REF86100 User Manual HumaCycler

HumaCycler
| User Manual
Cat No. 86100/1

REVISION LIST OF THE MANUAL
Rev. /DATE. REVISION DESCRIPTION
01/2015-06 First edition
02/2015-08 Addition of Packing List
SYSTEM VERSION
COPYRIGHT
Copyright 2015, Human Gesellschaft fr Biochemica und Diagnostica mbH, Wiesbaden,

Germany. All rights reserved.
No part of this documentation may be reproduced in any form, nor processed, copied or distrib-
uted by means of electronic systems, without prior permission of HUMAN in writing. Since all
precautionary measures were taken into account in producing these operating instructions, the
manufacturer accepts no responsibility for any errors or omissions. This includes any liability for
damage that could arise from possible incorrect operation based on this information. Subject to
changes without notice as result of technical development.
SERVICE UND SUPPORT

CONTENTS
TABLE OF CONTENTS
1 SAFETY INSTRUCTIONS 5
1.1INTRODUCTION 5
1.2 USER WARRANTY 5
1.3 INTENDED USE OF THE INSTRUMENT 5
1.4 GENERAL SAFETY WARNINGS 6
1.5 DISPOSAL MANAGEMENT CONCEPT 6
1.6 BIOHAZARD WARNING 7
1.7 INSTRUMENT DISINFECTION 7
2 SYSTEM DESCRIPTIONS 9
2.1 INTENDED USE 9
2.2FEATURES 9
2.3 FEATURES OF THE HCY4 SOFTWARE 10
3 TECHNICAL SPECIFICATIONS 11
4PREPARATION 13
4.1 TRANSPORT AND STORAGE CONDITIONS 13
4.2 NORMAL WORKING CONDITIONS 13
4.3 UNPACKING THE INSTRUMENT 13
4.4 REMOVAL OF THE FIXING PIN 13
4.5 POWER CORD CONNECTION 14
4.6 CONNECTION OF THE COMMUNICATION WIRE 15
5 SYSTEM INSTALLATION AND UNINSTALLING 17

5.1 SYSTEM ENVIRONMENT 17
5.2 SYSTEM INSTALLATION 17
5.3 SYSTEM UNINSTALLATION 20
6START 21
6.1 INSPECTION BEFORE STARTING 21
6.2 STARTING THE INSTRUMENT 21
6.3 STARTING THE SOFTWARE 22
7 ROUTINE UTILIZATION 25
7.1 ABSOLUTE QUANTIFICATION 25
7.1.1 Create a new Absolute Quantification Experiment 25
7.1.2 Detector Settings 26
7.1.3 Sample Information Settings 28
7.1.4 Plate Settings 29
7.1.5 Program Settings 31
7.2 PREPARATION OF THE PCR RUN 34
7.3 PCR RUN 35
7.3.1 Preparation of the Reagent Sample 35
7.3.2 Run a Fluorescence Curve 36
7.3.3 Run a Temperature Curve 38
7.3.5 Working State Indication Lamps on the Instrument 39
7.3.6 Occurrences during the run 40
7.4 DATA ANALYSIS 40
7.4.1 Result Analysis 41
7.4.2 Adjusting Parameters and Re-Analysis 48
7.5 DATA REPORT 51
7.5.1 Design the Report Template 51
7.5.2 Print Settings 53
7.5.3 Consolidated Report 54
7.5.4 Quality Control Summary 55
7.6 DATA EXPORT 56
7.6.1 Export to Database 57
7.6.2 Experiment Filing 57
7.6.3 Save Data Settings 58
7.6.4 Export Experiment Data to Text and Excel 59
7.6.5 Export Raw Data 59
7.7 DATA REVIEW 60
8 RELATIVE QUANTIFICATION 61
8.1 EXPERIMENT DESIGN 61
8.1.1 Create a New Relative Quantitative Experiment 61
8.1.4 Reaction Plate Settings 65
8.2 PREPARATION OF PCR RUN 68
8.3 PCR RUN 68
8.3.1 Run A Fluorescence Curve 69
8.3.2 Run A Temperature Curve 71
CONTENTS

8.4.2 Relative Quantification Data Analysis 76
8.4.3 Adjust Parameters and Re-Analysis 77
8.5 DATA REPORT 78
8.6 DATA EXPORT 80
8.7 DATA REVIEW 84
9 SINGLE-NUCLEOTIDE-POLYMORPHISM (SNP) DETECTION 85

