Comparison of Two Different Serological Viral Marker Testing Assays for Screening of Apheresis Donors: Which Assay Provides Optimum Safety for Transfusion?

While whole blood testing has evolved over the years, viral marker testing for plateletpheresis donors is still performed by Rapid Diagnostic Tests (RDT). Aim of this study was to compare diagnostic accuracy of RDT and Chemiluminescence Immunoassay (CLIA) in serological testing for HBsAg, anti-HCV and anti-HIV antibodies. A prospective, analytical study was conducted in the department of Transfusion Medicine at a tertiary healthcare center in India between September 2016 and August 2018. Samples were simultaneously tested by CLIA, RDT and a confirmatory test. Sensitivity, specificity, negative and positive predictive values and mean time taken to report results were calculated. A total of 102 (1.48%) of the 6883 samples were found to be reactive by either or both the assays. A total of 74 (1.08%) samples were HBsAg reactive, 23 (0.33%) were reactive for anti-HCV antibodies and 5 (0.07%) were reactive for anti-HIV I and II antibodies. A combined sero-prevalence of 1.05% (72) was observed; 0.78% (54) for HBsAg, 0.26% (18) for anti-HCV antibodies and none for anti-HIV I and II antibodies. Four (3.85%) reactive samples were missed by RDT and therefore sensitivity of RDT was quite less as compared to CLIA. RDT and CLIA both were found to have a statistically significant shorter turnaround time than confirmatory tests. There is increasing need to develop a safe donor screening strategy for plateletpheresis. CLIA offers an excellent alterative to RDT for viral marker testing in terms of sensitivity.

Implementation of an international barcode labeling standard, International Society of Blood Transfusion 128, and its integration with local regulations at a blood center in India: Step-by-step journey.

The International Society of Blood Transfusion (ISBT) 128 is an internationally endorsed, electronically readable labeling standard that provides a convenient and accurate means of identification, traceability, publication, and storage of information for blood and blood products. The authors' center recently registered with the International Council for Commonality in Blood Banking Automation (ICCBBA) and progressed to ISBT 128 labeling standard. This manuscript was written with the objective of sharing the authors' experience with respect to the implementation of ISBT 128 standards for whole blood donations and integration of ISBT 128 standards with Indian licensing regulations. The authors explore the process of implementation of ISBT 128 standards through a step-by-step journey that included facility registration with International Council for Commonality in Blood Banking Automation (ICCBBA), allotment of facility identification number, development of four-quadrant label for blood components, and integration of local regulatory requirements in the final "composite" label. Acknowledging the lack of any published report from India on ISBT 128 standards implementation, the authors wish to attempt help their peers in understanding and implementation of this global standard at their respective facilities.

Evaluation of quality matrix when practice changed from triple bags to quadruple (top and bottom) bags: In vitro analysis of blood components!

BACKGROUND: As a part of continuous quality initiatives, while moving from triple bags to quadruple bags, we undertook a study to compare platelet-rich plasma (PRP) and buffy-coat (BC) methods with respect to all blood components (red blood cells [RBCs], random donor platelet concentrate [RDPC], and fresh frozen plasma [FFP]) prepared by PRP and BC methods. MATERIALS AND METHODS: It was a prospective analysis of different physical and quality parameters of RDPC, RBC, and FFP prepared out of 100 whole blood (WB) donations. Of these, 50 WB units were processed by PRP method using Triple bags and 50 WB units by BC method, using quadruple (top and bottom) bags, with an attached integral filter. RESULTS: RBC prepared by BC method had higher hematocrit (61.3 ± 1.91% vs. 56.03 ± 3.37%; P < 0.05) and lower white blood cell (WBC) contamination (6.3 × 104 ± 6.1 vs. 5.41 × 105 ± 2.5; P < 0.05) in comparison to prepared by PRP method. Higher PLT yield (7.67 × 1010 ± 1.8 vs. 6.47 × 1010 ± 1.5; P < 0.05) and lower WBC count (8.24 × 103 ± 1.1 vs. 1.5 × 104 ± 2.1; P < 0.05) was observed in RDPC prepared by BC method than PRP derived. CD62P expression was lower in RDPC prepared by BC method (31.46 ± 9.7%; P < 0.05) as compared to PRP method (43.35 ± 12.5%; P < 0.05). The BC method also resulted in increased plasma yield (210.56 ± 18.54 ml vs. 187.92 ± 12.93 ml; P < 0.05) in FFP in comparison to PRP method. CONCLUSION: The blood components produced from WB by the BC method have laboratory variables suggestive of superior quality than those produced by the PRP method.

