Faculty Bibliography

BACKGROUND:Patients with sickle cell disease (SCD) frequently undergo prophylactic red blood cell (RBC) exchange transfusion and simple transfusion (RCE/T) to prevent complications of disease, such as stroke. These treatment procedures are performed with a target hemoglobin S (HbS) of ≤30%, or a goal of maintaining an HbS level of <30% immediately prior to the next transfusion. However, there is a lack of evidence-based instructions for how to perform RCE/T in a way that will result in an HbS value <30% between treatments. PRINCIPAL OBJECTIVE/UNASSIGNED:To determine whether targets for post-treatment HbS (post-HbS) or post-treatment HCT (post-HCT) can help to maintain an HbS <30% or <40% between treatments. MATERIALS AND METHODS/METHODS:We performed a retrospective study of patients with SCD treated with RCE/T at Montefiore Medical Center from June 2014 to June 2016. The analysis included patients of all ages, and data including 3 documented parameters for each RCE/T event: post-HbS, post-HCT, and follow-up HbS (F/u-HbS), which is the pre-treatment HbS prior to the next RCE/T. Generalized linear mixed model was used for estimating the association between post-HbS or post-HCT levels and F/u-HbS <30%. RESULTS:Based on our results, targeting post-HbS ≤10% was associated with higher odds of having events of F/u-HbS <30% between monthly treatments. Targeting post-HbS ≤15% was associated with higher odds of events of F/u-HbS < 40%. As compared to post-HCT ≤30%, a post-HCT >30%-36% did not contribute to more F/u-HbS <30% or HbS <40% events. CONCLUSIONS:For patients with SCD undergoing regular RCE/T for stroke prevention, a post-HbS ≤10% can be used as a goal to help maintain an HbS <30% for 1 month, and a post-HbS ≤15% allowed patients to maintain HbS <40%.

Importance/UNASSIGNED:There is clinical equipoise for COVID-19 convalescent plasma (CCP) use in patients hospitalized with COVID-19. Objective/UNASSIGNED:To determine the safety and efficacy of CCP compared with placebo in hospitalized patients with COVID-19 receiving noninvasive supplemental oxygen. Design, Setting, and Participants/UNASSIGNED:CONTAIN COVID-19, a randomized, double-blind, placebo-controlled trial of CCP in hospitalized adults with COVID-19, was conducted at 21 US hospitals from April 17, 2020, to March 15, 2021. The trial enrolled 941 participants who were hospitalized for 3 or less days or presented 7 or less days after symptom onset and required noninvasive oxygen supplementation. Interventions/UNASSIGNED:A unit of approximately 250 mL of CCP or equivalent volume of placebo (normal saline). Main Outcomes and Measures/UNASSIGNED:The primary outcome was participant scores on the 11-point World Health Organization (WHO) Ordinal Scale for Clinical Improvement on day 14 after randomization; the secondary outcome was WHO scores determined on day 28. Subgroups were analyzed with respect to age, baseline WHO score, concomitant medications, symptom duration, CCP SARS-CoV-2 titer, baseline SARS-CoV-2 serostatus, and enrollment quarter. Outcomes were analyzed using a bayesian proportional cumulative odds model. Efficacy of CCP was defined as a cumulative adjusted odds ratio (cOR) less than 1 and a clinically meaningful effect as cOR less than 0.8. Results/UNASSIGNED:Of 941 participants randomized (473 to placebo and 468 to CCP), 556 were men (59.1%); median age was 63 years (IQR, 52-73); 373 (39.6%) were Hispanic and 132 (14.0%) were non-Hispanic Black. The cOR for the primary outcome adjusted for site, baseline risk, WHO score, age, sex, and symptom duration was 0.94 (95% credible interval [CrI], 0.75-1.18) with posterior probability (P[cOR<1] = 72%); the cOR for the secondary adjusted outcome was 0.92 (95% CrI, 0.74-1.16; P[cOR<1] = 76%). Exploratory subgroup analyses suggested heterogeneity of treatment effect: at day 28, cORs were 0.72 (95% CrI, 0.46-1.13; P[cOR<1] = 93%) for participants enrolled in April-June 2020 and 0.65 (95% CrI, 0.41 to 1.02; P[cOR<1] = 97%) for those not receiving remdesivir and not receiving corticosteroids at randomization. Median CCP SARS-CoV-2 neutralizing titer used in April to June 2020 was 1:175 (IQR, 76-379). Any adverse events (excluding transfusion reactions) were reported for 39 (8.2%) placebo recipients and 44 (9.4%) CCP recipients (P = .57). Transfusion reactions occurred in 2 (0.4) placebo recipients and 8 (1.7) CCP recipients (P = .06). Conclusions and Relevance/UNASSIGNED:In this trial, CCP did not meet the prespecified primary and secondary outcomes for CCP efficacy. However, high-titer CCP may have benefited participants early in the pandemic when remdesivir and corticosteroids were not in use. Trial Registration/UNASSIGNED:ClinicalTrials.gov Identifier: NCT04364737.

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.

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-Jk^a 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

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

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.

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

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