9.1 CREATE A SNP EXPERIMENT 85
9.1.3 Plate Settings 88
9.2 PREPARATION OF PCR RUN 92
9.3 PCR RUN 92
9.3.1 Run A Fluorescence Curve 93
9.3.2 Run A Temperature Curve 94
9.4.2 Adjust the Parameter for Re-Analysis 100
9.5 DATA REPORT 101
9.5.1 Design the Report Template 101
9.5.2 Print Settings 103
9.5.4 Report Printing 104
9.6 DATA EXPORT 106
9.7 DATA REVIEW 110
10 GENE STUDY 113
10.1 CREATE GENE STUDY 113
10.2 GENE STUDY SETUP 114
10.3 GENE STUDY ANALYSIS 114
10.3.1 View Analysis Results 115
10.3.2 Analysis Settings 115
11 TOOL USAGES 117

11.1 GAIN SETTINGS 117
11.2 BLOCK SCAN METHOD 117
11.3 DETECTOR LIBARY 118
11.4 CUSTOMIZED DYES 118
11.5 CUSTOMIZED COLUMNS 119
11.6 COLUMN SELECTION 119
11.7 SAMPLE COLUMN LIBRARY 120
11.8 INSTRUMENT CALIBRATION PARAMETERS 121
11.9CROSSTALK 122
11.9.1 Measure Crosstalk Calibration Parameters 122
11.9.2 Measure Crosstalk Gain 123
12 SERVICE USAGE 125
13 OTHER FUNCTIONS 127

13.1 INSTRUMENT OPERATION 127
13.1.1Connect 127
13.1.2 Disconnect 127
13.2 INSTRUMENT INFORMATION 127
13.3 INSTRUMENT ALARM 128
13.4 SYSTEM MAINTENANCE 129
13.5 DATA QUERY 129
13.6 SYSTEM HELP 130
14MAINTENANCE 131
14.1 REGULAR CLEANING 131
15 ANALYSIS AND TROUBLESHOOTING 133
16APPENDIX 137
16.1 PACKING LIST 137
16.2 HUMACYCLER WIRING 138
Safety Instructions 5
1 SAFETY INSTRUCTIONS
1.1 Introduction
This manual is considered part of the instrument and must be available to the
operator and the maintenance personnel. For accurate installation, use and
maintenance, please read the following instructions carefully.
In order to avoid damage to the instrument or personal injury, carefully read
the GENERAL SAFETY WARNINGS, describing the appropriate operating pro-
cedures. Please contact your HUMAN authorised local Technical Service in the
event of instrument failure or other difficulties with the instrument.
1.2 User Warranty

HUMAN warrants that instruments sold by one of its authorised representa-
tives shall be free of any defect in material or workmanship, provided that this
warranty shall apply only to defects which become apparent within one year
from the date of delivery of the new instrument to the purchaser.
The HUMAN representative shall replace or repair any defective item within
this warranty period at no charge, except for transportation expenses to the
point of repair.
This warranty excludes the HUMAN representative from liability to replace
any item considered as expendable in the course of normal usage, e.g.: lamps,
valves, syringes, glassware, fuses, tubing etc.
The HUMAN representative shall be relieved of any liability under this warranty
if the product is not used in accordance with the manufacturers instructions,
altered in any way not specified by HUMAN, not regularly maintained, used with
equipment not approved by HUMAN or used for purposes for which it was not
designed.
1.3 Intended Use of the Instrument

The instrument must be used for its intended purpose (see paragraph 2). It must
[IVD]
be operated in perfect technical conditions, by qualified personnel, in such
working conditions and maintained as described in this manual, in the GENERAL
SAFETY WARNINGS. This manual contains instructions for qualified professional
operators.
6
1.4 General Safety Warnings

Use only chemical reagents and accessories specified and supplied by HUMAN
and/or mentioned in this manual. Place the product so that it has proper ven-
tilation.
The instrument should be installed on a flat, stationary working surface, that is
free of vibrations.
Do not operate in area with excessive dust.
Operate at temperature and at a humidity level in accordance with the specifi-
cations listed in this manual (chapter 3).
Do not operate this instrument with covers and panels removed.
Use only the power cord specified for this product, with the grounding conduc-
tor of the power cord connected to earth ground.
Use only the fuse type and rating specified by the manufacturer for this instru-
ment.
The use of fuses with improper ratings may pose electrical and fire hazards.
To avoid fire or shock hazard, observe all ratings and markings on the instru-
ment.
Do not power the instrument in environments that are potentially explosive or
at risk of fire.
Prior to cleaning and/or performing maintenance on the instrument, switch off
the instrument and remove the power cord.
Only cleaning materials described in this manual may be used, as other mate-
rials may damage parts. It is recommended to always wear protective clothing
and eye protection while using this instrument.
All warning symbols that appear in this manual must be carefully observed.
1.5 Disposal Management Concept

