Ashraf Dada

Antibody Response and Disease Severity in Healthcare Worker MERS Survivors

We studied antibody response in 9 healthcare workers in Jeddah, Saudi Arabia, who survived Middle East respiratory syndrome, by using serial ELISA and indirect immunofluorescence assay testing. Among patients who had experienced severe pneumonia, antibody was detected for >18 months after infection. Antibody longevity was more variable in patients who had experienced milder disease.

Recovery from the Middle East respiratory syndrome is associated with antibody and T cell responses

MERS-CoV T cell responses can be detected in MERS survivors in the absence of detectable virus-specific antibody. Breaking the camel virus’s back Middle East respiratory syndrome coronavirus (MERS-CoV) causes a potentially lethal zoonotic pneumonia that can transfer between individuals after initial exposure to an infected camel. Now, Zhao et al. dig deeper into the immune response in MERS-CoV survivors. They found that neutralizing antibody titers could predict protection in an animal model but that antibody levels were often transient. Moreover, both CD4+ and CD8+ T cells were induced after MERS-CoV infection, and these cells could be detected even in the absence of virus-specific antibody. These data suggest that T cells may be useful in detecting mild or subclinical infection and that epitopes recognized by these T cells may form the basis for future vaccine design and immunotherapy. The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pneumonia. MERS was recently identified as a candidate for vaccine development, but most efforts focus on antibody responses, which are often transient after CoV infections. CoV-specific T cells are generally long-lived, but the virus-specific T cell response has not been addressed in MERS patients. We obtained peripheral blood mononuclear cells and/or sera from 21 MERS survivors. We detected MERS-CoV–specific CD4+ and CD8+ T cell responses in all MERS survivors and demonstrated functionality by measuring cytokine expression after peptide stimulation. Neutralizing (PRNT50) antibody titers measured in vitro predicted serum protective ability in infected mice and correlated with CD4+ but not CD8+ T cell responses; patients with higher PRNT50 and CD4+ T cell responses had longer intensive care unit stays and prolonged virus shedding and required ventilation. Survivors with undetectable MERS-CoV–specific antibody responses mounted CD8+ T cell responses comparable with those of the whole cohort. There were no correlations between age, disease severity, comorbidities, and virus-specific CD8+ T cell responses. In conclusion, measurements of MERS-CoV–specific T cell responses may be useful for predicting prognosis, monitoring vaccine efficacy, and identifying MERS patients with mild disease in epidemiological studies and will complement virus-specific antibody measurements.

P136 Successful adeptness of donor exchange program in renal transplantation at King Faisal Specialist Hospital Jeddah

Aim Different methodologies were established to overcome HLA immunological barriers to match the highly increasing number of patients with end-stage renal disease in Saudi Arabia, which is currently ranged between 120 and 150 patients per million per annum. At our hospital, King Faisal Specialist Hospital Jeddah, we therefore successfully established the desensitization and ABOi renal transplantation. Recently we have started to use a donor exchange program. We hereby report about our experience to overcome the immunological incompatibilities by using this program. Methods We used an IT program with high grade on flexibilities by performing different options including simple two-pair exchanges, more complicated domino exchanges and chain donations. All recipients with willing donors and with high titer HLA antibodies and/or strong positive cross match were included in the program. HLA typing was performed by using One Lambda SSOr and SSP. HLA Antibody Identification, Single Antigen Class I/II and C1q test, was performed by One Lambda. T and B cell IgG XM is performed by FACSCanto II flow cytometer, where the cut-off for positive XM is determined based on normal human studies. Results 20 patients were transplanted successfully by using the donor exchange program within a period of 12 months. Considering a total number of 220 transplanted kidneys during this time, which makes our hospital as the largest center for renal transplantation in Saudi Arabia, we were able to increase the number of transplanted organs significantly by 9% and we continue to extend and optimize our histocompatibility services at the hospital. Conclusions Our experience demonstrates the high efficiency of the donor exchange program in overcoming immunological barriers in renal transplantation. This program provides in some cases also an alternative to the invasive and costly desensitization protocols. Further efforts and thoughts are going on to include deseased donors as well in this successful donor exchange program.

