Clinical Abstracts

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April 2004

Clinical pathology abstracts editors: Michael Bissell, MD, PdD, MPH, professor and director of clinical services and vice chair, Department of Pathology, Ohio State University Medical Center, Columbus, and Ronald Domen, MD, professor or pathology, medicine, and humanities, Penn State University College of Medicine, Hershey, Pa.

Clinical outcomes associated with intraoperative PTH assessment

The intraoperative quick parathyroid hormone assay is useful for determining when all hyperfunctioning parathyroid tissue has been removed during parathyroidectomy. Because intact PTH is rapidly cleared from the circulation and has a half-life of only a few minutes, intact PTH levels usually fall more than 50 percent after all hyperfunctioning parathyroid tissue has been removed. While this is well established for patients with solitary adenomas, the data for patients with multiple parathyroid tumors are limited. Recent reports have shown PTH levels decreasing by more than 50 percent in almost half of a series of patients with double adenomas after resecting only one abnormal gland despite another abnormal parathyroid gland remaining in the neck. The authors assessed the accuracy of preoperative technetium 99m sestamibi scintigraphy (MIBI) and ultrasonography examinations and whether the quick PTH assay improves the results of parathyroidectomy in patients with primary hyperparathyroidism who have had preoperative MIBI and ultrasonography. Such information is necessary to use a focused approach selectively for patients with sporadic primary hyperparathyroidism. The authors questioned whether the quick PTH assay improves the results of parathyroidectomy in patients with primary hyperparathyroidism by analyzing 115 unselected patients with primary hyperparathyroidism without a family history or multiple endocrine neoplasia but who had undergone parathyroidectomy. All 115 patients had successful operations without complications. Of these patients, 88 had solitary adenomas, 13 had double adenomas, one had triple adenomas, 12 had hyperplasia, and one had carcinoma. Overall, MIBI was correct in 72 percent (76/106), ultrasonography in 49 percent (49/99), and the quick PTH assay in 80 percent (92/115). For preoperative studies showing a single tumor, MIBI was correct in 83 percent (73/88), ultrasonography in 71 percent (45/63), and combined MIBI and ultrasonography in 95 percent (37/39). Adding the quick PTH assay in this subgroup did not improve the successful focused approach: 70 percent for MIBI, 65 percent for ultrasonography, and 87 percent for combined MIBI and ultrasonography. However, adding the quick PTH assay improved the overall success of parathyroidectomy (MIBI, 92 percent; ultrasonography, 86 percent; combined MIBI and ultrasonography, 97 percent), but at the cost of unnecessary further exploration (MIBI, 13 percent; ultrasonography, six percent; combined MIBI and ultrasonography, eight percent). The authors concluded that when the same solitary tumor is identified by MIBI and ultrasonography, a focused exploration can be done with a 95 percent success rate. Adding the quick PTH assay to MIBI or ultrasonography can improve the success rate but at a significant cost. General exploration of all parathyroid glands has the highest success rate at 100 percent.

Miura D, Wada N, Arici C, et al. Does intraoperative quick parathyroid hormone assay improve the results of parathyroidectomy? World J Surg. 2002;26:926–930.

Reprints: Dr. O.H. Clark, Dept. of Surgery, University of California, San Francisco/Mount Zion Medical Center, 1600 Divisadero St., San Francisco, CA 94143-1674

