Low Ionic Strength Saline Research Articles

When you think you should transfuse…don't!

A 48-year-old female presented to the emergency department with severe fatigue. Admission laboratory test results were hemoglobin 6.6 g/dL, platelet count 287,000/μL, and white blood cell count 25,200/μL. Lactate dehydrogenase was elevated at 898 U/L, haptoglobin was markedly decreased (< 31 mg/dL), indirect bilirubin was elevated (5.3 mg/dL), and the absolute reticulocyte count was low at 0.0050/μL. A sample was sent to the immunohematology reference laboratory. The direct antiglobulin test immunoglobulin G was negative; C3 was 1+. All cells were reactive at immediate spin phase, indirect antiglobulin testing (IAT) with polyethylene glycol, with low ionic strength saline, neat, prewarm, and in the solid phase. All cells were nonreactive at IAT-ficin. Additional testing included a cold antibody titer that was 1:4096 and thermal amplitude studies demonstrating reactivity of 2+ at 37°C. These results were consistent with a clinically significant anti-Pr and cold agglutinin disease (CAD). Although rituximab is effective in autoimmune hemolytic anemia, this may take weeks. The patient was treated with pegcetacoplan, a pegylated peptide that targets C3 inhibiting hemolysis. The patient was discharged on day 29 with a hemoglobin of 8 g/dL. This is a report of one of the first patients successfully treated with pegcetacoplan for CAD.

Collage of cases and brief review of the laboratory diagnosis and molecular testing in autoimmune haemolytic anaemia.

Autoimmune haemolytic anaemia (AIHA) is an acquired heterogenous clinical entity with variable presentations like acute haemolysis or mild, chronic haemolysis compounded with acute exacerbation in winters or fatal uncompensated haemolysis. A step-wise approach to the diagnosis and characterisation of AIHA should be undertaken, firstly the diagnosis of haemolysis followed by the establishment of immune nature with the aid of direct agglutination tests (DAT). Simultaneously the other causes of immune haemolysis need to be excluded too. In light of advancements in diagnostics, a wide array of investigations can be used like absolute reticulocyte count, bone marrow responsiveness index to establish the evidence of haemolysis, sensitive gel technology, enhanced DAT assays, e.g., modified DAT with low ionic strength saline solution (LISS) at 4°C, DAT assays utilizing reagents such as anti-IgA and anti-IgM and DAT by flowcytometry, to detect RBC bound autoantibodies (Abs) and monospecific DAT to establish immune causes of haemolysis and characterisation of the autoantibodies. The compensatory role of bone marrow and synchronous pathologies like clonal lymphoproliferation, dyserythropoiesis, fibrosis are important factors in the evolution of the disease and aid in the customisation of treatment modalities. The laboratory work up should aim to diagnose underlying diseases like chronic lymphoproliferative disorders, autoimmune disorders and infectious diseases. Also, tests like autoimmune lymphoproliferative syndromes (ALPS) screening panel and Next-generation sequencing (NGS) panel for RBC membrane disorders, RBC enzymopathies, and congenital dyserythropoietic aneamia have found their place. It is incumbent upon the clinicians to use the all-available diagnostic modalities for the accurate diagnosis, prognostication and customisation of the therapy.

Difficulties in identifying antibodies of high frequency antigens: A case report of anti-Holley antibody

Abstract Introduction/Objective The Dombrock blood group system consists of the Dombrock (Do), Gregory (Gya), Holley (Hy), and Joseph (Joa) antigens, of which, the Gya, Hy, and Joa represent high prevalence antigens. Anti-Hy antibodies have been implicated in hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Methods/Case Report We report a 22-year-old black female who presented with extensive burns following trauma. At admission the patient typed O, D-positive with a negative indirect antibody test (IAT). After receiving multiple units of red cells, plasma, and platelet products (&amp;gt;100 units total) the patient’s IAT, direct antiglobulin test (DAT) polyclonal, DAT IgG, and DAT C3 became seropositive (day 15 of ongoing transfusions). Elution was negative at all tested phases. An antibody rule-out panel was performed with pan-reactivity at polyethylene glycol (PEG)- anti-human globulin (AHG) (2+), Low ionic strength saline (LISS)-AHG (weak +), Ficin-AHG (2+), and negative with dithiothreitol (DTT)-PEG-AHG. Common DTT sensitive antigens were ruled out. Antigen typing displayed mixed field reactions secondary to transfusions. Hy-Jo(a-) confirmed cells were non-reactive at immediate spin and PEG-AHG. The patient was Dob positive, ruling out a null phenotype. Molecular testing could not be confirmed with serologic testing due to the extensive number of transfusions. Subsequent crossmatching demonstrated 1+ reactivity. The patient clinically tolerated the incompatible units. Her IAT resolved on day 29 of ongoing transfusions and remained negative throughout subsequent testing and transfusion. Results (if a Case Study enter NA) NA Conclusion Identification of antibodies to high frequency antigens is challenging due to the limitation of antigen negative sera in the setting of pan-reactive cells. Furthermore, blood contaminated with donor product limits the ability to perform serologic testing in the setting of mixed field reactivity. This case highlights the difficulty in identifying antibodies to high frequency antigens as well as the limitations in patient testing after extensive transfusion.

