Worldwide Network for Blood and Marrow Transplantation (WBMT) recommendations for establishing a hematopoietic stem cell transplantation program in countries with limited resources (Part II): Clinical, technical and socio-economic considerations.

The development of hematopoietic stem cell transplantation (HSCT) programs can face significant challenges in most developing countries because such endeavors must compete with other government health care priorities, including the delivery of basic services. While this is may be a limiting factor, these countries should prioritize development of the needed expertise to offer state of the art treatments including transplantation, by providing financial, technological, legal, ethical and other needed support. This would prove beneficial in providing successful programs customized to the needs of their population, and potentially provide long-term cost-savings by circumventing the need for their citizens to seek care abroad. Costs of establishing HSCT program and the costs of the HSCT procedure itself can be substantial barriers in developing countries. Additionally, socioeconomic factors intrinsic to specific countries can influence access to HSCT, patient eligibility for HSCT and timely utilization of HSCT center capabilities. This report describes recommendations from the Worldwide Network for Blood and Marrow Transplantation (WBMT) for establishing HSCT programs with a specific focus on developing countries, and identifies challenges and opportunities for providing this specialized procedure in the resource constrained setting.

A comparative study of the genetics of Behcet's disease in Iraq: international collaboration to transfer clinical and laboratory skills to Baghdad medical school and hospitals.

A collaborative project funded by the Iraqi Research Fellowship Programme of the Council for Assisting Refugee Academics was set up to transfer laboratory and clinical skills in between three clinical research teams in the UK, Jordan and Iraq. The project was set up to study the genetics of the potentially debilitating condition, Behçet's Disease (BD). Blood samples were collected from: 38 BD patients, 28 patients with oral ulcers not related to BD and 32 healthy controls. All samples were analysed using Micro SSP HLA Class I B locus kit (B locus, generic). Logistic regression analysis revealed that samples positive for HLA-B51 were 7.4 times more likely to have BD than the healthy control subjects. The results of this study make a valuable addition to the scientific literature. Additional valuable outcomes include the intellectual exchange and transfer of skills in between the collaborating teams, which led to the establishment of an international research collaboration.

ESHAP + fixed dose G-CSF as autologous peripheral blood stem cell mobilization regimen in patients with relapsed or refractory diffuse large cell and Hodgkin's lymphoma: a single institution result of 127 patients.

From 1996 to November 2004, 131 consecutive patients with relapsed or refractory diffuse large cell lymphoma (DLCL) and Hodgkin's lymphoma (HD) received ESHAP as mobilization chemotherapy before autologous peripheral blood stem cell transplant (ASCT). Patients received fixed dose G-CSF 300 microg SC bid starting 24-36 h after finishing mobilizing ESHAP. In all, four patients failed mobilization and are excluded. Characteristics of 127 patients: 68 males: 59 females. DLCL 49: HD 78. Initial stage I:II:III:IV:unknown was 15:34:33:42:3. Median age at ASCT 26 years. Median prior chemotherapy cycles were six [<6 (17 patients), 6-8 (90 patients), >8 (20 patients)]. Median ESHAP cycle used as mobilizer was third. Patients required 1, 2, 3, 4 apheresis were 93:25:8:1. Median total CD34+ cells/kg collected were 6.9 x 10(6) (DLCL 5.17 x 10(6) and HD 7.6 x 10(6)), patients weighing < or = 70 kg (93 patients) 6.54 x 10(6) and >70 kg (34 patients) 7.44 x 10(6) (P = 0.59), one apheresis (93 patients) 8.6 x 10(6)/kg and >1 apheresis (34 patients) 4.5 x 10(6) (P = 0.001). We conclude that ESHAP and G-CSF 300 microg SC bid is an effective mobilizing regimen even in patients >70 kg and most patients require only 1-2 apheresis.

Stem cell transplantation for patients with Fanconi anemia with low-dose cyclophosphamide and antithymocyte globulins without the use of radiation therapy.

In all, 22 patients with confirmed Fanconi anemia (FA) underwent stem cell transplantation (SCT) from HLA-matched, related donors at KFSHRC. Median age at SCT was 7.6 years (range, 2.5-14.6 years). Conditioning regimen consisted of cyclophosphamide (CY) 15 mg/kg/day intravenously (i.v.) for 4 consecutive days, in addition to equine antithymocyte globulins (ATG) given i.v. at 40 mg/kg/day for four doses pre-SCT. No radiation therapy was given. For graft-versus-host disease prophylaxis, we used cyclosporin at the standard doses; ATG was added at 20 mg/kg/dose i.v. on days 2, 4, 6, 8, 10, and 12 post-SCT (total of six doses). All patients engrafted and are alive and transfusion independent with a median follow-up time of 20.2 months (range, 3.3-59 months). One patient however developed a decrease in her WBC and platelet count. Her work-up revealed slightly hypocellular bone marrow, and a series of chimerism studies over 1 year confirmed that she has stable mixed chimerism; she remains transfusion independent. We conclude that low-dose CY without radiation therapy can be used satisfactorily in the conditioning of patients with FA undergoing related SCT.

