound in inpatients more than 70 years old.

In geriatric care, deep-vein thrombosis (DVT) is mostly diagnosed by noninvasive techniques. The objectives of this prospective study were: (1) to evaluate the power of ELISA plasma D-dimer assay versus ultrasound (US) in ruling out acute DVT of the lower limbs in symptomatic geriatric inpatients, and (2) to determine the most effective D-dimer cutoff value over the age of 70 years. Over a 10-month period, inpatients with suspected lower limb DVT simultaneously underwent US examination and ELISA plasma D-dimer assay. Noninclusion criteria were comorbid conditions able to modify the D-dimer level. Data were processed by receiver operating characteristic (ROC) curve analysis. In total, 150 patients (125 women, 25 men), average age 86.3 years (range 70-101) were included. A diagnosis of lower limb DVT was established in 53 patients (35.3%). With a 500 ng/mL D-dimer cutoff conventional value, DVT was ruled out in only five patients (3.3%), whereas a 750 ng/mL value ruled out DVT in 19 patients (12.7%) with a sensitivity of 98.1%, and a negative predictive value of 95.0%. The only false negative corresponded to a patient with a 15-mm thrombus in the distal calf. In inpatients above 70, an ELISA plasma D-dimer value smaller than 750 ng/mL is a rapid reliable noninvasive means to rule out lower limb DVT, if color flow Doppler ultrasound is not available on site.

[Biological diagnosis of hemoglobinopathies by phenotype analysis]. / Diagnostic biologique des hémoglobinopathies par analyse du phénotype.

Haemoglobinopathies are defined by the presence of qualitative and/or quantitative globin chains abnormalities. In most cases, laboratory diagnosis relies on phenotype analysis. Ethnic group, clinical data, complete blood count in absence of recent blood transfusion, iron status, should be taken into account for the diagnosis. Electrophoresis using an alkaline pH buffer system is used as an initial technique for the detection of haemoglobin variants, showing abnormal bands as compared with reference samples. Isoelectric focusing is more resolutive and could be preferred for this reason: If the migration pattern differs from normal, additional confirmatory tests, as citrate agar electrophoresis (acid pH), are required. Relative amounts of variant haemoglobin (Hb A2, F and S...) are quantified using chromatographic methods. Other tests, such as solubility tests, stability tests, Kleihauer-Betke test, are useful in specific cases. The diagnosis relies on clinical and laboratory findings. In the Paris area, the most common variant is Hb. S. Taalassemia traits are also very common. Screening of SS. S beta D. SO Arab, SD Punjab, SC, or thalassaemia major should be made early so that patients found to have one of these varieties should be referred to haemoglobinopathy centers.

[Myelodisplasic syndromes diagnosed in a geriatric hospital: morphological profile in 100 patients]. / Syndromes myélodysplasiques diagnostiqués dans un hôpital gériatrique: profil cytologique de 100 patients.

Myelodysplastic syndrome (MDS) is particularly common in geriatric practice. As few data are available in very elderly patients, we conducted a 54-month retrospective study in patients over 70 years with MDS diagnosed at Hôpital Charles Foix. Patients with cobalamine, folate or iron deficiency were excluded. Regarding biological and morphologic approaches, MDS patients were classified according to the FAB criteria. We then tempted to reclassify the patients according to the WHO criteria. The Bournemouth scoring system was used as a prognostic tool. During the study period, 100 patients were included, 29 males and 71 females, median age 86 +/- 7 years (70-103). At the time of bone marrow sampling, a peripheral blood cytopenia was documented in 64 patients, a bicytopenia in 27 patients and a pancytopenia in 9 patients. Isolated anaemia (Hb < 12 g/dL) was found in 60 patients and isolated thrombocytopenia (< 150 x 10(9)/L) in 4. Macrocytosis (MCV > 100 fL) was observed in 21 % of the cases. According to the FAB criteria, the 100 patients were classified as follows: refractory anaemia (RA): 79%; RA with ringed sideroblasts (RARS): 8%; RA with excess of blasts (RAEB): 8%; RAEB in transformation: 1%; chronic myelomonocytic leukaemia: 4%. According to the WHO classification, the patients were reclassified as follows: RA (unilineage) (with or without ringed sideroblasts): 10%; refractory cytopenia with multilineage dysplasia with or without ringed sideroblasts (RCMD): 73%; RAEB: 7% (RAEB-1 6%, RAEB-21%); MDS/Myeloproliferative disorder: 4%; unclassified (hypocellularity): 5%; acute leukaemia: 1%. In order to estimate prognosis at the time of the bone marrow aspirate, we calculated the Bournemouth'score: 8 patients scored 0,57 scored 1,25 scored 2,8 scored 3 and 2 scored 4. In this geriatric population, 83% cases of MDS are RA or RCMD (with or without sideroblasts); MDS with excess of blasts are uncommon. Thus, elderly patients under study with MDS were diagnosed at an earlier stage of the disease than younger ones from series published in the literature. Due to frequent comorbidities, geriatric patients may be symptomatic for a slight decrease of haemoglobin level. Therefore, elderly patients are investigated as soon as they present with moderate anaemia that may explain the early MDS diagnosis.