Prevalence of irregular red cell antibody in transfusion recipients vis-a-vis healthy blood donors attending a tertiary care hospital in North India.

CONTEXT:Alloimmunization by foreign red cell antigens is a matter of concern as it may lead to hemolysis in transfused patients as well as fetus of pregnant females.AIMS:This study aimed to perform a comparative analysis of prevalence and type of irregular antibodies in healthy donors, vis-a-vis blood transfusion recipients.SETTINGS AND DESIGN:Blood samples of 4000 individuals comprising healthy donors, exposed patients, and nonexposed patients were collected and were analyzed for irregular antibodies.MATERIALS AND METHODS:Commercially available three-cell antigen panel was used for the antibody screening. The samples positive in antibody screen were further subjected to an extended 11-cell panel for antibody identification in low-ionic strength saline with and without enzyme.STATISTICAL ANALYSIS:Statistical analysis was done using SPSS for Windows 15.0 program. Chi-square test was used for detecting statistical significance of exposure to red blood cell antigens in the formation of alloantibodies.RESULTS:Of the 4000 samples, antibodies were identified in 105 (2.6%) samples. Overall, nonexposed group showed a seropositivity of 0.36%, while the exposed group showed a seropositivity of 9.4%. Anti-D was the most common antibody found in 38 patients (33.3%). Anti-E was the most common antibody in males, while anti-D was the most common antibody in females.CONCLUSIONS:Since the risk of alloimmunization is more common in multitransfused patients, it is advisable to screen at least those cases for irregular antibodies.

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PREVALENCE OF IRREGULAR RED CELLANTIBODIES IN HEALTHY BLOOD DONORS ATTENDING A TERTIARY CARE HOSPITAL IN SOUTH INDIA

Introduction: Red cell antibodies that are found normally in human serum are considered naturally occurring and those are anti A and anti B. All other antibodies directed against RBC antigens are considered “unexpected or irregular". Aim: This study is aimed to evaluate the prevalence of the anti-red blood cell antibodies among healthy blood donors. Material and Methods: Antibody screening and identification was done using commercially available 3 cell and 11 cell reagent cells (0.8% Surgiscreen, Ortho Clinical Diagnostics Limited, USA and Low ionic Strength Saline Ortho Bliss with AHG Cassettes) in antihuman globulin phase. Results: A total of 36,684 donors were screened for the presence of irregular erythrocyte antibodies. Among these donors, twenty donors showed presence of alloantibodies in their serum (0.054%). Most frequent alloantibodies identified were from Lewis blood group system. The results showed statistically a higher prevalence of RBC alloantibodies in males than in females. Conclusion: Screening for presence of alloantibodies in donor blood is important to provide compatible blood products and to avoid transfusion reactions.

3168 - Comparision of Microcolumn Gel Assay and Tube Test for Identification of Anti-D Antibody among RH Negative Pregnant Women in Nepal