Role of blood smear morphology in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is thrombocytopenia associated with a microangiopathic hemolytic anemia. Differentiation from other causes of microangiopathic hemolytic anemia and thrombocytopenia, such as DIC, can be difficult, and there are no definitive tests to diagnose TTP. The presence of schistocytes, or fragmented red cells, is a hallmark of TTP but is not specific to it. There is little information on the reference ranges for the number of schistocytes in TTP, normal individuals, and patients with other causes of hemolysis. The authors of this study compared the number of schistocytes on the blood smears of TTP patients, non-TTP patients, and healthy subjects. The patient groups studied consisted of six patients with TTP, 28 with chronic renal failure, five with preeclampsia, five with mechanical heart valves, and 40 healthy blood donors. None of the non-TTP patients and healthy controls had evidence of hemolysis—that is, normal hemoglobin, reticulocyte count, serum LDH, and bilirubin—at the time of the study. The number of schistocytes per 2,000 red cells was counted using a Miller optical disk at 1,000-power magnification. Ten experienced observers noted the number of schistocytes per 1,000 red cells. Schistocytes were seen in 58 percent of healthy individuals (mean, 0.05 percent; range, 0 to 0.27 percent of red cells); 93 percent of chronic renal failure patients (mean, 0.21 percent; range, 0 to 0.6 percent; P<0.01); 80 percent of preeclamptic patients (mean, 0.25 percent; range, 0 to 0.45 percent; P<0.01); and 100 percent of patients with normally functioning prosthetic cardiac valves (mean, 0.18 percent; range, 0 to 0.48 percent). All of the TTP patients demonstrated schistocytes with a mean of five percent (range, 1.1 to 9.4 percent; P<0.001). In the patient groups studied, and in the absence of other causes of thrombotic microangiopathy, the authors concluded that an initial schistocyte count of greater than one percent strongly suggests a diagnosis of TTP in the absence of other known causes of thrombotic microangiopathy.

Burns ER, Lou Y, Pathak A. Morphologic diagnosis of thrombotic thrombocytopenic purpura. Am J Hematol. 2004;75:18–21.

Correspondence: Dr. Edward R. Burns, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461; eburns@aecom.yu.edu

Urine 8-hydroxyguanine in cancer patients

DNA lesions steadily accumulate with time in normal human cells due to endogenous factors that damage DNA, including ROS3 (superoxide anion, hydrogen peroxide, and hydroxyl radical) derived from oxidative respiration. Free radical attack on DNA generates a whole series of DNA damage, including modified bases. Hydroxyl radical attack on DNA leads to pyrimidine- and purine-derived base damage, some of which has considerable potential to damage the integrity of the genome. 8-hydroxyguanine (8-OH-Gua) is one of the most widely studied lesions of this type. Its presence in DNA leads to GC to TA transversion unless repairs are made before DNA replication. Thus 8-OH-Gua may lead to mutagenesis. Furthermore, there is a direct correlation between 8-OH-Gua formation and carcinogenesis in vivo. The products of repair of 8-OH-Gua in cellular DNA are likely excreted into the urine without further metabolism. The presence of the modified nucleoside (8-OH-dGuo) in urine is widely held to represent the primary repair product of oxidative DNA damage in vivo, presumably via nucleotide excision repair (NER). However, oxidatively damaged DNA bases are mostly repaired by the base excision repair (BER) pathway, although the NER pathway may also play a role in the repair of some oxidized bases in DNA. Therefore, assays that are able to determine the level of 8-OH-dGuo as well as the amount of 8-OH-Gua in urine may better reflect the oxidative damage of cellular DNA. Until recently, there has been no reliable assay for analyzing urine 8-OH-Gua; however, a new methodology—high-performance liquid chromatography prepurification followed by gas chromatography with isotope dilution mass spectrometric detection—allows for simultaneous determination of 8-OH-dGuo and 8-OH-Gua in the same urine sample. Using this method, the authors found that urinary excretion of 8-OH-Gua and 8-oxo-Guo does not depend on diet in humans. The authors determined whether the amount of 8-OH-Gua and 8-OH-dGuo excreted into urine was higher in 42 cancer patients suffering from metastasis of their primary tumors to bone than in a control group consisting of 38 healthy subjects. They found that the amount of the modified base, but not the nucleoside, excreted into urine was about 50 percent higher in the cancer patients than in the control group. Because the presence of the modified base in urine may represent the primary repair product of oxidative DNA damage in vivo, the results suggest that DNA glycosylases play an important role in removing the damage induced as a result of cancer development.

Rozalski R, Gackowski D, Roszkowski K, et al. The level of 8-hydroxyguanine, a possible repair product of oxidative DNA damage is higher in urine of cancer patients than in control subjects. Cancer Epidemiol Biomarkers Prev. 2002;11:1072-1075.