Immunoadsorption in ABO-incompatible kidney transplantation in adult and pediatric patients with follow-up on graft and patient survival: First series from India.

BACKGROUND: There are no published reports on desensitization protocol for ABO-incompatible kidney transplants using Immuno-Adsorption (IA) plasmapheresis from India. IA offers certain advantages including processing of larger plasma volumes, quicker reduction of isoagglutinin titers and no requirement of replacement fluids. AIMS AND OBJECTIVES: Authors' center evaluated success of desensitization protocol, and graft/patient outcomes when IA procedures were performed for desensitization in adult and pediatric ABO-incompatible kidney transplant patients. METHODS: Patients undergoing ABO-incompatible kidney transplant with use of IA were evaluated at tertiary care center in north India. Patient records for 2-years were collated from hospital information system (HIS) and procedure forms. RESULTS: Sixteen IA procedures were performed in five patients who underwent successful ABO-incompatible kidney transplant. Initial isoagglutinin IgG titer ranged from 32-512. Mean number of IA procedures performed to achieve the desired pre-transplant IgG titer ≤8 was 3.2. New IA column was used for each patient (and re-used for the same patient, if needed, after sterilization with Low temperature steam of formaldehyde). Mean plasma volume processed during each IA procedure was 4.5 times. No adverse events were observed during any IA procedure. All patients achieved successful desensitization. All patients continue to do well clinically with mean follow-up period of 8.8 months. Although IA was expensive, it offered advantages like specificity, larger plasma volume processing with desired reduction in titer, no 'replacement fluid' requirements and no adverse events in present case series. CONCLUSION: IA plasmapheresis was universally successful in decreasing the ABO-isoagglutinin titers to desired level in all prospective ABO incompatible kidney transplant patients.

Applying Donath-Landsteiner test for the diagnosis of paroxysmal cold hemoglobinuria.

61-year old male patient was admitted to the hospital with clinical picture of hemolytic anemia with hemoglobinuria. Patient was suspected to have Infectious Mononucleosis (IM) with Auto Immune Hemolytic Anemia (AIHA). DAT was positive with anti-C3d specificity. Donath Landsteiner (DL) test was positive; confirming Paroxysmal Cold Hemoglobinuria (PCH). The final diagnosis was IM with PCH. Patient was managed conservatively and discharged after seven days. DL test specifically detects a biphasic autoantibody (IgG type) that binds to RBCs at cold temperatures, and fixes complement on the RBC membrane. However RBCs are only lysed upon warming to 37C when complement cascade proceeds to completion.

Utility of grey zone testing strategy in transfusion transmissible infection testing in blood bank is of limited value!

Several blood banks use grey zone (GZ) phenomenon (defined as samples with optical density within 10% below the cut off in enzyme immuno-assay [EIA]/chemiluminescence immunoassay [CLIA]) to further augment blood safety. There is paucity of data regarding usefulness of GZ sample and its application in Transfusion Transmissible Infection (TTI) screening procedures in blood transfusion services. We looked at our GZ sample results and their confirmatory test results to verify if it adds to blood safety in our set-up? We performed a prospective analytical study on blood donors' samples over two years. All the donors' samples were screened for TTI using CLIA. Samples with signal/cut-off ratio between ≥0.90 and <1.00 were classified under GZ. They were re-tested in duplicate and submitted to confirmatory testing: Neutralization Test for HBsAg, Immunoblot for HCV, and Western blot for HIV. Among the 50,064 blood donors donating the blood during study period, 573 (1.14%) donors were reactive for HBsAg, HCV, and HIV. Forty-seven (0.1%) TTI samples were GZ, but none was "confirmed positive." The utility of GZ testing seems to be limited. However, this may be continued for sake of "erring on the side of caution" and since this only results in negligible wastage (0.1%) of blood units.

