bluuebunny/crossref_metadata_2025 huggingface dataset:
Dose-Dense Induction Followed by Autologous Stem Cell Transplant (ASCT) as 1st Line Treatment in Peripheral T-Cell Lymphomas (PTCL) - A Phase II Study of the Nordic Lymphoma Group (NLG).
Authors: d’Amore Francesco , Relander Thomas , Lauritzsen Grete , Jantunen Esa , Hagberg Hans , Anderson Harald , Cavallin-Ståhl Eva , Holte Harald , Østerborg Anders , Merup Mats , Hansen Mads , Telhaug Ragnar , Erlanson Martin , Kuittinen Outi , Gadeberg Ole , Delabie Jan , Sundström Christer , Ralfkiær Elisabeth , Vornanen Martine , Jensen Charlotte B.
Published: April 2020

Systemic PTCL, with the exception of alk-positive anaplastic large cell lymphoma (ALCL), have a poor prognosis. ASCT has been shown to have a favourable impact on relapsed PTCL. Therefore, the NLG designed a prospective multicenter phase II study to evaluate the impact of a dose-intensified induction schedule (6 courses of two-weekly CHOEP) consolidated in 1st PR/CR with high-dose therapy (BEAM) followed by ASCT in previously untreated systemic PTCL. This is the largest prospective PTCL-specific trial published so far. Newly diagnosed non-primary cutaneous PTCL cases aged 18–67 yrs were eligible for enrollment. Cases of alk-positive ALCL were excluded. From Oct 2001 to Feb 2006, 99 histologically confirmed PTCL cases were included in the study: PTCL unspecified (n=41), alk-neg ALCL (n=24), AILT (n=15), enteropathy-type (n=12), panniculitis-like (n=3), T/NK nasal-type (n=2), hepatosplenic (n=2). The M/F ratio was 1.8 and the median age 55 yrs (range 20–67 yrs). Although almost 2/3 of the cases presented with advanced-stage disease (62%), B-symptoms (61%) and/or elevated s-LDH (63%), the majority of them (65%) had a good performance score (WHO 0–1) at diagnosis. Of the 77 patients, where information was available for all 6 induction courses, 68 (88%) were in CR (31) or PR (37) after the 3rd and 66 (86%) after the 6th course. A total of 58 patients (75%) went through ASCT indicating that at least a fourth of this younger patient cohort has a primary refractory disease and fails therapy before reaching the transplant. Treatment-related toxicity after both induction and high-dose treatment was manageable. Of the 58 transplanted patients, 50 (86%) were still in remission at re-evaluation short after transplant. In 39 patients follow-up data one year post-transplant were available: 30 are still in CR and 9 have relapsed, suggesting that post-transplant relapses probably account for another 25% of the original patient cohort. In conclusion, the present data indicate that a time- and dose-intensified schedule is feasible and effective in previously untreated systemic PTCL. Continuous remissions are not uncommon, but a longer follow-up is needed to further characterize long-term remission rates and evaluate their impact on time-to-treatment failure and overall survival.
Increased Tendency of Venous Thrombosis in Patients with Hematology Malignancies with Over-Expression of TF and Heparanase.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: April 2020

Patients with hematology malignancies have an increased risk of deep vein thrombosis and lower limb lymphedema which is often increased by an intensive chemioterapy or by a myeloablative regimen that often requires multiple platelet-transfusion. Hypercoagulability in cancer patients is associated to phlogosis (the mRNA levels for pro-inflammatory cytokines IL-1, IL-8, LIF, MIP2-alfa were also increased), dysprotidemia, age, bed rest, cancer cell prothrombotic properties (including activation of blood coagulation), impairment of fibrinolytic pathways, alterations of endothelium, increase of the clotting activation markers, and up-regulation of such genes as those coding for transcription factors (c.fos EGR-1, ETO101, c-Myc, Fry-1), over-expression of TF, over-expression of heparanase and formation of a fibrin coat.

Both the over-expression of TF and the formation of the TF/VIIa complex induce multiple cellular responses that include proteolytic activity, calcium fluxes, p44/p42 mitogen activated protein kinase phosphorylation, and generation of thrombin. The over-expression of heparanase increased angiogenesis, tumor growth, and metastasis.

To investigate the incidence of VTE (venous thromboembolism), and DVT (deep venous thrombosis) in patients with hematology malignancies we have studied 22 patients: 7 with a myeloproliferative disorder (5 with polycythemia rubra vera, 2 with essential thrombocyhemia), 4 with lymphoma, and 11 with leukemia. No patients had suffered from venous or arterial thrombosis before diagnosis of cancer, 14 patients had a first symptomatic thrombosis occurring five months after diagnosis, 4 after starting cytoreductive treatment, 5 patients had a recurrent thrombosis during remission of their disease, no significant differences were found in the cumulative incidence of thrombosis after stratification of the patients respectiely to sex, age, and transient factors (surgery, immobilization, cateteri). For the diagnosis of DVT: CUS (compression ultrasonografy) was performed in all patients combined with D-Dimer levels. Besides we have studied the levels of TF and Heparanase (anti-heparanase antibodies IgG 2BK: HP3/17). The incidence of VTE/DVT has been demonstrated to be associated with the highest levels of TF and Heparanase.
Treatment of an Adult Patient with Hemophilia A and High Titer Inhibitor with Rituximab and Prednisone.
Authors: Rose Shelonitda S. , Greenberg David B. , Maceron Francis , Pappas Heather , Parvin Saidi
Published: April 2020