[ABO incompatibility and newborn haemolytic disease: two cases with major erythroblastosis]. / Incompatibilité foeto-maternelle ABO et maladie hémolytique du nouveau-né: à propos de deux observations avec érythroblastose majeure.

Two cases of newborn haemolytic disease because of ABO incompatibility are reported. In both cases, blood erythroblastosis exceeded 300 per 100 leukocytes at birth. Both newborns developed early jaundice and one presented with anaemia. Intensive phototherapy begun soon after birth was an effective treatment. The diagnosis of newborn jaundice and the patho-physiological basis of haemolytic disease because of ABO incompatibility are discussed.

[Evaluation of a new automatic hematological system: the Advia 70 (Bayer-Diagnostics)]. / Evaluation d'un nouvel automate de cytologie sanguine: l'Advia 70 (Bayer-Diagnostics).

The Advia 70 hematology system is a new automated analyser manufactured in Texas (USA) by MWI-DANAM and marketed by Bayer- Diagnostics in France. The throughput is 70 complete blood counts (CBC) with leukocyte differential counts (diff) per hour. Three sample modes are proposed: normal mode (180 muL of whole blood), sample saver mode (90 muL) or automatic sampler (180 muL). The principles used on Advia 70 are: 1) electrical impedance for cell counting (WBC, RBC, platelets, MCV) and RBC/plt sizing; 2) multi-dimensional optical system and absorbance for white cell differential. The Advia 70 has been evaluated in our laboratory over a three-week period. Within-run variations of blood counting (CBC & diff) were determined on normal and abnormal patient specimens whereas between run-variations were determined on normal controls. Linearity and detection limits were also tested. The Advia 70 system results were reproducible and reliable. The correlations between the various sample modes were very good. Contamination and store assays were performed. Thirty normal and abnormal samples were successively analysed on the Advia 70 system and on the Advia 120 system which methodologies are different (myeloperoxidase staining coupled with flow cytometry-nuclear lobularity analysis for WBC/Diff, flow cytometry and laser diffraction for RBC/platelets), and the results were compared: despite the different technologies used on both analysers, the correlations were very good (CBC and diff parameters). Finally, we evaluated the leukocyte differential flags on 72 patients samples with abnormal cells, using the blood smear examination as the reference method. In conclusion, the Advia 70 can be used in private and hospital laboratories.

[Cytochrome P450 2C9 polymorphisms (CYP2C9) and warfarin maintenance dose in elderly patients]. / Polymorphismes du cytochrome P450 2C9 (CYP2C9) et posologie à l'équilibre pour des patients âgés traités par warfarine.

PURPOSE: Allelic variants of the gene coding for cytochrome P450 isoform 2C9 (CYP2C9), 2C9*2 and 2C9*3, were shown to increase sensitivity to warfarin in adults. In the elderly, the maintenance dose is influenced by acquired factors including comorbidities and polymedication. The aim of our purpose was to investigate whether a genetic factor, such as cyp2c9 genotype, does influence the warfarin maintenance dose in very elderly patients. METHODS: In-patients treated with warfarin were recruited with the following inclusion criteria: i/ 75 years-old or over; ii/ a stable INR within the therapeutic range (INR 2.0-3.0). Genotypes were coded as numbers of alleles for each of the three polymorphisms, namely 2C9*1 (wild-type), 2C9*2, and 2C9*3. RESULTS: CYP2C9 genotype was performed in 126 patients, mean age 87 +/-6 years (75-103), 29 males-97 females. The mean daily dose of warfarin was 3.0 +/-1.4 mg, with 3.1 mg in patients with the wild-type *1/*1 genotype (n =80), 2.7 mg in *1/*2 heterozygotes (n =20), 2.9 mg in *1/*3 heterozygotes (n =18), 1.2 mg in *2/*2 homozygotes (n =2), 2.3 mg in compound heterozygotes *2/*3 (n =6). The relationships between dose and potential factors were assessed using the correlation coefficient test for age and Fischer exact tests for the categorical variables. The only factors significantly linked to the dose were the numbers of 2C9*1 and 2C9*2 alleles. CONCLUSION: In elderly patients, a genetic influence on response to warfarin does exist as in younger patients.

