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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.
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