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July 2003
Flow
cytometric monocyte phagocytic assay
and platelet transfusion
A significant proportion of the most closely HLA-matched platelet
transfusions administered to alloimmunized recipients fail, even
in the absence of nonimmunologic causes. Furthermore, platelet crossmatching
will not always be successful because not all antibodies may be
detected in platelet crossmatching and not all mismatches are clinically
significant. The authors developed a flow cytometric monocyte phagocytic
assay (FMPA) using 5-chloromethyl fluorescein diacetate (CMFDA)-labeled
platelets to better predict platelet transfusion outcomes. They
incubated CMFDA-labeled platelets with the serum of 12 patients
who had histories of multiple platelet transfusions and with the
serum of 21 controls. The platelets were then incubated with monocytes
and analyzed by flow cytometry. Monocytes that had phagocytized
platelets were detected with a CD14+ monocyte gate. These results
correlated well with one-hour and 24-hour CCIs. Nine of 10 positive
crossmatches in the group with high FMPA results showed low CCIs,
and six of seven negative crossmatches revealed high CCIs. The CCI
predictability of crossmatching in the group with high FMPA results
was high (88.2 percent). The authors concluded that this is a useful
method for predicting the outcome of platelet transfusion.
Lim J, Kim Y, Han K, et al. Flow cytometric monocyte
phagocytic assay for predicting platelet transfusion outcome. Transfusion.
2002;42:309-316.
Reprints: Dr. Chang-Suk Kang, #62 Yoido-dong, Youngdeungpo-gu, St.
Mary’s Hospital, Dept. of Clinical Pathology, College of Medicine,
Catholic University of Korea, Seoul, 150-713, Korea; cskang@cmc.cuk.ac.kr.
Significance
of increased circulating hyperchromic
red blood cells
An increase in the proportion of hyperchromic red blood cells in
an automated red blood cell analysis has become a consistent finding
in patients with hereditary spherocytosis. In this condition, mean
cell volume decreases slightly and mean cell hemoglobin (MCHC) increases
as red cells become progressively spherocytic. These findings can
help assess the severity of known cases of hereditary spherocytosis.
The authors noted increases in the proportion of hyperchromic red
cells in a small proportion of apparently healthy children in the
United Kingdom, so they undertook a study to determine whether such
children manifest other laboratory evidence of hereditary spherocytosis.
The authors performed blood and reticulocyte counts and Pink tests
on successive children with more than four percent hyperchromic
red cells and compared them with age- and MCHC-matched controls
and children known to have hereditary spherocytosis. Thirty-four
children with more than four percent hyperchromic red cells had
significantly increased absolute numbers of hyperchromic cells,
higher reticulocyte counts, higher MCHC and hemoglobin distribution
width values, and lower mean cell volume values than age-matched
and MCHC-matched controls. Pink tests were also higher but not to
a significant degree. The authors concluded that subjects with an
isolated increase in hyperchromic red blood cells may have a recessive
form of hereditary spherocytosis but lack the laboratory features
of clinically manifest HS.
Conway AM, Vora AJ, Hinchliffe RF. The clinical
relevance of an isolated increase in the number of circulating hyperchromic
red blood cells. J Clin Pathol. 2002;55:841-844.
Reprints: Dr. A. Vora, Dept. of Paediatric Haematology,
Sheffield Children’s Hospital NHS Trust, Sheffield S1O 2TH,
United Kingdom; ajay.vora@sch.nhs.uk.
Point
mutations of the McLeod phenotype
The McLeod phenotype is an X-linked condition characterized by the
absence of Kx surface antigen on red cells, weakened expression
of Kell system antigens, and acanthocytosis. McLeod red blood cells
lack the XK protein that carries Kx antigen and have greatly reduced
the amount of the Kell glycoprotein that expresses Kell system antigens.
People with the McLeod phenotype have a compensated hemolytic anemia,
elevated serum creatine kinase levels, and are prone to a variety
of neuromuscular disorders. The gene responsible for McLeod syndrome
is at the Xp21 locus and is designated XK. Gene deletions of various
sizes, point mutations leading to abnormal RNA splicing, single
base deletions or insertions leading to frameshifts, and premature
stop codons have been reported in people with the McLeod phenotype.
