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The objective of this study was to develop a diagnostic testing method to detect HbS, distinguish sickle cell homozygotes from heterozygotes, and overcome testing barriers encountered in laboratories in underdeveloped countries. Blood samples positive and negative for sickle cell were subjected to the standard hemoglobin solubility test followed by a variety of centrifugation and filtration procedures. Each procedure was evaluated for the ability to remove insoluble HbS from the sample. The hemoglobin types that remain (HbA, HbA2 and HbF) were measured spectrophotometrically or estimated visually allowing samples to be categorized into three genotypes (AA, AS and SS) as confirmed by hemoglobin electrophoresis. De-identified EDTA blood samples were obtained from Saint Louis University and Cardinal Glennon Children's hospitals and tested in the Department of Clinical Laboratory Science at Saint Louis University. The main outcome measures were turbidity of the solubility solution; color of the supernatant and the material on the surface of the solution following centrifugation; precipitate trapped on the filter paper; absorbance of the filtrate; and hemoglobin electrophoresis patterns. Centrifugation and filtration successfully separated HbS from HbA/A2/F allowing for the differentiation of seven sickle cell homozygotes from sixteen heterozygotes with a sensitivity and specificity of 100%. This method has the potential to reliably distinguish homozygous from heterozygous sickle cell patients and it is fast, inexpensive, and simple. These characteristics make Sickle Confirm a desirable method in developing countries like Haiti and Africa where sickle cell anemia is prevalent and modern diagnostic methods like electrophoresis, HPLC and nucleic acid testing are impractical.

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The objective of this study was to develop a simple, cost-effective method of HbF determination potentially useable in underdeveloped countries to determine sickle cell patient response to hydroxyurea treatment. Normal adult blood (HbA), cord blood (HbF), and a 50:50 mixture (HbA+F) were the three sample types used in procedure development. Normal blood samples were collected from the research team, and de-identified cord blood samples were provided by Cardinal Glennon Pediatric Research Institute, St. Louis, MO. The hematocrit of all blood samples was standardized to 35%. The method, based on the Kleihauer-Betke (K-B) test principle, used a citrate solution to selectively elute HbA from RBCs while HbF remained intracellular, and spectrophotometric absorbance of the eluate was the primary outcome measure. A procedure was developed and optimized utilizing a 395 nm wavelength, 30 sec centrifugation time, 6 min incubation time, 20 microL blood volume, and 0.07 M sodium citrate in a 0.06 M sodium phosphate buffer solution. Reproducibility was demonstrated (N = 39) with a mean HbA absorbance of 1.285 (SD 0.069), mean HbA+F absorbance of 0.690 (SD 0.050), and mean HbF absorbance of 0.035 (SD 0.005), also exhibiting linearity (r2 = 0.99). This simple, cost-effective method of HbF determination shows potential as a basis for determining sickle cell patient response to hydroxyurea treatment in underdeveloped countries.

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OBJECTIVE: The purpose of this study is to estimate the prevalence of sickle hemoglobin in northern Haiti. DESIGN: Sickle cell testing occurred from 2002-2009. Blood samples from 1035 subjects were collected for diagnostic purposes, de-identified, and made available for the study. SETTING: Bethesda Medical Center and Eben-Ezer Clinic in northern Haiti. SUBJECTS: Study subjects included prenatal patients, their companions, clinic staff and volunteers. All subjects were Haitian and selected to most closely represent healthy patients present at the clinic. Deidentification of the blood samples precluded the need for informed consent. MAIN OUTCOME MEASURES: Each subject was tested for sickle hemoglobin using a standard hemoglobin solubility test and results were recorded as either positive or negative. RESULTS: The estimated prevalence of sickle hemoglobin was 15.1% with a 95% confidence interval of 12.2-18%. CONCLUSIONS: These prevalence rates validate the clinical significance of sickle cell disorder, help guide clinical decisions, and suggest the need to develop intervention programs among the people of northern Haiti.