Neutrophils and lymphoid chimerism after adult living-related liver transplantation from a homozygous donor.

Chimerism and graft-versus-host disease (GVHD) pose significant risks to liver transplant patients. The risk of chimerism and GVHD is higher among cases of living-related liver transplant (LRLT). Donors homozygous at all HLA loci carry a higher risk for GVHD. Herein we present a case of LRLT. The recipient suffered from end-stage liver disease and received a right lobe graft from his son. After 8 months posttransplant, the patient developed profound bone marrow depression. The patient was negative for CMV, Brucella, HHV6, HHV8, HBV, HCV, and parvovirus. No skin or GI signs of GVHD were noted. The patient and donor were HLA typed by SSP. The donor was homozygous for all HLA loci while the patient shared the class II homozygosity and was class I heterozygous. Chimerism studies were prompted after noting that the neutrophil compartment of the patient was homozygous for all HLA loci. This initiated further studies of the PMN and lymphocytes by microsatellite analysis. A total 15 microsatellites were analyzed. The results suggest that the majority (75%) of the PMNs and 45% of the lymphocytes were of donor origin. The patient was treated with G-CSF; his WBC counts returned to normal. At 2.5 years posttransplant the patient had not developed GVHD, despite the large number of donor lymphocytes circulating in his bloodstream. The only complaint he had was severe arthritis, which was treated with steroids. It must be investigated whether this was the result of GVHD.

Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM.

CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on a wide range of cell types including B cells, macrophages, and dendritic cells. CD40 is the receptor for CD40 ligand (CD40L), a molecule predominantly expressed by activated CD4(+) T cells. CD40/CD40L interaction induces the formation of memory B lymphocytes and promotes Ig isotype switching, as demonstrated in mice knocked-out for either CD40L or CD40 gene, and in patients with X-linked hyper IgM syndrome, a disease caused by CD40L/TNFSF5 gene mutations. In the present study, we have identified three patients with an autosomal recessive form of hyper IgM who fail to express CD40 on the cell surface. Sequence analysis of CD40 genomic DNA showed that one patient carried a homozygous silent mutation at the fifth base pair position of exon 5, involving an exonic splicing enhancer and leading to exon skipping and premature termination; the other two patients showed a homozygous point mutation in exon 3, resulting in a cysteine to arginine substitution. These findings show that mutations of the CD40 gene cause an autosomal recessive form of hyper IgM, which is immunologically and clinically undistinguishable from the X-linked form.

The effect of fixation on detection of B-cell clonality by polymerase chain reaction.

It has been suggested that neutral buffered formalin (NBF)-fixed, paraffin-embedded, or fresh specimens might provide satisfactory DNA templates for polymerase chain reaction (PCR) assays used in establishing the clonality and presumptive B-cell lineage of lymphoma. The suitability of other fixatives used by hematopathologists, such as B5, is still undetermined. Thirty cases were identified from the files of the Cleveland Clinic Foundation, Cleveland Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangement by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 cases), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were studied. With use of consensus primers against the framework 3 (FR3) and FR2 regions of the VH gene, paired against JH primer(s), PCR analysis was performed. bcl-2/IgH translocation was also studied. Ten reactive lymphoid samples were used as controls, and 40 cases were evaluated. Successful amplification of a clonal proliferation was manifested as one or two discrete narrow bands in the appropriate size range. The sensitivity of detecting clonality was 95, 94, 67, 80, and 0% for NBF, Hollande's fixative, B5, ZF, and Bouin's fixative, respectively. Although NBF and Hollande's fixative were 100% specific, consistent false-positive results were a major problem with B5-fixed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishing B-cell clonality. The precipitating fixative B5 and Bouin's solution should not be used for this purpose until the issue of false-positive results is resolved.

Comparative analysis of interphase FISH and RT-PCR to detect bcr-abl translocation in chronic myelogenous leukemia and related disorders.

