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CAP Home > CAP Reference Resources and Publications > CAP TODAY > CAP Today Archive 2002 > July 2002 Clinical Abstracts
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  Clinical Abstracts





cap today

July 2002

Multicenter comparison of prestorage filtration leuko-reduction in red cells
Although the FDA continues to move forward with recommendations for the universal leukocyte reduction of red blood cells, in practice this remains controversial because of the expense involved. Relatively little data are available on the performance of white cell reduction filtration in routine practice vis-á-vis the FDA requirement of less than 5 x 10 6 WBC/unit, the Council of Europe recommendation, or the proposed FDA requirement of less than 1 x 10 6 WBC/unit. The authors enrolled in their study the 11 sites of the Viral Activation Transfusion Study, which is a prospective study of the impact of transfusion with and without leuko-reduction on survival and HIV viral load in HIV-1-infected subjects. Patients randomly assigned to undergo white cell reduction were required to receive red cells that were no more than 14 days old and that had undergone prestorage (that is, within 72 hours of collection) leuko-reduction filtration by a method devised to achieve a post-filtration WBC count of less than 5 x 106. Residual WBC quantitation was performed by polymerase-chain-reaction in the central VATS lab using frozen WBC-reduced red cell samples obtained at issue for transfusion. The authors studied 1,869 leuko-reduced red-cell units. The leuko-reduction filtration practices varied within and between sites. The mean residual WBC counts at the 11 sites showed significant differences. Among the WBC-reduced RBC units, 0.8 percent exceeded 5 x 106 WBC/unit, 8.3 percent exceeded 1 x 106 WBC/unit, and 14.3 percent exceeded 5 x 105 WBC/unit. The authors concluded that the residual WBCs and WBC-reduced RBC units varied within and between sites but that WBC reduction was successful because 99 percent and 91 percent of VATS WBC-reduced RBC units met United States and Council of Europe thresholds, respectively. Not every unit, however, will meet these guidelines, as indicated by a small but measurable failure rate.

Yomtovian R, Gernsheimer T, Assmann SF, et al. WBC reduction in RBC concentrates by prestorage filtration: multicenter experience. Transfusion. 2001;41:1030-1036.

Reprints: Dr. Roslyn Yomtovian, director, Blood Bank, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106;

Quantitating ANA antibody content using EIA
Whether autoantibodies are involved in the pathogenesis of rheumatologic disease and might, therefore, be related to changes in the clinical activity of the disease is a major question. It initially was observed that in systemic lupus erythematosus patients, anti-DNA antibodies fluctuated with disease activity, and in some patients, DNA antigen and antibody appeared in sequence in the circulation. This suggested that immune complexes of DNA antigen and antibody were formed and might be involved in disease pathogenesis. It subsequently was observed that SLE patients with high levels of antibodies to DNA and low levels of complement were more likely to have active disease, especially renal involvement, and that serial immunochemical observations of these factors might be useful in managing such patients. It was difficult, however, to determine the level of specific autoantibodies in blood or serum. This was addressed by an assay for antibodies to DNA (called the Farr assay), in which isotope-labeled DNA was mixed with serum, and immune complexes forming between labeled DNA and immunoglobulin were precipitated with ammonium sulfate. The Farr assay has been the basis for numerous publications reporting the association of changing levels of DNA antibody to disease activity in SLE. Interest in trying to equate antibody levels with disease activity has extended to antinuclear antibodies of other specificities, including anti-Sm, anti-SS-A/Ro, and anti-SSB/La. In some of these studies, antibodies to purified native or recombinant protein antigens were detected by enzyme immunoassays, but in many earlier studies, such methods as immunoblotting and immunoprecipitation were used. Results have been mixed. The authors studied whether EIA-ANA kits produced by some of the major manufacturers could be used to quantitate antibody content. They sent sera from the Centers for Disease Control and Prevention that was relatively monospecific and rigorously defined in terms of antibody specificities to the manufacturers. The sera were sent in a coded fashion to manufacturers who were not informed that the set of sera they received would contain individual antibodies at different dilutions. The authors then analyzed the data from these manufacturers using standard statistical methods. Twenty companies that were known to be major providers of EIA kits for ANA were asked to participate in the study. The manufacturers were briefed on the design of the study and told to use their own test kits to analyze the antibody specificities of the sera and to report the data in optical density units or an equivalent measurement. Analysts were blinded to the concentration of the antibodies and the specificities. Eleven of the 20 manufacturers agreed to participate initially but two subsequently withdrew. The commercial EIA kits can potentially quantitate specific autoantibody content to double-stranded DNA, SSB/La, Sm, and Scl-70. Certain deficiencies in these kits were also detected, however, with the most obvious being the lack of uniformly good performance. Kits from some manufacturers showed exceptional accuracy in three of their four antibody-specific kits and poor accuracy for the fourth. The authors concluded that it is important for clinicians to recognize the marked inter-manufacturer variations in the performance of EIA kits used in diagnosing SLE. Manufacturers need to be vigilant in their surveillance of kit performance and provide assurance that such surveillance is being done.

