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Red blood cell alloimmunization to antigens other than D, such as C, c, E, e, and antigens in the Kell, MNS, and Duffy blood group systems, has emerged as an important cause of hemolytic disease of the fetus and newborn (HDFN). Antibody screening for these antibodies is not routinely practiced for all antenatal patients in developing countries, mainly because of financial constraints. Here we report a rare case of HDFN due to dual antibodies to Rh and Kidd blood group system antigens: anti-E and anti-Jka. This case report highlights the importance of routine and regular antenatal screening of all pregnant women for proper monitoring and follow-up.

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. Immunohematology 2018; 34:57–60.

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. Immunohematology 2017;33:27–35.

Anti-Dia can mediate hemolytic disease of the fetus and newborn, but it is unclear if it can cause hemolytic transfusion reactions (HTRs). To date, there has only been one report of a possible immediate HTR attributed to anti-Dia. Our case report details an immediate HTR due to anti-Dia in a patient with pre-existing liver failure. This reaction triggered multi-organ failure, and the patient subsequently died. This case also highlights the importance of considering HTRs even when routine antibody screening has been unremarkable, particularly when electronic crossmatch is used, because of the potential for an alloantibody against a low-prevalence antigen. Immunohematology 2015;31:163–165.

Our facility changed antibody screening methods from a gel microcolumn–based test (ID-Micro Typing System Gel Test; Ortho Clinical Diagnostics, Inc., Raritan, NJ) to an automated solid-phase test (Galileo/Capture-R Ready-Screen [I and II], Immucor, Inc., Norcross, GA). To determine whether detection rates for commonly encountered clinically significant red blood cell antibodies differed as a consequence of this change, preimplementation and postimplementation antibody identification records were retrospectively reviewed. A statistically significant difference in the percentage of positive screening tests during the gel microcolumn testing period (73,903 total screens, 1.56% confirmed positive) versus the solid-phase screening period (80,242 total screens, 1.81% confirmed positive; p < 0.0002) was observed. The number of antibodies to K identified was significantly lower with solid phase than with gel (27% decrease; p = 0.004). It is unknown whether there is a statistical difference in delayed or hemolytic transfusion reaction rates as this was not evaluated. Immunohematology 2013;29:101–04.

Currently, the goal of testing for RBC antibodies is to use a method that will detect, if possible, all antibodies that are considered clinically significant and yet not detect antibodies of little clinical importance in transfusion or pregnancy. The focus of test method development has been on the more controllable variables of the first and second stages of agglutination. Tube test methods have been developed over the years to achieve shorter turnaround times for quicker test results and improved sensitivity, with the occasional negative impact on relevant results. The focus on improving efficiency through automation, and personnel resourcing challenges of the transfusion service, have led laboratories to select methods tailored to meet their needs. This review compares the newer methods used in the gel test and solid phase test with commonly used tube methods. Both of the newer methods were developed with future adaptation to automation in mind. Further literature reviews about antibodies detected in only one or two methods and their general lack of clinical relevance as well as the occasional rare examples that produce significant clinical effects on transfused patients are also discussed. Immunohematology 2006;22:196–202.

A variety of pretransfusion tests have been developed to improve the safety and effectiveness of transfusion. Recently, a number of traditional tests have been shown to offer limited clinical benefit and have been eliminated in many facilities. A survey of pretransfusion test practices was distributed to 116 hospital transfusion services. Routine test practices and facility size were analyzed. Ninety-one responses were received. Many smaller laboratories include tests such as anti-A,B, an autocontrol, and DAT, and immediate spin and 37°C microscopic readings. Nine percent never perform an Rh control with anti-D typing on patient samples. Various antibody screening and crossmatch methods are utilized. Individual laboratory test practices should be periodically assessed to ensure that they comply with standards, represent the recognized best practice, and are cost-effective. The survey responses indicate that many laboratories perform tests that are not necessary or cost-effective. These facilities should review their processes to determine which tests contribute to transfusion safety. Smaller facilities may be reluctant to change or lack the expertise necessary for this decision making and often continue to perform tests that have been eliminated in larger facilities. Consultation with larger hospital transfusion services may provide guidance for this change. Immunohematology 2003;19:1–6.

