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measures absorbance at 565 nm and 880 nm. Within
15-45 seconds, the instrument displays the hemoglobin
level in digital form.
Two studies of the HemoCue system have shown it to
be precise, accurate, and, within normal hemoglobin
levels, comparable to routine methods for hemoglobin
determination.2'5 In a comparison of hemoglobin levels
obtained by the HemoCue on capillary samples and by
the Hemalog® instrument using venous blood, coefficients of correlation were 0.96 for 116 unselected samples and 0.89 for 165 samples from the emergency unit.5
A comparison of the HemoCue and Coulter S-880®
yielded a coefficient of correlation of 0.99 for 78 venous
blood samples with hemoglobin levels between 110 and
170 g/L (11.0 and 17.0 g/dL).2
Data from the current study demonstrate that the
HemoCue compares favorably to routine methods for
the measurement of hemoglobin levels in nonanemic
children as well as in children with a variety of red blood
cell disorders, including sickle cell disease. Discrepancies of more than 10 g/L (1.0 g/dL) between the HemoCue and Coulter S-Plus III are uncommon and, when
present, are a result of a lower reading by the HemoCue.
Therefore, failure to detect anemia with the HemoCue is
highly unlikely, although low hemoglobin levels may
require confirmation. These data suggest that the HemoCue system for measuring hemoglobin levels is useful in both general pediatric and pediatric hematology
practice.
Acknowledgments. The authors thank the phlebotomists of the Department of Clinical Laboratories for their assistance in obtaining
blood samples and Joan Grady for the preparation of the manuscript.
Assistance with statistical analysis was generously provided by Henry
Drott, Ph.D., and Jeffrey Silber, M.D. The HemoCue instrument and
microcuvettes were supplied by Leo Diagnostics for the period of
study.
References
1. Bland JM, Altman DG: Statistical methods for assessing agreement between two methods of clinical measurement. Lancet
1986;1:307-310.
2. Bridges N, Parvin RM, von Assendelft OW: Evaluation of a new
system for hemoglobin measurement. American Clinical Products Review 1987;6:22-25.
3. Cornbleet PJ, Gochman N: Incorrect least-squares regression coefficients in method-comparison analysis. Clin Chem
1979;25:432-438.
4. Elion-Gerritzen WE: Analytical precision in clinical chemistry
medical decisions. Am J Clin Pathol 1980;73:183-195.
5. von Schenck H, Falkensson M, Lundberg B: Evaluation of "HemoCue," a new device for determining hemoglobin. Clin
Chem 1986;32:526-529.
6. Weisbrot IM: Statistics for the clinical laboratory. Philadelphia: JB
Lippincott, 1985: 88-89, 144-147.
Ultrastructural Detection of Herpes-Type Virions by Negativ
Staining in Oral Hairy Leukoplakia
KURT D. REED, MAJOR, USAF (MC), CRAIG B. FOWLER, MAJOR, USAF (DC),
AND ROBERT B. BRANNON, COLONEL, USAF (DC)
Hairy leukoplakia (HL) is a newly recognized virus-associated
lesion of oral mucosa that occurs in persons infected with
human immunodeficiency virus. Studies have demonstrated
Epstein-Barr virus within epithelial cells of HL. The authors
examined 12 cases of HL by transmission electron microscopy
to compare the use of a negative staining technique versus
routine plastic embedment for the detection of viruses.
Herpes-type virions were identified by both methods in 11
cases. One case had negative results for viruses by both
methods. Negative staining is a simple and rapid technique
that compares favorably with plastic embedment in the detec-
Received December 10, 1987; received revised manuscript and accepted for publication February 22, 1988.
The views of the authors are not necessarily those of the Department
of Defense or the United States Air Force.
Address reprint requests to Dr. Fowler: Department of Oral Pathology, SGDM, Wilford Hall USAF Medical Center, Lackland Air Force
Base, Texas 78236-5300.
