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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; ryomtovian@yahoo.com
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; fritzler@ucalgary.ca
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; johannes.oldenburg@ukb.uni-bonn.de
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; paelowps@nus.edu.sg
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;
gmychaskiw@anesthesia.umsmed.edu
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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