We compared the results of routine blood tests for 102 blood donors’ samples and 100 patients’ samples collected in spray-dried K2EDTA, spray-dried K3EDTA, and liquid K3EDTA blood collection tubes to evaluate the impact of changes in formulation of the anticoagulant (K2EDTA vs. K3EDTA), its application (liquid vs. spray- dried), and tube material (glass vs. plastic). Methods for ABO/D testing, antibody screening, and antibody identification included direct hemagglutination/microplate (Olympus® PK 7200) and gel column methods (Ortho ID-Micro Typing System™/Gel Test™). Additional studies on blood donors’ samples included time delayed antigen testing and antibody identification and half-draw/half- evacuated collections. Also, we compared the results of routine ABO/D testing and antibody screening for 50 patients’ samples collected in spray-dried K2EDTA and spray-dried K3EDTA and for an additional 50 patients’ samples collected in spray-dried K2EDTA tubes from two different manufacturers. All patients’ samples were tested in parallel by solid phase/microplate method (Immucor® ABS 2000) and the standard manual tube method. All test results for routine blood bank tests on donors’ and patients’ samples were concordant, demonstrating the equivalence of spray-dried K2EDTA, spray-dried K3EDTA, and liquid K3EDTA blood collection tubes for routine donor center or transfusion service testing.

It is not clear whether platelet-reactive antibody screening is clinically significant for patients facing frequent platelet transfusions. On the basis of data from 96 patients who had been examined for platelet-reactive antibodies by the mixed passive hemagglutination method for a variety of reasons, we investigated the following three issues retrospectively: (1) the relationship between platelet-reactive antibodies and the occurrence of problems in platelet transfusions, such as refractoriness or nonhemolytic reactions;(2) the influence of a history of transfusion on the production of those antibodies; and (3) the effect of screening for those antibodies on the prompt administration of appropriate platelet components. More than half of the platelet transfusion-related problems were associated with platelet-reactive antibodies. For patients with a history of transfusion, the mean period before a clinical problem occurred with platelet transfusions was 9 days,compared with 66 days for those without such a history. Accordingly, during the period, patients with a history of transfusions received fewer units of platelets and had fewer donor exposures than did patients without such a history. On the other hand, most patients who had been screened in advance for those antibodies received appropriate platelet components without delay, whereas an average of 10 days was needed before those who had not been screened received compatible platelets. The patients who had not been screened were transfused with 68 units of random platelets on average during the period. When frequent platelet transfusions are anticipated, especially for patients with a history of transfusion, screening for platelet-reactive antibodies beforehand would be helpful for prompt administration of appropriate platelets, although problems, such as the cost of those platelets and the burden on donors, remain to be resolved. Immunohematology 2002;18:104–108

Our institution has implemented two instruments, the Galileo and the Echo, that use different solid-phase red cell adherence assays for antibody screening in pretransfusion compatibility testing. During the initial implementation of these two instruments, we noticed very different problems: falsely positive results on the Galileo, and falsely negative results and lack of reproducibility on the Echo. Comparison of falsely positive antibody screen results from approximately equivalent numbers of samples run on the Galileo and samples tested by standard manual tube technique using low-ionic-strength saline enhancement showed a false-positive rate of 1.4 percent on the Galileo (defined as a positive screen with a negative panel). Testing using the Echo identified four cases of falsely negative antibody screens, (defined as a negative screen on a patient sample subsequently shown to be positive by the same method). In addition, we note a lack of reproducibility on the Echo, which emphasizes the importance of replicate testing during validation of automated antibody screening platforms. Immunohematology 2012;28:137–9.

The GLAM assay, a combined flow cytometric immunofluorescence test that simultaneously detects antibodies to granulocytes, lymphocytes, and monocytes, was used in the investigation of autoimmune neutropenia. This method avoids the need for a succession of immunofluorescence tests, first against granulocytes and then against lymphocytes, in order to distinguish granulocyte-specific from granulocyte/lymphocyte-reactive antibodies, such as noncomplement-fixing anti-HLA sera. Samples from 18 patients with a suspected diagnosis of autoimmune neutropenia were referred for investigation. Leukocytes were harvested in sufficient quantities from 10 of the patients such that neutrophil and lymphocyte direct antiglobulin tests (DATs) and antibody screening and identification could be undertaken (in one case the results were inconclusive). Only three of these 10 patients had DAT-positive granulocytes, and one of these three also had DAT-positive lymphocytes. One further patient demonstrated DAT-positive lymphocytes in the absence of granulocyte-bound IgG, despite a presumed diagnosis of autoimmune neutropenia, rather than pancytopenia. This was the only patient in this cohort who had demonstrable leukocyte antibodies reacting with lymphocytes but not granulocytes. Of the remaining eight patients (not evaluable for granulocyte or lymphocyte DATs), five had free leukocyte antibodies in the serum; three of these five had both granulocyte- and lymphocyte-reactive antibodies free in the serum and two only had granulocyte-specific antibodies.