Departments of Pathology and Oral Pathology, Wilford Hall
United States Air Force Medical Center,
Lackland Air Force Base, Texas
tion of herpes-type virions in HL. (Key words: Hairy leukoplakia; Ultrastructure; Epstein-Barr virus; Negative staining;
Human immunodeficiency virus) Am J Clin Pathol
1988;90:305-308
IN 1984, Greenspan and colleagues6 described the clinical and histopathologic features of hairy leukoplakia
(HL), a previously unrecognized virus-associated lesion
of oral mucosa. The lesions of HL appear as irregularly
outlined white plaques (leukoplakia) on the lateral or
ventral aspect of the tongue or, less commonly, the buccal mucosa. The surface may be corrugated or display
REED, FOWLER, AND BRANNON
306
epithelial tags or projections, hence the term "hairy"
leukoplakia. Flat lesions also occur.15 HL is seen virtually exclusively in persons infected with human immunodeficiency virus (HIV).58 Although most reported
cases are in homosexual men,1'5,8 it has been seen in
women,7 hemophiliacs,7'14 and transfusion recipients.37
Histopathologic changes of HL are similar to those
seen in flat warts of the skin and include hyperparakeratosis, acanthosis, koilocytosis, and nuclear pyknosis.
Most lesions contain Candida species. Ultrastructural
studies using plastic embedment techniques have demonstrated herpes-type virions within epithelial cells of
HL.1,6'916 Papillomavirus-like particles have also been
described.6 In this study we compare a simple and rapid
negative staining technique versus plastic embedment
for the ultrastructural detection of viruses in oral HL.
Materials and Methods
Patients
The study population was a group of patients with
serologic evidence of HIV infection documented by an
enzyme-linked immunosorbent assay and confirmed by
Western blot. Twelve patients had incisional biopsy of
tongue lesions clinically suspicious for HL while under
local anesthesia. All patients were male, and ages ranged
from 21 to 57 years. Two patients had clinical acquired
immune deficiency syndrome (AIDS) at the time of
biopsy.
Microscopic Examination
Light microscopic examination was performed on
formalin-fixed, paraffin-embedded tissue. Sections were
stained with hematoxylin and eosin (H and E) and periodic acid-Schiff (PAS).
Transmission electron microscopic examination was
performed on tissue fixed in 2% glutaraldehyde buffered
in 0.1 mol/L sodium cacodylate (pH 7.4), postfixed in
1% osmium tetroxide, dehydrated in a graded series of
acetone, and embedded in Polybed® 812 (Polysciences
Inc., Warrington, PA). One-micron-thick sections were
stained with toluidine blue and examined for epithelial
cells showing koilocytosis and nuclei with marginated
chromatin and central clearing. Corresponding thin sections were stained with uranyl acetate and lead citrate
and examined in a Zeiss® Model 10 electron microscope
at 60 kV.
A.J.C.P. • September 1988
For negative staining, a 2-3 mm3 piece of white surface epithelium was removed from the fresh biopsy specimen with a sterile blade. Care was taken to avoid including excess subepithelial connective tissue. The tissue
was placed in a porcelein dish with 200 tiL of distilled
water, minced with razor blades, and mashed with a
glass rod to form a suspension. A drop of suspension was
placed on a formvar-coated 200-mesh grid (Ernest F.
Fullam, Inc., Latham, NY) (average three grids per specimen) for 2 minutes. The grid was blotted dry from the
edge with filter paper and then floated on a drop of 2%
glutaraldehyde in 0.1 mol/L sodium cacodylate buffer
(pH 7.4) for 5 minutes. The grid was again blotted from
the edge with filter paper before it was floated on a drop
of 2% phosphotungstic acid (pH 7.0) for 30 seconds.
Excess stain was blotted from the edge with filter paper
and the grid allowed to air dry before examination.
