Clinical Pathology Department
The Clinical Pathology Department provides laboratory and patient care
support
services for clinical chemistry, hematology, immunology and microbiology
and phlebotomy. The Hematology Service offers consultation for Clinical
Center patients with hematologic disorders. There was a 6.5% increase in
test requests and a 7.6% increase in workload during Fiscal Year 1997 compared
to Fiscal Year 1996.
Departmental
- The plans for a molecular diagnostics laboratory were prepared by an
outside architectural firm in close collaboration with selected senior
staff members of the Department. These plans were finalized and a contract
has been awarded to initiate the renovations which are anticipated to be
completed in the Fall of 1997. This laboratory facility will initially
support molecular diagnostic tests in microbiology, mutation analysis in
immunodeficiency diseases and mutation analysis in selected malignant diseases.
- Departmental members have built screens for the new computer system
in consort with Soft Computer Consultants. In addition, instrument interfaces
are under way
and testing of the new laboratory information system (LIS) is anticipated
this Fall. Parallel testing of the new and current LIS systems will be
initiated and following a satisfactory testing program the new LIS system
will be taken live.
- An outside architectural firm was hired to provide plans for the core
laboratory space. The plans were developed with significant input from
the technical staff. These renovations will be delayed until the new LIS
is up and running and it is anticipated that the renovation work for the
core laboratory will begin in the late Spring of 1998. The plans for these
renovations call for a phased approach to the construction in order to
minimize disruption of the work flow in the clinical laboratory.
- The Chief of Clinical Pathology, Dr. Ronald J. Elin, retired in July
of 1997. Dr. Thomas A. Fleisher was appointed Acting Chief of Clinical
Pathology and assumed this position in July of 1997.· A departmental
retreat was held on July 31, 1997 involving all of the senior staff in
the Department together with the Laboratory Manager, the Chief Technologists,
the LIS Administrator and the Chief of the Phlebotomy Service. The emphasis
of
this retreat was on developing a stronger departmental identity, improving
the service component of Clinical Pathology and strengthening the research
program within the Department.
- A trial of providing services for veterinary animal samples was successfully
undertaken by the Chemistry and Hematology Services. Based on the results
of a questionnaire with regard to the needs for veterinary laboratory services
and the trial operation, a proposal to provide these services within Clinical
Pathology was developed and this has been approved by Dr. Gallin and will
be initiated following clarification of certain administrative issues.
- The reduction in overtime initiated during FY '96 was continued through
the effective staffing on weekends and evenings without the use of overtime
pay.
- Collaboration between CPD and Materials Management Department to institute
the use of computerized supply management through the Pyxis system to reduce
the quantities of items in the inventory and reduce the amount of storage
space required for supplies was successful.
Chemistry
Research
- The telomeric repeat amplification protocol (TRAP) was improved for
the
measurement of telomerase activity. Fluorescence detection enhanced the
speed of characterization and quantitation of TRAP products. Addition of
dUTP and uracil-N-glycosylase into the TRAP reaction sequence effectively
prevented interassay contamination by amplicons from previous telomerase
reactions. Discrete removal of red blood cells markedly improved detection
of telomerase activity in bloody specimens. The improved TRAP assay readily
detected telomerase activity in fewer than 50 tumor cells, and when tumor
cells constituted less than 1% of a cell mixture. The assay was linear
between 50 and 5000 tumor cells.· Clinical validation of the improved
telomerase assay began in collaboration with the Surgery Branch of the
NCI using bladder washings from patients at risk for developing bladder
and renal cell carcinoma.
- For standardization of results for DNA content, fluorescent reagents
were evaluated for DNA quantitation. Since these reagents cross-reacted
with RNA and proteins,
the quantitation of DNA required modifications.
- Oligonucleotide arrays were evaluated in collaboration with Beckman
Instruments, Inc. (confidentiality agreement: MTA# 0087597). Point mutations
were resolved in codon 12 of the K-ras oncogene, using either synthetic
oligonucleotides or PCR products as targets.
