accurate prediction of rare hemoglobin variants using a combination of high performance liquid chromatography, retention time and isoelectric focusing electrophoresis position

To investigate the predictive accuracy of using a combination of the high pressure liquid chromatography [HPLC] retention time and the relative isoelectric focusing [IEF] position to diagnose rare hemoglobin variants. A selected group of 40 patients with a rare beta-chain variant were assigned a presumed diagnosis following HPLC and IEF screening and then the variant identified in each case by DNA analysis. The study was conducted at the National Hemoglobinopathy Reference Laboratory, Oxford, United Kingdom, from August 2008 to October 2008. Thirteen out of 14 different variants were predicted accurately in 39 [97.5%] cases, compared to only one each for HPLC and IEF when used individually. A novel amplification refractory mutation system-polymerase chain reaction test was developed for Hb J-Baltimore and used successfully to provide a simple, rapid, and inexpensive diagnosis. The use of both HPLC retention time and isoelectric focusing position provides an accurate presumed diagnosis of a rare hemoglobin variant in the majority of cases. Amplification refractory mutation system-polymerase chain reaction test can provide a simple, rapid and inexpensive molecular diagnostic method for rare beta-chain variants

Expression of interleukin-2 receptors [IL-2R] and intracellular adhesion molecules-1 [ICAM-1] after allogeneic bone marrow transplantation

There is no specific diagnostic laboratory test for diagnosis of acute graft versus host disease [aGVHD] and sometimes it is a clinical diagnosis. Most investigators studying the relationship between IL-2 and aGVHD have obtained disappointing results. The expression and function of IL-2R and ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. This work was designed to investigate whether the observed phenotype pattern of IL-2R and ICAM-l may be useful for early diagnosis of aGVHD, in patients undergoing allogeneic peripheral blood stem cells transplantation [PBSCT]. Patients and Peripheral blood samples were obtained from 25 patients who underwent allogeneic PBSCT, and 13 patients with autologous PBSCT and 10 normal donors as control group. The expression of IL-2R and ICAM-1 were analyzed by flowcytometry using monoclonal antibodies against CD25 and CD54. There is statistically significant increase in the mean CD25 expression in patients with aGVHD [21.17 +/- 11.48] than patients with autologous PBSCT [5.01 +/- 2.62] or control group [1.4 +/- 0.51]. There was non significant difference [p > 0.05] in the CD 25 expression in patients with aGVHD grade I [15.11 +/- 13.9] and patients with grade 2-4 [mean 23.3 +/- 5.49]. There was significant increase [p < 0.01] in the number of patients with positive CD 25 expression with aGVHD grade 2-4 [3/3, 100%] than grade 1 [0%]. The percentage of CD54 was statistically significant higher [p < 0.05] in patients with allogeneic PBSCT [11.29 +/- 9.08] than in control group [1.34 +/- 0.47] and it was non significant with patients with autologous PBSCT [4.34 +/- 1.57]. There was non significant difference in the CD 54 expression in patients with aGVHD grade 1 [9.1 +/- 6.8] and patients with grade 2-4 [mean 16.3 +/- 12.2] [p > 0.05]. One patient 1/3 [33.3%] with positive expression of CD54 has aGVHD grade 1 and 2/3 [66.7%] have aGVHD grade 2-4 and this difference was statistically non significant [p < 0.2]. The increased level of CD25 in patients with allogeneic PBSCT is a useful marker for anticipating aGVHD and grade 2-4. CD54 is not a useful indicator for aGVHD or its grade

Adhesion molecules expression on B-cell chronic lymphocytic leukemia cells

Background and Abnormalities in the expression of cell adhesion molecules [CAM] are thought to influence the patterns of intranodal growth and hematogenous spread of malignant cells in Chronic Lymphocytic Leukemia [CLL]. Therefore, the characterization of some CAM phenotypic of the neoplastic clones in CLL may help to identify their role in staging and prognostic implications. In this work the expression of cell adhesion molecules of the Ig superfamily [ICAM- 1, CD54] and of selectin family [L-selectin-CD62L] of circulating malignant cells in patients with B-CLL were studied and whether the staging and tumor burden could be explained by their expression. Patients and Peripheral blood lymphocytes were obtained from 32 patients with B-cell chronic lymphocytic leukemia [B-CLL] at different stages, and from 13 healthy normal control. The expression of CAM was analyzed by fiowcytometry using mnonoclonal antibodies against CD54 and CD62L. There is significant differences in the mean expression of CD54 and CD62L [p < 0.001 and < 0.001] between CLL patients [13.25 +/- 8.72 and 24.6 +/- 10.6] and control group [3.66 +/- 1.27 and 1.69 +/- 0.24] respectively. Patients with positive ICAM-l 10/32 [31.2%] and L-Selectin 15/32 [46.9%] had more stage IV [7/10 and 10/15] than stage 11 [1/10-1/15] and stage III [2/10-4/15] respectively. There was non significant differences in the expression of CD54 and CD62L between patients who achieved in comparison to who failed remission. There was positive correlation between ICAM-1 and L-selectin expression and CD5, CDI9 and Rai stage [tau-b 0.359, 0.406, 0.254, p = 0.005, 0.001, 0.04] for ICAM-1 [tau-b 0.389, 0.254, 0.309, p = 0.002, 0.04, 0.001] for L-selection and there was no correlation regarding response to treatment. Patients with positive ICAM-1 and L-selectin expression had more advanced stage than patients with negative expression. There is positive correlation between ICAM-1 and L-selectin and stage of the disease and tumor burden and no correlation with response to treatment

