F. Vertessen

A myelodysplastic syndrome preceding acute lymphoblastic leukaemia

The bone marrow of a patient with pancytopenia showed dyserythro‐poiesis, dysmegakaryocytopoiesis and 13% blasts. The patient was hypertransfused and the pancytopenia resolved completely for 1 month, while the blastic infiltration in the bone marrow remained. Three months later a frank acute lymphoblastic leukaemia developed.

Haemostatic changes and acquired activated protein C resistance in normal pregnancy

Influence of changes in levels of coagulation factors and anticoagulants on acquired activated protein C (APC) resistance were studied in 40 healthy women during normal pregnancy. Factor VIII (FVIII), von Willebrand factor antigen (VWF:Ag), free protein S (FPS) and protein C were determined at 5–13, 14–26 and 27–40 weeks gestation and more than 6 weeks postpartum. APC anticoagulant activity was determined by measuring the activated partial thromboplastin time before and after adding human APC, expressed as the APC–sensitivity ratio (APC-SR). During the second and third gestation trimesters a significant increase (P < 0.05) in FVIII and VWF:Ag levels and a decrease in FPS levels were seen compared with the first trimester. Postpartum FVIII and VWF:Ag levels significantly decreased and FPS levels increased compared with the third trimester. Protein C levels remained unchanged during pregnancy and postpartum. Between increased FVIII and lowered APC-SR a trend of inverse correlation (r = −0.329; P = 0.076) occurred in the second trimester. No correlation was found between APC-SR and FPS or VWF:Ag levels. A remarkable finding is the strong inverse relationship between APC-SR and protein C levels (r ≤ −0.392; P < 0.05) during pregnancy and postpartum. This may indicate that anticoagulant activity of added human APC measured by activated partial thromboplastin time is diminished in the presence of high endogenous protein C levels. A possible hypothesis is steric hindrance by sample protein C of APC binding sites on target activated factor V and/or cofactor protein S. The clinical significance of this finding should be determined because it complicates the interpretation of lowered APC-SR.

Platelet prothrombinase activity, a final pathway platelet procoagulant activity, is overexpressed in type 1 diabetes: no relationship with mean platelet volume or background retinopathy

There is overwhelming evidence that platelets from diabetic individuals are hyperreactive, not only when mi crovascular complications are apparent, but already at an early stage of the disease. There is still controversy about the ques tion of whether primary hyperreactive platelets may contribute to the origin or progression of microangiopathy or whether diabetic platelet hyperfunctionality is just a logical conse quence of a continuous low-grade activation of platelets by contact with a diseased microvascular wall. As a consequence of platelet activation, the outer layer of its phospholipid mem brane is more procoagulant than in the quiescent state, stimu lating thrombin formation in plasma. This platelet function is called platelet procoagulant activity. We studied platelet pro thrombinase activity (PPA), a final pathway platelet procoagu lant activity of type 1 diabetic platelets, and looked for an eventual correlation with microvascular disease (background retinopathy) and mean platelet volume (MPV). Stypven clot ting times (SCTs), reflecting PPA expression, and MPV of citrated platelet-rich plasma (PRP), were measured in 21 pa tients with type 1 diabetes—10 with and 11 without back ground retinopathy—under clinically acceptable metabolic control and compared them to 20 disease-free voluntary con trols. We also compared PPA expression and MPV in diabetic individuals with and without retinopathy. With the SCT, a se lective test adapted for studying PPA in PRP, we found hyper expression of PPA in all diabetic patients. We found no differ ence in MPV between diabetic and control PRP. Comparing patients with and without background retinopathy we found no significant difference in PPA expression. From these results, we suggest that the phospholipid surface of diabetic platelets, more than the surface of normal control platelets, stimulate the expression of PPA. This diabetic platelet coagulant anomaly was not related to an increased platelet mass (higher MPV) nor to the presence of microangiopathy. We conclude that PPA hyperexpression is associated with patients with type 1 diabe tes, already occurring in an early stage of the disease, and not necessarily a consequence of early-stage microvascular disease, because the anomaly is also demonstrable, in the same degree, in patients with diabetes without microangiopathy.

