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B subgroup detection in a small hospital transfusion service [Case Report]

Elardo, E; Elbadri, N; Sanchez, C; Powell, V; Smaris, M; Li, Y; Jacobson, J; Hilbert, T; Hamilton, T; Wu, D W
The ABO blood group system includes phenotypes, or subgroups, that differ in the amount of A and B antigens present on the red blood cells (RBCs). These subgroups also differ in the A, B, or H substances present in secretions (for individuals who have the secretor phenotype). B subgroups are very rare and are less frequently reported than A subgroups. Usually, B subgroups are discovered during serologic testing when there is a discrepancy between RBC and serum grouping results. Subgroups of B are usually identified by a reference laboratory using molecular and adsorption-elution methods. This report details a case of a young, healthy, pregnant woman with a B subgroup detected by a small transfusion service using adsorption-elution methods. Serology and genotyping of the ABO gene was performed at a reference laboratory where the serology was consistent with a B subgroup, but no changes were identified in ABO gene sequencing. It is important to correctly identify B subgroups in donors and recipients to help resolve ABO discrepancies and potentially prevent ABO incompatibility in blood transfusion, thus minimizing transfusion reactions.
PMID: 34170644
ISSN: 0894-203x
CID: 4964882

Challenging the 3 day rule: A case with newly identified anti-e and anti-jka within 3 days from collection, prior to pre-transfusion specimen expiration [Meeting Abstract]

Li, Y; James, I; Simmons-Massey, K; Luo, X; Qu, S; Powell, V; Hamilton, T; Lifshitz, M; Wu, D; Hilbert, T; Nance, S
Background: According to the AABB, a pre-transfusion sample must be obtained within 3 days of transfusion if a patient has been transfused or pregnant in the preceding 3 months. Despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this 3 day window. To avoid issuing incompatible red blood cells (RBCs) to these patients, a new antibody screen (ABS) sample should be drawn and tested shortly before anticipated transfusion.
Aim(s): We report a case of a 60 y/o man who presented to the ED (hospital day 0, HD0) with a post-fall intracranial hemorrhage and multiple fractures. Anti-E and anti-Jka were identified after admission on a new specimen prior to current specimen expiration (<3 days).
Method(s): Specimen #1 (S1) was sent on HD0 for Type & ABS (T&S) and crossmatch (XM) of 2 RBCs. ABS and immediate spin XM were negative; there was no patient history. By HD9, he had 4 negative T&S specimens (HD0: S1; HD2: S2&3; HD6: S4) and had been transfused 4 RBCs (HD2: 2; HD5: 2) via electronic XM (EXM). At 1730 hr on HD9, 2 RBCs were requested and could have been issued via EXM since S4 was not expiring until midnight. However, given recent transfusions, BB staff first called the patient's nurse to review history. Patient was uncommunicative, but had scars suggesting past trauma or surgery. S5 was requested and received at 1801 hr.
Result(s): S5 showed anti-E and anti-Jka in plasma and eluate. His hemoglobin/hema-tocrit (H/H) decreased from 10.2 (14.0-17.5 g/dL)/30.1 (41.5-50.4%) on HD6 to 6.9/20.6 on HD9. During this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. Two E-Jk(a-) RBCs were transfused on HD9, which he tolerated well with an increase of hemoglobin from 6.9 g/dL to 8.6 g/dL. He did well post transfusion with stable H/H between 8.1/24.2.0 to 8.5/25.3. He was discharged on HD19. Repeat ABS on S4 was negative. Of the 4 RBCs transfused before S5, one was E+ and four Jk(a+). The family reported that he was injured 20 years prior and had been admitted to 3 hospitals, but was unaware of transfusion. Hospital #1 (H1) reported admissions 20 years ago (2 RBCs transfused) and 4 years ago; all ABS were negative. H2 admission was 5 years ago with positive ABS and inconclusive workup. H3 admission 4 years ago showed negative ABS. Summary/Conclusions: The patient developed a significant antibody response in less than 3 days from the specimen collection, likely a secondary immune response to sensitization from a transfusion 20 years earlier. A new specimen was requested prior to transfusion even though the existing sample (which was ABS Negative) had not expired. This approach identified new antibodies, preventing transfusion of incompatible RBCs, and a potentially serious hemolytic transfusion reaction. This case suggests that for high-risk patients, ABS more frequently than every 3 days may be beneficial. It is important to increase clinicians' and laboratorians' awareness of this issue
ISSN: 1423-0410
CID: 4022762

Subgroup B detection in a small hospital transfusion service [Meeting Abstract]

