Sepsis induced platelet activation releases platelet microparticles (PMPs). PMPs express phosphatidylserine (PS) and can serve as a scaffold for the prothrombinase complex, thereby promoting coagulation. Studies of PMPs in intensive care unit sepsis patients demonstrate mixed results, while the earliest changes and potential effects of clinical interventions remain understudied. We hypothesized PMPs would be associated with patient outcome and dysfunctional coagulation shortly after emergency department presentation with sepsis.

Cohort study of patients from a single center enrolled in a previously published randomized control trial comparing two early resuscitation strategies. Adults presenting to the emergency department (ED) with suspected infection, ≥2 SIRS criteria, and either systolic blood pressure <90 mm Hg or lactate >4 mmol/L were eligible. Triple positive platelet microparticles (PMPs) expressing phosphatidylserine and integrin complexes alphaIIb (CD41) and beta3 (CD61) were quantitated using plasma from the time of enrollment. The primary outcome was in-hospital mortality. Secondary outcomes included platelet count, disseminated intravascular coagulation (DIC), and prothrombin time (PT).

193 patients were enrolled and 184 had samples available. In-hospital mortality was 21%. 10 (5%) patients developed DIC. Median platelet count was 197 (IQR 135, 280) and PT was 13.2 (IQR 11.9, 16.8). Median triple positive PMP counts were 932 per μL (IQR 381, 1872). PMPs were significantly lower in non-survivors (575 vs 1128, p = 0.02) and non-significantly lower in DIC (387 vs 942, p = 0.17). PMPs demonstrated a positive linear association with platelet count (p < 0.001, R2 = 0.21). After adjusting for platelet count, PMPs were no longer significant predictors of mortality (p = 0.28). We observed no association between PMPs and PT.

Similar to patients enrolled later in the intensive care unit, PS-expressing PMPs are lower in emergency department sepsis non-survivors. These changes primarily reflect the degree of thrombocytopenia, and an independent prognostic role was not observed. Future studies should control for platelet count in assessment of PMP prognosis in sepsis.

OBJECTIVE

To evaluate the accuracy of platelet counts from various hematology analyzers using a reference immunologic method.

METHODS

We tested 403 samples with platelet counts less than 50 × 10(9)/L with the Advia (Siemens, Tarrytown, NY), Sysmex (Mundelein, IL), and Abbott (Santa Clara, CA) analyzers.

RESULTS

All methods showed a positive bias, especially at less than 20 × 10(9)/L and less than 10 × 10(9)/L. Undertransfusion risk ranged from 9.1% to 43.3 % in the groups below 20 × 10(9)/L and below 10 × 10(9)/L, respectively. For patients with optical counts more than 10 × 10(9)/L and CD61 less than 10 × 10(9)/L, 64.5% were transfused within 24 hours of the reported count, while 35.5% were transfused in more than 24 hours, after a subsequent optical platelet count of 10 × 10(9)/L or less was reported.

CONCLUSIONS

Although optical and impedance methods were shown to be falsely increased in severely thrombocytopenic samples, further studies are needed to determine if more accurate methods would be clinically useful.

We investigated the ability of endothelial cells (EC) to support hematopoiesis in contact and non-contact cocultures with isolated CD34+ or CD34/CD38low cells. In the absence of exogenous cytokines, umbilical vein EC (HUVEC) efficiently support proliferation of hematopoietic cells and generation of colony-forming cells (CFC). Cytokines (IL-6, LIF, G-CSF, GM-CSF, M-CSF, but not IL-1, IL-3, IL-7) were detected in HUVEC coculture supernatants. Neutralization of these cytokines profoundly inhibited the ability of EC supernatants to support the differentiation of hematopoietic progenitors and led to an accumulation of immature cells. Contact cocultures were significantly more efficient than non-contact cocultures. The expanded cell population essentially belonged to the myeloid and monocytic lineages. Contact cocultures generated cells expressing the CD61 or CD41 antigens. Interleukin-1alpha (IL-1alpha) augmented EC capacity to support hematopoiesis, this property resulting from the upregulation of cytokine expression. Glucocorticoids (GC) reduced this capacity by downregulating the biosynthesis of cytokines by EC and not by a direct effect on the progenitor cells. EC from the bone marrow microvasculature (BMEC) support the proliferation and the differentiation of hematopoietic progenitors. Synergistic increase in progenitor cells expansion and generation of CFC occurred when EC cocultures were added with exogenous cytokines. Supernatants of IL-1alpha-stimulated EC potentiated the effects of an association of IL-1, IL-3, IL-6, LIF, SCF, Flt3-ligand, TPO, G-CSF, GM-CSF, M-CSF and IL-11 on the proliferation of hematopoietic progenitors suggesting that EC may produce other soluble growth factors potentiating the action of the above set of cytokines.