Distal deep venous thrombosis in a hemophilia A patient with inhibitor and severe infectious disease, 18 days after recombinant activated factor VII transfusion.

We describe a 38 year old hemophilia A patient with a factor VIII inhibitor who was admitted to our Hematology Department in January 2001 with a seriously infected and bleeding perianal ulcer. To treat infection and bleeding the patient received broad spectrum antibiotics and recombinant activated factor VII (rFVIIa) (Novoseven(R)) for about 1 month (see detailed time of administration and dosing schedule of rFVIIa further in text). Eighteen days after his last rVIIa infusion the patient developed an ultrasound proven right calf vein thrombosis. In the whole period of admission, preceding the thrombotic event the patient biologically showed a picture of severe systemic inflammatory disease as indicated by persistent increased levels of D-dimer and fibrinogen (table). It is an interesting point of discussion whether the calf thrombosis was provoked as a consequence of rFVIIa infusion (with symptoms 18 days after the last infusion) or as a consequence of long-standing immobilization and severe inflammatory disease immobilization and severe infection are conditions well known for promoting venous thromboembolic disease.

Comparison of antiplatelet effect of loading dose of clopidogrel versus abciximab during coronary intervention.

Randomized clinical trials have evidently shown that the addition of thienopyridines or abciximab to standard aspirin results in a significant reduction of ischaemic complications after coronary stent implantation. A head-to-head comparison of these antithrombotic drug regimens during coronary intervention is, however, lacking, and this was the main aim of the present study. Thirty-nine patients with angina pectoris who were scheduled for coronary stent implantation were assigned to either group 1 (160 mg aspirin + 500 mg ticlopidine post-stent), group 2 (160 mg aspirin + abciximab + 500 mg ticlopidine post-stent) or group 3 (160 mg aspirin + loading dose (375/450 mg) clopidogrel pre-stent and 75 mg clopidogrel post-stent). A loading dose of 450 mg clopidogrel was found to be more effective than the standard loading dose of 375 mg. Platelet aggregation induced by 4 μmol/l adenosine diphosphate (ADP) was assessed in samples collected before intervention and 10 min, 4 h and 20 h after intervention. Before intervention, a moderate antiplatelet effect because of aspirin intake was observed (ADP aggregation level, ± 50%) in all study groups. After intervention, platelet aggregation tended to be enhanced in group 1 while it was strongly inhibited in the groups pre-treated with clopidogrel or abciximab: ADP induced an aggregation level early after intervention of 60 ± 12% in group 1 (ticlopidine post-stenting) versus 30 ± 10% in group 3 (loading dose clopidogrel) versus 3 ± 6% in group 2 (abciximab). Abciximab achieved a more complete inhibition of aggregation than clopidogrel (P = 0.007). The overall complication rate was low with only one major bleeding and one death due to side-branch occlusion with re-infarction occurring, both in the abciximab group. Platelet aggregation during coronary intervention is strongly inhibited by both abciximab and by high loading dose of clopidogrel. Although abciximab showed a stronger antiplatelet effect than clopidogrel, it remains to be established whether this ex vivo superiority of abciximab also translates into an overall clinical benefit in patients with elective stent implantation.

The evolution of platelet procoagulant activity of remnant platelets in stored platelet concentrates prepared by the platelet-rich plasma method and the buffy coat method