Reprints: Ryszard Olinski, Dept. of Clinical Biochemistry, ul. Karlowicza 24, 85-092 Bydgoszcz, Poland; ryszardo@aci.amb.bydgoszcz.pl

Clonal outbreak of Pseudomonas aeruginosa

Persistent airway infection by Pseudomonas aeruginosa among patients with cystic fibrosis is accompanied by deteriorating pulmonary function and reduced survival. The infection is believed to come from the environment, where patients acquire their own unique strains. Siblings with cystic fibrosis (CF) are frequently found to have identical isolates, but it is unusual to find unrelated patients with CF who share common strains. Previous studies proposing person-to-person transmission of P. aeruginosa have been criticized for omitting molecular typing techniques. However, when these methods have been employed, cross-infection of P. aeruginosa between patients has been detected in some CF clinics, suggesting the emergence of transmissible strains. Nevertheless, these observations are regarded as unusual and insufficient to warrant additional infection-control measures. From 1991 to 1995, while studying a cohort of 92 infants at the Royal Children’s Hospital, Melbourne, Australia, the authors observed that five unrelated cohort members died from severe lung disease before five years of age. A mucoid strain of P. aeruginosa had been identified in all five patients within 0.5 to 16 months of their deaths. Subsequently, pulsed-field gel electrophoresis testing of these and other P. aeruginosa isolates from this cohort revealed an identical macrorestriction pattern among eight of 27 infected subjects, including all five who died. The cluster of deaths in these young patients who shared a genotypically identical strain of P. aeruginosa was unexpected. To investigate the possibility of an outbreak and help guide infection-control policies, the authors performed a cross-sectional study to identify the distribution of the clonal strain within the CF clinic population using two independent genomic fingerprinting methods. They also determined the clinical status of children with P. aeruginosa. Between September and December 1999, 152 patients, aged 3.9 to 20.7 years, provided sputum for culture. P. aeruginosa was detected in 118 children (mean age, 13.5 years). The genotyping techniques were concordant, showing that 65 (55 percent) infected patients carried an indistinguishable or closely related strain. No distinctive antibiogram or environmental reservoir was found. Patients with the clonal strain were more likely than those with unrelated isolates to have been hospitalized for respiratory exacerbations in the preceding 12 months. This study demonstrates extensive spread of a single, clonal strain of P. aeruginosa in a large pediatric CF clinic. Whether this strain is also more virulent than sporadic isolates remains to be determined. The authors suggested that because transmissible strains could emerge elsewhere, other CF clinics should consider molecular methods of surveillance for cross-infection.

Armstrong DS, Nixon GM, Carzino R, et al. Detection of a widespread clone of Pseudomonas aeruginosa in a pediatric cystic fibrosis clinic. Am J Respir Crit Care Med. 2002;166:983–987.

Reprints: Dr. David Armstrong, Dept. of Pediatrics, Monash University, Monash Medical Centre, Clayton Rd., Clayton, Victoria 3168, Australia; d.armstrong@ southernhealth. org.au

Removing CMV from blood components using leukoreduction

Cytomegalovirus infection after blood transfusion is a potentially serious complication, particularly in immunocompromised patients. Methods to prevent or reduce post-transfusion CMV infection include selecting blood components for transfusion from CMV-seronegative donors or using leukocyte depletion filters prior to transfusion. Previous studies support both methods. The authors conducted a study in which they collected units of blood from CMV-seronegative donors, infected the mononuclear cells in vitro with CMV, and reintroduced the infected cells into each whole blood unit. Leukocyte filtration was performed. CMV load was assessed using quantitative CMV DNA real-time polymerase chain reaction. All leukoreduced units contained less than 5 x 106 leukocytes/U. Leukocyte filtration reduced the CMV load from a mean of 742 to a mean of 1.13 CMV genome copies/µL, a mean reduction of 2.81 ± 0.31 10log. For platelets, mean CMV levels before and after leukocyte filtration were 380 and 4.77 copies/µL, respectively, a mean reduction of 1.9 ±0.64 10log. The mean postfiltration plasma signal from whole blood and platelets was 1.52 (range, 0.05 to 3.72) and 6.19 (range, 1.09 to 12.8) copies/µL, respectively. It was not possible for the authors to compare CMV removal with residual leukocyte counts. Since the authors measured genome copies and not infectious virus, it is unclear if the residual levels of CMV seen in this study are sufficient to reliably prevent CMV infection in immunocompromised subjects. The authors noted that clinical studies continue to suggest that prestorage leukoreduction provides acceptable CMV-safe blood components but that incomplete CMV clearance by filtration suggests a need for additional studies.

