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October 2005
Editors:
Michael Bissell, MD, PhD, MPH
Ronald Domen, MD
Use of leukoreduction to remove Epstein-Barr virus from red blood cells
Acute leukemia in polycythemia vera
Value of non-invasive bilirubinometry
Clinical laboratory linearity
BNP as a presymptomatic screening test
Use of leukoreduction to remove Epstein-Barr virus
from red blood cells
Epstein-Barr virus, the causative agent of infectious mononucleosis,
has also been implicated in several other diseases, including Burkitt’s
lymphoma, post-transplant lymphoproliferative disorders, AIDS-related
lymphomas, and other lymphoproliferative diseases. Following infection,
Epstein-Barr virus (EBV) establishes life-long latency in B lymphocytes
in the majority of cases. EBV genomes can be detected in the circulation
of otherwise healthy individuals, and transfusion-transmitted EBV has
been reported following the transfusion of cellular blood components.
Leukoreduction filters can deplete greater than three logs (99.9 percent)
of white cells from blood components. Leukoreduction has already demonstrated
efficacy in reducing the number of cytomegalovirus (CMV) genomes in order
to provide CMV-safe blood components. The authors of this study examined
the efficacy of EBV removal from blood components by leukoreduction. Leukoreduction
by filtration was performed on 16 randomly selected, fresh AS-5 red blood
cell units. Filtrations were performed per routine procedure and protocol.
Pre- and postleukoreduction specimens were collected from each RBC unit,
and the mononuclear cells (MNCs) were isolated by Ficoll density gradient
centrifugation. DNA was extracted from the preleukoreduction CD19+ B-cell
pellet and from the postleukoreduction MNC pellet. EBV quantification
was performed by DNA amplification. The lower limit of detection was one
genomic copy of EBV DNA. EBV genomes were detected in CD19+ B lymphocytes
in 14 of 16 preleukoreduced RBC units. A median of 3.10 EBV genomic copies
(range, 0.18–96.84) per 105 CD19+ B lymphocytes was found (or approximately
a median of 2,325 EBV genomes [range, 135–72,630] per RBC unit).
EBV DNA was detected in one of 16 RBC units after leukoreduction. (Fifteen
units were negative.) The one positive postleukoreduced unit had the highest
EBV viral load before leukoreduction (72,630 EBV genomes per unit). These
results indicate that a four-log reduction of EBV genomes can be achieved
with leukoreduction of RBC units. The authors concluded that EBV genomes
can be removed from RBC components by leukoreduction through filtration
and that leukoreduction is probably the most effective and practical method
for reducing the risk of transfusion-transmitted EBV from cellular blood
components in patients who are at risk for primary EBV infection.
Qu L, Xu S, Rowe D, et al. Efficacy of Epstein-Barr virus removal by leukoreduction
of red blood cells. Transfusion. 2005;45:591–595.
Correspondence: Dr. Lirong Qu, Institute for Transfusion Medicine, 3636
Boulevard of the Allies, Pittsburgh, PA 15213; qul@
msx.upmc.edu
Acute leukemia in polycythemia vera
The clinical course of polycy themia vera is complicated by a variable
risk for transformation to myeloid metaplasia with myelofibrosis or acute
myeloid leukemia/myelodysplastic syndromes (AML/MDS). After 10 years of
polycythemia vera (PV), the incidence of AML/MDS ranges from five percent
to 15 percent, and the risk progressively increases over time. Previous
studies have attempted to assess risk factors that might predict progression
to AML/MDS, but consistently useful predictors have not been established.
The European Collaboration on Low-Dose Aspirin in Polycythemia Vera (ECLAP)
study prospectively monitored 1,638 PV patients and provided a comprehensive
evaluation of the risk of AML/MDS. Polycythemia vera patients were treated
to maintain their hematocrit value at 0.45 or less and their platelet
count at 400 ? 109/L or less. Additional data regarding clinical outcomes,
treatments, and laboratory tests were collected during the study. An ad
hoc committee of expert clinicians validated nonfatal and fatal events.
Patients were diagnosed with PV between 1964 and 2001 and were registered
in the study between 1997 and 2001. The median time from registration
to diagnosis was 3.5 years. The median age at diagnosis was 62.1 years
and at registration was 67.3 years. The median followup period was 2.8
years. Twenty-one cases of AML and one case of MDS (refractory anemia
with excess blasts in transformation [RAEB-t] with rapid progression to
AML) were diagnosed after a median of 8.4 years from the diagnosis of
PV. Three cases of AML were preceded by a spent phase with myelofibrosis.
