Cristina Sanz

Platelet-specific antibodies in HLA-immunized patients receiving chronic platelet support

BACKGROUND : In HLA‐alloimmunized patients, the unexpected failure of HLA‐matched platelet transfusions usually raises the suspicion about concomitant platelet‐specific antibodies. As the reported frequency of platelet‐specific antibodies in multitransfused patients varies widely, the aim of this study was to determine the prevalence of such antibodies in a population of chronic thrombocytopenic patients with HLA antibodies.

A model of the health and economic impact of posttransfusion hepatitis C: application to cost-effectiveness analysis of further expansion of HCV screening protocols.

BACKGROUND: Cost‐effectiveness analyses are needed to decide the value of further expansion of the screening protocols for HCV in blood donors. However, such analyses are hampered by imperfect knowledge of the health and economic repercussions of posttransfu‐sion hepatitis C (PTHC).

Prolonged Holding of Whole Blood at 22 C Does Not Increase Activation in Platelet Concentrates

Objectives: Platelets prepared after holding of whole blood overnight at 22 °C have a well‐preserved metabolism. However, the possibility that such prolonged incubation with active granulocytes may increase platelet activation has not been fully tested. Methods: We investigated this possibility by flow cytometric analysis of membrane glycoproteins (GPs) Ib and IIb/IIIa and the activation markers CD62P and CD63 in platelet concentrates (PCs) prepared from whole blood that was held for either 6 h without cooling plates (n = 20) or for 24 h on cooling plates of 1,4‐butanediol (n = 20). PCs were prepared by the platelet‐rich plasma method and analyzed on the second storage day. Results: Platelet yield and aggregation response to ristocetin, collagen and epinephrine + ADP were similar in both types of PCs, as was the mean fluorescence intensity for GPs Ib and IIb/IIIa. PCs prepared by the overnight‐hold method did not differ from those obtained 6 h after collection in the percentage of platelets expressing CD62P (12.3±6.2% vs. 14.1±4.0%; p > 0.1) or CD63 (9.8±6.4% vs. 8.8±3.6%; p > 0.1). Conclusion: Prolonged holding of whole blood at 22 °C prior to component preparation does not increase the level of platelet activation.

Influence of the Red Blood Cell Preparation Method on the Efficacy of a Leukocyte Reduction Filter

The performance of a leukocyte reduction bedside filter with different types of RBC concentrates was analyzed. Three types of RBCs were prepared: buffy‐coat‐depleted RBCs suspended in saline‐adenine‐glucose‐mannitol (SAGM)‐additive solution (BC‐RBCs; n = 20), RBCs suspended in SAGM‐additive solution without buffy coat removal (SAGM‐RBCs; n = 20), and RBCs drawn in CPDA‐1 conservative solution and processed for component preparation by the platelet‐rich plasma method (CPDA‐RBCs; n = 20). The units were filtered within 8 h of collection. One filter was used for every 2 units. High numbers of residual WBCs were found even in the units filtered first. Filtration of CPDA‐RBCs resulted in a higher residual WBC content than SAGM‐RBCs or BC‐RBCs (p = 0.0032 and p = 0.0002, respectively). The filter performance strikingly decreased when the WBC load per filter exceeded 4 × 109 or the platelet load was less than 100 × 109. We conclude that filter performance varies with the WBC and platelet content of the RBC concentrates. Under the experimental conditions assayed in this study CPDA‐RBCs are the least appropriate ones to be used for bedside leukocyte reduction.

Procoagulant Effect of Incompatible Platelet Transfusions in Alloimmunized Refractory Patients

The clinical effectiveness of platelet transfusion in refractory patients is still a subject of debate. We have evaluated the possible hemostatic effect of platelet transfusion in 16 alloimmunized thrombocytopenic patients whose platelet counts were less than 20,000/μl. Platelet concentrates were always obtained by apheresis procedures from incompatible donors. The posttransfusion platelet recovery was greater than 15% only in 3 cases. In the first 6 patients, measurements of bleeding time performed immediately before transfusion were in all cases longer than 30 min and did not change significantly 10 and 60 min after platelet transfusions. In all patients, ex vivo perfusion experiments with Baumgartners platelet adhesion model, using native nonanticoagulated blood, were performed immediately before and 10 and 60 min after transfusion. No difference in platelet deposition onto the subendothelial surface was observed after platelet transfusion. Unexpectedly, the deposition of fibrin on the subendothelial surface was statistically augmented in the posttransfusion studies. Quantification of thrombin‐antithrombin complexes (TAT) in plasma showed statistically significant elevations (p < 0.01) in the posttransfusion samples (31.9±12.6 vs. baseline 5.8±1.7 ng/ml), not justified by TAT levels in the transfused material (2.3±0.17 ng/ml). Transfusion of incompatible platelets to refractory patients may activate coagulation mechanisms in the absence of an increase in peripheral platelet count.