Development of inhibitors is a serious complication in hemophilia A patients (factor VIII deficiency) who receive plasma-derived or recombinant products. It occurs in about 20–30% of patients with severe or moderate hemophilia A. The presence of a high titer inhibitor (titer > 5BU), is associated with increased morbidity and mortality, complicates disease management, and increases cost of treatment. Treatments used for the management of acute bleeds include high doses of human FVIII, porcine FVIII, prothrombin complex concentrates, FVIII bypassing activity (FEIBA), and recombinant VIIa (rVIIa)). The induction of immune tolerance (IT)with daily high doses of FVIII has been reported to eradicate the inhibitor in 60–80% of patients; although it is a very costly procedure, the response appears to be dependent on the level of the inhibitor with patients with low inhibitor titer (<10 BU) responding better to IT treatment. To rapidly reduce/eradicate the alloimmune factor VIII antibodies immunomodulatory agents, including immunoglobulin, corticosteroids, cyclophosphamide, azathioprine, and plasmapheresis have been used. Rituximab, an anti-CD20 monoclonal antibody, eliminates circulating B cells and has been shown to reduce/eradicate alloimmune factor VIII antibodies in children with severe hemophilia A. We report the clinical course and response to rituximab treatment in a 24 year old male with moderate hemophilia A and high titer inhibitor. The patient presented with a massive retroperitoneal bleed with Hb 6.4g/dL and an inhibitor titer of 41 BU reaching a maximum of 111 BU over the next four weeks. For the acute bleeding episode, he received FEIBA and rVIIa. For the treatment of inhibitor, he was given rituximab 375 mg/m2 × 4 together with prednisone 1 mg/Kg daily. Prednisone was slowly tapered off over the next several weeks. Three months later, his inhibitor titer had decreased to 35 BU and remained below 15 BU for the next 30 weeks. The course of this patient’s response suggests that rituximab in combination with prednisone may rapidly reduce inhibitor titer in adult patients with hemophilia and high titer inhibitor. It is possible the rituximab therapy may replace IT therapy or be used as an adjunct to IT therapy. Also, the combination of rituximab and prednisone treatment may reduce/prevent the exposure of young patients to cytotoxic chemotherapy. Randomized clinical trials will be required to determine whether the combination of rituximab and prednisone is a better option compared to cytoxan and prednisone to reduce/eradicate inhibitors in young adult hemophilia patients with high titer inhibitor.

Figure Figure
TFPI (Tissue Factor Pathway Inhibitor) Is Present in Breast Cancer Tumor Cells.
Authors: Sierko Ewa , Sawicki Zbigniew , Butkiewicz Adrianna , Zimnoch Lech , Kisiel Walter , Wojtukiewicz Marek
Published: April 2020

Abnormalities in hemostatic system are common in breast cancer patients. The principal initiator of blood coagulation activation in cancer patients is tissue factor (TF). Tissue factor plays an important role in the processes of angiogenesis, invasion and metastatic dissemination. Tissue factor pathway inhibitor (TFPI) is the main inhibitor of TF-dependent pathway of blood coagulation. The catalytic activity of the TF/VIIa complex triggers activation of factor X, consequently thrombin generation and finally fibrin formation. Thrombin and fibrin facilitate cancer progression. Previous immunohistochemical studies revealed no expression of TFPI in cancer cells of different malignant tumor types (Werling et al, Thromb Haemost1993; 69: 366–369) The aim of the study was to evaluate the expression of TFPI protein and mRNA in tumor cells of breast cancer.

Breast cancer tissues were obtained during surgical treatment of 35 women. The patients undergoing surgery had not received any previous anticancer therapy. Tumor fragments were preserved in 4% paraformaldehyde/phosphate buffered saline and than embedded in paraffin. Immunohistochemical studies (avidin-biotin complex-ABC - technique) were performed using polyclonal antibodies against TFPI. In situ hybridization method employed single-stranded DNA oligonucleotide (probe sequence: 5′Biotin-CATCGCCCAGTGCAGCCTCCGTCAG-Biotin3′) directed against locus NM 006287 of TFPI mRNA.

Strong expression of TFPI protein was observed in cancer cell bodies in examined specimens. Weak expression of the above protein was observed in tumor infiltrating macrophages. Normal cells revealed much weaker staining for TFPI than cancer cells. Similarly, mRNA for TFPI was observed in cancer cells of all breast cancer tissues.

The above data indicate that TFPI is present in neoplastic cells of breast cancer and may play a role in neutralizing TF procoagulant and signaling functions.
DDAVP Versus vWF-Containing Factor Concentrate in a Patient with ET Associated Acquired von Willebrand Syndrome (AVWS).
Authors: Kruse-Jarres Rebecca , Leissinger Cindy A.
Published: April 2020

The patient is a 16 year old boy with a past medical history of atrial septal defect and juvenile migraine headaches. He had no unusual bleeding or bruising during his early childhood including circumcision. His family history was negative for bleeding diasthesis. At the age of 12, he was diagnosed with an intramuscular hematoma on MRI after a baseball injury. Four months later he had heavy bleeding after an accidental knife wound to his hand, requiring repeated suturing. At age 14, he had another intramuscular hematoma after baseball injury, impairing range of motion in his leg. At age 15, following another deep muscular hematoma,, he underwent a laboratory evaluation for the first time. His CBC was normal except for a platelet count of 1,216,000/μl and he was subsequently diagnosed with ET. He had an elevated PTT, with a normal PT. He had a FVIII level of 42%, vWF Ag of 43%, and vWF:RCoF < 20%. Multimer analysis showed relatively decreased to absent large multimers. To evaluate optimal treatment, a Stimate (DDAVP) challenge, as well as a vWF-containing concentrate (HumateP) infusion study were performed:

