P-Null Phenotype Due to a Rare Frame-Shift Mutation and with Allo-Anti-PP1Pk Causing a Severe Hemolytic Transfusion Reaction: A Case Report with Clinical Management.

INTRODUCTION: The identification of alloantibodies to high-frequency antigens (HFA) and subsequent transfusion management can be challenging and often poses a problem in finding the compatible blood for transfusion. The aim of this study was to investigate the specificity of the antibody to the HFA causing a hemolytic transfusion reaction (HTR) and procure the compatible blood unit for future transfusion. CASE PRESENTATION: A 4-year-old female met with a head injury that led to intracranial bleeding and surgical intervention was required to remove blood clots. In the face of anemia, blood transfusion was planned. The pretransfusion tests on her blood sample revealed the presence of a pan-reactive alloantibody with hemolytic properties. She was transfused with 10 mL of the least incompatible red blood cells (RBCs) to which she reacted with signs of clinical hemolysis, i.e., chill, rigor, fever, and hemoglobinuria, on 3 different occasions. Despite her anemia, she was managed by medical intervention only. Her antibody reacted with all RBCs tested, except autologous and P-null (p phenotype) cells. Her RBCs did not react with anti-PP1Pk, which corroborated her phenotype as P-null. The genomic study revealed she was hemi- or homozygous or for a deletion of 26-bp in A4GALTexon 3, previously reported as causing the P-null phenotype and designated A4GALT*01N.019. CONCLUSION: This report documents a rare case of the P-null phenotype with an alloanti-PP1Pk causing a severe HTR to transfusion of the trial dose of the least incompatible blood. The case is the first example of this specific A4GALTmutation found in India.

PIGG defines the Emm blood group system.

Emm is a high incidence red cell antigen with eight previously reported Emm- probands. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work suggests that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive. We investigated samples from a South Asian Indian family with two Emm- brothers by whole genome sequencing (WGS). Additionally, samples from four unrelated Emm- individuals were investigated for variants in the candidate gene. Filtering for homozygous variants found in the Emm- brothers and by gnomAD frequency of < 0.001 resulted in 1818 variants with one of high impact; a 2-bp deletion causing a frameshift and premature stop codon in PIGG [NM_001127178.3:c.2624_2625delTA, p.(Leu875*), rs771819481]. PIGG encodes for a transferase, GPI-ethanolaminephosphate transferase II, which adds ethanolamine phosphate (EtNP) to the second mannose in a GPI-anchor. The four additional unrelated Emm- individuals had various PIGG mutations; deletion of Exons 2-3, deletion of Exons 7-9, insertion/deletion (indel) in Exon 3, and new stop codon in Exon 5. The Emm- phenotype is associated with a rare deficiency of PIGG, potentially defining a new Emm blood group system composed of EtNP bound to mannose, part of the GPI-anchor. The results are consistent with the known PI-linked association of the Emm antigen, and may explain the production of the antibody in the absence of RBC transfusion. Any association with neurologic phenotypes requires further research.

A novel observation on grouping anomaly: The phenomena mimicking the Bel genetic variant of the ABO blood groups.

BACKGROUND: Discrepancy in "forward/reverse" grouping leads to confusion in assigning ABO group to a person. It could be genetic in nature and classified according to the presence/absent of antigen on red blood cell (RBC) vis-a-vis corresponding alloantibody in plasma. AIM: The aim of the study was to investigate the grouping anomaly found in a recently delivered woman who required transfusion. MATERIALS AND METHODS: A standard protocol for investigation was followed. RESULTS: A 27-year-old female, gravida 4, para 3, was grouped O on forward grouping, but her serum did not agglutinate Group B RBCs tested. Absorption-elution study gave an active eluate from her sensitized RBCs with anti-B or anti-A+B. Saliva showed H, but no B antigens indicating to her Bel phenotype. However, 2-week latter in the follow-up study, her serum revealed a presence of complement binding high titer anti-B. The problem of missing anti-B on the previous occasion was attributed to hemagglutination inhibition caused by accumulated complement macromolecules on RBCs that gave rise to physical hindrance in the formation hemagglutination clumps. CONCLUSION: The unusual case of erroneous reversed grouping was attributed to complement-mediated hemagglutination inhibition. The positive eluate obtained from sensitized RBCs of the mother was considered to be due to a contamination of fetal RBCs in maternal circulation entered during her postpartum phase of pregnancy. It could also be due to a conversion of H to B antigen no matter in trace amount by the fetal B group-specific transferase percolated into maternal circulation.