Safety Precautions
Procedures used in specimen collection and processing were in accordance with Centers for Disease Control
recommendations for prevention of HIV transmission
in health care settings.2
Results
All lesions occurred on the lateral or ventral aspect of
the tongue and had the typical white corrugated surface
of HL (Fig. 1). Size ranged from 3 mm to several centimeters in diameter. Light microscopic examination revealed acanthosis with irregular hyperparakeratosis,
koilocytosis of subcorneal keratinocytes, and frequent
pyknotic nuclei. Occasional cells had margination of
nuclear chromatin with central clearing. Fungal hyphae
consistent with Candida species were seen in the superficial epithelium in all cases. None of the lesions had
significant inflammation.
In 11 cases, electron microscopic examination with
the use of plastic embedment revealed herpes-type virions within nuclei and cytoplasm of superficial keratinocytes and within intercellular spaces. Virus particles in
nuclei were numerous and consisted of 95-100-nm nucleocapsids. Enveloped particles measuring 115-180
nm were seen in the cytoplasm and intercellular spaces
(Fig. 2). Fungal hyphae were seen in the cytoplasm of
some viral-infected cells. Papillomavirus-like particles
were not identified.
Negative staining revealed herpes-type virions in the
same 11 cases that had positive results by routine plastic
FIG. 1 (upper, left). Clinical photograph of oral hairy leukoplakia. Note thickened white mucosa along the lateral aspect of the tongue.
FIG. 2 (lower, left). Electron micrograph showing herpes-type virions in a keratinocyte. Nucleocapsids are seen
in the nucleus (arrow) and enveloped particles in the cytoplasm (arrowhead) (X70.000).
FIG. 3 (right). Negative staining of herpes-type virions. A (upper). Group of nucleocapsids
and empty capsids (X 100,000). B (lower). Enveloped particle (X200.000).
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REED, FOWLER, AND BRANNON
308
embedment. Nucleocapsids, empty capsids, and enveloped particles were found scattered among cellular
debris (Figs. 3A and B). In most cases 20-50 virions
were seen on each grid examined, but it was not unusual
for several hundred particles to be found on a single grid.
No case in which virus particles were found had fewer
than 10 unequivocal herpes-type virions per grid. Papillomavirus-like particles were not identified in any of the
specimens.
Discussion
Leukoplakia is a common finding of the oral mucosa
and frequently is the result of chronic physical irritation,
either from trauma or tobacco product use. A significant
number are idiopathic in nature.4 Often a biopsy must
be performed because leukoplakia may show evidence
of epithelial dysplasia or malignancy. The lesions of habitual cheek, lip, and tongue biting exhibit a marked
similarity to HL clinically and histologically. Other conditions manifesting as white keratotic lesions that should
be included in the differential diagnosis are white sponge
nevus, lichen planus, discoid and systemic lupus erythematosus, mucous patches of secondary syphilis, and
candidiasis.41719 HL is an important new addition to
this list and has significant prognostic implications for
persons at risk for development of AIDS. One survival
analysis showed the probability of AIDS developing in
patients with HL was 48% by 16 months and 83% by 31
months.8
Greenspan and associates used direct immunofluorescence to show that most cases of HL contain EBV, a
member of the Herpes viridae, within epithelial cells and
that other herpes viruses (herpes simplex virus, varicella-zoster virus, and cytomegalovirus) are absent.9 The
presence of EBV has also been demonstrated by DNA
hybridization.916 Although herpes viruses other than
EBV readily infect oral mucosa, the clinical and histologic features of lesions they produce do not resemble
those of HL. 101318 Therefore, detection of herpes virus
in lesions that are otherwise clinically and histologically
compatible with HL strongly supports that diagnosis
and excludes virtually any of the other causes of leukoplakia. We consider the laboratory diagnosis of HL to be
less certain in cases in which no evidence of EBV infection is found either ultrastructurally or by biochemical
methods.
Negative staining is a well-established method for detection of viruses in clinical specimens and has the advantages of simplicity and speed.'' This is especially true
with specimens that contain sufficient numbers of virus
particles so that additional steps to concentrate the virus
are unnecessary. Specimens of this type may contain
abundant cellular debris, some of which can closely resemble virions. Care must be taken to avoid misinterpretation, and results for specimens should not be con-
A.J.C.P. • September 1988
sidered positive unless unequivocal virions are identified, in this study we identified virions in 11 of 12 cases
of HL by use of a negative staining technique that required less than 20 minutes to perform. The morphologic characteristics of the virions were typical for a
member of the Herpes viridae12 and, combined with the
clinical and histopathologic findings, gave strong support for the diagnosis of HL.
