[From the bench to the clinic: The challenge of translating platelet production in vitro]. / Du laboratoire à l'usage clinique : le défi de la production de plaquettes in vitro.

Platelet transfusions, which are currently totally dependent on altruistic donations, are absolutely necessary to the treatment of patients with thrombocytopenia following trauma, surgery or other pathologies (especially malignancies). Producing platelets in vitro represent a major technological and scientific breathrough that would address logistical issues (supply chain, stock holding…) and medical concerns (compatibility and biosafety). The translation of this innovation will need to be accompanied by rigorous quality control, harmonised between laboratory when it comes to functionality and biosafety for use in the clinic.

In vitro-derived platelets: the challenges we will have to face to assess quality and safety.

Platelet transfusions are given to patients in hospital who have a low blood platelet count (thrombocytopenia) either because of major bleeding (following trauma or surgery) or because the bone marrow production of platelets is impaired often due to chemotherapy, infiltration with malignant cells, fibrosis or genetic disorders. We are currently entirely reliant on blood donors as a source of platelets in transfusion medicine. However, the demand for platelets continues to rise, driven by an aging population, advances in medical procedures and ever more aggressive cancer therapies, while the supply of blood donors continues to remain static. In recent years, several groups have made major advances toward the generation of platelets in vitro for human transfusion. Recent successes include results in both generating mature human megakaryocytes as well as in developing bioreactors for extracting platelets from these megakaryocytes. Platelets made in vitro could address several issues inherent to platelets derived from blood donors - the ability to scale up/down more flexibly according to demand and therefore less precarious supply line, reduction of the risk of exposure to infectious agents and finally the possibility of engineering stem cells to reduce immunogenicity. Here we define the quality control tools and suggest measures for implementation across the field for in vitro platelet genesis, to aid collaboration between laboratories and to aid production of the burdens of proof that will eventually be required by regulators for efficacy and biosafety. We will do this firstly, by addressing the quality control of the nucleated cells used to make the platelets with a particular emphasis to safety issues and secondly, we will look at how platelet function measurement are addressed particularly in the context of platelets derived in vitro.

Transcription factors in late megakaryopoiesis and related platelet disorders.

Cell type-specific transcription factors regulate the repertoire of genes expressed in a cell and thereby determine its phenotype. The differentiation of megakaryocytes, the platelet progenitors, from hematopoietic stem cells is a well-known process that can be mimicked in culture. However, the efficient formation of platelets in culture remains a challenge. Platelet formation is a complicated process including megakaryocyte maturation, platelet assembly and platelet shedding. We hypothesize that a better understanding of the transcriptional regulation of this process will allow us to influence it such that sufficient numbers of platelets can be produced for clinical applications. After an introduction to gene regulation and platelet formation, this review summarizes the current knowledge of the regulation of platelet formation by the transcription factors EVI1, GATA1, FLI1, NFE2, RUNX1, SRF and its co-factor MKL1, and TAL1. Also covered is how some platelet disorders including myeloproliferative neoplasms, result from disturbances of the transcriptional regulation. These disorders give us invaluable insights into the crucial role these transcription factors play in platelet formation. Finally, there is discussion of how a better understanding of these processes will be needed to allow for efficient production of platelets in vitro.

A role for adhesion and degranulation-promoting adapter protein in collagen-induced platelet activation mediated via integrin α(2) ß(1).

BACKGROUND: Collagen-induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α(2) ß(1) . Adhesion and degranulation-promoting adapter protein (ADAP) regulates α(IIb) ß(3) in platelets and α(L) ß(2) in T cells, and is phosphorylated in GPVI-deficient platelets activated by collagen. OBJECTIVES: To determine whether ADAP plays a role in collagen-induced platelet activation and in the regulation and function of α(2) ß(1). METHODS: Using ADAP(-/-) mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. RESULTS AND CONCLUSIONS: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP(-/-) platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α(2) ß(1) -selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α(2) ß(1), was reduced in ADAP(-/-) platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP(-/-) platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α(2) ß(1). In addition, we found that ADAP(-/-) mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.

The mitogen-activated protein kinase signaling pathways: role in megakaryocyte differentiation.

