Katherine McGrath

Effect of peripheral-blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy

The haematopoietic growth factor (HGF), granulocyte colony stimulating factor (G-CSF; filgrastim) substantially shortens the period of severe neutropenia that follows high-dose chemotherapy and autologous bone marrow infusion by stimulating granulopoiesis. Filgrastim also increases numbers of circulating progenitor cells. We have studied the ability of filgrastim to mobilise peripheral blood progenitor cells (PBPC) and assessed their efficacy when infused after chemotherapy on recovery of neutrophil and platelet counts. Seventeen patients with non-myeloid malignant disorders received filgrastim (12 micrograms/kg daily for six days) by continuous subcutaneous infusion. Numbers of granulocyte-macrophage progenitors in peripheral blood increased a median of 58-fold over pretreatment values, and numbers of erythroid progenitors increased a median of 24-fold. Three leukapheresis procedures collected a mean total of 33 (SEM 5.7) x 10(4) granulocyte-macrophage progenitors per kg body weight. After high-dose chemotherapy in 14 of the patients (busulphan and cyclophosphamide), these cells were used to augment autologous bone marrow rescue and post-transplant filgrastim treatment. Platelet recovery was significantly faster in these patients than in controls who received the same treatment apart from the infusion of peripheral blood progenitors; the platelet count reached 50 x 10(9)/L a median of 15 days after infusion of haematopoietic cells in the study patients compared with 39 days in controls (p = 0.0006). The accelerated neutrophil recovery associated with filgrastim treatment after chemotherapy was maintained. Subsequently, 10 patients received filgrastim-mobilised PBPC without marrow after high-dose chemotherapy. The rate of platelet and neutrophil recovery in these patients was at least equal to that observed in the patients receiving PBPC and bone marrow.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors influencing 20‐hour increments after platelet transfusion

The 20‐hour posttransfusion platelet count determines transfusion policy for patients requiring platelet support, and yet factors influencing the 20‐hour count have been poorly defined. The clinical factors influencing both the 1‐ and 20‐hour corrected count increment (CCI), were studied in 623 human leukocyte antigen (HLA)‐unmatched platelet transfusions in 108 patients. The 1‐ and 20‐hour CCIs were highly correlated (r = 0.67, p less than 0.001). On average, the 20‐ hour CCI was 64 percent of the 1‐hour CCI. Multiple linear regression analyses identified splenectomy, bone marrow transplantation, disseminated intravascular coagulation, administration of amphotericin B, palpable spleen, and HLA antibody grade as the major factors influencing the 20‐hour posttransfusion CCI. Platelet‐specific antibodies, number of concurrent antibiotics, clinical bleeding, and temperature did not significantly influence the 20‐hour posttransfusion CCI. The 1‐hour CCI was the only significant factor influencing the 20‐ hour CCI in a regression model containing the 1‐hour CCI and the above factors. Thus, the same clinical factors exert a major influence on the CCI at both 1 and 20 hours after platelet transfusion, with no evidence that any factor has more influence at 20 hours after transfusion than at 1 hour.

Analysis of cerbB2 expression using a panel of 6 commercially available antibodies

&NA; Results are presented of a study comparing cerbB2 (neu or Her2) expression as assessed immunohistochemically in breast neoplasia using a panel of 6 commercially available antibodies. The antibodies were examined utilizing conventional formalin fixed paraffin embedded tissue, and compared with molecular analysis of gene amplification. The aim was to determine the practical utility of each antibody, assessing ease of use, specific and non-specific staining characteristics, and expense, thus allowing a specific recommendation as to antibody of choice for immunohistochemical assessment of cerbB2 expression. Reassuringly, amongst the 38 breast lesions (36 carcinomas, 2 fibroadenomas) subjected to immunohistochemistry (IHC) with the panel of 6 antibodies (Ab), no gross discrepancy of staining pattern was seen. Of the 38 cases, 10 were positive (26%), where at least one Ab demonstrated clear cytoplasmic membrane staining. Of a total of 45 breast lesions (43 carcinomas, 2 fibroadenomas), including all those examined by IHC, the total number of cases showing cerbB2 amplification by DNA analysis was 14 (31%). Using the DNA amplification as a base line for comparison, one Ab (No. 4) was found to stain 6 of the 14 cases of breast carcinoma that were assessed as showing amplification at the DNA level. Four Abs (1,3,5,6) stained 5 of these cases. However, Abs 3,4 and 6 displayed artefactual cytoplasmic staining (in the absence of membrane staining) that precluded the practical use of these reagents. Therefore, based on additional considerations of cost and ease of use, Ab No. 1 was finally chosen for recommendation from the 6 Ab panel.

Stimulation of PAI-1 expression in endothelial cells by cultured vascular smooth muscle cells.

