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 Department of Transfusion Medicine

FY '97 has been another year of growth for the Department of Transfusion Medicine (DTM). With a talented staff and state-of-the-art facilities devoted to the highly regulated industry of blood manufacture, the practice of transfusion medicine, and the science of clinical and basic research, the DTM is uniquely poised to contribute to the mission of the Clinical Center. DTM staff have applied their knowledge and expertise to improving the quality of patient care in many arenas, including blood safety, blood component therapy, hematopoietic transplantation, gene therapy, immunotherapy, and transfusion transmissible diseases.

Research Highlights

Blood Services Section (BSS)

Research activities of the BSS have focused on five general areas: (1) studies to optimize the safety and efficacy of apheresis donations by healthy volunteer blood donors; (2) studies to optimize the yields of cytapheresis procedures through modifications of commercially available apheresis devices and through the development of novel mobilization regimens for apheresis donors; (3) studies of the effect of blood irradiation on the function and survival of cellular blood elements; (4) kinetic studies of radiolabeled leukocytes and their trafficking patterns in various disease states; and (5) controlled trials of the efficacy of therapeutic apheresis in various disease states.

Evaluation is underway of the safety and efficacy of a new plateletpheresis instrument and modification of its parameters so that it routinely produces a high-yield plateletpheresis component that is profoundly leukocyte reduced. DTM was one of seven national field sites for evaluation of this device (Amicus Cell Separator, Baxter, Inc.). Collection of a platelet-
pheresis component that is leukoreduced prior to storage should improve the quality of the product by decreasing the incidence of cytokine-associated transfusion reactions, and should decrease transfusion costs by avoiding the need for expensive bedside leukoreduction filters.

A series of controlled, prospective studies comparing several pre-donation drug mobilization regimens that markedly increase the yield, in vivo survival, and function of granulocyte concentrates collected for transfusion was completed in FY '97. The combination of a single oral dose of dexamethasone plus a single injection of G-CSF given the day prior to donation was found to be significantly better that either drug alone, yielding a 2.5 to 3.5-fold increase in the granulocyte content of the product. Side effects of the combination drug regimen were well tolerated. The ability to collect a markedly more cellular granulocyte concentrate is likely to bring this infrequently used transfusion product back into circulation.

There is an ongoing evaluation of the clinical efficacy of G-CSF-mobilized granulocyte transfusions in improving outcome in neutropenic patients with life-threatening infections. Twelve Clinical Center patients received prolonged courses of daily G-CSF mobilized granulocyte transfusions in FY '97. In all six of the neutropenic patients, prompt clinical improvement in life-threatening infections (five patients had disseminated fungal infection) was seen. In the six non-neutropenic patients with CGD, the clinical effect of such transfusions could not be discerned in the absence of a prospective randomized trial. The next step in the systematic evaluation of this new transfusion component is a randomized, controlled, prospective study of the efficacy of G-CSF mobilized granulocyte transfusions in patients with aplastic anemia and life-threatening infections. This study is expected to begin patient accrual in 1998.

A study characterizing the distribution of irradiation dose in blood components treated with prophylactic gamma irradiation to prevent transfusion-associated graft-versus-host disease in the recipient was completed. Dose inhomogeneity was found to be significantly greater with free-standing cesium irradiators than with linear accelerator devices. Canister size and canister geometry markedly affected the distribution of dose. Dose homogeneity could be improved with placement of Styrofoam or Lucite "spacers" at the bottom of the irradiation canister to elevate the blood component into the mid-portion of irradiation field where the dose was more uniform. DTM has incorporated these findings into its standard blood irradiation practices.

Two collaborative, controlled, randomized trials demonstrating the clinical efficacy
of plasma exchange versus intravenous immunoglobulin or placebo in accelerating the neurologic and psychiatric recovery of children with Sydenham's chorea or other forms
of childhood obsessive-compulsive disorder were also completed.

Analysis of the efficacy of LDL-apheresis using dextran sulfate adsorption chroma-
tography (Liposorber LA-15 System, Kaneka, Japan) in stabilizing or causing regression
of atherosclerosis in patients with familial hypercholesterolemia, Type IIa is on-going.
The short-term efficacy of such treatment in acutely lowering LDL cholesterol levels
was documented and published by the multicenter study (in which DTM participated)
two years ago; the current study is evaluating the effect of therapy on long term progression of atherosclerotic disease and cardiovascular morbidity and mortality.A study was completed to evaluate the effect of storage time, storage temperature and filtration temperature on the efficacy of white cell removal from red cell units. The degree
of white cell removal was significantly greater in red cell units undergoing poststorage rather than prestorage filtration using a standard bedside filter. In poststorage filtered units, neither the temperature nor rate at which filtration was performed affected the degree of leukoreduction, although a greater range of residual WBCs was seen with bedside filtration. These findings suggest that optimal leukoreduction of red cell units is accomplished in the transfusion labor-
atory rather than at the bedside, and that white cell removal by filtration is least effective if performed immediately after the units are collected.

