Robert V. Pierre

Age-associated aneuploidy: loss of Y chromosome from human bone marrow cells with aging.

Direct bone marrow chromosome studies were carried out on 165 males, 3 months to 94 years of age. Forty‐one males were found to have 45,X cell lines in their bone marrow. The number of males exhibiting Y chromosome loss correlated with age, but the degree of Y chromosome loss did not correlate with age. The loss of Y chromosome appears to be a permanent event. The longest follow‐up period in this study was 13 months. The incidence of 45,X cell lines in the bone marrow is much greater than that in peripheral blood leukocyte cultures. The presence of 45,X cell lines in the bone marrow or peripheral blood of aging males should be recognized as a common event, and the finding should not be considered evidence for the presence of a specific disease state.

Cytogenetic Studies in 174 Consecutive Patients With Preleukemic or Myelodysplastic Syndromes

Routine cytogenetic studies were done in 174 consecutive patients with preleukemic or myelodysplastic syndromes (PL/MDS): 5 had the 5q - syndrome, 2 had refractory cytopenia, 43 had refractory anemia, 38 had refractory anemia with ringed sideroblasts, 69 had refractory anemia with excess blasts, 6 had refractory anemia with excess blasts in transition, and 11 had chronic myelomonocytic leukemia. Successful chromosome studies were accomplished in 167 patients (96%); 64 (37%) had a chromosomally abnormal clone. Abnormal clones were most common among patients who had refractory anemia with excess blasts (45%), refractory anemia with excess blasts in transition (60%), and chronic myelomonocytic leukemia (45%); they were least common among patients with refractory anemia (32%) and refractory anemia with ringed sideroblasts (21%). The two patients with refractory cytopenia had normal cytogenetic results. Each patient with the 5q - syndrome had a 5q-chromosome, as this is a prerequisite for the diagnosis. The two most common structural abnormalities were deletion of part of a chromosome 5 long arm (17 patients) and deletion of part of a chromosome 20 long arm (8 patients). Nonspecific structural abnormalities of chromosomes 1, 3, 6, and 17 were also common. The most common numeric abnormalities were monosomy 5 (7 patients), monosomy 7 (4 patients), loss of the Y chromosome (9 patients), and trisomy 8 (20 patients). No chromosome abnormalities were specifically associated with any PL/MDS classification.

Pseudothrombocytopenia: a laboratory artifact with potentially serious consequences

When a low platelet count is unexpected or is unaccompanied by signs or symptoms of hemorrhage, pseudothrombocytopenia should be suspected. The platelet number and morphologic features should be examined on a smear prepared from blood anticoagulated with EDTA. Platelet counts should be repeated either by obtaining a finger-stock specimen with an ammonium oxalate Unopette and counting by phase microscopy or by collecting both an EDTA- and a sodium citrate-anticoagulated venous sample, performing a platelet count on both test tubes, and examining a blood smear from each test tube. If a Coulter Model S Plus IV or V or the Technicon H6000 is used for performance of platelet counts, examination of the histogram display of the Coulter instrument or the peroxidase X-Y display of the Technicon H6000 should alert the instrument operator to the presence of EDTA-induced platelet clumping and prevent the reporting of a spuriously low platelet count.

Hematologic manifestations associated with deletions of the long arm of chromosome 20

We investigated 20 patients with hematologic disorders who had a clone of cells with a deletion of most of a chromosome #20 long arm (20q-) in the bone marrow. Three patients had polycythemia vera (PV), 6 had acute nonlymphocytic leukemia (ANLL), 8 had preleukemia (PL), and 3 had other myeloproliferative disorders. In our laboratory, a 20q- chromosome is found in 6% of patients with PV, 3% of patients with ANLL, and 1% of patients with PL. Among the 6 patients with ANLL and a 20q- abnormality, 3 had erythroleukemia. There were no apparent clinical differences among our patients with 20q- chromosomes compared with other patients with similar disorders who did not have a 20q- chromosome. The breakpoint of the 20q- anomaly, in each instance, was in band 20q11, but it occurred near the centromere at 20q1101 in 16 patients and at the distal part of this band at 20q1109 in 4 patients. Three of the 4 patients with a breakpoint at 20q1109 had PL.

