Michelle L. Brereton

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF) on megakaryopoiesis in normal subjects and patients with myelodysplasia and acute myeloid leukaemia

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 7 d 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.

The use of digital 'virtual slides' in the quality assessment of haematological morphology: Results of a pilot exercise involving UK NEQAS(H) participants

We report the results of a pilot study assessing the use of digital ‘virtual slides’ in haematological quality assessment. Conducted together with the UK National External Quality Assessment Scheme for General Haematology, the study involved 166 separate participants, using the format of a typical assessment exercise. The results revealed substantial concordance of observations made using digital slides with those reported in previous glass slide surveys that used identical cases. Participant feedback strongly supported the use of electronic slides in teaching and assessment roles. Our results suggest roles for this new electronic resource in external quality assessment (EQA), education and continuing professional development.

The effect of the chemokine rhMIP-1α, and a non-aggregating variant BB-10010, on blast cells from patients with acute myeloid leukaemia

The effects of recombinant macrophage inflammatory protein 1α (rhMIP‐1α) on the proliferation of leukaemic blast cells from patients with acute myeloid leukaemia was assessed. Using the previously described [3H]thymidine incorporation index assay, the response of autonomous and growth factor responsive AML blast cells to the chemokine rhMIP‐1 was measured. In the case of autonomous proliferators, rhMIP‐1α had no inhibitory effect  on [3H]thymidine incorporation and in 4/6 cases [3H]‐thymidine incorporation was stimulated by rhMIP‐1α. In the presence of stem cell factor (SCF), a majority (8/9) of the samples which responded to this growth factor were inhibited when rhMIP‐1α was included in the assay medium. Similar results were obtained with GM‐CSF‐responsive samples; however, when these two cytokines were combined, only 3/14 were significantly inhibited. In the presence of human placental conditioned medium (HPCM), rhMIP‐1α significantly inhibited [3H]thymidine incorporation in only 2/10 of HPCM‐responsive samples. In methylcellulose assays rhMIP‐1α had no consistent effect on colony/cluster formation in the presence of either GM‐CSF + SCF or HPCM. Similar results were obtained with BB‐10010, a mutant of rhMIP‐1α which has defined aggregation properties in solution. These data suggest that autonomously proliferating AML cells, and also some AML samples which require cytokines to proliferate, are non‐responsive to the growth inhibitors rhMIP‐1α and BB‐10010 in the presence of multiple growth factors.

Expression of AML1/MTG8 transcripts in clonogenic cells grown from bone marrow of patients in remission of acute myeloid leukaemia with t(8;21)

Patients in long‐term remission of acute myeloid leukaemia (AML) M2 with t(8;21) after chemotherapy, with or without bone marrow transplantation, are known to retain residual cells which express AML1/MTG8 transcripts in bone marrow, detectable by RT‐PCR. In order to determine whether these residual cells are clonogenic, we have grown remission bone marrow samples in standard semi‐solid culture and picked individual CFU‐GM and BFU‐E colonies which were then analysed for the expression of AML1/MTG8 transcripts using a rapid specific RT‐PCR technique. Nine patients were tested in remission, six between 1 and 83 months post chemotherapy, one 103 months post autologous bone marrow transplant and one 41 months post allogeneic bone marrow transplant. One of these patients also had quantitation of AML1/MTG8 transcripts on five occasions after recovery from each course of chemotherapy and at the end of treatment. There was a significant correlation between the percentage of positive colonies and the level of AML1/MTG8 transcripts. Between two and 80 CFU‐GM and between two and 21 BFU‐E colonies were analysed from each patient sample; 0–23% CFU‐GM and 0–17% BFU‐E colonies were found to express AML1/MTG8 transcripts suggesting that these residual cells are clonogenic in vitro and that the cell of origin is a multipotent myeloid progenitor.

