Saroj Singh

Evaluation of Cd64 Expression on Neutrophils as an Early Indicator of Neonatal Sepsis

Background: Enhanced neutrophil CD64 (nCD64) expression is likely to be useful in diagnosis of neonatal sepsis. This study evaluated the diagnostic efficacy of nCD64 expression as an early indicator of neonatal sepsis. Methods: Sixty neonates (culture positive, 24; negative, 36) with suspected sepsis and 30 controls were studied prospectively. CD64 expression was evaluated flow cytometrically on neutrophils and monocytes. Mean and median nCD64 expression, mean and median monocyte CD64/nCD64 (M/N CD64) ratios were computed. Results were correlated with blood culture and other conventional indices of sepsis. Results: The sick neonates had significantly higher mean and median nCD64 expression compared with controls. Monocyte CD64 values did not differ significantly among the groups. Both mean and median M/N CD64 ratios were significantly lower in the former group. Culture-positive neonates had significantly higher mean and median nCD64 values and significantly lower mean and median M/N CD64 ratios than clinically indistinguishable but culture-negative neonates. Both groups were significantly different with respect to these indices from normal controls. Median M/N CD64 ratio was the best discriminant by virtue of highest area under the receiver operator characteristic curve (0.903), with sensitivity and specificity of 91.7% and 88.9%, respectively. Conventional indices were inferior, both singly and in combination. Conclusions: Enhanced nCD64 reported as median M/N CD64 ratio is a highly sensitive marker of culture-positive neonatal sepsis. It additionally identifies a separate group among culture-negative sick neonates and may be useful to guide antibiotic administration especially in these neonates.

The clinical risk index of babies (CRIB) score in India.

Objective : To assess the usefulness of clinical risk index of babies (CRIB score) in predicting neonatal mortality in extremely preterm neonates, compared to birth weight and gestation.Methods : 97 preterm neonates with gestational age less than 31 weeks or birth weight less than or equal to 1500 g were enrolled for the prospective longitudinal study. Relevant neonatal data was recorded. Blood gas analysis results and the maximum and the minimum FiO2 required by babies in first 12 hours of life were noted. Mortality was taken as death while the baby was in nursery. The prediction of mortality by birth weight, gestational age and CRIB score was done using the Logistic model, and expressed as area under the ROC curve.Results : The area under the ROC curve for birth weight, gestational age and CRIB score was almost the same, the areas being 0.829, 0.819 and 0.823 respectively. Hence CRIB score did not fare better than birth weight and gestational age in predicting neonatal mortality.Conclusion : The CRIB score did not improve on the ability of birth weight and gestational age to predict neonatal mortality in the study.

Lactate: creatinine ratio in babies with thin meconium staining of amniotic fluid

BackgroundACOG states meconium stained amniotic fluid (MSAF) as one of the historical indicators of perinatal asphyxia. Thick meconium along with other indicators is used to identify babies with severe intrapartum asphyxia. Lactate creatinine ratio (L: C ratio) of 0.64 or higher in first passed urine of babies suffering severe intrapartum asphyxia has been shown to predict Hypoxic Ischaemic Encephalopathy (HIE). Literature review shows that meconium is passed in distress and thin meconium results from mixing and dilution over time, which may be hours to days. Thin meconium may thus be used as an indicator of antepartum asphyxia. We tested L: C ratios in a group of babies born through thin and thick meconium, and for comparison, in a group of babies without meconium at birth.Methods86 consecutive newborns, 36 to 42 weeks of gestation, with meconium staining of liquor, were recruited for the study. 52 voided urine within 6 hours of birth; of these 27 had thick meconium and 25 had thin meconium at birth. 42 others, who did not have meconium or any other signs of asphyxia at birth provided controls. Lactate and creatinine levels in urine were tested by standard enzymatic methods in the three groups.ResultsLactate values are highest in the thin MSAF group followed by the thick MSAF and controls. Creatinine was lowest in the thin MSAF, followed by thick MSAF and controls. Normal babies had an average L: C ratio of 0.13 (± 0.09). L: C ratio was more among thin MSAF babies (4.3 ± 11.94) than thick MSAF babies (0.35 ± 0.35). Median L: C ratio was also higher in the thin MSAF group. Variation in the values of these parameters is observed to be high in the thin MSAF group as compared to other groups. L: C ratio was above the cutoff of 0.64 of Huang et al in 40% of those with thin meconium. 2 of these developed signs of HIE with convulsions (HIE Sarnat and Sarnat Stage II) during hospital stay. One had L: C Ratio of 93 and the other of 58.6. A smaller proportion (20%) of those with thick meconium had levels above the cutoff and 2 developed HIE and convulsions with L: C ratio of 1.25 and 1.1 respectively.ConclusionIn evolving a cutoff of L: C ratios that would be highly sensitive and specific (0.64), Huang et al studied it in a series of babies with severe intrapartum asphyxia. Our study shows that the specificity may not be as good if babies born through thin meconium are also included. L: C ratios are much higher in babies with thin meconium. It may be that meconium alone is not a good indicator of asphyxia and the risk of HIE. However, if the presence of meconium implies asphyxia then perhaps a higher cut-off than 0.64 is needed. L: C ratios should be tested in a larger sample that includes babies with thin meconium, before L: C ratios can be applied universally.

