Conventional Tube Technique Research Articles

The role of conventional tube and column agglutination technique in predicting hemolysis in various clinical conditions

Introduction: The correlation of positive result in direct antiglobulin test (DAT) with in vivo hemolysis is important for the patient management. This study compares the correlation of tube-DAT and -DAT by column agglutination technique (CAT) with in vivo hemolysis and reviews the appropriateness of the requests for DAT in patients with various clinical conditions. Patients and Methods: A total of 250 consecutive patients referred for DAT were enrolled for the study. Laboratory indicators of hemolysis were obtained from the computerized hospital data management system. Hemolysis in patients was graded using predetermined criteria. DAT was done using CAT and tube techniques and graded on the agglutination pattern. Results: Among 250 patients studied, 160 patients were from the hemato-oncology unit, and 47 had an autoimmune disease. Laboratory evidence of hemolysis was seen in 170 patients (severe: 45.88% and moderate: 54.12%). Positive DAT by tube technique was seen in only 41 out of 65 patients whose DAT was positive by CAT. Hemolytic anemia was the single largest cause in this group. Positive predictive values (PPVs) of tube and CAT were comparable. On comparing the strength of agglutination with the in vivo-hemolysis, 1+ reaction by CAT had low PPV. Conclusion: The present study shows that mere DAT positivity does not always indicate in vivo hemolysis. Hence, while recommending the routine use of CAT for DAT, we suggest to incorporate the strength of agglutination reaction in laboratory report for the ease of interpretation.

Comparison of micro column technology with conventional tube methods for antibody detection.

BACKGROUND AND OBJECTIVES:Conventional tube technique (CTT) has been the mainstay for antibody detection in pretransfusion testing. There have been rapid technological advances in blood banking and methodology of crossmatch has been modified to improve the sensitivity of these tests and to enable automation. This study was done to compare the efficacy of three crossmatch techniques: CTT, tube low-ionic-strength-saline indirect antiglobulin test (tube LISS-IAT), and micro column technology (MCT) used in the blood bank serology laboratory.MATERIALS AND METHODS:In this prospective study, 150 samples from patients who had received two or more transfusions on two different occasions (with at least 72 h between two transfusions) were subjected to cross match by three different techniques – CTT, LISS-IAT, and MCT.RESULTS:A total of 16 cases with antibodies were identified in 150 patients. Out of 16 cases, 14 were clinically significant (anti-c = 5, anti-K = 4, anti-E = 2, anti-S = 2, anti-Jka = 1) and 2 nonclinically significant antibody cases (anti-Lea). MCT detected all the 14 clinically significant antibody cases and no case of nonclinically significant antibody. Tube LISS-IAT detected 14 antibody cases including 2 cases of non-clinically significant antibody but failed to detect 1 case of anti-c and the only case of anti-Jka. CTT detected only 10 antibody cases including 2 cases of non-clinically significant antibody and but failed to detect 3 cases of anti-c, 1 case of anti-K, 1 case of anti-E, and the only case of anti-Jka.CONCLUSION:MCT was found to be most efficacious when compared to CTT and tube LISS-IAT in detecting clinically significant red cell antibodies; although MCT missed 2 cases of Lea antibody which were detected by CTT and LISS-IAT.

Comparison of antibody titers using conventional tube technique versus column agglutination technique in ABO blood group incompatible renal transplant.

INTRODUCTION:Measurement of alloantibody titer to a red cell antigen (ABO titers) is an integral part of management of ABO incompatible kidney transplants (ABOiKT).MATERIAL AND METHODS:There are different methods of titer estimation. Alloantibody detection by tube titration and Gel agglutination columns are accepted methodologies. It is essential to find the difference in titers between the two methods so as to set the 'cut-off' titer accordingly, depending upon the method used.RESULTS:We did a prospective observational study to compare and correlate the ABO titers using these two different techniques – conventional tube technique (CTT) and the newer column agglutination technique (CAT). A total of 67 samples were processed in parallel for anti-A/B antibodies by both tube dilution and column agglutination methods. The mean titer by conventional tube method was 38.5 + 96.6 and by the column agglutination test was 96.4 + 225. The samples correlated well with Spearman rho correlation coefficient of 0.94 (P = 0.01).CONCLUSION:The column agglutination method for anti A/B titer estimation in an ABO incompatible kidney transplant is more sensitive, with the column agglutination results being approximately two and half fold higher (one more dilution) than that of tube method.

