Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS.

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

Assessment of normal erythropoiesis by flow cytometry: important considerations for specimen preparation.

INTRODUCTION: The extension of quantitative flow cytometric studies to the erythroid lineage in patients with suspected myelodysplastic syndrome has prompted a reassessment of cell surface antigen expression during normal erythropoiesis. Erythropoiesis in normal and pathologic bone marrows was studied to determine the expected antigenic relationships of maturing erythroid cells. METHODS: A total of 200 bone marrow specimens were evaluated by multidimensional flow cytometry (MDF). Samples were prepared using either NH4 Cl lysis or Ficoll density gradient separation. RESULTS: Normal erythroid development is described as a two-step process observable with the intensity relationships between CD235a, CD71, CD45, CD105, CD34, CD117, and CD36. The variability of these intensities (CV) was determined. A comparison of processing techniques determined lysis is the optimal analytic technique for the analysis of early-stage erythroid cells. Nucleic acid staining with DRAQ5 revealed that Ficoll allows for the analysis of reticulocytes and mature erythrocytes otherwise eliminated by lysis. CONCLUSION: These data demonstrate while lysis alters the light scatter characteristics of erythroid precursors, it did not alter quantitative antigen expression or nucleic acid content. The expected variability in antigen intensities is defined. These studies provide a basis for a comparison of erythroid development between normal individuals and those with erythroid dysplasia associated with myelodysplastic syndromes.

Beyond dorsal penile nerve block: a more humane circumcision.

OBJECTIVE: To explore techniques that can be utilized in addition to the dorsal penile nerve block (DPNB) to further reduce the neonate's stress and pain from routine circumcision, and thus make the procedure more humane. SETTING: Level 1 nursery at a community hospital. SUBJECTS: Eighty healthy, term, newborn male infants scheduled for routine neonatal circumcision. STUDY DESIGN: Prospective and randomized; double blind and placebo controlled for the study solutions. METHODS: Four statistically similar groups of 20 were studied. The control group included infants circumcised using: a) a rigid plastic restraint board; b) standard DPNB; and c) a pacifier dipped in water to comfort the infant. Each study group differed from the controls in one variable including: 1) using a specially designed, physiologic circumcision restraint chair; 2) pH buffering of lidocaine hydrochloride used for DPNB; and 3) offering a pacifier dipped in a 24% sucrose solution during the DPNB and circumcision. Behavioral observations were recorded and compared for each group starting before the injection of lidocaine hydrochloride and continuing through the completion of the circumcision. Plasma for cortisol levels were collected 30 minutes after the circumcision. RESULTS: Neonates circumcised on the new restraint chair showed a significant decrease in distress scores (>50%) compared with the control group on the rigid molded-plastic restraint. The pacifier dipped in sucrose had a distress-reducing effect during both the post-DPNB injection and circumcision periods. The infants who were injected with the buffered lidocaine showed no differences in distress from the controls. The plasma cortisol levels were not significantly affected by any additional technique and were comparable to the levels previously reported. CONCLUSIONS: When neonatal circumcisions are performed routinely, they should be done as humanely as possible. This study demonstrates that, when used in conjunction with DPNB, a pacifier dipped in 24% sucrose and a more comfortable, padded, and physiologic restraint can be useful in decreasing distress and pain.

Dampening of adrenocortical responses during infancy: normative changes and individual differences.

Changes in cortisol and behavioral responses were examined longitudinally in 83 infants (39 girls, 44 boys) tested at their well-baby exams with inoculations at 2, 4, 6, and 15 months (72 infants completed all testing). Another sample of 2-, 4-, and 6-month-olds (n = 18 per age) received mock exams without inoculations to determine early developmental changes to the exam procedures. Behavioral distress was coded every 30 sec during the exam, a 5-min inoculation period, and a 20-min recovery period. Salivary cortisol was obtained upon arrival at the clinic and 25 min after the beginning of the inoculation period. To the exam-inoculation procedures, cortisol responses were high at 2 months, decreased significantly between 2 and 4 months, remained comparable between 4 and 6 months, and then declined again between 6 and 15 months. Between 6 and 15 months there was also a decrease in pretest cortisol. By 15 months, significant increases in cortisol from pre- to posttest were no longer observed for most infants. To the exam-only procedures, cortisol responses decreased between 2 and 4 months, and by 4 months most infants failed to show pre- to posttest increases in cortisol. Behavioral distress decreased between 2 and 6 months, but increased again at 15 months. While crying and cortisol were modestly correlated during the 2- to 6-month exam-inoculation procedures, at 15 months no significant correlations were obtained. Behavioral and hormonal reactions thus followed different ontogenetic paths and may provide different information about infant functioning. There was some evidence that the emergence of the circadian rhythm in cortisol might be related to the early decrease in cortisol response.