- A semi-automated and non-isotopic method was improved to detect K-ras
mutations by using PCR followed by single-stranded conformational polymorphism
analysis (PCR-SSCP), and to characterize PCR products derived from asymmetric
PCR.
- Using SSCP as an enrichment strategy, mutant PCR products were detected
in mixed samples when the mutant DNA constituted less than 0.1% of a mixed
DNA population. Major differences were found between two ion-selective
electrode methods for ionized magnesium (Nova and AVL) in serum samples
with normal total magnesium concentration and abnormally low Nova ionized
magnesium result. Most of these specimens showed a normal AVL result and
the inter-method difference appeared
to be associated with a particular patient rather than a specific diagnosis.
- Clinically important differences were found between the Nova and AVL
ion-selective electrode methods for the measurement of serum ionized magnesium
in apparently healthy smokers as well. Only results with the Nova method
showed a dose-dependent decrease in response to increasing number of cigarettes/day.
Besides, the Nova results inversely correlated with the white blood cell
count (WBC).
- The serum ionized magnesium results with the Nova method inversely
correlated with the serum thiocyanate (SCN-) concentration.
This SCN--related interference appeared to be equimolar and
significantly decreased the Nova ionized magnesium results at concentrations
commonly found in smokers. Since serum SCN- ordinarily
is the metabolite of cyanide that is inhaled with tobacco smoke and ingested
withcyanogenic foods, further, some drugs also are known to increase serum
SCN- levels, the observed interference greatly limits the clinical
utility of this method for measuring serum ionized magnesium.
- The serum total magnesium concentrations positively correlated with
the AVL ionized magnesium results but not with the Nova results in chronic
alcoholics, indicating instrument-dependent detection of an altered ionized
magnesium status.
- The serum ionized magnesium concentrations measured with the Nova ion-selective
electrode exceeded the concentration of total magnesium in patients who
transiently developed severe to profound hypomagnesemia due to cisplatin
or interleukin-2
(IL-2) therapies, suggesting an error in the measurement of low ionized
magnesium concentrations. Three so-called 'direct' and two calculation-based
methods, including a recently re-formulated 'direct' method (Magnetic LDL-C,
Polymedco) and a new, calculation-based indirect method were evaluated
for the estimation of LDL-C in human sera. Diagnostically important differences
were found between an in-house and a commercial enzyme-linked immunosorbent
assay (ELISA) for the measurement of serum anti-oxidized LDL autoantibodies
in human subjects.
- A new technique for quantitative determination of lipoprotein(a)-cholesterol
(Lp[a]-C) concentration was evaluated and tested for possible associations
with
Lp(a) concentration and apo(a) isoforms in human sera.
- The association of apo(a) isoforms and the type and titer of anti-cardiolipin
antibodies (ACA) with premature thromboembolic events was studied in patients
with systemic lupus erythematosus (SLE). Independent of risk assessment
with traditional ACA measurements, determination of genetically determined
apo(a) isoforms
improved the prediction of premature thromboembolism in these patients.
- Colorimetric methods (Randox Labs.) were evaluated for quantitative
assessment of superoxide dismutase, glutathione peroxidase, and the total
antioxidant status. Reference intervals were established in healthy adult
subjects. Because of analytical limitations, the method for total antioxidant
status was considered unacceptable
for diagnostic use. A significant correlation was discovered between glutathione
peroxidase and ionized magnesium in females and between glutathione peroxidase
and age in males.
- A colorimetric method for reduced glutathione (GSH) and an HPLC method
for GSH and GSSG (oxidized glutathione) were developed by modifying previously
published techniques. A commercial colorimetric kit (Calbiochem) was also
evaluated for GSH. The commercial colorimetric method gave GSH results
that were twice as high as those obtained with the other two methods, resulting
in a twice as high reference interval for GSH as those published in the
literature.
Operational
- The following new tests were offered during FY '97: CSF lactate, serial
parathyroid hormone (PTH), and specimen processing (with no testing in
CPD).