Review of the UK NEQAS (H) digital morphology pilot scheme for continuing professional development accessed via the internet

UK NEQAS (H) developed and instigated a pilot scheme for digital morphology, which was accessed by participants over the internet in order to assess the viability of using high quality images as an educational tool for continuing professional development. The pilot scheme was trialled over a 2‐year period with eight releases totalling 16 morphology cases. Digital images allowed participating individuals to examine and comment on exactly the same cells and compare their findings with those of other participants, consensus data from traditional glass slide surveys and expert opinion. Feedback from participants on their experience was then relayed back to the development team by UK NEQAS (H) in order to drive the educational format and to ensure that any new scheme would meet the requirements of the users.

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) on megakaryopoiesis in patients with aplastic anaemia

Recombinant human megakaryocyte growth and development factor (rHuMGDF), a truncated form of the Mpl ligand, stimulates megakaryopoiesis both in vitro and in vivo. We describe the in vitro effect of pegylated recombinant human MGDF (PEGrHuMGDF) alone and in combination with other haemopoietic growth factors (G‐CSF, GM‐CSF, IL3, IL6, erythropoietin, SCF) on megakaryopoiesis in bone marrow from 11 normal subjects and 19 patients with aplastic anaemia (AA). We used semi‐solid cultures to assess megakaryocyte colony growth (CFU‐Mk) and 7 d suspension cultures to assess production of platelet glycoprotein IIIa (CD61) positive cells. CFU‐Mk growth from normal marrow increased 3–4‐fold and CD61+ve cells in suspension culture increased 8–10‐fold with the addition of 10 ng/ml PEGrHuMGDF. In normal subjects growth factor combinations further increased responses in suspension culture, PEGrHuMGDF + SCF, PEGrHuMGDF + IL3 and PEGrHuMGDF + SCF + IL3 + Epo (P < 0.05). IL6, GM‐CSF, G‐CSF or Epo added with PEGrHuMGDF did not consistently give this increase. CFU‐Mk growth from AA marrow remained very low in the presence of PEGrHuMGDF, with or without the addition of other growth factors. CD61+ve cells in suspension culture were, however, increased in the presence of PEGrHuMGDF alone in 12/19 AA cases. Of the 12 patients responsive to PEGrHuMGDF, nine were tested with additional growth factors and further responses were seen in six. In the AA cases PEGrHuMGDF+SCF and PEGrHuMGDF+SCF+IL3+Epo gave the highest responses. These data suggest that PEGrHuMGDF, alone or in combination with SCF and/or IL3, can enhance megakaryocyte proliferation in some patients with aplastic anaemia and may therefore have a role in the treatment of thrombocytopenia in these cases.

Megakaryopoiesis in vitro in myelodysplastic syndromor and development es and acute myeloid leukaemia: effect of pegylated recombinant human megakaryocyte growth and development factor in combination with other growth factors

Pegylated recombinant human megakaryocyte growth and development factor (PEG‐rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG‐rHuMGDF combined with granulocyte colony‐stimulating factor (G‐CSF), granulocyte–macrophage CSF (GM‐CSF), interleukin 3 (IL‐3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61‐positive cells in suspension cultures increased with PEG‐rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL‐3 and/or SCF were added to PEG‐rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU‐Mk growth was poor overall, but could be enhanced by PEG‐rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG‐rHuMGDF can be augmented by IL‐3 and SCF in many MDS and AML patients.

Do We Know Why We Make Errors in Morphological Diagnosis? An Analysis of Approach and Decision-Making in Haematological Morphology

Summary Background The laboratory interpretation of blood film morphology is frequently a rapid, accurate, and cost-effective final-stage of blood count analysis. However, the interpretation of findings often rests with a single individual, and errors can carry significant impact. Cell identification and classification skills are well supported by existing resources, but the contribution and importance of other skills are less well understood. Methods The UK external quality assurance group in haematology (UK NEQAS(H)) runs a Continued Professional Development scheme where large digital-images of abnormal blood smears are presented using a web-based virtual microscope. Each case is answered by more than 800 individuals. Morphological feature selection and prioritisation, as well as diagnosis and proposed action, are recorded. We analysed the responses of participants, aiming to identify successful strategies as well as sources of error. Findings The approach to assessment by participants depended on the affected cell type, case complexity or skills of the morphologist. For cases with few morphological abnormalities, we found that accurate cell identification and classification were the principle requirements for success. For more complex films however, feature recognition and prioritisation had primary importance. Additionally however, we found that participants employed a range of heuristic techniques to support their assessment, leading to associated bias and error. Interpretation A wide range of skills together allow successful morphological assessment and the complexity of this process is not always understood or recognised. Heuristic techniques are widely employed to support or reinforce primary observations and to simplify complex findings. These approaches are effective and are integral to assessment; however they may also be a source of bias or error. Improving outcomes and supporting diagnosis require the development of decision-support mechanisms that identify and support the benefits of heuristic strategies while identifying or avoiding associated biases. Funding The CPD scheme is funded by participant subscription.

