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Pre-transplantation work-up on a patient with end stage renal disease using Single Antigen Bead (SAB) testing showed significant anti-HLA-B*44:02 (5,000 MFI) and anti-HLA-B*44:03 (1,000 MFI) antibodies, with persistence on quarterly testing. No significant Class II anti-HLA antibodies were present. The patient received a potential offer from a living unrelated-donor expressing HLA-B*44:02. Based on the presence of anti-HLA-B*44:02 antibody, the crossmatch (XM) was predicted to be positive. However, the actual fluorescence cytometry crossmatch (FCXM) was negative. FCXM and Complement Dependent Cytotoxicity-XM (CDC-XM) studies with three surrogate donors who expressed HLA-B*44:02 (and no other potential confounding HLA types) were also negative. Additional assays were performed for detecting anti-HLA antibodies. Immucor® LSA® SAB analyses also revealed presence of anti-HLA-B*44:02 and anti-HLA-B*44:03 antibodies. However, One Lambda® Antigen Trays, C1q analysis, and iBeads®, did not detect elevated anti-HLA-B*44:02 and/or anti-HLA-B*44:03 antibodies. An extensive evaluation of all exposed and non-exposed epitopes expressed by the patient and the donor was performed to identify the non-shared epitopes between them. The donor specific antibody (DSA) pattern detected would be expected to conform to non-shared epitopes; however, non-shared "exposed" epitopes were not present in the DSA antibody pattern. Whereas, the apparent DSA antibody pattern consisted of antibodies to "non-exposed" epitopes. Altogether, it was concluded that the anti-HLA-B*44:02 antibody detected by SAB testing was directed against some denatured component(s) (non-exposed) of the HLA antigen attached to the SAB, and would not be clinically significant. The patient received the transplant and the post-transplant course has been uneventful for greater than 5 years. This case emphasizes: (1) A significant number of SAB may have denatured HLA molecules attached to them (2) The DSA and non-DSA anti-HLA antibody patterns should be evaluated for the expected epitope components to determine the clinical relevance. Additional testing should be considered to help with these analyses (3) The FCXM remains the "gold standard" for making the final decision to transplant, since the "stringent" use of a "predicted" positive XM using apparent DSA detected by SAB analysis may exclude XM-compatible donors.

Platelet refractoriness or lack of platelet increase after platelet transfusion is seen in patients receiving chronic platelet transfusion support. Antibodies may develop against human platelet antigens (HPA) and/or against HLA class I antigens. Crossmatch (XM) compatible platelets or HLA-identical or HLA-compatible platelets are typically used to manage transfusion refractoriness. We aimed to determine if percent calculated Panel Reactive Antibody (% cPRA) against class I HLA antigens could predict percent positive platelet XM when looking for compatible transfusion products.A retrospective review of all platelet XM performed at our institution between 2008-2012 was performed, and patient characteristics recorded. For each patient, the percentage of all positive platelet XM performed was calculated and compared with the corresponding % cPRA levels against class I HLA antigens.Mean and median % positive platelet XM for all 50 patients tested in the period 2008-2012 were 61% and 60% (range 0-100%), respectively. Mean and median % cPRA levels were 66% and 68% (range 0-100%), respectively. No correlation was seen between age, sex, race, or diagnosis and positive platelet XM results.The results of our study indicate that the % cPRA correlates well with the % positive platelet XM. Thus, a higher % cPRA alerts the blood bank that additional platelets will be required for XM and/or that it would be beneficial to request HLA-identical or compatible units.

Radiologic procedures form one of the cornerstones of diagnosis in hematological malignancies. Various radiological procedures are essential for the diagnosis, assessment of the therapeutic response, to identify disease relapse, and to evaluate for complications pre- and post-transplantation. The significance of radiology and radiological procedures in stem cell transplantation is expected to grow. In this chapter, we discuss the radiologic procedures and review the key imaging modalities and implications related to pediatric hematopoietic stem cell transplant (HSCT). We will also discuss the role of radionuclide Imaging for patients undergoing hematopoietic stem cell transplantation with a brief review of use of radio-labeled MIBG imaging in children with Neuroblastoma.