The applicable local regulations governing disposal must be observed. It is the
users responsibility to arrange for proper disposal of the individual components.
All parts which may contain potentially infectious materials must be disinfect-
ed by suitable, validated procedures (autoclaving, chemical treatment) prior to
disposal. Applicable local regulations for disposal must be carefully observed.
The instruments and electronic accessories (without batteries, power packs etc.)
must be disposed of according to the applicable local regulations for the dispos-
al of electronic components.
Batteries, power packs and similar power sources must be removed from elec-
tric/electronic parts and disposed of in accordance with applicable local regula-
tions.
HumaCycler | User Manual

Safety Instructions 7
1.6 Biohazard Warning

Analytical instruments for in vitro diagnostic application involve the handling
of human samples and controls which should be considered at least potentially
infectious. Therefore every part and accessory of the respective instrument
which may have come into contact with such samples must equally be consid-
ered as potentially infectious.
The BIOHAZARD warning label must be affixed to the instrument prior to
first use with biological material!
Figure 1
Biological Hazard Symbol
1.7 Instrument Disinfection

Before performing any servicing on the instrument it is very important to thor-
oughly disinfect all possibly contaminated parts. Before the instrument is re-
moved from the laboratory for disposal or servicing, it must be decontaminated.
Decontamination must be performed by authorised well-trained personnel, and
in observance of all necessary safety precautions.
8

SYSTEM DESCRIPTIONS 9
2 SYSTEM DESCRIPTIONS
This chapter describes the applications, the instrument and software features of
the Real-Timer PCR instrument HumaCycler.
2.1 Intended Use

The 96-well fluorescent PCR detection system HumaCycler allows the real-time
detection of amplified DNA.
Application areas for the use of the HumaCycler include the clinical diagnosis of
pathogens, cancer, genetic diseases and research of the human genome, foren-
sics, oncology, epidemiology, zoology and botany.
The HumaCycler belongs to IVD medical equipment, which uses the
Polymerase-Chain- Reaction to perform quantitative or relative analysis of
genes, mutations or Melting Curve analysis in clinical laboratories.
2.2 Features
-- Unique patented advanced module bottom fluorescent detection techno-
logy.
-- User friendly interface with flexible program settings, analyses and repor-
ting using the stored parameters.
-- Ease of use and flexible running interface for smooth operation.
-- Advanced thermo-electric technology ensures fast and steady heating and
cooling of the ultra-fast heat cycling system.
-- It can create a temperature gradient with 4 thermo electric modules.
-- Stable and accurate 1...36C gradient function for optimized PCR conditions.
-- The advanced fiber optic transmission technology makes the photo-electric
detection system very sensitive and reliable.
-- Maintenance-free, long life LED excitation light source.
-- Precise optical path system and ultra-sensitive PMT system provide the most
accurate and sensitive fluorescent detection.
-- The instrument has high linear range up to 10 orders of start DNA copies
without serial dilution.
-- Automatic hot-lid technology needs no manual opening/closing and en-
sures constant pressure of the hot-lid used with different height reaction
tubes or plates.
10
2.3 Features of the HCy4 Software

-- Parameter setting-up function (including temperature, time, cycles, hea-
ting/cooling rate, selection of detection channel and yield of photo-electric
amplification tube).
-- Sample material record and batch function (sample no., sample name and
sample data).
-- Document running display function (heat cycle data, fluorescence detection
data and real-time display of each data during PCR runs).
-- Detection data analysis function (The analysis function can be independent-
ly used without connectivity to the instrument).
-- Analysis result output function. It may output the analysis result to various
types of document, e.g. EXCEL, TXT documents. It is possible to run an en-
quiry and print out analysis result, modify the printing format and select/
de-select items to print.
-- Document storage function (setting up data, running data and analysis re-
sults).
-- Fault protection and alarm function.

TECHNICAL SPECIFICATIONS 11
3 TECHNICAL SPECIFICATIONS
Specification/Model HumaCycler (4 channel system)
Sample capacity 96x0.2 ml tubes (suitable for single tubes, 8 strip
tubes and 96-well fully, half and non- skirted plate
Detection channel F1 F2 F3 F4
Applicable dye FAM VIC Cy5 ROX
SYBR Green I HEX Texas Red
JOE
TET
Temperature range
of block working 4...105C (minimum devision 0.1C)
Heating/cooling rate 4C/s (max.)