Tan EM, Smolen JS, McDougal JS, et al. A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificities. II: Potential for quantitation of antibody content. J Rheum. 2002;29:68-74.

Reprints: Dr. M.J. Fritzler, Faculty of Medicine, HRB 410B, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N, 4N1, Canada;

Somatic mosaicism in hemophilia A
The most common severe hereditary bleeding disorder in humans is hemophilia A, which affects one in 5,000 male newborns. The condition results from a defective or absent factor VIII (FVIII) protein, which leads to varying degrees of hemorrhage. The gene for FVIII spans 186 kb on chromosome Xq28 and consists of 26 exons. A wide variety of defects affect this gene. Forty percent of all mutations in severe hemophilia A are caused by the intron 22 inversion, and another 30 to 35 percent of severe hemophiliacs carry point mutations of the nonsense or missense type, which are randomly distributed throughout the gene. Ten percent of mutations comprise small deletions or insertions, and five percent comprise large deletions. Autoradiography is relatively insensitive at detecting mutation rates. The detection limit for Southern blot analysis is 10 to 20 percent. Allele-specific PCR and other special methods are expected to be more sensitive. The authors analyzed 61 families in which members had sporadic severe hemophilia A and known FVIII gene defects. They used allele-specific PCR. Eight (13 percent) of the 61 families showed somatic mosaicism of varying degrees, which was confirmed by a mutation-enrichment procedure. All of the mosaics were found in families with point mutations (eight of 32 families). In the subgroup of eight families with CpG transitions, 50 percent had mosaicism. In contrast, the authors did not find mosaics in 13 families with small deletions/insertions or in 16 families with intron 22 inversions. The authors concluded that mosaicism may be a fairly common event in hemophilia A. Consequently, risk assessment in genetic counseling should consider the possibility of somatic mosaicism in families with apparently new mutations, especially families with the subtype of point mutations.

Leuer M, Oldenburg J, Lavergne J-M. Somatic mosaicism in hemophilia A: a fairly common event. Am J Hum Genet. 2001;69:75-87.

Reprints: Dr. Johannes Oldenburg, Institute of Experimental Hematology and Transfusion Medicine, University of Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany;

ApoA1 gene promoter region and Lp(a) levels
Genetic and environmental factors influence plasma high-density lipoprotein cholesterol and apoA1 levels, with apoA1 being the predominate protein associated with the HDL cholesterol particle. Evidence links polymorphisms in the apoA1 gene to coronary artery disease prevalence and incidence. A common polymorphism described in the apoA1 promoter region consists of a G to A substitution at the 75 bp upstream of the transcription start site. This polymorphism consistently has been found to be associated with HDL cholesterol levels in several populations. Studies of the association of apoA1 polymorphisms with lipid quantitative traits have, for the most part, been conducted in adult populations. These studies have had to take into consideration confounding factors for lipid profiles, such as smoking, diet, and medication. The authors of this study analyzed neonatal cord blood samples in order to be free of these confounding environmental effects. They hoped to measure genotype-specific association of the apoA1 polymorphisms with cord plasma lipid levels. They examined two DNA polymorphisms in the promoter region of the apoA1 gene on cord plasma level of Lp(a). This was done in more than 1,000 male and female newborns from three major ethnic groups in Singapore: Chinese, Malays, and Asian Indians. The frequency of the A allele, at -75 bp in the Indians, was significantly lower than for the Chinese and Malays. The two sites that were studied were not subject to linkage disequilibrium. Both polymorphic sites were not associated with lipid factors, except for Lp(a) levels in the Asian Indians. The AA and CC homozygotes were significantly associated with Lp(a) levels. The associations were specific to the male Indian neonates. These genetic variations at the -75 and +83 bp locations explained 6.9 percent and 7.2 percent, respectively, of the total variability of the plasma Lp(a) levels at birth in the Asian Indians. The effects of the two polymorphisms appeared to be additive, and the composite genotypes were able to explain 14 percent of the Lp(a) variance. The authors found significant ethnic- and gender-specific influence of the apoA1 gene on Lp(a) levels at birth and that this effect is independent of gene environment interactions.