Immunophenotypic analysis of T‐acute lymphoblastic leukemia. A CD5‐based ETP‐ALL perspective of non‐ETP T‐ALL

T‐cell antigens [CD5,CD1a,CD8] define early T‐cell precursor acute lymphoblastic leukemia (ETP‐ALL). To understand immature T‐ALL of which ETP‐ALL is part, we used these antigens to subcategorize non‐ETP T‐ALL for examining expression of myeloid/stem cell antigens (M/S) and clinical features. Using CD5 (+/−) to start categorization, we studied 69 routinely immunophenotyped patients with T‐ALL. CD5− was a homogenous (CD8,CD1a)− M/S+ ETP‐ALL group (n = 9). CD5+ cases were (CD8,CD1a)− pre‐T‐ALL (n = 22) or (CD8,CD1a)+ (n = 38) thymic/cortical T‐ALL; M/S+ 20/22 (90.91%) in former and 22/38 (57.89%) in latter (P = 0.007). ETP‐ and pre‐T‐ALL together (CD1a−,CD5−/+ immature T‐ALL group) were nearly always M/S+ (29/31; 93.55%). In multivariate analysis, only ETP‐ALL predicted poor overall survival (P = 0.02). We conclude (i) CD5 negativity in T‐ALL almost always means ETP‐ALL. CD1a and CD8 negativity, as much as CD5, marks immaturity in T‐ALL, and the CD5+/−/CD1a−/CD8− immature T‐ALL group needs further study to understand the biology of the T‐ALL–myeloid interface. (ii) ETP‐ALL patients may be pre‐T‐ALL if CD2+; CD2+, conversely, CD5−/CD1a−/CD8− pre‐T ALL patients are ETP‐ALL. (iii) Immunophenotypic workup of T‐ALL must not omit CD1a, CD5, CD8 and CD2, and positivity of antigens should preferably be defined as recommended for ETP‐ALL, so that this entity can be better evaluated in future studies of immature T‐ALL, a group to which ETP‐ALL belongs. (iv) ETP‐ALL has poor prognosis.

Using gross national product to calculate acceptable immunisation costs deploying cost-effectiveness calculations in reverse

1. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497 2. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498 3. Discussion and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

Evaluation of the Protection Provided by Hepatitis B Vaccination in India

ObjectiveIn India, Hepatitis B vaccination is recommended at 6 wk except for hospital-deliveries. The authors examined protection afforded by the birth dose.MethodsA case-control study was done. HBsAg and HBcAb were tested in 2671 children, 1 to 5 y and HBsAb was evaluated in a subset of 1413 children. Vaccination history was recorded. Cases were HBsAg carriers. In another analysis, children who got infected (HBsAg and/or HBcAb positive) were considered as cases. Exposed were the unvaccinated. In another analysis, exposed were those vaccinated without the birth dose.ResultsThe odds ratio (OR) for HBsAg positivity with birth vaccination was 0.35 (95% CI 0.19–0.66); while with vaccination at 6 wk was 0.29 (95%CI 0.14–0.61), both compared to unvaccinated. Birth vaccination has no added protection when compared to the unvaccinated. Unvaccinated children in index study had HBsAg positivity of 4.38%. The number needed to treat (NNT) to prevent one case of HBsAg positivity was 32.6 (95% CI, 20.9 to 73.6). The odds of getting HBV infection was 0.42 (CI 0.25–0.68) with birth dose and 0.49 (CI 0.30–0.82) without the birth dose compared to the unvaccinated. Protective antibody (HBsAb) was present in about 70% of the vaccinated. In the unimmunised, in the first 2 y HBsAb protection was present in 40%. The odds ratio (OR) for HBsAb in the fully vaccinated between 4 and 5 y was 1.4 (95%CI 0.9–2.18) compared to the unvaccinated.ConclusionsThe present study lends support to the pragmatic approach of the Government to vaccinate babies born at home starting at 6 wk.

