Biology Reference
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storage; number of freeze/thaw cycles; and variations in collection and pro-
cessing across biospecimen sets.
Blood is the most frequently analyzed bodily fluid, and the ease with which
it can be sampled makes it a logical choice for biomarker applications. The
levels of individual blood proteins represent a summation of multiple, dis-
parate events that occur in every organ system. Blood contains proteins
shed by the affected tissue as well as proteins that reflect secondary sys-
temic changes. In addition, the blood proteome depends on many other
factors governing the actual state of the whole organism that may not be
related to the monitored disease, complicating the evaluation and perti-
nence of the data obtained. Another factor that complicates the analysis
of plasma/sera is the wide range of protein amounts and isoforms. Plasma
and sera are highly complex mixtures containing high amounts of many dif-
ferent proteins with a wide dynamic range, spanning 12 orders of magni-
tude from albumin to the lowest abundance, often most clinically relevant,
proteins such as cytokines and their receptors
[5,6]
. The 22 most abundant
proteins constitute approximately 99% of the plasma proteome, whereas
the remaining 1% of the plasma proteins are medium and low abundance
proteins
[6]
. Thus, both depletion of the predominant proteins and subse-
quent fractionation of the proteome are usually required to allow the detec-
tion of low abundance proteins. Unfortunately, the steps involved in sample
preparation may result in the loss of proteins of interest during the deple-
tion step
[7]
. Considering that most clinically relevant plasma biomarkers
belong to the low abundance plasma protein fraction and have concentra-
tions 10
[6-8]
times lower than those of albumin
[5]
, highly sensitive detec-
tion methods are required.
451
Urine samples represent an alternative to plasma/sera samples for bio-
marker discovery. Urine has three main advantages compared to plasma/
sera: (i) it can be obtained in large quantities; (ii) the protein mixture is far
less complex and the variation in protein abundance is low
[8]
; and (iii) it
is more stable than plasma
[9]
. However, a limitation is that urine yields
better information about diseases in the organs directly involved in its pro-
duction and excretion, such as the kidneys, as the proteins are produced
mainly from kidney function (~70%) and partially by glomerular filtration of
plasma proteins (~30%)
[8]
; thus, urine is less informative for other systemic
diseases.
An ideal schedule of sample collection post-HSCT will contain both calen-
dar- and event-driven collection. Based on currently validated biomarkers,
we propose a cost-effective collection for plasma/sera that contains calen-
dar samples: pre-HSCT, day 14, day 21, day 28, and day 100 post-HSCT. Days
14 and 21 post-HSCT are to capture GVHD before clinical signs occur, day 28
is to capture samples of non-GVHD patients at matched time points to those
of GVHD patients, and day 100 is to predict chronic GVHD (cGVHD). Event-
driven samples should include onset of complications (e.g., aGVHD) during
the 48-h window of treatment initiation, onset of cGVHD, and onset of other
complications that can either mimic GVHD or pose a difficult diagnosis and
for which biomarkers should still be discovered, such as idiopathic pneumo-
nia syndrome, sinusoidal obstructive syndrome, and sepsis. Sample quality,
acquisition, and storage should be followed as specified above.
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