Stimate (DDAVP) trail Stimate (DDAVP) trail Humate P trail Humate P trail

The patient demonstrated a good response to DDAVP, with greater than 2-fold baseline levels of FVIII and VWF:RCo persisting after 4 hours. Multimer studies showed increases of low and intermediate multimers after administration of DDAVP. Following infusion of Humate P (53 vWF:RCo U/kg), VWF:Ag levels increased appropriately; however, VWF:RCo and FVIII levels increased to a lesser extent and fell to baseline by 4 hours. Platelet counts remained stable with both Stimate and Humate P. These results suggest that the patient’s platelets have an especially high affinity for high molecular weight (HMW) multimers, explaining the lack of HMW multimers on baseline analysis in this patient. The shortened response duration following Humate P infusion suggests rapid clearance due to platelet binding of the larger (and more functional) multimers contained in the concentrate. The longer duration of response to DDAVP, which releases his own low and intermediate multimers (but not HMW multimers) into circulation, indicates that DDAVP may be a superior treatment option in patients with AVWS associated with ET.
Increased Thrombin Generation Capacity in Septic Patients with DIC.
Authors: Kawasugi Kazuo , Shirasaki Ryousuke , Noguti MIzuho , Tashiro Haruko , Gotoh Moritaka , Shirafuji Naoki
Published: April 2020

Variety clinical conditions may cause systemic activation of coagulation, ranging from insignificant laboratory changes to severe disseminated intravascular coagulation (DIC). DIC consists of widespread systemic activation of coagulation, resulting in diffuse fibrin deposition in small and midsize vessels. However, little is known about thrombin generation capacity in patients with DIC. To investigate the thrombin generation capacity, we measured thrombin generation in septic patients with DIC (n=20) and acute promyelocytic leukemia (APL)-induced DIC (n=5). Thrombin generation was determined by the Throbogram-Thrombinoscope assay (Thermo Electron Corporation, Netherlands). The analyzed TG parameters ware the peak of thrombin activity (Peak) and endogenous thrombin potential (ETP).

The thrombin antithrombin complexes (TAT) levels were higher in both DIC patients as reported by others. In the septic patients with DIC, we found significant elevations in peak of thrombin activity and ETP as compared with normal controls (determined in 17 healthy males and 14 healthy female). However, the peak of thrombin activity and ETP levels were severely decreased by 60% n the APL patients with DIC. There was slightly correlation between the ETP and TAT levels in septic patients with DIC. Also, there was correlation between the ETP and bleeding tendency in APL patients with DIC.

These results suggest that assess of thrombin generation capacity may be helpful in the making the diagnosis in septic patients with DIC. It appears that assess of ETP may contribute to evaluate bleeding tendency in APL patients with DIC.

Sepsis with DIC APL with DIC Controls (n=31) *(P<0.05) significantly different controls Peak (nM) 325.3 ± 55.5* 88.2 ± 47.2* 281.2 ± 40.9 ETP (nM.min) 1 ± 326.1* 652.1 ± 274.5* 1469.2 ± 227.3 TAT (μg/ml) 28.4 ± 17.5* 22.5 ± 15.3* <3
Safety and Efficacy of Von Willebrand Factor/Factor VIII Concentrate (Humate-P®) for Prophylaxis of Excessive Bleeding during Elective Surgery in Patients with von Willebrand Disease.
Authors: Bernstein Jonathan , Gill Joan Cox , Leissinger Cindy A. , Di Paola Jorge , Ragni Margaret V. , Shopnick Rinah , Valentino Leonard A. , Friedman Carl , Knaub Sigurd ,   Humate P Study Group
Published: April 2020

The safety, efficacy, and optimal dosing of a von Willebrand Factor/Factor VIII concentrate (Humate-P®) were evaluated in an open-label, uncontrolled study in patients with von Willebrand disease (VWD) undergoing elective surgery. During an initial pharmacokinetic (PK) phase, a detailed profile of FVIII:C, VWF:RCo, and VWF:AG was obtained for each patient after an infusion of 60 IU VWF:RCo/kg as Humate-P. Individual PK values were used to calculate subsequent loading and maintenance doses. Hemostatic efficacy was characterized using a 4-point scale (excellent, good, moderate/poor, or none) at several time points following surgery. Forty-two adults and children were enrolled in the study (17 VWD type 1; 6 type 2; 13 type 3; 6 type 2M), and 35 of these patients underwent a surgical procedure (classified as 3 oral, 7 minor, and 25 major). The median loading dose administered was 55.6 IU/kg (range 17.4 to 135.3 IU/kg). For patients with more severe VWD (baseline VWF:RCo<12 IU/dL), the median loading dose administered was 70.9 IU/kg (range 38.6 to 135.3 IU/kg). The dosing interval was 8 or 12 hours in most subjects (4 were dosed every 6 hours), and treatment duration ranged from 1 to 6 days depending on surgery type. Effective hemostasis (investigator- rated as “excellent” or “good”) was noted in 91.4% (32/35) of subjects immediately after surgery, 100% (35/35) of subjects 14 days after surgery, and 100% (34/34) of subjects evaluated 24 hours after the last infusion (primary endpoint). Mean blood loss was less than expected, and four patients required transfusions, related to their surgery. Only six adverse events were considered possibly treatment related: headache (3), itching, nausea, and dizziness (1). These results demonstrate that von Willebrand Factor/Factor VIII concentrate is safe and effective in the prevention of excessive bleeding during and after elective surgery in adult and pediatric patients with von Willebrand disease.
Safety and Efficacy of Activated Recombinant FVII in Uncontrolled Bleeding Episodes.
Authors: Gunduz Eren , Akay Olga Meltem , Teke Hava Uskudar , Gulbas Zafer
Published: April 2020

Activated recombinant FVII (rFVIIa, NovoSeven) has been successfully used to control bleeding episodes in a variety of medical and surgical conditions including bleeding episodes secondary to over-anticoagulation, trauma, intracerebral hemorrhage, post operative bleeding and other refractory bleeding disorders. We report our experience in the use of rFVIIa to control bleeding episodes after failure of conventional measures.

