Protocols to Dissolve Amorphous Urate Crystals in Urine.

OBJECTIVE: Amorphous urate crystals can obscure significant findings during a routine urinalysis. There is no standardized protocol to minimize their effect. MATERIALS AND METHODS: We tested 210 urine specimens. Three specimens had high red blood cell (RBC) or white blood cell (WBC) counts. Fifty-six specimens formed amorphous urates. Sediment from these specimens was treated with 50 mM sodium hydroxide (NaOH) at a 1:2 and/or 1:4 dilution. We warmed 22 specimens with crystals at various temperatures. RESULTS: Amorphous urate crystals formed in concentrated urine with an acidic pH. Adding 50 mM NaOH dissolved amorphous urates, revealing the presence of underlying bacteria and yeast, but WBC and RBC counts were grossly decreased. Prewarming unspun specimens to 60°C for 90 seconds dissolved most amorphous urates. CONCLUSION: The protocol to eliminate amorphous urate crystals is to prewarm the specimen before testing. Adding 50 mM NaOH to sediment dissolves amorphous urates to enhance the visibility of bacteria and yeast but has a deleterious effect on WBC and RBC.

Strategies for Sustainability of University-Based Medical Laboratory Sciences Programs.

The COVID-19 pandemic has taken a major toll on the economy and funding for public education. For that reason, the pandemic has a worrisome effect on the sustainability of university/college based Medical Laboratory Sciences MLS training programs. Stakeholders of university-based MLS programs include university administrators, students, clinical affiliates and faculty. Each group has specific goals and challenges that affect the sustainability of the program. This report details strategies that can be used to satisfy the goals specific to key stakeholders that lead to sustainability. These strategies apply in pandemic times and in the back-to-normal future.

Does race alter the relationship between Hba1c and glucose in type 2 diabetes?

OBJECTIVE: Hemoglobin Alc (HbAlc) is the standard measurement of glycemic control, and the HbAlc value can be used to estimate average glucose using a formula. Several studies suggest that the relationship between average glucose and HbAlc may be different for Blacks. This project enrolled non-Hispanic black and white individuals with type 2 diabetes and evaluated the relationship between HbAlc and blood glucose. METHOD: 22 black and 29 white adults with type 2 diabetes were included in the analysis. Approximately 42 measurements (fasting and postprandial glucose) were collected over three months and compared to HbAl1 of the third month. The effect of race was evaluated by ANCOVA and X2 analysis testing the slope and intercepts simultaneously for HbA1c and its relationship to fasting glucose and to postprandial glucose. RESULTS: The relationship between HbAlc and glucose was not statistically significantly different between Blacks and Whites (ANCOVA: P = 0.968 for fasting glucose, P = 0.428 for postprandial glucose), allowing us to calculate estimated fasting and postprandial glucose disregarding race. For fasting glucose, the linear regression is FGmgiadl = (18.939 X HbAlc%) - 1.864, R2 = 0.586, P < 0.0001. For postprandial glucose, the linear regression is In(PPG mg,dl) (1.261 X In(HbA1c%)) + 2.555, R2 = 0.614, P < 0.0001. Predicted values for postprandial glucose based on HbA1c were similar to estimated average glucose values reported by ADAG. CONCLUSION: This study reinforces the A1c-Derived Average Glucose (ADAG) group finding that the relationship between HbA1c and glucose is similar in non-Hispanic black and white adults with type 2 diabetes.

Sedimentation by gravity stabilizes plasma glucose for up to 60 minutes.

OBJECTIVE: Glucose levels decrease in whole blood in vitro, but there are several methods that minimize the loss, including special tubes and ice. This study evaluated whether sedimentation by gravity in an upright position was a viable alternative. DESIGN: Lithium heparinized blood was collected from 20 individuals without a diagnosis of diabetes. The samples were allowed to sediment at ambient temperature and were tested in quadruplicate at 30 minute intervals. A Repeated Measures ANOVA compared the means of each time-point. RESULTS: Plasma glucose results were not statistically different between 30 minutes and 60 minutes after collection (p = 0.156). At 90 minutes after collection, glucose was significantly different than the initial glucose readings (p <0.001). Each reading thereafter also showed a statistically significant difference from the initial reading. CONCLUSIONS: Samples for glucose measurement are stable in lithium heparin for no longer than 60 minutes when held in an upright position prior to centrifugation.

Reporting variant hemoglobins discovered during hemoglobin A1c analysis - common practices in clinical laboratories.

BACKGROUND: Patients with variant hemoglobins may receive inaccurate results by some HbA(1c) methods. We examined reporting practices of clinical laboratories with respect to variant hemoglobins and limitations of methodology. METHODS: A survey of reporting practices was published in LabMedicine, and circulated to directors of Clinical Laboratory Sciences programs. Websites of reference laboratories were reviewed. RESULTS: One hundred thirty-five laboratories from 42 US states responded. 61.5% of those laboratories report only HbA(1c) value and reference interval; 5% of laboratories include methodology. 51% of laboratories use IE-HPLC, 47% use immunoassay and 2% use boronate affinity chromatography. Of laboratories using IE-HPLC, 39% routinely report the presence of hemoglobin variants, and 10% report variants only if they cause interference with the test. Of laboratories using immunoassay, only one appends the disclaimer that elevated HbF interferes with test results. All of the major reference laboratories report methodology on their websites; only 2 can detect hemoglobin variants. Six out of 7 reference laboratories state limitations of methodology on their websites. CONCLUSIONS: There is no standardized reporting format for HbA(1c) that includes methodology, test limitations or notification of variant hemoglobins. An algorithm for detection and reporting of variant hemoglobins and test methodology is proposed based on best practices.