Visconti MR, Pennington J, Garner SF, et al. Assessment of removal of human cytomegalovirus from blood components by leukocyte depletion filters using real-time quantitative PCR. Blood. 2004;103:1137– 1139.

Correspondence: Lorna M. Williamson, Division of Transfusion Medicine, National Blood Service, Long Rd., Cambridge CB2 2PT, England; lorna.williamson@nbs.nhs.uk

Phylogenetic analysis of Salmonella, Salmonella, E. coli with gyrB gene sequence

Shigella and Salmonella are pathogens that cause gastroenteropathy in humans. Alimentary infections are mostly caused by Salmonella, which has a broad distribution throughout the natural world and a widespread occurrence in animals, particularly poultry and swine. Shigella spp. are metabolically inactive biogroups of Escherichia coli, and some E. coli strains can cause diarrhea similar to that caused by Shigella. E. coli, Shigella, and Salmonella belong to the family Enterobacteriaceae. It would be useful to classify these types of bacteria to aid the treatment of bacterial infections. PCR has been used to determine the evolutionary relationships of bacteria by analyzing the nucleotide sequences of various genes, including 16S/23S rRNA, housekeeping genes, and invasion genes. In particular, 16S rRNA sequences have been used widely to construct bacterial phylogenetic relationships or to detect pathogenic bacteria. Bacterial analysis by 16S rRNA has become popular because these sections of RNA are conserved and easy to sequence. However, the classification of closely related species of bacteria—for example, Shigella spp. and E. coli—is difficult to achieve through 16S rRNA analysis. As an alternative, the authors of this study carried out phylogenetic analysis of about 200 strains of Salmonella, Shigella, and E. coli using the nucleotide sequence of the gene for DNA gyrase B (gyrB), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for classifying Salmonella, Shigella, and E. coli, in which Salmonella forms a cluster separate from but closely related to Shigella and E. coli. In comparison with 16S rRNA analysis, the gyrB sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for bacteria, the gyrB gene might be useful for differentiating between closely related species of bacteria, such as Shigella spp. and E. coli. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable gyrB sequence region can identify unknown bacteria to the species level. The authors concluded that gyrB sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for classifying closely related bacterial species.

Fukushima M, Kakinuma K, Kawaguchi R. Phylogenetic analysis of Salmonella, Shigella, and Escherichia coli strains on the basis of the gyrB gene sequence. J Clin Microbiol. 2002;40:2779–2785.

Reprints: Ryuji Kawaguchi, 5-6-50 Shinmachi, Hinoshi, Tokyo 191-0002, Japan; ryukaw@srl.srl-inc.co.jp