The authors sequenced the coding and flanking intron regions of
XK from four unrelated male individuals with the McLeod phenotype
and nonchronic granulomatous disease and compared this with the
wild type sequence. They found point mutations at a different 5’
splice site than previously reported and in the coding region, where
an amino acid substitution results that abolishes cell surface expression,
providing another mechanism for the McLeod phenotype. The authors
concluded that the McLeod phenotype may be caused by several different
mutations.
Russo DCW, Lee S, Reid ME, et al. Point mutations
causing the McLeod phenotype. Transfusion. 2002;42:287-293.
Reprints: Dr. David C.W. Russo, New York Blood Center, 310 E. 67th
St., New York, NY, 10021; drusso@nybc.org.
Can
a single measurement replace the lipid profile?
Deciding whether to treat patients at risk for coronary artery disease
is determined based on measurement of total cholesterol, triglycerides,
and high-density lipoprotein cholesterol, the calculation of low-density
lipoprotein cholesterol and the total cholesterol/HDL cholesterol
ratio, and the presence of other risk factors. Most patients do
not reach their designated target lipid concentrations, and the
authors speculated that this may be in part because of the inherent
complexity of the lipid panels’ interpretation. They also
point out that other indices have been suggested to be better predictors
of risk on initial diagnosis and at followup. The best studied of
these is plasma apolipoprotein B (apo B), which is present in each
of the different atherogenic particles in serum (VLDL, LDL, intermediate-density
lipoprotein, and lipoprotein (a)). The authors conducted a study
to determine whether apo B measurement alone would lead to the same
categorization of risk as the full lipid profile. The concordance
or discordance between these two approaches was determined on 215
patients at their first and last clinic visits. Both high-risk and
low-risk groups showed high concordance (88 percent at the first
visit to the clinic and 92 percent at the last visit for the high-risk
group and 76 percent at the first visit and 78 percent at the last
visit for the low-risk group). Discordance was present only in those
with high triglycerides and normal apo B, a group in which little
independent evidence points to a substantially increased risk of
vascular disease. The authors concluded that these data raise the
possibility that, at least for high-risk patients treated with statins,
followup could be simplified and expenses reduced if only apo B
were measured.
Miremadi S, Sniderman A, Frohlich J. Can measurement
of serum apolipoprotein B replace the lipid profile monitoring of
patients with lipoprotein disorders? Clin Chem. 2002;48:484-488.
Reprints: Jiri Frohlich, St. Paul’s Hospital Healthy Heart
Program, #180-1081 Burrard St., Vancouver, British Columbia, V6Z
1Y6, Canada; jifr@interchange.ubc.ca.
A
molecular approach to determining blood plasma viscosity
The measurement and management of blood plasma viscosity are important
issues in the development of artificial blood and in the use of
plasma expanders in blood replacement. The traditional techniques
for this measurement are mechanical and include the use of capillary
shear, falling-ball, and rotational shear in viscometers. These
approaches have limitations when applied to plasma and have not
proven fully practical. The authors proposed that the use of viscosity-sensitive
fluorescent molecules, known as fluorescent molecular rotors, may
solve this problem. These are molecules whose absorption and re-emission
of fluorescent light is a function of their degree of twistedness.
The authors examined a series of human plasma samples at graded
viscosities modified by the addition of pentastarch. Viscosities
were determined with the Brookfield viscometer and the new method.
After calibration and scaling, the molecular rotor measurements
deviated by less than 1.8 percent from the mechanical methods. The
authors concluded that molecular rotors are suitable for fast, low-volume
biofluid viscosity measurements and are comparable to mechanical
viscometers in accuracy and precision.
Haidekker MA, Tsai AG, Brady T, et al. A novel
approach to blood plasma viscosity measurement using fluorescent
molecular rotors. Am J Physiol Heart Circ Physiol. 2002;282:H1609-H1614.