BACKGROUND

Megakaryopoiesis occurs as a result of a complex interaction between hemopoietic cells, stromal cells, hemopoietic growth factors and extracellular matrix (ECM). Megakaryocyte growth and development factor (MGDF) or thrombopoietin is the main growth factor that induces megakaryocyte commitment. The role of adhesion proteins in the generation of megakaryocytic progenitors has not been well studied.

METHODS

We isolated CD34(+) cells from umbilical cord (UC) blood and cultured them in serum-free medium containing IL-3, IL-6, SCF and MGDF, with or without fibronectin. Cultures were sampled at Days 4 and 8 for cell count, immunophenotyping by flow cytometry, and megakaryocyte colony-forming units (CFU-Mk) assay.

RESULTS

Fibronectin in synergy with MGDF increased both total and CD34(+) cell count. Immunophenotyping of the CD34(+) and CD34(-) cells showed that the percentage expression of CD61 and CD41a on CD34(-) cells was increased by fibronectin compared with CD34(+) cells by Day 8 of culture. The CFU-Mk assay confirmed that fibronectin increased generation of megakaryocytic progenitors by 2.4-fold by Day 8 of culture, but the absolute number was less than that of Day 4 culture. The cells in the CFU-Mk colonies derived from culture containing fibronectin were larger than those from cytokine-only culture.

CONCLUSIONS

It was concluded that the addition of fibronectin to the culture system enhanced MGDF, induced ex vivo expansion of megakaryocytic progenitors from CD34(+) cells, as well as increasing their maturation towards later stages of megakaryocyte differentiation.

BACKGROUND

There is little information in the literature on the structural basis mediating gingival cell adhesion to the surface of titanium abutments. We cultured gingival fibroblasts on a titanium abutment creating as closely as possible the in vivo state. We analyzed the constitutive and transforming growth factor (TGF) beta-induced expression of the adhesion molecules CD44, CD49b, CD49c, CD51, CD54, and CD61 and extracellular matrix (ECM) components fibronectin, laminin and collagen IV.

METHODS

Three totally edentulous patients underwent implant treatment to anchor the mandibular denture on 2 implants. Gingival mucosa cell specimens were collected from the mandible during the first surgical stage and the gingival fibroblast cultures were prepared. Cells were cultured for 48 hours with or without isoforms TGF-beta1, TGF-beta2, and TGF-beta3. The expression of adhesion molecules and ECM components was analyzed by immunofluorescence staining and flow cytometry.

RESULTS

The addition of TGF-beta isoforms to the cell culture over the incubation period had little effect on cell growth rate, but significantly influenced cell orientation, which changed from a sun-burst pattern in control conditions to a more elongated organization and perpendicular to abutment surface. In all fibroblast preparations, a marked expression of CD44 and a moderate positivity for anti-CD49b and CD49c were found. By contrast, CD51, CD54, and CD61 expressions were negligible. When fibroblasts were cultured for 48 hours in the presence of TGF-beta, the expression of most of the receptor molecules increased. The cells expressed constitutively moderate levels of laminin and fibronectin and low amounts of collagen IV. By contrast, treatment with any one of the 3 TGF-beta isoforms greatly enhanced the expression levels of fibronectin, laminin, and, especially, collagen IV.

CONCLUSIONS

TGF-beta not only seems to affect the orientation of the cultured gingival fibroblasts, but also to induce a clear-cut modification of their adhesion molecule expression.