Panel reactive antibody (PRA) reduction protocols are used to decrease anti-HLA antibodies with concomitant PRA monitoring as a measure of successful treatment prior to organ and haploidentical blood and marrow transplant (BMT). We hypothesized that the more sensitive flow cytometry (FC) based assays for PRA [FlowPRAA female patient with myelodysplastic syndrome and a high HLA class I PRA [90% PRA and cPRA by complement-dependent cytotoxicity (CDC) assay and Flow PRA] required allogeneic BMT. Baseline HLA Class I and class II antigen typing was performed and a matched sibling donor was identified. Although baseline anti-HLA class I and class II antibodies measured by FC and CDC revealed no donor specific antibodies (DSA), the decision was made to attempt antibody desensitization to facilitate platelet transfusion during BMT. FC and CDC assays were performed to determine anti-HLA class I antibodies and cPRA/%PRA prior to starting desensitization and at the end of desensitization. Over the course of desensitization and BMT, a total of 194 apheresis platelet units underwent cross-match (XM) using Capture-PHigh PRA by FC or CDC assays correlates with a high % of XM-positive (incompatible) platelet units. When the CDC PRA fell to 2% after desensitization, platelet XM incompatibility fell from 100% to 63% positive (incompatible). When the FC PRA fell to 5% the positive platelet XM fell to 5%.Antibody desensitization facilitated platelet transfusion. PRA determination by FC appeared better correlated than determination by CDC with the ability to find XM-compatible platelets.

Acute graft versus host disease (GVHD) following orthotopic liver transplantation (OLT) is a rare but severe disease. GVHD diagnosis can often be delayed, since the symptoms are non-specific. We present a case of a 62 year-old male with end-stage liver disease who underwent OLT. There were no major peri-transplant complications. Patient presented ~4 weeks post-OLT, with fever,cough, throat discomfort, and rash; and was treated for suspected infection with broad-spectrum antimicrobials. ~5 weeks post-OLT, patient became pancytopenic, and a skin biopsy was suspicious for GVHD. Bone marrow biopsy was performed and revealed extreme pancytopenia with necrosis. The individual pre-OLT patient and donor types were compared at the serologic equivalent and high-resolution (HR) HLA-type, without clear indication of risk for GVHD, and or graft rejection. HR HLA-typing was performed on bone marrow cells obtained post-OLT and compared with the pre-OLT specimen to demonstrate donor lymphoid chimerism. HR typing results from the patient’s bone marrow post-OLT indicated the presence of all four alleles, and along with the clinical features,confirmed the diagnosis of GVHD. This case demonstrates howHR HLA typing can aid in rapid diagnosis and early recognition of GVHD post-OLT.

Omenn syndrome comprises a rare form of combined immunodeficiency with TH2-type features of eosinophilia and elevated IgE. Previous studies have led to reports of restricted heterogeneity in the T lymphocyte repertoire, and in vitro cloned T lymphocytes have been shown to produce IL-4 and IL-5. We hypothesized that (1) T cell receptor beta V(D)J DNA sequence analysis would confirm and further define the putative restricted heterogeneity, and (2) increased production of IL-4 and IL-5 should be found in nonstimulated T lymphocytes, if the molecular pathogenesis of Omenn syndrome is an uncontrolled TH2 state. We report the results of molecular analyses of T lymphocytes from an untreated 3-month-old patient. Oligoclonal T cell receptor beta variable gene usage was found. Sequence analysis revealed sets of identical V(D)J sequences, each in-frame, with apparently normal N-diversification and no obvious antigen combining site motif. From fresh, nonstimulated lymphocytes, proinflammatory TH1 cytokines could be detected, but TH2 cytokines could not, so that a simple TH1/TH2 paradigm cannot explain the eosinophilia and elevated IgE in Omenn syndrome. Our studies fully document for the first time at the molecular level that clonally expanded populations of T lymphocytes are present in Omenn syndrome.

The term myelodysplasia (MDS) refers to a group of bone marrow failure syndromes which are relatively rare in childhood. The pathogenesis of MDS is unknown, but a variety of chromosomal, molecular, and cytochemical abnormalities have been reported. We describe a 4-month-old female with MDS who presented with severe neutropenia and refractory anemia with excess blasts (RAEB). Bone marrow progenitor cell assays showed decreased erythroid and myeloid colony formation as compared to normal marrow, and the patient's serum further diminished colony formation of both her own and control marrow. These observations suggested the presence of a soluble factor inhibitory to hematopoiesis. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total RNA derived from the patient's bone marrow mononuclear cells revealed highly elevated tumor necrosis factor-α (TNF-α) mRNA levels. Using a similar RT-PCR profile, TNF-α mRNA levels were found to be elevated in two other children with myelodysplasia. We conclude that TNF-α is produced in large amounts by bone marrow mononuclear cells of children with MDS, and we hypothesize that TNF-α plays an important role in the pathophysiology of the ineffective hematopoiesis observed in MDS. © 1997 Wiley-Liss, Inc.

