bluuebunny/crossref_metadata_2025 huggingface dataset:
Ex Vivo Expansion of Frozen Cord Blood Products without Selection.
Authors: McNiece Ian K. , Harrington Jenny , Kellner Joshua , Turney Jennifer , Shpall Elizabeth J.
Published: April 2020

Ex vivo expansion of cord blood products (CB) has been proposed as an approach to increase the number of cells available from a single CB unit. We and others have reported the requirement of CD34 selection for optimal expansion of CB products, however, the selection of frozen CB products results in significant losses of CD34+ cells with a median recovery of 43% (range 6 to 203%, N=40) and low purities resulting in decreased expansion. Therefore we explored approaches to expand CB without prior selection and have described the use of co-culture of CB mononuclear cells (MNC) on mesenchymal stem cells (MSC). In the present study we have evaluated the expansion of clinical CB products (provided by Duke University CB Bank CB). MNC were obtained after ficol separation of RBCs and 10% of the CB product was cultured on preformed layers of MSC in T150 flasks containing 50ml of defined media (Sigma Aldrich) plus 100 ng/ml each of rhSCF, rhG-CSF and rhTpo. After 6 days of culture, the non adherent cells were transferred to a Teflon bag and a further 50 ml of media and GFs added to the flask. Again at day 10, non adherent cells were transferred to the Teflon bag and media and growth factors replaced. At day 12 to 13 of incubation the cells were harvested, washed and total nucleated cell (TNC) counts and progenitor assays performed. In three separate experiments we have achieved greater than 20 fold expansion of TNC with a median of 22, and a median expansion of GM-CFC of 37 fold. Morphologic analysis demonstrated the expanded cells contained high levels of mature neutrophils and neutrophil precursors. In vivo studies in NOD/SCID mice also demonstrated that the expanded cells maintained in vivo engraftment potential. Clinical studies are being designed to evaluate the in vivo potential of CB MNC products expanded on MSC.
Recombinant TAT-HOXB4 Protein Promotes Ex Vivo Expansion of Primitive Human Hematopoietic Cells.
Authors: Krosl Gorazd , Giard Marie-Pier , Krosl Jana , Humphries R. Keith , Sauvageau Guy , Roy Denis-Claude
Published: April 2020

The clinical application of therapeutic protocols depending on hematopoietic stem cell (HSC) transplantation for long term reconstitution with donor-derived HSCs, particularly in patients previously exposed to intensive radiation or chemo-therapy, or when grafts are purged of infiltrating malignant or alloreactive T cells, can be severely hampered by limited numbers of HSCs in the graft. In mouse bone marrow transplantation models, engineered overexpression of HOXB4 has been one of the most potent stimulator of HSC expansion identified to date. The simple addition of soluble recombinant TAT-HOXB4 protein was also recently reported to enable rapid in vitro expansion of mouse HSCs that retain their in vivo proliferation and differentiation capacity. To test the feasibility of using TAT-HOXB4 as a stimulator of human HSC expansion, we performed a series of experiments using CD34+ populations isolated from healthy volunteers. The CD34+ cell populations were cultured in X-Vivo medium supplemented with Stem Cell Factor (300 ng/mL) and G-CSF (50 ng/mL) in the presence or absence of TAT-HOXB4 protein (50 nmol/L) for 4 days. In response to TAT-HOXB4, total numbers of mononuclear cells demonstrated a modest but distinct 2-fold increase compared to controls. TAT-HOXB4 treatment had the largest proliferation enhancing effect on more primitive cell populations such as CFU-GEMM, BFU-E and BFU-Meg, whose numbers increased 26.5 ± 1.4 fold (mean±S.D.), 2.2 ± 0.7 fold and 2.1 ± 0.2 fold, respectively, over their input values, and 19.1 ± 1.3 fold, 2.7 ± 0.7 and 31 ± 3.4 fold, respectively, compared to growth factor only controls. In response to TAT-HOXB4, the total numbers of CD34+CD38-Lin- cells increased 2.1 ± 0.7 fold above their starting numbers compared to a 1.5 ± 0.5 fold loss of this population in control cultures. HSC numbers were enumerated at the beginning, and after a 4-day TAT-HOXB4 treatment period using a NOD/SCID repopulation assay. In response to 50 nM TAT-HOXB4, NOD/SCID repopulating cell (SRC) numbers increased ~2-fold over their input values, compared to a 9-fold loss in control cultures without TAT-HOXB4. These results show that recombinant TAT-HOXB4 protein has the capacity to rapidly induce ex vivo expansion of primitive human bone marrow populations, and suggest that optimization of treatment conditions will rapidly lead to clinically useful expansion of human HSCs.
Most of the Short Term Repopulating Cells in Human Mobilized Peripheral Blood Do Not Have a Side Population (SP) Phenotype.
Authors: Fischer M. , Schmidt M. , Klingenberg S. , Eaves C. , von Kalle4 C. , Glimm4 H.
Published: April 2020