Investigation of the Complete Suite of the Leucine and Isoleucine Isomers: Toward Prediction of Ion Mobility Separation Capabilities.

In this study we investigated 11 isomers with the molecular formula C6H13NO2 (m/z 131) to ascertain the potential of utilizing drift tube ion mobility mass spectrometry to aid in the separation of isomeric mixtures. This study of small molecules provides a detailed examination of the application of uniform field ion mobility for a narrow scope of isomers with variations in both bond coordination and stereochemistry. For small molecules, it was observed that in general constitutional isomers are more readily separated by uniform field mobility in comparison to stereoisomers such as enantiomers or diastereomers. Diastereomers exhibited differences in their collision cross section (CCS), but were unresolvable in a mixture, whereas the enantiomers studied did not exhibit statistically different CCS values. A mathematical relationship relating the CCS to resolving power was developed in order to predict the required ion mobility resolving power needed to separate the various isomer classes. For the majority of isomers evaluated in this study, a uniform field-based resolving power of 100 was predicted to be sufficient to resolve over half (∼60%) of all hypothetical isomer pairs, including leucine and isoleucine, whereas their stereoisomers (d- and l-forms) are predicted to be significantly more challenging, if not impossible, to separate by conventional drift tube techniques.

Distribution of DI*A and DI*B Allele Frequencies and Comparisons among Central Thai and Other Populations.

Alloantibodies to the Diego (DI) blood group system, anti-Dia and anti-Dib are clinically significant in causing hemolytic transfusion reactions (HTRs) and hemolytic disease of the fetus and newborn (HDFN), especially in Asian populations with Mongolian ancestry. This study aimed to report the frequency of the DI*A and DI*B alleles in a Central Thai population and to compare them with those of other populations previously published. Altogether, 1,011 blood samples from unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok were included. Only 391 samples were tested with anti-Dia by conventional tube technique. All samples were genotyped for DI*A and DI*B alleles using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The DI phenotyping and genotyping results were in 100% concordance. The DI*A and DI*B allele frequencies among 1,011 Central Thais were 0.0183 (37/2,022) and 0.9817 (1,985/2,022), respectively. Allele frequencies were compared between Central Thai and other populations. Our data shows that DI*A and DI*B allele frequencies are similar to Southeast Asian, Brazilian, Southern Brazilian and American Native populations; whereas, these frequencies significantly differ from those reported in East Asian, Italian, Alaska Native/Aleut, Hawaiian/Pacific Islander and Filipino populations (P<0.05), corresponding to the results of a matrix of geometric genetic distances. This study confirms that the prevalence of DI*A and DI*B alleles among Central Thais is similar to Southeast Asians and different to others populations of the world. A PCR-based identification of DI genotyping should overcome some of the serological limitations in transfusion medicine and provides a complementary tool for further population-genetic studies.

Antibody Titers Study in Group O Blood Donors: Tube and Column Agglutination Techniques

Background: O blood group transfusions to patients of all blood groups has continued since long. Clinical significance of ABO antibody titre in ABO-I kidney transplantation is well known. Few studies done on group O blood/ apheresis donations, for detecting ABO antibody titers in collected plasma components. The passively acquired antibodies may destroy recipient’s own red cells and tissue grafts, cause acute hemolysis, hemoglobinemia, jaundice, progressive anemia, spontaneous agglutination, positive direct antiglobulin test and increased osmotic fragility of the patient’s red cells. Objective: To evaluate agglutinin levels in group O blood donations. Group O donor population, randomly selected and titrated using tube technique and gel card technique to identify titer levels for Anti A, Anti B, Anti AB antibodies. Both IgM and IgG titer levels evaluated. Methods: Plasma samples from 200 randomly selected blood group O donors were tested by ABO antibody titration using conventional tube technique and AHG gel card column agglutination technique (CAT). ABO antibody levels categorized as those higher than 16 and those lower than 16. After treatment with Dithiothretiol (DTT) for characterization of only IgG class, titres levels were again tested in same O group blood/apheresis donors. Statistical analyses performed using various tests. Results: Males constituted 88% of O group donors studied and 12% were females. ABO antibody titer categorized as 0 to ≤16 and titer >16 for both IgM and IgG antibody for Anti A, Anti B and Anti AB. Both test tube and CAT used for testing. Estimates of prevalence of titers by CAT: Anti A IgM ≤ 16 in 62%; >16 in 38% (p-value 16 in 68% (p-value 16 in 31% (p-value 16 in 65% (p-value 16 in 70% (p-value 16 in 73% (p-value 16 in 56% (p-value 16 in 56% (p-value 16 in 49% (p-value 16 in 64% (p-value 16 in 66% (p-value 16 in 96% (p-value < 0.001). No significant co-relation found between age and titer or gender and titer, by both technologies. Mean anti-A and anti-B and anti-AB titers in group O plasma were, respectively,163.28,113.42 and 166.77 for IgM antibody and 174.50,152.98 and 311.63 for IgG antibody by tube method and 34.01,33.30 and 63.77 for IgM Antibody,108.41,103.10 and 272.46 for IgG antibody by CAT (p < 0.0001). Conclusion: Study confirms that titration of ABO antibodies in blood banks will increase safety in non-identical ABO transfusions and transplants. No significant correlation established between titer and age or titer and gender.