Incremental Value of the Leukocyte Differential and the Rapid Creatine Kinase-MB Isoenzyme for the Early Diagnosis of Myocardial Infarction

Acute electrocardiographic ST-segment elevation is highly predictive of myocardial infarction in patients who present with prolonged chest pain [1, 2].However, although most patients with infarction do not have ST elevation [2, 3], they do have a substantial in-hospital mortality [4]. These patients cannot currently be distinguished in the emergency department from patients with unstable angina and other acute chest-pain syndromes [5]. As a result, patients with infarction but no ST elevation and patients with unstable angina have been grouped together in clinical trials [6]. The identification of patients who will have an infarction but who do not have ST-segment elevation may permit more appropriate triage and therapy for these patients. Markers reported for the early identification of acute myocardial infarction include initial clinical variables [7], rapid creatine kinase-MB measurements [8], creatine kinase-MB isoforms [9], myoglobin [10], troponin T [11], and cortisol [12]. The value of the total leukocyte count for the diagnosis of myocardial infarction has long been recognized [13]. Elevation of the total leukocyte count is probably caused by the increase in cortisol levels during an acute stress reaction [14]. The potential value of the leukocyte differential has probably been obscured by the great variation in the traditional manual leukocyte differential, which has a coefficient of variation of 20% to 30% [15]. Widely available, fast, and accurate automated hematology analyzers have greatly decreased the analytical variation for leukocyte differentials [16]. Although the leukocyte differential is influenced by corticosteroid use and other stressful illnesses, an initial study of patients presenting with chest pain to the emergency department suggested that many of these other causes could be identified by simple clinical assessment. In our initial study, we also found that myocardial infarction, but not unstable angina, often altered the leukocyte differential by elevating the neutrophil count and decreasing the lymphocyte count. These changes thereby caused relative lymphocytopenia and granulocytosis, frequently without an elevation of the total leukocyte count (unpublished observations). Because the automated leukocyte differential is widely available, fast, and inexpensive, it offers an attractive combination of incremental value for the diagnosis of infarction with little incremental cost. We therefore conducted a prospective, blinded study to assess the potential incremental value of the leukocyte differential as an early diagnostic tool. We examined whether relative lymphocytopenia, abnormal total creatine kinase levels, or abnormal rapid creatine kinase-MB measurements were useful for the early diagnosis of acute myocardial infarction in patients presenting to the emergency department with chest pain. Methods Patients The study group of 384 patients was selected from 511 consecutive patients aged 20 years or older who presented to the emergency department with anterior or left lateral chest pain between 8 March and 31 August 1992 and from whom initial blood samples were obtained (Figure 1). Eleven patients who presented twice and three patients who presented three times during this period were included separately for each visit. Exclusion criteria were the following: infection in the previous week detected by history or initial clinical evaluation (34 patients); exogenous glucocorticoid use within the previous month (25 patients); documentation of malignancy in the previous 5 years (20 patients); transfer from other hospitals after diagnosis (17 patients); major trauma, major gastrointestinal bleeding, surgery, dialysis, or resuscitation in the previous week (8 patients); and insufficient data to exclude myocardial infarction (17 of 20 patients dismissed from the emergency department who did not return after 24 hours for blood tests as part of the protocol and 6 patients who were admitted but had only one determination of the creatine kinase level because of low clinical suspicion). Of the 127 patients who were excluded, 4 patients had evidence of infarction (see below). Figure 1. Flow diagram showing patient selection and the prevalence of ST-segment elevation and positive serum markers (defined as both a relative lymphocytopenia and an elevated rapid creatine kinase-MB level) in patients with and patients without myocardial infarction. The study protocol was approved by the Mayo Clinic institutional review board. In the protocol, the leukocyte differential was identified only as test X. Only five of the investigators and one representative of the institutional review board knew the identity of test X. Patients who were not admitted were asked to return the next day as part of the protocol for the measurement of creatine kinase levels and leukocyte counts, but only 3 of 20 such patients returned. To determine the value