Introduction: Pregnant women with a Rh-negative blood group may form Rh-antibodies if fetus has a Rh-positive blood. This sensitisation is likely to occur during birth, but can also happen in late pregnancy. These antibodies can cause anaemia or even death for future babies with RH positive blood from the mother. In pregnant women who are known to be RhD alloimmunized or for whom the prenatal antibody result is positive,anti-D concentration measurement is necessary,both to assess the risk to the fetus and to guide clinical decision-making. Methods: The study was conducted in the lab of Central blood transfusion centre from February to July 2015. During this period a total of 114 samples of RH negative women were processed. Anti-D antibody identification and titration is done by using indirect antiglobulin test by Conventional tube method using indirect antiglobulin technique in normal saline. Then parallel identification was done by Microcolumn gel assay, recommended by AABB manual using indirect antiglobulin technique in LISS (Low Ionic Strength Saline) solution. Results: Among a total of 114 pregnant women with RH negative blood processed during the study period, more than one-third (36%) were of blood group O, one-quarter were blood group A (25%), followed by blood group B (23%) and blood group AB (16%). Patients with blood group O showed the highest proportion of anti-D antibody positive cases (17%) . No case of anti-D antibody was detected among women with blood group AB. Among 114 pregnant women, 13 (11.04%) sample from Rh negative pregnant women were positive for anti D antibody. When comparing the titration approaches, the microcolumn gel assay showed more frequent significant titer value (1:1024) than that of tube method titration value. Among 13 samples positive for anti D antibody, gel method could identify positive for all 13 cases while Tube method showed positive anti D antibody in 12 cases only. Moreover, titre value shown by gel method was higher than that of tube method in all cases. Conclusion: The proportion of anti-D antibody positive cases are still higher in Nepal. Early detection and prevention is required to prevent hemolytic disease of Newborn. To identify anti D antibody, microcolumn gel assay is sensitive than tube method and can be a best alternative to tube method in our setting.

Low-ionic-strength saline solution–antiglobulin test (LISS-AGT)

The use of low-ionic-strength saline (LISS) solution as an enhancement for antibody screening and crossmatching was first described by Löw and Messeter in 1974. This method allowed for a reduced incubation time while maintaining adequate specificity and sensitivity of the antiglobulin test (AGT). Since then, the LISS-AGT tube method has been widely used in antibody detection and identification, as well as compatibility testing. As initially described, the method used red blood cells suspended in LISS. Modifications of the method led to development of the commercially prepared LISS additive solutions in use today. The LISS-AGT can be used effectively to detect alloantibodies of all major blood groups in antibody detection, antibody identification, and crossmatching procedures.

Erythrocyte Alloimmunization and Autoimmunization among Blood Donors and Recipients visiting a Tertiary Care Hospital.

The ultimate aim of pretransfusion testing is the acceptable survival of donor red cells in recipient's body and antibody detection plays a critical role in achieving the same. The cornerstone of antibody detection method is detecting an unexpected antibody as against the expected antibodies of ABO blood group system. Autoantibodies can also interfere with the detection of clinically significant alloantibodies. To study the frequency of alloantibodies and autoantibodies in the healthy blood donors and patient population visiting our hospital. The Column Agglutination Technology (CAT) was used for ABO RhD blood grouping, Direct Antiglobulin Test (DAT), Autocontrol (AC), Indirect Antiglobulin Test (IAT) and red cell antibody screening and the unexpected reactions in any of these tests were recorded for further evaluation. Ethylene Diamine Tetra Acetic Acid (EDTA) blood samples were used for all these tests for both blood donors and admitted patients. The CAT was exercised for the blood grouping (using ABD-Reverse Diluent cassettes) and antibody screening (using 0.8% Surgiscreen, Ortho Clinical Diagnostics Limited, USA and Low Ionic Strength Saline Ortho BLISS with AHG cassettes) on the automated immunohaematology platform ORTHO AutoVue® Innova system (Ortho Clinical Diagnostics Limited, USA). Among all blood donors (n=6350), seven (0.11%) donors had showed unexpected reaction. Of these, four had positive antibody screen (three having naturally occuring antibodies 2=anti-M, 1=anti-Lea and 1=inconclusive) and the other three had positive DAT. Of all the patient samples (n=6136) screened for irregular red cell antibodies, four (0.06%) patients were found to have unexpected reaction revealing one (0.02%) with anti-M antibody and the other three (0.05%) had autoantibodies in their serum. The combined prevalence for both blood donor and recipient population (n=12,486) was found to be 0.11% at our center. The alloimmunisation among patient population was found to be lower than many other studies worldwide as our hospital does not cater to multitransfused or transfusion dependant patients with haematological disorders and majorly elective surgery patients with no history of previous blood transfusions visit our hospital.