P019 HLA-Matched platelet transfusion rescued the life of a child with severe thrombocytopenia

A four-year-old female child who was initially diagnosed with myelodysplastic syndrome was admitted in the bone marrow work up. A fully matched HLA allogeneic bone marrow transplantation was performed. Post-transplant the patient received multiple platelets transfusions to manage extreme thrombocytopenia, but without any positive effects. The platelet count was zero/ μ l and the life of the patient was seriously in danger. At our center, we define the refractory for a patient as an increase of platelet count of less than 10 k after the transfusion of at least two fully functioned platelet units containing a sufficient number of platelets ( > 2 × 10 11 per unite). As a further therapeutic attempt, the young girl received Prednisolone 4 mg/kg/day for 4 days and IVI g 1 g/kg/day for 2 days. However, the medications did not render any response and the count of platelet was still zero and the life of this child was still threatened as she developed rapidly and increasingly petechial exanthema with a massive hemorrhagic in the skin. The HLA-workup showed that the patient developed multiple HLA antibodies against HLA class I antigens (B8. B55. B27. B18. B56. B7. B47. B13. A69. B54. B41. B37. B46. B35. B75. Cw10. B67. B76. Cw1. B53. B64. A68. Cw8. Cw9. B65. Cw14. A68. A34. Cw7. A66. A43). The screening for platelet specific antibodies against human platelet antigens (HPA) was negative. Using our in–house established platelet HLA-matched program, corresponding HLA-matched platelet units donated by 4 selected donors was given over several sessions. The child responded excellent to most of the given HLA-matched platelets. After the second transfusion, the platelets count raised to 51 × 10[c]/ μ l. Generally, we consider an increase in platelet count of more than 30 k per transfused unit as a sufficient transfusion. After the clinical symptoms and platelet count were stabilized the patient was discharged with a platelet count of 40 × 10[c]/ μ l. This case highlights the importance and the benefits of platelet matched HLA-program in rescuing the life, decreasing the mortality rate as well as the stay in the hospital and the treatment cost.

P035 Important role of HLA laboratory in the diagnosis of HLA associated diseases

A 43 years old female patient was introduced to our polyclinic and the treating physician referred her to our HLA-lab for HLA-B27 typing. According to the ordering physician the patient is suffering from back pain, stiffness in the morning, degenerative changes, facet Arthropathy and her hip was also affected, hence we were suspicious that the patient was suffering from ankylosing spondylitis. The blood pressure was normal, BMI 29, WBC 6,849/L, HB 146 g/dl, Glc 5.5 mmol/L, PTH 56.6 ng/L, anti gliedin 3.88 mg/L, CRP 3.46 mg/L. According to published studies HLA-B27 is present in 85% of patients with ankylosing spondylitis. This means that HLA-B27 positive individuals are approximately 85 times at more risk to develop ankylosing spondylitis compared to the general population. However, in this case our HLA-typing, which was performed for HLA-Class I by using SSO for low/intermediate resolution, showed a negative result for HLA-B27. The negative HLA-typing results for HLA-B27 in this female patient do not totally exclude the presence of ankylosing spondylitis because 15% of affected patients do not carry this HLA allele. However, the absence of HLA-B27 in this patient makes the diagnosis of ankylosing spondylitis unlikely. This interpretation is supported by the fact, that Ankylosing spondylitis predominantly affects male patients and not females in the age  > 30 years old. In addition, the described clinical symptoms could be caused by many other various diseases. At our lab we usually performed the testing of all HLA class I alleles even when only single allele requested. In this case we have observed that the patient has beside HLA-B27 HLA-B51 also, which is very strongly associated with Behcet’s disease. Behcet’s disease has similar clinical symptoms to ankylosis spondylitis. Our positive results for HLA-B51 directed the treating physicians to perform further investigations to confirm Behcet’s disease instead of ankylosing spondylitis. The presented case highlights the important role of HLA laboratory in the diagnosis of HLA associated diseases.