Conventional tube testing was used for antibody screening and titration in D– pregnant women in our hospital until the recent introduction of the gel test. In this study we assessed the sensitivity of the gel test in our setup and tried to establish a correlation between these tests for determining antibody titer. We collected 652 blood samples from 223 antenatal D– women during a span of 1 year. The samples were tested separately by the conventional tube technique and the gel test for antibody detection and titration. The tube test detected 84 (12.8%) positive samples as compared with 93 (14.2%) by gel test, indicating the latter to be more sensitive (p < 0.01). The gel test picked up weakly reactive anti-D that the tube test missed. We did not use any enhancing media such as LISS in titration studies performed by either method in an effort to establish a correlation. However, much higher titers (one- to fivefold) were obtained by the gel test with no clear correlation with the corresponding tube values. When comparing the titer values to the finding of hydrops on ultrasound and Liley’s chart OD reading on amniocentesis, a value of less than 128 (i.e., 64) by gel test corresponded to normal results. Through this study, we thus conclude that the gel test is more sensitive for antibody detection, although a linear correlation could not be established for titers. Clinical correlation may point toward a critical titer of 64 for the gel test, but further studies need to be done to support this finding. Immunohematology 2010;26:174–77.

Pretransfusion tests must detect antibodies that can shorten the life of red blood cells (RBCs). Some studies have demonstrated the existence of clinically significant antibodies detected at 37°C in saline that are not detected by the indirect antiglobulin test (IAT) when the conventional tube test is used. Our aim was to determine whether these antibodies, detected with a 37°C saline tube test, are also detected when a sensitive column gel agglutination method is used. The 2373 pretransfusion samples were tested as they were received (from May 1998 to December 1999), in 37°C saline and by IAT using the DiaMed gel system. The screening tests were performed using 50 μL of 0.8% low-ionic-strength saline suspended RBCs and 50 μL of plasma. The tests were examined for agglutination and hemolysis. Two hundred and thirty three samples (9.81%) were reactive by IAT and 88 (3.70%) by 37°C saline. All 88 samples reactive by 37°C saline also reacted by IAT. These data indicate that 37°C saline is not an essential pretransfusion procedure when the DiaMed gel test is used. Immunohematology 2002;18:13–15.

Our blood bank routinely screens donors for antibodies using a solid-phase red cell adherence (SPRCA) assay. Positive results are then confirmed using a tube technique with polyethylene glycol (PEG) enhancement due to reported higher specificity than with SPRCA. Over a 5-month period, 49,084 donor serum or plasma samples were tested using the SPRCA assay. Further identification of positive samples was performed using a PEG enhancement method. Testing was performed with strict adherence to the manufacturers’ inserts. Of 49,084 samples, 313 (0.64%) were positive by the SPRCA assay. Of these, 99 (31.6%) samples remained positive when tested with PEG enhancement. The remaining 214 (68.4%) were negative, giving specificity for the SPRCA assay of 99.6 percent (48,985/ 49,199). We report a high specificity for antibody screening using the SPRCA assay. However, it is cost effective to perform a confirmatory tube test with PEG enhancement because 214 SPRCA assay samples were interpreted as having a negative antibody screen, thus allowing the release of valuable blood components for transfusion. Immunohematology 2001;17:14–16.

The gel system has been reported to be more sensitive and specific than the conventional tube indirect antiglobulin test (IAT) for antibody screening. However, a major concern about the gel system is its cost. A cost analysis study was therefore conducted at our hospital. The gel system costs more than the conventional tube IAT per test; however, the total staff and reagent costs per year were about equal, because of staff savings. Workload and cost per patient requiring blood were reduced using the gel system. Immunohematology 1996;12:30-32.