BSS and TSL senior staff members are the primary investigators on a multicenter study
of the acquisition of hematopoietic stem cells for second transplants from G-CSF stimulated donors participating in the NMDP that began in FY '97. The objectives of the study are to monitor the immediate and long-term safety and sequelae of G-CSF administration in healthy, volunteer donors, to compare donor tolerance of, psychosocial response to, and adverse effects of first donations of marrow versus second donations of PBSCs, and to monitor the outcome of unrelated-donor PBSC transplants in terms of time to engraftment, incidence of acute and chronic GVHD, and disease free and overall survival. Donor accrual and data analysis are ongoing.

Transfusion Services Laboratory (TSL)

Investigations of autoimmunity in patients with a congenital defect in the gene responsible for programmed cell death, fas, continue. Patients with this syndrome, autoimmune lymphoproliferative syndrome (ALPS), experience splenomegaly and lymphadenopathy, hypergammaglobulinemia, autoimmunity and B cell lymphocytosis. They often have autoimmune phenomena including warm autoimmune hemolytic anemia, neutropenia, and thrombocytopenia. Patients with ALPS are being tested for antibodies to red cells, platelets, neutrophils and HLA antigens. They are also being typed for HLA class I and II antigens. The results of the antibody tests and HLA typings are being correlated with clinical features.

A protocol to investigate drugdependent hemolysis has been initiated. Drugdependent antibodies are a rare but serious cause of hemolytic anemia, but little is known of the nature of the antigens recognized by the drugdependent antibodies. The purpose of these studies is to use serologic and biochemical techniques to determine the nature of the antigens recognized by drug-dependent antibodies. Proteolytic and glycolytic enzymes will be used todetermine if the antigens are located on carbohydrates or proteins. Immunoprecipitation or monoclonal antibody capture assays will be used to more precisely define the molecules identified by the drug-dependent antibodies. To confirm the identity of the epitope, the antibody will be tested against red cells that lack the suspected target molecule. Tolmetin, cefotetan and quininedependent antibodies will be tested.

Polymorphisms of Fcgreceptor III (FcgRIII, CD16) are being investigated. Two very similar genes encode for FcgRIII. FcgRIIIA is expressed on NK cells and macrophages and FcgRIIIB on neutrophils. FcgRIIIB has two clinically important polymorphisms, NA1 and NA2. We developed a new method to genotype NA1 and NA2 with PCR and restriction enzymes. The method will be used to compare the NA1 and NA2 genotypes of Japanese, African American and white people. If unusual genotypes are identified, donor neutrophils will be phenotyped and the molecular basis of the polymorphism will be determined. Initial studies show that the NA1 and NA2 gene frequencies in Japanese are 0.70 and 0.30 compared to 0.30 and 0.70 in African Americans and whites.

TSS will collaborate with BSS in studies to investigate the yield of granulocytes from donors whose granulocytes are mobilized under different medical regimens and to determine the efficacy of granulocyte transfusions for neutropenic adults with bacterial or fungal infections. These studies are discussed above in the report of the Blood Services Section. Succinctly, the purpose of the donor study is to determine how a single dose of GCSF or dexamethasone or G-CSF plus dexamethasone influences granulocyte yield and the deleterious side effects seen with multidose GCSF. In addition, the data obtained will be used to determine the minimum interval between each donation and the cells collected will be used to investigate the storage of GCSF mobilized granulocytes concentrates. In the second study, eligible patients will be randomly assigned to receive conventional antimicrobial therapy with or without GCSF mobilized granulocyte transfusions. The response of their survival will be monitored.

Kazuhiko Matsuo, M.D. joined the LSS as a Fogarty Fellow in August. His research will be directed toward the structure and function of neutrophil specific antigens and drug-dependent RBC antibodies. Three new SBB students started new projects in the LSS. One project is investigating the effects of biotinylation on the expression of RBC antigens, another is comparing the results of RBC phenotyping using the gel and the tube agglutination assays and the third is developing methods to test for granulocyte specific antibodies using stored granulocytes.

HLA Laboratory (HLA)

The human leukocyte antigens (HLA) are the most polymorphic genes in the human genome. Knowledge about HLA polymorphisms in relation to peptidebased, T cellrestricted vaccination protocols is important for understanding T cell recognition and for improving T cell antigenspecific vaccination strategies. The HLA laboratory has developed highresolution polymerase chain reaction (PCR) techniques for typing HLA class II antigens and heteroduplex techniques for highresolution analysis of HLAA2 subtypes (the dominant allele in the U.S. population). The lab has defined models to study the heterogeneity of expression of HLA class I molecules in different tissues and described mechanisms by which some tissues may escape immune recognition. It is also exploring techniques aimed at exemplifying the relevance of the polymorphisms of HLA with regard to T cell activation and inactivation.
The ultimate goal is to identify the steps occurring in response to T antigenspecific vaccination and their correlation with clinical responses.