Direct antiglobulin test positivity in multi-transfused thalassemics.

Introduction:Red cell allo- and auto-immunization is a well recognized problem in multi-transfused thalassemic patients. We conducted this study on 301 multi-transfused thalassemic patients under the Thalassemia Transfusion Programme of Advanced Pediatric Centre of PGIMER.Aims and Objectives:The study was designed to determine the frequency of alloimmunization and autoimmunization in multi-transfused thalassemic patients and to establish the specificity of alloantibody to red cell antigens, if alloimmunization is detected.Materials and Methods:The antibody screening was performed by the conventional tube technique using commercially available three cell screening panel (Diamed Switzerland) by saline, low ionic strength solution (LISS) and albumin indirect antiglobulin test (IAT). Samples with alloantibodies were then tested with red cell identification panel to determine the alloantibody specificity. Autoantibody screening was performed by direct antiglobulin test (DAT) during pre-transfusion testing.Results:Of the 301 patients, 52 (17.28%) were found to have antibodies (-allo and –autoantibodies). A total of 11 red cell alloantibodies were detected in 10 patients and the specificities were anti-Kell in 6(54.5%), anti-D in 2(18.2%), anti-c in 1(9.1%) and a combination of anti-E (9.1%) and anti-Jkb in 1 (9.1%) patients. DAT was positive in 48 (15.9%) patients. The frequency of autoantibody was significantly higher in alloimmunized group as compared to non-alloimmunized group (60% V/s 14.4%). Also, the pre-transfusion hemoglobin was significantly lower in the immunized group (8.5 gm/dl V/s 9.0 gm/dl; p=0.03) than the non-immunized group.Conclusion:Based on these observations, we suggest antigen typing of all thalassemia major patients for ABO, Rh and Kell antigens before initiating transfusion therapy. Also, screening for allo- and auto-antibodies at regular intervals should be done prior to each transfusion.

Comparison Between Conventional and Automated Techniques for Blood Grouping and Crossmatching: Experience from a Tertiary Care Centre.

Context:The routine immunohematological tests can be performed by automated as well as manual techniques. These techniques have advantages and disadvantages inherent to them.Aims:The present study aims to compare the results of manual and automated techniques for blood grouping and crossmatching so as to validate the automated system effectively.Materials and Methods:A total of 1000 samples were subjected to blood grouping by the conventional tube technique (CTT) and the automated microplate LYRA system on Techno TwinStation. A total of 269 samples (multitransfused patients and multigravida females) were compared for 927 crossmatches by the CTT in indirect antiglobulin phase against the column agglutination technique (CAT) performed on Techno TwinStation.Results:For blood grouping, the study showed a concordance in results for 942/1000 samples (94.2%), discordance for 4/1000 (0.4%) samples and uninterpretable result for 54/1000 samples (5.4%). On resolution, the uninterpretable results reduced to 49/1000 samples (4.9%) with 951/1000 samples (95.1%) showing concordant results. For crossmatching, the automated CAT showed concordant results in 887/927 (95.6%) and discordant results in 3/927 (0.32%) crossmatches as compared to the CTT. Total 37/927 (3.9%) crossmatches were not interpretable by the automated technique.Conclusions:The automated system shows a high concordance of results with CTT and hence can be brought into routine use. However, the high proportion of uninterpretable results emphasizes on the fact that proper training and standardization are needed prior to its use.