Heng C-K, Low P-S, Saha N. Variations in the promoter region of the apolipoprotein A-1 gene influence plasma lipoprotein(a) levels in Asian Indian neonates from Singapore. Pediatr Res. 2001;49:1514-1518.

Reprints: P-S Low, Dept. of Paediatrics, National University of Singapore, 5, Lower Kent Ridge Rd., Singapore 119074;

Hyperbaric oxygen and sickle cell morphology
Hyperbaric oxygen is used in some institutions for the empiric treatment of acute sickle cell crisis. Little has been published about using hyperbaric oxygen in this way, but physicians who undertake this therapy report nearly immediate cessation of pain after patients are exposed to hyperbaric oxygen. One possible mechanism of action might be an effect on the morphology of erythrocytes during a sickle cell crisis. The authors examined the in vitro effect of hyperbaric oxygen on the morphology of sickled red blood cells. They conducted a prospective in vitro study of 10 children known to be homozygous for hemoglobin S, with each child's sample serving as its own control. Blood samples were obtained during routine visits to a university sickle cell clinic, and they were exposed to room air to achieve maximum sickling. Each sample was then divided into control and study aliquots, and the study portions were placed in a research hyperbaric chamber with 100 percent oxygen at three atmospheres absolute pressure for 15 minutes. Smears were then prepared for these samples at regular intervals and examined for morphology by technicians who were blinded about the details of the study. Percentages of normal cells, sickle cells, and sickle forms were reported. The authors found that hyperbaric oxygen appeared to have no effect on sickle cell morphology. They concluded that other mechanisms may account for the beneficial clinical effects of hyperbaric oxygen in sickle cell crisis, although in vivo studies are warranted.

Mychaskiw II G, et al. In vitro effects of hyperbaric oxygen on sickle cell morphology. J Clin Anesth. 2001;13:255-258.

Reprints: Dr. George Mychaskiw II, Dept. of Anesthesiology, University of Mississippi School of Medicine, 2500 N. State St., Jackson, MS 39216-4505;

Rapid disease progression in HIV-infected infants
The levels of vertical transmission of HIV-1 are significant in underdeveloped countries and inner-city communities. More than 20 percent of infants infected with HIV-1 develop CDC class C disease, which is equivalent to AIDS, or they die before 18 months of age. Early antiretroviral therapy for HIV-1-infected infants has been recommended, particularly for those whose disease progresses rapidly. Studies have suggested different surrogate markers for assessing risk of rapid progression in pediatric populations. These markers include HIV-1 RNA levels, lymphocyte subsets, and serum proteins. In HIV-1-infected adults, decreased levels of CD3-positive T-cells occur approximately 1.5 to 2.5 years before AIDS develops and independent of CD4-positive T-cell counts. The authors, therefore, hypothesized that analysis of CD3-positive T-cells in HIV-1-infected infants could be used to assess the risk of rapid progression in a large cohort of subjects. They studied peripheral blood lymphocytes from HIV-1-infected infants (up to six months of age) and looked for an association between lymphocyte subsets and rapid progression. Rapid progression was defined as AIDS or death before 18 months of age. In 32 infants with rapid progression, CD3-positive T-cell counts were 3,093 cells/µL at younger than one month of age, 3,092 cells/µL at one to three months, and 2,062 cells/µL at three to six months. In 49 infants with nonrapid progression, CD3-positive T-cell counts were maintained at approximately 4,000 cells/µL for at least six months. CD3-positive and CD4-positive T-cell counts were significantly associated with rapid progression. The authors concluded that a decreased CD3-positive T-cell count may be used to predict rapid progression in HIV-1-infected infants (relative risk, 2.16; P=.001).

Chinen J, Easley KA, Mendez H, et al. Decline of CD3-positive T-cell counts by 6 months of age is associated with rapid disease progression in HIV-1-infected infants. J Allergy Clin Immunol. 2001;108:265-268.

Reprints: Dr. Javier Chinen, Texas Children’s Hospital, 6621 Fannin St. (MC:1-3291), Houston, TX 77030




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