A rare case of naturally occurring allo anti-Jk(a) missed in manual screening test on tube method.

BACKGROUND: Antibodies to the Kidd blood group are mainly red blood cell (RBC) immune, but a few reports on naturally occurring antibodies have been documented. AIM: The aim of this study is to study the anti-Jk(a) for its unusual reactivity with different serological methods. MATERIALS AND METHODS: Donor's plasma was tested with RBCs from in house donors and commercial panels by manual and automated devices. RESULTS: A 36-year-old male blood donor with naturally occurring anti-Jk(a) is detected by solid-phase assays and the gel card technique but not by the tube method. The IgG antibody with the titer of >32 was not a complement-fixing hemolysin, showed a reduced reactivity with enzyme-treated RBCs, and was detectable through 8 months' follow-up period. The donor was typed as (Jk(a-). CONCLUSION: An unusual naturally occurring anti-Jk(a) detected by solid-phase red-cell adherence but not reacting by tube technique reflected on the sensitivity of the methods used.

A resource-conserving serologic and highthroughput molecular approach to screen for blood donors with an IN:-5 phenotype.

CONCLUSIONS: The Indian blood group system (ISBT 023) comprises one lowprevalence antigen, Ina (IN:1), and five high-prevalence antigens: Inb (IN:2), INFI (IN:3), INJA (IN:4), INRA (IN:5), and INSL (IN:6). The antigens are located on the single-pass trans-membrane glycoprotein encoded by the CD44 gene. The present study was designed to identify the prevalence of the INRA- (IN:-5) phenotype and the frequency of its associated allele (IN*02.- 05) to inform us of the probability of finding antigen-negative donors and to assess the risk of antibody formation in transfusion recipients. Buffy coats were extracted from EDTA-anticoagulated whole blood samples, collected with consent from 5261 random blood donors in Surat, Gujarat, India. Standard serologic methods were performed with a modification allowing the use of antiserum generated by recycling the antibody augmented from the test already performed. A real-time polymerase chain reaction- based assay was devised to genotype c.449G>A (p.Arg150His) single nucleotide variation in exon 5 of the CD44 gene. None of the 411 donors tested by serology or the 5261 donors tested molecularly were positive for the IN:-5 phenotype or the allele (IN*02.-05), respectively. The allele frequency estimate ranged from less than 1 in 10,522 (0.01%) to 1 in 3203 alleles (0.03%) in the study cohort (95% confidence interval, Poisson distribution). The absence of this rare allele in the present survey could be due to an ethnic difference, since the donors mostly came from the Hindu community, and the only case of the IN:-5 phenotype was found in the Muslim community. The p.150His variant may be either restricted to the index case family or only found in the Muslim community. Further studies in local subpopulations may provide more information on the frequency of p.150His and its immunogenicity in transfusion recipients if occurring among blood donors.

High thermal amplitude auto-anti-"N" complicating pretransfusion compatibility tests on a patient with malaria and bladder cancer.

BACKGROUND: Antigen "N" is a high-frequency antigen of the MNS blood groups and carried on glycophorin B that is resistant to enzymatic cleavage by trypsin, and provides differential diagnosis of its antibody specificity to N being present of glycophorin A. Naturally occurring IgM antibodies to N are known to be clinically insignificant, as against the IgG counterpart with clinical relevance. AIM: Auto-anti-"N" association with the bladder cancer was explored for its clinical significance as well as its interference in grouping anomaly. MATERIALS AND METHODS: A warm environment was created while blood sampling for the laboratory work up as the patient had a high-titer auto-cold agglutinin causing spontaneous hemagglutination. The antibody was tested by standard serological methods with the red cell, antisera, and enzymes prepared in house or obtained commercially. RESULTS: The case was admitted to hospital with high fever and hematuria; he was diagnosed with malaria and bladder cancer. He required transfusions in the face of severe anemia. His blood sample posed problems in compatibility tests due to autoantibody present. Serological workup revealed its specificity as anti-"N." CONCLUSION: Auto-anti-"N" as a cause of severe anemia could not be attributed to, for concurrent malarial infection. However, its presence may have some association with the underlying malignant condition.

INRA, a new high-frequency antigen in the INDIAN (IN023) blood group system.