Technical development projects include: (1) Dr Kicklighter has developed a technique
to differentiate HLAA2 subtypes using heteroduplex typing. Heteroduplex typing has several advantages over the standard typing method that uses sequence specific primers in that it can identify new alleles and is simpler. The technique will detect single nucleotide differences between alleles. (2) Dr Lee is developing a method to identify and monitor the frequencies of clones of cytotoxic T lymphocytes (CTL) through the characterization of
their unique T cell receptors. The techniques being used include directed heteroduplex
analysis, PCR and northern blotting. (3) Dr Bettinotti has sequenced the DNA from five patients with unusual HLAA phenotypes. At least one new allele has been identified.
She also is devolping a method to use heteroduplex analysis of HLACw antigens.
(4) Toni Simonis has been comparing the HLA-A*02 allele frequencies in North American caucasians, North American afroamericans, North American hispanics and Taiwanese.
(5) Kathy Barracchini is developing a computer program to determine which HLA alleles will react with any PCR primer of 18 to 22 nucleotides.

Cell Processing Section (CPS)

Based on the results of a preclinical trial of a new 2-step immunoadsorption system (CellPro) for T cell depletion last year, CPS moved forward to incorporate this system into a clinical trial in allogeneic PBSC transplantation. To date, 10 patients have been transplanted on thisprotocol, and we are generating valuable data in a comparison of processing pooled apheresis collections vs. fresh processing. This study will be completed in the next fiscal year.

Following completion last year of a pilot study of a new assay method (Volumetric capillary cytometry, Biometric Imaging, Inc.) for quantitating CD34 hematopoietic progenitor cells in mobilized peripheral blood and clinical products, we further evaluated this method using plastic capillaries and a modified computer software analysis system during this fiscal year. We anticipate incorporating this methodology into our laboratory's activities to reduce the number of STAT flow cytometry assays performed. In addition, we will be evaluating a new assay for detection of CD3+ T cells in hematopoietic progenitor cell products.

Based on the success of 10 preclinical runs, plans have been formulated to incorporate CD34+ cell selection on peripheral blood progenitor cells using the new automated Baxter Isolex 300i into two NIH clinical protocols. One protocol was started in June 1997 and uses positive selection as part of a 2-step process for tumor cell depletion from autologous PBPCs in breast cancer patients. The second protocol, to begin in FY '98, will use the Isolex 300i to accomplish T cell depletion of PBPC collections in patients with chronic ITP prior to high dose chemotherapy and autologous transplantation.

Two additional physician-investigators joined the CPS staff with primary duties in research and development. They are Scott Donnelly, M.D., in a three year IRTA fellowship position, and Anne Husebekk, M.D. in a one year position as a Visiting Scientist, on sabbatical from
her university hospital position in Tromso, Norway.

Infectious Diseases Section (IDS)

The focus of the infectious diseases section in 1997 was again on hepatitis viruses C and G, and resulted in 10 publications with three others in-press. In 1996, IDS investigators were part of the team that discovered the hepatitis G virus (HGV), and this year expanded the initial investigations by studying the clinical relevance of this agent. In a major study published in the New England Journal of Medicine, we demonstrated that infection with HGV was present in 1%-2% of the blood donor population, a rate five-fold higher than that for the hepatitis C virus (HCV) and 10-20 fold higher than that for the hepatitis B virus (HBV). We also established that the agent was readily transmitted by blood transfusion and that it could lead to persistent infection in the recipient. Despite the ease of transmission, we could not find a specific association between this agent and transfusion-associated hepatitis and, surprisingly, found no correlation between HGV and biochemical evidence of hepatitis. Indeed,there was a striking dissociation between the level of virus and enzyme levels indicative
of hepatic inflammation. In other studies we showed that HGV did not worsen the clinical course of coexistent hepatitis C and, indeed, there is preliminary evidence that HGV may
aid in the clearance of HCV when both agents are simultaneously transmitted. We have
also shown that while HGV is highly prevalent in both intravenous drug users and dialysis patients, there was no correlation with liver disease in these at-risk patient populations.
At the present time, it is unclear whether HGV is actually a hepatitis virus or whether it has pathogenicity for other organ systems. Most recently, we have studied the antibody response to HGV. While there is no antibody assay that is indicative of viral replication and useful as
a blood screen, one can now measure antibody to the viral envelope. This anti-E2 antibody is
a recovery antibody and allows one to measure total exposure to HGV. That rate of exposure is very high, being about 3% in the general population and as high as 85% in IV drug users. We have also found that about 85% of HGV infected individuals eventually recover from their infection as opposed to hepatitis C where 85% remain chronic viral carriers.