HIV viral load and the menstrual cycle

Human immunodeficiency virus-1 RNA is present in the genital tract secretions of most HIV-infected women, but little is known about viral dynamics within the female genital tract or about potential variation of genital tract viral load through the menstrual cycle. Studies attempting to relate plasma viral load and levels of HIV in the female genital tract have produced inconsistent results. Several lines of evidence suggest that hormonal variations throughout the menstrual cycle could have an impact on viral dynamics in the female genital tract: Cyclic estrogen and progesterone levels may affect immunologic control of HIV replication. In a number of published studies in which cervical and vaginal secretions have been assessed for HIV-1 in the context of the menstrual cycle, most studies did not document ovulation, nor was the sampling taken at each distinct phase. The authors of this study decided to determine the vaginal, cervical, and plasma viral load throughout the menstrual cycle in women who are HIV-positive. They conducted a prospective cohort study on 14 HIV-positive women with ovulatory menstrual cycles whose mean age was 32.7 years, median HIV helper cell count value was 355, and median plasma viral load was 24,000 copies/mL. A total of 301 duplicate cervical and vaginal viral load samples were taken at four stages—menstrual, follicular, periovulatory, and luteal—during two consecutive cycles. No statistically significant difference in plasma viral load was noted throughout the menstrual cycle, but there was a significant decrease in genital tract viral load at the periovulatory phase (vagina, P=.018; cervix, P=.007). Vaginal and cervical viral load were correlated (r=0.582; P<.001). Although the plasma viral load remained constant throughout the menstrual cycle, the genital viral load decreased at the periovulatory phase. These results suggest that local factors may affect the genital viral load compartment independent of plasma viral load.

Money DM, Arikan YY, Remple V, et al. Genital tract and plasma human immunodeficiency virus viral load throughout the menstrual cycle in women who are infected with ovulatory human immunodeficiency virus. Am J Obstet Gynecol. 2003;188: 122–128.

Reprints: Dr. Deborah Money, Division of Maternal Fetal Medicine, Dept. of Obstetrics and Gynecology, UBC, Rm. 2H30, 4500 Oak St., Vancouver, BC V6H 3N1 Canada; dmoney@cw.bc.ca

Increased glutathione levels as a marker of longevity

The authors have, over several decades, investigated the tissue distribution and function of glutathione during the life spans of different organisms, including the mosquito, mouse, rat, and human, with particular emphasis on the total glutathione profile in blood during aging. These findings led the authors to regard total glutathione as an accessible index of longevity and overall body glutathione. In one study, the authors had documented blood glutathione deficiency in more than half of healthy, elderly subjects 60 to 79 years old, compared with normal levels in the 20- to 39-year-olds who made up the reference group. The criterion for deficiency was a subnormal concentration of erythrocytic glutathione, accounting for 99.5 percent of that in blood. It is, therefore, a superior index of deficiency compared with plasma, which contains only trace levels of glutathione. In a related experiment, the authors found glutathione deficiency in 36 percent of newly admitted hospital patients with such chronic conditions as cardiovascular or liver disease and diabetes mellitus. These findings suggested that, in contrast, high total glutathione levels in elderly subjects would be associated with excellent physical and mental health. In the current study, the authors assessed blood total glutathione levels and physical and mental health status in healthy elderly women and compared these values with previous findings from representative groups of healthy subjects from the region and with values used nationally. The authors recruited 87 white women who ranged in age from 60 to 103 years and who reported that they felt healthy. The subjects’ health was verified with physical examinations, clinical chemistry profiles, psycho-social assessments, and blood total glutathione determinations. This evaluation was performed in three waves over a five-year period. The values were compared with those from representative individuals regionally and nationally. The results verified that these subjects were in top physical and mental health. The authors also found that subjects of all ages had very high blood total glutathione levels in the first and second waves but normal levels in the third wave. These findings confirm that high blood total glutathione concentrations and excellent physical and mental health are characteristics of long-lived women.

Lang CA, Mills BJ, Lang HL, et al. High blood glutathione levels accompany excellent physical and mental health in women ages 60 to 103 years. J Lab Clin Med. 2002; 140: 413–417.

Reprints: Dr. Calvin A. Lang, Dept. of Biochemistry, MDR 412, University of Louisville, Louisville, KY 40292; calang@ louisville.edu