DDAVP enhances the ability of blood monocytes to form rosettes with activated platelets by increasing the expression of P-selectin sialylated ligands on the monocyte surface

Summary. The mechanism through which DDAVP (1‐deamino‐8‐d‐arginine vasopressin) promotes blood coagulation is not completely understood. As blood monocytes have been identified as a target for DDAVP, we investigated whether this drug increased monocyte adhesion to activated platelets, which would result in the close intercellular contact that is necessary for a juxtacrine effect on platelets and/or endothelium at sites of vascular injury. Monolayers of non‐confluent monocytes adhered to glass slides were incubated with thrombin‐activated, formaldehyde‐fixed platelets before and after the adherent monocytes were stimulated with DDAVP or n‐formyl‐methyl‐leucyl‐phenylalanine (fMLP). The number of platelets involved in rosettes with monocytes was quantified, and the effect of DDAVP or fMLP on the monocyte surface expression of P‐selectin ligands and CD11b/CD18 was assessed. DDAVP or fMLP increased the number of activated platelets involved in rosettes with monocytes by 2·8‐ and 4·9‐fold respectively. EDTA and inhibitors of the P‐selectin/counter‐receptor interaction decreased the platelet numbers in rosettes by 80–90%, whereas inhibitors of the integrin‐mediated adhesion reduced rosettes by 40–50%. Blocking the P‐selectin glycoprotein ligand‐1 (PSGL‐1) with the monoclonal antibody, Pl‐1, decreased the platelet numbers in rosettes by only 50%. In contrast, surface expression of the sialylated ligands of P‐selectin and, to a lesser extent, of CD11b/CD18 increased upon monocyte activation with DDAVP or fMLP, whereas it decreased slightly with PSGL‐1. These results indicate that DDAVP enhanced the ability of blood monocytes to bind activated platelets, mainly by increasing the expression of P‐selectin sialylated ligands on the monocyte surface. A similar effect was achieved with fMLP.

Massive intravascular haemolysis in a patient with Clostridium perfringens sepsis

Massive intravascular haemolysis is a life-threatening complication of clostridial infection. Prompt recognition of this entity and early antibiotic therapy offers the only possibility of preventing an otherwise fatal outcome [1]. We present the case of a patient with massive haemolysis as a result of infection with Clostridium perfringens. Gross haemolysis in the blood samples taken for pretransfusion compatibility testing, and reactivity of the patient’s red blood cells (RBCs) with the lectin Arachis hypogaea, alerted the blood bank to direct clinicians caring for the patient towards the correct diagnosis. A 79-year-old-diabetic man was admitted to our hospital because of a 6-h history of fever, weakness and elimination of red dark urine. His temperature was 37·5 °C. Jaundice and a distended and tender abdomen were found on physical examination. The haemoglobin (Hb) level was 9·2 g/dl, haematocrit (Hct) 0·25 l/l, mean cell volume (MCV) 93 fl, reticulocytes 5%, white blood cell (WBC) count 40 × 109/l (80% neutrophils and 6% band cells) and platelets 59 × 109/l. Other abnormal laboratory data included creatinine 1·4 mg/dl [normal range (NR): 0·5–1·3 mg/dl], total bilirubin 10 mg/dl (NR: 0·2–1 mg/dl) and lactate dehydrogenase (LDH) 19 000 U/l (NR: 250–450 U/l). Two packed RBC units were requested from the blood bank, where the severity of the haemolysis was ascertained upon visual examination of blood samples. Direct and indirect antiglobulin tests were negative. Testing of the patient’s RBCs with the Gamma Lectin System (Gamma Biologicals, Houston, TX) showed strong reactivity with Arachis hypogaea, but not with Glycine soja, Salvia sclerae or Salvia horminum, indicating Th or Tk activation. This finding raised the suspicion of massive haemolysis secondary to clostridial infection. Gram staining of a peripheral blood smear revealed abundant Gram-positive rods. Haemoglobinuria, but not intact RBCs, was found in a urine sample. High-dose intravenous penicillin G and clindamicyn were started. Two days later, C. perfringens grew in blood cultures. Complementary explorations, including abdominal ecography and a battery of tumour-associated antigens, were suggestive of an underlying pancreatic neoplasm. Despite intensive medical treatment, the patient deteriorated rapidly and died 4 days later. Necropsy was not performed. Haemolysis associated with clostridial infection is thought to be the result of α-lecithinases released by the bacteria. Although it is not clear whether clostridial neuraminidase contributes to haemolysis, desialylation of the RBC membrane leads to a variable degree of T activation that can be easily detected with the lectin panels usually available in blood banks [2]. T activation has been previously described in two patients with severe haemolysis caused by infection with C. perfringens [3,4], but not in a third patient [5]. Although the frequency of T activation in patients with extensive haemolysis and C. perfringens infection has not been documented, supernatants from cultures of C. perfringens induced T activation in 26 out of the 29 different strains tested, 24 of which led also to RBC haemolysis (M. P. E. Poulsen, cited in ref. [2]). This is the second case of massive haemolysis caused by C. perfringens infection detected in our hospital during the last 2 years [1]. In both cases, inspection of blood samples taken for pretransfusion compatibility testing allowed the blood bank to raise the alarm about the severity of the haemolysis. In this setting, testing the patient’s RBCs with appropriate lectins may be of help in achieving a prompt diagnosis.

Platelet activation and interval between plateletpheresis

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