Superior Mediastinal Syndrome : A Rare Presenting Feature of Acute Myeloid Leukemia

Superior mediastinal syndrome (SMS) is an uncommon manifestation of malignant neoplastic disease in children. The commonest neoplastic cause of SMS is Non Hodgkin lymphoma. Acute myeloid leukemia (AML) as a cause of SMS is extremely uncommon in childhood. The authors hereby report a case of a child with AML who presented with SMS at their hospital. This report also highlights the importance of flowcytometry as a diagnostic modality in hematological malignancies.

Comparison between photostability of Alexa Fluor 448 and Alexa Fluor 647 with conventional dyes FITC and APC by flow cytometry

Sir, Flow cytometry involves conjugation of an antibody to a fluorochrome and analysis of its expression. The intensity and quality of fluorescence depend on a large number of factors namely antigen density, fluorolabeling techniques, instrument properties, and also on the type of fluorochrome. With the new age of synthetic dyes, better and brighter fluorochromes are constantly added to the existing palette so making the photostaining much simpler. Recent studies have shown that synthetic dyes are brighter and more photostable than the conventional dyes.1-6 Mahmoudian J et al7 used mouse antihuman nestin monoclonal antibody conjugated to Alexa Fluor 568 and fluorescein isothiocyanate (FITC) dyes and evaluated their photobleaching and photostability profiles by immunohistochemistry and demonstrated that Alexa Fluor dye had brighter fluorescence and more photostability than its counterpart FITC. We intended to explore the photostability as well as the intensity and resolution of expression of antigens using the conventional dyes and compare these parameters with those of newer synthetic dyes for their prospective use in clinical flow cytometric service. Peripheral blood was collected in EDTA from healthy subjects (n = 10), and 5 sets of each of the 10 samples were prepared. All the sets were immediately processed by the standard stainlysewash method, and 0.5 × 106 cells were labeled with CD45conjugated Alexa Fluor 488 (AF488), Alexa Fluor 647 (AF647), FITC, and allophycocyanin (APC) (Beckman Coulter, USA) as per manufacturer’s recommendations. Briefly, the samples were incubated for 20 minutes at room temperature in dark followed by lysis with ammonium chloridebased solution and washed twice with phosphatebuffered saline. The cells were finally suspended in 0.5% paraformaldehyde solution. The acquisition and analysis of data were performed on Gallios flow cytometer (10 colors, 4 lasers, Beckman Coulter (BC), Hialeah, FL, USA). For instrument setup, Flow Check fluorospheres (BC, Hialeah, FL, USA) were used for alignment and Flow Set beads (BC, Hialeah, FL, USA) for the voltage setting. One of 5 sets of all samples was run immediately after staining, and analysis was performed using Kaluza software (BC, Hialeah, FL, USA). This reading was taken as reference reading for comparison of loss in mean fluorescent intensity (MFI) and signaltonoise ratio (SNR) in subsequent runs. The remaining 4 sets of samples were exposed to photo source helium lamp with 36 watts fluorescent light, and 20 000 events were acquired on flow cytometer at 1 hour, 4 hours, 15 hours, and at 24 hours, respectively. The MFI and SNR for CD45 were checked on the gated population of normal lymphocytes. The changes, that is, the % fall in the MFI and % fall in the SNR with respect to different fluorochromes and time, were compared using Friedman’s multiple comparison test using CD45+ normal lymphocytes at 0hour time point as reference. The photostability of FITC and AF488, APC, and AF647 was analyzed at different time points and is shown in Table 1. The % loss in MFI with respect to 0hour time point ranged from 8.19% to 63.64% and 1.19% to 33.05% in FITC and AF488, and this difference was statistically significant (P = .0006). Similarly, the % loss in MFI ranged from 37.14% to 71.97% for APC and 17.29% to 54.85% for AF647, respectively, over a period of 24 hours and was statistically significant (P ≤ .0001; Table 1). The % fall in SNR with respect to 0hour time point ranged from 5.54 to 68.6 for FITC and 4.15 to 38.74 for AF488, and this difference was statistically significant (P ≤ .0001). Similarly, the % fall in SNR ranged from 21.94 to 62.62 for APC and 21.64 to 47.40 for AF647, respectively, over a period of 24 hours and was statistically significant (P ≤ .0001; Table 1). Thus, AF488and AF647conjugated antibodies had better photostability in terms of lower % fall in MFI at all the four time points; wherein, although the MFI gradually decreased with time, the fall was more pronounced in FITC and APC resulting in higher % fall in MFI (Figure 1). In addition, AF488and AF647conjugated antibodies were demonstrated to exhibit better resolution in terms of higher SNR at all the time points evaluated in this study where the background signals were constant but signal intensity decreased with time more in FITC and APC resulting in lower SNR (Figure 1). We have demonstrated that the synthetic dyes namely AF488and AF647conjugated antibodies had better resolution and photostability than APCand FITClabeled antibodies. Thus, the samples labeled with antibodies tagged to synthetic dyes can withstand some time delay and accidental exposure to light prior to acquisition on a flow cytometer. Alternatively, these antibodies tagged to synthetic dyes with better photostability and resolution offer better logistic solutions to transportation, storage, and reporting in high throughput laboratories.