Safety assessment of RhD-positive red cell transfusion in RhD-negative liver-transplant recipients: Single-centre report from India.

BACKGROUND & OBJECTIVES: The number of blood components required during a liver-transplant surgery is significant. It is challenging for blood transfusion services to provide the required RhD-negative red blood cells (RBCs) for recipients during the peri-operative period. This retrospective study presents safety data of transfusing RhD-positive RBCs in RhD-negative living donor liver-transplant (LDLT) recipients during the peri-operative period with six-month follow up for risk of developing alloantibodies. METHODS: All RhD-negative patients who underwent LDLT and were transfused ABO-compatible but RhD-positive RBC units between January 2012 and May 2018 were included in the study. Twenty one RhD-negative patients who received a total of 167 RhD-positive RBCs peri-operatively were chosen for alloantibody screening. All the patients were started on triple immunosuppression drugs as per the standard hospital protocol. Blood grouping, cross-match and antibody screening were done by column agglutination technique. RESULTS: Post-transplant antibody screen (weekly for 12 wk) was negative, and none of the patients developed anti-D alloantibodies till their last follow up (mean 21 months). INTERPRETATION & CONCLUSIONS: Our observations suggest that it may be safe to use RhD-positive RBCs peri-operatively in RhD-negative LDLT recipients with low risk of alloimmunization.

Site-specific conjugation of native antibody.

Traditionally, non-specific chemical conjugation, such as acylation of amines on lysine or alkylation of thiols on cysteines, are widely used; however, they have several shortcomings. First, the lack of site-specificity results in heterogeneous products and irreproducible processes. Second, potential modifications near the complementarity determining region (CDR) may reduce binding affinity and specificity. Conversely, site-specific methods produce well-defined and more homogenous antibody conjugates, ensuring developability and clinical applications. Moreover, several recent side-by-side comparisons of site-specific and stochastic methods have demonstrated that site-specific approaches are more likely to achieve their desired properties and functions, such as increased plasma stability, less variability in dose-dependent studies (particularly at low concentrations), enhanced binding efficiency, as well as increased tumor uptake. Herein we review several standard and practical site-specific bioconjugation methods for native antibodies, i.e., those without recombinant engineering. First, chemo-enzymatic techniques, namely transglutaminase (TGase)-mediated transamidation of a conserved glutamine residue and glycan remodeling of a conserved asparagine N-glycan (GlyCLICK), both in the Fc region. Second, chemical approaches such as selective reduction of disulfides (ThioBridge) and N-terminal amine modifications. Furthermore, we list site-specific antibody-drug conjugates (ADCs) in clinical trials along with the future perspectives of these site-specific methods.

Application of sequential serological testing strategy for detection of Hepatitis B surface antigen (HBsAg) for diagnosing HBV infection.

The diagnostic accuracy of any serological test for detection of HBsAg is not 100%. We hypothesized that the sequential testing strategy proven for anti-HIV laboratory diagnosis should also apply to other infectious disease markers like HBsAg. Therefore, we evaluated the diagnostic accuracy of these strategies, I (single test), II (two tests in sequence), III (three tests in sequence) for diagnosis in patients and blood donors and compared it to the confirmatory test for HBsAg (Neutralization Test). Samples were initially tested for HBsAg by A1- Enhanced Chemiluminescent Immuno Assay (ECLIA). Initial reactive (aliquoted donor/patient) samples were reflexly tested by A2- Enzyme Linked Fluorescence Assay (ELFA) and A3- Immuno Chromatography Assay (ICA) assays. Confirmatory neutralization assay was performed on all initial reactive samples. Four strategies (I, II A, II B, and III) that were used in this analysis were; I = A1, IIA = A1 + A2, IIB = A1 + A3, and III = A1 + A2 + A3. The results of all four strategies were compared to Gold Standard (Neutralization Test). A total of, 112, 011 blood samples (75,111 patient samples and 36,900 whole blood donor samples) were initially tested for HBsAg by A-1 (CLIA). Amongst the tested samples, 1,296, 1,188, 1,078, 1,074 samples were found to be reactive by strategy I, IIA, IIB, III respectively. We observed that the PPV (Positive Predictive Value) of Strategy III > Strategy IIB > Strategy IIA > Strategy I. Sequential serological testing strategy comprising of initial sensitive test followed by more specific test increases the diagnostic accuracy of test report as compared to a single test.

