Poonam Coshic

Nucleic acid testing for blood banks: An experience from a tertiary care centre in New Delhi, India

INTRODUCTIONnBlood safety is a challenging task in India; with a population of around 1.23 billion and a high prevalence rate of HIV (0.29%), HBV (2-8%) and HCV (≈ 2%) in general population. Nucleic acid testing (NAT) in blood donor screening has been implemented in many developed countries to reduce the risk of transfusion-transmitted viral infections (TTIs). NAT shortens this window period, thereby offering blood centers a much higher sensitivity for detecting viral infections.nnnMATERIALS AND METHODSnRoutine ID-NAT for HIV-1, HCV and HBV was started from June 2010 at AIIMS blood bank by the Procleix® Ultrio® Assay (Novartis Diagnostics, USA) a multiplex NAT, which allows the simultaneous detection of HIV-1, HCV, and HBV in a single tube. During the period of 27 months from June 2010 to August 2012, around 73,898 samples were tested for all the three viruses using both ELISA (by Genscreen Ultra HIV Ag-Ab(BIO-RAD), Hepanostika HCV Ultra & HBsAg Ultra(Biomerieux) and Nucleic acid testing. The comparative results of both the assays are being presented here in this study.nnnRESULTSnOut of 73,898 samples, 1104 samples (1.49%) were reactive by NAT. out of these 1104 samples, 73 were reactive for HIV-1 (0.09%), 186 were reactive for HCV only (0.25%), 779 (1.05%) were reactive for HBV only, and around 66 (0.08%) were HBV-HCV co-infections. There was one HIV, 37 HCV, 73 HBV and 10 HBV-HCV co-infection cases that were not detected by serology but reactive on NAT testing, with a combined yield of 1 in 610 donations (total 121 NAT yields).nnnCONCLUSIONnNAT could detect HIV, HBV and HCV cases in blood donor samples that were undetected by serological tests. NAT can interdict a large number of infected unit transfusions and thus help in providing safe blood to the patients.

Retrospective evaluation of adverse transfusion reactions following blood product transfusion from a tertiary care hospital: A preliminary step towards hemovigilance

Background: The goal of hemovigilance is to increase the safety and quality of blood transfusion. Identification of the adverse reactions will help in taking appropriate steps to reduce their incidence and make blood transfusion process as safe as possible. Aims: To determine the frequency and type of transfusion reactions (TRs) occurring in patients, reported to the blood bank at our institute. Materials and Methods: A retrospective review of all TRs reported to the blood bank at the All India Institute of Medical Sciences, between December 2007 and April 2012 was done. All the TRs were evaluated in the blood bank and classified using standard definitions. Results: During the study period a total of 380,658 bloods and blood components were issued by our blood bank. Out of the total 196 adverse reactions reported under the hemovigilance system, the most common type of reaction observed was allergic 55.1% (n = 108), followed by febrile non-hemolytic transfusion reaction (FNHTR) 35.7% (n = 70). Other less frequently observed reactions were Anaphylactoid reactions 5.1% (n = 10), Acute non-immune HTRs 2.6% (n = 5), Circulatory overload 0.5% (n = 1), Transfusion related acute lung injury 0.5% (n = 1), Delayed HTRs 0.5% (n = 1). Not a single case of bacterial contamination was observed. Conclusion: The frequency of TRs in our patients was found to be 0.05% (196 out of 380,658). This can be an underestimation of the true incidence because of under reporting. It should be the responsibility of the blood transfusion consultant to create awareness amongst their clinical counterpart about safe transfusion practices so that proper hemovigilance system can be achieved to provide better patient care.

Blood and blood component therapy in neonates

Blood component therapy is a very common intervention practiced in newborns; nearly 85% of extremely low birth weight (ELBW) babies get transfusions during their hospital stay. However, there are no set guidelines for transfusion of blood component therapy in newborns. This protocol includes available types of blood components, their methods of preparation, indications and side effects of transfusion, in relation to newborns.