The paper reviews literature updates on the organization and specific features of megakaryocytopoiesis, the mechanisms of its regulation, possible ways for platelets to occur from megakaryocytes. It characterizes thrombocytopoiesis, by describing the detailed structural organization of platelets and gives data on the relationship of the morphofunctional features of platelets to the ploidy of megakaryocytes that give rise to the latter. The structure and clinical value of a number of platelet activation markers, such as CD41/CD61, CD42, CD62p, platelet-leukocyte aggregates, microvesicles, are described.

False-positive diagnosis of lymph nodes occurs when a benign element in a lymph node, or in its capsule, is interpreted as metastatic carcinoma. This report describes a patient with breast carcinoma who had megakaryocytes in axillary sentinel lymph nodes mimicking metastatic carcinoma. The patient had no history of a hematologic disease, and we found no evidence of a concurrent hematopoietic disorder. The megakaryocytes were reactive for CD31, CD61, and von Willebrand factor, but not for cytokeratin (AE1/AE3). Megakaryocytes should be added to the list of benign histologic abnormalities that may simulate metastatic carcinoma in a sentinel lymph node.

OBJECTIVE

Placental fibrin deposits in patients wih recurrent spontaneous abortion (RSA) indicate an exaggerated haemostatic response. This 'hypercoagulability' may involve pro-coagulant factors such as circulating microparticles (MPs). We investigated the relationship between circulating pro-coagulant MPs and systemic coagulation in RSA patients.

METHODS

Platelet- and endothelial cell-derived microparticles (PMPs, EMPs) were evaluated by flow cytometry in RSA patients (n = 51) and compared to controls (n = 24) using annexin V (total numbers of MP), and antibodies against CD61, CD63 and CD62P (PMP), as well as CD144 and CD62E (EMP). Prothrombin fragment 1 + 2 (F(1+2)) and thrombin generation were determined to assess the pro-coagulant potential of MP.

RESULTS

Numbers of annexin V-binding MP were nearly similar in RSA patients and controls. However, a subgroup of ten RSA patients (10/51; 20%) presented with MP concentrations >10,000 x 10(6)/L, compared to only one women out of the control group (1/24; 4%; P = 0.038). Neither PMP and EMP nor F(1+2) and thrombin generation differed significantly within the study population.

CONCLUSIONS

The present study shows that circulating MPs are not directly associated with the extent of systemic coagulation activation in RSA patients. We hypothesize that increased numbers of circulating MPs either are only indirectly associated with coagulation during pregnancy of RSA patients, or affect abortion via mechanisms independently from hypercoagulation.

In order to identify the cause of maternal deaths due to thrombotic thrombocytopenic purpura (TTP) and identify future preventative measures we retrospectively reviewed all maternal deaths due to TTP in London from 2003 to 2008 that were confirmed on post-mortem examination. There were three maternal deaths due to sudden cardiac arrest within 1, 5 and 2 days of presentation, respectively. Post-mortem findings revealed they all died with intramyocardial microvascular thrombosis, the thrombi being characteristically platelet rich (CD61+, fibrin-). Platelet thrombi in the coronary microvasculature are the cause of early sudden death in TTP in pregnancy. If TTP cannot be excluded in the differential diagnosis of thrombotic microangiopathies of pregnancy, then plasmapheresis should be instituted as a medical emergency. ADAMTS-13 levels are helpful in making the diagnosis, but results should not be awaited before instituting plasmapheresis.

The authors present information on the presence of adhesive proteins on membranes of myeloma and precursor cells isolated from bone marrow and blood from a group of 33 patients examined by fluorescent flow cytometry. They also compare the density of integrins (CD29, CD49e, CD41, CD51 and CD61) and adhesive proteins from the group of "homing" receptors (CD44) and IgG "superfamily" (LFA-1, LFA-3, ICAM-1, N-CAM) and their changes after a single oral dose of a mixture of proteolytic enzymes (Wobe Mugos, Wobenzym, MUCOS Pharma, FRG). The authors observed a significant drop of CD29, CD54 (ICAM-1), CD58 (LFA-3) after Wobe Mugos, CD49, CD51, CD58 after Wobenzyme. The insignificant decline of density of CD44 on cells, as well as of the soluble receptor of CD44 after oral administration of proteolytic enzymes in serum, incl. the mentioned changes of integrins and other adhesive proteins, indicate the importance of enzyme preparations in the supporting treatment of malignant processes.