The t(9;22)(q34;q1 1) between the abl and bcr genes plays a pivotal role in the diagnosis and pathogenesis of chronic myelogenous leukemia (CML). Its detection is routinely accomplished by Southern blot analysis and karyotyping. Interphase fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) are emerging molecular techniques that offer viable alternatives. We analyzed 40 samples of peripheral blood and bone marrow (CML, 16; acute myelogenous leukemia, 6; acute lymphoblastic leukemia [ALL], 1; chronic lymphoblastic leukemia, 2; myelodysplasias, 4; myeloproliferative disorders, unclassified, 3; nonleukemic hematologic malignancies, 3; hypercellular bone marrow, 1; normal control samples, 2; and K562 cell line samples, 2) for the presence of bcr-abl fusion gene and its messenger RNA (mRNA) transcript by FISH and RT-PCR, respectively. We compared the results with results of Southern blot analysis and karyotyping when available. Cost analysis was performed. Thirty-three samples were evaluable by FISH; 14 of 14 evaluable CML samples and one ALL sample were positive for bcr-abl by FISH (100%). The other 15 evaluable samples were negative; 16 of 16 (100%) and 13 of 16 (81%) of CML cases were positive for bcr-abl mRNA by RT-PCR (chemiluminescent blot method) and RT-PCR (colorimetric method), respectively. The ALL sample was positive by both RT-PCR methods. All other samples were negative by RT-PCR (chemiluminescent blot method), and all but 1 case of myeloproliferative disorder tested negative by RT-PCR (colorimetric method). We conclude the utility of FISH and RT-PCR is associated with certain limitations, such as insufficient RNA for RT-PCR and the occasional absence of internal positive FISH control signals. However, each procedure offers (with a high concordance rate) a specific and cost-effective alternative to Southern blot analysis and karyotyping and improved turnaround time for the detection of bcr-abl fusion gene or its mRNA transcript.

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.

The use of amplified variable number of tandem repeats (VNTR) in the detection of chimerism following bone marrow transplantation. A comparison with restriction fragment length polymorphism (RFLP) by Southern blotting.

Chimerism analysis after allogeneic bone marrow transplantation (alloBMT) allows detection of early marrow engraftment, disease relapse, and graft rejection. Our objective was to do retrospective and prospective studies of chimerism analysis by restriction fragment length polymorphism (RFLP) by Southern blotting and variable number of tandem repeats (VNTR) by polymerase chain reaction (PCR) to compare and contrast the methods. The retrospective group comprised 46 samples from 26 patients previously analyzed by RFLP, while the prospective group contained 34 samples from 25 patients. Using four different VNTR primers (D1S80, D17S30, D1S111, and APO-B), the recipient and donor samples amplified by the PCR were screened for unique banding patterns. The VNTR primer with the unique banding pattern was used to detect chimerism in each sample. A total of 635 VNTR analyses were performed. Interpretation was blinded for previous RFLP results. A comparison between the VNTR and RFLP results and a cost analysis of the two procedures were done. A unique VNTR banding pattern was present in 49 of 51 patients (identical twins in one case). The VNTR analysis showed complete chimerism in 68 samples, mixed chimerism in 9, and recurrences in 2. This agreed with the RFLP results in 64 (80%) of 80 samples. Failure to detect 1% to 10% of recipient DNA accounted for 15 (VNTR, 8; RFLP, 7) discordances. Follow-up revealed all donor DNA in five cases, decreasing quantities of recipient DNA in two cases (six samples), and no additional studies available in four cases. In one case, VNTR detected a complete chimerism when the DNA was insufficient for RFLP analysis. The cost analysis revealed an approximately 50% savings with the use of VNTR; VNTR is a viable alternative to RFLP in the detection of chimerism after bone marrow transplantation and offers substantial cost savings, faster turnaround time, easier preparation of the DNA, smaller DNA requirements, and the elimination of radioisotopes and cumbersome restriction enzymes.

The use of isotypic control antibodies in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets by flow cytometry. Are they really necessary?

OBJECTIVE: Isotypic control reagents are defined as irrelevant antibodies of the same immunoglobulin class as the relevant reagent antibody in a flow cytometry panel. The use of the isotypic control antibody has been advocated as a necessary quality control measure in analysis of flow cytometry. The purpose of this study was to determine the necessity of an isotypic control antibody in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets. MATERIALS AND METHODS: We performed a prospective study of 46 consecutive patient samples received for lymphocyte subset analysis to determine the need for the isotypic control. For each sample, a sham buffer (autocontrol) and isotypic control reagent were stained for three-color immunofluorescence, processed, and identically analyzed with Attractors software. The Attractors software allowed independent, multiparametric, simultaneous gating; was able to identically and reproducibly process each list mode file; and yielded population data in spreadsheet form. RESULTS: Statistical analysis (Fisher's z test) revealed no difference between the CD3+ autocontrol and CD3+ isotypic control (correlation = 1, P < .0001) or between the CD3+, CD4+ autocontrol and the CD3+, CD4+ isotypic control (correlation = 1, P < .0001). The elimination of the isotypic control reagent resulted in a total cost savings of $3.36 per test. Additionally, the subtraction of isotypic background can artifactually depress population enumeration. CONCLUSIONS: The use of an isotypic control antibody is not necessary to analyze flow cytometric data that result in discrete cell populations, such as CD3+ and CD3+, CD4+ lymphocyte subsets. The elimination of this unnecessary quality control measure results in substantial cost savings.