Elardo, E; Sanchez, C; Powell, V; Smaris, M; Li, Y; Jacobson, J; Hilbert, T; Hamilton, T; Wu, D
Background: An ABO blood group subtype is called a subgroup and/or variant. Subgroups of ABO are often distinguished by decreased amounts of A, B or O (H) antigens on red blood cells. Blood type A appears to have the most variation in subgroups. In general, serologic distinction between A1 and other subgroups of A is based on the ability of anti- A1 lectin (an extract of Dolichos biflorus seeds) to agglutinate A1 red blood cells (RBC) but not cells of other A subgroups. As A2 is most common A subtype and is frequently detected at regular hospital transfusion services. Definitive identification of ABO subgroups is usually performed at a reference laboratory using molecular techniques and special immunohematology methods. Here we report an interesting case where a subgroup B was detected by an astute technologist using manual immunohematology tests and conventional reagents at our hospital transfusion service. Our hospital transfusion service received a blood specimen requesting a type and screen from a pregnant woman with no known medical problems. Testing the specimen showed a front type of O and a back type of B, with a negative antibody screening.
Aim(s): To resolve the typing discrepancy and to correctly Identify the ABO blood type of the pregnant patient.
Method(s): In an attempt to enhance the typing reactions with the patient's RBCs and plasma, mixtures of the front typing and back typing were incubated separately for 15 minutes at room temperature. This however did not result in any change in reactivity. The technologist became suspicious of a possible B subgroup as this was a healthy young woman and it would be rare for such a young patient to exhibit missing antibodies in the reverse type. He then performed a DAT on the specimen to assure that nothing was coating the patient's RBCs which might interfere with further testing. The DAT was negative for IgG and C3d. He subsequently performed an absorption on vigorously washed patient RBC) with anti-B reagent from Ortho. The reaction mixture was incubated at room temperature (RT) for 15 minutes, followed by an elution. The eluate was added to washed A cells, B cells, and screening cells (Ortho), and then incubated at room temperature for 15 minutes. The mixtures were washed with eluate washing solution. Anti-IgG Coombs reagent was added, the tubes were centrifuged, and results were recorded.
Result(s): A cells: Negative B cells: 2 + Screen cell #1: negative Screen cell #2: negative Screen cell #3: negative Summary/Conclusions: Based on the manual tests the technologist designed and performed, he was able to demonstrate the 2 + reactivity with type B reagent RBCs and concluded that the patient's blood type is a B subgroup. The patient's specimen was then sent to New York Blood Center Immunohematology Reference Laboratory for further confirmation and identification. The B group of the patient's RBCs was not detectable again by the conventional immunohematology tests. Molecular tests (PCR-RFLP) and full gene sequencing were performed. A B allele (*B1.01) and an O1 allele (*O1.09) were identified, which predicts a B weak subtype. The molecular testing confirms the conclusion made by our technologist using conventional manual immunohematology tests
ISSN: 1423-0410
CID: 4784112

Effects of Serial Phlebotomy on Vascular Endothelial Function: Results of a Prospective Double-Blind Randomized Study

Jelani, Qurat-Ul-Ain; Harchandani, Bhisham; Cable, Ritchard G; Guo, Yu; Zhong, Hua; Hilbert, Timothy; Newman, Jonathan D; Katz, Stuart D
INTRODUCTION/BACKGROUND:Blood donation has been proposed as a potential therapy to reduce risk of cardiovascular disease, but the effects of phlebotomy on vascular function in human subjects have not been well characterized. AIMS/OBJECTIVE:We conducted a prospective randomized double-blind study to determine the effects of serial phlebotomy on vascular endothelial function in the brachial artery. 84 iron-replete, non-anemic subjects were randomly assigned to one of three study treatment groups: 1) four serial phlebotomy procedures each followed by intravenous infusion of placebo normal saline; 2) four serial phlebotomy procedures each followed by intravenous infusion to replete lost iron; and 3) four serial sham phlebotomy procedures each followed by intravenous infusion of placebo normal saline. Assigned phlebotomy procedures were conducted at 56-day intervals. We measured brachial artery reactivity (BAR, %) in response to transient oxidative stress induced by oral methionine with high-resolution duplex ultrasound imaging before and one week after the fourth study phlebotomy. RESULTS:Before phlebotomy, oral methionine decreased BAR by -2.04% (95% CI -2.58, -1.50%), p<0.001) with no significant difference between groups (p=0.42). After phlebotomy, the BAR response to oral methionine did not significantly change between groups (p=0.53). Brachial artery nitroglycerin-mediated dilation did not change in response to phlebotomy. CONCLUSIONS:Four serial phlebotomy procedures over six months with or without intravenous iron supplementation did not alter vascular endothelial function in the brachial artery when compared with sham phlebotomy.
PMID: 30341986
ISSN: 1755-5922
CID: 3370152