This study compared the performance of polyethylene glycol (PEG) and low-ionic saline solutions (USS) as enhancement media for routine use in a large transfusion service, A PEG additive solution (PEG plus LISS) was compared to a LISS additive (LISS plus polymers) and to an albumin-indirect antiglobulin test (A-IAT). Fifty serum samples containing clinically significant alloantibodies and fifty samples without alloantibodies were tested. Following an acute hemolytic transfusion reaction (HTR) involving an anti-K that was not detected with LISS but was retrospectively found to be reactive with PEG, an additional 151 samples received for antibody screening were prospectively evaluated in parallel using PEG and LISS. PEG detected all clinically significant antibodies in the 50 previously tested samples, with mean reactivity scores greater than LISS or A-IAT. In the prospective study, PEG detected 35 clinically significant antibodies and 10 clinically insignificant antibodies, while LISS detected only 15 clinically significant antibodies and 33 clinically insignificant antibodies. PEG appears to increase detection of significant antibodies while decreasing detection of insignificant antibodies. PEG was therefore substituted for LISS as an enhancement medium and has been in routine use for 12 months, with no reported acute or anamnestic HTRs in 6,353 transfusions.

The BioVue™ column agglutination technology (CAT) was evaluated simultaneously with standard tube test (STT) methodology for use in indirect antiglobulin testing (IAT) and direct antiglobulin testing (DAT). One thousand thirty-five blood specimens were used for the IAT comparison, and 44 blood specimens were used for the DAT comparison. Both polyspecific antiglobulin and anti-IgG antiglobulin reagents were used in the tube testing and the CAT testing. For IAT, specificity was 100 percent for CAT and 99.6 percent for STT; sensitivity was 97.9 percent for CAT and 100 percent for STT. In addition, a 67 percent labor savings was realized with CAT versus STT. Specificity and sensitivity of both methodologies were 100 percent for the DAT. BioVue™ proved to be a reliable and efficient alternative to standard test tube methods for doing IATs and DATs. Immunohematology 1993;9:78.

The Olympus PK™ CMV system (OHA) for the detection of antibodies to cytomegalovirus (CMV) was evaluated for use on the Olympus PK7100 automated pretransfusion blood testing instrument. Indeterminate reactions were obtained with 18 (1.2%) of the 1,495 samples originally tested, of which 9, 6, and 3 were OHA positive, indeterminate, and negative, respectively, on retest. The OHA was found to have a concordance of 99.7 percent when tested in parallel with the CMV Scan latex agglutination test (LAT). There were four discrepant results in the 1,495 donor plasmas tested by the two methods. Two of the three samples that were positive by the LAT and negative by the OHA test were also negative with the IMx enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT). The OHA failed to detect antibodies to CMV in a sample that was positive by the IMx ELISA, CFT, and LAT. The IMx ELISA, which was regarded as the arbiter of true positivity, yielded a sensitivity and specificity of 99.9 percent and 100 percent respectively, for the Olympus PK™ CMV system. Forty percent of 1,807 random blood donors in this region had antibodies to CMV. CMV testing can be performed simultaneously with blood grouping and red cell antibody screening on the Olympus PK7100. Immunohematology 1992;8:41–43.

Two solid phase methods of antibody detection, Capture-R (CR) and Capture-R Ready-Screen (RS), were compared to determine their acceptability for use in prenatal antibody screening. Ninety-six serum samples, screened using a saline antiglobulin test, were coded and tested by CR and RS at two laboratory sites using a blinded study design. Thirty of the samples were free of antibody, and 66 samples contained antibody. Parallel testing was also performed in both laboratories on 648 prenatal samples. The sensitivity and specificity of CR, based on the 96 previously screened samples, was 95 percent and 90 percent, respectively, and the sensitivity and specificity of RS was 90 percent and 89 percent, respectively. Antibodies detected only by CR included anti-K(1), -Ch(2), -Jka(1), -Lea(1), -Fya(1), -Mca(1), and -e(1). Antibodies detected only by RS included anti-Jka(5) and -E(1). The sensitivity and specificity of CR for the 648 prenatal samples was 100 percent for each, while the sensitivity and specificity of RS was 97 percent and 100 percent, respectively. Both CR and RS are acceptable techniques for prenatal antibody screening.