Stress reactivity and attachment security.

Seventy-three 18-month-olds were tested in the Ainsworth Strange Situation. These children were a subset of 83 infants tested at 2, 4, 6, and 15 months during their well-baby examinations with inoculations. Salivary cortisol, behavioral distress, and maternal responsiveness measures obtained during these clinic visits were examined in relation to attachment classifications. In addition, parental report measures of the children's social fearfulness in the 2nd year of life were used to classify the children into high-fearful versus average- to low-fearful groups. In the 2nd year, the combination of high fearfulness and insecure versus secure attachment was associated with higher cortisol responses to both the clinic exam-inoculation situation and the Strange Situation. Thus, attachment security moderates the physiological consequences of fearful, inhibited temperament. Regarding the 2-, 4-, and 6-month data, later attachment security was related to greater maternal responsiveness and lower cortisol baselines. Neither cortisol nor behavioral reactivity to the inoculations predicted later attachment classifications. There was some suggestion, however, that at their 2-month checkup, infants who would later be classified as insecurely attached exhibited larger dissociations between the magnitude of their behavioral and hormonal response to the inoculations. Greater differences between internal (hormonal) and external (crying) responses were also negatively correlated with maternal responsiveness and positively correlated with pretest cortisol levels during these early months of life.

First time experiences in infancy: when they appear to be pleasant, do they activate the adrenocortical stress response?

The effect of a positive first-time experience on adrenocortical activity was examined in 48 6- to 13-month-old infants who took part in two sessions of a YMCA-like mother-infant swim class. Experience was manipulated by comparing Novice swimmers with infants who previously had taken a swim class, and by examining responses to first versus second class session. In addition, the effects of temperamental fear of novelty were examined by maternal report prior to the first session. Preswim and Postswim cortisol levels were compared to measures obtained at the same time of day in two comparison groups, one tested at home and one tested in a playroom at the University. The results yielded no evidence of an adrenocortical stress response (elevated cortisol) to the swim sessions for either the Novice or Experienced swimmers. Infant temperamental fearfulness predicted behavioral but not adrenocortical responses during swimming. Postswim cortisol levels were significantly lower than posttest levels in the two baseline comparison groups, and lower postswim cortisol levels were associated with less negative and more positive emotional behavior and engagement of the swimming experience. These results are consistent with the hypothesis that emotions serve as a major pathway influencing adrenocortical activity, and provide no support for the uncertainty hypothesis of adrenocortical activation.

The stressfulness of separation among nine-month-old infants: effects of social context variables and infant temperament.

This manuscript reports on the results of 2 experiments dealing with behavioral and adrenocortical responses to separation among 9-month-old human infants. In both experiments the social context of separation was manipulated. The results of Experiment 1 yielded evidence of a statistically significant adrenocortical response to 30 min of separation under conditions in which the substitute caregiver responded sensitivity to infant distress, but was busy and relatively noninteractive when babies were not distressed during the separation period. Altering the behavior of the substitute caregiver such that she was warm, responsive, and interactive throughout the separation produced a significant reduction in adrenocortical activity and in negative affect. In fact, these measures were not significantly different than those obtained when the mother and infant remained together in the playroom (No Separation). In Experiment 2, the effects of group versus singleton care were examined using the less stressful mode of substitute caregiving as described above. No significant condition differences in behavioral distress or cortisol were found. Furthermore, neither condition elicited a significant increase in cortisol over basal levels. Finally, these data provide evidence that maternal reports of infant Distress to Limits temperament, using Rothbart's Infant Behavior Questionnaire, predict adrenocortical responses to separation, while reports of Fear of Novelty do not.