Diagnostic Accuracy of Flow Cytometric DNA Index in Saudi Children with B Cell Acute Lymphoblastic Leukemia.

B cell Acute Lymphoblastic Leukemia (B-ALL) is one of the most common types of cancer diagnosed in children in Saudi Arabia. Cytogenetic investigations, including karyotyping and FISH, are used to determine the incidence and prognostic significance of chromosomal abnormalities in B-ALL. However, in ALL, accurate identification of the morphology of chromosomes is sometimes not achieved. Flow cytometric DI may have the advantage of being technically fast, using either fresh or frozen samples to correctly stratify the patient into the appropriate risk group for treatment. In this study, we evaluated the reliability and validity of using fixed samples instead of fresh samples to determine aneuploidy in cancer cells using a DNA index. The results of the DNA index obtained by flow cytometry were compared with those of conventional cytogenetic analysis to validate the accuracy. Fixed samples (n = 72) from children diagnosed with B-ALL at King Fahad Medical City in Riyadh between 2017 and 2019 were investigated. The results showed strong and statistically significant positive correlation between DNAI-FCM and conventional cytogenetic analysis (p = 0.000 < 0.01). The DNA index value by flow cytometry was proportional to the cytogenetic study in 94.36% (67) of the cases, while discrepancy was observed in 5.6% (four) cases. Our findings highlight the ability of the DNA index method to provide complementary information for the accurate diagnosis of aneuploidy in patients with B-ALL.

Clinical laboratory services for primary healthcare centers in urban cities: a pilot ACO model of ten primary healthcare centers.

BACKGROUND: Primary healthcare centers (PHC) ensure that patients receive comprehensive care from promotion and prevention to treatment, rehabilitation, and palliative care in a familiar environment. It is designed to provide first-contact, continuous, comprehensive, and coordinated patient care that will help achieve equity in the specialty healthcare system. The healthcare in Saudi Arabia is undergoing transformation to Accountable Care Organizations (ACO) model. In order for the Kingdom of Saudi Arabia (KSA) to achieve its transformational goals in healthcare, the improvement of PHCs' quality and utilization is crucial. An integral part of this service is the laboratory services. METHODS: This paper presents a pilot model for the laboratory services of PHC's in urban cities. The method was based on the FOCUS-PDCA quality improvement method focusing on the pre-analytical phase of the laboratory testing as well as the Saudi Central Board for Accreditation of Healthcare Institutes (CBAHI) gap analysis and readiness within the ten piloted primary healthcare centers. RESULTS: The Gap analysis, revealed in-consistency in the practice, lead to lower the quality of the service, which was seen in the low performance of the chosen key performance indicators (KPI's) (high rejection rates, lower turn-around times (TAT) for test results) and also in the competency of the staff. Following executing the interventions, and by using some of the ACO Laboratory strategies; the KPI rates were improved, and our results exceeded the targets that we have set to reach during the first year. Also introducing the electronic connectivity improved the TAT KPI and made many of the processes leaner. CONCLUSIONS: Our results revealed that the centralization of PHC's laboratory service to an accredited reference laboratory and implementing the national accreditation standards improved the testing process and lowered the cost, for the mass majority of the routine laboratory testing. Moreover, the model shed the light on how crucial the pre-analytical phase for laboratory quality improvement process, its effect on cost reduction, and the importance of staff competency and utilization.

Progressive Ataxia and Neurologic Regression in RFXANK-Associated Bare Lymphocyte Syndrome.

OBJECTIVE: To identify the genetic cause of a late-onset immunodeficiency and subacute progressive neurodegenerative disease affecting cognition, motor, visual, and cerebellar systems in a patient with a family history of 2 younger siblings with an early-onset immunodeficiency disease. METHODS: Physical examinations, immunologic, brain MRI, whole-exome sequencing, and segregation studies were used to identify the genetic and neuroimmunologic etiology of disease in this family. RESULTS: We identified a homozygous loss-of-function (LOF) mutation (c.271+1G>C) in the RFXANK gene in the index patient and one of his younger affected siblings. Biallelic mutations in the RFXANK gene are known to cause bare lymphocyte syndrome (BLS) type II, complementation group B. The clinical and immunologic investigations were consistent with a clinical diagnosis of BLS type II. MRI demonstrated global cerebral and cerebellar atrophy with white matter signal changes in the index case. CONCLUSIONS: In addition to BLS type II, our study has expanded and further characterized the phenotype associated with the LOF of RFXANK to include progressive neurodegenerative disease. Our study also provides evidence for the impact of LOF on brain development and function. Thus, early bone marrow transplantation, as a standard of care for BLS, could prove to be protective against the neurologic phenotypes in this group of patients.