Interleukin-1 beta (IL-1β)-the most potent pro-inflammatory is responsible for a broad spectrum of immune and inflammatory responses, it induces T-cell and B-cell activation and consequently the synthesis of other pro-inflammatory cytokines (such as IFN-γ and TNF). IL-1β induces the formation of blood platelet-leukocyte aggregates (PLAs), which suggests that IL-1β significantly affects the cross-talk between blood platelets and the immune response system, leading to coronary thrombosis. The aim of our study is to investigate the effect of flavonolignans (silybin, silychristin and silydianin) on the IL-1β-induced interaction between platelets and leukocytes, as well as on the expression and the secretion of pro-inflammatory factors. Whole blood samples were pre-incubated with commercially available flavonolignans (silybin, silychristin and silydianin) in a concentration range of 10-100 µM (30 min, 37 °C). Next, samples were activated by IL-1β for 1 h. Blood platelet-leukocyte aggregates were detected by using the double-labeled flow cytometry (CD61/CD45). The level of produced cytokines was estimated via the ELISA immunoenzymatic method. IFN-γ and TNF gene expression was evaluated using Real Time PCR with TaqMan arrays. We observed that in a dose-dependent manner, silybin and silychristin inhibit the IL-1β-induced formation of blood platelet-leukocyte aggregates in whole blood samples, as well as the production of pro-inflammatory cytokines-IL-2, TNF, INF-α, and INF-γ. Additionally, these two flavonolignans abolished the IL-1β-induced expression of mRNA for IFN-γ and TNF. Our current results demonstrate that flavonolignans can be novel compounds used in the prevention of cardiovascular diseases with dual-use action as antiplatelet and anti-inflammatory agents.

OBJECTIVE

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder caused by increased platelet destruction and impaired platelet production. Antibody binding to megakaryocytes may occur in ITP, but in vivo evidence of this phenomenon is lacking.

METHODS

We determined the proportion of megakaryocytes bound with immunoglobulin G (IgG) in bone marrow samples from primary patients with ITP (n = 17), normal controls (n = 13) and thrombocytopenic patients with myelodysplastic syndrome (MDS; n = 10). Serial histological sections from archived bone marrow biopsies were stained for CD61 and IgG. IgG binding and the number of bone marrow megakaryocytes were determined morphologically by a hematopathologist with four assessors after a calibration exercise to ensure consistency.

RESULTS

The proportion of ITP patients with high IgG binding (>50% of bone marrow megakaryocytes) was increased compared with normal controls [12/17 (71%) vs. 3/13 (23%), P = 0.03]. However, the proportion of ITP patients with high IgG binding was no different than thrombocytopenic patients with MDS [12/17 (71%) vs. 7/10 (70%), P = 1.00]. IgG binding was associated with increased megakaryocyte numbers. Like platelet-associated IgG, megakaryocyte-associated IgG is related to thrombocytopenia but may not be specific for ITP.

CONCLUSIONS

Mechanistic studies in ITP should focus on antibody specificity and include thrombocytopenic control patients.

Embryonic megakaryopoiesis starts in the yolk sac on gestational day 7.5 as part of the primitive wave of hematopoiesis, and it continues in the fetal liver when this organ is colonized by hematopoietic progenitors between day 9.5 and 10.5, as the definitive hematopoiesis wave. We characterized the precise phenotype of embryo megakaryocytes in the liver at gestational day 11.5, identifying them as CD41⁺⁺CD45-CD9⁺⁺CD61⁺MPL⁺CD42c⁺ tetraploid cells that express megakaryocyte-specific transcripts and display differential traits when compared to those present in the yolk sac at the same age. In contrast to megakaryocytes from adult bone marrow, embryo megakaryocytes are CD45^- until day 13.5 of gestation, as are both the megakaryocyte progenitors and megakaryocyte/erythroid-committed progenitors. At gestational day 11.5, liver and yolk sac also contain CD41⁺CD45⁺ and CD41⁺CD45^- cells. These populations, and that of CD41⁺⁺CD45^-CD42c⁺ cells, isolated from liver, differentiate in culture into CD41⁺⁺CD45^-CD42c⁺ proplatelet-bearing megakaryocytes. Also present at this time are CD41^-CD45⁺⁺CD11b⁺ cells, which produce low numbers of CD41⁺⁺CD45^-CD42c⁺ megakaryocytes in vitro, as do fetal liver cells expressing the macrophage-specific Csf receptor-1 (Csf1r/CD115) from MaFIA transgenic mice, which give rise poorly to CD41⁺⁺CD45^-CD42c⁺ embryo megakaryocytes both in vivo and in vitro In contrast, around 30% of adult megakaryocytes (CD41⁺⁺CD45⁺⁺CD9⁺⁺CD42c⁺) from C57BL/6 and MaFIA mice express CD115. We propose that differential pathways operating in the mouse embryo liver at gestational day 11.5 beget CD41⁺⁺CD45^-CD42c⁺ embryo megakaryocytes that can be produced from CD41⁺CD45^- or from CD41⁺CD45⁺ cells, at difference from those from bone marrow.