A total of 5 patients (Behcet’s disease 1, over-anticoagulation with warfain and asprin 2, tuberculosis 1, and acute lymphoblastic leukaemia 1) were included in this analysis. The median age of the patients is 41. The bleeding episodes included massive hemoptysis 1, subdural hematoma 2, hemothorax and injury to intercostal artery following pleural biopsy 1 and cytomegalovirus-induced gastroenteritis in 1 patient. All patients failed to respond to conventional therapies prior to rFVIIa. The bleedings stopped with an average dose of 75 mcg/kg and with the average number of 2 doses of rFVIIa. No safety issues were encountered.

Conclusion: The use of rFVIIa is effective in the control of refractory bleeding episodes in patients with a variety of underlying medical or surgical conditions. In this small experience, a dose similar to that approved for patients with hemophilia and inhibitors appears to be effective to control bleeding within a short period of time after administration of rFVIIa. No safety issues were encountered in this group of patients. Further studies to evaluate the utility and appropriate dose and frequency of rFVIIa in refractory bleeding disorders are warranted.
JAK2 V617F in 3 Patients with Venous Thromboembolism (VTE) Followed during a Long Period of Time.
Authors: Conard Jacqueline , Marzac Christophe , Juin Fabrice , Horellou Marie-Helene , Calendini Ors’Antone , Casadevall Nicole
Published: April 2020

Hereditary thrombophilia and myeloproliferative disorders (MPD) are risk factors of VTE. The JAK2 V617F mutation may be found in polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis. We report three women referred to our out-patient clinic for a thrombophilia screening. JAK2 mutation was studied using wild type FRET probes and subsequent analysis of the melting curve (James et al, Leukemia2006; 20: 350–353). Patient 1, 75 year-old (yo), had pulmonary embolism (PE) and proximal deep vein thrombosis (DVT) at 68. We detected heterozygous FV Leiden (FVL) and FII 20210A mutations. In 2005, she had internal jugular vein thrombosis after surgery (platelet count > 600 G/L). ET was suspected in 2006. Patient 2, 44 yo, on oral contraception, had PE and proximal DVT at 33 after sclerotherapy, recurrent massive PE requiring emergency embolectomy and proximal DVT during pregnancy. Heterozygous FVL was detected in 1995 (platelet count was 432 G/L) and ET in 2000. Patient 3, 65 yo, with PV detected in 1995, had a proximal DVT one year later. Heterozygous FVL was detected. She is the only patient treated with hydroxyurea. Results of blood cell counts and JAK2 V617F/JAK2 WT ratios are shown in the table. Conclusion 1. JAK2 mutation was found in 3 patients with VTE, platelet count >400 G/L and hereditary thrombophilia; 2. the risk of VTE related to JAK2 needs further investigation; 3. JAK2 V617F increased over time in 2 of 3 cases; 4. its early detection might improve the diagnosis and treatment of MPD and VTE.

Patient Year Platelet (G/L) Hemoglobin (g/dl) Hematocrit (%) Leukocytes (G/L) JAK2 ratio 1 2000 431 15.3 45.9 8.6 10%     2006 448 13.9 43.3 10.4 50% 2 1997 541 14.1 42.6 6.1 25% 2005 796 14.0 42.8 7.4 15% 3 1997 501 14.1 42.6 4.6 15% 2003 609 13.6 42.1 5.8 20% 2006 485 12.8 37.9 4.8 50%
Tumor Site and Metastases as Predictors of Venous Thromboembolism Recurrence among Active Cancer Patients with Incident Deep Vein Thrombosis or Pulmonary Embolism: A Population-Based Cohort Study.
Authors: Chee Cheng E. , Heit John A. , Ashrani Aneel A. , Petterson Tanya M. , Farmer Sara A. , Bailey Kent R. , Melton L. Joseph
Published: April 2020

Background: Active cancer is an independent predictor of recurrent venous thromboembolism (VTE; hazard ratio=2.2–4.2). However, whether the risk of VTE recurrence among active cancer patients can be further stratified by tumor site or presence of metastases is uncertain.

Objectives: To estimate and compare the cumulative incidence of VTE recurrence by tumor site and presence of metastases among active cancer patients with incident VTE.

Study Design and Population: Retrospective cohort study of Olmsted County, MN residents with active cancer who lived >1 day after a first-lifetime deep vein thrombosis or pulmonary embolism over the 35-year period, 1966–2000 (n=418). 25 patients with oral (6), bone (1), skin (4), soft tissue (3), eye (1) and other/unknown (10) were excluded, leaving 393 remaining patients.

Measurements: Baseline demographic and clinical characteristics, tumor site, presence and location of tumor metastases, and date of first recurrent VTE. Tumor site and metastases were tested as potential predictors of time-to-recurrent VTE using the Cox proportional hazards model.

Results: 114 of 393 patients with active cancer developed recurrent VTE over 764 person-years of follow-up. The cumulative incidence of recurrent VTE at seven, 30, 90, and 180 days, 1, 5, and 10 years was 1.9%, 10.3%, 18.3%, 22.3%, 28.7%, 44.5%, and 53.5%, respectively. After adjusting for age and gender, tumor site was an independent predictor of first VTE recurrence (p<0.01). The hazard of VTE recurrence was over 2-fold higher for lung cancer patients compared to the lowest recurrence group (kidney, bladder and other genitourinary cancer; HR=2.04, 95% CI: 0.88, 4.70, p=0.096), and the hazard for brain and pancreatic cancer was nearly 2-fold higher (HR=1.9 for each, p=0.22, 0.24, respectively). The hazard of recurrence among hematological; breast and ovarian; gastric, colon, and liver; and prostate cancer patients did not differ significantly from the lowest recurrence group. Univariately, the presence of metastases at cancer diagnosis was not associated with recurrent VTE (HR=1.16; 95% CI: 0.79, 1.69, p=0.45).