Characteristics and Outcome of Direct Antiglobulin Test-Negative Hemolytic Anemia: A Case Series

▪ BACKGROUNDNon-hereditary hemolytic anemia of unknown etiology after an exhaustive work-up is commonly known as direct antiglobulin test (DAT)-negative hemolytic anemia. Due to its rarity, limited data are available regarding this condition. We performed this retrospective study to determine the frequency, associated medical conditions, treatments used, and clinical outcome of patients with DAT- negative hemolytic anemia. METHODSWe included adult (age >18 years) patients with DAT-negative hemolytic anemia seen at Mayo Clinic from 1997-2015 who met all of the following criteria: a) hemoglobin (Hb)< 12 g/dL; b) presence of hemolysis (reticulocytosis, elevated lactate dehydrogenase, decreased haptoglobin, and/or elevated indirect bilirubin); c) DAT-negative; and d) unknown etiology of hemolytic anemia after extensive work-up appropriate for clinical context. Remission status after appropriate therapy for perceived hemolysis was defined as follows: a) complete remission (CR; Hb ≥12g/dL and normalization of at least one previously abnormal hemolytic marker); b) partial remission (PR; Hb: 10-11.9 g/dL with evidence of ongoing hemolysis); c) no remission (NR; neither CR nor PR). In order to calculate the relative frequency of DAT-negative hemolytic anemia, we also determined the number of DAT-positive hemolytic anemia cases during the same time period using the above criteria for hemolytic anemia. RESULTSDuring the 19-year study period, 24 and 484 patients were diagnosed with DAT- negative and DAT-positive hemolytic anemia, respectively, a ratio of 1:20. Among those with DAT-negative hemolytic anemia, the majority were males (67%) and the median age at diagnosis was 70 years (range, 22-87). Six patients (25%) had pre-existing autoimmune conditions including immune thrombocytopenia (n=3), Crohn’s disease (n=2), and Grave’s disease (n=1). All patients had negative DAT by conventional method with one patient turning positive at 104 days after initial diagnosis. Enhanced DAT technique using 4°C low-ionic strength saline wash was performed in one case and was negative. In general, peripheral blood smear showed polychromasia, occasional spherocytes, and no schistocytes or bite cells. Additional work-ups performed based on clinical context and proportion of patients tested (all results negative or normal) were as follows: hemoglobin electrophoresis (54%), flow cytometry for paroxysmal nocturnal hemoglobinuria (92%), glucose 6-phosphate dehydrogenase level (67%), osmotic fragility test/eosin-5’-maleimide stain for hereditary spherocytosis (54%), other red blood cell enzymes (46%), computed tomographic scan of the body to look for occult malignancy (46%), and bone marrow biopsy (50%). The median hemoglobin at diagnosis was 8.5 g/dL (range, 4.5-11.8). Fourteen patients (58.3%) required blood transfusion during the course of their disease. Sixteen patients (67%) received prednisone as initial therapy with 19% CR and 38% PR response rates. Subsequent therapies included: rituximab (n=6; 40% PR), cyclophosphamide (n=2; 100% NR), mycophenolate mofetil (n=1; 100% NR), intravenous immunoglobulin (n=2; 100% NR), and splenectomy (n=6; 50% CR and 33.3% PR), respectively. The median follow-up for all cases was 32.9 months (range, 2-433). Six patients (25%) developed hematologic malignancies after a median follow-up of 8.7 months (range, 0.7-29.3), which included primary myelofibrosis (n=2), myelodysplastic syndrome (n=1), mantle cell lymphoma (n=1), non-Hodgkin lymphoma-not otherwise specified (n=1), and T-cell large granular lymphocyte leukemia (n=1). At last follow-up, 9 patients (38%) were in CR and 8 (33%) in PR. Relapses occurred in 67% and 0% of patients who received prednisone and splenectomy, respectively. CONCLUSIONAt our institution, DAT-negative hemolytic anemia was approximately 20 times less common than DAT-positive hemolytic anemia. Most patients were treated with either immunosuppressive agents with or without splenectomy. Splenectomy was the most effective treatment. At least a quarter of the patients were eventually diagnosed with hematologic malignancies mostly within a year of DAT-negative hemolytic anemia diagnosis. Routine upfront evaluation for hematologic malignancies might be necessary for these patients. DisclosuresNo relevant conflicts of interest to declare.

Outcomes and Management of Red Blood Cell Transfusions in Multiple Myeloma Patients Treated with Daratumumab

