Platelet concentrates produced from whole blood using the Atreus processing system

Background  The Atreus 2C+ system automates whole blood (WB) processing into a red cell concentrate, plasma and buffy coat (BC) suitable for platelet concentrate (PC) manufacture. This study compared the quality of PC made from BC using the Atreus, with those made by a manual method.
Study design and methods  WB was collected into Atreus disposables or standard bottom and top processing packs and held without active cooling for 26 h at 22 ± 2°C before processing, either with the Atreus, or using a centrifuge and press. BC were rested for 3 h and then 4 BC were pooled with one unit of plasma, mixed, centrifuged and pressed to make a pooled PC. The PC were analysed for quality markers to day 9 of storage.
Results  Platelet quality was good in both Atreus 2C+ derived PC and control units throughout storage. Metabolic markers (pH, ATP and HSR) and activation markers (CD62P, sCD62P, annexin V binding, microparticles, GP IIb/IIIa) did not differ between the Atreus and control units. Atreus-derived PC had significantly lower platelet yields (302 ± 59 × 10⁹ platelets/unit; mean ± standard deviation, n  = 8) than control PC (411 ± 76 × 10⁹ platelets/unit; P  < 0·01), but met the UK guidelines for platelet yield
Conclusion  From these in vitro data, PC produced from buffy coats prepared using the Atreus appear suitable for clinical use, and WB may be held at ambient temperature overnight without the use of active cooling devices. Optimizing the secondary processing conditions to handle Atreus 2C+ derived BC may increase the platelet yield.     

Effect of Haemostatic Control Resuscitation on mortality in massively bleeding patients: a before and after study

Background and Objectives   Evidence supporting the use of platelets and plasma in resuscitation of massive bleedings is questionable. Current consensus guidelines recommend restrictive use. Our aim was to determine the effect of changing the transfusion practice on 30-day survival in massively bleeding patients.
Materials and Methods   Consecutive adult patients receiving more than 10 units of red blood cells (RBC) within 24 h 2 years prior to (2002–2003) and 2 years after (2005–2006) a change in transfusion practice were included. In 2004, we implemented Haemostatic Control Resuscitation (HCR) with preemptive use of platelets and plasma, administered in transfusion packages, comprising 5 units of RBCs, 5 units of fresh-frozen plasma and 2 units of platelet concentrates (PC), when massive bleeding occurred or upon arrival at the emergency room and thereafter directed by thrombelastography throughout the peri- and postoperative period.
Results   In 2005–2006, the 442 patients received more PCs within 24 h from admission [mean 5·0 (SD 4·2) vs. 1·7 (2·0); P  < 0·0001] and had a smaller decrease in platelet count during the bleeding episode [91·5 (81·2) vs. 119·7 (100·8) × 10⁹/l; P  = 0·0025] than the 390 patients treated in 2002–2003. Thirty-day mortality was reduced in 2005–2006 (20·4% vs. 31·5%; P  = 0·0002) and at 90-day (22·4% vs. 34·6%; P  < 0·0001) as compared to 2002–2003.
Conclusions   In patients who experience massive bleeding, HCR with platelets and plasma, as guided by thrombelastography, is associated with improved survival. While confirmation from a randomized controlled trial is urgently needed, HCR may be considered in these patients.     

Evaluation of Apheresis Platelet Concentrates Collected with a Reduced (30-ml) Collection Chamber with Resuspension and Storage in a Synthetic Medium

Recently, the CS-3000® Plus Blood Cell Separator with the TNX-6 platelet separation chamber insert has been furnished with a small-volume (30-ml) collection chamber. In this study, a platelet synthetic medium containing glucose and bicarbonate (PSM) was used for resuspension and storage of this highly concentrated platelet product. Eighteen donors participated in a paired study design where each participant donated platelets on two occasions, once following collection in a standard chamber with resuspension and storage in plasma and once following collection in the new chamber with resuspension and storage in PSM. Substantially higher total platelet counts were obtained using platelets collected in the small chamber and stored in PSM as compared to control (4.4±0.9times10¹¹ vs. 3.5±0.9times10¹¹ platelets, p<0.01 by paired t test). After 5 days of storage, PSM-stored platelets demonstrated higher ATP levels, less lactate dehydrogenase in the supernatant and increased lactate production with resulting lower pH at day 5 of storage (6.94±0.15 vs. 7.08±0.09, p<0.05). There were no statistically significant differences of the survival by multiple-hit estimation of PSM-stored as compared to plasma-stored platelets as determined by ¹¹¹In labeling and infusion. A slight decrease in the initial percent recovery with the additive-suspended as compared to suspended plasma cells was noted: 50±8 versus 54±9%, respectively (p<0.05). In conclusion, the CS-3000 Plus/TNX-6 apheresis system with a new reduced-volume collection chamber and an additive solution provides a plasma-poor and highly concentrated platelet product with satisfactory in vivo viability and in vitro functional characteristics after 5 days of storage.     