BACKGROUND: The INDIAN blood group system comprises 4 antigens sensitive to enzymes and 2-aminoethyl isothiouronium bromide (AET). AIM: The patient's antibody was investigated for its specificity to the high-frequency antigens (HFA) of this system. MATERIAL AND METHODS: Low ionic strength solution (LISS)-tube/LISS-indirect antiglobulin test (IAT) methods were used. The patient's red blood cells (RBCs) were tested with antisera to HFA. Her antibody was tested with RBCs lacking the HFA. Furthermore, it was tested with RBCs as untreated or treated with enzyme or AET. The genetic sequence was studied for mutation in CD44 gene that encodes INDIAN antigens. RESULTS: The patient was grouped A1B, RhD+, antibody screening test positive, direct antiglobulin test negative. A negative autocontrol test had suggested to the alloantibody being present. Antibody had agglutinated RBCs in LISS-tube at RT and by LISS-IAT at 37°C. The RBCs of the 11-cell panel, those lacking HFA and from 50 random donors, were agglutinated by her antibody indicating its specificity to the HFA, though the RBCs of Lu (a-b-)/In (Lu) type showed a weaker reaction. The patient's RBCs were agglutinated by antisera to a number of the enzyme-sensitive HFA, including those of INDIAN blood groups. The antibody showed reduced reactivity with the RBCs treated with papain, chymotrypsin, and AET but resistant to trypsin and dithiothreitol. The patient's genetic sequence revealed a novel homozygous mutation 449G>A in exon 5 of CD44. CONCLUSION: The antibody to enzyme sensitive HFA was tested for serological and molecular genetics studies and found to be directed to the novel HFA, named as INRA of the INDIAN blood group system and was assigned a numerical symbol IN: 005 by the International Society of Blood Transfusion (ISBT).

The prozone effect exerted by the complement-binding anti-Lea on anti-D.

BACKGROUND: Prozone phenomenon is seen with very high-titer antibodies in an immune serum. AIM: The prozone effect on anti-D by a low-titer anti-Lea was investigated associated with neonatal jaundice. MATERIALS AND METHODS: Standard methods were used in investigations. RESULTS: The child was born at full-term developed mild jaundice. With weak direct antiglobulin test+, her indirect serum bilirubin was progressed to 27.5 mg/dL in 48 h. Anti-D and anti-Lea were detected in the mother. Both these antibodies were detected in the child's serum though the eluate from red blood cells (RBCs) contained only anti-D. Mother's anti-D was masked by anti-Lea if the RBCs possessed both the antigens together. Anti-D was revealed only with D-positive RBCs lacking Lea or if the serum was modified by mixing with Lea+ saliva or was heated at 56°C or fortified with citrate solution. CONCLUSION: An anti-D showed prozone effect exerted by the complement-fixing anti-Lea in the test.

Assessment of quality of platelets preserved in plasma and platelet additive solution: A Malaysian experience.

BACKGROUND: A use of platelet additives solution (PAS) improves storage conditions so as to give increased shelf life to platelets and to maintain hemostatic function. OBJECTIVE: The present study was aimed to compare in vitro quality of platelet rich plasma (PRP)-derived platelet concentrate (PC) during extended period of storage in plasma and in additive solution (Composol PS and Fresenius). STUDY DESIGN: Randomized 19 PCs each were used in the study for plasma and PAS as the storage medium. The measurement parameters, including pH, total white blood cell (WBC) count, total platelet count, and platelet activation rate, were studied on day 1, day 5, and day 8 of the storage period. The sterility test was carried out on the eighth day of storage. RESULTS: pH of PC suspended in PAS was significantly lower as compared to that in plasma (P < 0.001) for all the three days of sampling. The WBC count, both in plasma and in PAS, showed an acceptable values of being <0.2 Χ 10(9) /unit during the storage period. Platelet count in PAS was higher as compared to that in plasma, though it was not statistically significant. While both the groups showed increased platelet activation rate during the storage, the PCs suspended in PAS showed significantly higher platelet activation rate (p0.001). Results from sterility test showed no bacterial growth in the PCs in both the groups. CONCLUSION: Most parameters studied on platelet storage in suspending medium of native plasma and PAS remained well within the acceptable limits. However, the pH values and platelet activation rate significantly differed in PAS as compared with plasma.

Unusual spontaneous cold auto-hemagglutination phenomenon in blood units stored under blood bank condition: A retrospective analysis.