BACKGROUND: Whereas ex vivo expanded megakaryocytic progenitor cells have been investigated for their ability to support platelet regeneration, the question whether more mature platelet-like particles expanded from hematopoietic progenitor cells may be useful for transfusion purposes remains largely elusive. METHODS: Human peripheral blood progenitor cells (PBPCs) were enriched using surface expression of CD34 by immunoselection. CD34+ enriched PBPCs were expanded ex vivo in serum-free medium supplemented with cytokines. As a proof-of-principle, distribution of expanded CD61+ particles was analyzed after transfusion into Non-Obese Diabetic/ Severe Combined Immunodeficiency (NOD/SCID) mice. RESULTS: Highest ex vivo expansion for CD41+/CD61 + cells was achieved when medium was supplemented with SCF, TPO and IL-3. During expansion culture, CD34 marker expression decreased from 85 to 2-8%, while megakaryocytic cells appeared and CD41 and CD61 expression increased from 3 to about 30%. After transfusion of the expanded cells in NOD/SCID mice, CD61 + cells located mainly to bone marrow and to a lesser degree to spleen, but also circulated in blood. CONCLUSIONS: Platelet-like particles using cytokine-substituted serumfree medium can be generated efficiently from CD34+ expansion cultures, but mainly home to hematopoietic tissue.

A clinical goal for ex vivo expansion of cord blood (CB) CD34(+) cells is to shorten the period of neutropenia and thrombocytopenia following myeloablative therapy and transplantation. Prolongation of cytokine expansion leads to the production of greater numbers of cells, and should have an impact on neutrophil and platelet recovery. Furthermore, expansion of CD34(+) cells should support the continued production of neutrophils and platelets in the 6-week period following transplantation. We tested these hypotheses by characterization of the kinetics (human CD45(+) cells in the blood) and phenotype (CD45, CD34, CD61, CD33, CD19 and CD3) of human engraftment in the non-obese diabetic severe combined immunodeficient mouse (NOD-SCID) following 7 or 14 d of ex vivo expansion of CB CD34(+) cells. Mice transplanted with 14 d cells showed greater percentages of human CD45(+) cells in the blood, bone marrow and spleen than mice transplanted with unexpanded cells or 7 d cells. Prolonging cytokine exposure of CD34(+) cells and transplantation with increasing numbers of input cells facilitated the production of absolute numbers of CD34(+), CD33(+), CD61(+) and CD19(+) cells in vivo. Furthermore, analysis of SCID engrafting potential showed that prolongation of culture duration facilitates in vivo expansion of CD45(+), CD34(+) and CD19(+) cells after transplantation. It is anticipated that prolonged (2 weeks) ex vivo culture of CB will have a beneficial clinical effect.