The multidrug resistance transporter, ABCG2, is expressed in primitive hematopoietic stem cells from a variety of sources. These cells are detected in dual wave-length fluorescent FACS profiles as a “side population” (SP cells) on the basis of their ability to efflux the fluorescent dye, Hoechst 33342. We have previously shown that 2 types of human short term repopulating cells (STRC) can be enumerated by limiting dilution analysis of their efficient ability to regenerate exclusively myeloid cells after 3 weeks (STRC-Ms), or both myeloid and lymphoid cells after 6–12 weeks (STRC-MLs) in NOD/SCID-b2microglobulin-/- (b2m-/-) mice. Previous findings also implicated these STRCs as determinants of the rapidity of early hematologic recovery in patients transplanted with cultured mobilized peripheral blood (mPB) cells. Here we asked whether any human STRCs have an SP phenotype and hence whether the isolation of SP cells would retain the rapid repopulating activity of a clinical transplant. CD3- SP and non-SP cells were isolated by FACS from low-density (LD) mPB cells after Hoechst staining and transplanted at limiting dilutions into 117 sublethally irradiated b2m-/- mice. The numbers and types of human hematopoietic cells present in the bone marrow of these mice were subsequently monitored by FACS analysis of bone marrow cells aspirated serially, 3, 8 and 12 wks post-transplant. A verapamil-sensitive SP population was reproducibly detected in all 5 patients’ samples studied (0.039 ± 0.012% of the CD3- LD cells). The in vivo assays failed to detect either STRC-Ms or STRC-MLs in the SP fraction and all these activities were obtained from the non-SP cells. If even a single recipient of the largest dose of SP cells transplanted had been positive, this would have detected 10% of the STRCs present. Thus, >90% of all STRC-M and STRC-ML in mPB are non-SP cells. However, 4 of 40 mice transplanted with SP mPB cells produced some B-lymphoid cells only starting 12 wks post-transplant. However, this result is difficult to interpret since subjecting the STRC-Ms to the Hoechst 33342 staining and FACS isolation procedure alone eliminated their ability to generate megakaryocytic progeny in vivo, although this did not occur when these cells were just stained for CD34 and then isolated by FACS. In addition, the differentiation behaviour of STRC-MLs was not affected by the Hoechst staining and subsequent FACS isolation procedure. In summary, we demonstrate that purification of SP cells depletes human mPB transplants of STRCs, thereby raising serious concerns about the safety of any clinical use of SP cell-enriched transplants as stem cell support after myeloablation. Our results also suggest that the staining and enrichment procedure for isolating SP human cells may differentially affect the lineage potential of some types of STRCs, including those whose activity may be indispensable for rapid and multi-lineage hematologic recovery.
Phase II Study of a Single Pegfilgrastim Injection as Adjunct to Chemotherapy to Mobilize Stem Cells into Peripheral Blood of Resistant/Relapsed Lymphoma Patients.
Authors: Lemoli Roberto M. , Isidori Alessandro , Tani Monica , Bonifazi Francesca , Zinzani Pier Luigi , Curti Antonio , Motta Maria Rosa , Rizzi Simonetta , Giudice Valeria , Farese Oriana , Rovito Manuela , Alinari Lapo , Conte Roberto , Baccarani Michele
Published: April 2020

The primary end point of the study was the successful mobilization of a target cell dose of 2 x 106 CD34+ cells/kg in lymphoma patients receiving ifosfamide, epirubicin and etoposide (IEV) chemotherapy and a fixed dose (6 mg) of pegfilgrastim given as single subcutaneous injection. An open-label phase II study including 25 relapsed or refractory patients (Hodgkin’s disease=4; aggressive non-Hodgkin’s lymphoma=21) was conducted to evaluate the efficacy of pegfilgrastim, in combination with salvage chemotherapy, mobilizing CD34+ stem cells into peripheral blood. Following chemotherapy, all patients had grade 4 neutropenia with a median duration of 1.5 days (1–3). Pegfilgrastim treatment was well tolerated and only 2/25 patients required pain-control medication. CD34+ cells were mobilized in all patients. The median (range) peak value of peripheral blood CD34+cells after IEV chemotherapy and pegfilgrastim was 141/microL (12.8–386) and occurred almost invariably on day +14 (13–16). Twenty three/25 patients underwent a single apheresis to collect a median of 8.7 CD34+cells/Kg (1.8–17.3). Twenty four/25 patients (96%) reached the target cell dose of 2 x 106 CD34+ cells/kg. High concentrations of circulating CD34+ cells (> 50/microL) were observed for several days after the achievement of the peak value. All patients have been transplanted with pegfilgrastim-mobilized CD34+ cells and all of them showed a rapid and sustained engraftment after high-dose chemotherapy. Our results show that pegfilgrastim as adjunct to chemotherapy is a predictable and highly effective mobilization regimen in lymphoma patients
Isolation of CD56+ Natural Killer Cells Using the CliniMACS® MARRS Tubing Set, a Newly Developed Tubing Set Including a Cross-Flow Filtration Module for Removal of Unbound Reagent.
Authors: Huppert Volker , Wolf Inka , Duwendag Julia , Arendt Andreas , Schmitz Jurgen
Published: April 2020

Use of Natural Killer (NK) cells in cellular immunotherapy requires processing of large numbers of NK cells in a closed system consisting of clinical grade components. We have previously developed a two step NK cell isolation process in which the CliniMACS® plus Instrument and CliniMACS® CD3 and CD56 Reagents are used for CD3+ cell depletion and subsequent CD56+ cell enrichment, respectively. We have recently optimized the CD3 depletion step for very large scale cell processing by development of the CliniMACS® Depletion Tubing Set (“DTS”) and appropriate cell separation software, reducing the processing time of the CD3 depletion step by 50%. We now have developed the CliniMACS® MARRS Tubing Set (MARRS = MAgnetic Reagent Removal System), a tubing set which includes a cross-flow filtration module for removal of unbound magnetic reagent after magnetic labelling. The cross-flow filtration module allows to directly connect a magnetically labelled cell product to the tubing set without centrifugational washing steps, reducing the processing time by 45 minutes. We isolated CD56+ cells from buffy coats as well as apheresis harvests using the CliniMACS® MARRS Tubing Set as compared to the standard CliniMACS® Tubing Set. Both tubing sets (MARRS vs. standard) yielded CD56+ cells of comparable purity (94.1% vs. 92.3%), comparable depletion of CD56−CD3+ T cells (3.2 log vs. 3.0 log) and comparable recovery (72.9% vs. 76.7%) with a trend towards better performance of the CliniMACS® MARRS Tubing Set. To assess the functionality of CliniMACS® separated CD56+ cells we performed flow cytometric cytotoxicity tests against the MHC class I negative target cell line K562. Specific killing could be increased from <2% (unseparated sample) to 47% (MARRS) and 53% (standard) at an effector to target ratio of 50:1. Thus, K562 killing cells are efficiently enriched and cytotoxicity is similar for both CD56+ cells isolated using the standard CliniMACS® Tubing Set and the CliniMACS® MARRS Tubing Set. We additionally assessed whether the killing efficiency of CD56+ cells correlates with NK receptor expression (NCR, KIR, NKG2D, costimulatory molecules) on different NK and NKT subsets. Results of these analyses will be presented. We conclude that the CliniMACS® MARRS Tubing Set can be used to efficiently isolate functional CD56+ cells from buffy coats or medium scale apheresis harvests (e.g. non-mobilized donors), clearly reducing total processing time. For larger scale NK cell selection using apheresis harvests (e.g. mobilized donors) the combination of Depletion Tubing Set for CD3+ cell depletion and the standard CliniMACS® Tubing Set for CD56+ cell enrichment is suitable.
FOXP1, a Highly Expressed Gene in a Subset of DLBCL, Is Targeted by a Recurrent t(3;14)(p13;q32).
Authors: Wlodarska Iwona , Vandenberghe Peter , Nooien Peet , Hagemeijer Anne , Marynen Peter , De Wolf-Peeters Chris
Published: April 2020