[Myelodysplastic syndromes. Diagnosis and care in patients over 70 years of age]. / Les syndromes myélodysplasiques. Diagnostic et prise en charge des patients de plus de 70 ans.

A FREQUENT CONDITION IN GERIATRICS: Myelodysplastic syndromes comprise a group of bone marrow disorders characterized by abnormal hematopoetic stem cell clones. They are generally observed after 60 years of age with a peak frequency in the 70-80 year age group. Prevalence is probably underestimated and diagnosis is commonly made in geriatric wards. DIAGNOSIS: Clinical presentations vary widely, making diagnosis a difficult task. Cytology studies play a predominant role. Recent progress in our understanding of myelodysplastic syndromes have been achieved with cytogenic and molecular biology techniques, although the diagnostic procedures and management of elderly subjects have changed little. DISEASE COURSE AND TREATMENT: Prognosis of myleodysplastic syndromes is highly variable and mainly depends on the type of syndrome according to the international FAB classification. Therapeutic management in patients over 70 is limited in most cases to symptomatic treatment, principally with transfusional support.

[Excess antivitamin K in elderly hospitalised patients aged over 70. A one-year prospective survey]. / Surdosages en antivitamine K dans une population de patients hospitalisés âgés de plus de 70 ans. Enquête prospective sur un an.

INTRODUCTION: Antivitamin K treatments (AVK) are related to increased morbidity and mortality, notably in elderly patients. The International Normalized Ratio (INR) should be well controlled and its stabilisation within the therapeutic range help to prevent the haemorrhagic complications. METHODS: A one-year prospective survey on all the cases of excess AVK was conducted in hospitalised patients aged over 70. RESULTS: During the study period, 225 hospitalised patients treated with AVK (mean age 84 years) were identified: 62% received warfarin, 19% fluindione, 8% acenocoumarol and 11% received several successive AVK. During this period, 1.904 INR measurements were recorded: 97 (5.1%) were > or =5.0 and 12 (0.63%) were > or =9.0. In all, 59 patients (23.1%) exhibited one or several episodes of excess AVK (INR > or =5.0) and 57 exhibited a target INR of 2.5. Three patients died of accidental haemorrhage--two of them due to intra-cerebral bleeding--among the 59 patients with excess AVK. In three cases, the INR was greater than 7.0 at the time of the accident. DISCUSSION: In half of the cases of excess, the episode occurred during the month following initiation of treatment with AVK. In nearly two thirds of cases, a change had been made in drug therapy in the 10 days preceding the excess, with the prescription of a drug enhancing the effect of the AVK: anti-infection agents (antibiotics and anti-fungals) and amiodarone were the drugs most frequently involved. Oral or intravenous vitamin K1 was administered in only 19% of cases. CONCLUSION: In very old patients treated with oral anticoagulants, certain risk factors must be identified: the initiation period of treatment, the occurrence of an intercurrent disease and the subsequent change in the drug therapy. INR monitoring should be intensified in order to detect any excess and, if detected, ensure the optimal management of the patient.

[Targeting bone marrow with the help of polyalkylcyanoacrylate nanoparticles]. / Ciblage de la moelle osseuse à l'aide de nanoparticules de polyalkylcyanoacrylate.

Using a mouse model, we examine drug targeting towards bone marrow. One cytotoxic (doxorubicin) and one stimulating (rhG-CSF), bound to polyalkylcyanoacrylate nanoparticles, were studied. Histological studies, using a fluorescence microscope, showed rapid capture of nanoparticles by bone marrow macrophages and granulocytes as soon as 15 minutes after injection into the blood stream. Doxorubicin nanoparticles, administered at a dose of 11 mg/kg were more toxic than free doxorubicin on all blood and marrow cell lines. Moreover, the choice of the nature of the polymer had an influence on toxicity: doxorubicin polyisohexylcyanoacrylate nanoparticles were more toxic than polyisobutylcyanoacrylate particles. Quantification of doxorubicin in bone marrow has confirmed these results. The bone marrow concentrations observed demonstrated that there was a high level of targeting towards the bone marrow that would be very interesting to use for a stimulating drug. Nevertheless, rhG-CSF nanoparticles did not show better efficacy than free rhG-CSF.