BackgroundDaratumumab (DARA) is a human IgG1 kappa monoclonal antibody that targets cells expressing CD38, which is highly expressed in multiple myeloma (MM) cells. In the phase 2 MMY2002 (Sirius) trial, treatment with single agent DARA resulted in a 29% overall response rate, with a median PFS of 3.7 months in heavily pretreated MM patients (Lonial S. J Clin Oncol. 2015; 33(suppl): abstrLBA8512). However, CD38 is also expressed on red blood cells (RBCs) and DARA binding to RBCs results in pan-reactivity on RBC panel testing using an indirect antiglobulin test (IAT). Recently, it was reported that treating RBCs with dithiothreitol (DTT) removes DARA to allow for accurate antibody testing (Chapuy CI, et al. Transfusion. 2015;55(6pt2):1545-1554) but also denatures Kell antigen in the process. To date, little is known about the impact of DARA interference with IAT on the outcomes of patients requiring packed RBC (PRBC) transfusions.MethodsThe frequencies of PRBC transfusions and any transfusion-related adverse events from all 124 patients treated on the Sirius study were obtained from the clinical database as of the data cut off of January 9th, 2015. Although details of transfusions were not prospectively collected, surrogate outcomes of transfusion reactions within 4 weeks of the transfusion were analyzed including change in hemoglobin (Hb), elevated bilirubin, fever, and hypotension. In addition, we evaluated IAT results and transfusion outcomes of the 8 patients treated at our institution.ResultsDuring the study, 47 patients received 147 transfusions with a total of 235 units of PRBCs. To date, no transfusion-related reactions were noted. Of the 47 patients, 8 patients were treated at our institution. One patient showed multiple alloantibodies (anti-E, -K, -Jkb, -Fya, -Fy3, -S, Knops) on IAT prior to DARA. This individual and 6 others agglutinated all RBCs on panel testing with weak reactivity at the antihuman globulin phase of testing. Reactivity was enhanced by gel testing compared to reactivity in low ionic strength saline and was unaffected by enzymatic treatment with ficin. Six out of 7 patients had a negative autocontrol. The one patient with a positive autocontrol had a weakly positive direct antiglobulin test with IgG. Six out of these 7 patients with panagglutinin had a positive result on the first IAT after initiation of DARA, ranging from 7 to 175 days (median 49). One patient did not have an IAT after initiation of DARA.A total of 9 leuko-reduced, irradiated, phenotypically matched RBCs were given to 3 patients during DARA treatment, and additional 9 units were given to 3 patients after DARA completion while their IATs remained positive. All transfusions resulted in an appropriate rise in Hb (median 1.0 g/dL, range 0.5-2.7) without any associated transfusion reactions. None of the patients made new unexpected RBC alloantibodies while receiving phenotypically matched RBCs.ConclusionFor the 7 patients at our site with IAT testing after DARA treatment, all demonstrated pan-reactivity on RBC panel testing. Treatment with DTT removes this interference but also denatures Kell antigen in the process. Most hospitals do not use DTT as part of routine immunohematology workups; therefore, we recommend obtaining a red cell phenotype prior to initiating DARA treatment and providing phenotypically matched blood thereafter to avoid resultant difficulties in new alloantibody identification and delays in providing compatible PRBCs. Based on a database query of all 124 patients as well as all patients at our site, PRBC transfusions did not appear to be associated with complications. In a MM patient population that will frequently require PRBC transfusion, blood bank and hematologist/oncologist awareness of these findings is important to minimize errors or delays in providing compatible PRBCs. DisclosuresChari:Novartis: Consultancy, Research Funding; Millennium/Takeda: Consultancy, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Consultancy, Research Funding; Biotest: Other: Institutional Research Funding; Array Biopharma: Consultancy, Other: Institutional Research Funding, Research Funding. Jagannath:Celgene: Honoraria; BMS: Honoraria; MERCK: Honoraria; Novartis Pharmaceuticals Corporation: Honoraria; Janssen: Honoraria. Catamero:Celgene: Honoraria, Other: Lecturer; Onyx: Other: Lecturer; Millennium/Takeda: Other: Lecturer. Verina:Celgene: Other: Lecturer. Doshi:Janssen: Employment. Feng:Janssen: Employment. Uhlar:Janssen: Employment. Khan:Janssen: Employment. Ahmadi:Janssen: Employment. Voorhees:Millennium/Takeda and Novartis: Honoraria; Array Biopharma, Bristol-Myers Squibb, Celgene, GlaxoSmithKline, and Oncopeptides: Consultancy; Celgene, GlxoSmithKline, and Oncopeptides: Research Funding.

