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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.