Sensitivity of individual and mini-pool nucleic acid testing assessed by dilution of hepatitis B nucleic acid testing yield samples

Introduction: For nucleic acid testing (NAT) of blood donations, either the blood samples can be pooled together in a batch of six or eight prior to testing (mini-pool-NAT [MP-NAT]), or the tests can be run on every individual sample (individual donor-NAT [ID-NAT]). It has been debated in various studies whether pooling of samples results in decreased sensitivity of detection as the volume of individual samples gets lesser in a pool. The objective of this study was to investigate the effect of dilution on the sensitivity of tests. Materials and Methods: The study was performed on nine plasma samples which were hepatitis B reactive exclusively by Procleix Ultrio Plus and not by Procleix Ultrio or serology. These nine exclusive UltrioPlus ID-NAT yield samples were diluted in 1:2, 1:4. 1:6 and 1:8 dilutions using previously tested negative plasma and each dilution of every sample along with archived undiluted sample were retested in three replicates with Procleix Ultrio Plus Assay. Results: Among NAT yield samples, 88.88% of the samples were detected when retested in ID-NAT in undiluted form. Samples with higher viral load (sample 5 and 6) were detected by all dilutions. When samples with viral load below 20 IU/mL were tested in dilutions of 1:6 or 1:8, only 9 out of 27 replicates (33.33%) were detected. This means that more than 67% of low viral load samples were missed by MP-NAT of 1:6 or 1:8 dilution out of total NAT yield samples. Conclusion: Individual Donor NAT is ideal methodology for NAT as dilution due to pooling may miss samples with low viral load as evident in this study.

Modulation of cardiac autonomic tone in non-hypotensive hypovolemia during blood donation.

Non-hypotensive hypovolemia, observed during mild haemorrhage or blood donation leads to reflex readjustment of the cardiac autonomic tone. In the present study, the cardiac autonomic tone was quantified using heart rate and blood pressure variability during and after non-hypotensive hypovolemia of blood donation. 86 voluntary healthy male blood donors were recruited for the study (age 35xa0±xa09xa0years; weight 78xa0±xa012xa0kg; height 174xa0±xa06xa0cms). Continuous lead II ECG and beat-to-beat blood pressure was recorded before, during and after blood donation followed by offline time and frequency domain analysis of HRV and BPV. The overall heart rate variability (SDNN and total power) did not change during or after blood donation. However, there was a decrease in indices that represent the parasympathetic component (pNN50xa0%, SDSD and HF) while an increase was observed in sympathetic component (LF) along with an increase in sympathovagal balance (LF:HF ratio) during blood donation. These changes were sustained for the period immediately following blood donation. No fall of blood pressure was observed during the period of study. The blood pressure variability showed an increase in the SDNN, CoV and RMSSD time domain measures in the post donation period. These results suggest that mild hypovolemia produced by blood donation is non-hypotensive but is associated with significant changes in the autonomic tone. The increased blood pressure variability and heart rate changes that are seen only in the later part of donation period could be because of the progressive hypovolemia associated parasympathetic withdrawal and sympathetic activation that manifest during the course of blood donation.

Cell-intrinsic genetic regulation of peripheral memory-phenotype T cell frequencies

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while recently activated -B and -CD4 T cell frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets, suggesting genetic regulation of memory lymphocyte frequencies. To explore this possibility further, we screened effector memory (EM)-phenotype T cell frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4EM and/or CD8EM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.

P066HLA-A*02 repertoire in three defined population groups from North and Central India: Punjabi Khatries, Kashmiri Brahmins and Sahariya tribe

Aim The allelic family of HLA-A*02 with a repertoire of approximately 951 known alleles represents the predominant and most heterogeneous group at the HLA-A locus. This remarkable diversity signifies the evolutionary relevance of this allelic family. Its population specific diversity is attributed to environmental factors and pathogen pressure and can be harnessed in biology and medicine; particularly in disease association and HLA based vaccine approaches. We therefore investigated the HLA-A*02 repertoire in three defined population groups including a Central Indian tribe Sahariya (ST, Nu202f=u202f100) and two North Indian caste populations viz Punjabi Khatries (PK, Nu202f=u202f250) and Kashmiri Brahmins (KB, Nu202f=u202f160). Methods Luminex based high resolution rSSO method was utilized for HLA genotyping. Results were also confirmed using high resolution PCR-SSP and/or next generation sequencing (NGS) based approach. Results HLA-A*02 was observed with an overall high phenotypic/ allelic frequency in these populations. However, within A*02 repertoire, differences were observed among the three population groups evaluated. A total of 6 alleles were observed (A*02:01, *02:03, *02:05, *02:06, *02:07 and *02:11) in the caste groups (PK and KB) compared to four (except *02:05 and *02:07) in the tribals (ST). A striking observation was the high occurrence of A*02:11 i.e. 33–38% in caste groups and extremely high, more than 80% in tribals. Globally, this allele is rarely observed and is present with low frequencies in limited ethnic groups. The primordial A*02:01 allele, which is the representative A*02 allele in most ethnicities was observed as the second most predominant allele (∼30% in caste groups and ∼10% in tribals). Conclusions Extreme high prevalence of A*02:11 in Sahariya Tribes may be representation of ancient Indian genetic pool and in caste populations the observed A*02 repertoire may be a consequence of genetic drift, natural selection and/or admixture from different races.