A study on Rh incompatibility and frequency of weak D among blood donors and patients at a tertiary care referral teaching hospital in Tirupati, Andhra Pradesh

Background: Rh D antigen is the next most important after ABO antigens in the field of transfusion medicine. Weak D refers to reduced expression of D antigen on the red blood cell that requires an extended testing with indirect antiglobulin test (IAT) to get detected. Clinical importance of weak D arises when labelling the donor and patient, as the donor is labelled as D positive, patient as D negative. Methods: In our center all blood donor and patient samples are tested for ABO and Rh D by conventional tube technique using two anti-D reagents; anti-D immunoglobulin M (IgM) monoclonal and a blend of anti-D IgM and immunoglobulin G (IgG). The blood samples which were negative for agglutination by immediate spin method were further tested for weak-D using IgG anti-D in the IAT phase with low ionic strength solution (LISS)/Coombs’gel card. Results: A total of 46,654 blood samples were tested (22,326 donors and 24,328 patients) during the period January 2012 to August 2014. Among these 43,771 (93.82%) were Rh D positive and remaining 2,883 (6.18%) were Rh-D negative. A total of 30 individuals (16 donors and 14 patients) were weak D positive constituting 1.04% of Rh-D negatives and 0.06% of total individuals screened. Conclusions: This study shows the prevalence of weak D antigen in our population who are representative of Rayalaseema region of Andhra Pradesh. It also stresses the need to identify individuals with variant D (rather than weak or partial D) and to inform them about their status as donor and recipient of blood/organ.

Phenotype frequencies of blood group systems (Rh, Kell, Kidd, Duffy, MNS, P, Lewis, and Lutheran) in blood donors of south Gujarat, India

Background:This is the first study on phenotype frequencies of various blood group systems in blood donors of south Gujarat, India using conventional tube technique.Material and Methods:A total of 115 “O” blood group donors from three different blood banks of south Gujarat were typed for D, C, c, E, e, K, Jka, Lea, Leb, P1, M, and N antigens using monoclonal antisera and k, Kpa, Kpb, Fya,Fyb, Jkb, S,s, Lua, and Lub antigens were typed using polyclonal antisera employing Indirect Antiglobulin Test. Antigens and phenotype frequencies were expressed as percentages.Results:From the 115 blood donor samples used for extended antigen typing in the Rh system, e antigen was found in 100% donors, followed by D [84.35%], C [81.74%], c [56.32%], and E [21.74%] with DCe/DCe (R1 R1, 40.87%) as the most common phenotype. k was found to be positive in 100% of donors and no K+k- phenotype was found in Kell system. For Kidd and Duffy blood group system, Jk(a+b+) and Fy(a-b-) were the most common phenotypes with frequency of 52.17% and 48.69%, respectively. In the MNS system, 39.13% donors were typed as M+N+, 37.39% as M+N-, and 23.48% as M-N+. S+s+ was found in 24.35% of donors, S+s- in 8.69%, and S-s+ as the commonest amongst donors with 66.96%. No Lu(a+b+) or Lu(a+b-) phenotypes were detected in 115 donors typed for Lutheran antigens. A rare Lu(a-b-) phenotype was found in 2.61% donors.Conclusion:Data base for antigen frequency of various blood group systems in local donors help provide antigen negative compatible blood units to patients with multiple antibodies in order to formulate in-house red cells for antibody detection and identification and for preparing donor registry for rare blood groups.

Frequencies and specificities of red cell alloantibodies in the Southern Thai population.

