Comparison of a modified flow cytometry osmotic fragility test with the classical method for the diagnosis of hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia. The flow cytometric test using eosin-5'maleimide (EMA) is a well-established diagnostic method. However, in order to improve HS detection, it is recommended that EMA and an osmotic fragility test (OFT) both be performed. OFT is time consuming and labor intensive. We used a flow cytometric (FOFT) adaptation of the classical OFT reported by Yamamoto. We compare the FOFT to the classical OFT including practical data and propose options for simplifying this method. METHODS: Suspected and known HS patients and controls were tested by the following methods: EMA, OFT, and FOFT including some modifications. RESULTS: The FOFT method is robust and correlates to loss of red blood cells. OFT and FOFT gave similar results in healthy controls and four HS patients. Normal range for FOFT in 70 adults is shown and can be used as a reference value. Neonates should have their own normal range defined. Overnight sample incubation at 37°C did not add information to the FOFT results. CONCLUSION: Our modified Yamomoto FOFT can replace the classic OFT as the addition to EMA for the diagnosis of HS. The use of flow cytometry in both these methods requires small sample volume, is reproducible, simpler, and produces results more rapidly.

Cooperation between BRCA1 and vitamin D is critical for histone acetylation of the p21waf1 promoter and growth inhibition of breast cancer cells and cancer stem-like cells.

Carriers of germline mutations in the BRCA1 gene have a significant increased lifetime risk for being diagnosed with breast cancer. The incomplete penetrance of BRCA1 suggests that environmental and/or genetic factors modify the risk and incidence among mutation carriers. Nutrition and particular micronutrients play a central role in modifying the phenotypic expression of a given genotype by regulating chromatin structure and gene expression. The active form of vitamin D, 1α,25-dihydroxyvitamin D3, is a potent inhibitor of breast cancer growth. Here we report that two non-calcemic analogues of 1α,25-dihydroxyvitamin D3, seocalcitol (EB1089) and QW-1624F2-2, collaborate with BRCA1 in mediating growth inhibition of breast cancer cells and breast cancer stem-like cells. EB1089 induces a G1/S phase growth arrest that coincides with induction of p21waf1 expression only in BRCA1-expressing cells. A complete knockdown of BRCA1 or p21waf1 renders the cells unresponsive to EB1089. Furthermore, we show that in the presence of ligand, BRCA1 associates with vitamin D receptor (VDR) and the complex co-occupies vitamin D responsive elements (VDRE) at the CDKN1A (p21waf1) promoter and enhances acetylation of histone H3 and H4 at these sites. Thus, BRCA1 expression is critical for mediating the biological impact of vitamin D3 in breast tumor cells.

Historical data decrease complete blood count reflex blood smear review rates without missing patients with acute leukaemia.

INTRODUCTION: The availability of historical data decreases the rate of blood smear review rates in outpatients, but we are unaware of studies done at referral centres. In the following study, we determined the effect of historical data on the rates of peripheral blood smears over a 3-month period and then the detection rate of patients with acute leukaemia. METHODS: All results of complete blood counts (CBCs) tested on three ADVIA 120 analyzers at the regional Rabin Medical Centre, Beilinson Campus over a 3-month period were accessed on a computerised laboratory information system. Over a 3-month period, we determined the proportion of total CBC and patients with criteria for a manual differential count and the actual number of peripheral blood smears done. Finally, we determined the proportion of 100 consecutive patients with acute leukaemia detected using our criteria that included limiting reflex testing according to historical data. RESULTS: Over the 3-month period, there were 34,827 tests done in 12,785 patients. Without historical data, our smear rate would have been 24.5%, but with the availability of historical data, the blood smear review rate was 5.6%. The detection rate for cases of acute leukaemia was 100%. CONCLUSIONS: We conclude that the availability of previous test results significantly reduces the need for blood smear review without missing any patients with acute leukaemia.

BRCA1-dependent Chk1 phosphorylation triggers partial chromatin disassociation of phosphorylated Chk1 and facilitates S-phase cell cycle arrest.

Chk1 phosphorylation by the PI3-like kinases ATR and ATM is critical for its activation and its role in prevention of premature mitotic entry in response to DNA damage or stalled replication. The breast and ovarian tumor suppressor, BRCA1, is among several checkpoint mediators that are required for Chk1 activation by ATM and ATR. Previously we showed that BRCA1 is necessary for Chk1 phosphorylation and activation following ionizing radiation. BRCA1 has been implicated in S-phase checkpoint control yet its mechanism of action is not well characterized. Here we report that BRCA1 is critical for Chk1 phosphorylation in response to inhibition of replication by either cisplatin or hydroxyurea. While Chk1 phosphorylation of S317 is fully dependent on BRCA1, additional proteins may mediate S345 phosphorylation at later time points. In addition, we show that a subset of phosphorylated Chk1 is released from the chromatin in a BRCA1-dependent manner which may lead to the phosphorylation of Chk1 substrate, Cdc25C, on S216 and to S-phase checkpoint activation. Inhibition of Chk1 kinase by UCN-01 or expression of Chk1 phosphorylation mutants in which the serine residues were substituted with alanine residues abrogates BRCA1-dependent cell cycle arrest in response replication inhibition. These data reveal that BRCA1 facilitates Chk1 phosphorylation and its partial chromatin dissociation following replication inhibition that is likely to be required for S-phase checkpoint signaling.