Megakaryopoiesis is a process by which bone marrow progenitor cells develop into mature megakaryocytes (MKs), which in turn produce platelets required for normal hemostasis. The mitogen-activated protein kinases (MAPKs) family comprises four main groups of proteins: extracellular signal-related kinases (ERKs) (ERK1/2 or p44/p42), ERK5, p38MAPKs (alpha, beta, gamma, delta) and c-Jun amino-terminal kinases (JNKs) (JNK 1, 2, 3). These intracellular signaling pathways play a pivotal role in many essential cellular processes including proliferation and differentiation. The purpose of this review is to summarize our current knowledge on the role of MAPKs in MKs, specifically regarding differentiation in immortalized cell lines and primary MKs. A critical role of the MEK (MAPK kinase)-ERK1/2 pathway in MK development has been demonstrated although the details remain controversial. There is at present no functional evidence for a role of p38MAPKs whereas the role of JNKs and ERK5 in MK development is not known. Characterization of these molecular event cascades remains crucial for the understanding of the megakaryopoiesis process.

Immunologic and structural analysis of eight novel domain-deletion beta3 integrin peptides designed for detection of HPA-1 antibodies.

BACKGROUND: The single-nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen-1 (HPA-1) system encoding a Leu (HPA-1a) or Pro (HPA-1b) at position 33. HPA-1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires alpha(IIb)beta3 integrin from the platelets of HPA-genotyped donors. OBJECTIVES: We set out to define the beta3 integrin domains required for HPA-1a antibody binding and produce recombinant soluble beta3 peptides for HPA-1 antibody detection. METHODS: We designed two sets (1a and 1b) of four soluble beta3 domain-deletion peptides (deltaSDL, deltabetaA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA-1a-specific phage antibodies were defined by analyzing binding patterns to the beta3 peptides and canine platelets, and models of antibody-antigen interfaces were derived. Specificity and sensitivity for HPA-1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT). RESULTS: Fusion of recombinant proteins to calmodulin resulted in high-level expression in Drosophila S2 cells of all eight beta3 peptides. Testing of FMAIT samples indicated that deltabetaA-Leu33 is the superior peptide for HPA-1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA-1a antibodies was confirmed by the study of HPA-1a phage antibody footprints and the reactivity pattern of clinical samples with the four beta3-Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. CONCLUSIONS: Soluble recombinant beta3 peptides can be used for detection of clinical HPA-1a antibodies.

A modified rapid monoclonal antibody-specific immobilization of platelet antigen assay for the detection of human platelet antigen (HPA) antibodies: a multicentre evaluation.

BACKGROUND: The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay is the cornerstone technique for the detection and identification of human platelet antigen (HPA) antibodies. However, the original technique described by Kiefel and colleagues requires approximately 8 h adding to diagnostic delay. Moreover, proficiency exercises indicate that there are substantial variations in the MAIPA protocol, and that these may account for interlaboratory differences in sensitivity and specificity. STUDY DESIGN AND METHODS: A review of current MAIPA assay protocols from six laboratories together with performance in quality-assessment schemes identified several key variables potentially affecting the assay results. An optimized protocol was derived and assay time reduced to 5 h. The modified rapid MAIPA (MR-MAIPA) assay was evaluated using 61 samples with a range of HPA antibodies typically encountered in cases of fetomaternal alloimmune thrombocytopenia (n = 22), post-transfusion purpura (n = 8), platelet refractoriness (n = 7) and other platelet immune conditions (n = 24). The sensitivity of the assay was assessed using three international standards and the recombinant HPA-1a antibody CamTran007. The results obtained were compared with the original findings obtained with the local MAIPA assays. In addition, four different glycoprotein IIb/IIIa capture monoclonal antibodies were evaluated for their effect on assay sensitivity. RESULTS: Complete concordance was found between the original MAIPA results and those obtained with the new assay when testing a selected panel of clinical samples. The modified assay had nanogram level sensitivity for the detection of HPA-1a antibodies and titration of HPA-1a and HPA-5b antibody sensitivity standards yielded end-points equal to or greater than the mean recorded in international workshops. CONCLUSION: The MR-MAIPA assay offers improved turnaround for the detection of HPA antibodies without loss of sensitivity.

Non-secretory multiple myeloma with multinucleated giant plasma cells.

We report a case of non-secretory multiple myeloma with unusual cytological features. The plasma cells were multinucleated and contained up to forty nuclei. All nuclei had regular outlines without multilobulated and convoluted slopes. DNA content measurement demonstrated that all nuclei of uni- and multinucleated cells were diploid. All plasma cells contained cytoplasmic alpha chain but light chains and their corresponding transcripts were absent. There is no clear explanation concerning multinuclearity. In addition, hypotheses regarding non-secretion of immunoglobulin in non-secretory multiple myeloma and in other B-cell neoplasias are discussed.

Laboratory evaluation of the Abbott Cell DYN 3500 5-part differential.