Conclusions: Tumor site is an independent predictor of VTE recurrence after adjusting for age and gender. In particular, lung, brain and pancreatic cancer patients have the highest risk for recurrence and these patients should be considered for secondary prophylaxis. Disease severity, as reflected by metastatic disease at cancer diagnosis, is not a predictor of recurrence.
Detection of Antiphospholipid Antibodies Using a Novel Multiplex Test Method: Correlation with ELISA Methods.
Authors: Janatpour Kim A. , Gosselin Robert C. , Owings John T. , Wun Ted
Published: April 2020

-Background: Laboratory criteria for the antiphospholipid antibody syndrome (APS) include detection of the lupus anticoagulant (LA) by coagulation based tests, or detection of either anti-cardiolipin (aCL), or anti-B2-glycoprotein I (aB2GPI) antibodies by enzyme-linked immunoabsorbant assay (ELISA). Multiplex testing enables simultaneous measurement of multiple serum antibodies with small beads coated with target antigens and small sample volumes (5 μL). We compared the concordance of a prototype multiplex method to standard ELISA methods for measuring IgG and IgM antibodies directed against CL or B2GPI.

Methods: Samples from patients with suspected APS were tested for IgG and IgM aCL and aB2GPI antibodies using standard ELISA methods (ELISA, Bio-Rad Laboratories, Hercules, CA) and by a prototype multiplex bead set analyzed on the BioPlex 2200 (Bio-Rad Laboratories, Hercules, CA). The multiplex method used beads coated with either CL alone, B2GPI alone, or CL-B2GPI complex. Results were normalized using ratios (obtained result/upper limit of normal). A positive result was defined as a ratio of > 1. For CL, the multiplex test is considered positive if positive results are present using beads with either the CL alone or the CL-B2GPI complex.

Results:

Table 1. Concordance of ELISA vs Multiplex tests for antiphospholipid antibody detection Multiplex Test Results Concordance (%) ELISA Test Results Positive Negative Positive Negative All Results aCL-IgG Positive (n=52) 30 22 58 - Negative (n=232) 32 200 - 86 82 aCL-IgM Positive (n=42) 32 10 76 - Negative (n=241) 34 207 - 85 84 aβ2GPI-IgG Positive (n=34) 20 14 59 - Negative (n=249) 17 232 - 93 89 aβ2GPI-IgM Positive (n=61) 31 30 51 - Negative (n=219) 22 197 - 90 81

Conclusions: The overall concordance of multiplex results with ELISA results was good (81–89%). Negative multiplex results showed excellent correlation with ELISA (85–93%). Positive multiplex results had variable correlation with ELISA (51–76%). Evaluation of clinically significant positive results and clinical outcome measures is needed to determine clinical sensitivity and specificity of the multiplex test. Further refinement of the multiplex platform may provide an efficient and cost-effective assay for antibodies associated with the antiphospholipid syndrome.
Inactivation of Human White Blood Cells in Red Blood Cell Products Using Mirasol® Pathogen Reduction Technology (PRT).
Authors: Fast Loren D. , Marschner Susanne , DiLeone Gilbert , Doane Suzann K. , Goodrich Raymond P.
Published: April 2020

The transfusion of blood products can result in transmission of pathogens and immunological consequences such as transfusion associated graft-versus-host disease and alloimmunization. Previous studies have shown that exposure of platelet concentrates to riboflavin and light (Mirasol PRT treatment) causes irreparable modification of nucleic acids that results in inactivation of a wide range of pathogens as well as inhibition of the immunological responses mediated by the WBC present in platelet concentrates. Initial studies investigated the effects of Mirasol PRT treatment of RBC concentrates on WBC function. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation from control or test non-leukoreduced RBC units exposed to riboflavin and varying doses of light. These PBMNC were tested for their ability to be activated in response to PMA, to proliferate in response to PHA or allogeneic stimulator cells, and to stimulate proliferative responses of allogeneic responder cells. While lower light doses were sufficient to inhibit activation and proliferation, higher energies (30J/ml RBC) were required for inhibition of antigen presentation. These Mirasol treatment conditions did not induce crossmatch incompatibility, methemoglobin (MetHb) levels stayed within an acceptable range (2.6% + 1.5) and hemolysis was below 1% during storage. Interestingly, MetHb levels decreased to background levels during storage at 4°C, suggesting that the activity of the enzyme NADH methemoglobin reductase was not compromised by the exposure to riboflavin and light. These treatment conditions also reduced levels of Gram positive and negative bacteria to the limits of detection and reduced infectivity of both enveloped and non-eveloped viruses by > 4 logs on average in packed, washed RBC units. In summary, Mirasol PRT is able to functionally inactivate WBCs and pathogens in washed RBC products without adversely affecting the quality of the RBCs. This novel pathogen reduction therapy for RBC products complements the Mirasol PRT treatment for platelets and offers inactivation of pathogens in cellular blood components using a single technology.
Procedural Apheresis Instrumentation Algorithm Minimizes Platelets Loss during Peripheral Blood Stem Cells Collection.
Authors: Richa Elie M. , Burgstaler Edwin A. , Greiner Carl W. , Bryant Sandra C. , Ege Hilmi , Gastineau Dennis A. , Winters Jeffrey L.
Published: April 2020

Introduction:

Peripheral blood hematopoietic progenitor cell collection is a more popular source of stem cells for transplantation than bone marrow harvest. A number of automated cell separators exist which can be used to perform mononuclear cell collections. These separators collect varying amounts of other cells during the collection process, including them in the mononuclear cell product. Because of this, collection of mononuclear cells can result in significant reductions in platelet count and loss of red blood cells during the procedure. In order to minimize platelet transfusion support, we developed an algorithm to optimize mononuclear cell collections while minimizing platelet losses using the Gambro COBE Spectra and the Baxter Amicus

Methods:

Previously, patients were assigned randomly to any available apheresis machine. Starting August 2nd 2002, patients with a WBC count higher than 35×10 (9)/L were preferably assigned to Amicus for 5 hour collection with processing 1000 ml/cycle or as second choice Spectra for 5 hours run with maximum draw rate 65 ml/Min., for both instruments. If the WBC was lower than 35×10 (9)/L, the preferred machine would be Amicus for 5 hours run at 1400 ml/cycle with maximum draw rate 90ml/Min. or Spectra for 5 hours run with maximum draw 100 ml/Min. We reviewed the difference of pre and post procedure platelet counts of non Hodgkin’s Lymphoma patients undergoing stem cells collection for 6 months before and after the implementation of the above protocol from February 2002 till February 2003. All CBC were performed on the same Coulter analyzer.

Results:

14 patients (2 on Amicus and 12 on Spectra) underwent stem cells collection before August 2nd, 2002. 13 patients (8 on Amicus and 5 on Spectra) were collected after this date. The Fisher’s exact test was used for this study. The median and mean differences in the first group were 141.5 ×10(9)/L and 135 ×10 (9)/L respectively (95% CI, p 0.0183). The second group had a median and mean of 45 ×10 (9)/L and 51 × 10(9)/L (95% I, p 0.0183). The minimum and maximum differences were 36 × 10(9)/L and 200 × 10(9)/L respectively in the first group and −19 ×10(9)/L and 121×10(9)/L for the second group. There was no difference in the numbers of the collections days in both groups in regard to the type of automated cell collector.

Discussion:

The implemented algorithm showed a net advantage. Even though none of the patients required blood transfusions, there was lesser drop of platelets counts with the new settings.
Hemolysis in an Infant Post Small Bowel Transplant: A Case Report.
Authors: Rhamy Jennifer F. , Parks Jodi A. , Houseworth Julie L. , Seidel Judy , Slayten Jay A. , Waxman Dan A. , Smith Daniel S.
Published: April 2020

Introduction: A 4-month-old male infant received a small bowel transplant. He was readmitted at 9 months of age (m.o.a.) with marrow suppression and severe hemolytic anemia. Serologic evaluation demonstrated both cold and warm autoantibodies as well as two alloantibodies (anti-K and -E). T-activation was also present. The patient was unresponsive to Rituxan and IVIG, so therapeutic plasma exchange (TPE) was initiated at 12 m.o.a. A regimen of immunosuppression, IVIG and TPE was mildly successful in abating hemolysis. A third alloantibody was then identified (anti-Jka) at 13 m.o.a., and the patient continued to hemolyze. This case report describes the investigation of severe hemolytic anemia associated with small bowel transplantation.

Methods: Spreadsheets were generated with hematocrit plotted against these variables: Rituxan, IVIG, TPE, RBC transfusions, and serologic findings. Both venous and buccal samples were DNA-typed for RBC antigens. A venous sample was evaluated for chimerism at two different time points after a discrepancy was observed between buccal and venous DNA typing.

Results: The patient’s condition declined precipitously at 9 m.o.a., and stabilized at 10 and 11 m.o.a., then worsened again. Graphs of the hematocrit did not correlate with any one variable. Slight improvement was observed post-TPE, but hemolysis was observed during the procedures. Blood priming was required for all TPE. A heated circuit was devised to avoid extracorporeal cold agglutination. Samples sent for DNA analysis of the RBC antigens showed donor/recipient chimerism in the venous blood. Two of three “alloantibodies” (anti-E and Jka) were probably of donor graft origin against pt RBC. The third antigen was absent in both patient and donor DNA, and the antibody (anti-K) was likely formed in response to transfusion by either pt or donor lymphocytes. There is also a broadly reactive autoantibody. Genetic analysis showed 10% chimerism at 10 m.o.a., followed by 2% chimerism at 13 m.o.a. A decrease in chimerism was qualitatively associated with some decrease in hemolysis.

Conclusion: No one scenario explains all of this patient’s symptoms. The presence of chimerism suggests generation of autoantibodies and production of two alloantibodies by passenger lymphocytes. Alternately, the patient’s own autoimmune response, or a combination of both processes may play a causative role. Reduction of circulating cold and warm autoantibodies and alloantibodies formed by passenger lymphocytes plus priming with alloantigen-negative, least incompatible RBCs may account for the slight improvement seen with TPE.
Ex Vivo Culture and Amifostine Effects in Human Cord Blood.
Authors: Ryoo Hun-Mo , Bae Sung Hwa , Lee Kyung Hee , Hyun Myung Soo
Published: April 2020

Background

Human cord blood(CB) is the third most promising source of hematopoietic stem cells to the bone marrow and peripheral blood for replacing depressed bone marrow function in high dose chemotherapy in patient with cancer. The possibility of cord blood transplantation in adults was limited by the amount of cord blood that could be collected. Cord blood transplantation after ex vivo expansion with cytokines have already been tried in adults. Amifostine is a phosphorylated aminothiol that affords broad cytoprotection from the myelosuppressive effects of antineoplastic agents.

Objectives

The purposes of this study were to investigate expansion of progenitor and myeloid cells after ex vivo culture of mononuclear cells(MNCs) in umbilical cord blood with growth factor and characterize hematopoietic activities of amifostine.