Chromosomal translocations involving the immunoglobulin heavy chain genes cluster (IGH) at 14q32 have been found in up to 70% of B-non Hodgkin’s lymphoma. These aberrations lead to deregulation of putative oncogenes by their juxtaposition with IGH enhancer elements. So far, more than 20 genes affected by t(14q32) have been identified in B-cell malignancies. The most frequently rearranged genes include BCL1/11q13, BCL2/18q21, BCL6/3q27 and CMYC/8q24.

We report here a novel t(3;14)(p13;q32) involving IGH, as shown by FISH, in three lymphoma cases. One of these cases was diagnosed as gastric MALT-type lymphoma, the remaining two cases as diffuse large B-cell lymphoma with a focal nodular growth pattern reminiscent of follicular lymphoma.

In order to identify the gene targeted by the t(3;14)(p13;q32) we performed FISH analysis of the first case and narrowed down the breakpoint to RP11-154H23 at 3p13 that showed a split signal on both derivative chromosomes. This BAC clone covers the FOXP1 gene. Using a pair of probes selected for the 5′ and 3′ ends of FOXP1, we demonstrated rearrangement of the gene by dual color FISH in the remaining two cases. An analysis of FOXP1 expression in present cases is being performed. FOXP1 (Forkhead box-P1) is a winged-helix transcription factor that acts as a transcriptional repressor. It is expressed in a wide variety of normal and neoplastic tissues, including lymphomas. FOXP1 has been shown to be expressed in normal activated B-cells using genomic-scale expression profiling, and in B-cells within and outside the germinal center by immunohistochemistry. Its physiological role in lymphocytes, however, is unclear. Recent studies showed that FOXP1 is strongly expressed in a subset of DLBCL. Interestingly, high expression of FOXP1 carries an independent prognostic significance, what suggests a possible role of the gene in the biology of this group of lymphoma. Although genomic rearrangements of FOXP1 have not been demonstrated so far in cancer, rearrangements of other members of FOX gene family in various pathological conditions have been reported. The involvement of FOXP1 in t(3;14)(p13;q32) found in two DLBCL cases indicates that this translocation may underlie a strong expression of FOXP1 in at least part of these lymphomas. Its rearrangement in a case of gastric lymphoma is intriguing, but additional investigations are required to find out whether t(3;14)(p13;q32) is a recurrent aberration in MALT lymphoma. Further molecular, immunophenotypic and clinical studies of the three cases with t(3;14)(p13;q32) are under progress.
Chromosomal Rearrangements Affecting ABL1 in T-Cell Acute Lymphoblastic Leukemia.
Authors: Cools Jan , Graux Carlos , Boogaerts Marc , Vandenberghe Peter , Wlodarska Iwona , Marynen Peter , Hagemeijer Anne
Published: April 2020

T-cell Acute Lymphoblastic Leukemia (T-ALL) is a heterogeneous entity with several biologically distinct subtypes that differ in clinical outcome. Cytogenetics in T-ALL shows recurrent involvement of TCR a/d (14q11) and TCR b (7q34) rearrangements. The Philadelphia translocation, encoding the BCR-ABL1 (BCR-ABL) fusion gene, is typically found in chronic myeloid leukemia (CML) and precursor B-cell acute lymphoblastic leukemia (B-ALL), but is exceptionally rare in T-ALL. To study the potential involvement of ABL1 gene rearrangements in T-cell malignancies, we screened 90 T-ALL cases by fluorescence in situ hybridization (FISH), using BCR and ABL1 probes. No BCR-ABL1 rearrangements were detected, but 5 patients showed amplification of ABL1 and are described in a separate abstract and 2 patients showed a chromosomal rearrangement affecting ABL1. The first patient (male, age 16, 455x109 WBC/L, 99% blasts, cortical T immunophenotype) showed a cryptic t(9;14)(q34;q32). This was demonstrated in 7/12 metaphases and 60% of nuclei using FISH with the 5′/3′-ABL1 probes RACE-PCR detected a fusion between EML1 at 14q32 and ABL1 at 9q34. The fusion transcript joins exon 1–18 of EML1 to exon 2 of ABL1 and generates an ORF of 4926 nt. The functional characterization of the fusion protein is ongoing. The second patient (male, age 16, 25x109 WBC/L, 35% blasts, mature T-cell immunophenotype) showed a 47, XY, +10[5], idem, inv(9)(p21q34)[2}]/46, XY[9] karyotype. One third of the metaphases are tetraploid with the same distribution of abnormalities. The split signals obtained with the 5′-/3′ABL1 probes in the inv(9) subclone point to an ABL1 rearrangement that is being characterized at the present. These result suggest that ABL1 rearrangements are a rare but recurrent event in T-cell malignancies and support a role of ABL1 in T-cell biology. The therapeutic potential of ABL1 inhibitors for treating this group of T-ALL needs to be explored.
AML1-FOG2 Fusion Protein in Myelodysplasia.
Authors: Chan Edward , Comer Elisha , Brown Frank , Richkind Kathleen , Holmes Melissa , Chong Beng , Shiffman Roger , Zhang Dong-Er , Li Yanjun , Chan Rebecca , Heiber Devan , Vance Gail , Ramsey Heather , Hromas Robert
Published: April 2020