We report the case of a 78-year-old man who presented with acute myeloid leukaemia showing subpopulations of cells expressing platelet-associated markers and the presence of a pan-myeloid component, besides glycophorin A-positive cells. Most of the immature cells had a proerythroblast-like morphology and we classified this case as an FAB-M6 variant, as suggested by Bain (1). According to the WHO classification, this leukaemia fulfilled the criteria of'AML with multilineage dysplasia' (2). Immunophenotyping characteristics showed two distinct aberrant subpopulations, a young pan-myeloid (CD45+ with low density, CD34+, CD117+, CD13+, CD33+, partial cytoplasmic myeloperoxidase (MPO)+) population with platelet-associated markers (CD41+, CD42+, CD61+) and a CD45+, CD117+, CD34- population with partial CD235a positivity indicative for erythroid maturation. This case belongs to the group of 'early' erythroblastic leukaemias where a subset of progenitor cells present with erythroid-megakaryocyte bipotentiality or are blocked at an early BFU-E (burst-forming unit erythrocyte)-like stage of erythroid differentiation (11, 12, 13).

Patients with chronic myelogenous leukemia (CML) present typically with an elevated white blood cell count (WBC) and cytogenetic or molecular genetic evidence of t(9;22)/BCR-ABL1 fusion gene. Rarely, CML patients may present with a normal or mildly elevated WBC and are asymptomatic, and we describe 7 patients in this study. The WBC in these patients ranged from 3.6 to 14.3 K/mm(3) with 50% to 73% granulocytes and 0% blasts. In all patients, t(9;22)(q34;q11.2) was detected by conventional cytogenetics, and BCR-ABL1 fusion was shown, supporting the diagnosis of preleukemic CML (pre-CML). We compared these patients with a group of 5 cases of CML in chronic phase (CML-CP) and 5 bone marrow specimens with a leukemoid reaction (n=5). Reticulin, CD34, and CD61 immunostains were performed on all bone marrow biopsy specimens. Peripheral blood absolute basophilia (≥200/mm(3)) was noted in only 4 of 7 pre-CML cases, whereas it was present in all CML-CP cases and absent in leukemoid reaction cases. The mean ±SD of microvascular density of pre-CML cases (10.0 ± 4.3 vessels/200× field) was twice that of leukemoid reaction cases (5.0 ± 1.0) (P=.02; Student t test) but similar to that of CML-CP cases (12.5 ± 3.6). Microvessels in pre-CML, highlighted by CD34, were tortuous with abnormal branching, although to a lesser extent than those found in CML-CP. Microvessels in leukemoid reaction were generally straight. The percentage of small, hypolobated megakaryocytes, highlighted by CD61 in pre-CML, was 40%, 3 times that found in leukemoid reaction cases (13%) but less than that of CML-CP cases (86%). We conclude that pre-CML should be suspected in patients with a normal to mildly elevated WBC and absolute basophilia. Bone marrow examination can usually distinguish pre-CML from a leukemoid reaction based on the percentage of small, hypolobated megakaryocytes; microvascular density; and morphologic features.

Mpl ligand is a recently cloned haemopoietic growth factor that stimulates megakaryopoiesis in vitro and in vivo. We describe the in vitro effect of a truncated form of Mpl ligand, recombinant human megakaryocyte growth and development factor (rHuMGDF), on megakaryopoiesis in bone marrow from normal subjects and patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We used both semi-solid and suspension culture techniques to assess the effect of pegylated (PEG) rHuMGDF on megakaryocyte colony growth (CFU-Mk) and on the production of CD61+ cells in 7d suspension cultures. PEG rHuMGDF increased CFU-Mk growth and CD61+ cell production in a dose-dependent fashion in all normal marrows tested. Normal CFU-Mk growth was increased threefold with the addition of 10 ng/ml PEG rHuMGDF to cultures and CD61+ cells were increased 8-10-fold by the same dose. Although increased CFU-Mk growth was only seen in 1/10 AML and 6/16 MDS marrows, CD61+ cell numbers in suspension culture were increased in 9/13 AML and 12/15 MDS samples, responses ranged from very limited to normal magnitude. There was no correlation between platelet count and CFU-Mk number, CD61+ cell number or response to PEG rHuMGDF. We did not find any increased CFU-GM colony or cluster growth in response to PEG rHuMGDF and the CD61+ cells produced in suspension culture had features of megakaryocytic differentiation. These data suggest that PEG rHuMGDF can enhance megakaryocyte proliferation in some patients with MDS and AML, and may have a role in the treatment of thrombocytopenia in these patients.