Comparison of a column agglutination technology-based automated immunohematology analyzer and a semiautomated system in pretransfusion testing.

INTRODUCTION: Semi-automated equipment using Column agglutination technology (CAT) is widely used where centrifugation and incubation are automated but substantial amount of the work is still executed manually. Larger laboratories are moving towards automation to eliminate errors, reducing exposure to bio-hazardous samples, assuring traceability, reliability, turnaround time (TAT) and throughput. Moving towards automation and greater reliability, we therefore, decided to install an automated immunohematological (IH) analyzer "Vision". In this study we evaluated reliability and performance before clearing "Vision" for routine use. MATERIALS AND METHODS: Study was conducted in the Department of Transfusion-Medicine. The primary objective was to assess the reliability of results and compare with routine use semi-automated BIOVUE system (Reference system). Secondary objective was to evaluate the performance (TAT and throughput) of the Vision to handle routine and emergency workload. RESULTS: Total of 1276 known samples were used to assess 2640 pre-transfusion tests (1229 ABO/Rh D typing; 1229 antibody screening; 54 antibody identification; 86 crossmatch and 42 DAT). All 1229 ABO Rh typing results were concordant between the two systems. Overall agreement between the Vision IH analyzer and reference system for ABO Rh typing was 99.95%. All antibody screening, crossmatch and DAT results were concordant between the two systems. TAT of Vision was substantially shorter than the reference system for all test profiles. CONCLUSION: Based on study results, Vision was approved for routine use in laboratory. It was found to be reliable with considerably shorter TAT and capable of handling high throughput of immunohematological tests.

Applying newer parameter Ret-He (reticulocyte haemoglobin equivalent) to assess latent iron deficiency (LID) in blood donors-study at a tertiary care hospital in India.

BACKGROUND: It is important to detect Latent Iron Deficiency (LID) to prevent development of an overt iron deficiency anemia. Early detection is difficult by using conventional hematological and biochemical parameters. Soluble transferrin receptor (sTfR) is presently the gold standard for diagnosing LID. We evaluated the utility of Reticulocyte Hemoglobin Equivalent (Ret-He), a newer hematological parameter, to predict LID in blood donors as compared to sTfR. METHODS: This was a randomized prospective study performed on 501 donor samples over a period of three-months. All donors were included after administering medical history questionnaire and a brief physical examination in accordance with national guidelines (Hb ≥12.5). Additional samples were collected during donation according to the institutional standard operating procedure (SOP). All hemograms were performed on the Sysmex XE-2100 analyzer which included Ret-He. sTfR was measured in batch assays by ELISA (Biovendor, Czech Republic). Ret He <28 pg and sTfR≥3µg/ml were used to diagnose LID. Serum Iron, Total Iron Binding Capacity (TIBC) and Serum Ferritin were also measured simultaneously. RESULTS: Of the 501 blood donors, sTfR and Ret-He detected LID in 148 and 135 donors respectively. In comparison to sTfR, Ret-He had sensitivity of 92.7%, a specificity of 97.16%, PPV of 93.1% and NPV of 96.3%. Serum Ferritin, TIBC and serum Iron had comparatively lower sensitivity of 87.16%, 79.7% and 77.7% respectively. CONCLUSION: Ret-He can be used as a routine screening test to detect LID in blood donors. This could provide an opportunity to make appropriate and timely interventions like dietary changes or drug supplementation.