Context:Detailed reports of red cell alloantibody frequencies and specificities in the Thai population are limited. The aims of this study were to determine the specificity and compare the frequency of alloantibodies detected using column agglutination technology (CAT) and conventional tube techniques in blood donors and previously transfused patients.Settings and Design:We retrospectively reviewed antibody screening and identification records for two time periods: January-December 2006 during which conventional tube techniques were used and January 2008-December 2009 when CAT was used.Results:The overall prevalence of alloantibodies in both patients and donors when using conventional tube techniques was 0.7%, for patients only was 0.9% and donors 0.6%. The most frequent antibodies detected in both groups were anti-Lea, anti-Mia, anti-Leb, anti-P1 and anti-E. When using CAT, alloantibodies were found in 0.8% of patients and 0.13% of donors with the five most common alloantibodies found in patients were anti-Mia, anti-E, anti-Lea, anti-c and anti-Leb respectively. Similarly the common alloantibody specificities in donors were anti-Lea, anti-Mia, anti-Leb, anti-M and anti-D.Conclusions:One of the most commonly identified alloantibodies in the Thai population studied was anti-Mia suggesting that Mia positive red cells should routinely be included in antibody screening and identification in this population. For antibody screening and identification, CAT method detected immune and warm alloantibody (ies) more frequently than that associated with conventional tube techniques.

Blocked D phenomenon.

The blocking of D antigen sites by IgG anti-D in severe cases of Haemolytic Disease of the new born (HDN) is not a new phenomenon1. The coating maternal anti-D prevents the agglutination of the D positive red cells (RBC) by the IgM typing reagent anti-D. Blocking tests were actually the first one used to prove the existence of non-agglutinating IgG antibodies1–3. However the total blocking of D antigen sites by IgG resulting in false negative D typing when a human IgM anti-D was used are rare. In Issitt’s 1981 book, Applied Blood Group Serology, 2nd edition, he says that he surveyed the staff in his own laboratory and found, that even workers having over 30 years of work experience between them, had identified only four cases of blocking anti-D. Issitt4 commented that this phenomenon, much loved by teachers of immunohaematology, is far more likely in theory than encountered in practice. Where it does occur, the anti-D does not necessarily have to be of a high titre. Sulochana et al.5 described a case of blocking anti-D in 2008 with a maternal IgG anti-D titre of 32, the IgM titre was 1,024. In this case the maternal and baby’s RBC were initially grouped as B RhD negative in the local hospital. Due to profound jaundice and signs of kernicterus the baby was transferred to the neonatology department of the Medical College Hospital. Despite three exchange transfusions with B RhD negative blood the Direct Antiglobulin Test (DAT) remained positive and free anti-D was still detectable in the baby’s plasma. Anti-D with a titre of 32 was eluted from the baby’s RBCs. Antenatal grouping and atypical antibody screening had not been performed so an early opportunity to both detect and quantify the anti-D had been missed. This would have informed on monitoring and preventative management through the pregnancy. In this issue of “Blood Transfusion” Verma et al.6 describe a case of blocked D in RhD haemolytic disease of the foetus. At 20 weeks gestation the maternal anti-D titre was found to be 256 by conventional tube technique. Subsequent ultrasound screening showed the foetus to be hydropic and percutaneous umbilical blood sampling confirmed the findings of foetal anaemia (Hb 54 g/L and haematocrit 13.9%). The foetal RBC grouped as RhD negative with a 4+ DAT, an eluate yielded anti-D, showing the D typing to have been blocked. Successful intrauterine transfusion (IUT) was performed in that post transfusion the Hb had incremented to 141 g/L and Hct 41.8%. An icteric baby was delivered, again grouping as RhD negative, but on this occasion due to the ORhD negative blood used for the IUT(s). The blocking phenomenon is not limited to anti-D. Other blood grouping failures have been reported e.g. two cases of false negative K1 typing of foetal cells due to blocking maternal IgG anti-K7,8. Lee et al.7 reported on a case of K1 blocking and showed evidence that various antenatal anti-K1 samples with a titre of 256 or greater can exhibit the blocking of K1 antigens. The number of K1 antigen sites per RBC is in the range 4,000–18,000. The author has also shown the blocking of Fya antigen sites with high titre HIMA-19 (human-murine) anti-Fya in a simulated experiment (unpublished observation). False negative typing results caused by potent maternal IgG antibodies blocking antigen sites are not common when using modern monoclonal blood grouping reagents, especially anti-D. With the immunogenicity of D antigens being only second to ABO antigens, accuracy in RhD typing is critical in transfusion medicine. British Committee for Standards in Haematology (BCSH)9 antenatal grouping and screening guidelines provide guidance on the most secure way to identify potentially harmful cases of HDFN, the outcomes of which may include evidence of RhD antigen blocking. The performance of a cord DAT is also advocated. The performance of an eluate on subsequent DAT positive samples and antibody identification panel will provide the answer to both the causative antibody and the antigen expression of the neonate’s RBC.

