Kristin Jochmans

Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies.

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.

Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy.

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.

Different expression of adhesion molecules on CD34+ cells in AML and B-lineage ALL and their normal bone marrow counterparts

Abstract:  The expression of adhesion molecules on CD34+ cells in acute myeloid leukemia (AML) and B‐lineage acute lymphoblastic leukemia (B‐lineage ALL) was compared with that on the myeloid and B‐lymphoid CD34+ cells in normal bone marrow. Bone marrow aspirates of 10 patients with AML, 8 patients with B‐lineage ALL and of 6 healthy volunteers were examined. The phenotype of the CD34+ cells was determined with a double immunofluorescence method and flow cytometry. CD34+ cells in AML and B‐lineage ALL showed a lower expression of VLA‐2 and VLA‐3 and a higher expression of ICAM‐1 and LFA‐3 than their normal bone marrow counterparts. AML CD34+ cells had less L‐selectin but more VLA‐5 on their surface membrane than normal myeloid CD34+ cells. B‐lineage ALL CD34+ cells showed an overexpression of LFA‐3. In individual patients deficiencies or over‐expression of the β1 integrin chain, VLA‐4, PECAM‐1 or HCAM also occurred. An abnormal adhesive capacity of the leukemic cells may influence their proliferation, their localisation and apoptosis. An aberrant expression of adhesion molecules may be used for the detection of minimal residual leukemia in these patients.

An immunogold-silver staining method for detection of cell surface antigens in cell smears.

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.

Pearson marrow pancreas syndrome: a molecular study and clinical management

Human mitochondrial DNA (mt DNA) lesions can cause a heterogeneous group of mitochondrial degenerative disorders. We report on a 5‐year‐old patient suffering from the full‐blown picture of Pearson syndrome. His symptoms started in the first year of life with failure to thrive, followed by chronic diarrhoea and lactic acidosis at 18 months of age. Analysis of mitochondrial DNA revealed large amounts of mt DNA molecules with a 2.7 kb deletion in all tissues examined. The diagnosis of Pearson syndrome was made initially in the absence of haematological disturbances. In the following months neutropenia, sideroblastic anaemia and hypoparathyroidism developed. Daily administration of dichloroacetate (DCA) and bicarbonate controls the lactic acidosis, while episodic treatments with filgastrim (Neupogen®) reverse episodes of severe neutropenia. Calcium and vitamin D supplementation compensate for the hypoparathyroidism. Chronic administration of DCA and supportive treatment for a long period help to stabilize patients with multiorgan dysfunction.

Development of thrombotic thrombocytopenic purpura after a single dose of gemcitabine