Temperatur range 0.1C (full-range), (55C typical value 0.1C)
Temperatur accuracy 0.2C (full-range), (55C typical value 0.1C)
Temperatur uniformity 0.4C (full-range), (55C typical value 0.3C)
Temperatur range of
hot-lid working 30...110C (adjustable, default 105C)
Repeatability of fluorescent
intensity detection 5%
Running mode Continuous running
Operation system Windows2000/XPSP2/Vista/
Power supply 100...240V, 50/60Hz 600W
Dimensions 430mm395mm352 mm
Weight 28kg
12

PREPARATION 13
4 PREPARATION
This chapter describes the transport and storage conditions, the removal of
Fixing Pin, installation/unloading of the HCy4 software and preparations.
4.1 Transport and Storage Conditions

Environmental temperature: -20C...55C
Relative humidity: 80%
4.2 Normal Working Conditions

Environmental temperature: 10C...30C
Environmental relative humidity: 70%
Power supply: 100...240V, 50/60Hz, 600W
! Before using the instrument,
please make sure the wor-
king conditions meets the requi-
4.3 Unpacking the Instrument rements mentioned above. The
Remove all the parts from their package. power socket shall be a 3-hole so-
Before unpacking the instrument, please make sure that all items, which are cket and properly earthed.
listed in the packing list, are included in the shipping box. In case of damage or
missing items, please contact the supplier immediately.
4.4 Removal of the Fixing Pin

In order to prevent the inner moving parts to shift and collide during transport,
they were secured with a Fixing Pin before leaving Human. After the instrument
is placed, the Fixing Pin must be removed from its location on the right side of
the instrument by unscrewing it counter clockwise. Then, it must be inserted
into the hole at the back of the instrument (Figure 2) and fully tightened by
clockwise rotation to release the locking mechanism inside the instrument (Fig-
ure 3). Only after correct installation of the Fixing Pin, the instrument may be
switched on; otherwise, the instruments temperature control system is able to
run, but the X-Y axis scanning system isnt able to move.
14
Figure 2
1 Fixing Pin on the right side

of the instrument.
2 Fixing Pin in the unlocker
port.
Figure 3
Please keep the Fixing Pin tightened during the normal operation of the instru-
ment. Before moving the instrument, turn the instrument on and wait until the
self-test action is completed. Shut down the instrument normally and remove
the Fixing Pin from the back of the instrument and insert it into the hole on the
right side of the instrument and tighten it.
4.5 Power cord connection
! If the supplied power cord

connection is loose, it should
be replaced with one of the same
Only the power cord supplied with the instrument should be used. To connect
the instrument to the energy source, ensure that the instrument is in the OFF
position. After the connection, the power cord should be checked to ensure a
type and specification . tight contact with the instrument socket; otherwise it should be replaced.

PREPARATION 15
4.6 Connection of the communication wire

One of the supplied communication wires and communication converter boxes
(USB, RS232C, Bluetooth) should be used. One end of the communication con-
version box is connected to the DB15 communication interface at the back of
the instrument, the other end is connected with the computer USB, RS232C or
Bluetooth interface. After connection, screws should be securely tightened.
The communication converter box is built with special circuits and must not be
opened.
16

SYSTEM INSTALLATION AND UNINSTALLING 17
5 SYSTEM INSTALLATION AND UNINSTALLING
5.1 System Environment

Operating system: Windows 2000/XPSP2/VISTA/7
Runtime environment: Net Framework 4.0
Other software: PDF Reader, EXCEL, TEXT
Minimum requirements:
Processor: Intel Core i3
Memory: 2 GB
Hard Disc: 10 GB
5.2 System Installation

The provided HCy4 software is specific for each instrument and should not be
used for other instruments.
To install the software, insert the provided CD into the disc drive of the
computer, click onto the HCy4 software file and follow the instructions to install
it:
Double click on the server file ( .exe) to start the installing process. Follow the
instructions on the screen.
a. Choose the location of the HumaServer.
Figure 4
18
b. Select the start menu folder.
Figure 5
c. Start the installation process of the HumaServer.
Figure 6

SYSTEM INSTALLATION AND UNINSTALLING 19
After that, install the HCy4 Software by clicking on the corresponding .exe file.
a. Select the destination folder.
Figure 7
b. Select the start menu folder.
Figure 8
20
c. Start the installation process of the HCy4 software.
Figure 9
Operating System
Double click on the HumaCycler shortcut (HCy4) on the desktop
The software will be opened and is ready to work with.
5.3 System Uninstallation

Click Control Panel Add/Delete Programe HCy4 Uninstall.