Abstract Platelets stored as concentrates are gradually activated (storage lesion), a process associated with changes in the expression of platelet procoagulant activity (PPCA). The aim of the present study was to evaluate the evolution of PPCA and the mean platelet volume (MPV) of stored platelets prepared according to the platelet-rich method (PRM) and the buffy coat method (BCM). Using the platelet factor 3 availability clotting test (PF3AT) on appropriately diluted concentrate samples, we found a decrease in PPCA expression of remnant platelets as a function of storage time (0.025<p<0.01 between day 1 and 7) in PRM-derived but not in BCM-derived platelet concentrates. Using the PF3AT reduction test we found a more important clotting time reduction in samples obtained from BCM than in samples obtained from PRM platelet concentrates, suggesting a higher PPCA expression of BCM platelets, not significant after 1 day but highly significant after 3 days (p<0.0005) and after 7 days (p<0.0005) of storage, as compared with PRM platelets. For both PRM and BCM concentrates there were no significant MPV changes as a function of storage time, but at any storage day the MPV of BCM concentrates was significantly higher (p<0.0005) than the MPV of PRM concentrates. We conclude that the decrease of PPCA expression in PRM-derived concentrates as a function of storage time is in agreement with the gradual decrease of the platelet activation status in PRM concentrates during storage. There are probably several factors or variables causing platelets of BCM concentrates to express higher PPCA than those of PRM concentrates. Higher PPCA expression in BCM concentrates may be explained by an intrinsic platelet property, such as a difference in MPV between the two kinds of concentrates, or it may be related to an extrinsic factor such as different storage media, e.g., undiluted autologous plasma in PRM concentrates versus Plasmalyte A-diluted autologous plasma in BCM concentrates. Whether the difference in PPCA expression of remnant platelets in PRM and BCM concentrates is just an in vitro laboratory finding or may have consequences for the therapeutic efficiency of the concentrates is an interesting, still unresolved question.

Performance evaluation of the PENTRA 60C+ automated hematology analyzer and comparison with the ADVIA 2120

The PENTRA 60C+ hematology analyzer provides a complete blood cell (CBC) count, including a five‐part differential (5‐DIFF) count and two leukocyte subpopulations, i.e. large immature cells (LIC’s) and atypical lymphocytes (ALY’s). We evaluated its analytical performance and assessed agreement with the ADVIA 2120, in order to install the analyzer in a small satellite hematology laboratory. First we assessed repeatability, reproducibility and carry‐over to evaluate the analytical performance. Then we used Pearson correlation coefficients, Passing and Bablok regression analysis and a graphical approach (n = 209) to evaluate agreement with the ADVIA 2120. Repeatability and reproducibility were excellent for the majority of CBC and 5‐DIFF count parameters. Carry‐over was negligible. Our data showed very good correlation for most CBC count parameters. Lower correlation coefficients were observed for red cell distribution width, mean corpuscular volume and mean platelet volume. As compared to the ADVIA 2120, the 5‐DIFF count performed very well. Agreement was poorer for low‐level eosinophils and basophils. Furthermore, the PENTRA 60C+ was equally able to identify pathological blood samples through the determination of LIC’s and ALY’s. Therefore, the PENTRA 60C+ is an eligible blood cell counter to be operational in a satellite laboratory setting.

Clinical relevance of clopidogrel unresponsiveness during elective coronary stenting: Experience with the point-of-care Platelet Function Assay–100 C/ADP

BACKGROUND Early identification of nonresponders to clopidogrel may be important in identifying subgroups of patients that might be at risk for future thrombotic events. METHODS We prospectively assessed postclopidogrel platelet reactivity in 250 consecutive patients scheduled for elective percutaneous coronary intervention (PCI). All patients received dual antiplatelet therapy with 160 mg aspirin and a 300 mg loading dose of clopidogrel >12 hours before PCI. A platelet aggregation test was performed at the time of the intervention using a point-of-care assay, the Platelet Function Assay (PFA-100C/ADP; Dade-Behring, Deerfield, IL). Nonresponders were defined as having a PFA closure time of <71 seconds under dual oral antiplatelet therapy, reflecting normal platelet reactivity. Myonecrosis post-PCI constituted the primary end point and was defined as the release of creatine kinase-MB >1x the upper limit of normal on a sample taken 12 to 24 hours after intervention. The secondary end point was a composite end point of major adverse cardiac events including death, myocardial infarction, and stent thrombosis after 6 months. RESULTS The PFA closure time was available in 242 patients and ranged from 31 to 300 seconds with a mean value of 147 seconds. Nonresponders represented 7% (17/242) of the cases. Myonecrosis post-PCI occurred in 29 patients (12%) and was more common in nonresponders than in normal responders (29% vs 11%, respectively; P = .03 on multivariate analysis). Major adverse cardiac events at 6 months occurred in 13 patients (1 sudden death possibly related to stent thrombosis and 12 post-PCI myocardial infarctions) and were more common in the nonresponder group (12% vs 5%, respectively; P = .06 on multivariate analysis). CONCLUSIONS Unresponsiveness to clopidogrel as assessed by the point-of-care test PFA-100C/ADP is an independent major risk factor for thrombotic complications after coronary intervention.