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OBJECTIVE: To develop a micro-erythrocyte sedimentation rate (ESR) system with potential to be self-administered by patients at home using capillary blood. DESIGN: For each subject, three tubes of blood were collected in ethylenediaminetetraacetic acid (EDTA), centrifuged, and the cells separated from the plasma. Plasma was pooled and divided into three aliquots, two of which were spiked with defined amounts of fibrinogen creating a normal ESR and two distinct degrees of ESR acceleration. Fifteen hundred microliters of pooled autologous cells were resuspended in 1500 microL of the three prepared autologous plasmas to standardize hematocrit values. ESRs were performed using the Westergren method and four potential micro-ESR systems, utilizing a micro-hematocrit tube, S/P capillary blood gas tube, Natelson blood collecting tube, and Caraway micro blood collecting tube. SETTING: Saint Louis University, St. Louis MO. PATIENTS/SAMPLES: Twenty-eight volunteers between the ages of 18 and 60 years with no underlying conditions participated in the study. INTERVENTIONS: Hematocrit was standardized to approximately 40% for all samples and fibrinogen concentrations of approximately 200 mg/dL, 382 mg/dL, and 563 mg/dL were achieved for each subject. MAIN OUTCOME MEASURES: ESRs were measured in mm/hour and in percentage. Micro-ESR values were plotted versus the paired Westergren ESR value and the data were analyzed using Pearson's r correlation. RESULTS: When compared to the Westergren ESR method, the following correlation coefficients were achieved: S/P tube (r = 0.808), Caraway tube (r = 0.797), Natelson tube (r = 0.719), and micro-hematocrit tube (r = 0.655). CONCLUSION: Three of the four micro-ESR methods achieved correlation coefficients acceptable to the authors, with the potential of being converted into a capillary ESR system for in-home use.

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DATA SYNTHESIS: Chronic myelocytic leukemia (CML) was initially described in 1845 and is considered one of the first leukemias to be discovered. Diagnosis of CML was dramatically improved with the discovery of the Philadelphia chromosome by Nowell and Hungerford in 1960. However, the rudiments of our understanding of the molecular cause of CML began in 1973 when Janet Rowley discovered that the Philadelphia chromosome is a reciprocal translocation between chromosomes 9 and 22. The leukemogenic mechanisms of CML were hypothesized 20 years later when it was discovered that the t(9;22) translocation produced a fusion gene involving the BCR gene from chromosome 22 and the ABL protooncogene from chromosome 9 [corrected] Multiple breakpoints in BCR produce fusion genes that are translated into chimeric protein products of different lengths that are associated with different leukemic subtypes. CONCLUSION: Although CML has a rich history of interest to hematologists, it also represents a leukemogenic paradigm to the molecular biologist. Nearly all malignancies result from a series of mutagenic events, which culminate in full malignant transformation. However, it appears that CML results from a single mutagenic event involving the t(9;22) translocation leading to the development of the BCR/ABL fusion gene and the corresponding fusion protein. The successful transcription and translation of the BCR/ABL fusion protein led researchers to carefully study its involvement in leukemogenesis. The BCR/ABL fusion protein exhibits increased and constitutive tyrosine kinase activity that differs depending on which BCR breakpoint is expressed, resulting in varying clinical presentations.

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DATA SYNTHESIS: Chronic myelocytic leukemia (CML) was initially described in 1845 and is considered one of the first leukemias discovered. Effective approaches to therapy were not instituted until arsenic was first administered in 1865. Since then, four major therapeutic milestones have been achieved; the development of alkylating agents like busulphan and 6-thioguanine in 1953, alpha interferon in 1983, bone marrow transplantation in 1986, and tyrosine kinase inhibitors in 1998. The discovery that the protein product of this fusion gene expresses constitutive tyrosine kinase activity prompted the synthesis of a designer drug, imatinib mesylate, which binds the fusion protein and neutralizes the tyrosine kinase activity. Molecular methods of detecting BCR-ABL transcripts are showing promise in confirming drug resistance and predicting patient outcomes in response to imatinib mesylate therapy. Evidence of drug resistance can guide physicians in selecting alternative therapeutic approaches early in the course of the disease to potentially rescue non responders. Although the success of clinical trials has been dramatic, drug resistance and disease relapse are issues to be considered. CONCLUSION: The discovery that the BCR/ABL fusion protein exhibits increased and constitutive tyrosine kinase activity led investigators to develop an inhibitor to this activity. The synthesis of imatinib mesylate, currently marketed as Gleevec(TM) or Glivec(R), is in stage III clinical trials and has proven to be the most successful antileukemic drug to date. As in CML, an understanding of the leukemogenic mechanisms involved in other leukemias will provide the groundwork for the development of therapeutic interventions tailored to the specific molecular defects identified, eventually rendering obsolete the shotgun approaches to massive cell killing produced by chemotherapy.