Core binding factor (CBF) participates in specification of the hematopoietic stem cell and functions as a critical regulator of hematopoiesis. Translocation or point mutation of AML1, which encodes the DNA-binding subunit of CBF, plays a central role in the pathogenesis of acute myeloid leukemia and myelodysplasia. We characterized the translocation t(X;21)(p22.3;q22.1) in a patient with myelodysplasia that fuses AML1 to the novel partner gene Friend of GATA-2 (FOG2). The reciprocal gene fusions AML1-FOG2 and FOG2-AML1 are both expressed. AML1-FOG2, which fuses the DNA-binding domain of AML1 to most of FOG2, represses transcription from the promoter of a hematopoietic target of AML1. AML1-FOG2 retains a motif that recruits the corepressor C-terminal binding protein (CtBP) and both proteins associate in a protein complex. These results suggest a role for CtBP in AML1-FOG2 transcriptional repression and implicate coordinated disruption of the AML1 and GATA developmental programs in the pathogenesis of myelodysplasia.
Single-Dose Pegfilgrastim after Chemotherapy Is Highly Effective in Enhancing the Mobilization of Autologous CD34+ Peripheral Blood Stem Cells in Patients with Lymphoid Malignancies and Solid Tumors.
Authors: Kroschinsky Frank , Hoelig Kristina , Platzbecker Uwe , Schleyer Eberhard , Ordemann Rainer , Haack Astrid , Schaich Markus , Bornhaeuser Martin , Ehninger Gerhard
Published: April 2020

The administration of myelosuppressive chemotherapy followed by daily injections of granulocyte-colony stimulating factor (G-CSF) is the common procedure to mobilize autologous CD34+ peripheral blood stem cells (PBPC). Pegfilgrastim (NeulastaTM, Amgen Inc., Thousand Oaks, USA) is a covalent conjugate of filgrastim and polyethylene glycol with an increased elimination half-life due to decreased serum clearance. Whereas a single injection of pegfilgrastim (PEGFIL) has been shown to be equivalent to daily filgrastim in enhancing neutrophil recovery after chemotherapy, the experiences with PEGFIL in mobilization of PBPC are limited.

We report 40 pts (22 male, 18 female, median age 53 years) who had a PBPC mobilization treatment for Hodgkin′s lymphoma (n=3), non-Hodgkin′s lymphoma (n=13), multiple myeloma (n=16), acute lymphoblastic leukaemia (n=3) or solid tumors (n=5). The mobilization regimen consisted of disease specific chemotherapy and a single subcutaneous injection of 6 mg PEGFIL administered 48 hours after the end of cytotoxic treatment (day 0). CD34+ cells in the peripheral blood (PB-CD34) were measured if white blood cells (WBC) exceeded 1.0 Gpt/L after nadir. PBPC collections started at a PB-CD34 cell count >10/μl and were performed as large-volume apheresis (4x blood volume) using a Cobe Spectra (Gambro BCT Inc.). Additional conventional filgrastim (FIL) was given at a dose of 2x5μg/kg if PB-CD34 count was found to be <10/μl. Blood samples for pharmacokinetics were taken in 9 pts.

The median start of aphereses was on day +9 after the administration of PEGFIL and on day +15 after start of chemotherapy regimen, respectively. Median PB-CD34 peak was 74/μl (range 9–565/μl). The median PBPC yield was 7.6 x 10^6 CD34+ cells/kg (range 1.5–88.1). The target cell dose to be collected (≥ 2.5 x 10^6 CD34+ cells/kg) was achieved in 36 (90 %) pts, in 29 pts (72.5 %) ≥ 4.0 x 10^6 CD34+ cells/kg could be obtained with a single collection. Additional FIL administrations were necessary in 7 patients (17.5 %) for 2 to 6 days. All of them were heavily pretreated including a previous autologous transplant in two of these patients. PEGFIL was well tolerated except for moderate bone pain which occurred in all patients.

The mean values (± SD) for peak plasma concentration of PEGFIL (cmax), time to reach the maximum plasma level (tmax) and elimination half-life (t1/2) were 154 (± 83) ng/ml, 56 (±21) hours and 23 (± 9) hours, respectively.

We conclude that a single dose of 6 mg PEGFIL after chemotherapy is safe and highly effective in enhancing the mobilization of CD34+ PBPC for stem cell collection. Further investigations are warranted, including comparison with non-pegylated G-CSFs and in combination with antiadhesive agents.
G-CSF as a Potent Mitogen Augments the Elimination of CML Stem Cells by Imatinib Mesylate In Vitro.
Authors: Jorgensen Heather G. , Copland Mhairi , Allan Elaine K. , Eaves Allen C. , Eaves Connie J. , Jiang Xiaoyan , Holyoake Tessa L.
Published: April 2020