Methods

MNCs were cultured and ex vivo expanded into myeloid progenitors by using hematopoietic growth factors(IL-1β, IL-3, IL-6, G-CSF, GM-CSF, SCF, EPO) which are known to stimulate differentiation and proliferation of myeloid progenitors. MNCs exposed to the appropriate amount of amifostine for 15 min were cultured in semisolid media and harvested at 24h intervals, and then apoptosis was assessed by flow cytometry.

Results

Myeloid colonies were successfully produced from MNCs. Maximal expansion was obtained with the combination of IL-3+SCF+G-CSF+GM-CSF. SCF was thought to be the most important growth factor for expansion of myeloid progenitor. Pretreatment with amifostine for 15 min stimulated formation of hematopoietic colonies at clinically relevant concentrations ranging from 1 to 100μM. Increase in colony number compare to control were comparable after pretreatment with amifostine (10μM), and CFU-GEMM and BFU-E were highly responsive. Further enhancement of colony was not observed after prolonging the duration of pre-incubation exposure to 1, 8 and 24 hours. Amifostine enhanced IL-1 and IL-3 induced formation of CFU-GEMM and BFU-E. Incubation of MNCs with amifostine in suspension culture increased recovery of secondary colonies. Treatment with amifostine retarded cell loss and apoptosis, and promoted cell survival at 24, 48 and 72 hours in cytokine-deficient medium.

Conclusions

In conclusion, cord blood MNCs can be successfully expanded into myeloid progenitors by using hematopoietic growth factors. This investigation extend the previously recognized hematologic effects of amifostine, and indicate that in addition to its cytoprotective properties, amifostine is a potent stimulant of hematopoietic progenitor growth.
The Effects of Heavy Ion Particles on Human Megakaryocytopoiesis and Thrombopoiesis.
Authors: Kashiwakura Ikuo , Takahashi Kenji , Monzen Satoru , Eguchi-Kasai Kiyomi , Toki Tsutomu , Abe Yoshinao
Published: April 2020

Heavy ion particles provide unique properties in radiotherapy. However, they have also been shown to pose high risks for both work at nuclear facilities and astronauts participating in space missions. In a previous study, we demonstrated that in radio-sensitive megakaryocyte progenitor cells, namely colony-forming unit megakaryocytes (CFU-Meg), a degree of X-ray-induced damage was prevented by post-treatment with several cytokines. In this study, we analyzed the effects of heavy ion particles on megakaryocytopoiesis and thrombopoiesis.

The CD34+ CFU-Meg were isolated from human placental and umbilical cord blood using a magnetic isolation kit and then were exposed to a carbon ion beam (LET=50 KeV/mm). They were cultured in a serum free medium supplemented with a thrombopoietin (TPO) alone or a combination of TPO plus other cytokines including stem cell factor, interleukin-3 (IL-3) and Flt3-ligand. The number of CFU-Meg was calculated by a plasma clot technique. The differentiation into megakaryocytes (CD41+) and the release of platelets (CD42a+) in a liquid culture were both analyzed by flow cytometry. The increase of gamma-H2AX, a marker of DNA double-strand breaks (DSBs) was also detected by flow cytometry.

The sensitivity of CFU-Meg to a carbon ion beam was found to be extremely high and could not be lowered by any type of cytokines unlike X-rays. However, treatment with TPO plus IL-3 potentially induced megakaryocytopoiesis and thrombopoiesis at 14 days after the exposure to a carbon ion beam at 2 Gy. The cytokine treatment enhanced the induction of gamma-H2AX in X-ray-irradiated CD34+ CFU-Meg but not in a carbon ion beam-irradiated one.

These results show that not only the downregulation of death signals, but also the repair of DSBs was less strongly promoted by cytokines in CFU-Meg exposed to a carbon ion beam than X-rays. Different treatments therefore are required to protect against megakaryocytopoiesis and thrombopoiesis damage by heavy ion particles.
Growth Factor Independent Succession of Hemangiopoietic Cell Development and Gene Activation Patterns in Murine Embryonic E14 Stem Cells.
Authors: Peters Stefan O. , Seifert Torsten , Middeke Mareike , Fiedler Anja , Vetters Katja , Wagner Thomas , Stoelting Stephanie
Published: April 2020

Introduction:

We use murine embryonic E14 stem cells (ESC) to study cellular and molecular events that occur during early hemangioblastic development. Cells were precultured in standard medium (DMEM, 20% FCS) for 6 days and then kept as embryoid bodies (EBs) for 0, 2, 5, 10, 15, 20, 25, 30 days either in standard or medium supplemented with VEGF or hematotropic cytokines (G-CSF, IL-3, IL-6, SCF, Flt3). Suspended cells were analyzed for CD 31, flk1, CD144, CD133, CD13 and CD 90 positivity by flow cytometry. CD31 and CD13 stained embryoid bodies were examined by fluorescent microscopy. We here focus gene activation studies on pathways implying endothelial and hematopoetic development according to literature including gene array data. cDNA was analyzed for ERG, EGR1, FLI1, HoxD3, PECAM, FLK1, VE-Cadherin, Pu-1, GATA2, SCL, Epo, LMO4, HoxB4, LMO2, and cyclin D1 activation by TaqMan PCR.

Results:

28% of cells show endothelial CD31 positivity in day 6+0 EBs. In contrast, hematopoietic CD90 positivity cannot be detected at day 6+0 and are firstly found at low levels in 2% at day 6+5. Endothelial CD31 positivity increases significantly at day 6+2 to as much as 58% and remains very high at day 6+5. Thereafter, at day 6+10 and later, the proportions of cells showing CD31 positivity drop to a constant level of about 15%. Hematopoietic development remains low throughout all timepoints and amount to maximally 10% CD90 positivity at day 6+10 declining at later time points. Low level of VEGF could be detected in ESC supernatant as early as day 6+0, endothelial cells could not be further augmented by adding VEGF to the culture. ‘Hematotropic’ cytokines occasionally show to lead to minor increases of hematopoietic cell fractions but not to lead to a shift to earlier hematopoiesis.