Comparison of megakaryopoiesis in vitro of paired peripheral blood progenitor cells and bone marrow harvested during remission in patients with acute myeloid leukaemia

We have studied paired peripheral blood progenitor cells (PBPC) and bone marrow (BM) samples from 12 acute myeloid leukaemia (AML) patients following intensive chemotherapy, and assessed direct granulocyte–macrophage colony‐forming units (CFU‐GM), erythroid burst‐forming units (BFU‐E), megakaryocyte CFU (CFU‐Mk) numbers and the production of CD61+ (platelet glycoprotein IIIa) cells in suspension culture in response to various haemopoietic growth factor combinations. We found that CFU‐GM and BFU‐E numbers per 105 mononuclear cells were similar in both AML PBPC and BM harvests; CFU‐Mk numbers, however, were significantly higher in PBPC than BM. In addition, the higher total white cell count of the PBPC harvests meant that PBPC have much higher numbers of total progenitors per collection. CD61+ cell numbers in suspension cultures of AML PBPC and BM were lower than those of harvested normal marrow. However, response to pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) both alone and in combination with other growth factors was qualitatively similar to that of normal BM. As with normal BM, response to PEGrHuMGDF alone did not increase further with addition of granulocyte colony‐stimulating factor (G‐CSF), granulocyte–macrophage CSF (GM‐CSF), interleukin 6 (IL‐6) or erythropoietin (EPO) in the AML PBPC and BM. Further responses over PEGrHuMGDF alone were seen when added with stem cell factor (SCF) or with a combination of SCF + IL‐3 + EPO in both AML PBPC and BM cultures; however, the magnitude of the response was greater in the PBPC cultures. Response to PEGrHuMGDF + IL‐3 was seen in the PBPC cultures but not in the AML BM. These data suggest that, in AML patients, there are proportionally more megakaryocyte progenitor cells in the mobilized PBPC than in the BM harvests, which would explain the more rapid platelet recovery following PBPC autografts.

Web-Based Virtual Microscopy of Digitized Blood Slides for Malaria Diagnosis: An Effective Tool for Skills Assessment in Different Countries and Environments

Background Morphological examination of blood films remains the reference standard for malaria diagnosis. Supporting the skills required to make an accurate morphological diagnosis is therefore essential. However, providing support across different countries and environments is a substantial challenge. Objective This paper reports a scheme supplying digital slides of malaria-infected blood within an Internet-based virtual microscope environment to users with different access to training and computing facilities. The feasibility of the approach was established, allowing users to test, record, and compare their own performance with that of other users. Methods From Giemsa stained thick and thin blood films, 56 large high-resolution digital slides were prepared, using high-quality image capture and 63x oil-immersion objective lens. The individual images were combined using the photomerge function of Adobe Photoshop and then adjusted to ensure resolution and reproduction of essential diagnostic features. Web delivery employed the Digital Slidebox platform allowing digital microscope viewing facilities and image annotation with data gathering from participants. Results Engagement was high with images viewed by 38 participants in five countries in a range of environments and a mean completion rate of 42/56 cases. The rate of parasite detection was 78% and accuracy of species identification was 53%, which was comparable with results of similar studies using glass slides. Data collection allowed users to compare performance with other users over time or for each individual case. Conclusions Overall, these results demonstrate that users worldwide can effectively engage with the system in a range of environments, with the potential to enhance personal performance through education, external quality assessment, and personal professional development, especially in regions where educational resources are difficult to access.