START 21
6 START
6.1 Inspection before Starting

Please, insert the power cord, turn the instrument on and prove the following
items:
-- Control, whether the voltage of the power supply is consistent with the sys-
tem requirements.
! Before the initial use of the
instrument, take out the Fix-
ing Pin correctly and insert and
-- Control the power cord for the correct and reliable insertion into the power tighten the pin into the unlocker
socket. port on the back; otherwise, the
-- Control the communication converter box for the correct tightening inser- instrument would be unable to
tion into the instrument and the cord for correct and reliable connection to perform fluorescence scanning.
the computer. (For removal of Fixing Pin, refer
-- Control the update shift switch "MODE" of communication converter is set to section 4.4 "Removal of Fixing
to normal mode. Pin").
-- Control the environmental conditions meet the required tolerances.
6.2 Starting the Instrument

In order to ensure an effective connection and communication between the
instrument and the computer, the instrument shall be started in the following
order:
1st step: Start the computer

2nd step: Turn the instrument on using the power switch on the back of
the instrument.
3rd step: Press the Run Switch button on the front cover of the instrument
to start the instrument
4th step: Start the software by clicking the start menu Program
HCy4 or double click the short cut icon HCy4 on the desktop.
22
6.3 Starting the Software

Run the program and the system window will be displayed.
Figure 10
After the software was started, the User Login interface appears. Enter your
login data and click OK.
If customers using the HCy4 software for the first time, please enter User name:
admin and Password: admin and press OK.
Figure 11
Click Service User Management Add User and enter your login data.

START 23
Figure 12
The following window will pop up:
Figure 13
To add a new user click Add and the following window will pop up:
Figure 14
24
Enter user name, password and confirm the password. To determine the differ-
ent permissions, click onto the corresponding fields and click OK.
Another option to log in is click Service User Login.
Figure 15
To change your password click Change Password.
Figure 16
Change your password and confirm the new one.
Figure 17

ROUTINE UTILIZATION 25
7 ROUTINE UTILIZATION
7.1 Absolute Quantification

Start

Design Experiment

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to design a new absolute quantification experiment

and covers the inspection of the item settings, sample information, reaction
plate and program settings.
7.1.1 CREATE A NEW ABSOLUTE QUANTIFICATION EXPERIMENT

a. Click Absolute on the HCy 4 software interface or
b. Click New Absolute on the toolbar or
c. Click File New Absolute on the menu bar
Figure 18
26
7.1.2 DETECTOR SETTINGS

1. Click Setup Detector
Figure 19
2. Enter the experiment properties

Figure 20
-- Experiment name: Is generated automatically but can be modified.

-- User name: User name can be added to refer the experiment to operator.
-- Comment: Add any comments.
3. Detector Settings
Set up the detector, assay, dye and colour.
For modifying the detector setup the following items can be changed:
a. Add/Delete detector: A further target can be added or deleted.
Enter the name of the target into the corresponding field.
Figure 21

b. Add/Delete Assay: A further assay can be added or deleted.
Users can also conduct Add, Modify and Delete operations in the item library.
Add the detector to the Detector Library: click Add Detector From Library the
Detector Library window will pop up select the detector in the window to be
added.
Figure 22
4. Set up a Reference Dye

If it is necessary to add a Reference Dye for the run, add the name of the dye in
the corresponding from the list.
Figure 23
28
7.1.3 SAMPLE INFORMATION SETTINGS

1. Click Setup Sample
Figure 24
2. Add Samples
Figure 25
a. Itemized addition: Input ID in Sample ID press Enter

or
b. Batch addition: click Batch Add the Batch Add window will pop up.
Figure 26
Insert the start Sample ID and add the numbers of the batch samples.