- Serum cortisol was added to the battery of tests available on a routine
basis on
weekends and holidays.
- The following tests were modified or improved: vancomycin, prolactin
transferrin, aldolase, uric acid, creatinine (from Jaffe to enzymatic),
free thyroxine (FT4), and thyrotropin (thyroid-stimulating hormone,
TSH).
- During FY '97, three mid-volume Hitachi 917's were replaced with two
Hitachi 917 analyzers, a Dade aca IV was replaced with anaca
Star analyzer, and two Beckman Synchron CX3's were replaced with a more
advanced Synchron CX3 Delta Clinical System. The new instruments are expected
to improve both efficiency and turn-around times. For spectrophotometric
analysis of different hemoglobin species in whole blood, the IL 482 CO-oximeter
was replaced with an IL-682 CO-oximeter.
- Support of the Bedside Glucose Monitoring Program continued by monitoring
of all quality control activities such as CAP proficiency testing, split-sample
comparison of results by trained nursing and laboratory personnel, verification
of these results with the laboratory's glucose method, and checking of
all glucose monitors before routine use.
- Since both the former in-house candidate method Abbott TDx and the
current binding assay in the reference lab (MML) for urinary free cortisol
exhibited interferences from drugs and steroid metabolites, a method with
the quality of a reference technique is now being developed by using high
performance liquid chromatography (HPLC) technology.
- Possible interferences due to hemolysis, hyperbilirubinemia and lipemia
on the current 'direct' and calculated (Friedewald) methods for the measurement
of low-density lipoprotein cholesterol (LDL-C) were further investigated.
Interference patterns were identified that aid in correct clinical interpretation
of LDL-C results obtained by various techniques.
- A joint study with the Hematology Service evaluated an automated urinalysis
workstation (Yellow IRIS) for spinal fluid analysis.
- Evaluation of new firmware for the Yellow IRIS reagent strip reader
for urinalysis was completed. Implementation of this firmware allows replacement
of the existing reagent strip reader (BMD CUA) and consolidation of all
urinalysis tasks onto one instrument.
Honors, Awards, and Scientific Recognition
- ·Andrew Hruszkewycz served as a member on the Council on Clinical
Chemistry
for the American Society of Clinical Pathologists (ASCP).
Hematology
Research
- Evaluation of the possible causes of the bleeding diathesis in Hermansky
Pudlak Syndrome showed that one-third of patients have abnormally low von
Willebrand factor activity in their platelets. Long term evaluation of
the clinical history of these patients will be necessary to assess whether
the low values will predict the severity
of bleeding in these patients.
- Monoclonal antibodies which have been developed to study platelet activation
were used to evaluate the thrombocytopenia in some patients with von Willebrand's
disease; the results indicate that platelet activation is present in some
of these patients and is a cause for the thrombocytopenia.
- An assay to measure plasma glycocalicin was developed which will identify
platelet destructive processes; patients with HIV and thrombocytopenia
have been assessed
to help determine whether their thrombocytopenia is caused by increased
destruction or decreased production of platelets.
- A group of 30 patients with Idiopathic Inflammatory Myopathy have been
studied
to determine whether assays for hypercoagulability will aid in the evaluation
of the clinical disease activity over time; abnormalities have been observed
in the levels
of inhibitors of coagulation and in thrombin-antithrombin complexes.
- The mutation present in a family with an abnormal von Willebrand factor
which does not bind factor VIII properly has been inserted into the gene
for von Willebrand factor, expressed in cultured cells, and assessed for
its binding activity. The expressed protein has a moderate defect in binding
factor VIII.
- Clinical evaluations of 6 patients with thromboembolic disease have
revealed that they have hereditary resistance to activated Protein C and
carry the mutation for
factor V Leiden in their factor V genes.
- Flow cytometry has been utilized to evaluate the efficacy of on-going
treatment in patients with Large Granular Lymphocytic Leukemia.
