Performance characteristics of consensus approaches for small and minor paroxysmal nocturnal hemoglobinuria clone determination by flow cytometry.

BACKGROUND: Evaluation of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM) is not standardized and is associated with consistent inter-laboratory variability. METHODS: In order to rule out the influence of particular approach in generating final results, we analyzed the performance characteristics of individual consensus strategies for small to intermediate (1%-20%) and minor (<1%) PNH clones within the white blood cell (WBC) and red blood cell (RBC) compartments with sensitivity up to 0.1%. RESULTS: Coefficient of variation (CV) for precision/reproducibility analysis ranged from 0.67%/1.49% to 2.56%/3.09% for granulocytes, from 0.93%/3.09% to 7.76%/12.06% for monocytes and from 0.41%/4.73% to 6.53%/5.1% for RBCs. Coefficient of determination (r2) for linear regression analysis ranged from 0.95 to 0.99, Wilcoxon ranks test showed no statistically significant differences (p>0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from -0.18 to 1.24. CONCLUSIONS: Our results confirmed very good performance characteristics for precision and reproducibility analysis, excellent correlation and favorable agreement between strategies, suggesting that reported inter-laboratory variability is related mainly to incorrect performance and/or insufficient experience with PNH testing by flow cytometry, rather than to relevant limitations of any particular approach.

Intra- and interlaboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high-sensitivity paroxysmal nocturnal hemoglobinuria testing.

BACKGROUND: Sutherland et al. recently published the Practical Guidelines for high-sensitivity detection of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM), containing concise protocols for PNH testing. METHODS: Using this approach, we studied the intra- and interlaboratory variability observed in a multicenter study in which fresh blood samples containing three clinically relevant PNH clone sizes within the granulocytic, monocytic, and red blood cell (RBC) populations were shipped to each participating center. RESULTS: Coefficients of variation (CVs) for precision/reproducibility analysis ranged from 0.01%/0.02% to 0.48%/0.45% (big clone), from 0.69%/1.52% to 4.24%/5.80% (small-intermediate clone), from 1.47%/3.91% to 15.01% /17.83% (minor clone) for PNH white blood cells (WBCs) and from 0.24%/0.48% to 1.76%/1.83% (big clone), from 0.80%/1.14% to 2.39%/4.45% (small-intermediate clone), from 1.09%/3.36% to 10.54%/10.23% (minor clone) for PNH RBCs, respectively. Linear regression analysis showed excellent performance correlation between centers (r > 0.99), Wilcoxon rank test revealed no statistically significant differences for PNH granulocytes, monocytes, and RBCs (P > 0.05%), Bland-Altman analysis demonstrated good performance agreement for all target PNH clones (mean bias ranging from -1.47 to 0.71). CONCLUSION: Our results demonstrate very good intra- and interlaboratory performance characteristics for both precision and reproducibility analyses and excellent correlation and agreement between centers for all target PNH clone sizes. Our data confirm the reliability and robustness of the recently published Practical Guidelines approach for high sensitivity PNH testing by flow cytometry and suggest that such an approach represents an excellent basis for standardization of PNH testing by flow cytometry.

Zucker diabetic fatty rat: a new model of impaired cutaneous wound repair with type II diabetes mellitus and obesity.

Impaired diabetic wound healing is an important current medical issue, mainly concerning patients recovering from complicated operations or patients with ulcers on their feet. The obese Zucker diabetic fatty rat, with a mutation in leptin receptors, may be a good choice for studying impaired wound healing. Male and female rats were fed a diabetogenic high-fat diet. Wound size changes of air-exposed excisional 2 cm circular wounds were measured until Day 10. Wound tissue was analyzed morphologically, histologically, and immunohistochemically. The hydroxyproline content in the granulation tissue (GT) was determined. mRNA expression was assayed by DNA-array analysis and real-time reverse transcription-polymerase chain reaction. Wound-size changes were retarded in diabetic rats and differed between the sexes. Diabetic wounds were characterized by impaired contraction, abundant crust production, increased inflammation, and pus formation. On Day 10, the GT contained a significantly increased amount of intercalated fat tissue and showed an irregular arrangement of GT and collagen fibers. Interestingly, the length of new epithelium was increased in diabetic wounds. The concentration of hydroxyproline in the GT of diabetic animals was significantly decreased to about one half when compared with the nondiabetic controls. The expression of interleukin-6, myeloperoxidase, stromelysin-1, and collagenase-3 was increased in the GT of diabetic rats on Day 10, while the expression of type I collagen and elastin was decreased. Taken together, Zucker diabetic fatty rats exhibited impairments in wound-size reduction, inflammatory response, tissue organization, and connective tissue turnover and are thus proposed as a new model for studying impaired repair.