Notification and counselling of hepatitis positive blood donors, their immediate emotional response, contact-testing and their follow-up: Study from a tertiary care hospital!

INTRODUCTION: Post-donation counselling informs donors of unusual test results. Timely notification and counselling regarding their Transfusion Transmitted Infection (TTI) status is necessary for early clinical intervention in the donor and reducing risk of transmission. We share our experience with respect to Hepatitis B (HBV) and Hepatitis C (HCV) positive donors who were counselled and followed-up for clinical outcome. MATERIALS AND METHODS: It was prospective 2-year study in TTI positive blood donors. Confirmed positive HBV/ HCV donors were notified to attend the donor-clinic or to visit local hepatologist for further management. At donor clinic, donor's immediate emotional response was observed; donors were offered contact-testing, associated risk factors were noted, counselled, referred to hepatologist, treated and followed-up for clinical outcome. RESULTS: Of 481 donors (0.91%) confirmed positives, 351 were contacted telephonically; 280 promised to attend donor clinic and 71 were referred to their local hepatologist. 145 donors attended the donor clinic, eventually. Most common immediate emotional response noted were 'feeling of fear' (55.2%) and 'disbelief' (35.2%). Most common associated risk factor was history of medical treatment/ injections without knowledge of sterilisation. Five donors availed contact testing and four (spouses in all four cases) came out positive. Of 98 donors contacted post-counselling; 89 went to hepatologist. No medication was advised to seven donors (low viral load), 59 donors completed treatment course and 23 donors were undergoing treatment at time of follow-up. Nine donors opted for alternative treatment or "no treatment". CONCLUSION: Donor-clinic proved beneficial to substantial number of donors and their families.

Advantages of type and screen policy: Perspective from a developing country!

INTRODUCTION: The authors' center recently changed their pretransfusion testing protocol from "conventional" type and screen (TS) with anti-human globulin (AHG) crossmatch (Policy A) to TS with immediate-spin (IS) crossmatch (Policy B). Red blood cell (RBC) units were issued after compatible IS crossmatch as and when required instead of AHG crossmatch. This study was conducted to compare the effects of change of policy from A to B over 1-year period on crossmatch-to-transfusion (C/T) ratio, RBC issue turnaround time (TAT), outdating of RBC, man-hours consumption, and monetary savings. MATERIALS AND METHODS: This was a comparative, prospective study conducted by the Department of Transfusion Medicine of a tertiary hospital-based blood bank in Northern India. The Policy B was implemented in the department from January 2014. Relevant retrospective data for comparison of the previous 1 year, when Policy A was practiced, were derived from hospital information system. RESULTS: 23909 and 24724 RBC units transfused to patients admitted to the hospital during respective 1-year period of practice for Policy A and B. There was significant reduction in C/T ratio (1.94 vs. 1.01) and RBC issue TAT (79 vs. 65 min) with Policy B. Expiry due to outdating reduced (37 vs. zero) along with man-hours (16% reduction) and monetary (33% reduction) savings. CONCLUSION: Use of 'TS with IS crossmatch' policy provides multiple advantages to all the stakeholders; blood banker, clinician, patient, and the hospital management.

Evaluation of new indigenous "point-of-care" ABO and Rh grouping device.