Objective: DiGeorge syndrome is characterized by developmental defects of the heart, parathyroid glands, and thymus. The objective of this study was to determine whether T-cell function spontaneously improves in patients with DiGeorge syndrome who have profoundly depressed T-cell proliferative responses to mitogens at presentation, regardless of the T-cell count. Study design: We conducted a retrospective chart review of eight patients with DiGeorge syndrome who had no proliferative responses to mitogens on presentation. Results: Despite lack of responsiveness of the patients' peripheral blood lymphocytes to mitogens, T cells were occasionally detected, and the patients' cells often responded to IL-2 and in mixed lymphocyte reactions. Unresponsiveness to mitogens and clinical immunodeficiency persisted without immune-based therapy. One patient is alive and well after immunoreconstitution from thymic transplantation. The others either died early of complications of their disease such as gastroesophageal reflux with aspiration (2 patients) or infection (2 patients) or died after attempts at immunorestorative therapy with IL-2, thymus transplantation, or bone marrow transplantation (3 patients). Conclusion: Eight patients with DiGeorge syndrome who were first seen with no mitogen responsiveness did not improve spontaneously. We recommend HLA-identical bone marrow transplantation or thymic transplantation for these patients as soon as the diagnosis is confirmed. (J Pediatr 1998;132:15-21)

Objective: To determine the relative frequencies of the different genetic forms of severe combined immunodeficiency (SCID) and whether there are distinctive characteristics of the particular genotypes. Study design: The demographic, genetic, and immunologic features of 108 infants with SCID who were treated consecutively at Duke University Medical Center were analyzed. Results: Eighty-nine subjects were boys and 19 were girls; there were 84 white infants, 16 black infants, and 8 Hispanic infants. Forty-nine had X-linked SCID with mutations of common cytokine receptor gamma chain (γ c ) , 16 had adenosine deaminase (ADA) deficiency, 8 had Janus kinase 3 (Jak3) deficiency, 21 had unknown autosomal recessive mutations, 1 had reticular dysgenesis, 1 had cartilage hair hypoplasia, and 12 (all boys) had SCID of undetermined type. Deficiency of ADA caused the most profound lymphopenia; γ c or Jak3 deficiency resulted in the most B cells and fewest natural killer (NK) cells; NK cells and function were highest in autosomal recessive and unknown types of SCID. Conclusions: Different SCID genotypes are associated with distinctive lymphocyte characteristics. The presence of NK function in ADA-deficient, autosomal recessive, and unknown type SCIDs, and low NK function in a majority of γ c and Jak3 SCIDs indicates that some molecular lesions affect T, B, and NK cells (γ c and Jak3), others primarily T cells (ADA deficiency), and others just T and B cells. (J Pediatr 1997;130:378-87)

BONE MARROW TRANSPLANTATION FOR SEVERE COMBINED IMMUNODEFICIENCY: ANALYSIS AT 15 YEARS ♦ 40

PA was noted to have erythrodermatitis and alopecia soon after birth. These symptoms persisted and by age 2 months, lymphadenopathy, heptosplenomegaly, chronic neutropenia (0-100 cells/μl), and lymphopenia were present. Multiple hospitalizations due to infections ensued. Treatment with G-CSF(10-20 μg/kg/day) gave no definitive improvement in PMN counts (0-200 cells/μl), but precipitated thrombocytopenia. CID was diagnosed because of poor lymphocyte enumeration and proliferation studies, and absence of Ig production. Biopsy of skin, lymph node, and bone marrow (BM) revealed excessive numbers of histiocytes. On repeated studies, malignancy could not be detected and 4 separate chromosomal analyses were normal (nl). BM exhibited an apparent myeloid arrest. It was hypothesized that a serum inhibitory factor was suppressing PMN, and possibly lymphocyte, development. PA's serum enhanced patient and control lymphocyte proliferation to mitogens above that of nl serum. PA's serum inhibited both patient and control BM cell growth in soft agar progenitor assays, whereas patient colony formation was near-normal in the presence of nl serum. Serum TNF-α was 59 pg/ml (nl < 20 pg/ml) by ELISA assay. RT-PCR of RNA from BM mononuclear cells disclosed elevated levels of TNF-α mRNA. Normalizing the TNF-α levels with concurrently amplified β-actin by calculating ratios of densitometric readings gave 0.45±0.07 and 0.13±0.03 (p < 0.01) for PA and control samples, respectively. Comparisons with 20 other cytokines and growth factors revealed no differences between PA and controls. Previous studies have implicated that TNF-α is a potent inhibitor of myeloid development. We believe that this is the first report demonstrating that elevated TNF-α in the bone marrow is associated with severe neutropenia in a patient. The process leading to dysregulation of TNF-α production (from histiocytes?) could be responsible for both CID and neutropenia in PA, and perhaps plays a role in other conditions where neutropenia is a prominent feature.