Since HGV did not prove to be the major agent responsible for cases of apparent viral hepatitis that could not be explained by hepatitis viruses A to E (non-A-E or cryptogenic hepatitis), we are currently entering into a viral discovery program to investigate the possibility of a new hepatitis agent. This is being accomplished through a collaborative research and development agreement (CRADA) with industry.

Studies on HCV have concentrated on the immune response to this agent and the neutralizing domain that is critical to viral containment or escape. Working with Drs. Patrizia Farci and Robert Purcell of NIAID, we had previously shown that neutralizing antibodies to HCV are developed, but that they are highly strain specific, transient and generally incapable of preventing the emergence of neutralization-resistant strains that result in chronic infection.
In this year's studies, we have shown that the critical site for HCV neutralization is located
in the hypervariable region of the envelope protein. It is the marked variation in this region
of the genome that allows the virus to continually escape immune attack and to result in persistent infection in the vast majority of patients. This finding bodes poorly for effective vaccine development because the target of induced antibody will be constantly shifting.

In other studies of HCV, we have described with Drs. Farci and Purcell that HCV is a
rare cause of fulminant, fatal hepatitis and in studies with Dr. David Thomas at Johns Hopkins have shown that in HCV and HIV coinfected patients, HCV viral titers increase as the patientsbecome increasingly immunodeficient, but that the increase in viral load does not correlate with worsening liver disease. This suggests that liver damage in HCV infection is due to immunologic damage rather than a direct cytopathic effect of the virus.

An interesting study of HIV infection was performed in the chimpanzee model. We inoculated a chimp with HIV and then obtained weekly blood samples to determine the interval to the first appearance of virus and the first detection of antibody to the virus. We found that there was a 5 week interval (window) until virus could be detected by culture
and by measurement of plasma RNA and cellular DNA. Antibody was not detectable for
an additional 3 weeks. In a second experiment. we inoculated window period samples (post-exposure weeks 3 and 4) into a second animal and found that these samples were not infectious. Infectivity could not be demonstrated until the fifth week sample when the first animal was culture positive and PCR positive. This study has major implications because it suggests that the infectious window for HIV is shorter than previously suspected. Further, it suggests that donor screening for HIV-RNA could obliterate the infectious window and perhaps totally prevent HIV transmission by blood transfusion. Further studies to solidify this observation are in progress.

This year saw the continuation of two large-scale, long-term epidemiologic studies. The first is a prospective study of transfusion-associated hepatitis (TAH). We have now followed approximately 700 transfusion recipients since the initiation of second generation anti-HCV screening of blood donors in 1992. Not a single patient has developed HCV infection and only one of 700 had any form of hepatitis, this being a very mild case of unexplained etiology. Based on hepatitis C incidence prior to HCV donor screening, we would have expected 35 hepatitis cases in this population. Thus there has been a dramatic decline, and indeed near eradication, of transfusion associated hepatitis in the wake of anti-HCV screening.

The second ongoing study is an analysis of HCV infection in asymptomatic blood donors. We have now enrolled over 400 HCV-infected donors and 200 controls into long-term follow-up and have studied the routes of transmission and clinical, biochemical and histologic outcome of their infection. As reported last year 42% of donors were infected as the result of distant intravenous drug use, 27% as the result of blood transfusion and small proportions as the result of other percutaneous exposures such as tattoos or occupation-related needle sticks.
A surprising finding was the very strong correlation between HCV infection and cocaine snorting and this association was highly significant independent of IVDU. We postulate thatthe use of shared snorting devices and bleeding from irritated nasal mucous membranes might represent a covert means of blood-borne viral spread. This year has seen a large increase in the number of patients undergoing liver biopsy in collaboration with the liver service, NIDDK. We have shown that hepatitis C-associated liver disease was very mild in the vast majority of patients and that only 13% had severe histologic lesions despite a mean duration of infection that exceeded 15 years.

IDS is engaged in a collaborative study with the National Children's Medical Center
to determine the proportion of children who were infected with HCV prior to HCV donor screening and the outcome of that infection in this young population. The goal is to determine if the natural history of HCV infection is the same in children as it is in adults. If so, then treatment emphasis might switch to this younger population whose long anticipated life-span might put them at increased risk for the very slowly evolving severe chronic sequelae
of HCV infection. The data from this study are too preliminary to report at this time.