Size dependent platelet subpopulations: relationship of platelet volume to ultrastructure, enzymatic activity, and function

A method for the separation of platelets on the basis of their size has been developed using counterflow centrifugation. Platelets were separated, free of plasma proteins and other cells, into seven subpopulations. The smallest-sized platelets, designated as Fraction 1, had a mean platelet volume (MPV) of 3.94 ± 0.60 μm³ (SD). Each successive fraction had a progressively larger MPV. The MPV for the largest-sized platelets, designated Fraction 7, was 8.19 ± 0.64 μm³. The MPV for the original platelets prior to fractionation was 6.57 ± 0.61 μm³. The mean density of Fraction 1 platelets was 1.067 ± 0.002 g/cm³, while Fraction 7 had a mean density of 1.072 ± 0.001 g/cm³. Transmission electron microscopy demonstrated that Fraction 1 had 4.3 ± 0.9 dense bodies per platelet, and Fraction 7 had 12.6 ± 2.4 dense bodies per platelet. Platelet LDH activity showed that the Fraction 1 platelets had 4.77 ± 0.92 iu per 10¹⁰ platelets; Fraction 7 platelets had 14.88 ± 1.23 iu per 10¹⁰ platelets. The LDH activity in the platelets before separation into subpopulations was 9.47 ± 1.45 iu per 10¹⁰ platelets.     

A randomized study of buffy coat platelets in platelet additive solution stored 1–5 versus 6–7 days prior to prophylactic transfusion of allogeneic haematopoietic progenitor cell transplant recipients

Background and Objective  Storage of platelets > 5 days provides improved availability, logistical management and decreased outdating. Promising results on in vitro parameters and on in vivo post-transfusion recovery and survival of autologous platelets in healthy volunteers have earlier been shown. To provide additional verification, randomized patient transfusion studies are needed.
Materials and Methods  Sixty allogeneic haematopoietic progenitor cell transplant recipients were randomized to receive buffy-coat (BC) platelets stored in platelet additive solution (PAS) for 1–5 days the first time a prophylactic transfusion was needed after transplantation, followed the second time by platelets stored for 6–7 days or vice versa. The corrected count increment (CCI) for 1 and 24 h were calculated.
Results  CCI 1 h and CCI 24 h were higher for platelets stored 1–5 days as compared to 6–7 days, 10·4 ± 5·1 vs. 7·4 ± 3·8 ( P <  0·001) and 5·4 ± 4·1 vs. 2·6 ± 2·6 ( P <  0·001), respectively. Time to next platelet transfusion was significantly longer after a transfusion of platelets stored for 1–5 days as compared to platelets stored for 6–7 days: 2·2 ± 1·1 vs. 1·6 ± 0·8 days, respectively ( P <  0·005). No differences in bleeding events and no transfusion reaction were recorded.
Conclusion  The advantage of an extension of platelet storage time beyond day 5 should be balanced against the increased need for platelet transfusions that may occur and the conceivable risk of transfusion failure.     

Improved leucoreduction of red blood cell units prepared after a 24-h hold with the platelet-rich plasma method using newly developed filters