Regulation of endothelial cell (EC) plasminogen activator inhibitor type-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (TPA) and urokinase-type plasminogen activator (UPA), by various stimuli has been well characterized. We report the upregulation of secreted and intracellular PAI-1 in human umbilical ECs when cocultured with human smooth muscle cells (SMCs) on amniotic membranes or incubated with SMC conditioned medium (CM) under serum-free conditions as determined by enzyme-linked immunosorbent assay. Cocultured human umbilical vein ECs and SMCs, or human umbilical artery ECs and SMCs, displayed a 73% and 68% increase, respectively, in released PAI-1. SMC-derived stimulatory factor release showed tissue specificity, since only human aortic, umbilical vein, and umbilical artery SMCs upregulated PAI-1 synthesis, whereas SMCs from human mammary artery, pulmonary artery, and saphenous vein did not. Stimulation of EC PAI-1 by SMC CM was both time and concentration dependent, with as much as five- and fourfold increases in supernatants and lysates, respectively. PAI-1 synthesis and activity in ECs from other vascular beds were also upregulated by SMC CM. Northern blot analysis paralleled the protein results, showing as much as a 2.7-fold increase in specific EC PAI-1 mRNA expression after incubation with SMC CM for 8 hours. PAI-1 stimulatory activity in SMC CM was completely abolished by boiling or incubation with protamine sulfate and was reduced by transient acidification or heparin-Sepharose pretreatment by 33% or 48%, respectively. The stimulatory factor(s) appeared to have a molecular mass of 23 kD as determined by gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

γ-interferon counteracts interleukin-1α stimulated expression of urokinase-type plasminogen activator in human endothelial cells in vitro

Abstract The effect of γ-interferon (γ-IFN) on the interleukin-1α (IL-1γ) induced stimulation of urokinasetype plasminogen activator (u-PA) expression in human foreskin microvascular endothelial cells (HFMEC) and in human umbilical vein endothelial cells (HFMEC) was investigated. When γ-IFN and IL-1α were added to the cells simultaneously, γ-IFN inhibited the IL-1α induced increase in uPA antigen production in both HFMEC and HUVEC in a dose dependent fashion, with a maximum inhibitory effect achieved between 2.0 and 20.0U/ml of γ-IFN. Pretreatment of HFMEC with γ-IFN for 1 hour before addition of IL-1α resulted in a significant reduction in u-PA synthesis. However, when HFMEC were pretreated for 8 hours with γ-IFN before the addition of IL-1α the reduction in u-PA production was even more significant. When γ-IFN was added to HFMEC 1 hour after IL-1α, a significant inhibition in u-PA synthesis was seen. In contrast only a slight inhibition in IL-1α induced u-PA production was seen when γ-IFN was added to the cells 8 hours after IL-1a. γ-IFN also inhibited significantly the IL-1α induced increase in u-PA specific mRNA in HUVEC and HFMEC.

Guidelines for the collection, processing, storage and of administration of hemopoietic stem and progenitor cells for transplantation. Report of the Working Party on Hemopoietic Stem Cell Processing, Hematology Discipline Advisory Committee, The Royal College of Pathologists of Australasia

Division of Hematology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, South Australia*, Division of Haematology-Oncology, The Queen Elizabeth Hospital, Woodville, South Australia?, Department of Hematology-Oncology, Royal Melbourne Hospital, Parkville, Victoria:), Department of Hematology, Prince of Wales Hospital, Randwick, New South Wales~b and Hunter Valley Pathology Service, Hunter Valley, New South Walesll, Australia

Regulation of plasminogen activator inhibitor type 1 in cultured smooth muscle cells by interleukin 1α and tumour necrosis factor-α

Summary Objective: Studies on the regulation of the fibrinolytic system of smooth muscle cells (SMC) are of importance for our understanding of the formation and stabilization of intravascular thrombi and the migration and proliferation of SMC during arterial injury and atheromatous plaque development. Results: We report here the stimulation of plasminogen activator inhibitor type-one (PAI-1) synthesis in cultured human mammary artery smooth muscle cells (HMASMC) by the macrophage inflammatory cytokines, interleukin 1α (IL-1α) and tumour necrosis factor-α (TNF-α). SMC responded to IL-1α and TNF-α treatment with a time- and dose-dependent increase in PAI-1 protein of up to 1.9- and 4.2-fold, respectively, as determined by ELISA. Maximum cytokine effects were achieved at 14 pM IL-1α and 10 nM TNF-α. Analysis of mRNA showed that 3.4 kb and 2.4 kb PAI-1 mRNA transcripts increased maximally by over 5- and 3-fold, respectively, after TNF-α treatment and by over 3- and 2-fold, respectively, after IL-1α treatment, compared to respective, untreated controls. IL-1α and TNF-α did not affect urokinase-type plasminogen activator (u-PA) synthesis while IL-1α decreased tissue-type plasminogen activator (t-PA) in conditioned medium. Conclusion: These results suggest that the fibrinolytic potential of SMC can be regulated by IL-1α and TNF-α which may be released by activated monocytes/macrophages, endothelial cells or SMC themselves during the development of atherosclerosis, vasculitis or vascular injury. Increased PAI-1 would suggest an antifibrinolytic tendency in the SMC microenvironment in response to IL-1α and/or TNF-α exposure.