All cases of AML/MDS were fatal within six months of diagnosis. Compared
to patients without AML/MDS, patients who subsequently developed AML/MDS
were more likely to have had white blood cell counts exceeding 12 ? 109/L
at registration (P=0.0733). Likewise, patients with AML/MDS were more
likely to be older (P=0.0036) and to have had longer disease duration
(P=0.0067) as well as a previous hemorrhagic event (P=0.0116) and a lower
total blood cholesterol level (P=0.0157). They also were more likely to
have been treated with more than one cytoreductive drug (P=0.0002) and
to have experienced more frequent use of P32 (P=0.0014), busulphan (P=0.0003),
and pipobroman (P=0.0246). Phlebotomy, hydroxyurea, and interferon were
not associated with AML/MDS. Although the relationship between high white
blood cell counts and progression to AML/MDS was not statistically significant,
the high WBC count may be indicative of a different proliferation pathway
in patients who are at risk for AML/MDS. The authors concluded that additional
study is needed to determine the efficacy and safety of cytoreductive
treatments in PV patients.
Finazzi G, Caruso V, Marchioli R, et al. Acute leukemia in polycythemia
vera: an analysis of 1638 patients enrolled in a prospective observational
study. Blood. 2005;105:2664–2670.
Correspondence: R. Marchioli, ECLAP Co-ordinating Centre, Consorzio Mario
Negri Sud, Via Nazionale, 6630 Santa Maria Imbaro, Italy; marchioli@negrisud.it
Value of non-invasive bilirubinometry
Other than routine screening for inborn errors of metabolism, the most
frequent laboratory test performed in the normal newborn nursery is a
total serum bilirubin measurement. The Minolta/Hill-Rom Air-Shields Transcutaneous
Jaundice Meter JM-103 uses two wavelengths and a dual optical path system.
The principle of operation involves the formation of two beams, one of
which reaches only the shallow areas of the subcutaneous tissue while
the other penetrates the deeper layers. The differences between the optical
densities are detected by blue and green photocells. The measurement of
bilirubin accumulated primarily in the deeper subcutaneous tissue should
decrease the influence of other pigments in the skin, such as melanin
and hemoglobin. Another transcutaneous device, the BiliChek, uses multiple
wavelengths, and a close correlation was found between transcutaneous
bilirubin (TcB) and total serum bilirubin (TSB) measurements in mixed
racial populations. The authors conducted a study to evaluate the JM-103.
They studied a convenience sample of 849 newborns from three hospitals
who were 35 weeks of gestation or older. These infants had TSB levels
measured on clinical indication and TcB levels obtained within one hour
of the TSB levels. The population was 59.2 percent white, 29.8 percent
black, 4.5 percent East Asian, 3.8 percent Middle Eastern, 1.6 percent
Indian/Pakistani, and 1.1 percent Hispanic. A close correlation was found
between TSB and TcB values in all of the population groups: white (n=503,
r=.949); black (n=253, r=.822); and East Asian, Indian/Pakistani, and
Hispanic (n=93, r=.926). In the black population, the correlation was
less close than in the other groups, and differences between the TcB and
TSB measurements tended to increase with rising TSB values. JM-103 values
differed from TSB values by 3 mg/dL or more in two percent of white, 3.2
percent of other, and 17.4 percent of black infants. In the black infants,
the JM-103 value was always greater than the TSB value. The authors found
that TcB measurements using the JM-103 jaundice meter correlate very closely
with TSB levels over the range of TSB encountered in this study. Because
only 3.3 percent of infants had TSB values greater than 15 mg/dL (257
?mol/L), more data are needed in this range of TSB concentration. The
correlation in black infants was not as close as in other groups, but
because the tendency in blacks is for the JM-103 to overestimate serum
bilirubin levels, dangerous clinical errors are unlikely to occur. The
measurement technique is rapid and simple, and it is easy to perform repeated
measurements over time, thus reducing the likelihood of error. The authors
concluded that TcB measurements with the JM-103 meter should eliminate
the need for most serum bilirubin levels in newborn infants of at least
35 weeks gestation, although serum bilirubin measurements are still required
when considering treatment with phototherapy or exchange transfusion.
Maisels MJ, Ostrea EM, Touch S, et al. Evaluation of a new transcutaneous
bilirubinometer. Pediatrics. 2004;113:1628-1635.
Reprints: M.J. Maisels, Dept. of Pediatrics, William Beaumont Hospital,
3601 W. 13 Mile Rd., Royal Oak, MI 48073-6769; jmaisels@beaumont.edu
Clinical laboratory linearity
According to NCCLS EP6-A, a quantitative analytical method is said to
be linear when the analyte recovery from a series of sample solutions
(measured value) is linearly proportional to the actual concentration
or content of the analyte (true value) in the sample solutions. The points
at the upper and lower limits of the analytic measurement range that acceptably
fit a straight line determine the linear range. In some assays, the instrument
response versus concentration of sample solutions is not linearexample,
competitive radioimmunoassays have a parabolic-shaped instrument response
when plotted against concentration and a sigmoid-shaped curve when the
response is plotted against the logarithm of the concentration. The responses
may be transformed using a four-parameter logistic formula or other formula,
such as logit-log. The test results from this transformation should be
linearly proportional to the true value of the analyte in the sample solutions.