Background and Objectives  A previous study indicated that the extension of whole blood (WB) storage from 8 to 24 h at 20–24 °C before the processing of platelet-rich plasma (PRP)-depleted red blood cell (RBC) units had a negative effect on the efficacy of leucoreduction filters. In this study, we further characterized the phenomenon and tested the leucoreduction capacity of two newly developed filters.
Materials and Methods  Whole blood was stored at 20–24 °C and processed at 4-h intervals between 8 and 24 h postcollection. Components were leucoreduced before storage. Efficacy of novel filters to leucoreduce 24-h-hold PRP-depleted RBC units was also evaluated.
Results  Using a conventional filter, the mean residual white blood cell (WBC) counts in leucoreduced PRP-depleted RBCs were comparable in units prepared within 12 h from collection but gradually increased upon extended preprocessing storage from 0·36 ± 0·03 at 12 h to 0·46 ± 0·21, 0·76 ± 0·54 and 1·72 ± 1·76 × 10⁶ per unit at 16, 20 and 24 h, respectively. However, the mean residual WBC content in 24-h-hold RBCs was reduced to 0·60 ± 0·39 × 10⁶ and 0·46 ± 0·13 × 10⁶ per units using RC2D and the prototypes B-1582 rev B filters, respectively.
Conclusion  For PRP-depleted RBC units, the extension of the WB room temperature storage from 8 to 24 h before processing is likely to require the introduction of newly developed filters having an increased leucoreduction capacity in order to meet the maximal residual WBC guideline in the RBCs.     

Application of the intravenous glucose tolerance test and the minimal model to patients with insulinoma: insulin sensitivity (Si) and glucose effectiveness (Sg) before and after surgical excision

Background  The unmodified frequently sampled intravenous glucose tolerance test (FSIGT) has not previously been used to assess insulin/glucose kinetics in patients with insulinoma.
Objective  To measure insulin sensitivity (Si) and glucose effectiveness (Sg) by means of the FSIGT in patients with insulinoma, before and after surgical removal of the tumour.
Subjects and methods  FSIGTs were performed in five patients, before and approximately 3 months post-surgery, and in 11 controls. Si and Sg were estimated using Minimal Model computer analysis of dynamic glucose and insulin data.
Results  Si was lower in insulinoma patients before, compared with after surgery (3·37 ± 0·62 vs. 6·24 ± 1·09 SE [×10⁻⁴] min⁻¹µU⁻¹ ml, P  < 0·05). Sg was similar in patients pre- and post-surgery (3·0 ± 0·67 vs. 2·4 ± 0·6 [×10⁻²] min⁻¹, NS).
Conclusions  Insulin sensitivity improves after excision of an insulinoma. Glucose effectiveness is not influenced by chronic hyperinsulinaemia and hypoglycaemia.     

Measurement of the density of human platelets and its relationship to volume

S ummary . New methods are described for platelet isolation and buoyant density determination using low-speed centrifugation in continuous density gradients of Percoll. The conditions used do not induce loss of granule or cytoplasmic markers and enable reproducible platelet frequency distributions to be obtained in linear density gradients. Such frequency distributions are normal with a mode of 1·0645 ± 0·0015 g cm⁻³ (mean ± SD, n = 20). Platelets fixed in 0·1% glutaraldehyde show a modal density of 1·0712 ± 0·0005 g cm⁻³. Content of protein, lactate dehydrogenase, beta-thromboglobulin and ³H-serotonin correlate closely with platelet numbers throughout the density distribution.
The frequency distribution of platelet volume between 2·2 and 21 fl fits a log normal model and cell volume in density subfractions from the most dense to the least dense also approximate log normality. There is a positive correlation between mean platelet volume and buoyant density with a small increment between the least and the most dense extremes. Platelet subfractions separated by volume using a FACS II cell sorter differ substantially from each other in cell volume but the difference in mean density of four different volume fractions is negligible. In discontinuous density gradients of Stractan factors other than platelet density must influence the separation of platelets, as rebanding of platelets from interfaces shows a wide variation in buoyant density when analysed in continuous gradients.
It is concluded that analysis of platelet buoyant density in continuous Percoll gradients supports the view that platelet density, like platelet volume, is determined primarily during thrombocytopoiesis and that volume and density are largely independent elements of platelet heterogeneity.     