- Forty-two patients have received urokinase through their venous access
devices (VAD's) to relieve obstruction caused by a fibrin sheath; a portion
of the group
also received heparin. With follow-up studies, it was shown that the probability
of reocclusion of a reopened catheter was only 0.28 within 6 months.
- Intra-clot, pulse-sprayed tissue plaminogen activator has been administered
in 28 patients to treat VAD-related thromboses; clots recurred in 6 patients,
they could
not be lysed in 8 patients, and the veins remained open in 12 patients.
- Histidine-rich glycoprotein (HRGP), a plasma protein which may promote
hyper-
coagulability, has been purified, and assays have confirmed its anti-heparin
and plasminogen-binding activity. Further assessment is being carried out
to understand its function, particularly with regard to thrombosis.
- The stoichiometry of the platelet-heparin-PF4-IgG interaction has been
investigated as part of a study to understand the mechanisms of thrombocytopenia
in Heparin-induced Thromboctopenia.· Both follow-up and accrual
of patients has continued with Aplastic Anemia patients for treatment with
antithymocyte globulin and cyclosporine; the durability of response, incidence
of late complications and long-term prognosis are evaluated.
Operational
- The major portion of the arrangements for the administration of the
performance of testing for animal species was structured by the Hematology
Service. We also made major adjustments in our instrumentation (Cell Dyn),
evaluating and specifyng the electrical gains necessary to accurately assess
blood counts on 12 different animal species.
- A comparison evaluation of the Iris and Cell Dyn for assessment of
body fluids was completed which showed that the current manual methods
are most satisfactory at present.
- Evaluation of fibrinogens and thrombin times in a fully automated format
was completed and initiated for use in the main coagulation area (MDA's).
Mixing studies were also automated and transferred to the MDA.
- A group of technologists completed training and are currently interpreting
differentials for all bone marrow smear specimens.
- A new ordering system for scheduling bone marrow tests through the
Central Ordering System was put in place.
- An extensive evaluation was completed on the Variant Hemoglobin system
for evaluation of abnormal hemoglobins and glycosylated hemoglobins. Case
histories with graphics and nstructional questions and an algorithm for
replacement therapy for hemophilia were placed on the hematology web pages.
Immunology
Research
- Characterization of monocytes using a panel of monoclonal antibodies
was performed in patients with recurrent mycobacterial infections. Two
patients in this latter group were identified that have a defect in the
gamma interferon receptor alpha chain.Evaluation of the possible expression
of this receptor in amniocytes was under-
taken as a possible pre-natal screen for this disorder.
- Extensive lymphocyte immunophenotypic characterization of patients
with the
autoimmune lymphoproliferative syndrome (ALPS) and their family members
was undertaken.
- Evaluation of eosinophils using a panel of monoclonal antibodies and
a specific gating procedure was undertaken in patients with hypereophilia
and other conditions with ncreased levels of circulating eosinophils.
- ·The effects of IL-10 therapy on the peripheral blood lymphocytes
in patients with psoriatic arthritis was initiated.
- A neutrophil oxidative burst assay was used to evaluate the persistence
of allogeneic granulocytes following transfusion in patients with chronic
granulomatous disease.
- sCharacterization of genetic defects in the common gamma chain gene
characteristic of X-linked severe combined immunodeficiency by dideoxy
finger printing (ddF)
was initated in collaboration with NHGRI and initial work to establish
a non-
radioactive method to perform this genetic screening was undertaken.
Operational
- An assay to evaluate anti-thyroid peroxidase was added to the test
menu and a
screening test for antibodies to extractable nuclear antigens (ENA) was
added to economize on reagents for specific ENA antibody testing.
- A new automated ELISA instrument was evaluated which should be more
reliable
and have greater capacity than the current ELISA system.
- A standard flow cytometric assay for neutrophil oxidase activity was
offered on a weekly basis for patient and carrier testing. This assay identified
one patient with CGD who had been previously felt to be normal based on
an NBT screening test.