Clinical and reference lab characteristics of patients with suspected direct antiglobulin test (DAT)-negative immune hemolytic anemia

Abstract Clinical evidence of warm autoimmune hemolytic anemia is present in 1 percent to 10 percent of patients whose direct antiglobulin test (DAT) is negative. The clinical underpinnings associated with DAT-negative immune hemolysis are poorly understood, and the current study aimed to further define the clinical characteristics associated with this form of anemia. A 19-question survey, requesting clinical information about each patient, was retrospectively mailed to all referring labs that had sent patient samples for an enhanced DAT evaluation from January 2011 through June 2013. An enhanced DAT evaluation involved a standard DAT and DATs performed using gel, polyethylene glycol, and 4°C low-ionic strength saline wash. We obtained detailed clinical information from 57 patients with an enhanced DAT investigation. Eighteen of these 57 patients (31.6%) were found to have a positive DAT, 11 (19.3%) of which were found to have a positive enhanced DAT (2 were positive by enhanced methods and negative by standard methods). The reported mean nadir hemoglobin for all 57 patients was 7.8 g/dL (range 3.2–12.7), and lactate dehydrogenase was 827.8 U/L (range 136–6917). Thirty-seven (88.1%) presented with a haptoglobin &lt;10 mg/dL, and 21 (48.8%) reported spherocytes on peripheral smear. About half of the respondents reported using steroids as treatment for the anemia, and 4 of the 18 DAT-positive respondents (23.5%) changed their treatment plan because of the reference laboratory results. One patient died as a result of the reported hemolytic anemia (2.0%). We conclude that immune hemolysis detected by enhanced DAT methods is relatively common, and enhanced DAT methods are valuable tools in the diagnosis and management of patients with DAT-negative hemolytic anemia. Immunohematology 2015;31:108–115.

Frequencies and specificities of "enzyme-only" detected erythrocyte alloantibodies in patients hospitalized in austria: is an enzyme test required for routine red blood cell antibody screening?

The aim of this study was to determine the frequencies and specificities of “enzyme-only” detected red blood cell (RBC) alloantibodies in the routine antibody screening and antibody identification in patients hospitalized in Austria. Routine blood samples of 2420 patients were investigated. The antibody screening was performed with a 3-cell panel in the low-ionic strength saline- (LISS-) indirect antiglobulin test (IAT) and with an enzyme-pretreated (papain) 3-cell panel fully automated on the ORTHO AutoVue Innova System. The antibody identification was carried out manually with an 11-cell panel in the LISS-IAT and with an enzyme-pretreated (papain) 11-cell panel. In total 4.05% (n = 98) of all patients (n = 2420) had a positive RBC antibody screening result. Of them 25.51% (25/98) showed “enzyme-only” detected specific or nonspecific RBC alloantibodies. Rhesus and Lewis system antibodies were found the only specificities of “enzyme-only” RBC alloantibodies: all in all 4.8% (4/98) were detected with anti-E, 3.06% (3/98) with anti-Lea, 3.06% (3/98) with anti-D after anti-D prophylaxis and 1.02% (1/98) with anti-e. In total, 14.29% (14/98) showed a nonspecific RBC alloantibody result with the enzyme test. The results of the present study demonstrate that a high number of unwanted positive reactions with the enzyme technique overshadows the detection of “enzyme-only” RBC alloantibodies. (Trial Registration: K-37-13).

Indirect antiglobulin test-crossmatch using low-ionic-strength saline–albumin enhancement medium and reduced incubation time: effectiveness in the detection of most clinically significant antibodies and impact on blood utilization

Abstract Indirect antiglobulin test-crossmatch (IAT-XM) using enhancement media such as low-ionic-strength saline (LISS) and polyethylene glycol (PEG) usually requires 15 minutes of incubation. These methods are necessary when testing samples from blood recipients who have a higher risk of alloimmunization. In emergency situations, IAT-XM can be time-consuming and can influence presurgery routine, resulting in more red blood cell (RBC) units being tested and stored to avoid the transfusion of uncrossmatched ones. The objective of this study was to evaluate the performance of a LISS-albumin enhancer to intensify antigen-antibody reaction after 5 minutes of 37°C incubation and compare this performance with that of other enhancers, gel, and conventional tube testing. Second, the study evaluated the impact of this method’s implementation in the C:T ratio (crossmatched to transfused RBC units) of a transfusion laboratory. Ninety serum samples containing alloantibodies of potential clinical significance were tested against phenotyped RBCs using four different methods: (1) tube with LISS-albumin enhancer (5 minutes of incubation), (2) tube with LISS-albumin and PEG (15 minutes of incubation), (3) gel, and (4) conventional tube method (60 minutes of incubation). In parallel, the study compared the C:T ratio of a tertiary-hospital transfusion laboratory in two different periods: 3 months before and 3 months after the implementation of the 5-minute IAT-XM protocol. The use of LISS-albumin with 5 minutes of incubation exhibited the same performance as LISS-albumin, PEG, and gel with 15 minutes of incubation. Conventional tube method results were equally comparable, but reactions were significantly less intense, except for anti-c (p = 0.406). Accuracy was 100 percent for all selected methods. After the implementation of the 5-minute IAT-XM protocol, the C:T ratio fell from 2.74 to 1.29 (p &lt; 0.001). IAT-XM can have its incubation time reduced to 5 minutes with the use of LISS-albumin enhancement. We suggest this strategy should be used to quickly prepare RBC units for surgical patients, keeping transfusion safety without compromising blood supplies. Immunohematology 2014;30:1–5.