IDS is also part of a long-term retrospective-prospective, collaborative study of transfusion-associated hepatitis. After a mean follow-up of 20 years, less than 10% of patients with transfusion-associated hepatitis have developed clinical, biochemical or histologic evidence of severe liver disease.

The Molecular Diagnostics Lab of IDS has been engaged in more basic research involving mycoplasma, quantitative analysis of viral genomes and their clinical correlation's, evaluation of nucleic acid vaccines for HCV and gene therapy for hepatitis C infection. Studies of mycoplasma have been collaborative with Dr. Shyh-Ching Lo of the Armed Forces of Pathology and have led to the discovery of several novel mycoplasma agents. Our laboratory has developed serologic tests for these agents that have allowed screening of diverse populations. We have found a high prevalence of antibodies to M. penetrans in patients with HIV infection and Kaposi's sarcoma, and a high prevalence of M. genitalium in patients with non-gonococcal urethritis. Sexual transmission of M. genetalium significantly correlates with sexual transmission of HIV. We have also shown that both M. penetrans and M. genetalium are transmissible through blood transfusion. In 1997, our lab and Dr. Lo's lab tested over 6,000 paired specimens from patients who suffered the Gulf War Syndrome. Our controlled studies did not support the early evidence that mycoplasma might play a causative role in the Gulf War Syndrome. We are currently studying whether mycoplasma contribute to the development of neoplasia in those chronically infected. We have shown that some species ofmycoplasma can transform cells in vitro after long-term cocultivation and also that several oncogenes were activated.

Studies of the feasibility of nucleic acid vaccination for the treatment and prevention
of hepatitis C were continued this year. The typical immune response in HCV infection is incapable of clearing the virus. Nuclei acid immunization could boost the immune response in that endogenously expressed proteins could be recognized by HLA class-I specific molecules and expressed as cell surface peptides that are linked to MHC and could stimulated cytotoxic lymphocytes to attack viral-infected cells. We are using the mouse model to evaluate this approach and in the past year have prepared several different plasmid constructs
of the HCV core gene and have shown that these can induce antibodies to the HCV core (nucleocapsid) in the mouse. We are now developing methods to assess cell mediated
immunity to the expressed protein.

This past year saw the initiation of studies to develop a gene-therapy strategy for HCV infection. It is our goal to establish functionally measurable gene expression in hematopoetic cells using virally-mediated delivery of the HCV core gene, to demonstrate lineage-specific gene expression and to demonstrate the efficacy of lineage-specific gene expression by assess-ment with CTL and tumor killing assays directed to HCV-specific epitopes. Ultimately, this method will be evaluated as a therapeutic modality.

Service Accomplishments

Blood Component Therapy

The DTM collects and processes 98% of the blood components transfused in the Clinical Center. Ensuring the quality, safety, and effectiveness of these components requires significant input from each section of the department. Activities that are involved in the manufacture of blood for transfusion include donor recruitment, donor screening and selection, blood collection and apheresis, component processing, laboratory testing, labeling, storage and distribution.

There were approximately 4,600 units of whole blood collected and 2,500 plateletpheresis procedures performed, the latter of which yielded which 23,000 units of platelets. These numbers are slightly lower than last year. Transfusions of red blood cells decreased by 12% this year to 4,158 and, after five years of steady increase, the demand for platelets finally begn to drop with 17,076 units transfused. This can be attributed in part to stability in the number of patients receiving allogeneic marrow transplants on the NHLBI(2W) transplant service, which has been operating at the same bed occupancy rate for two years. The increased use of peripheral blood stem cells (PBSC) over bone marrow for
hematopoietic grafting on most NHLBI and NCI protocols has also had a significant effect.
A preliminary analysis suggests that recipients of PBSCs require fewer RBC and platelet transfusions than bone marrow transplant recipients because of more rapid engrafting.

In contrast, the number of granulocyte concentrate transfusions more than doubled, increasing from 124 to 287. Provision of a completely new transfusion component, G-CSF-mobilized granulocyte concentrates, began in July 1996 and interest in its use is expected to increase into FY '98. As described in the research highlights for BSS, studies conducted by DTM documented the safety and efficacy of administering G-CSF to normal donors, with a resulting four-fold increase in granulocytapheresis yields.

DTM began two new initiatives in FY '97 aimed at producing high quality leukocyte reduced platelets for transfusion. These components are indicated for patients with aplastic anemia, patients undergoing marrow transplantation, and any patient in whom avoidance of HLA alloimmunization or CMV transmission is critical. Use of this leukoreduced apheresis product also prevents the need to use an expensive bedside leukoreduction filter, and is predicted to save the Clinical Center about $45,000 per year. The first initiative involves the acquisition of a new plateletpheresis device, the Amicus Cell Separator (Baxter), which produces a high-yield platelet product with an extremely low level of white cell contamination. The second initiative involves in-laboratory filtration by TSL staff prior to issue. This procedure has been shown to yield a better, more standardized component as compared to bedside filtration.