of blood samples and electrocardiograms for the early diagnosis of acute myocardial infarction, we used only the initial measurements. Definition of Myocardial Infarction A final diagnosis of myocardial infarction was made if one of the following two criteria was met: 1) standard creatine kinase-MB isoenzyme level greater than 9.6 ng/mL within 48 hours [73 patients] or 2) sudden unexplained death within 72 hours of presentation (1 patient). The absence of myocardial infarction was defined by the absence of these criteria. In five patients, the final clinical (discharge) diagnosis differed from that defined above. Clinical records for these patients were reviewed by two experienced cardiologists blinded to the results of test X. One patient had a previously defined myopathy. In the other four patients, creatine kinase-MB levels were mildly elevated, and no further evidence of infarction was observed. These patients were considered by the panel to not have infarction and were categorized as such for the analysis. Initial electrocardiograms were reviewed independently by one cardiologist (who was blinded to the patients history, clinical diagnosis, and blood test results) for the presence of diagnostic ST-segment elevation, defined as an elevation of more than 0.1 mV in two electrically contiguous leads. Electrocardiograms showing ST-segment elevation that was not seen on previous electrocardiograms from our institution or ST elevation in patients for whom previous electrocardiograms were not available were considered positive. Electrocardiograms showing no ST-segment elevation or an elevation that was unchanged from previous electrocardiograms were considered negative. Laboratory Measurements Peripheral blood leukocyte counts were estimated with a widely available automated hematology analyzer, Coulter STK-R (Hialeah, Florida) [16]. This instrument uses approximately 10 000 cells per differential to produce, within minutes, a precise three-part leukocyte differential consisting of granulocytes, lymphocytes, and monocytes. The normal ranges for leukocyte concentrations were defined as the central 95th percentile for 150 healthy adults and have been used since 1984 [16]: 45.8% to 73.7% for granulocytes and 20.3% to 46.7% for lymphocytes. The total creatine kinase activity was determined on the Kodak Ektachem E700XR (Eastman Kodak Company, Rochester, New York) [17]. The upper limits of normal were determined to be 176 U/L for women and 336 U/L for men on the basis of values from 160 healthy adults. Creatine kinase-MB levels were measured on the Magic Lite system (CIBA Corning Diagnostics, Medfield, Massachusetts) using a two-site chemiluminescent immunoassay method; the cut-off value for the detection of myocardial infarction was 9.7 ng/mL [18]. After this initial clinical testing, serum specimens were frozen at 70C until they were thawed for measurements of cortisol and rapid creatine kinase-MB levels. Rapid creatine kinaseMB measurements were done on the Stratus II system (Baxter Diagnostics, Inc., Miami, Florida) using a two-site fluorometric immunoassay [19]. The upper limit of normal was chosen to be 4.7 ng/mL, which is three standard deviations greater than the mean value for 73 patients with cardiac disorders but no current myocardial infarction [20]. Cortisol levels were tested by RIA (Diagnostics Products Corporation, Los Angeles, California). The upper limit of normal was 690 nmol/L (25 g/dL) at 0800 h [20]. The amount of the sample was insufficient for the determination of the total creatine kinase level in one patient without an infarction, for the determination of rapid creatine kinaseMB levels in 9 patients (1 with infarction and 8 without infarction), and for the measurement of cortisol levels in 8 patients without infarction. Laboratory variables were measured without knowledge of clinical or other laboratory results. Statistical Analysis We used the Wilcoxon rank-sum test to assess whether patients with and patients without infarction differed significantly for individual variables. We determined the sensitivity and specificity for individual variables using the abnormal thresholds of greater than 10.4 109/L (in women) or 10.9 109/L (in men) for total leukocyte counts and of greater than 689.75 nmol/L (25 g/dL) for serum cortisol levels, as well as the cut-off values described above for total creatine kinase levels, rapid creatine kinase-MB levels, granulocytosis, and lymphocytopenia. Because exogenous estrogen replacement therapy increases cortisol levels, cortisol values from 29 women (4 with infarctions) who were receiving estrogens were excluded from the analysis of cortisol results. Sex, age, abnormal total creatine kinase levels, abnormal rapid creatine kinase-MB levels, and relative lymphocytopenia were entered into a multivariate logistic regression analysis to predict the presence of infarction in the 345 patients without electrocardiographic ST-segment elevation and for whom complete blood test data were available (10 patients with incomplete data were