BACKGROUND: Erycard 2.0 is a "point-of-care" device that is primarily being used for patient blood grouping before transfusion. MATERIALS AND METHODS: Erycard 2.0 was compared with conventional slide technology for accuracy and time taken for ABO and Rh forward grouping result with column agglutination technology (CAT) being the gold standard. Erycard 2.0 as a device was also evaluated for its stability under different storage conditions and stability of result till 48 h. In addition, grouping of hemolyzed samples was also tested with Erycard 2.0. Ease of use of Erycard 2.0 was evaluated with a survey among paramedical staff. RESULTS: Erycard 2.0 demonstrated 100% concordance with CAT as compared with slide technique (98.9%). Mean time taken per test by Erycard 2.0 and slide technique was 5.13 min and 1.7 min, respectively. After pretesting storage under different temperature and humidity conditions, Erycard 2.0 did not show any deviation from the result. The result did not change even after 48 h of testing and storage under room temperature. 100% concordance was recorded between pre- and post-hemolyzed blood grouping. Ease of use survey revealed that Erycard 2.0 was more acceptable to paramedical staff for its simplicity, objectivity, and performance than conventional slide technique. CONCLUSION: Erycard 2.0 can be used as "point-of-care" device for blood donor screening for ABO and Rh blood group and can possibly replace conventional slide technique.

Changing Trends in Therapeutic Plasmapheresis: An Indian Perspective.

Indications for therapeutic apheresis (TA) are dynamic; they keep changing and expanding because of introduction of newer treatment modalities and accumulating evidence in the form of case-reports, case-series and original articles. We evaluated our therapeutic plasmapheresis (TP) data and compared this data with an earlier published Indian report for indications, frequency, response rate and adverse reactions. We conducted a retrospective analysis of all TP procedures performed from January 2014 to June 2016 in department of Transfusion Medicine at a tertiary care hospital. All TP procedures performed for various clinical disorders including neurological, hematological, renal, hepatic and rheumatologic indications were included in the study. Analysis was performed with respect to demography, procedure details and response. 187 patients (118 Males and 69 females) underwent 683 TP procedures during study period. According to 2013 ASFA guidelines, 99, 59 and 29 patients belonged to category I, II and III respectively. In comparison with the earlier report, the number of patients and procedures have increased, indications have changed, response rate is comparable, and the frequency of adverse reactions have decreased. In the last decade there has been increase in number and spectrum of indications for therapeutic apheresis and frequency of procedures. The response rate and safety of these procedures have also improved.

Applications of selected cells in immunohematology in a developing country: case studies.

CONCLUSIONS: When an antibody is detected, its specificity should be determined and its likely clinical significance should be assessed. When one antibody has been identified, it becomes necessary to confirm the presence of additional significant antibodies to ensure that compatible blood is provided to the patient. To perform this confirmation, specific reagent red blood cells (RBCs) are selected; these are called selected cells. Though the most common use of selected cells is for antibody confirmation, they can also be used for several other immunohematologic applications. In a developing country like India, the performance of antibody screening for unexpected antibodies on a routine basis is a comparatively new phenomenon, and those laboratories performing advanced immunohematologic testing would need to use selected cells to arrive at an accurate conclusion. This report defines selected cells and enumerates sources of these RBCs. Detailed immunohematologic applications are discussed with applicable case studies.

Co-infection of blood borne viruses in blood donors: A cross-sectional study from North India.

BACKGROUND: There are several studies on prevalence of individual infectious disease markers (mono-infection) in donors but none on prevalence of coinfection. Co-infection is significant as it leads to accelerated disease progression. We, therefore, evaluated the prevalence of co-infection among blood donors. MATERIALS AND METHODS: The cross-sectional analysis was conducted in blood donors. All donors were tested for anti-HIV I and II, HBsAg, anti-HBC IgM, anti-HCV, Malaria and syphilis by chemiluminescence and ID-NAT assay. All reactive donor samples were confirmed by using confirmatory assays. Donors were grouped as mono-infected and co-infected. The student t-test was used for comparison. RESULTS: During the study period, a total of 106,238 blood donors were tested. Mean age of donors was 34.2 years and 94.2% of blood donors were males. 1776 (1.67%) donor samples were confirmed serologically reactive. 1714 (1.61%) samples were reactive for single marker (mono-infected) while 62 (0.05%) donors' samples exhibited co-infection. 18 donors were positive for HBV+HCV followed by HIV +syphilis (14). CONCLUSION: We report for the first time the prevalence of different co-infection patterns in blood donors. Co-infection influence the disease progression; it would be important to investigate the co-infection prevalence in larger sample size.