CSF LDH isoenzymes in Guillain-Barré syndrome

Lactic dehydrogenase is present in most tissues and body fluids, including cerebrospinal fluid. Patients with an intracranial pathology, including malignancy or bacterial infection, have increased CSF LDH activity compared with healthy subjects (normal, 40 U/mL). No reports have been published regarding CSF LDH activity in Guillain-Barré syndrome (GBS). In this study, the authors presented a series of nine children with a diagnosis of GBS in whom CSF showed a distinctive LDH isoenzyme pattern. They collected CSF samples from nine patients, aged 15 months to nine years, with suspected GBS, who underwent routine lumbar puncture at an inpatient department of the authors’ tertiary pediatric center. The authors analyzed the CSF specimens for total LDH isoenzyme activity, comparing them to samples from 15 patients with normal results. Mean total LDH activity was 33.33 (6.63) U/L. All patients had significantly increased LDH-3 isoenzyme compared with controls. LDH-3 was the predominant fraction, accounting for more than 50 percent of total LDH activity and present in more than twice the percentage of LDH-1 or LDH-2. In contrast, the control group had high percentages of mainly LDH-1 and LDH-2. The authors concluded that GBS is apparently associated with a distinct LDH isoenzyme pattern in CSF. More studies are needed to confirm the rise in LDH-3, as serial CSF analyses are unavailable, and to determine the optimum time of analysis when this finding first becomes detectable.

Nussinovitch M, Prais D, Finkelstein Y, et al. Lactic dehydrogenase isoenzymes in cerebrospinal fluid of children with Guillain-Barré syndrome. Arch Dis Child. 2002;87:255–257.

Reprints: Dr. M. Nussinovitch, Dept. of Pediatrics “C”, Schneider Children’s Medical Center of Israel, Petach Tikvah, Israel; moshen@post.tau.ac.il

Lamellar body fetal lung maturity assessment in diabetic pregnancy

Accurate assessment of fetal lung maturity is very important in treating complicated pregnancies. The current gold standard for determining fetal lung maturity is the evaluation of phospholipids—that is, the lecithin/sphingomyelin (L/S) ratio—from amniotic fluid. The presence or absence of the more stable phospholipid, phosphatidylglycerol, may also play an important role in treatment decisions, particularly with pregnancies that are complicated by diabetes mellitus. Lamellar body counts have been introduced as an alternative method of predicting fetal lung maturity. Lamellar bodies are lamellated phospholipids that carry surfactant and are secreted into the amniotic fluid by fetal type II pneumocytes. They can be measured in the platelet channel of an automated hematologic cell counter. A major advantage of lamellar body counts is that results may be obtained rapidly with only 0.5 mL of fluid. The authors reviewed lamellar body counts and fetal lung status in the pregnancies of diabetic women at their institution to confirm that lamellar body counts are a valid, rapid screening test for fetal lung maturity in pregnancies that are complicated by diabetes mellitus and to determine a lamellar body count cutoff value that would correlate well with the L/S ratio. The authors reviewed a prospectively collected perinatal database from November 1992 through October 1999 to identify pregnancies that were complicated by diabetes mellitus for which fetal lung maturity studies had been performed within 72 hours of delivery. Lamellar body counts were correlated with L/S ratio and phosphatidylglycerol values. The sensitivities and specificities of various lamellar body count cutoff values were calculated with the L/S ratio and phosphatidylglycerol values as indicators of fetal lung maturity. Receiver operating characteristic curves were used to determine the lamellar body count that indicated fetal lung maturity. The authors’ neonatal database was reviewed for this same time period to obtain all cases of respiratory distress syndrome. The maternal data were compared with the neonatal data to determine whether respiratory distress syndrome had developed in an infant who had undergone fetal lung maturity testing. Lamellar body counts were correlated with L/S ratio (r=0.51; P<.001) and phosphatidylglycerol values (r=0.57; P<.001) in 90 diabetic pregnant patients. A lamellar body count of 37,000/µL was found to have a sensitivity of 80 percent and a specificity of 100 percent in predicting fetal lung maturity by standardized methods of phospholipid analysis. No cases of neonatal respiratory distress syndrome were noted in this study population. The authors concluded that the lamellar body count is a valid, rapid screening test for determining biochemical fetal lung maturity in pregnancies that are complicated by diabetes mellitus.

DeRoche ME, Ingardia CJ, Guerette PJ, et al. The use of lamellar body counts to predict fetal lung maturity in pregnancies complicated by diabetes mellitus. Am J Obstet Gynecol. 2002;187:908–912.

Reprints not available from the authors.