IVE (ifosfamide, epirubicin and etoposide) is a more effective stem cell mobilisation regimen than ICE (ifosphamide, carboplatin and etoposide) in the context of salvage therapy for lymphoma

Two commonly used chemotherapy regimens for lymphoma salvage therapy were compared: ICE (ifosphamide, carboplatin and etoposide) ± rituximab and IVE (ifosfamide, epirubicin and etoposide) ± rituximab, for their efficacy in mobilising peripheral blood stem cells for autologous transplantation. Significant differences were observed between the cohorts in terms of number of patients mobilising the stipulated minimum >2 × 10⁶ CD34⁺/kg (99·2% in IVE group versus 83% in ICE group: P  =   0·0002) and also in terms of the number of patients achieving the predetermined target of >5 × 10⁶ CD34⁺/kg, both in total and during the first apheresis procedure (72% in IVE versus 51% in ICE group and 49% in IVE versus 7% in ICE group: P  =   0·02 and P  <   0·0001 respectively). This analysis of two similar groups of patients treated within a single-centre appears to demonstrate that the IVE regimen is a more effective stem cell mobilisation regimen than ICE in the context of salvage therapy for Hodgkin and non-Hodgkin lymphoma, allowing more patients to achieve the target CD34⁺ cell collection and proceed to high-dose therapy and autologous stem cell transplantation.     

Haematological reconstitution following high dose and supralethal chemo-radiotherapy using stored, non-cryopreserved autologous bone marrow

S ummary . Eighteen patients with small cell carcinoma of the lung received high dose cyclophosphamide (180–200 mg/kg) intensification following five pulses of 'CHOP' chemotherapy (cyclophosphamide 750 mg/m² i.v., adriamycin 50 mg/m² i.v., vincristine 1·4 mg/m² i.v., prednisolone 40 mg orally for 5 d). They received infusions of autologous bone marrow which had been stored at 4°C for 34 h. Pancytopenia was predictable in onset and its duration acceptable. Recovery of neutrophils to greater than 1·0 × 10⁹/l was achieved in 17·5 ± 0·9 d (mean ± SEM) and platelets to greater than 100 × 10⁹/l in 17·5 ± 0·8 d. Four patients with acute myeloid leukaemia in complete remission received intensification with the supralethal combination of cyclophosphamide and total body irradiation followed by infusion of autologous marrow which had been stored at 4°C for 54 h. Haematological reconstitution in these patients was acceptable but slower (greater than 1·0 × 10⁹/l neutrophils between days 26 and 40; greater than 20 × 10⁹/l platelets between days 23 and 77). Except in one case, normal peripheral counts were attained in all patients.
It is concluded that bone marrow stored at 4°C for up to 54 h is a simple and practical source of viable stem cells which have the capacity for acceptable haematological reconstitution.     

Not every PRP-gel is born equal Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet®, RegenPRP-Kit®, Plateltex® and one manual procedure

Background  The rationale for using topical platelet gel therapy is to provide the healing tissues with concentrated platelet-derived factors. Several systems are available to prepare platelet-rich plasma (PRP) and from these, the platelet gel. These systems produce two- to six-fold platelet and growth factor-enriched concentrations. The bioavailability of growth factors in tissue healing depends on the amount of growth factors stored in platelets but a portion of these is lost during platelet manipulation. Very few data have been reported on the kinetics of growth factor release from PRP-gels. The aim of this study is to assess the growth factor recovery and its bioavailability to tissues in four different PRP and PRP-gel preparation techniques.
Materials and methods  Three commercially available devices (Fibrinet®, RegenPRP-Kit®, Plateltex®) and one manual procedure (home made, HM) were evaluated with reference to resulting platelet concentration, growth factor content and the kinetics of growth factor release from gel.
Results  Platelet concentration increased from 1·65- to 4·4-fold in comparison with whole blood initially used. The final platelet concentration (× 10³/µl) was: Fibrinet 1358 ± 419, Regen 430 ± 109, HM 1196 ± 188, and Plateltex 1160 ± 164. A high variation (5- to 27-fold) was found in growth factor concentration in relation to the method used and also a high variation in the kinetics of growth factor release from gels.
Conclusions  Similar methods for platelet gel preparation revealed different performances concerning growth factor recovery and the kinetics of its release from the gel. It is unclear whether these noticeable differences are important for clinical management.     