A Successful Selective Concurrent Audit of Platelet Utilization in a Large Academic Hospital [Meeting Abstract]

Simmons-Massey, Kellie; James, Ian; Mamone, Linda; Liu, Shiguang; Luo, Xunda; Grant, Michelle; Pudhota, Abhiraj; Afronz, Tanjila; Shah, Saumil; Shah, Alay; Miller, Maureen; Wu, Ding Wen; Hilbert, Timothy; Li, Yanhua
ISSN: 0003-2999
CID: 3727542

Lipids and dyslipoproteinemia

Chapter by: Hilbert T; Lifshitz MS
in: Henry's clinical diagnosis and management by laboratory methods by McPherson RA; Pincus MR [Eds]
[S.l.] Saunders-Elsevier, 2007
pp. 200-218
ISBN: 1416002871
CID: 4479

Cloning and expression of the cDNA encoding the human homologue of the DNA repair enzyme, Escherichia coli endonuclease III

Hilbert TP; Chaung W; Boorstein RJ; Cunningham RP; Teebor GW
We previously purified a bovine pyrimidine hydrate-thymine glycol DNA glycosylase/AP lyase. The amino acid sequence of tryptic bovine peptides was homologous to Escherichia coli endonuclease III, theoretical proteins of Saccharomyces cerevisiae and Caenorhabditis elegans, and the translated sequences of rat and human 3'-expressed sequence tags (3'-ESTs) (Hilbert, T. P., Boorstein, R. J., Kung, H. C., Bolton, P. H., Xing, D., Cunningham, R. P., Teebor, G. W. (1996) Biochemistry 35, 2505-2511). Now the human 3'-EST was used to isolate the cDNA clone encoding the human enzyme, which, when expressed as a GST-fusion protein, demonstrated thymine glycol-DNA glycosylase activity and, after incubation with NaCNBH3, became irreversibly cross-linked to a thymine glycol-containing oligodeoxynucleotide, a reaction characteristic of DNA glycosylase/AP lyases. Amino acids within the active site, DNA binding domains, and [4Fe-4S] cluster of endonuclease III are conserved in the human enzyme. The gene for the human enzyme was localized to chromosome 16p13.2-.3. Genomic sequences encoding putative endonuclease III homologues are present in bacteria, archeons, and eukaryotes. The ubiquitous distribution of endonuclease III-like proteins suggests that the 5,6-double bond of pyrimidines is subject to oxidation, reduction, and/or hydration in the DNA of organisms of all biologic domains and that the resulting modified pyrimidines are deleterious to the organism
PMID: 9045706
ISSN: 0021-9258
CID: 8383

Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide

Hilbert TP; Boorstein RJ; Kung HC; Bolton PH; Xing D; Cunningham RP; Teebor GW
We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny
PMID: 8611553
ISSN: 0006-2960
CID: 57363


Radiation and oxidation agents damage the pyrimidine bases of DNA. The resulting modified bases can be recognized by enzymes which effect the removal of base lesions, leaving an apurinic site in the DNA backbone. We have been studying three distinct classes of pyrimidine base damage: a) saturation of the 5,6-ethylenic bond to produce the corresponding glycol, b) hydration of the 5,6 double bond to produce the corresponding 6-hydroxy-5,6-dihydroderivative, and c) oxidation of the methyl group of thymine and 5-methylcytosine to produce the corresponding hydroxymethylated compound
ISSN: 0021-7689
CID: 54134


Boorstein, RJ; Hilbert, TP; Cunningham, RP; Cadet, J; Teebor, GW
UV irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates which are repaired by bacterial and mammalian DNA glycosylases (Biochemistry, 28: 6164, 1989). E. coli endonuclease III was used to quantitate the formation and stability of these hydrates in poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254 nm light at pH 8.0, 2.2% of Cyt residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these photoproducts, poly(dG-dC) was incubated in solution for up to 24 hours after UV irradiation. Cytosine hydrate was stable at 4-degrees-C and decayed at 25- degrees-C, 37-degrees-C and 55-degrees-C with half lives of 75, 25 and 6 hours, respectively. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4-degrees-C and at 25- degrees-C, and decayed with a half life of 6 hours at 37- degrees-C and less than one half-hour at 55-degrees-C. Uracil hydrate and uracil were also shown to be formed in irradiated poly(dG-dC). These experiments demonstrate that far-UV induced cytosine hydrate may persist in DNA for prolonged time periods and also undergoes deamination to uracil hydrate which, in turn, undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase
ISSN: 0021-7689
CID: 32153