Severe combined immunodeficiency (SCID) is a rare, fatal syndrome of diverse genetic etiology characterized by profound deficiencies of T and B cell function. In the U.S., it affects primarily male infants. Some are due to mutations in the genes encoding adenosine deaminase (ADA), the common gamma chain (Yc) of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, or Janus kinase 3 (Jak3). However, for a significant proportion of SCID's, the molecular basis remains unknown. In an effort to identify unique phenotypic and/or functional features that could help identify fundamental causes, we analyzed the clinical, genetic and immunologic features of 95 SCID infants who presented consecutively to this institution between August of 1965 and December of 1995. Eighty (84%) were male and 15 (16%) female; 74 (78%) were white, 13 (14%) black, and 8 (8%) Hispanic. The 95 infants fit 7 categories: 16 (17%) were ADA deficient; 6 (6.3%) had Jak3 deficiency; 16 (17%) had autosomal recessive inheritance of unknown molecular cause; 1 (1.2%) had reticular dysgenesis; 1 (1.2%) had cartilage hair hypoplasia; 38 (40%) had X-linked SCID; and 17 (18%) (all males) had SCID of unknown type. Numbers of total lymphocytes and the various lymphocyte phenotypes distinguished the groups: ADA deficient SCID's had the lowest numbers of total lymphocytes; B cells were present in highest number and NK cells in lowest number in X-linked and Jak3-deficient SCIDs. Mean numbers of NK cells were highest in autosomal recessive and unknown types of SCID. All types were profoundly deficient in T cells. NK function was tested in 60 and found to be normal in 25. Although the family history was positive in only 34% of X-linked cases, it is likely that as many as two-thirds of the boys with SCID have mutations in Yc. The presence of NK function in ADA deficient, autosomal recessive, and some SCID's of unknown type, and abnormally low NK numbers and function in a majority of X-linked and Jak3-deficient SCID's implies that some molecular lesions affect T, B and NK cells (Yc and Jak3 mutations), but that others affect only T cells (ADA deficiency), and others T and B cells (possible recombinase abnormalities). The findings in this group of SCID's, the largest reported in the U.S., argue against a common lineage for T and NK cells and may provide clues as to the molecular bases of SCID in the 35 (37%) infants who did not have X-linked SCID, Jak3 deficiency or ADA deficiency.

JH presented at 5 months of age with FTT, and respiratory distress due to parainfluenza virus type III (PFIII). Our evaluation led to a diagnosis of SCID. Respiratory symptoms improved with long-term nebulized Ribavirin and supportive care, but GI tract adenovirus (AV) resulted in continuing management problems (cramps, diarrhea, FTT). For definitive therapy JH received a maternal-donor, T-cell-depleted bone marrow stem cell transplant(no pre-conditioning chemotherapy). T lymphocyte function reconstituted during the subsequent 3-4 months, with elimination of the PFIII. GVHD affecting the bone marrow developed, with ensuing life-threatening pancytopenia. Worsening GI symptoms and increase in stool AV were noted with anti-GVHD therapy(corticosteroids and cyclosporin A). We hypothesized that exacerbation of the AV infection was in part due to the immunosupressive treatment and, additionally, that the immune response generated against the AV was resulting in simultaneous activation of maternal T lymphocytes capable of causing and/or worsening the GVHD. Cognizance that systemic AV infections can be catastrophic in immunocompromised patients prompted us to escalate therapy. Recent studies indicating successful and less toxic usage of oral Ribavirin for the treatment of various viruses led us to consider this over parenteral therapy. Low dose therapy, 20 mg/kg/day was initiated, with equivocal results. One week later, dosing of 200 mg/kg/day resulted in immediate clinical improvement (reduction in vomiting, loose stools, abdominal cramping, and improved feeding). Stool AV was quantified by enumerating viral particles via electron microscopy. Post-treatment viral load was decreased to 20% of that of immediate pre-treatment levels. To our knowledge, this is the first report of oral Ribavirin therapy for GI tract AV infection. While long-term effects of Ribavirin remain unknown, clinical improvement in JH demonstrated that oral Ribavirin can be of benefit as anti-viral therapy in immunodeficient patients.