Augmentation index in resistance to thyroid hormone (RTH)

Objective  Resistance to thyroid hormone (RTH) is associated with a varied clinical presentation. The cardiac effects of RTH have been described but vascular function has yet to be fully evaluated in this condition. We have measured the arterial function of those with RTH to assess any vascular changes.
Design  An observational study.
Patients  Twelve RTH patients were recruited from the thyroid clinic (mean value ± SD), age 40·8 ± 18·7 years; BMI 27·2 ± 4·2 kg/m² and compared with 12 healthy, euthyroid, age-matched controls (age 41·4 ± 19·3; BMI 24·8 ± 4·4 kg/m²) with no history of cardiovascular disease. No interventional measures were instituted.
Measurements  Arterial stiffness was measured using pulse wave analysis at the radial artery. Thyroid function, fasting lipids and glucose were also measured on the same occasion in both patients and controls.
Results  The corrected augmentation index, a surrogate marker of arterial stiffness was significantly higher in patients compared with controls (21·0% ± 14·1% vs. 5·4% ± 18·2%, P  < 0·03). Low density lipoprotein cholesterol (LDL-cholesterol) levels were also significantly elevated in patients compared with controls (3·0 ± 0·6 vs. 2·1 ± 0·5 mmol/l; P  < 0·002).
Conclusion  RTH patients show evidence in this study of increased augmentation index consistent with an increase in arterial stiffness compared with euthyroid controls. They also demonstrate elevated LDL-cholesterol levels. Both these measures may lead to increased cardiovascular risk.     

Comparison of a novel viscous platelet additive solution and plasma: preparation and in vitro storage parameters of buffy-coat-derived platelet concentrates

Background and Objectives  We developed a viscous platelet additive solution (PAS) based on MacoPharma's SSP+ but containing hydroxyethyl starch to address the poor osmotic balance and low yield associated with conventional PAS for the storage of buffy-coat platelet concentrates (PC).
Materials and Methods  Pools of four buffy-coats were made into leucoreduced PCs ( n  = 5) suspended either in plasma or viscous PAS. After determination of platelet recoveries, the PCs were stored under standard conditions. On days 1, 2, 3, 5, 7 and 9, PCs were tested for mean platelet volume, platelet concentration, soluble protein concentration, CD62 expression, platelet morphology, partial pressure of oxygen and partial pressure of carbon dioxide, glucose and lactate concentration, pH, extent of shape change, and hypotonic shock response (HSR).
Results  Platelets were prepared with greater ease using the viscous PAS and had improved platelet yield. PCs stored in either plasma or viscous PAS displayed similar storage characteristics to day 9. On days 7 and 9 of storage, platelets stored in viscous PAS displayed significantly lower ( P <  0·05) CD62 expression and higher HSR scores than those stored in plasma.
Conclusion  Alteration of the viscosity of PAS improves platelet recovery during processing and may prolong platelet quality at the later stages of storage.     

The effect of pathogen reduction technology (Mirasol) on platelet quality when treated in additive solution with low plasma carryover

Background and Objectives Pathogen reduction technologies (PRT) for platelets are now compatible with both plasma and platelet additive solutions (PAS). The aim of this study was to examine the effect of PRT on the platelet storage lesion, in the presence of PAS with low plasma carryover. Materials and Methods PRT‐treated (Mirasol) and untreated buffy coat‐derived platelet concentrates prepared in 28% plasma/PAS‐IIIM were evaluated using in vitro cell quality parameters on days 1, 2, 5, and 7 post‐collection. Results At day 5, there were no significant differences between control and PRT treated platelets for swirl, viability, pO₂, pCO₂, mean platelet volume and adenosine diphosphate‐induced aggregation. PRT treatment did not affect the functional integrity of the mitochondria. However, PRT resulted in a decrease in pH and enhancement of platelet glycolysis and activation, evidenced by increased glucose consumption and lactate production rates, increased expression of CD62P, CD63, annexin V staining and increased secretion of cytokines (P < 0·05). Hypotonic shock response and aggregation in response to collagen were also significantly reduced in PRT treated platelets (P < 0·05). Conclusion Despite the observed differences in platelet metabolism and activation observed following PRT treatment in PAS and low plasma carryover, the results suggest that treatment and storage of platelets in PAS is no more detrimental to platelets than treatment and storage in plasma.     