Imatinib mesylate (IM; Glivec®, Novartis) induces an impressive frequency of complete cytogenetic remission in patients with Chronic Myeloid Leukemia (CML) but molecular disease persists in most cases. We have previously demonstrated survival and accumulation of quiescent Ph+ CD34+ cells in the presence of IM in vitro. Using Q-RT-PCR analysis of FACS-purified subsets, we have since found that granulocyte-colony stimulating factor receptor (G-CSFR) transcript levels are significantly elevated in these primitive quiescent CML cells relative to their normal counterparts. Indeed CD34+ CML cells proliferated in response to exogenous G-CSF (20 – 200 ng/mL). Both total and quiescent CD34+ cell numbers were measured to test the effects of G-CSF with IM. Primary CFSE-labelled, CD34-enriched CML cells were cultured in serum-free medium for 3 cycles of treatment, 96 hours each (12 days in total). Nine treatment conditions were compared: (1) incubation alone (no G-CSF or IM), (2) continuous G-CSF alone, (3) continuous IM alone, (4) pulsed IM (final 72 hr of each 96 hr cycle), (5) continuous G-CSF with pulsed IM, (6) pulsed G-CSF (for first 24 hr) then washed out before pulsed IM, (7) pulsed G-CSF alone, (8) pulsed G-CSF with continuous IM, and (9) continuous G-CSF and IM. Pulsed G-CSF with continuous or pulsed IM, and continuous or pulsed IM alone, reduced total viable cell numbers more than continuous G-CSF with or without IM. Notably, at the end of 3 treatment cycles, pulsed G-CSF before pulsed or continuous IM reduced the number of persisting quiescent CD34+ CML cells 5-fold more than continuous IM alone (p<0.03, n = 6). Thus, use of a pulsed G-CSF and IM regimen in vivo may offer a novel therapeutic strategy for the improved elimination of primitive quiescent CML cells that are relatively IM-insensitive. A clinical protocol incorporating this strategy has been given ethical approval, and is being tested in a pilot study in the UK.
Evaluation of Physiologic and Pathologic Alterations of Coagulation and Fibrinolysis Using a New Global Assay.
Authors: Goldenberg Neil A. , Hathaway William E. , Jacobson Linda , Manco-Johnson Marilyn J.
Published: April 2020

BACKGROUND: The Clot Formation and Lysis (CloFAL) assay, modified from prior global assay methods by He et al., 1999, and Smith et al., 2003, involves activation of coagulation and fibrinolysis in platelet-poor plasma via addition of a physiologic reactant solution in a multi-well microassay plate. Continuous spectropohotometric data analysis allows measurement of kinetic absorbance changes of the plasma sample over three hours, which yields a unique clot formation and lysis curve. Using parameters of the curve, coagulation and fibrinolytic indices (CI and FI) are calculated relative to a simultaneously run pooled normal plasma standard.

METHODS: Platelet-poor plasma obtained from pregnant women at term (n=24), neonatal cord blood (n=29), and healthy children (n=22) were analyzed using the CloFAL assay. Healthy adult (n=22) plasma samples, as well as those of individuals with factor deficiencies, were obtained commercially. Fibrinolytic alterations in vitro were also investigated. Intra-assay coefficients of variation (CVs) for normal controls ranged from 3–12% for all assay parameters, with inter-assay CVs of 5–15%.

RESULTS: Representative CloFAL curves for healthy adults and children, pregnant women, and newborn infants are shown in Figure 1. Coagulation potential (measured by median CI) was significantly increased, while fibrinolytic capacity (measured by median FI) was markedly decreased, in pregnant women as compared to healthy adults (CI: 239% vs. 115%, FI: 59% vs. 95%; P<0.001 for each). By contrast, CI was decreased, and FI notably increased, in neonatal cords versus children, although the former comparison did not achieve statistical significance (CI: 58% vs. 69%, P=0.09; FI: 210% vs. 142%, P<0.001). The influence of deficiencies of coagulation factors and fibrinolytic regulators upon CloFAL parameters was also investigated. The greatest impact upon CI occurred with severe deficiency of fibrinogen or factors II, V, VII, VIII, IX, or X. Furthermore, CI was sensitive to deficiencies of factor VIII and fibrinogen in a concentration-dependent manner. In addition, FI was increased by PAI-1 deficiency and by inhibition of thrombin-activatable fibrinolysis inhibitor (TAFI) activation, and was zero in the setting of aminocaproic acid treatment. Further studies revealed that the influence of heparin concentrations of up to 2 U/mL in plasma was completely reversible by heparinase treatment of samples prior to assay.

CONCLUSION: These results indicate that the CLoFAL assay is reproducible and analytically sensitive to known physiologic and pathologic alterations in coagulation and fibrinolysis. The application of this global assay to patients with a variety of disorders of thrombosis and hemostasis is currently ongoing.

Figure Figure
Pediatric Reference Intervals for Ten Coagulation Assays.
Authors: Flanders Michele M. , Crist Ronda A. , Roberts William M. , Rodgers George M.
Published: April 2020

There is a lack of reliable pediatric reference intervals for many clinical laboratory tests. In 2002, the Children’s Health Improvement through Laboratory Diagnostics (CHILDx) organization initiated a project to collect blood and urine samples from healthy children 7 – 17 years of age with the goal of establishing reference intervals for many laboratory tests. The purpose of the present study was to determine pediatric reference intervals for ten coagulation proteins associated with common bleeding and thrombotic disorders. All assays were functional except for vonWillebrand factor antigen. All were measured according to manufacturer specifications and standard methods using the STA-R coagulation analyzer (Diagnostica Stago), with the exception of the ristocetin cofactor assay, which was performed on the BCS (Dade Behring). Samples used to establish adult reference intervals were purchased from George King Bio-Medical, Precision Biologic, and also drawn in-house. At each age of life, 62 individuals (31 girls/31 boys) were drawn for a minimum of 124 individuals for each age group. Reference intervals were established based on a nonparametric method (NCCLS C28-A).

RESULTS: 1. Although pediatric PTT values do not differ from adult values, the mean pediatric PT values are about 1 sec longer, 2. Pediatric FIX levels trend upward until ages 16-17 when adult levels are reached, 3. FVIII, FXI, RCF and vWFAg demonstrate higher reference values in younger ages, 4. The lower limit of pediatric AT levels is significantly higher than adults, 5. The lower limit of pediatric protein C levels is significantly lower than adults, however, this difference is not seen for protein S levels. In conclusion, a number of significant differences between pediatric and adult reference intervals have been found supporting the use of these newer reference intervals.