Role of gel test and flow cytometry in diagnosis of Coombs’ negative autoimmune haemolytic anaemia

This study evaluated the use of both gel test (GT) and flow cytometry (FC) techniques in the detection of red blood cell-bound immunoglobulin G (IgG) and, hence, the diagnosis of autoimmune haemolytic anaemia (AIHA) in cases that were negative by the conventional tube technique (CTT) direct antiglobulin test (DAT). The study enrolled 50 clinically diagnosed AIHA patients with CTT Coombs' negative test and 55 control subjects. GT and FC were performed on both groups. Ninety-two percent (46/50) of patients were positive by GT. As for FC assay, the Receiver Operating Characteristic curve determined that a cut-off of 17.5% fluorescence was the best value for interpreting FC-DAT positively in the group of patients with haemolytic anaemia (100% sensitivity and specificity), while the best cut-off for mean fluorescence intensity (MFI) was 1.74, with 76% specificity and 96% sensitivity. Both mean percent fluorescence and MFI were significantly higher among patients when compared to controls (P < 0.001). FC assay results showed no statistically significant correlations with patients' laboratory data or GT grades (P > 0.05). Flow cytometry is more sensitive than GT for assessing CTT-DAT-negative AIHA. We propose that FC percent fluorescence cut-off values should be employed to determine the Coombs' negative AIHA cases.

Automation in Immunohematology

There have been rapid technological advances in blood banking in South Asian region over the past decade with an increasing emphasis on quality and safety of blood products. The conventional test tube technique has given way to newer techniques such as column agglutination technique, solid phase red cell adherence assay, and erythrocyte-magnetized technique. These new technologies are adaptable to automation and major manufacturers in this field have come up with semi and fully automated equipments for immunohematology tests in the blood bank. Automation improves the objectivity and reproducibility of tests. It reduces human errors in patient identification and transcription errors. Documentation and traceability of tests, reagents and processes and archiving of results is another major advantage of automation. Shifting from manual methods to automation is a major undertaking for any transfusion service to provide quality patient care with lesser turnaround time for their ever increasing workload. This article discusses the various issues involved in the process.

Titers of ABO antibodies in group O blood donors

BackgroundPlasma components of group O blood donations are rarely submitted to ABO antibody titrations even though it is well known that passively acquired antibodies may destroy the recipient's own red cells and tissue grafts.ObjectiveThus, group O donations stratified by gender and age were randomly titrated to identify the best source of products for apheresis and exsanguinous transfusion.MethodsSamples from 603 blood donors were tested by ABO antibody titration using the conventional tube technique at room temperature. ABO antibody levels higher than 64 were considered high. After correction for gender, statistical analyses were performed using the Fisher exact and Kruskal-Wallis tests.ResultsMost donors in the blood bank were male (65.7%). ABO antibody titers ranged from 1 to 2048. The estimations of prevalence for the titers were: anti-A,B < 128 = 86.9% and = 128 = 2.16%; Anti-A = 128 = 9.29% and anti-B = 128 = 4.81%. Low mean titers for both anti-A and anti-B antibodies were found in over 50-year-old men (p-value = 0.040). High anti-B antibody levels were found in young women (p-value = 0.002).ConclusionThis study confirms that over 50-year-old O group men should be selected as blood donors in non-identical ABO transfusion situations. Also, titration of ABO antibodies in blood banks will increase safety in non-identical ABO transfusions.

Multi‐centre evaluation of pre‐transfusional routine tests using 8‐column format gel cards (DG Gel®)