Therefore, the curve of the instrument response, which can be parabolic
or sigmoid-shaped, should not be confused with linearity between the measured
value and the true value. In 1988, the CAP Instrumentation Resource Committee
began offering linearity Surveys as a tool for evaluating linearity. The
linearity program provides pre-prepared, analyte-spiked human samples
(mostly serum and some urine) covering the full, expected operating range
for the analytes being tested for linearity. Lyophilized samples were
used initially, but analyte-spiked serum and urine samples that do not
require dilution are now being used, eliminating imprecision from manual
pipetting. Since the Survey samples are made to specific analyte target
values, the data analysis verifies calibration within preset tolerances
for the participants, unlike the use of stored patient samples, which
can only be used to evaluate linearity. These Surveys also allow comparison
across laboratories and methods. The authors described the theory and
procedural steps of each linearity evaluation. They then evaluated the
statistical methods for each procedure. The authors found that visual
assessment, although simple, is subjective. The lack-of-fit error and
the 1986 NCCLS EP6-P G test are sensitive to imprecision and assume that
the data are first order. Regression analysis, as developed as the polynomial
method, is partly based on the experiences of the CAP Instrumentation
Resource Committee and has proved to be a robust statistical method. The
authors concluded that their findings provide general guidelines for handling
non-linear results from a linearity evaluation. Handling linearity data
in an objective manner will help clinical laboratorians to improve the
quality of the tests they perform.
Jhang JS, Chang CC, Fink DJ, et al. Evaluation of linearity in the clinical
laboratory. Arch Pathol Lab Med. 2005;128:44–48.
Reprints: Dr. Martin H. Kroll, VA Medical Center, Pathology and Laboratory
Medicine, SVC (113), 4500 Lancaster Rd., Dallas, TX 75216; martin.kroll@med.va.gov
BNP as a presymptomatic screening test
Preclinical systolic dysfunction is common and is associated with progression
to heart failure and increased mortality. Half of patients with heart
failure have normal ejection fraction, with diastolic dysfunction as the
presumed cause of heart failure symptoms in these patients. Diastolic
dysfunction too is common and is predictive of heart failure and death.
Although more data are needed before screening for and treatment of preclinical
ventricular dysfunction (PCVD) can be recommended in the general population,
an adequate screening test is needed before efficacy of screening strategies
can be evaluated. Several studies have evaluated the predictive characteristics
of brain natriuretic peptide (BNP) for detecting preclinical systolic
dysfunction in different settings and have come to different conclusions.
Although a study in a clinical population suggested that BNP may have
value for detecting diastolic dysfunction, no study has evaluated BNP
for its value in detecting preclinical diastolic dysfunction in the general
population. The authors of this study assessed the ability of BNP to detect
preclinical systolic or diastolic dysfunction in the population and in
a high-risk subset (65 years or older and with known cardiovascular disease).
Because BNP is higher in female subjects and increases with age among
those without cardiovascular disease, they also sought to determine if
age and gender influence the predictive characteristics or discriminatory
value of BNP. Finally, they explored the implications of the predictive
characteristics of BNP for screening, accounting for the prevalence of
PCVD in the population. The authors measured BNP, systolic and diastolic
ventricular function, and clinical parameters in 2,042 randomly selected
residents of Olmsted County, Minn., who were 45 years or older. For preclinical
systolic dysfunction, the areas under the receiver operating characteristics
curve were higher for those with more severe (0.82 to 0.92) than any (0.51
to 0.74) systolic dysfunction and were similar in men and women and in
younger and older subjects. For preclinical diastolic dysfunction, the
areas under the receiver operating characteristics curve were higher for
those with moderate-to-severe (0.74 to 0.79) than any (0.52 to 0.68) diastolic
dysfunction and were similar regardless of age or gender. Optimal discriminatory
values of BNP varied with age and gender. Considering the prevalence of
preclinical systolic or diastolic dysfunction and the predictive characteristics
observed, using BNP to screen for PCVD would necessitate echo in 10 percent
to 40 percent of those screened, with most confirmatory echocardiograms
being negative, and would miss 10 percent to 60 percent of those affected.
The authors concluded that BNP is a suboptimal screening test for PCVD.
Redfield MM, Rodeheffer RJ, Jacobsen SJ, et al. Plasma brain natriuretic
peptide to detect preclinical ventricular systolic or diastolic dysfunction:
a community-based study. Circulation. 2004;109:3176–3181.
Reprints: Dr. Margaret M. Redfield, Guggenheim 9, Mayo Clinic, 200 First
St. SW, Rochester MN 55905; redfield.margaret@mayo.edu
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