LYMPHOSARCOMA CELL LEUKEMIA.

Abstract The clinical and hematologic manifestations of twenty-three patients with lymphosarcoma (LSA) cell leukemia are reviewed. Eleven of these patients had the acute or subacute form of the disease; twelve had chronic lymphosarcoma cell leukemia. The morphologic hallmark of lymphosarcoma cell leukemia is an atypical, large lymphocyte with coarsely reticulated nuclear chromatin and a single prominent nucleolus. The maturity of these cells correlates with the chronicity or acuteness of the leukemic process. The degree of bone marrow involvement appears to constitute the chief difference between lymphosarcoma presenting as lymphoma and lymphosarcoma cell leukemia. Superimposed hemolysis, intercurrent infections and hemorrhagic phenomena were frequent complications. Prognosis could be related to cell maturity. Acute lymphosarcoma cell leukemia has a prognosis comparable to that of acute lymphocytic leukemia; chronic lymphosarcoma cell leukemia has a prognosis which appears to be intermediate between that of acute and chronic lymphocytic leukemia. Differences in clinical manifestations, course and response to therapy warrant the separation of acute and chronic lymphosarcoma cell leukemia from acute and chronic lymphocytic leukemia.

The efficacy of direct, 24-hour culture, and mitotic synchronization methods for cytogenetic analysis of bone marrow in neoplastic hematologic disorders

Bone marrow aspirates from 90 patients suspected of having a hematologic disorder were processed by using different cytogenetic methods to determine if any procedure was more likely to reveal a chromosomally abnormal clone or produce better-quality metaphases. All specimens were processed by a direct technique and 24-hr culture without mitogens; 50 specimens were also processed by an amethopterin mitotic synchronization method. In each case, the microscope slides were coded by the processing technologist and analyzed by two other experienced cytogenetic technologists. The results were not known to any of the investigators until all 90 specimens were analyzed. With the exception of one specimen, in which a chromosomally abnormal clone was identified only in the direct preparation, no apparent differences were found in the karyotypes among the three methods. Also, the differences in the quality or number of metaphases found among the three methods were not statistically significant; however, 24-hr unstimulated cultures produced more metaphases than the mitotic synchronization procedure. The greatest source of discordance was caused by one test yielding either no metaphases or an uncertain result when the other tests produced a successful study. We suggest that in routine practice at least two different methods should be used, and it may be best if at least one of these methods is a direct technique.

A possible specific chromosome marker for monocytic leukemia: three more patients with t(9;11)(p22;q24) and another with t(11;17)(q24;q21), each with acute monoblastic leukemia.

An apparently balanced 9;11 reciprocal translocation with break points most likely at 9p22 and 11q24 was found in 3 patients with acute monocytic leukemia [M5 in the French-American-British (FAB) classification schema]. This translocation was not observed in 6 other patients with M5 acute nonlymphocytic leukemia (ANLL) or in chromosome studies on 143 patients with other types of ANLL. This study supports the previously published suggestion that such 9;11 translocations may be associated with some patients with M5 ANLL. In this report, we have also included a patient with M5 ANLL who had an 11;17 translocation with break points apparently at 11q24 and 17q21. Perhaps this is a variant translocation of chromosome No. 11, which may also be associated with monocytic leukemia.

Microchromosomes in human preleukemia and leukemia

Microchromosomes have been observed in direct bone‐marrow chromosome preparations in 9 patients. Seven of these patients had overt myelomonocytic leukemia, one had a preleukemic syndrome initially and subsequently had acute leukemia, and one is believed to be in the preleukemic phase of myelomonocytic leukemia. It is suggested that the microchromosomes in these cases are marker chromosomes which identify a clone or clones of leukemic cells. Microchromosomes appear to be frequent findings in myelomonocytic leukemia.

Deletions of chromosome 13 in malignant hematologic disorders

Thirteen patients with a hematologic disorder and an interstitial deletion of part of a chromosome #13 were evaluated to determine if any specific clinical manifestations are associated with these cytogenetic anomalies. Our results suggest that these anomalies occur in approximately 1.7% of patients with a chromosomally abnormal clone and a hematologic disorder. They may occur as the sole chromosome anomaly (8 of our patients) or with other abnormalities (5 of our patients). The breakpoints are not always the same, but band 13q14 always seems to be lost. At the time of chromosome analysis, 5 patients had a history of myelofibrosis or agnogenic myeloid metaplasia, 2 had acute nonlymphocytic leukemia, 2 had a myelodysplastic syndrome, one had polycythemia vera, one had sideroblastic anemia, one had acute lymphocytic leukemia, and one had an undifferentiated myeloproliferative disorder.