Age N PT PTT F VIII F IX F XI 7–9 186 13.1–15.4* 27–38 78–199* 71–138* 70–138 10–11 124 12.9–15.5* 27–38 83–226* 72–159* 63–137 12–13 124 13.1–15.2* 27–38 74–205* 73–152* 65–130* 14–15 124 12.9–15.4* 26–35 69–241* 80–162 57–125* 16–17 121 12.6–15.9* 26–35 63–225* 85–175 64–160 Adult 125 12.3–14.4 26–38 56–190 78–184 56–153 Age AT RCF VWF Ag PC PS-Male PS-Female * The t-test of the means, F-test of the SD, or both is statistically different (p< 0.05) from adult reference values. 7–9 96–135* 51–172* 62–176 71–143* 64–141 58–154 10–11 92–134* 61–195* 61–201* 76–146* 68–150 68–140* 12–13 92–128* 47–183* 61–186* 68–162* 65–143 60–150 14–15 95–135* 50–215* 57–204* 69–170* 66–149 53–147* 16–17 94–131* 47–206* 51–211 70–170* 75–157* 51–150* Adult 76–128 44–195 51–185 83–168 66–143 57–131
BEX1, a Marker for Acute Myeloid Leukemia Cell Lines with MLL Rearrangements.
Authors: Quentmeier Hilmar , Dirks Wilhelm G. , Geffers Robert , Uphoff Cord C. , Drexler Hans G.
Published: April 2020

Patients with acute leukemias carrying MLL rearrangements have a poor prognosis. The tumor cells show characteristic gene expression profiles with increased levels of selected HOX genes (1). We have shown that acute lymphoblastic leukemia (ALL) cell lines with and without MLL rearrangements could likewise be recognized by analysis of HOX gene expression (2). In contrast, MLL wild-type (MLLwt) and mutant (MLLmu) acute myeloid leukemia (AML) cell lines could not be distinguished by analysis of HOX genes, because even wild-type cell lines had a high expression background (2). It was our aim to find out whether MLLwt and MLLmu AML cell lines could be discriminated on the basis of gene expression - other than HOX genes. We performed gene expression analysis with pooled RNAs of MLLmu (n=8) and MLLwt (n=8) cell lines applying high density oligonucleotide Genechips from Affymetrix (HG-U133A). Defensin alpha4 (83x), defensin beta1 (32x), cathepsinG (9x) and FLT3 (7x) genes were overexpressed in MLLmu cell lines, stabilin1 (82x) and galectin10 (55x) in MLLwt cell lines. PCR analysis with individual (non-pooled) cDNAs of the 16 cell lines showed that none of the above genes was exclusively expressed by MLLmu or MLLwt cells. Thus, pooling RNAs has a major disadvantage: many PCRs have to be performed to establish faithful expression profiles for individual samples. BEX1 finally proved to be a gene that was exclusively overexpressed in one group of cell lines. It had been an interesting candidate already after oligonucleotide chip analysis, being both overexpressed (18x) in MLLmu cell lines, and - in contrast to the genes listed above - not a marker of myeloid differentiation. BEX1 is reportedly expressed in brain, testis and ovary, but not in peripheral blood leukocytes, lymph node and bone marrow (3). By RT-PCR analysis we showed that 7/8 MLLmu and 0/8 MLLwt cell lines expressed BEX1. Screening a panel of 54 hematopoetic cell lines gave the same result: BEX1 expression was restricted to MLLmu AML cell lines: 8/11 (73%) MLLmu AML cell lines expressed BEX1, but 43 other hematopoetic cell lines (including Hodgkin′s disease, anaplastic large cell lymphoma, ALL and MLLwt AML cell lines) tested negative. BEX1 expression may depend on the type of MLL rearrangement or the histological background of the cells, as MLLmu ALL cell lines (0/5) also tested negative. It has been shown in the mouse system, that BEX family members may be involved in cell signalling processes. Thus it will be interesting to elucidate whether BEX1 also participates in proliferative/antiapoptotic signalling processes in MLLmu AML cell lines.
Seasonal Variation in New Adult Acute Myeloid Leukemia.
Authors: Campbell Lynda J. , Farish Stephen J. , White Joanne S.
Published: April 2020

Investigations into seasonal trends for the onset of leukemia have produced conflicting results. The majority of studies demonstrating seasonal variation have done so for childhood ALL and have suggested a role for an infectious agent. Few investigations have studied adult AML and none have looked at the potential differences between various AML subtypes. An infectious etiology for adult AML will more likely be reflected by cases that are truly de novo and that have not arisen by transformation from a pre-leukemic phase or from prior exposure to a carcinogenic agent. The Victorian Cancer Cytogenetics Service is the central reference laboratory for cytogenetic analysis of all new adult leukemias in the state of Victoria. We have investigated seasonal trends in the diagnosis of 635 cases of new adult AML (≥15 yrs) received for cytogenetic analysis during a five-year period, March 1999 – Feb 2004. Cases of new AML that were secondary in nature were excluded. Cases were subdivided into three recognised prognostic risk groups (favourable, intermediate and adverse) based on cytogenetic findings according to the published criteria of the UK Medical Research Council. The favourable risk group (FR) consisted of t(15;17), inv(16)/t(16;16) and t(8;21) (n = 95). The adverse risk group (AR) included complex karyotypes, abnormalities of 3q, monosomy 7 and loss of chromosome 5 or part of 5q (n = 118). The intermediate risk group (IR) consisted of all other cytogenetic findings including normal results (n = 422). The cytogenetic abnormalities comprising the FR group are rarely associated with secondary AML whereas the converse is true of the AR group. Initial descriptive statistics demonstrated that the greatest trend towards seasonal variation was in the FR group with a peak occurrence in Spring. Spectral analysis was subsequently performed on the data in order to detect if any underlying pattern was genuinely present for the three prognostic groups. The outcome of this analysis supported the possibility of an approximately 12 month cycle for the FR group with the potential peak around the month of October, the middle month of a southern hemisphere Spring. The relatively short time frame of only 5 years limited the interpretation of the analysis. However, these results suggest a possible seasonal trend in the incidence of new adult AML presenting with cytogenetic abnormalities associated with a favourable prognosis. Analysis of data covering a greater time period is proposed to further investigate these preliminary findings.
Protection Against Graft-Versus-Host Disease and Retention of Graft-Versus-Tumor Effect after Allogeneic BMT in Mice Conditioned with TLI and Anti-Thymocyte Serum.
Authors: Pillai Asha , Teo Pearline , Strober Samuel
Published: April 2020