Can mobilized unprocessed whole blood replace leukapheresis product for autologous peripheral blood stem cell transplantation in patients with lymphoma? implementation of pre-apheresis CD34 analysis

High dose chemotherapy [HDC] with peripheral blood stem cell transplantation [PBSCT] is increasingly used therapeutic option for patients with hematological malignancies. This requires leukaphresis and cryopreservation of at least 1-2 x 10 [6]/kg CD34+ cells. As the leukapheresis procedures is expensive, time-consuming, and involve several technical and psychological problems for the patients. The aim of this study was to develop a simplified, safe, and cost effective peripheral blood stem protocol for collection of at least 1-2 x 10 [6]/kg CD34+ stem cells with liquid preservation at 4°C which may be suitable for patients refusing leukapheresis or in areas lacking cryopreservation facilities. Patients and Twelve patients with relapsed lymphomas underwent stem cell mobilization with rhG-CSF subcutaneous 10 ug/kg/day for 5 days. At the day of stem cell harvest white blood cells [WBCs], mononuclear cells [MNCs] and CD34+ cell count were estimated from peripheral blood. According to absolute count of CD34+ cells in the peripheral blood, CD34+ cells in 1000 ml blood and/patient kg were calculated. Patients with CD34+ cells < 1 x 10 [6]/kg, leukapheresis was performed, while extraction of 1000 ml mobilized whole blood by 2 phlebotomies was performed for patients with calculated CD34+ cells > 1 x 10 [6]/kg. Six patients were candidate for whole blood collection by phlebotomy with liquid preservation while leukapheresis and cryopreservation were necessary to reach the target stem cell dose for another 6 patients. Hematological recovery after high dose chemotherapy was compared between both groups. Pre-apheresis MNCs, and CD34+ cells count were higher in six patients candidate for leukapheresis than patients candidate for unprocessed mobilized whole blood collection [27.7 +/- 3.4 x 10 [9]/L, and 26.6 +/- 6.9 x 10 [6]/L] versus [39.5 +/- 5.7 x 10[9]/L, 89.8 +/- 21.9 x 10 [6]/L] respectively and this difference was statistically significant [p < 0.01 and 0.001] respectively. The apheresis product and collected mobilized unprocessed whole blood both exceed the minimum target cell dose for auto transplantation. The patients with leukapheresis received product contained more MNCs and CD34+ cells [2.9 +/- 0.3 x 10 [8]/kg and 2.15 +/- 0.5 x 10 [6]/kg] higher than patients received collected mobilized whole blood [2.6 +/- 0.4 x 10 [8]/kg and 1.28 +/- 0.3 x 10 [6]/kg] respectively and this difference was statistically significant for CD34 [p < 0.01]. There was non significant difference between patients who received apheresis products for neutrophil and platelets recovery [p > 0.07 and 0.057], where the days to reach ANC >/= 0.5 x10 [9]/L was 10.3 +/- 1.5 [8-12] days and for platelet >/= 20 x10 [9]/L was 11.3 +/- 3.01 [7-15] while patients received whole blood the ANC was 12.3 +/- 1.9 [9-16] and for platelets was 16.0 +/- 4.3 [8- 20] days but this was statistically non significant. In some patients with CD34+ cell count more than 65/uL measured in peripheral blood at the day of stem cell collection and if the total CD34+ cell/kg count in one liter blood exceed target stem cell dose of 1 x 10 [6]/kg, mobilized unprocessed whole blood collected by phlebotomy with liquid preservation can replace leukaphresis product for stem cell autotransplantation. The mobilized unprocessed whole blood with liquid preservation was able to reconstitute bone marrow after HDC like cryopreserved mobilized PBSC with lower cost and effort