Quantitative comparison of immunohistochemical staining intensity in tissues fixed in formalin and Histochoice.

Formaldehyde fixatives have traditionally been used to preserve tissues as they impart excellent morphological preservation. Formaldehyde fixes tissue by cross linking, a process which can reduce the antigenicity of tissue and weakens the intensity of immunohistochemical stains. Preliminary studies have shown that Histochoice tissue fixative offers equal or greater staining intensity than neutral buffered formalin (NBF). This study compares these fixatives quantitatively and presents the results in unambiguous statistical terms. Tissue samples were collected, bisected, and fixed in NBF or Histochoice. The sections were stained with a total of 21 antibodies, and color images were collected. The hue, saturation, and value were determined for each positive pixel and an ANOVA performed. Small differences in hue were noted in 8 of 21 cases. Histochoice-fixed tissue gave a greater mean saturation than NBF with 57.1% of the antibodies tested. No significant difference in the saturation was detected in 28.6% of the cases; NBF gave higher mean saturation levels with only 14.3% of the antibodies. Histochoice-fixed tissue was found to give lower values in 66.7% of cases than those prepared with NBF, indicating darker staining. These results show that Histochoice produces staining intensity that is comparable, and in many cases superior, to formalin.

Flow cytometric immunophenotyping of non-Hodgkin's lymphomas and related disorders.

More than two decades have past since the recognition of non-Hodgkin's lymphomas (NHLs) as neoplasms of the immune system. During that time, a vast literature and knowledge base regarding the immunophenotypic and functional characteristics of neoplastic lymphocytes has been developed. Despite the accumulated wealth of knowledge, there remains no consensus as to the exact role of immunotypic and genotypic ancillary procedures in the evaluation of a lymph node biopsy. We review selected literature in this regard and provide an overview of the role of multicolor flow cytometry in establishing the diagnosis of specific NHL and related disorders.

De novo light-chain deposition disease in a cadaver renal allograft.

The deposition of immunoglobulin (Ig) light chains after renal transplantation most commonly occurs as a manifestation of recurrent multiple myeloma or recurrent light chain nephropathy. We report the development of de novo light chain deposition disease (LCDD) in a cadaveric renal transplant recipient 16 years after transplantation with no evidence of prior multiple myeloma or LCDD and no current evidence of myeloma or lymphoproliferative malignancy.

Histopathologic effects of laser radiation on the human prostate.

High levels of applied laser irradiation to the prostate will carbonize or vaporize tissue, and may cause explosive expansion of superheated tissue water. Lower levels, used most often to relieve obstruction caused by benign prostatic hypertrophy, will cause coagulation necrosis. This effect is apparent within 1 h of application. In contrast to the canine, in which laser-coagulated prostate sloughs in 2 to 3 weeks leaving a smooth cavity, in the human necrotic tissue is sloughed irregularly over a period ranging up to 12 weeks. This difference is attributed to the dominantly glandular nature of the canine prostate, and the dense fibromuscular composition of the human prostate stroma. Sloughing is accomplished by surface liquefaction, cavitation of the necrotic coagulum, and to a lesser degree, formation of granulation tissue at the margins. As often occurs at the margin of spontaneous infarcts in the prostate, squamous metaplasia may be prominent at the margins of laser-induced coagulation necrosis.

Recurring phyllodes tumor in aberrant breast tissue of the vulva.

A 20-year-old nulligravida woman presented with bilateral cystic nodules of the vulva, diagnosed after simple excision as benign phyllodes tumor. Breast tissue, which also displayed fibrocystic changes, was also recognized. Follow-up at 2 months revealed no evidence of disease; however, 8 months after surgery she returned with a new mass in the vulva. This was excised and found to be recurrent phyllodes tumor. Although somewhat more cellular than the first lesions, it also was deemed to be benign based on histological features supported by flow cytometric DNA studies.