Leukemic priming of resting NK cells is killer Ig-like receptor independent but requires CD15-mediated CD2 ligation and natural cytotoxicity receptors.

Resting human NK cells require a two-stage activation process that we have previously described as "priming" and "triggering." NK-sensitive tumor cells provide both priming and triggering signals. NK-resistant tumors evade lysis, mostly by failure to prime; however, we recently reported a tumor cell line (CTV-1) that primes resting NK cells but fails to trigger lysis. In this article, we report two additional leukemia cell lines that prime NK cells but are resistant to lysis. Tumor-mediated NK priming is via CD2 binding to a ligand within CD15 on the tumor cell. NK-resistant RAJI cells became susceptible to NK lysis following transfection and expression of CD15. Blockade of CD15 on K562 cells or on CD15(+) RAJI cells significantly inhibited lysis, as did blockade of CD2 on resting NK cells. NK priming via CD2 induced CD16 shedding, releasing CD3ζ to the CD2, leading to its phosphorylation and the subsequent phosphorylation of linker for activation of T cells and STAT-5 and synthesis of IFN-γ. Blockade of C-type lectin receptors significantly suppressed the tumor-mediated priming of NK cells, whereas blockade of Ig-superfamily-like receptors had no effect at the NK-priming stage. Tumor priming of resting NK cells was irrespective of HLA expression, and blockade of HLA-killer Ig-like receptor interactions did not influence the incidence or degree of priming. However, CD15-CD2 interactions were critical for NK priming and were required, even in the absence of HLA-mediated NK inhibition. Tumor-mediated priming led to a sustained primed state, and the activated NK cells retained the ability to lyse NK-resistant tumors, even after cryopreservation.

Tumor-primed human natural killer cells lyse NK-resistant tumor targets: evidence of a two-stage process in resting NK cell activation.

NK cells are defined as those cells that lyse tumor cells without priming. In this study, we show that the preincubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias, and solid tumors even when HLA-matched, allogeneic or autologous. The primed NK cells remained nonresponsive to HLA-C matched and mismatched normal mononuclear cells from multiple donors. CD69, a known NK trigger receptor, was shown to be the predominant trigger on the tumor-primed NK cells because lysis was blocked with the rCD69 protein. The lack of lytic activity against normal hemopoietic cells implied that the ligand for CD69 is tumor restricted, and this was confirmed by experiments using fluorochrome labeled rCD69. It has been recently shown that resting NK cells require prior stimulation with IL-2 before triggering by all known NK-triggering ligands. In this study, we show that a tumor cell can provide the NK priming signal independently of IL-2. These data provide evidence for two NK evasion strategies for tumor cells, namely the prevention of priming (type1 evasion) and failure to trigger (type 2 evasion). Most NK-resistant cell lines are type 1 and fail to prime resting NK cells but are lysed by IL-2-primed NK cells. In contrast, CTV-1 cells prime resting NK cells but fail to trigger (type 2), and coincubation with CTV-1 primes for triggering by type 1 NK-resistant tumor cells. These tumor-activated NK cells lyse a broad spectrum of tumor cells with a degree of specificity never previously reported.

Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity.

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric alpha/alpha form are more cytotoxic than their CD8- counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8- NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8alpha+ cells after lysis of the same targets. We report that cross-linking of the CD8alpha chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK-cell apoptosis from which CD8alpha+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK-cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti-MHC Class I antibody. Thus, in contrast to the CD8- subset, CD8alpha+ NK cells are capable of sequential lysis of multiple target cells.