Clinical Services

The DTM component infusion service, begun in 1991 as a simple outpatient red cell
and platelet transfusion service, has expanded to include infusions of cryopreserved, thawed mononuclear cell components. The PBSC infusion service, initiated two years ago as a service to institutes performing hematopoietic transplants, performed 90 stem cell infusions, about the same as last year. However, infusions of peptide activated lymphocytes for cancer treatment protocols and genetically altered, cryopreserved lymphocytes in syngeneic anti-HIV immunotherapy protocols have tripled from FY '96 to FY '97, and are expected to continue to increase next year.Therapeutic apheresis procedures performed as part of clinical trials increased 20% in
FY '97, reflecting growth in the number of lymphocyte and monocyte apheresis components collected for ex vivo manipulation, immunotherapy, tumor vaccine programs, and gene-marking and gene therapy trials. Gene-marking and gene therapy trials continued to be
active in patients with HIV infection (syngeneic immune reconstitution), breast cancer
(MDR trial), chronic granulomatous disease (p47phox correction), Gaucher disease (glucocerebrosidase gene) and Fanconi anemia (FACC gene). Activity in clinical trials of plasma exchange, secondary plasma processing, and red cell exchange was about the same
as in FY '96, with 68 plasma exchanges performed in children with obsessive-compulsive disorders, adults with autoimmune disorders, patients with TTP following marrow trans-
plantation, and patients with familial hypercholesterolemia.

As in previous years, use of the autologous red cell donation service was minimal;
autologous donations decreased from 57 to 38 in FY '97. Most of these units were donated by allogeneic marrow donors participating in NMDP or NHLBI marrow donation protocols. Directed donations, discouraged by the DTM, were stable at 36 units per year, suggesting continued confidence in the safety of the DTM blood supply by Clinical Center patients and their families.

Laboratory Services

The RBC tube agglutination test was replaced by the gel agglutination test for performing red cell antibody screening and crossmatching. The gel system is more efficient, generates less liquid waste and can be automated in the future. The performance of the direct antiglobulin test (DAT) as part of all routine RBC antibody type and screen requests was discontinued. The immunohematology reference area of TSL introduced a new service in FY '97 to determine the genotype of platelet specific antigens. TSL now performs both platelet antibody testing and phenotyping.

The number of tests performed by the HLA Laboratory this year went up 27 %. This represents a 22% growth in class I antigen typings and a 35% growth in class II antigen typings. Increases in the number of active protocols and in the number of tests requested by the NCI accounted for much of the new demand. In addition, the introduction of new molecu-lar HLA-Cw and HLA-DR assays resulted in increased requirements for quality assurance testing. In order to ensure all ordering of tests is appropriate, Dr Marincola has begun to review protocols that request HLA testing but were not involved in direct patient care. Thedevelopment of molecular tests for HLA antigens has continued. The laboratory now tests HLA-Cw alleles using PCR and sequence specific primers. The PCR primers used to test
for class II antigens were redesigned to improve the efficiency and resolution of the assay.

The Transfusion Transmitted Viruses Laboratory (TTVL) offers laboratory analyses for the viral markers of hepatitis and retroviral infection. The TTVL performed 118,700 assays during FY '97, a 4.2% increase over the previous year. During this period, TTVL also began to offer new services such as polymerase chain reaction (PCR) testing for HCV RNA. At present, this service is primarily provided for investigators with liver disease protocols. In
the third quarter of this year, responsibility for testing of serum ALT level as part of blood screening was transferred to the Chemistry Section of the Clinical Pathology Department. This transfer was considered as part of a Clinical Center-wide effort to gain efficiencies in service operations. The TTV Laboratory of DTM continues to maintain responsibility for compliance with blood bank specific regulatory requirements.

The Cell Processing Laboratory performs CBC's for the purpose of donor qualification as well as more extensive determinations of cellular content for the purpose of product characteri-zation and acceptance. While the large number (>20,000) of automated cell counts remained relatively steady from FY '96 to FY '97, there was a 71% increase (from 136 to 232) in
the time-consuming manual PI assay for quantitation of low WBC content of leukodepleted products. Also, there were dramatic increases in the number of endotoxin assays performed (from 18 to 110) and mycoplasma cultures sent out (from 31 to 125). These data are consistent with the increased number of clinical components prepared for protocols involving complex processing and long-term culture.

