Siemens Coat-A-Count TSH IRMA Manual Del Usuario página 5

Label sixteen TSH Ab-Coated Tubes
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A (nonspecific binding) and B through
H ("maximum binding") in duplicate.
Label additional TSH Ab-Coated
Tubes, also in duplicate, for controls
and patient samples.
If Total Counts tubes are required for
data reduction, label two plain
(uncoated) 12x75 mm polystyrene
tubes T (total counts) in duplicate.
Calibrators
A (NSB)
B
C
D
E
F
G
H ("MB")
Pipet 200 µL of each calibrator,
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control and patient serum sample into
the tubes prepared.
Pipet directly to the bottom.
Samples expected to contain TSH
concentrations greater than the
highest calibrator (60 µIU/mL) should
be diluted in the zero calibrator before
assay. The use of disposable-tip
micropipets is recommended, to avoid
carryover from sample to sample.
Positive displacement pipets and
automatic pipettor-diluters should be
used only if the possibility of carryover
has been evaluated and found to be
insignificant.
Add 100 µL of
3
tube.
Pipet directly to the bottom, and
make sure that sample and tracer are
thoroughly mixed, without foaming. A
repeating dispenser is recommended.
Set the (optional) T tubes aside for
counting at step 6; they require no
further processing.
Shake at room temperature (15–28°C)
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for 2 hours on a rack shaker set at
200 strokes a minute.
Decant thoroughly. Add 2 mL
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Buffered Wash Solution to each tube.
Wait 1 to 2 minutes, then decant
Coat-A-Count TSH IRMA (PIIKTS-8, 2010-11-04)
µIU/mL
0
0.15
0.5
1.5
4
15
30
60
125
I TSH Ab to every
thoroughly. Again add 2 mL Buffered
Wash Solution, wait 1 to 2 minutes,
and decant thoroughly.
Removing all visible moisture will
greatly enhance precision. After the
second wash, using a foam decanting
rack, decant the contents of all tubes
(except the T tubes) and allow them to
drain for 2 to 3 minutes. Then strike
the tubes sharply on absorbant paper
to shake off all residual droplets.
Count for 1 minute in a gamma
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counter.
In multi-head gamma counters, the
(optional) Total Counts tubes should
be separated from the remaining
assay tubes by at least one space, to
minimize the possibility of spillover.
Calculation and Quality Control
To calculate results (in terms of
concentration units) from a log-log
representation of the calibration curve, first
correct the counts per minute (CPM) of
each pair of tubes by subtracting the
average CPM of the nonspecific binding
tubes (calibrator A):
Net Counts = (Average CPM) minus (Average
NSB CPM)
Then determine percent binding (relative
to that of the highest calibrator) – here
called "%B/MB" – of each pair of tubes as
a percent of "maximum binding," with the
NSB-corrected counts of the highest
calibrator taken as 100%:
Percent Bound = (Net Counts / Net MB Counts)
× 100
Using 4-cycle log-log graph paper, plot
Percent Bound versus Concentration for
each of the nonzero calibrators, and draw
a curve approximating the path of these
points. (Connect the calibration points with
arcs or straight line segments. Do not
attempt to fit a single straight line to the
data.) Concentrations for controls and
unknowns within range of the nonzero
calibrators may then be estimated from
the calibration curve by interpolation. An
additional plot of Percent Bound versus
Concentration for the three lowest
calibrators on linear-linear graph paper
may be used for interpolation near zero
dose.
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