The study reports the performance of the Abbott CD3500 automated hematology analyzer for the enumeration and delineation of leukocyte populations for both adult and pediatric samples, and the ability of this instrument to detect the presence of abnormal cells. Samples from 542 individual patients either attending medical practitioners or during hospitalization were examined and then subdivided for the purposes of this study into 106 samples from newborn infants (< days), 145 samples from older children (15 days to 14 years) with non-oncologic disorders, 100 samples from normal adults, and 191 samples from oncology patient (97 adults and 94 children). The leukocyte differentials provided by both the Abbott CD3500 and the Coulter STKS were compared with those obtained from conventional morphology (two observers, total of 400 leukocytes). The sensitivities and specificities of the blast, immature granulocyte (IG) and NRBC "flags" were also determined. For the non-oncology adult (n=100) and pediatric (n=145) cohorts, automated differentials were given in all samples with the CD3500, whereas the STKS did not provide a differential analysis for 20 of the 145 (14%) pediatric samples, 11 of these were absent for no obvious reason. However, for the evaluable cases, the performances of the CD3500 and the STKS were broadly similar and generally correlated well with the manual reference procedure. The results for the newborn samples were less consistent with wider 95% confidence intervals (CI) noted. For example, the CD3500 (which reported a differential for all 106 samples studied) gave CI values of +/-14.4% for neutrophils, +/-14.6% for lymphocytes and +/-8.1% for monocytes. For comparison, the STKS (which did not provide a differential in 15% of 79 samples analyzed; insufficient material being available from the remaining 27 of 106 newborn samples) gave CI values of +/-21.9% for neutrophils, +/-23.5% for lymphocytes and +/-8.2% for monocytes. For all samples, the sensitivity of the blast flag on the CD3500 was 85% with a specificity of 91% (STKS: sensitivity, 75%; specificity, 85%); the sensitivity of the CD3500 IG flag was 72% with a specificity of 76% (STKS: sensitivity, 75%: specificity, 73%); and the sensitivity of the NRBC flag was 43% with a specificity of 94%(STKS sensitivity, 37%; specificity, 88%). This study confirms competitive performance levels for the CD3500 in the analysis of normal adult samples and suggests positive performance advantages in the study of neonatal, pediatric, and leukopenic samples.

[Evaluation of Abbott CD 3500 in severe leukopenia]. / Evaluation du CD 3500 Abbott dans les leucopénies profondes.

Leucopenia patient follow-up remains, in terms of laboratory turnaround, a heavy workload due to the leucoconcentrations necessary for evaluating leucocytic formulas. We tested the CD 3500 with the objective of defining its' analytical performances and routine practice. 101 leucopenia samples (< 2.10(9)/l) procured from the onco-hematology department (adults and children) were studied during a 1 month period. The leucocytic formula obtained after leuconcentrations was our reference. The alarm sensitivity, as a whole, was of 97% for a 15.5% specificity. The correlation coefficients (Cell-Dyn/microscope) for polynuclears, lymphocytes and monocytes were respectively 0.889, 0.925 and 0.926. The correlation coefficients observed in both following subgroups: < or = 0.5.10(9)/l and > 0.5.10(9)/l were superposable. In 95% of the cases, the numeric value difference between the two methods attained a maximum of 21% for all neutrophils and lymphocytes and 13% for the monocytes. There was an excellent concordance between both methods for eosinophilia and basophils with confidence intervals of +/-8.8% and +/-2.2%. In practice, we feel that the use of a CD 3500 in post chemotherapy leucopenia, is perfectly adaptable and appreciated for leucocytic formulas, as well as a good exit for aplasia.

Immunological detection of myeloperoxidase in poorly differentiated acute leukemia.

We performed immunocytochemical detection of myeloperoxidase (MPO), using monoclonal antibody MPO-7, in 15 consecutive cases of adult acute leukemia (AL) unclassified by conventional cytological and cytochemical criteria and 7 AML-M1 with less than 10% of cytochemically MPO-positive blasts. In AL with negative MPO cytochemistry the anti-MPO reaction was positive in 5 of the 15 patients with 3, 3, 7, 11 and 45% positive blasts respectively. In AML-M1, immunocytochemistry was positive in a larger percentage of blasts than cytochemistry in 2 cases. Immunological detection of myeloid surface markers was positive in all 15 cases of unclassified AL (including the 10 AL with negative anti-MPO reaction). Eleven of the 22 patients from this study had mixed lymphoid-myeloid phenotype. Discrepancy between immunological MPO detection and light cytochemistry was more frequent in patients with mixed immunophenotype than in patients without lymphoid markers. No relationship between MPO-antigen positivity and clinical or biological features was seen. These findings confirm immunological detection of MPO as useful for the diagnosis of poorly differentiated AL. The high incidence of inactive MPO detectable only by immunocytochemistry in mixed lineage AL needs to be confirmed.