Insert the sample names and adapt the sampling time if necessary.
3. Delete Sample
a. Itemized deletion: select the sample to delete, click Delete the
selected sample is deleted.
b. Delete all: click Clear All all samples are deleted.
4. Import/Export Sample Information

a. Click Import Sample Info the File Import window will pop up import
sample information file in CSV format.
b. Click Export Sample Info the Save As window will pop up the sample
information will be exported in CSV file format.
7.1.4 PLATE SETTINGS

1. Plate Preparation for an Absolute Quantification
Figure 27
2. Click Setup Plate
a. Select a plate well

b. Select a sample by clicking on the empty field in the corresponding row
checkmark appears and the sample color appears in the well.
30
Figure 28
c. Mark all the detectors that should be detected.

d. Determine the sample properties (Unknown, Standard, Positive,
Non-Template Control).
e. Add the concentrations of the standards (e.g. 1.00e+07, 1.00e+06).
f. Select the concentration units.
Figure 29
Users can also do a right click into one well of the plate to Copy, Paste and Add
New Detector. Adding a new detector will open the Edit Detector Library win-
dow.

In addition, users can zoom-in, zoom-out and reset the plate.

To arrange samples automatically, click Sample Auto Arrange and determine
the order of the samples.
Figure 30
7.1.5 PROGRAM SETTINGS

1. Click Setup Program
Figure 31
2. Run Program Setup

Click Program the default temperature profile is opened.
32
Figure 32
a. To create a new cycling program, users can add new Hold Stage, Cycling
Stage or Melting Stage by clicking on the button Add Stage and selecting
the corresponding stage to add. Click Delete to delete selected stages.
b. To insert new steps, select the step before or after a new step should be
inserted. Click Add Step. Click Delete to delete selected steps.
Figure 33
c. To view the program in a table form, click Display With Table. A new win-
dow will pop up and the details of the current experiment will be dis-
played in a table.
d. To set up the temperatures and times of the hold stage, cycling and melt-
ing stages enter temperatures and times directly or use the arrows.

Figure 34
e. Enter the hot-lid temperature and liquid volume.

f. Enter the cycle numbers.
The setup can be saved as template file. Click File Save As Save as Template...
Figure 35
34
...or click Save As Template in the lower tool bar.
Figure 36
The file will be stored in the template folder.
7.2 Preparation of the PCR RUN
Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
Before running a PCR reaction, users should:

-- Ensure that appropriate materials are used.
-- Ensure that the arrangement of the PCR plate is consistent with the setting
layout of the plate described in section 7.1.4.

7.3 PCR RUN

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to run the PCR and how to operate the fluorescence
and temperature curves.
! Caution: Before starting, read the instructions to start the instrument carefully and follow
the procedure for a correct start of the system. Before running the program, push the
module smoothly until the locking sound is heard and the alarm lamp is switched off. If the
module is pulled out or is improperly closed, the software will produce a pop-up warning and
the alarm lamp will light up. In this case, the temperature program can be run but the fluore-
scence scanning data could become invalid.
7.3.1 PREPARATION OF THE REAGENT SAMPLE

For the preparation of a PCR run with the HumaCycler, 0.2 ml PCR tubes, 8-tubes
strips or 96-well PCR plates should be used to perform the PCR reaction. The
recommended reaction volume is 10l...50l for an optimal reaction.
-- The tubes, tube strip or 96 well PCR plate must have an optically clear bot-
tom.
-- Centrifugation step: Before placing the reaction tubes into the instrument,
spin down the reaction tubes briefly to ensure that the reagent is at the bot-
tom of the reaction tube and the reagent/sample mix is free of bubbles.
36
-- If individual tubes or strip tubes are used and the sample quantity is less
than the maximum capacity of the instrument, it is recommended to distri-
bute the sample tubes across the block as far as possible. This will create
even pressure across the hot-lid, ensuring consistent pressure on all the
tubes during the PCR run, which greatly improves temperature consistency
across all sample tubes.
Figure 37
Correct! The sample is on the bottom of the PCR tube.
Figure 38
Wrong! For the left tube use higher spin speed.

For the right tube use longer spin time.
7.3.2 RUN A FLUORESCENCE CURVE

1. Click Run Fluorescence Curve
Figure 39
2. Click Start Run

Figure 40

3. Operating Confirmation
a. Modify hot-lid temperature and liquid quantity (sample volume) if re-
quired.
b. Modify Gain (baseline) parameter settings if required.
c. Modify Target Fluorescence Value settings if required.
Figure 41
Users can select Reference Gain, if they analysed the gain settings for the corre-
sponding dyes (see HumaCycler Installation Kit REF 86150).
4. After starting the PCR run, Users can:

a. Skip the current stage
b. Add cycles
c. Delete cycles
d. Stop the run
5. Plot Display Settings

a. Assay item
b. Plot color
38
Figure 42
To get back to a running experiment if users review other files, tools etc. click
Service See Running Experiment.
Figure 43
7.3.3 RUN A TEMPERATURE CURVE