Stem Cell Services

CPS continued to support over 30 NIH clinical protocols sponsored by NCI, NHLBI, NIAID, NINDS, and NCHGR during this fiscal year. These included clinical trials in hema-
topoietic transplantation, gene therapy, and immunotherapy, including autologous tumor vaccines. CPS support of cellular therapeutic protocols consists of processing, storage, and final component preparation of all specialized cellular components for patient use, all of which required GMP handling and tracking. The total number of cellular components prepared and/or handled by the CPS increased by 21% in FY '97 from 474 to 575. The bulk of the increase was due to a dramatic increase in tumor vaccine components, including antigen presenting cells (APCs) and peptide activated lymphocytes (PALs), from 109 to 222. Accrualof patients on two of the five active NCI study protocols being done at the Naval Medical Center Oncology Program account for this increase. Also noteworthy is the substantial activity related to cryopreservation of research vials associated with those protocols. Because this workload exceeded the section's capacity based on staffing, we were forced to discontinue this valuable research service in August 1997. The total number of containers thawed and prepared for infusion by the CPS during FY '97 was 1,001.

Construction of the new GMP Cell Processing Facility, the largest multi-use cell processing facility of its kind, was completed in February 1997. Clinical cell processing operations began on a limited basis in the new space in late May 1997, and we anticipate that all clinical cell processing operations will be transferred to the new facility by the end of this fiscal year. In addition to serving the needs of the Institutes, this facility has become a prototype in its design, validation and operation. Over 90 extramural scientists representing academic institutions and biotechnology firms from all over the world as well as numerous intramural scientists have already visited the facility. In the next fiscal year, we will focus on establishing and improving operational support systems including cleaning, air handling, and environmental monitoring with an emphasis on streamlining the handling of supplies and documentation of facility functions.

The number of peripheral blood stem cell collections in FY '97 was 140, the same as for the previous year. The majority of these were autologous collections in patients with breast cancer, myeloma, lymphoma, and sarcoma; 40 of these PBSC collections were obtained from healthy donors as a source of stem cells for allogeneic transplant.

Blood Component Support for Basic Research

There was a dramatic increase in the number of requests from NIH investigators to collect blood components from healthy donors for research use in FY '97. Requests for research whole blood components increased 22%, from 961 to 1,168 and requests for
research apheresis components increased 122%, from 1,749 to 3,884. Termination of the volunteer donor apheresis program at the NCI Frederick Cancer Facility in September 1996 and transfer of the apheresis activities of that program to the BSS section of DTM accounted for much of this new demand.

CPS continued to provide specialized cellular preparations from normal donors and patients for use by over 50 NIH investigators for research. Specific research uses rangedfrom basic studies on cellular physiology to pre-clinical process development for anticipated clinical trials. These preparations included over 202 large-scale separation procedures for isolation of lymphocytes and monocytes and for CD34+ hematopoietic cells, with an increase of approximately 25% over FY '96.

In addition, DTM provided almost 4,000 buffy coats prepared from whole blood donations to research laboratories throughout NIH.

National Marrow Donor Program

The NIH Marrow Donor Center, a participant in the National Marrow Donor Program (NMDP), has continued to experience dramatic growth in the size of its unrelated-donor registry, currently at 45,000 participants. Minority representation in the NIH marrow donor registry continues to be one of the largest in the country, at 27% of overall participants. During FY '97, 24 NIH marrow donors underwent marrow harvest, bringing the total number of NIH-donor harvests to 170 (out of a total of 5,900 NMDP harvests performed nationwide).

Information Systems

DTM continues to work closely with the Information Systems, Clinical Pathology and Anatomic Pathology departments in the planning, design, installation, training, and validation of a new laboratory information system (LIS) to serve the entire hospital. Substantial supervisory and staff time has been allocated to this project which will replace outdated legacy systems in some areas, and manual, paper-driven systems in others. Final implementation of the labora-tory and transfusion service modules is expected to occur in FY '98. The donor module is still in the design phase and must be approved as a medical device by the FDA before it is released to the Clinical Center. DTM serves as the primary consultant to the vendor on this design project.

Two major software applications are being developed in-house by ISD to address the special needs of the Cell Processing Section and the HLA Laboratory. Staff in both of these areas have invested significant time working with ISD in the development of user requirements and in the design phase. The first module of the Cell Processing application, including protocol administration and scheduling, was delivered in September 1997 for beta testing by CPS. The HLA project will convert and enhance an application originally designed by DTM staff using off-the-shelf DBMS software.

Training and Education

The ACGME accredited two year training program in transfusion now supports three fellows this past year and will increase to four next year. Because of the increasing complexity of the clinical rotations, the program was expanded in order to provide fellows with a broader and more in-depth clinical training experience while still allowing ample time for them to fulfill the research goals of the program.

The AABB accredited Specialist in Blood Banking Program continues to support three students. Two graduates from this year's class received national recognition with an AABB Fenwal award for the outstanding research that they completed at the NIH.