The new assay has been shown to be reliable in assaying samples that are
shipped
to the NIH by overnight courier.
- Four color flow cytometry has been evaluated to provide special capabilities
in selected studies.
- Support for two Institute investigators was provided on research stored
samples in screening for the presence of rheumatoid factor in one study
and identifying EBV seronegative individuals for another investigator.
Microbiology
Research
- Studies of Helicobacter-like organisms are ongoing to help further
define the various species, and their role as agents of human infection.
Focusing on the urease enzyme gene, we have amplified, cloned and sequenced
a region of the ure B gene, which
has revealed considerable variability when compared to other available
sequences
for this group of bacteria. The ure B gene should prove to be useful for
identification
and differentiation of fastidious or nonculturable Helicobacter,
as well as provide information on the prevalence and significance of different
species.
- Extensive studies were completed to modify and validate a commercially
available
kit for Legionella PCR that was designed for environmental samples in order
for us
to use it as a test for clinical specimens. Appropriate processing to eliminate
PCR inhibitors was determined and has resulted in a sensitive yet specific
diagnostic
assay, which should enhance our laboratory's ability to make a timely diagnosis
of this infection.
- PCR for the diagnosis of cytomegalovirus (CMV) in blood was successfully
developed, with a sensitivity of detection as low as three to five viral
genome equivalents. The assay has been further refined into a competitive
quantitative PCR assay which may be of greater clinical utility for either
predicting the likelihood of progression
to serious CMV disease, or to look at response to antiviral therapies.
Preliminary comparisons have demonstrated that this assay has greater sensitivity
and permits
an earlier detection of CMV than either p65 antigenemia detection or shell
vial
CMV culture. In addition to implementing our in-house assay, we will also
be
evaluating a new commercial assay just recently released but approved for
research use only.
- A PCR assay for mycobacteria using primers targeting a segment of the
16S ribosomal RNA gene permits us to detect all members of the genus Mycobacterium
whileexcluding other common bacteria. Individual species will then be identified
using specific europium-labeled fluorescent probes. Coupled with a simplified
sample preparation technique, this method could greatly reduce the time
to detection of mycobacterial infections, as well as provide early presumptive
species identification for patient care.
- A PCR method has been developed for the detection of microsporidia
in stool
specimens. Using restriction endonucleases on the PCR product to identify
isolates
to species was not sufficiently sensitive and reliable, so we have combined
PCR with the technique of single strand conformation polymorphism (SSCP)
to identify the genus and species of each positive patient. Preliminary
studies show this to be a promising method.
- Cultivation of microsporidia using a shell vial system containing either
a fibroblast
or epithelial cell line suggests replication occurs in either cell line.
By staining cells, microsporidial inclusions can be detected, but we hope
to develop a more definitive PCR method to quantitate the amount of microsporidial
DNA present. This assay would then allow us to look at the effects of disinfectants
and environmental conditions on growth of the organism. Such an assay would
also provide us with an in
vitro method for susceptibility testing of antimicrosporidial drugs, an
important
step towards improving treatment of patients infected with microsporidia.
- Rapid culture of BK virus, PCR assay and cytological examination for
BK virus are under study. Detection of this virus may be of use in the
management of bone marrow transplant patients, and we are looking for the
most clinically useful BK assay.
- · Enhancing the growth of CMV in shell vial cultures would lead
to faster as well as more sensitive culture results. Incorporation of either
indo- or bromo-deoxyuridine
or hexadimethrine bromide into the shell vial is being examined as a possible
modification to improve CMV growth.
- A PCR procedure using degenerate primers was used to amplify a fragment
from chromosomal DNA from a virulent strain of M. tuberculosis (H37Rv).
The primers were based on known sequences of LpxA/LpxD genes from various
organisms, in
an attempt to look at a region associated with the virulence or "cord"
factor, known
to reside in the lipid component of the cell wall. The product is thus
far specific forM. tuberculosis, and has a sequence that shows a
perfect match with a gene proposed to encode a possible membrane associated
protein with ATPase motif. Further studies
of the location and function of this gene are underway.