Reprints: M.A. Haidekker, University of Missouri-Columbia, Dept.
of Bioengineering, Food Science and Engineering Unit, 215 Agricultural
Engineering Bldg., Columbia, MO 65211.
Use
of ELISA to detect West Nile virus
In the absence of proven therapy, vector control is of primary importance
in preventing outbreaks of West Nile virus infection. Serologic
surveillance of sentinel or wild birds or mosquitoes, or all of
them, is the most effective approach. Such programs are similar
for West Nile virus and the related St. Louis encephalitis virus.
In areas where both viruses are present, serologic monitoring tests
must distinguish them unequivocally. The authors developed an antigen
capture immunoassay to detect West Nile virus antigen in infected
mosquitoes and avian tissues. The assay detected purified West Nile
virus at a concentration of 32 pg/0.1 mL, and antigen in infected
suckling mouse brain and laboratory-infected mosquito pools could
be detected when the West Nile virus titer was 102.1 to 103.7 PFU/0.1
mL. The assay identified 12 of 18 (66.7 percent) blindly coded field-collected
mosquito pools (n=100) that were TaqMan-positive. This is compared
with 10 of 18 (55.5 percent) detected by reverse transcriptase PCR.
The assay performed similarly in 73 organ homogenates from naturally
infected American crows. The recommended West Nile virus antigen
capture protocol, which includes a capture assay followed by a confirmatory
inhibition assay used to retest presumptive positive samples, could
distinguish between the West Nile virus and St. Louis encephalitis
virus in virus-infected mosquito pools and avian tissues. The authors
concluded that this assay shows adequate sensitivity and specificity
for surveillance of West Nile virus activity in mosquito vectors
and avian tissues and is easy to perform and relatively inexpensive
compared with the TaqMan assay.
Hunt AR, Hall RA, Kerst AJ, et al. Detection of
West Nile virus antigen in mosquitoes and avian tissues by a monoclonal
antibody-based capture enzyme immunoassay. J Clin Microbiol.
2002;40:2023-2030.
Reprints: Ann R. Hunt, Division of Vector-Borne
Infectious Diseases, Centers for Disease Control and Prevention,
P.O. Box 2087, Ft. Collins, CO, 80522-2087; arh4@cdc.gov.
Quantitative
fluorescent PCR vs. FISH for detecting
common aneuploidies
Although conventional cytogenetics and fluorescent in situ hybridization
remain a gold standard method for the prenatal detection of common
aneuploidies, they are time consuming and labor intensive and require
more samples than does quantitative fluorescent-polymerase chain
reaction. The authors evaluated the use of QF-PCR as a diagnostic
tool for rapid prenatal diagnosis in the Greek population. They
extracted DNA from amniotic fluid, chorionic villus samples, and
fetal blood and tissue samples using a simple, rapid protocol. They
then used an automated laser fluorescent sequencer to measure fluorescent
multiplex PCR products of single tandem repeats located on chromosomes
13, 18, 21, X, and Y. All samples were analyzed with at least two
polymorphic markers for chromosomes 13, 18, and 21, and one for
the X chromosome. The amelogenin gene locus was used for sexing.
The analysis was performed on 1,100 samples, and 25 chromosomal
aberrations were identified, including trisomy 13, 18, 21, XYY,
triploidies 69,XXX and 69,XXY, and one Turner mosaic. All but three
results were consistent with conventional cytogenetics. One mosaic
was missed. Most bloodstained samples were analyzed successfully.
Bili C, Divane A, Apessos A, et al. Prenatal diagnosis
of common aneuploidies using quantitative fluorescent PCR. Prenat
Diagn. 2002;22:360-365.
Reprints: C. Bili, Alfalab, Medical Institute of
Research and Diagnosis, Anastasiou 8, Athens 11524, Greece; alab@leto.grn.
Clinical
pathology abstracts editors
Michael Bissell, MD, PhD, MPH, professor and director of clinical
services and vice chair, Department of Pathology, Ohio State University
Medical Center, Columbus.
Ronald Domen, MD, professor of pathology, medicine, and humanities,
Penn State University College of Medicine, Hershey, Pa.
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