Cessation of menstruation improves the correlation of FPG to hemoglobin A1c in Caucasian women.

BACKGROUND: Anemia is known to cause spurious hemoglobin A1c (HbA1c) results. The effect of menstruation on HbA1c was tested by correlating it to FPG in non-anemic premenopausal and in menopausal women. METHOD: Non-diabetic, non-obese middle-aged Caucasian women were classified as premenopausal or menopausal. Hemogram, FPG, and A1c results were obtained. RESULTS: Hemoglobin concentrations were lower in the premenopausal group. FPG showed a poor correlation to A1c value overall (r = 0.251, p = 0.001) which was improved by multiplying the A1c % by the total hemoglobin concentration to create an absolute A1c value (r = 0.362, p = 0.000). When the data was sorted by menopause status, the correlation of FPG to Absolute A1c improved (r = 0.463, p = 0.000) in the menopausal women, but remained low (r = 0.283, p = 0.005) in the premenopausal women. CONCLUSIONS: Menstruation may be a significant factor affecting the accuracy of A1c concentrations.

Screening for diabetes: sensitivity and positive predictive value of risk factor total.

OBJECTIVE: Screening for diabetes is recommended for individuals > or =45 years of age, or earlier if they manifest > or = one specific risk factors. This study examined the sensitivity and positive predictive value (PPV) of risk factor total for identifying individuals with diabetes and prediabetes. DESIGN: Subjects were interviewed to assess the presence of risk factors. Fasting plasma glucose levels were obtained. SETTING: The study occurred at a health fair in Greensburg, PA. PATIENTS: Six hundred sixty-one Caucasians between the ages of 19 and 100. RESULTS: Using the criterion of screening individuals with > or = one risk factors detected 100% of both diabetics and prediabetics. This dropped to 91.2% when screening individuals with > or = two factors. The PPV of the risk factor total was poor (80% of individuals with a total of four factors were not diabetic). The ability of the risk factor total to predict individuals with impaired glucose metabolism (prediabetics + diabetics) was considerably better, and increased almost linearly with the risk factor total. Of the subjects with normal glucose values, the mean glucose increased as the risk factor total increased. CONCLUSION: While the sensitivity of using > one risk factor as an algorithm to screen is 100% for identifying diabetics, the PPV of risk factor analysis for identifying diabetics is poor. The same algorithm works well to identify at-risk individuals, presumably allowing early intervention and education.

Mutations in lozenge and D-Pax2 invoke ectopic patterned cell death in the developing Drosophila eye using distinct mechanisms.

Mutations in the lozenge gene of Drosophila melanogaster elicit a pleiotropic set of adult phenotypes, including severe compound eye perturbations resulting from the defective recruitment of photoreceptors R1/6 and R7, cone and pigment cells. In this study, we show that excessive patterned apoptosis is evident at the same developmental stage in these lozenge mutants. In lozenge null mutants, apoptosis occurs prior to lozenge-dependent cell fate specification. A second gene, D-Pax2, genetically interacts with lozenge. Interestingly, D-Pax2 mutants also exhibit increased cell death, but slightly later in development than that in lozenge mutants. Although expression of the caspase inhibitor p35 eliminates death in both lozenge and D-Pax2 mutants, the lozenge mutant eye phenotypes persist because other normal Lozenge functions are still lacking. D-Pax2 eye phenotypes, in contrast, are dramatically altered in a p35 background, because cells that normally differentiate as cone and primary pigment cells are subsequently transformed into secondary pigment cells. This study leads us to propose that Lozenge, aside from its known role in gene regulation of cell-specific transcription factors, is required to contribute to the repression of cell death mechanisms, creating a permissive environment for the survival of undifferentiated cells in early eye development. Lack of lozenge expression increases the likelihood that an undifferentiated cell will initiate its default death program and die prematurely. The ectopic cell death evident in D-Pax2 mutants appears to arise from the cell fate transformation of cone cells into secondary pigment cells, either autonomously or as a result of defective signalling.

Yan regulates Lozenge during Drosophila eye development.

The Drosophila eye offers an excellent opportunity to understand how general developmental processes are subtly altered to result in specific cell fates. Numerous transcription factors have been characterized in the developing eye; most of these are active in overlapping subsets of cells. Mechanisms used to regulate transcription factors act at many levels, and include competition for cognate binding sites, post translational modification, transcriptional regulation and cofactor availability. In undifferentiated cells of the larval eye imaginal disc, the transcriptional repressor Yan outcompetes the transcriptional activator Pointed for ETS binding sites on the prosperoenhancer. During differentiation, the Ras signaling cascade alters the Yan/Pointed dynamic through protein phosphorylation, effecting a developmental switch. In this way, Yan and Pointed are essential for prospero regulation. Hyperstable Yan (ACT) cannot be phosphorylated and blocks prospero expression. Lozenge is expressed in undifferentiated cells, and is required for prospero regulation. We sequenced the eye-specific enhancer of lozenge in three Drosophila species spanning 17 million years of evolution and found complete conservation of three ETS consensus binding sites. We show that lozengeexpression increases as cells differentiate, and that Yan (ACT) blocks this upregulation at the level of transcription. We find that expression of Lozenge via an alternate enhancer alters the temporal expression of Prospero, and is sufficient to rescue Prospero expression in the presence of Yan (ACT). These results suggest that Lozenge is involved in the Yan/Pointed dynamic in a Ras-dependent manner. We propose that upregulated Lozenge acts as a cofactor to alter Pointed affinity, by a mechanism that is recapitulated in mammalian development.