Advances in immunohaematology laboratory practice to improve performance, cost-effectiveness and patient safety are desirable. To perform a multi-centre evaluation of the 8-column Grifols DG Gel(®) cards and reagent system to assess its performance, suitability and adaptability to the daily blood transfusion laboratory routine in the United Kingdom. A total of 4281 immunohematological analyses {1825 ABO/D grouping, 1921 antibody screening, 75 Rh phenotyping and K antigen determination, 361 antibody identification and 99 neonates [ABO/D and DAT (direct anti-globulin test)]} were performed on 2255 specimens. All cases were run in parallel with the reference method of each laboratory (DiaMed-ID(®) cards or conventional tube technique in some cases). Concordant results between Grifols DG Gel(®) system and the reference method were obtained in 97·7% of tests. For ABO grouping by the Grifols DG Gel(®) system, sensitivity was 99·95%, specificity was 99·96%, predictive positive value (PPV) was 99·89% and predictive negative value (PNV) was 99·98%. For D grouping, sensitivity was 99·78%, specificity was 100%, PPV was 100% and PNV was 99·78%. For antibody screening, sensitivity was 90·63%, specificity was 99·94%, PPV was 99·32% and PNV was 99·15%. Of the Rh subgroups and K types, results were 100% concordant. For antibody specificity detection, accuracy was 96·95% for Grifols DG Gel(®) system and 95·29% for DiaMed-ID(®) system. For the newborn tests, concordant results were obtained in 100% of ABO/D grouping and in 89·9% of DAT. The Grifols DG Gel(®) 8-column system is reliable and safe for routine tests performed in the immunohaematology laboratory.

Functional consortia for cresol-degrading activated sludges: Toxicity-to-extinction approach

The conventional roll tube and plating techniques are typically time consuming and can culture in vitro only a small fraction of microbes in natural microflora. This study utilizes a novel, simple, and rapid method, the toxicity-to-extinction approach, to obtain the minimal functional consortium that can effectively degrade meta- (m-), para- (p-), and ortho- (o-) cresols. The original sludge had 16 major bands by denaturing gradient gel electrophoresis (DGGE). Microbial diversity decreased as the cresol concentration increased. The functional strains acquired under toxic stress by dosed cresols that individually degraded m-, p-, and o-cresols were identified. Catechol 1,2-dioxygenase (C12D) and catechol 2,3-dioxygenase (C23D) activities in cell-free extracts were determined spectrophotometrically and were correlated with noted changes in microbial communities under cresol stress. The proposed toxicity-to-extinction approach is feasible for isolating a functional consortium from sludge for cresol degradation.

Comparison of gel test and conventional tube test for antibody detection and titration in D-negative pregnant women: study from a tertiary-care hospital in North India

Abstract 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 &lt; 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.

Cut‐off value of red‐blood‐cell‐bound IgG for the diagnosis of Coombs‐negative autoimmune hemolytic anemia

Direct antiglobulin test (DAT)-negative autoimmune hemolytic anemia (Coombs-negative AIHA) is characterized by laboratory evidence of in vivo hemolysis, together with a negative DAT performed by conventional tube technique (CTT) in clinically suspected AIHA patients. The immunoradiometric assay (IRMA) for red-blood-cell-bound immunoglobulin G (RBC-IgG) can be used to diagnose patients in whom CTT does not detect low levels of red cell autoantibodies. We investigated the diagnostic cutoff value of the IRMA for RBC-IgG in Coombs-negative AIHA and calculated its sensitivity and specificity. Of the 140 patients with negative DAT by CTT referred to our laboratory with undiagnosed hemolytic anemia, AIHA was clinically diagnosed in 64 patients (Coombs-negative AIHA). The numbers of Coombs-negative AIHA and non-AIHA patients changed with age and gender. The cutoff values were determined from receiver operating characteristic (ROC) curve according to age and gender. The IRMA for RBC-IgG proved to be sensitive (71.4%) and specific (87.8%) when using these cutoffs. Using these cutoffs for 41 patients with negative DAT referred to our laboratory in 2006, all the pseudonegative cases were treated with steroids before the test. The 31 untreated cases could be grouped using one cutoff value of 78.5 and showed 100% sensitivity and 94% specificity, independent of gender and age. Results indicate that RBC-IgG could become a standard approach for the diagnosis of Coombs-negative AIHA, when measured before treatment.

Comparative study of blood cross matching using conventional tube and gel method

Background: Conventionally tube method is used for compatibility and cross matching in transfusion medicine. Methods: A comparative study was carried out to evaluate the efficacy of conventional tube and gel technique. Result: Compatibility testing was performed on 1000 blood samples by conventional test tube method and DiaMed gel method. The results were analysed. Conclusion: The gel method was found to be a rapid and reliable procedure without controls. There was no requirement of wash phase in indirect antiglobulin test and sensitivity and specificity was comparable to spin tube method. We conclude that this technique is a better substitute for spin tube method.