Real-Time Compensation in Flow Cytometry: A Real Need of Time

Dear Sir, Compensation is done to prevent spectral overlap and spill-over during data acquisition in fluorescence activated cell sorting (FACS). Spectral overlap occurs because the fluorophores used in flow cytometry emit photons of multiple energies and wavelengths, where emission spectra overlap, fluorescence from more than one fluorochrome may be detected in a channel [1–3]. With newer advanced machines with more lasers and many more detectors or ‘‘channels’’, the importance of compensation cannot be undermined. The conventional single Antibody (Ab) stained cell tube run before standardization of a protocol experiment, and getting a desired compensation settings is not sufficient in this age of multicolor and multi-tube experiments, real time compensation takes care of other problems apart from compensation like steric hinderance and tube specific ‘‘spill overs’’. In our experience, even if an experimental protocol is standardized beforehand with single tube run experiments, a ‘‘real time’’ compensation setting contributes significantly to the results. Hence we recommend after standardization of protocol by single Ab experiments for compensation, a tube specific compensation should be applied and voltage and gain settings adjusted during acquisition to simulate real time data collection. This procedure of real time compensation has better results as compared to single tube runs in standardized protocols as explained in Fig. 1. To elaborate this point we acquired data from two sets of experiments one which was run on standardized settings (single Ab tube experiment) of protocol and the other when compensation was done in real time while acquisition (Figs. 1 and 3). The difference in voltages in different channels was also compared (Fig. 2). The compensation algorithm needs to be performed both on positive population and a negative population (Fig. 1). For best compensation, selection of controls is important— either internal control with similar fluorescence as that of the test cells or external beads can be used, when added to the same tube which have surface test markers acting as controls. Internal control should have adequate adherence to fluorochromes and the individual samples should have the same carrier particles for the fluorochromes, [3–5] this can be done by using known positive controls and comparing their fluorescence [4]. In our experiment, the voltage and compensation settings of single Ab tube (Fig. 2) were saved in the general

Effect of processing time on analysis of rare event in minimal residual disease study

Editor, Rare event analysis was started with the analysis of the fetal red blood cells in maternal circulation in a rare percentage.[1,2] Flow cytometry (FCM) has been used to analyze numerous rare cell populations in blood or bone marrow from circulating endothelial cells to cancer stem cells and became an important means in analysis of blood cancer.[2‐7] Initially, FCM was used only for lineage deciding in leukemia; but by years, it was developed for morphological assessment of blood and bone marrow to detect minimal residual disease (MRD), with molecular analysis used in treatment decisions of pediatric B‐acute lymphoblastic leukemia and more recently acute myeloid leukemia.[8‐13] The FCM is helpful in the detection of small tumor population to the level of 0.01% in lymph proliferative disorders.[9,10] MRD detection has been associated with time to relapse in the acute leukemia;[8,9‐11,13] there are several technical and interpretative hurdles that need to be addressed before a laboratory undertakes such an analysis. For acute myeloid leukemia (AML), there are few well‐validated protocols, and this is complicated by the fact that this disease is often composed of multiple clones at presentation and can be affected by antigen modulation during treatment.[10,11] In MRD, the time of sample processing in the rare event analysis was important because cells were very less (>0.01%) so it was important to correlate the time to get maximum number of cells and also validate staining procedure and fluorochrome. A total of four AML MRD processed samples were used in the study, previously diagnosed for AML on morphology. Immunophenotyping done at Lab Oncology, Dr. BRAICH, All India Institute of Medical Sciences, New Delhi. For flow cytometric analysis, at least 200,000 were acquired at follow‐up in all cases from each tube, and data were stored in list mode. The procedure was repeated after 15 h, and data were collected. For all the specimens, five‐color FCM was performed on Coulter FC500 instrument (Beckman Coulter [BC], Hialeah, FL, USA). Combinations of 5 antibodies in 7 different tubes,