DTM continues to provide a variety of training and educational opportunities to professionals and students from the NIH community, universities and other institutions. The 16th Annual DTM Symposium on Immunohematology and Blood Banking was well received, with over 300 people attending. It covered several topics, including the development of blood substitutes, new blood products, immunology and cell processing. In addition to the annual symposium, DTM provides educational services throughout the year, through formal lecture series, mentoring programs, and hands-on technical training. TSL and HLA personnel taught an FAES course on immumohematology. BSS and TSL staff provided blood administration training to all Clinical Center patient care staff reponsible for blood transfusion. CPS and BSS staff have begun development of infusion guidelines and an associated training program for novel transfusion products that will target both DTM and Clinical Center nursing personnel. Charles Carter participated as an NIH preceptor in the Presidential Classroom Project
for high school students. DTM provided clinical, technical and research training to clinicians, researchers, fellows, residents, medical technologists, and students from within and outside of the NIH. Demand for this service is high, and DTM continues to receive a great number
of requests to provide training to both local and international students and professionals.

Honors and Awards

Several DTM personnel were recognized for their contributions to science and their professions in FY '97. Harvey Alter was the recipient of First Bill T. Teague Lectureship, Gulf Coast Regional Blood Center, Houston, Texas. Susan Leitman received the Charles E. Walter Memorial Award of the Mid-Atlantic Association of Blood Banks. De Tan received the Clinical Center fellows award for excellence in biomedical research. Jaime Oblitasreceived the MAPAVI 1996 Northern Virginia Volunteer Community Award for his efforts
in recruiting Hispanic donors to participate in solid organ and bone marrow transplant registries. Mr. Oblitas also received the 1996 Recognition Award for his assistance in planning
the Third International Transplant Congress in Washington, D.C. Gamma Biologics awarded Lisa Pinkney an SBB student scholarship, and Laura Barreda and Janet Smith were awarded AABB Fenwal scholarships for research completed during their SBB training. The HLA Laboratory received a perfect score on the UCLA International Cell Exchange again, and
it is the only laboratory out of 300 to do so for 8 consecutive years.

DTM staff continued to influence national standards setting and policy making through participation on a number of government, accrediting agency and professional society committees. These included the Board of Directors of the American Association of Blood Banks (AABB), numerous committees and work groups of AABB, the Board of Directors of the American Society of Apheresis (ASFA), the Board of Directors of the National Marrow Donor Program (NMDP) and committees of NMDP, the FDA Blood Products Advisory Committee, and others. The long list of invited lectures that were presented in FY '97 at meetings around the world attest to the unique contributions that DTM personnel have
made in their respective fields.

Department of Transfusion Medicine Service Statistics

Table 1. BSS Service Activities, FY '95 ­ FY '97 (Projected)  Service Category  1995 1996 1997  % of change: 1996-1997  Patient apheresis 1,163 1,257 1,419 13  1,163  1,257 1,419 13  Donor apheresis for transfusion 2,918 3,100 2,856 -8  2,918 3,100 2,856 -8  Donor apheresis for research 1,636 1,624 1,733 4  1,636 1,624  1,733 4  Clinical trial (therapeutic) apheresis procedures 634 768 916 20  634 768 916 20  Whole blood collected for research 904 961 1,168 22  904 961 1,168 22  Whole blood collected for transfusion 5,086 4,911 4,536 -7  5,086 4,911 4,536 -7  Components transfused in Blood Services 76 189 162 -14  76 189 162 -14

Table 2. Blood Components Transfused, FY '95 ­ FY '97 (Projected)  Service Category  1995 1996 1997  % of change: 1996-1997  RBC  4,553 4,737 4,158 -12.2  APLT  16,227 19,339 17,076 -11.7  FFP  454 1,117 685 -38.7  GRANS  58 124 287 131.5  BONE MARROW  54 36 24 -33.3  LYMPHS  74 115 181 57.4  PBSC  69 122 110 -9.8  CRYO  314 180 367 103.9

Table 3. Components Prepared by TSL, FY '95 ­ FY '97 (Projected)  Component  1995 1996 1997  % of change: 1996-1997  RBC  5,033 4,782 4,380 -8.4  FFP  561 679 831 22.4  CRYO  121 100 107 7.0  BUFFY COATS  4,487 4,147 3,919 -5.5  GRANS  31 105 317 201.9  PLATELETS, PHERESIS  23,089 26,365 22,718 -13.8  PRE-FILTERED RBC  204 895 1,748 95.3

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Questions about the Clinical Center? OCCC@nih.gov Or call: (301) 496-2563 National Institutes of Health, Warren Grant Magnuson Clinical Center, Bethesda, Maryland 20892. Last Modified 3/98