A comparative-study of latex d-dimer reagents

Abstract D-dimers are terminal fragments resulting from plasmin digestion of cross-linked fibrin. They are assayed in citrated plasma by agglutination of latex particles coated with monoclonal antibodies directed against D-dimer epitopes. In the following study we have compared six latex D-dimer reagents in several experiments: reactivity with freshly drawn and frozen plasma samples, with so-called “D-dimer standards”, with increasing fibrinogen concentration, with fibrinogen and fibrin split products prepared in fresh normal samples and with “purified” D and E fragments. We have also tested the influence of several diluting fluids and the effect of freezing and thawing. We have found remarkable titre differences of D-dimers between the six reagents in fresh and frozen samples. The reagents do not recognize, or in a titre different from the labelled value, ELISA standards from Ortho and Stago. They do not react with fibrinogen and fibrinogen split products. Self prepared fibrin split products are recognized by all latex reagents but with amazing titre differences, also depending on the plasmin digestion time. According to our experiments the titre is dependent on the diluting fluid used. None of the tested latex reagents react with pure E fragments but all react, in a rather strong way, with pure D fragments except one. We have tested six commonly available D-dimer latex reagents showing quite different titre reading of fresh and frozen plasma samples and of self prepared fibrin split products. Perhaps these results can be explained by differences in specificity and/or affinity of the coating monoclonal antibodies for the D-dimer cross-link. Perhaps some of the used monoclonal antibodies are also sensitive to the presence of early and intermediate fibrinolysis products and not only to terminal fragments (D-dimers). The reaction of all but one of the reagents with purified D fragments is in favour of a lack of specificity of the monoclonal antibodies for the D-dimer cross-link. We conclude that D-dimer titres of plasma samples assayed by latex reagents have no absolute value making interlaboratory comparison of D-dimer results impossible especially when different reagents are used. The only meaningful application of such reagents is the follow up of the D-dimer titre in a same patient for example during therapy.

RETICULOCYTE ENUMERATION ON BAYER ADVIA 120 IS NOT INFLUENCED BY MALARIA PARASITES.

Address for correspondence : Jan Van den Bossche Grote Steenweg, 271 bus 6 2600 Berchem Recent fully automated haematology analysers are capable of generating reticulocyte counts. This reticulocyte count is based on ribonucleic acid staining and flow cytometric analysis to differentiate reticulocytes from red blood cells. There is marked improvement in the precision and cost-effectiveness of this automated method compared to the manual method . It has been demonstrated that red blood cell inclusions, like HowellJolly bodies 3 and malaria parasites , may react with nucleic acid dyes and can falsely increase the reticulocyte count. During the summer of 2000, 30 samples of 10 patients infected with malaria were analysed to study the possible interference of parasites on our automated reticulocyte count. Blood samples were analysed on a Bayer Advia 120 haematology analyser (Tarrytown, USA) and manual reticulocyte counts were done following brilliant Cresyl blue staining. Results were reported as % reticulocytes for both methods. For malaria investigations, thick and thin smears were prepared from EDTA anticoagulated blood. The percentage of malaria infected red blood cells was calculated from a Wright stained thin blood smear. Correlations are presented in figure 1 (next page). We found a good correlation between automated and manual reticulocyte counts 2 (correlation coefficient : r = 0.95) and no correlation between our automated reticulocyte count on Bayer Advia 120 and the number of malaria infected red blood cells present in the sample(r =0.07). Hoffman and Pennings found a pseudoreticulocytosis on Abbott Cell Dyn 4000 due to the presence of malaria parasites. The reticulocyte function of different haematology analysers uses different nucleic acid dyes and different incubation times. Therefore the interference of malaria parasites on reticulocyte enumeration can be dependant on the type of haematology analyser. To conclude, the automated reticulocyte enumeration of Bayer Advia 120 was comparable to the manual method and was not influenced by the presence of malaria infected red blood cells.