Dear Editor, Gemcitabine is a nucleoside analogue which was first approved for the treatment of unresectable pancreatic carcinoma. The indications for chemotherapy with gemcitabine have expanded to include the treatment of various types of cancer. Primary side effects include myelosuppression, flu-like symptoms and in rare cases, gemcitabine-associated thrombotic thrombocytopenic purpura (GTTP). The incidence of GTTP ranges from 0.015% to 1.4%. The risk for GTTP increases with a cumulative dose (above 20,000 mg/ m or more than 18 doses) and rarely occurs before 7 months of treatment [1]. Gemcitabine-associated thrombotic thrombocytopenic purpura might occur with lower doses of gemcitabine when using it in combination with other chemotherapeutic agents [2–4]. Patients heavily pretreated with other chemotherapeutic agents before gemcitabine also tend to have a higher risk of developing GTTP [5]. The pathophysiology of this complication is currently unknown, but it is thought to involve endothelial-cell injury, resulting in thrombotic microangiopathy. Both immune and nonimmune mechanisms have been proposed. The time interval between initiation of chemotherapy and the onset of GTTP seems to indicate that the clinical presentation represents the result of cumulative injury, which overwhelms the endothelial repair mechanisms [1]. In contrast to currently available data, we report a patient who developed GTTP after the administration of a single dose of gemcitabine in monotherapy and who had not received other chemotherapy before. A 71-year-old woman with a carcinoma of the pancreas underwent a Whipple duodenopancreatectomy. The tumour and four – tumour free – lymph nodes were removed. Four months later, a local relapse was diagnosed; subsequently, she underwent radiotherapy with a cumulative dose of 50 Gy to the site of recurrence. Six months later, multiple liver metastases were diagnosed, as well as diffuse peritoneal carcinomatosis with an associated ascitic effusion. The patient experienced cancer-related fatigue, anorexia, weight loss and diffuse abdominal pain. Her performance status had declined to 60% according to the Karnofsky score. Palliative chemotherapy was initiated with intravenous gemcitabine (at a total dose of 2,700 mg by a 30-min intravenous infusion). Five days after administration of the first dose, the patient was taken into hospital because of a rapid deterioration of her general condition and an unclear story of haematemesis and haematochesis. Medical imaging and gastroscopy showed no active bleeding. Laboratory analysis revealed a severe haemolytic anaemia (decreased haemoglobin, elevated indirect bilirubin, elevated LDH, decreased haptoglobin and schistocytes on peripheral blood smear), deteriorating renal function (elevated creatinine and urea), thrombocytopenia and a normal coagulation profile (normal prothrombin time and activated partial thromboplastin time). None of these abnormal laboratory findings were present 5 days before the start of the gemcitabine treatment (Table 1). After the diagnosis of GTTP, plasmapheresis and haemodialysis were considered, but taken into account the palliative context and poor prognosis of the underlying Ann Hematol (2008) 87:495–496 DOI 10.1007/s00277-007-0429-9

Growth factor receptor profile of CD34+ cells in AML and B‐lineage ALL and in their normal bone marrow counterparts

Abstract: Leukaemic cells show a low clonogenic activity and a heterogeneous proliferative response to growth factors. We investigated whether this could be due to an altered expression of growth factor receptors on the leukaemic precursors. Receptors for G‐CSF, stem cell factor (SCF), IL‐3, IL‐6 and IL‐7 were detected on CD34+ cells in AML and B‐lineage ALL with monoclonal antibodies and flow cytometry. The expression was compared with that on myeloid and B‐lymphoid CD34+ cells in normal bone marrow. Leukaemic CD34+ cells expressed the same receptors as their normal counterparts. AML and B‐lineage ALL could be distinguished by the growth factor receptor profile of their CD34+ cells. SCFR, G‐CSFR and IL‐6Rα were found in AML, IL‐7R in B‐lineage ALL and IL‐3Rα in both. IL‐3Rα was upregulated in AML and B‐lineage ALL CD34+ cells, while samples with low or high expression were present for the other receptors. This variable expression could correlate with the heterogeneous response of leukaemic cells to growth factors. Functional studies on isolated CD34+ cells are needed to investigate this further.

Plasma D-dimer concentrations in different clinical conditions.

Abstract D-dimers (DD), specific degradation products of crosslinked fibrin, are markers for activation of plasma coagulation and/or fibrinolysis. DD results below the cut-off level are proven to be useful to rule out the probable diagnosis of deep venous thrombosis (DVT) and/ or pulmonary embolism (PE). Our objective was to demonstrate that positive DD occur in many diseases and certain physiological conditions as high age and pregnancy and to look for gradations in positivity between different pathological conditions. We wanted to investigate the request attitude of our clinicians concerning DD. Positive DD results still confuse some physicians. Retrospectively, we examined medical records of 574 consecutive patients, in whom plasma DD were measured in daily routine. Both outpatients (n=423) and inpatients (n=151) were included. We noted their clinically predominant disease. Measurement of DD concentration is too often requested by clinicians, in any medical condition, and is not always clinically relevant. The relation of a positive result and the clinical problem is sometimes not understood. Overall, we found 64% DD positivity with a median concentration of 775 μg/L. We found elevated DD concentrations in various clinical conditions. Positive DD are of limited clinical value and concentrations should be correlated with disease, age or, if pregnant, with duration of pregnancy.