Functional characteristics of apheresis-derived platelets treated with ultraviolet light combined with either amotosalen-HCl (S-59) or riboflavin (vitamin B2) for pathogen-reduction

Background  To examine if different pathogen-reduction technologies (PRTs) induce different degrees of platelet (PLT) storage lesion.
Design  Twenty-seven split triple-dose apheresis PLTs were PRT treated using ultraviolet light with either riboflavin (M) or psoralen (I) or remained untreated (C). Samples taken on days (d) 0 to 8 were analysed for PLT count, blood gas (pH, pO₂ and pCO₂), metabolism (lactate, glucose, ATP content), in vitro function [swirling, hypotonic shock response (HSR) and aggregation], activation (p-selectin expression) and cellular integrity (JC-1 signal, annexin A5 release).
Results  Platelet counts of all study groups remained unchanged during storage indicating that PRT treatment did not induce relevant cell lysis. Although M units demonstrated the highest values for HSR until d5, PRT treatment lowered all parameters examined with significant differences to untreated controls by d7 of storage. During final storage, M was significantly superior over I for HSR, aggregation with TRAP-6 as agonist (collagen was similar), annexin A5 release and JC-1 signal. Regarding blood gas and metabolic analysis, the most evident effect of PRT was an elevated glycolytic flux combined with higher acidity due to increased lactate accumulation. Most likely due to impaired O₂ consumption, pH and ATP decreased more rapidly in I relative to C and M.
Conclusion  Pathogen reduction technology-treated PLTs remained comparable to untreated units throughout 7 days of storage. Mitochondria-based oxidative respiration appeared up-regulated after the riboflavin-based PRT. Compared to the psoralen-based PRT, this resulted in significantly better ATP maintenance and in vitro function during the last storage period (d7, d8).     

Antiphospholipid antibodies in adults with immune thrombocytopenic purpura

To determine the clinical significance of antiphospholipid antibodies (aPL) in patients with immune thrombocytopenic purpura (ITP), anticardiolipin (aCL) (IgG and IgM) and lupus anticoagulant (LA) were sought at diagnosis in 215 ITP adults with platelets <50 × 10⁹/l. aPL (aCL and/or LA) were detected in 55 patients (26%): aCL alone in 39 (18%), aCL and LA in 15 (7%) and LA alone in one (0·5%). LA was significantly associated with high IgG-aCL levels ( P  =   0·001). Among age, sex, initial platelet count, bleeding score, acute or chronic ITP outcome, only younger age was significantly associated with LA-positivity (mean age 29 ± 14 years vs. 45 ± 20 years, P  =   0·002). After a median follow-up of 31 months, 14/215 (7%) patients developed thrombosis (four arterial, 10 venous and/or pulmonary embolism); four of them (29%) had high aCL levels and LA. Multivariate analysis significantly associated thrombosis events only with age [hazard ratio (HR)   =   1·6; 95% confidence interval (CI): 1·2–2·4], LA (HR: 9·9; 95% CI: 2·3–43·4) or high IgG-aCL level (HR: 7·5; 95% CI; 1·8–31·5). Although the thrombosis rate was low, the significant associations between thrombosis and LA or high aCL level suggest that aPL should be tested at ITP diagnosis.     

A population pharmacokinetic model for pegylated-asparaginase in children

We analysed 1221 serum activity measurements in 168 children from the Berlin-Frankfürt-Münster acute lymphoblastic leukaemia studies, ALL-BFM (Berlin-Frankfürt-Münster) 95 and ALL-BFM REZ, in order to develop a pharmacokinetic model describing the activity-time course of pegylated (PEG)-asparaginase for all dose levels. Patients received 500, 750, 1000 or 2500 U/m² PEG-asparaginase on up to nine occasions. Serum samples were analysed for asparaginase activity and data analysis was done using nonlinear mixed effects modelling (NONMEM Vers. VI, Globomax, Hanouet, MD, USA). Different linear and nonlinear models were tested. The best model applicable to all dosing groups was a one-compartmental model with clearance (Cl) increasing with time according to the formula: Cl=Cl_i * e ^{(0·0793 * t )} where Cl_i = initial clearance and t  = time after dose. The parameters found were: volume of distribution ( V ) 1·02 ± 26% l/m², Cl_i 59·9 ± 59% ml/d per m² (mean ± interindividual variability). Interoccasion variability was substantial with 0·183 l/m² for V and 44·7 ml/d per m² for Cl, respectively. A subgroup of the patients showed a high clearance, probably due to the development of inactivating antibodies. This is the first model able to predict the activity-time course of PEG-asparaginase at different dosing levels and can therefore be used for developing new dosing regimens.     