Cytologic, immunologic, and cytogenetic studies were performed on the blast cells of a newborn with Down syndrome and transient myeloproliferative disease. This hematologic disorder is uncommon, and occurs primarily in infants with Down syndrome. This boy presented with a high white blood cell count and a high percentage of blast cells, without anemia or thrombocytopenia. Chromosome analysis showed a constitutional trisomy 21 without any other clonal abnormality. A three-color flow cytometric analysis was performed and revealed two different CD45 dim, CD34(+), CD117(+), CD56(+) immature subpopulations: the normal immature myeloid precursor and an immature blast cell population that expressed CD41, CD42, CD61, CD36, CD13, CD1a, and CD2. We postulate that this population could be the leukemic precursor involved in the acute megakaryoblastic leukemia frequently observed in children with Down syndrome.

During the last decade, biannual quality controls were performed in the Netherlands focusing on the immunophenotyping of leukaemic haematological malignancies. All results on 48 specimens obtained by 18-34 laboratories were analysed. The interlaboratory variability and percentages of discordant results from 30 markers were measured by assessing false positive or negative (cut-off 10%) results in comparison with median results of the group. The quality of the immunophenotypic diagnoses obtained from the interpretation of these markers in relation to clinical data was evaluated by scoring them as 'correct', 'minor fault', 'major fault', 'not based upon the markers used', and 'no diagnosis', CD3, CD8, CD19, CD61 and Sm lambda had the lowest percentage discordancy (sum of total negative and positive discordant values 5-7.5% of assays): CD13, CD15, cyCD22, CD33 and TdT scored worst with 14-20% cumulative discordancy. The analysis of each diagnosis yielded 78% acceptable immunophenotypic conclusions (correct 54% and minor fault 24%). It appeared that the major faults in immunophenotyping were caused by suboptimal antibody selection and erroneous interpretation of the results obtained, rather than by technical errors. Large differences per diagnostic category were observed, with the best scores for mature B-cell leukaemias, AMLs and common-ALL, and the poorest scores for T-cell malignancies which were correctly diagnosed in only 24-60% of specimens. Mature T-NHL and T-PLL were mistakenly diagnosed as T-ALL by 40% of the centres. Misinterpretation of TdT immunofluorescence or omitting this marker contributed significantly to these wrong diagnoses. A median of 4% of immunophenotypic diagnoses were not based on a correct panel of antibodies, but upon the morphology of the accompanying blood smear, and was often flawed by overinterpretation. In conclusion, both the technical performance of immunophenotyping of haematological malignancies in The Netherlands and the procedure by which a final diagnosis is obtained needs improvement, especially for T-cell malignancies.

Morphologic, immunologic, cytogenetic, and clinical features were studied in 9 cases of acute undifferentiated leukemia (AUL). These patients were unclassifiable by FAB criteria, they were CD34+ and did not express myeloid- or lymphoid-associated antigens (CD13, CD33, CD14, CD15, CD61, CD19, CD10, CD22, CD7, CD2, CD5, CD3). Clonal abnormalities were seen in 8 of 9 cases. Del(5q) as the sole anomaly was observed in 3 cases; +13 was the primary change in 3 cases, and isolated trisomy 12 was found in 1 patient. A complex karyotype with trisomy 12q, in association with del 17p and trisomy 21q was detected in 1 case. One patient with 5q- relapsed with refractory anemia with excess of blasts; the presence of dysgranulopoiesis and a few blasts with possible monocytoid morphology in the remaining 2 patients point to a "myeloid nature" of these leukemias. Analysis of cytologic features in our 3 patients with +13, in combination with previously reported cases, suggests the occurrence of immature stem cell involvement with limited differentiation potential, possibly more along the myeloid than the lymphoid lineage. The significance of trisomy 12q in this subset of leukemia remains elusive; some clues of minimal differentiation towards the myeloid lineage in our cases are provided by positivity for the CD117 (c-kit) antigen and by relapse with acute myeloid leukemia without maturation (M1) in one patient. We conclude that, with presently available diagnostic techniques, AUL is a rare subset of leukemia, in which cytogenetic changes are confined to a few chromosomes, with prevalent involvement of 5q and of chromosomes 13 and 12. Chromosome findings may be of value in clinical practice, especially in those cases with "myeloid-oriented" karyotype.