BACKGROUND: Cold agglutinins (CA) are benign naturally occurring low titer autoantibodies present in most individuals. Those with moderate strength are found in infections, malignancies or autoimmune conditions with diagnostic importance. AIM: Present report deals with CA that brought spontaneous hemagglutination in blood units stored at 2-6°C. STUDY DESIGN: Over 32 months period between July 1993 and December 1995, blood units were inspected for spontaneous cold auto-hemagglutination (SpCA) phenomenon. The plasma from these units was separated and investigated for serological specificity using in house red cell panel and standard serological methods. RESULTS: Among 51,671 blood units, 112 units showed SpCA phenomenon. A rising trend seen in first half of study period significantly fell in remaining half. Specificities of the antibodies detected include anti-I (27), anti-i (53), anti-Pr (21) with remaining few being undetermined specificity. Absorption of serum using enzyme-treated red cells revealed a presence of anti-Pr among the cases, the two of which with new specificities that reacted preferentially with red cells from either new-born or adults and were tentatively named as anti-Pr(Fetal) and anti-Pr(adult), respectively. While 9 cases showed optimum reaction at neutral pH of 7, 68 (62%) cases reacted at pH 5.8 through 8.0, 28 (26%) cases preferred an acidic pH 5.8 and 4 cases opted an alkaline pH 8. Of 28 cases with antibodies preferentially reacting in acidic medium, 17 (60%) cases were anti-i and 7 (25%) cases were anti-Pr. CONCLUSION: Unique SpCA phenomenon observed in blood units stored under blood bank conditions seems to be due to CA developed in response to vector-borne infectious agents. Majority of the cases displayed their specificities, otherwise are rare to be encountered.

Prevalence of Diego blood group antigen and the antibody in three ethnic population groups in Klang valley of Malaysia.

BACKGROUND: Diego blood group antigen, Di(a), is very rare among Caucasians and Blacks, but relatively common among the South American Indians and Asians of Mongolian origin. The antibody to Di(a) is clinically significant to cause hemolytic disease in a new-born or hemolytic transfusion reaction. OBJECTIVES: This study was designed to determine the prevalence of Di(a) antigen among the blood donors from the three major ethnic groups in Klang Valley of Malaysia as well as to find an incidence of an antibody of the Diego antigen, anti-Di(a), in a tertiary care hospital to ascertain the need to include Di(a+) red cells for an antibody screen cell panel. MATERIALS AND METHODS: Serological tests were performed by column agglutination technique using commercial reagents and following instruction as per kit insert. RESULTS: Di(a) antigen was found with a frequency of 2.1% among the Malaysians donors in three ethnic groups viz, Malay, Chinese and Indian. It was present among 1.25% of 401 Malay, 4.01% of Chinese and 0.88% of 114 Indian origin donors. None of the 1442 patients, including 703 antenatal outpatients, had anti-Di(a) in serum. CONCLUSION: The prevalence of Di(a) antigen was found among the donors of all the three ethnic background with varying frequency. Inclusion of Di(a+) red cells in routine antibody screening program would certainly help in detection of this clinically significant antibody and to provide safe blood transfusion in the Klang Valley, though the incidence of antibody appears to be very low in the region.

A profile of rare bloods in India and its impact in blood transfusion service.

From transfusion point of view, a rare blood is the one which lacks a high-frequency antigen as well as the one who lacks multiple common antigens and such blood donations help in transfusion to those recipients having alloantibodies to corresponding antigens. In India, we have about four such kinds of phenotypes potential enough to pose problems in providing blood to the recipients having these phenotypes. Besides, there are other four kinds of rare bloods that pose seldom problems in blood supply, though some of these may cause problems in interpretation of results on assigning proper blood groups for a person.

Development of blood transfusion service in Sultanate of Oman.

BACKGROUND: Sultanate of Oman is geographically situated in south-west of Asia, having common borders on western side by the land with United Arab Emirates, Saudi Arabia and Yemen and with the Arabian Sea and the Gulf of Oman in the east and the north respectively. The country enjoys one of the best health care facilities including blood transfusion services in the region. STUDY DESIGN: Information was collected through informal personal interviews, digging out the past records, and the report presentations at various forums. RESULTS: A modest start by providing blood units through import, the country is now self-reliant on procuring blood units from voluntary non-remunerate blood donors within the sultanate. A steady growth of blood banks is witnessed in every aspect of blood banking including blood collection, blood processing and supply. Various modalities are adapted in promoting voluntary blood donation programme. CONCLUSION: Sultanate of Oman has created one of the best blood transfusion services in the region in providing safe blood for transfusion through voluntary donation, a use of blood components and irradiating blood products.