1. Click Run Temperature Curve
Figure 44

2. Click Start Run
Figure 45
The same setting can be modified as mentioned in 7.3.2.
The temperature curve shows the profile of the temperature during the cycling
process.
Figure 46

Users can only review the program settings but cannot make any modifications
or changes during the PCR run.
7.3.5 WORKING STATE INDICATION LAMPS ON THE INSTRUMENT

The panel at the upper right of the instrument is fixed with 3 indicator lamps.
These lamps are related to the system state during the run of the PCR program:
Running: The indicator lamp lights green, which denotes that the entire ma-
chine is operating.
Alarm: The indicator lamp lights yellow, which denotes that the module is
notfully pushed in and not locked into the right position for a cor-
rect operation.
40
! For prolonged shutdown,

switch off the flip switch at
the back of the instrument and at
Error: The indicator lamp lights red, which denotes that the instrument
has detected an error.
the power socket. When turned The front cover of the instrument is fixed with a self-locking Run Switch button
on again, the hot-lid and module to control if internal control system is energized:
will revert to the default settings .
-- Run Switch: running/standby switch.
-- After pressing this button, the green indicator lamp is lit on the instrument,
the internal system is energized and the instrument is ready to run the pro-
! The Run Switch button is for

the ease of operation and is
merely used for temporary or
gram.
-- If you press the button again, the green indicator lamp goes off, the internal
system of the instrument is de-energized and the system is in the standby
short term shutting down of the mode.
control system. When the sys-
tem is in the standby mode, the
instruments internal alternating 7.3.6 OCCURRENCES DURING THE RUN
current (AC) circuit remains tur- -- The hot-lid temperature sensor alarm is activated.
ned on . -- The temperature sensor alarm is activated.
-- The environmental temperature sensor alarm is activated.
-- The module temperature sensor alarm is activated.
-- The module sensor short-circuit or short-circuit alarm is activated.
! In case the temperature alarm

is activated during the run,
the HumaCycler will terminate
7.4 Data Analysis
Start
the current program. The instru-
ment should be switched off and Experiment Design
re-started .
PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End

This section describes how to analyze the results of the PCR run and how to
adjust the parameters for the re-analysis. In addition, it covers the analysis of
amplification and standard curves.
7.4.1 RESULT ANALYSIS
7.4.1.1 Amplification Plot Analysis

1. Click Analysis Amplification Plot
Figure 47
2. Analyze the Amplification Plots

a. To show the amplification plots of samples, click onto the corresponding
well of the plate. The color of an amplification curve is identical to the color
of the corresponding sample. In addition, the amplification plots of the
dyes can be displayed.
b. The plot type is shown in a linear format and can be switched to the loga-
rithmic format.
c. The target dyes can be shown separated from each other or at the same
time.
Figure 48
42
3. Analyze the Reaction Plate

a. The default setting to analyze a plate means, all wells are selected. To view
an amplification plot of a special well, select the well on the plate.
b. To analyze the well table, click Well Table. All Ct values and calculated con-
centrations are listed.
c. To analyze the summary of the results click Result Summary. The plate
with the Ct values and calculated concentrations in the wells is shown (Fig-
ure 50).
d Users can zoom-in, zoom-out and reset the reaction plate.
Figure 49
Figure 50

4. Set up Plot Settings

a. To analyze the amplification plots of a target dye, click Assay.
b. To set up the threshold, mark Threshold in the empty field.
c. To set up an automatic baseline, mark Auto Baseline in the empty field.
If the threshold value was not marked as automatic, users cannot set up an au-
tomatic baseline.
Figure 51
7.4.1.2 Standard Curve Analysis

1. Click Analysis Standard Curve
Figure 52
The standard curve of the assay, which is generated based upon the CT values
and concentration of the used standards, is shown. Several features of the
standard curve are shown, e.g. the correlation of the Ct values or the efficiency
of the PCR reaction.
Figure 53
44
2. Analyze the reaction plate

centrations are listed (Figure 50).
with the Ct values and calculated concentrations in the wells is shown.
d. User can zoom-in, zoom-out and reset the reaction plate.
Figure 54
Figure 55

To export a standard curve for the usage in other experiments click File Export
Standard Curve.
Figure 56
Select the folder for the standard curve data. Data can be imported into other
experiments by selecting Analysis Analysis Settings Standard Curve
Settings Use Standard Curve imported from another experiment Import.
Figure 57
7.4.1.3 Melting Curve Analysis