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DATA SOURCES: Current literature. DATA SYNTHESIS: Acute lymphoblastic leukemia (ALL) is a stem cell disorder characterized by an overproduction of lymphoblasts in the bone marrow that eventually spill into circulation, producing lymphocytosis. As with the other acute leukemias, the most common symptoms experienced by patients include fatigue, bleeding, and recurrent infections resulting from the suppression of normal hematopoiesis in the bone marrow by the accumulating blasts. ALL primarily affects children and exhibits the best response to standard chemotherapy as compared to acute myeloblastic leukemias (AML). Further, remission rates are highest among ALL patients, many of whom are experiencing sustained remissions suggesting cure. In light of early treatment successes, researchers began to investigate modifications of standard treatment regimens to accommodate variability in weight, age, and response to therapy among children with ALL. Individualized treatment plans were implemented where some patients received a reduced intensity course of therapy to minimize drug toxicity while others received drug intensification to maximize response. More recently, research efforts have been directed at the elucidation of leukemogenic mechanisms implicated in ALL to identify specific protein mutants that can be used to design drugs tailored to interfere with the activity of these mutant protein targets. Identification of chimeric proteins produced from chromosomal translocations and gene expression profiles from microarray analyses are the primary techniques used to identify the potential therapeutic targets. CONCLUSION: Several reliable prognostic indicators have been identified and are being used to improve therapeutic planning and outcome prediction in ALL patients. Individualized treatment regimens have been developed based on the specific characteristics of each patient to minimize treatment related adverse events and maximize response. Through the use of cytogenetic, molecular, and microarray testing, ALL classification schemes have improved and potential therapeutic targets have been identified. It is anticipated that the next major advance in the treatment of ALL will involve the use of designer therapies developed to specifically interfere with particular molecular abnormalities producing the leukemogenic aberration to the normal signal transduction pathways.

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OBJECTIVE: Determine the minimum concentration of plasma fibrinogen needed to stimulate the aggregation of platelets, collected from normal subjects, using ADP. DESIGN: Platelet rich plasmas (300 x 10(9) platelets/L) were made and adjusted to final fibrinogen concentrations of 75, 19, 5, and 0 mg/dL using fibrinogen free serum. Each fibrinogen concentration in all twelve subjects was aggregated with ADP SETTING: Research laboratory in the Department of Clinical Laboratory Science at Saint Louis University. PARTICIPANTS: Twelve healthy volunteers of both genders, between the ages of 18 and 60 years who were not pregnant and weighed at least 110 pounds were included in the study. Subjects were excluded from the study if they had ingested aspirin within one week prior to blood collection. In addition, subjects with a history of bleeding disorders such as afibrinogenemia, hypofibrinogenemia, von Willebrand disease, and Bernand-Soulier disease were rejected from the study. MAIN OUTCOME MEASURES: Platelet aggregation tracings were analyzed for amplitude and compared across plasma fibrinogen concentrations. In addition, the type of curve (monophasic vs. biphasic), smoothness and aggregation stability were also noted. RESULTS: The results show that aggregation occurred with every dilution of fibrinogen tested and that the amplitude of the aggregation curves appears not to be dependent on plasma fibrinogen. CONCLUSIONS: The results indicate that platelets from healthy individuals previously exposed to normal fibrinogen levels will aggregate equally well in decreasing plasma fibrinogen concentrations and even in the absence of plasma fibrinogen using ADP as the aggregator.

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