Detection of HLA antibodies by platelet crossmatching techniques

Thirty‐seven monospecific HLA antibodies directed against all common HLA‐A and ‐B loci and reactive by the microlymphocytotoxicity assay (LCT) were tested against platelets carrying the corresponding antigen by three platelet crossmatch methods, the platelet immunofluorescence test (PIFT), platelet enzyme‐linked immunosorbent assay (P‐ELISA), and platelet radioimmunoassay (P‐RIA). Positive reactions were obtained with the PIFT in 67 percent, the P‐ELISA in 41 percent, and the P‐RIA in 49 percent of 85 cell‐serum pairs. The same cell‐serum combinations gave 49 percent positive reactions in the lymphocyte immunofluorescence test. Three multispecific HLA antisera were positive in nine of nine cell‐serum combinations by all four methods. Thirteen transfusions were given to eight patients with known HLA antibodies. All donor‐recipient pairs were LCT positive, six were PIFT positive, and seven were PIFT negative. Three of seven PIFT‐negative and none of six PIFT‐positive transfusions were successful. Thus, platelet crossmatching is less sensitive than the LCT for the detection of complement‐binding monospecific HLA antibodies. The platelet crossmatch, however, is able to identify some potentially successful HLA‐incompatible donors for patients with multispecific HLA antibodies and limited access to HLA‐ identical donors.

Lack of In Vitro Cross-Reactivity Predicts Safety of Low-Molecular Weight Heparins in Heparin-Induced Thrombocytopenia

Alternative anticoagulation in patients with heparin-induced thrombocytopenia (HIT) is often problematic. The relatively high cross-reactivity rate reported for the low-molecular-weight heparins (LMWH) has discouraged their use in this setting. This study has investigated the safety of using the LMWH Fragmin, based on a negative heparin-dependent platelet aggregation test using the latter, in patients with proven HIT. Fifty-three evaluable patients with clinical and laboratory evidence of HIT were evaluated for cross-reactivity with Fragmin using a Fragmin-dependent platelet aggregation test. In 20 of 38 patients who showed no in vitro cross-reactivity. Fragmin was substituted for unfractionated heparin. The outcome of these 20 patients was evaluated and compared to that of the remaining 33 patients, in whom anticoagulates were ceased or warfarin or Orgaran was used. Eighteen of 20 patients treated with Fragmin increased their platelet count by ≥50 x 109/l from a mean nadir of 57.9 ± 4.7 x 109/l within 2.8 ± 0.29 days following substitution of Fragmin for unfractionated heparin. Twenty-eight of the 33 remaining patients who did not receive Fragmin increased their platelet count by ≥50 x 109/l from a mean nadir of 53.0 ± 4.8 x 109/l within 3.0 ± 0.29 days. In seven patients (two treated with Fragmin), response could not be evaluated due to death within 36 h of cessation of heparin or discharge from hospital. The results indicate that in vitro cross-reactivity testing employing a heparin-dependent platelet aggregation assay can be safely used to select patients with HIT for further anticoagulation with LMWH. Key Words: Fragmin—Crossreactivity—Heparin-induced thrombocytopenia.

A review of 100 consecutive patients with spontaneous or recurrent thrombosis

One hundred consecutive patients referred to the Royal Melbourne Hospital, Diagnostic Haematology Department for investigation of a possible hypercoagulable state were analysed 1) to determine the prevalence of congenital deficiencies of the naturally occurring anticoagulant protein C, protein S, antithrombin III and plasminogen, 2) the prevalence of acquired disorders, eg: antiphospholipid syndrome and abnormal fibrinolytic activity, and 3) whether there were clinical features associated with the inherited disorders that would identify high risk patients to allow more effective application of these tests. All patients referred were screened by the following tests: FBE, PT, APTT, thrombin time, fibrinogen, D-dimer, filtered APTT, lupus anticoagulant screening by dilute thromboplastin time and dilute Russell Viper venom time, protein C functional assay, protein S free and bound, anthithrombin III functional assay and plasminogen. Anticardiolipin assays were also performed. The age range of patients was 12-93 years, median 44 years. Both venous and arterial thrombi were studied, ratio 6:1. Abnormalities of the fibrinolytic system occurred in 35%, lupus anticoagulants in 13%, deficiencies of antithrombin III in 2%, protein C in 2% and plasminogen 2%. Neither age, documented family history, nor gender were useful in identifying patients with a detectable abnormality. The incidence of deficiencies of naturally occurring anticoagulants were similar to those previously reported. Protein S deficiency was not detected. The antiphospholipid syndrome and reduction in post stress fibrinolysis were the most frequently detected abnormalities. This suggests the need for greater understanding of the role of the fibrinolytic system in recurrent thrombosis.