Allogeneic red blood cell adsorption for removal of warm autoantibody

Abstract Adsorption studies are usually required to confirm or rule out the presence of underlying alloantibodies in samples containing warm autoantibody. Allogeneic adsorptions are necessary if the patient has been recently transfused. Most commonly, allogeneic adsorptions are performed using a trio of phenotyped reagent red blood cells to rule out clinically significant alloantibodies to common antigens. The adsorbing cells may be used untreated or treated with enzymes or with ZZAP before adsorption. Adsorption may also be performed using enhancement such as low-ionic strength saline or polyethylene glycol added to the mixture. Multiple adsorptions may be necessary to remove strongly reactive autoantibodies. Allogeneic adsorptions will not detect alloantibodies to high-prevalence antigens. Immunohematology 2014;30:153–155.

The Importance of Antibody Detection and Identification in the Chronically Transfused Patient

### Patient: A 44-year-old, African-American female with sickle cell disease (SCD) presented for evaluation of pulmonary hypertension. ### Past Medical History: Sickle cell disease, pulmonary hypertension, hepatitis C (genotype 2), iron overload secondary to transfusion, hypothyroidism, and human leukocyte antigen (HLA)-alloimmunization due to multiple blood product transfusions for a simple transfusion every 3 weeks. ### Past Immunohematology History: Patient’s RBCs phenotyped as group A, Rh positive, C-E+c+e+, K:-1, Fy(a−b−), Jk(a+b−), and S-s+. Red blood units transfused were ABO-compatible and negative for C and K antigens as per hospital policy to transfuse Rh- and K-matched RBC to patients with SCD. One month later, anti-Swain-Langley (Sla) was identified (Figure 1). This antibody is considered clinically insignificant and reactivity was observed when testing with polyethylene glycol (PEG) and low ionic-strength saline (LISS) showed <1+ reactivity with all cells tested except for the Sla antigen negative red cell. Sla is an antigen in the Knops blood group system and is destroyed by 0.2 M dithiothreitol (DTT). This technique was used, and the chemically-treated cells were used to rule out additional antibodies. Care must be taken when performing rule outs using 0.2 M DTT-treated cells because antigens other than Sla are destroyed. Antigens usually denatured or altered include Yta, JMH, Knops, LW, Kell, Lutheran, Dombrock, and Cromer blood group antigens.1 The patient remained on C and K antigen negative restrictions and received units that were crossmatch-incompatible due to the anti-Sla. Approximately 3 years later, an antibody screen was performed (Figure 2), and anti-S was suspected. Additional testing confirmed the new antibody. After the identification of anti-S the patient was restricted to receive phenotypically-matched (ABO/Rh-compatible, C, K1, Fya, Jkb, and S negative) RBC units. It was decided not to give units negative for the Fyb antigen since it is common …

Utility of adsorption techniques in serological evaluation of warm autoimmune haemolytic anaemia.

Various adsorption techniques are available to remove serum autoantibodies and subsequently detect the underlying alloantibody in previously transfused patients with autoimmune haemolytic anaemia. We planned to establish a suitable adsorption technique in our transfusion service which can remove all autoantibodies and detect underlying alloantibodies rapidly, cheaply and effectively. We evaluated 71 direct antiglobulin test-reactive patients with warm AIHA over a period of 20 months. Twenty-three of these 71 patients who had a previous history of blood transfusion or pregnancy and were confirmed carriers of autoantibodies (indirect antiglobulin test-reactive) were considered for the adsorption study. Depending on the adequacy of samples, history of blood transfusion and severity of anaemia either autoadsorption or alloadsorption or both using polyethylene glycol (PEG) or low ionic strength saline (LISS)-papain were performed. Underlying alloantibodies were detected in 7 of the 23 patients (30.4%) and all these were specific to Rhesus antigens. The mean number of alloadsorptions for complete autoantibody removal using PEG was 1.43 which was significantly lower than the 3.9 using the LISS-papain method (p<0.05). The mean time required by PEG alloadsorption and LISS-papain alloadsorption for autoantibody removal was 93.6 minutes and 177.7 minutes, respectively (p<0.05). Discordant results were not observed in any case and identical alloantibodies were detected by both the techniques. We found that the PEG method is a rapid, cheap and effective way to remove autoantibodies and detect underlying alloantibodies.