- Two commercial procedures have been evaluated for yeast susceptibility
testing
and found that the E test was more difficult to interpret than the other
method, a rapid colorimetric test. This latter method correlates well with
the more laborious reference method, and we can now offer testing on clinical
isolates as a research tool for
investigators.
- A review of bronchoalveolar lavage results has been initiated with
the Cytopathology Section of the Laboratory of Pathology, NCI, to compare
the yield of diagnoses of infectious etiologies as performed by the Microbiology
Laboratory and by the Cytopathology Laboratory. Such a review will help
to define what would be optimal handling of BAL specimens to facilitate
rapid as well as sensitive procedures for
each group of respiratory pathogens.
- PCR amplification of a portion of the genome of rapidly-growing mycobacteria
and Nocardia, followed by restriction fragment length polymorphism
(RFLP) analysis
is being developed for use in the diagnostic laboratory to identify these
organisms. Preliminary results show the technique to provide identification
much more rapidly than conventional methods, and suggest that it will also
permit more accurate discrimination among the species.
- No standardized susceptibility testing procedures exist for rapidly-growing
myco-
bacteria and Nocardia. We have compared several different methods
and will
concentrate on further development of a microdilution procedure. We are
currently involved in a multi-institution collaborative study looking at
reproducibility of
methods for testing rapidly-growing mycobacteria.
Operational
- The infectious diseases molecular diagnostics section has continued
to develop methods and optimize procedures for service tests. An in-house
developed PCR based technique for the detection of CMV in cerebrospinal
fluid specimens was validated, and became the first PCR test to be offered
and performed within the Service. The addition of HSV and VZV PCR for spinal
fluids is expected to be added within the year. In addition, a Legionella
PCR assay is ready for implementation. and work is already underway to
define and validate Pneumocystis and Mycobacterial PCR assays.
- Methods utilizing molecular epidemiology have become state-of-the-art
for academic hospitals. We have kept current with the field by making methods
such as pulsed field gel electrophoresis (PFGE) available to quickly determine
if bacterial isolates are being spread by patient contact. This has been
particularly valuable in looking at the occurrence of oxacillin-resistant
Staphylococcus aureus and vancomycin-resistant enterococci that
have been encountered in Clinical Center patients. The versatility
of molecular typing, however, now allows us to investigate potential outbreaks
of almost any organism, such as members of the Enterobacteriaceae or Pseudomonas
species. Along with PFGE, we have introduced typing by a random amplified
polymorphic DNA (RAPD) procedure, which is more versatile, faster, and
simpler than PFGE. These valuable tools will allow us to track organisms
in our hospital in a timely, accurate, and reliable way that has not been
available to us in the past. We have already performed typing on multiple
occasions for the Hospital Epidemiology Service and for patient-care clinicians
concerned about particular organisms in
their patient population. We will be setting up a repository by computer
scanning
of all patterns from the organisms that we test, so that a Clinical Center
profile can
be established for various pathogens. This will give us a permanent record
that will permit more efficient tracking of strains and documentation of
trends.
- Members of the Microbiology Senior Staff presented lectures on antibiotic
resistance and on molecular epidemiology for a week long training course
(three sessions/yr) offered by the Hospital Epidemiology Service for physicians
in infectious disease training programs here as well as at other local
hospitals. In addition, we continued to offer daily patient-care and teaching
Service Rounds, and our two-week course in basic diagnostic microbiology
for NIAID clinical associates and trainees from other local area programs
(always filled from September through May each year). During this past
year we have also provided short training sessions for visiting scientists
from Tamil Nadu, India; Thessaloniki, Greece; Riyadh, Saudi Arabia; and
New Delhi, India.
Phlebotomy
Operational
- Collaboration between two phlebotomists, the Laboratory Manager and
staff from
the Special Photography Unit of the FBI has resulted in a video device
to aid in
the location of veins in patients that are difficult to draw.