Characterization of P2x1 purinoreceptors on rat platelets: effect of clopidogrel

This study aimed to determine the binding characteristics of [³H]α,β-Me-ATP, a specific ligand of the P2x1 receptors to rat platelets, and to investigate the effect of clopidogrel, a thienopyridine compound which has been found to selectively inhibit ADP-induced platelet aggregation and adenylyl cyclase ex vivo . Binding of [³H]α,β-Me-ATP to rat platelets was time-dependent and saturable. Scatchard analysis of the saturation binding data indicated that [³H]α,β-Me-ATP bound to one population of specific binding sites with high affinity ( K _D  =  23.6 ± 1.6 n m ; B _max = 690 ± 24 fmole/10⁸cells) ( n= 3). Unlabelled α,β-Me-ATP as well as 2-MeS-ADP and ADP competitively inhibited the specific binding of [³H]α,β-Me-ATP with IC₅₀ values of 19.0 ± 6.6, 103 ± 20 and 1120 ± 80 n m respectively ( n= 3). Other nucleotide analogues such as ATP, ATP-γS, UTP and GTP also antagonized [³H]α,β-Me-ATP binding. When administered orally (10 mg/kg, p.o.), clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation but did not affect the binding of [³H]α,β-Me-ATP to rat platelets ex vivo. In vitro , α,β-Me-ATP did not induce the aggregation or shape change of rat platelets and did not interfere with ADP-induced platelet aggregation.     

The presence of CD56/CD16 in T-cell acute lymphoblastic leukaemia correlates with the expression of cytotoxic molecules and is associated with worse response to treatment

Some cases of T-cell acute lymphoblastic leukaemia (ALL) express markers found in natural-killer (NK) cells, such as CD56 and CD16. Out of 84 T-cell ALL cases diagnosed at our Institution, CD56 and/or CD16 was detected in 24 (28·5%), which we designated T/NK-ALL group. Clinical features, laboratory characteristics, survival and expression of cytotoxic molecules were compared in T/NK-ALL and T-ALL patients. Significant differences were observed regarding age (24·9 vs. 16·4 years in T/NK-ALL and T-ALL, respectively, P  =   0·006) and platelet counts (177 × 10⁹/l vs. 75 × 10⁹/l in T/NK-ALL and T-ALL, respectively, P  =   0·03). Immunophenotypic analysis demonstrated that CD34, CD45RA and CD33 were more expressed in T/NK-ALL patients, whereas CD8 and terminal deoxynucleotidyl transferase were more expressed in T-ALL patients ( P  <   0·05 ) . The mean overall survival (863 vs. 1869 d, P  =   0·02) and disease-free survival (855 vs. 2095 d, P  =   0·002) were shorter in patients expressing CD56/CD16. However, multivariate analysis identified CD56/CD16 as an independent prognostic factor only for DFS. Cytotoxic molecules were highly expressed in T/NK-ALL compared to T-ALL. Perforin, granzyme B and TIA-1 were detected in 12/17, 4/17 and 7/24 T/NK-ALL patients and in 1/20, 0/20 and 1/20 T-ALL respectively ( P  <   0·001, P  =   0·036 and P  =   0·054). Therefore, the presence of CD56/CD16 was associated with distinct clinical features and expression of cytotoxic molecules in the blasts.     

IL-17-producing CD4+ T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome

Immunological responses are increasingly recognised as being important in the initiation and progression of myelodysplastic syndrome (MDS). Indeed, autoimmune diseases commonly occur in association with MDS, particularly in subtypes with a low risk of leukaemic transformation. This study showed for the first time that the numbers of CD3⁺ CD4⁺ IL-17 producing T cells (Th17) were markedly increased in low risk MDS compared with high risk MDS ( P  < 0·01). An inverse relationship between the numbers of Th17 cells and naturally occurring CD4⁺CD25^high FoxP3⁺ regulatory T cells (Tregs) were also described. The Th17:Tregs ratio was significantly higher in low risk disease ( P  < 0·005) compared with high risk MDS and was correlated with increased bone marrow (BM) apoptosis ( P  < 0·01). Tregs from MDS patients suppressed interferon-γ (IFN-γ) secretion by effector CD4⁺ T cells but had no effect on interleukin (IL)-17 production. In addition, the serum levels of IL-7, IL-12, RANTES and IFN-γ are significantly elevated in low risk MDS, while inhibitory factors, such as IL-10 and soluble IL-2 receptor, are significantly higher in high risk disease. The 'unfavourable' Th17:Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and the improved response to immune suppression in patients with low risk MDS compared to those with high risk disease.