Quantitative PCR show that genes implying endothelial development (ERG, EGR1, FLI1, HoxD3, PECAM, FLK1, VE-Cadherin) precede characteristic ‘hematopoetic’ gene (Pu-1, GATA2, SCL, Epo, LMO4, HoxB4, LMO2, cyclin D1) activation. Addition of supplemntal VEGF surprisingly downregulate many of the ‘endothelalial’ genes. Main expression patterns remain unaffected by adding cytokines to the culture.

Summary:

We show on cellular and molecular levels that endothelial cell development in EBs precedes hematopoietic development and that the addition of cytokines does not affect this sequence.
Stimulation of Human Hematopoiesis: The Androstene Link.
Authors: Groothuis Jessie R. , Stickney Dwight R. , Malik Ajay , Garsd Armando , Reading Christopher , Dowding Charles , Frincke James
Published: April 2020

5-Androstene-3β, 17β-diol (AED) is a naturally occurring member of the androstene series of steroids. In models of chemotherapy and radiation-induced mylosuppression, AED expanded hematopoietic stem and progenitor cells (HSPC), decreased need for clinical support and increased survival. We studied hematopoietic activity of AED vs. Placebo in healthy adult (20–64 years, n=9) and elderly (65–72 years, n=9) subjects. AED was safe and well tolerated. Complete blood counts were serially obtained between study days 1–56. Bone marrow (BM) samples were obtained on days -1 and 7. AED produced significant increases in average neutrophil counts (NT) and platelet counts (PLT) as compared to placebo. Adult NT significantly increased between days 2–7 (40–73%, p=0.05) and persisted through day 28 (41.5%); adult PLT increased (19.9%) by day 14, and achieved significance by day 21 (15.6%, p=0.01) and increases persisted through day 28 (19.4%, p=0.02). In elderly subjects, NT significantly increased by 56–96% (days 4–6, p=0.05) which persisted through day 28 (30.7%). Fewer elderly subjects demonstrated robust PLT responses; maximum increase in PLT was seen on day 14 (31.5%) and persisted through day 28. BM samples at day 7 showed increased cellularity in AED but not placebo-treated subjects which was consequently reflected as an increase in peripheral blood NT and PT in AE-treated subjects. BM phenotype changes and maturation potential were estimated from immunophenotyping and CFU assay in vitro. BM precursor populations were elevated in most treated subjects on day 7; this reflected an activity of AED on precursor cells that resulted in a decrease in the CD34+CD38− and CFU-F populations and an increase in erythrocyte (BFU-E, CFU-E), and granulocyte (CFU-GM) precursors and megakaryocytes. Elderly patients (but not adults) had increased levels of two angiogenic factors; vascular endothelial growth factor and plasminogen activator inhibitor-1 in plasma (day 5) and in BM (day 7). These findings suggest that AED has a regenerative role in elderly BM and that the stimulation of angiogenic factors precedes an increase in BM cellularity which in turn leads to an increase in circulating terminally differentiated blood elements.
Fresh and Cryopreserved Umbilical Cord Blood Side Population Cells Have Comparable Immunophenotype and Function but Are Not Characterised by ABCG2 Expression.
Authors: Alt Ruediger , Pelz-Ackermann Oliver , Egger Dietmar , Niederwieser Dietger W. , Cross Michael
Published: April 2020

The side population (SP) phenotype resulting from efflux of the fluorescent dye Hoechst 33342 has been associated with high quality stem cells from both hematopoietic and non-hematopoietic organs. While expression of the transporter proteins MDR1 and ABCG2 has been strongly implicated in the SP phenotype of murine bone marrow stem cells, relatively little is known about the SP in human umbilical cord blood. The aim of this study was to investigate the relationship between the expression of common stem cell markers, the SP phenotype and the expression of the MDR1 and ABCG2 transporters in both fresh and cryopreserved umbilical cord blood. Assays of long-term Cobblestone Area Forming Cell (CAFC) activity at week 13 confirmed a 22 fold enrichment (p=0,002) of CAFC potential in the sorted SP (0,11% +/−0,06%, n=7) compared to the control living lymphocyte population subjected to Hoechst-free preparation and sorting (0,005% +/−0,0023%, n=6). SP cells from fresh (n=3) and cryopreserved (n=4) blood samples performed equally well in CAFC assays (p=0,28). A multiparameter FACS analysis involving a combination of four colors plus SP controlled with verapimil revealed the CD133+ and CD34+ cells to be distributed towards the upper part of the SP plot and to extend into the verapimil-sensitive SP. However, only 20,5% +/−15,6% (n=9) of the SP cells expressed either CD34 or CD133. ABCG2+ cells were found to be distributed throughout the main population but to be specifically absent from the side population. Fresh and cryopreserved blood samples were similar in all of these respects. In contrast to the SP cells, the MACS-purified ABCG2+ population failed to grow in cytokine-supplemented media and yielded no colonies in the CAFC assay. Semi-quantitative RT-PCR assays comparing cDNA prepared from the SP to that from the entire mononuclear population revealed a moderate but consistent enrichment of MDR1 mRNA in the SP, but no enrichment of ABCG2 mRNA. A similar comparison of the MACS-separated CD133+ (n=4) and CD133− (n=3) cells revealed the CD133+ population to be enriched (4 fold) for MDR1 but depleted (19-fold) for ABCG2 mRNA. We conclude that ABC transporters other than ABCG2 are responsible for the SP phenotype in human UCB; that MDR1 remains a potential candidate in this respect and that the frequency, immunophenotype and CAFC function of SP cells from fresh and cryopreserved blood are indistinguishable.