How I manage cold agglutinins

ararely cause accelerated destruction of mismatched red blood cells (RBCs), and it is not necessary to detect examples of these antibodies that only react below body temperatures. The extent to which cold agglutinins can interfere with the results of pretransfusion antibody detection and compatibility tests is evident from a study by Garratty 1 on the importance of anticomplement reagents in immunohematology. With a low-ionic-strength saline (LISS) method that included room temperature incubation and polyspecific (anti-IgG+ C3) antiglobulin serum, the rate of unwanted positive tests (due primarily to the detection of cold-reactive auto- and alloagglutinins) was on the order of 1.41 percent! Omitting the room temperature incubation phase and use of anti-IgG reduced the unwanted positive rate reduced to 0.1 percent.

Ｐｒｅｗａｒｍ ｔｅｃｈｎｉｑｕｅはＲｈ系抗体の検出率を低下させる

Prewarm technique (PW), a method of warming reagents and samples before reaction, is used to prevent the effect of cold-reactive antibodies on the detection of warm-reactive antibodies. However, a recent report indicates the possibility that PW may attenuate the agglutination of red blood cells (RBC) induced by warm-reactive antibodies. In this study, we determined the effect of PW on detection efficiency for Rh system antibodies. We used 38 samples with Rh system antibodies (29 samples with anti-E antibody, 6 with anti-D antibody, 2 with anti-c antibody and 1 with anti-e antibody) which showed a positive reaction on 3 kinds of indirect antiglobulin tests (IAT) including 37°C saline IAT, low ionic strength saline (LISS)-IAT and the Polyethylene glycol (PEG)-IAT or bromelin method.Reduction of antibody titers induced by the prewarm technique was observed in 46.7% samples and 36.1% of samples tested by 37°C saline IAT and PEG-IAT, respectively. This reduction was observed more frequently in samples tested by LISS-IAT and the bromelin method (60.7% in LISS-IAT and 62.9% in the bromelin method). In samples with lower antibody titers (less than 2-fold), more than 30% of samples showed a negative reaction in LISS-IAT and the bromelin method. We then examined the effect a warming period on the Rh system antibodies-agglutination score. A decrease in agglutination score was observed on warming for 5min and the extent of this decrease was enhanced at 120min. In addition, we warmed the RBC suspension and plasma separately, and examined their effect on agglutination score. Since a decrease in agglutination score was observed only when the RBC suspension was warmed, warming of RBC may cause a decrease in warm-reactive antibodies reactivity. These results indicate that PW may attenuate not only cold-reactive antibodies-induced RBC agglutination but also warm-reactive antibodies-induced RBC agglutination.

Micro–Column Affinity Test and Gel Test: Comparative Study of Two Techniques for Red Cell Antibody Screening

Background and Objectives: We describe the results of a comparative evaluation of a gel test (ID Micro Typing) and a micro–column affinity test (MCAT, Cellbind Screen) for red cell antibody screening and identification under routine conditions. Materials and Methods: 3,000 serum samples of patients from the Mannheim University Hospital were tested in parallel by means of the gel test and the MCAT, using the low–ionic–strength–saline indirect antiglobulin test and the protein G affinity technique, respectively. Test cells used were the same in all tests. In addition, we performed titration studies with all detected antibodies as well as with 59 frozen sera containing antibodies of known specificity. Results: A total of 154 antibodies (5.1%) were detected, 149 by gel test and 147 by MCAT. The overall sensitivity and specificity of the gel test was 96.8 and 96.5% and of the MCAT 95.5 and 97.2%. No significant differences between the gel test and MCAT were found when the titer scores of all 213 (fresh and frozen) antibodies were used to check the results. The mean scores for the gel test and the MCAT were 26.8 and 28.5, respectively. For anti–Fy^a and anti–Kell, a significantly higher titration score could be obtained in the MCAT, whereas anti–Lu^a showed a significantly higher score with the gel test. Conclusion: For the screening of unexpected red blood cell antibodies, the MCAT is as sensitive as the gel indirect antiglobulin test. The sensitivity and specificity of the two systems are more or less the same although it seems that IgM antibodies are better detected by the gel test.