Influence of a critical single nucleotide polymorphism on nuclear receptor PXR-promoter function†

The Pregnane and Xenobiotic Receptor (PXR; NR1I2) is a ligand‐modulated transcription factor that belongs to the nuclear receptor superfamily. It is expressed at higher levels primarily in liver and intestine as compared to the levels in several other organs. It is activated by a broad spectrum of xenobiotics and endobiotics. The primary function of PXR is to regulate the expression of drug metabolizing enzymes and transporters and prevent the accumulation of toxic chemicals in the body, thereby maintaining bodys homeostasis. In this study, we identified a C/T single nucleotide polymorphism at position −831 from the transcriptional start site of the PXR gene promoter and examined the functional significance of this variant using both the luciferase reporter gene assays and electrophoretic mobility shift assays (EMSA). Transient transfection experiments showed that the T‐allele was associated with significantly greater transcriptional activity than the C‐allele of SNP rs3814055. These results indicate that the −831C/T polymorphism has a direct effect on transcriptional regulation of PXR gene. This allelic variation may be a potential genetic marker that can help identify individuals at higher risk for Inflammatory Bowel Disease (IBD).

Low-density lipoprotein apheresis in a pediatric patient of familial hypercholesterolemia: Primi experientia from a tertiary care center in North India

Familial hypercholesterolemia (FH) is an autosomal dominant disorder due to mutation of apolipoprotein-B receptor gene causing severe dyslipidemia. Lifestyle modification and medical treatment attenuate the disease progression, but as these fail to control the blood cholesterol levels, low-density lipoprotein (LDL) apheresis comes forth as a treatment option. To the best of our knowledge, the following is the very first case of pediatric FH being treated by LDL-apheresis to be reported from India. A severely malnourished female child presented with yellowish skin lesions over different parts of the body, viz., bilateral Achilles tendon, both knees, elbows, both pinnae, and outer canthus of both eyes. She had a strong family history of borderline hypercholesterolemia and was diagnosed as a case of FH. She was maintained on diet modification. LDL-apheresis was planned as the cholesterol levels were not controlled with the diet modificationt. However, unavailability of an appropriate kit in India for LDL-apheresis led to the use of the modified PL1 kit meant for therapeutic plasma exchange procedures. We conducted two sessions of LDL-apheresis. After the first session, the LDL-cholesterol (LDL-C) level fell by 75.9% and the total cholesterol fell by 73.5%. A second procedure led to a decline in total cholesterol level by 18.6% and LDL-C by 19.46%. Subsequently, she was advised diet modification and statin therapy with regular follow-up after every 6 months. Thus, the cascade filtration technique is a safe and effective treatment option for removing the undesired lipoproteins.

Decreased Spontaneous Baroreflex Sensitivity as an Early Marker for Progression of Haemorrhage

IntroductionBlood donation provides an ideal setup for assessment of cardiovascular responses to mild hypovolemia for understanding the underlying mechanisms.AimTo evaluate cardiovascular responses in time and magnitude by estimating the spontaneous baroreflex sensitivity (BRS) during and after donation of 450xa0ml of blood.MethodsContinuous beat-to-beat blood pressure and lead II ECG was recorded before, during and after blood donation in 54 healthy volunteers (age 34.7 ± 5.08 years; weight 77.9 ± 8.20 kg), followed by offline analyses of baroreflex sensitivity.ResultsThe systolic, diastolic or mean blood pressures did not change during or after the blood donation. Decrease in pulse pressure and increase in heart rate was observed post donation. The spontaneous BRS decreased during [8.68 (6.038–12.69) ms/mmHg] and after blood donation [9.401 (6.396–11.59) ms/mmHg] as compared to the baseline [12.83 (6.884–18.18) ms/mmHg] with a significant decrease in α-HF on spectral analysis.ConclusionMild blood loss (450xa0ml) results in non-hypotensive haemorrhage with a decrease in spontaneous BRS before the rise of heart rate during blood donation.