RBD PDD/DLB / NCNP Collaboration / Reference Range

(Huebecker et al. 2019, PMID 31703585)

Fig. B Significant reduction in GM1a and GD1a levels in serum from PD patients and significant reduction in all measured glycosphingolipids, except GlcCer, in serum from RBD patients. Levels of GlcCer (a), LacCer (b), Gb3 (c), Gb4 (d), GM3 (e), GM2 (f), GM1a (g) and GD1a (h) were determined in serum samples from control subjects (n = 15), PD patients (n = 30) and age-matched RBD patients (n = 30) with NP-HPLC (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, one-way ANOVA). Data are presented as mean ± SD

NCNP collaboration for RBD (& PD) (Yuji)

• NCNP just provides sample and we pay for their ‘maintenance of biobank’

NCNP, Dr. Yuji Takahashi — Molecular BM discovery using CSF and PBMC from PD and RBD patients

	MBM discovery
	H Kamiguchi
	Translational R

Summary	REM sleep behavior disorder (RBD) is thought to be a reliable prodromal stage of Parkinson's disease. In order to discover novel biomarkers for early therapeutic intervention, clinical CSF and PBMC samples from RBD and PD patients have been collected and multiple omics analysis will be performed.
Deliverable	Biomarker for early therapeutic intervention and patient segmentation
Impact on portfolio	Biomarker for PD
Milestone	1. Sample collection from RBD, PD and HC 2. Multiple omics analysis using cells in CSF and PBMC 3. Identification of biomarkers
Period	Feb 2019-Feb 2021 (48 month)
Funding	0.12 OKY at Sep 2019 (IMM, immunology unit) 0.12 OKY at Sep 2020 (NS DDU)

Reference range (normal range)

• Values within the reference range (WRR) are those within the normal distribution and are thus often described as within normal limits (WNL).
• limits of the normal distribution are called the upper reference limit (URL) or upper limit of normal (ULN) and the lower reference limit (LRL) or lower limit of normal (LLN)
• Definition
  • = Standard ranges

Data distribution approach	Normal distribution parametric	If skewed Non-parametric
	the central 95% of the data
	95% of the total population 95% Prediction interval = mean +/- 2 SD	2.5 - 97.5 percentiles of the population
	Outliers are removed from the calculation

• Cf) Outliers are results that differ from the mean by more than 3 SD or differ from other results by more than 30%, outliiers are best identified by visual inspection,
• Sample size
  • NCCLS recommends testing at least 120 (120 is the number needed to calculate the 90% confidence limit of the upper and lower reference limits using non-parametric statistics.) patient samples for establishment of a statistically significant reference interval. Other experts recommend a minimum of 200 samples to ensure stable lower and upper reference limits.
• Verification of a reference interval
  • http://www.clinlabnavigator.com/reference-ranges.html|Verifying a reference range is different than establishing a reference range. For an FDA approved test method, the clinical laboratory can adopt the manufacturer’s stated reference range if its patient population gives similar results to the manufacturer’s package insert.
  • A total of 40 samples, 20 from healthy men and 20 from healthy women, should be tested and the results compared to the published reference range. The results should be evenly spread throughout the published reference range and not clustered at one end. If 95% of the results fall within the published reference range, it can be accepted for use. If the manufacturer’s reference range cannot be validated, the laboratory needs to establish its own reference range.
  • Verification of the reference range is also useful when it is too difficult to collect a large number of samples to establish a new reference range. In this situation it is permissible to determine whether the manufacturer’s stated reference range, your existing reference range, or a reference range established by another neighboring laboratory using the same instrument and reagents is applicable.

Methods

CLSI. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline - Third Edition. CLSI EP28-A3C (2010).	reference intervals are determined using test results from a cohort of healthy reference individuals chosen from the population. The bounds of the reference interval are the values corresponding to the 2.5th and 97.5th percentiles of the distribution of test results3. The reference cohort must consist of a 'sufficient' number of carefully selected and apparently healthy individuals who meet specific inclusion and exclusion criteria3. This way of sampling from a healthy cohort and establishing reference intervals from their test results is referred to as the 'direct' or 'conventional' method3.
	The reference range is then derived mathematically by taking the average value for the group and allowing for natural variation around that value (plus or minus 2 standard deviations from the average
	95% prediction interval (95% PI)
	90% confidence interval of a standard reference range limit

Retina

	Normal anatomy	Native (endogenous) aSYN in healthycontrol retinas is most abundant in the photoreceptor outersegments, in the outer plexiform layer and in neurons andtheir processes in the inner nuclear layer and inner plexiformlayer.
		relatively low numbers of dopaminergic amacrine cells in the retinathe fraction of dopaminergic cell bodies in the inner nuclear layer is limite
F	PD patients	https://www.michaeljfox.org/grant/retinal-pathology-parkinsons-disease-implications-vision-and-biomarkers Nicolás Cuenca, PhD Assistant Professor at University of Alicante Location: Alicante Spain phosphorylated alpha-syn in the retina, in specific types of cells including cell bodies, dendrites and axons. A clear correlation between the amount of phosphorylated alpha-syn present in retina and in brain was observed, a decrease in the numbers of dopamine cells and a reduction of their synaptic contacts with their postsynaptic neurons.a definite decrease in the numbers, the ganglion cells, as well as the loss of their axons in the optic nerve
		there are no ASYN ligands available for retinal imaging.
dis		https://www.retinalphysician.com/issues/2023/january-february-2023/advances-in-retinal-biomarkers-and-retinal-tracers
		October 2022, Amydis Inc, a privately held company, announced that patient enrollment for the phase 1/2a Prospective Randomized Open, Blinded Endpoint (PROBE) trial was under way. The trial is designed to evaluate "retinal tracer", a fluorescent, nonradioactive retinal imaging agent (AMDXP-2011P) that targets protein

Uncertain Spans

location	text/status	reason
Fig. B caption	exact significance asterisks per panel	Stars are small in the figure and were transcribed from the figure caption verbatim; per-panel significance comparisons should be cross-checked from the body_full evidence crop.
Reference-range parametric/skewed table	row layout	The middle and lower rows of the small table span both columns based on visible cell ruling, but vertical separators are faint; intent of the multi-bullet cell vs. shared rows pending manual review.
Retina table column 1	leftmost cell labels (F, dis)	The leftmost cells of the retina table are partly clipped at the page edge; visible fragments suggest the column holds short markers.
Amydis row	trailing word protein	The bottom row is clipped at the page edge after targets protein; the sentence continues into the next photo.