P260 Accurate risk assessment for DQB1 donor specific antibodies avoid overlooking of relevant DSA

Aim Interpretation of DQB1 HLA antibodies has to be accurate especially in the coincidently presence of Donor Specific Antibody (DSA) and mismatch in DQA1 between recipient and donor. A sensitive and specific method as solid phase analysis can help a lot when there is a proper interpretation. In this review, due to presence of two and more DQB1 beads for the same antigen in all test kits offered in the market, interpretation of positive HLA antibody for DQB1 will be discussed considering strength and Mean Florescent Intensity (MFI). Methods HLA Typing was performed by One Lambda Sequence specific oligonucleotide revers and sequence specific primers (SSOr/SSP). DSA was performed by Solid phase antibody assay using Class II Single Antigen beads with positive HLA Flow XM. HLA DSA for DQB1 was reviewed carefully and accurately for 5 pairs waiting for allogeneic renal transplant. More than one bead for the same DQB1 DSA has to be checked for the presence of combined antibody against donor DQA1 mismatches. Results It was found that DQB1 DSA has to be assigned according to their DQA1 mismatches and it shows good correlation with positivity in B cell flow cytometer XM in all 5 cases. Conclusions This accurate assignment and procedure might be recommended in the interpretation of DQB1 DSA. Accurate pre and post transplantation workup, including immunological risk assessment for DQB1 DSA, could influence the outcome of organ transplantation significantly.

LBP20: RELEVANCE OF STRONG POSITIVE DONOR SPECIFIC ANTIBODIES AND FLOW CROSSMATCH WHILE C1q TEST NEGATIVE IN RENAL TRANSPLANTATION

Aim In this study we present a case of a kidney recipient with pre-formed high titer anti donor DSA to one allelic mismatch antigen of a sibling donor. Pre-transplant the DSA appeared to be clinically significant based on strong positive crossmatch (XM) testing in both T- and B cell XM. Methods HLA typing is performed by SSO for low/intermediate resolution and SSP for high resolution. DSA is determined by One Lambda BScreen Single Antigen and C1q Screen assay. T and B cell IgG XM is performed by CANTO flow cytometer, where our cut-off for positive XM is determined based on normal human studies. Results Both donor recipient pairs were typed using low resolution class I and high resolution class II HLA typing. Five out of the twelve alleles (−A ∗ 31, B ∗ 51, DRB1 ∗ 11:01, DQA1 ∗ 05:05 and DQB1 ∗ 03:01) were considered the mismatched antigen based on the available data. Flow crossmatch result was Positive for bothT (+209 MCS) & B (+224 MCS) cell IgG flow crossmatch. DSA titer for the allelic mismatched antigen, B ∗ 51 was 10360 MFI. C1q-SA was despite high DSA titer and crossmatch clear negative. Anti- A ∗ 31, DRB1 ∗ 11:01, DQA1 ∗ 05:05 and DQB1 ∗ 03:01 were negative based on our cut off. The Kidney was transplanted and functioned immediately and continues to function well one month after transplantation. No modifications have been made in the standard immunosuppressive protocol used in our hospital (Induction: Basiliximab and Methylprednisolon; Maintenance: Mycophenolate mofetil, Prednisone and Tacrolimus) and the patient is without any signs of AMR. Conclusion The interpretation of DSA, flow crossmatch in this case was more accurate and predictive when result of solid phase antibody testing was considered along with the high resolution HLA typing. However, further studies and more data in C1q are needed allow renal transplantation and clarify the clinical relevance in patients with high DSA titers with positive crossmatch and negative C1q Testing.