We conclude that electron microscopic examination
with the use of negative staining is a simple and rapid
method that compares favorably with plastic embedment for the detection of herpes-type virions in oral
hairy leukoplakia.
Acknowledgments. The authors thank Judy Ellsworth and David
James for their excellent technical assistance.
References
1. Belton CM, Eversole LR: Oral hairy leukoplakia: ultrastructural
features. J Oral Pathol 1986; 15:493-499.
2. Centers for Disease Control: Recommendations for prevention of
HIV transmission in health-care settings. Morbidity and Mortality Weekly Report 36(suppl 2S):1-17.
3. Dylewski J, Prchal J: Oral "hairy" leukoplakia: a clue to HIV-1
exposure. Can Med Assoc J 1987;136:729-730.
4. Eversole LR: Clinical outline of oral pathology: diagnosis and
treatment. 2nd ed. Philadelphia: Lea & Febiger, 1984:7-25.
5. Eversole LR, Jacobsen P, Stone CE, Freckleton V: Oral condyloma planus (hairy leukoplakia) among homosexual men: a
clinicopathologic study of thirty-six cases. Oral Surg
1986;61:249-255.
6. Greenspan D, Greenspan JS, Conant M, Petersen V, Silverman S,
de Souza Y: Oral "hairy" leucoplakia in male homosexuals:
evidence of association with both papillomavirus and a herpesgroup virus. Lancet 1984;2:831-834.
7. Greenspan D, Hollander H, Friedman-Kein A, Freese UK,
Greenspan JS: Oral hairy leukoplakia in two women, a hemophiliac, and a transfusion recipient. Lancet 1986;2:978-979.
8. Greenspan D, Greenspan JS, Hearst NG, et al: Relation of oral
hairy leukoplakia to infection with the human immunodeficiency virus and the risk of developing AIDS. J Infect Dis
1987;155:475-481.
9. Greenspan JS, Greenspan D, Lennette E, et al: Replication of
Epstein-Barr virus within the epithelial cells of oral "hairy"
leukoplakia, an AIDS-associated lesion. N Engl J Med
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10. Kanas RJ, Jensen JL, Abrams AM, Wuerker RB: Oral mucosal
cytomegalovirus as a manifestation of the acquired immune
deficiency syndrome. Oral Surg 1987;64:183-189.
11. Miller SE: Detection and identification of viruses by electron microscopy. J Elec Micro Tech 1986;4:265-301.
12. Palmer EL, Martin ML: An atlas of mammalian viruses. Boca
Raton, Florida: CRC Press, 1982:111-118.
13. Phelan JA, Saltzman BR, Saltzman GH, Klein RS: Oral findings
in patients with acquired immunodeficiency syndrome. Oral
Surg 1987;64:50-56.
14. Rindum JL, Schiodt M, Pindborg JJ, Scheibel E: Oral hairy leukoplakia in three hemophiliacs with human immunodeficiency
virus infection. Oral Surg 1987;63:437-440.
15. Schiodt M, Greenspan D, Daniels TE, Greenspan JS: Clinical and
histologic spectrum of oral hairy leukoplakia. Oral Surg
1987;64:716-720.
16. Sciubba JJ, Schwartz MH: Hairy leukoplakia and Epstein-Barr
virus (abstract). Oral Surg 1987;64:563.
17. ShklarG: Oral leukoplakia. N Engl J Med 1986;315:1544-1545.
18. Silverman S Jr.: AIDS update: oral findings, diagnosis, and precautions. J Am Dent Assoc 1987; 115:559-563.
19. WHO Collaborating Center for Oral Precancerous Lesions: Definition of leukoplakia and related lesions: an aid to studies on
oral precancer. Oral Surg 1978;46:518-539.