In a completely MHC-mismatched model of bone marrow transplantation [C57BL/6 (H-2b) donors into BALB/c (H-2d) hosts], we have developed a technique of non-myeloablative host conditioning using fractionated total lymphoid irradiation (TLI) and and anti-thymocyte serum (ATS) that prevents lethal graft-versus-host disease (GVHD). We have previously reported that this GVHD prevention is dependent on the secretion of IL-4 and on host regulatory NKT cells. In the current study, we assessed the graft-versus-tumor (GVT) effect of BMT to determine whether the GVT effect remains intact in this non-myeloablative conditioning model. Male BALB/c mice were given fractionated TLI (17 doses of 240 cGy each), 3 doses of ATS, and subsequently received intravenous infusion of 50 x 106 bone marrow and 60x 106 splenocytes from C57BL/6 donors, with and without the BCL1 B-cell lymphoma. Animals were observed for minimum of 100 days, and underwent autopsy at death to assess for sub-clinical evidence of GVHD or tumor infiltration.

TLI/ATS-conditioned mice achieved a high percentage of donor chimerism, in the range of 50–100% in all lineages. TLI/ATS-conditioned hosts uniformly survived without signs of GVHD beyond day +100. By contrast, hosts conditioned with a single dose of 800cGy total body irradiation (TBI) died of acute GVHD (severe diarrhea, hunched back, weight loss) by day +21. When TBI/ATS or TBI-treated mice receive bone marrow, splenocytes, and BCL1 lymphoma, all hosts died with signs of acute GVHD by day +28. TLI/ATS-conditioned hosts receiving marrow, splenocytes and tumor cells showed no evidence of disease progression by day +100 and either cleared tumor idiotype completely or had persistence of low-intensity staining for tumor idiotype (tumor dormancy). TLI/ATS-conditioned hosts given BCL1 tumor cells without allogeneic BMT all succumbed to tumor. TLI/ATS hosts receiving bone marrow plus BCL1 without splenocytes all died by day +108 with high circulating BCL1 tumor burden and no clinical evidence of GVHD.

The data indicate that peripheral donor T cells are necessary to maintain a robust graft-versus-tumor effect after TLI/ATS conditioning, and that complete donor chimerism is not a requirement for tumor eradication. In conclusion, using the TLI/ATS non-myeloablative conditioning regimen, it is possible to maintain a clinically significant graft-versus-tumor effect without inducing GVHD despite a high dose of infused donor peripheral T cells.
Treatment for Acute Myelogenous Leukemia by Low Dose Irradiation Based Conditioning and Hematopoietic Cell Transplantation from Related and Unrelated Donors.
Authors: Niederwieser Dietger W. , Hegenbart Ute , Sandmaier Brenda M. , Maris Michael B. , Stuart Monic , Greinix Hildegard , Cordonnier Catherine , Rio Bernard , Gratwohl Alois , Lange Thoralf , Pönisch Wolfram , Grommisch Leante , Storer Barry , Maloney David , McSweeney Peter , Chauncey Thomas , Agura Ed , Blume Karl , Storb Rainer
Published: April 2020

Conventional allogeneic hematopoietic cell transplantation (HCT) is effective treatment for AML, but its use has been restricted to younger patients in good medical condition. However, AML is increasingly common with advancing age, and older patients have more adverse prognostic features than younger patients. The vast majority of older patients who reach a CR with chemotherapy relapse within two years from diagnosis and die within three years. The situation is similar in patients with secondary AML or with AML in CR2, more advanced remission status or in partial remission, where median duration of disease-free survival (DFS) is generally <12 months. The use of low-dose irradiation-based preparative regimens has allowed extending allografting to, and exploring graft-versus-tumor effects in, older and medically infirm patients.

The present study included 122 patients with AML ineligible for conventional transplants. Their disease stages ranged from CR1 to resistant disease, and their median age was 57.5 (range 17–74) years. The patients were given 2 Gy total body irradiation (TBI) on day 0 with and without preceding fludarabine (30 mg/m2/d from days -4 to -2). After HCT, cyclosporine, 6.25 mg/kg, was given twice daily from day -3, and mycophenolate mofetil, 15 mg/kg, twice daily from day 0. HLA-matched related donors were used in 58, and HLA-matched/partially matched unrelated donors in 64 patients. Among 121 (one died to early after transplant) evaluable patients, 116 (95.8%) had durable engraftment. Cumulative incidences of acute GvHD grade II-IV were 33 % after related and 42% after unrelated HCT. With a median observation time of 17 (range 4–57) months, 58 patients were alive, 53 of whom were in complete remissions. Cumulative non-relapse mortalities (NRM) were 12% and 27%, and the cumulative relapse mortalities were 47% and 34% at 2 years for related and unrelated recipients, respectively. Two-year overall survival (OS) was 41%, and DFS was 35%, with stages of the disease and cytogenetic risks being major determinants for outcomes. Patients transplanted in CR1 had a 2-year OS of 40% after related and 57% after unrelated HCT. We concluded that HCT from related and unrelated donors after low-dose TBI is promising therapy for elderly patients and medically infirm younger patients with AML not eligible for conventional HCT. Due to the lower relapse rates, results in patients with unrelated donors were better than results in patients with related donors. Results have encouraged studies of the role of low-dose irradiation-based conditioning and allogeneic HCT in the primary treatment of patients with AML ≥60 years.
Acquired Hemophilia Caused by Non-Hemophilic Factor VIII Gene Variants.
Authors: Tiede Andreas , Eisert Roswith , Czwalinna Andreas , Miesbach Wolfgang , Scharrer Inge , Ganser Arnold , von Depka Mario
Published: April 2020