ALIFE2 study: low-molecular-weight heparin for women with recurrent miscarriage and inherited thrombophilia - study protocol for a randomized controlled trial

BackgroundA large number of studies have shown an association between inherited thrombophilia and recurrent miscarriage. It has been hypothesized that anticoagulant therapy might reduce the number of miscarriages and stillbirth in these women. In the absence of randomized controlled trials evaluating the efficacy of anticoagulant therapy in women with inherited thrombophilia and recurrent miscarriage, a randomized trial with adequate power that addresses this question is needed. The objective of the ALIFE2 study is therefore to evaluate the efficacy of low-molecular-weight heparin (LMWH) in women with inherited thrombophilia and recurrent miscarriage, with live birth as the primary outcome.Methods/DesignRandomized study of LMWH plus standard pregnancy surveillance versus standard pregnancy surveillance alone.Study population: pregnant women of less than 7 weeks’ gestation, and confirmed inherited thrombophilia with a history of 2 or more miscarriages or intra-uterine fetal deaths, or both.Setting: multi-center study in centers from the Dutch Consortium of Fertility studies; centers outside the Netherlands are currently preparing to participate.Intervention: LMWH enoxaparin 40 mg subcutaneously once daily started prior to 7 weeks gestational age plus standard pregnancy surveillance or standard pregnancy surveillance alone.Main study parameters/endpoints: the primary efficacy outcome is live birth. Secondary efficacy outcomes include adverse pregnancy outcomes, such as miscarriage, pre-eclampsia, syndrome of hemolysis, elevated liver enzymes and low platelets (HELLP syndrome), fetal growth restriction, placental abruption, premature delivery and congenital malformations.Safety outcomes include bleeding episodes, thrombocytopenia and skin reactions.DiscussionAfter an initial period of slow recruitment, the recruitment rate for the study has increased. Improved awareness of the study and acknowledgement of the need for evidence are thought to be contributing to the improved recruitment rates. We aim to increase the number of recruiting centers in order to increase enrollment into the ALIFE2 study.The study website can be accessed via www.ALIFE2study.org.Trial registrationThe ALIFE2 study was registered on 19 March 2012 under registration number NTR3361

Intravenous thrombolysis with recombinant tissue plasminogen activator in a stroke patient treated with apixaban

Apixaban is increasingly used in clinical practice (1), but data on the bleeding risk in patients treated with recombinant tissue plasminogen activator (rt-PA) while taking apixaban are nonexistent. A 74-year-old right-handed man presented with abrupt onset of global aphasia. He was known with a partial right hemianopsia secondary to a left occipital intracerebral hemorrhage five-years earlier and with paroxysmal nonvalvular atrial fibrillation treated with apixaban 5 mg bid. The National Institutes of Health Stroke Scale (NIHSS) score was 8. Noncontrast computed tomography (CT) of the brain showed no signs of acute intracranial pathology. Perfusion-CT revealed hypoperfusion in the territory of the left middle cerebral artery (Fig. 1a). An ostial stenosis of the left internal carotid artery was diagnosed on CT angiography (Fig. 1b). After informed consent by proxy, i.v. rt-PA therapy (0·9 mg/kg; total dose 81 mg) was administered at 4·5 h after symptom onset and 8·5 h after apixaban intake. Platelet count, prothrombin time, activated partial thromboplastin time, and fibrinogen levels were normal, as was creatinine clearance. The patient experienced an excellent recovery (NIHSS score 1) without signs of new infarction or intracranial hemorrhage on repeat CT. As apixaban is commonly used in patients with elevated stroke risk (1), therapeutic decision-making with regard to thrombolytic therapy may not uncommonly pose problems in the near future. Our case report illustrates that further study on the safety of rt-PA in this patient population is justified. Ann De Smedt*, Melissa Cambron, Koenraad Nieboer, Maarten Moens, Robbert-Jan Van Hooff, Laetitia Yperzeele, Kristin Jochmans, Jacques De Keyser, and Raf Brouns