The development of inhibitory antibodies against coagulation factor VIII (FVIII) in patients without congenital hemophilia A is a rare disorder known as acquired hemophilia. The etiology of the disease remains obscure. In approximately half of the patients, acquired hemophilia is associated with autoimmune disease, malignancy, multiple transfusions or the postpartum period. The remaining patients present without concomitant disorders. We hypothesized that anti-FVIII inhibitor formation might result from allo-immunization against antigenetically different FVIII variants in some patients with acquired hemophilia. The FVIII gene sequence was analyzed in 18 patients with acquired hemophilia and in two kindred of female patients with postpartal inhibitors. A hemizygous missense mutation (E2004K) was found in a male patient who developed an inhibitor after receiving multiple blood transfusions. This mutation has not been reported in the HAMSTeRS database and was not found in 50 male and 50 female healthy blood donors. It does not cause inherited hemophilia A as demonstrated by the patient’s unremarkable bleeding history before the onset of acquired hemophilia and a FVIII activity of >100 % after eradication of the inhibitor. Peptides containing E2004 or K2004 scored among the top 1 % of A3 domain-derived peptides presented on the patient’s major histocompatibility complex (MHC) class II allele, HLA-DRB1*0101. A further single amino acid substitution (D1241E) was found in four male patients and three female patients. This substitution resides in the FVIII B domain and represents a previously unknown polymorphism as it was also found in eight out of 50 male and in 17 out of 50 female controls. Comparing the allelic frequency of FVIII gene variants in patients and controls, these were equally frequent in female patients (3/18 or 17 %) and controls (18/100 or 18 %, P=0.74), but significantly more frequent in male patients (5/9 or 56 %) compared to male controls (8/50 or 16 %, P<0.01). Peptides containing D1241 or E1241 scored among the top 2 % of B domain-derived peptides presented on some MHC class II alleles (HLA-DRB1*0701 and *1501), but were unlikely to be presented on other alleles (*0101, *0301, *0401, *1101). In summary, we report allo-immunization against antigenetically relevant FVIII variants presented in a suitable MHC class II background as a novel potential mechanism of inhibitor development. Thus, exposure to ‘foreign’ FVIII variants during pregnancy or transfusion may elicit an immune response that eventually cross-reacts with ‘self’ FVIII in some patients resulting in acquired hemophilia. Similar inhibitory immune reactions may also cause loss of efficacy in patients receiving other therapeutic proteins, e.g. erythropoietin, or multiple transfusions.
Pathology of Bone Marrow in Human Herpes Virus 8 (HHV8) - Associated Multicentric Castleman’s Disease.
Authors: Bacon Chris M. , Miller Robert F. , Noursadeghi Mahdad , McNamara Christopher , Du Ming-Qing , Dogan Ahmet
Published: April 2020

Human Herpes virus 8 (HHV8) associated multicentric Castleman’s disease (MCD) is an unusual multifocal lymphoid hyperplasia induced by HHV8 infected B cells and associated with a characteristic systemic syndrome attributed to raised levels of IL-6. Most cases develop on a background of immunosuppression, often as a result of human immunodeficiency virus (HIV) infection. Despite the haematological problems at presentation and the difficulties in initial diagnosis, the bone marrow appearances of MCD have not been so far described. In this study we examined the pathology of bone marrow in 13 patients with MCD, 11 of whom had HIV infection, with a view to identifying features that may be helpful in early diagnosis. Patients typically presented with fever, lymphadenopathy, hepatosplenomegaly and cytopaenias. Bone marrow aspirates showed mild to moderate trilineage dysplasia, a mild plasmacytosis, and a mild eosinophilia similar to that seen in HIV infected patients without MCD. Bone marrow biopsies showed hypercellularity, architectural disorganisation, variably prominent dysplasia especially in megakaryocytes, mild eosinophilia, and a polytypic plasmacytosis representing 5–20% of all cells. Interestingly, two cases showed marked megakaryocytic and granulocytic hyperplasia with reticulin fibrosis, similar to the effects of IL-6 on the marrow in experimental systems. Importantly, in 3 cases there were small lymphoid follicles typical of MCD in diagnostic nodal specimens. Depleted germinal centres were surrounded by mantle zones containing scattered large plasmablasts which expressed HHV8 latent nuclear antigen (LNA) and showed lambda immunoglobulin light chain restriction. Furthermore, varying numbers of dispersed interstitial HHV8-LNA positive plasmablasts were present in 11/13 cases. Double immunohistochemical staining confirmed the B cell phenotype of these plasmablasts. The presence of these cells was highly specific for MCD as rare single HHV8 positive cells were identified in only 4 of 66 control bone marrow biopsies from HIV positive patients. Each of these 4 patients had Kaposi’s sarcoma and 1 also had a primary effusion lymphoma. HHV8 positive cells were not identified in bone marrow biopsies from 23 other HIV positive patients with lymphoma. These results suggest that the presence of HHV8 positive plasmablasts in bone marrow biopsies, within characteristic lymphoid follicles and/or the interstitium, is highly specific and sensitive for MCD. As the examination of bone marrow is the first diagnostic test performed in virtually all MCD patients, the features described in this study should greatly enhance the chances of early diagnosis by either providing the tissue diagnosis or prompting a lymph node biopsy and further investigations. Furthermore, the HHV8 positive cells within the bone marrow may play an important pathogenetic role in the haematological disturbances typically seen in MCD.