1. Click Analysis Melting Curve
2. Melting Curve Analysis
a. Analyze the fluorescence curve by selecting the target.
46
Figure 58
b. Analyze the Derivative Curve.
Figure 59

c. Set up the color by selecting well or target.
Figure 60

with the CT values and calculated concentrations in the wells is shown.
d. User can zoom-in, zoom-out and reset the reaction plate.
Figure 61
If the calculation of the melting temperatures is needed, click Tools Tm

Calculator.
48
Figure 62
Enter the sequence of the forward and reverse primers, the average tempera-
ture and annealing temperature and click Calculate.
Figure 63
7.4.2 ADJUSTING PARAMETERS AND RE-ANALYSIS

All data are calculated based upon the Default Negative Judgment Settings,
either by concentration or Ct value.

Figure 64
The lowest detectable Ct value is 45; the lowest calculated concentration is 1.
1. Click Analysis Settings the Analysis Settings dialog box will pop up.
Figure 65
Users can adapt many features for reanalyzing the data:

50
a. Adjust the start cycle and end cycle of the baseline.

b. Adjust Ct analysis algorithm.
c. Set up the use of S fitting.
d. Set up the stage to use for Ct analysis.
e. Set up the automatic threshold value.
f. Adjust Advanced setting.
g. Adjust the Standard curve settings.
To import another standard curve from a recent run, change the standard curve
settings and import the standard curve file form the recent experiment.
Figure 66
After finalizing the adjustments, click Apply Analyse Settings.

7.5 Data Report

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to design the data report and covers the design of the
report template and print settings.
7.5.1 DESIGN THE REPORT TEMPLATE

1. Click Report Report Template Editor the Report Designer window will
pop up.
Figure 67
52
Reports can be generated for Absolute Quantification and Single-Nucleo-

tide-Polymorphism (SNP) experiments. The data report consists of controls that
can be added, modified and deleted by users. Available controls include Static
Text, Dynamic Text, Line, Static Image, Amplification Curve and Quantification
Analysis Results.
Figure 68
To insert the required information into the report, click Used Controls, mark the
field to insert the information and go to the appearance interface and add the
information on the right side in the corresponding field.
Figure 69

The inserted information appears in the report. To change the appearance, de-
sign and layout, click on the feature that you want to change and a scroll bar
appears on the right side. Choose your design by clicking on it. In addition, users
can delete selected controls; either click Delete Selected Controls on the toolbar
or click right on the corresponding field.
To preview the adapted report click Preview on the toolbar.
Figure 70
7.5.2 PRINT SETTINGS

1. Click Report Print Template Setting the Print Template Setting window
will open.
Figure 71
Users can set up the report name, operator, investigator, the amplification plot
color, default report template and paper size.
Figure 72
54
7.5.3 CONSOLIDATED REPORT

1. Click Report Consolidated Reports the Consolidated Report window will
pop up.
The Consolidated Report includes the basic, sample and plate information, am-
plification and standard curve, plate information, etc.
Figure 73
To adapt the consolidated report to the users needs, change the features on the
right side by clicking on the feature to insert or delete and checkmarks appear.

Figure 74
To change the appearance of the report, use the options on the toolbar. Users
can zoom in or zoom out of the report, change the page with or more than 1
page could be shown.
Figure 75
To print the report click Report Report Print.
Select the items to print.
7.5.4 QUALITY CONTROL SUMMARY

1. Click Report QC Summary
Figure 76
56
2. Analyze the Quality Control Summary.
Figure 77
The summary shows the amplification plots and if the minimum requirements
are fulfilled by using checkmarks.
7.6 Data Export

Start

Design Experiment

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to save and export data and covers exporting to a da-
tabase, experiment filing and exporting the experiment data to EXCEL and TXT.

7.6.1 EXPORT TO DATABASE

Click Data Summary Export to Database the Save File dialog box will pop
up save the exported database file of the HumaCycler.
Figure 78
7.6.2 EXPERIMENT FILING

1. Click Data Summary Experimental Archive Storage Folder the Experi-
mental archive storage directory window will pop up set up the storage
path of file.
Figure 79
Figure 80
To change the location of the storage folder Click Change and select the folder to
archive your data.
2. Experiment Filing
Click Data Summary Archived Experiment the experiment will be exported

to the archive folder. The suffix of the archived experiment file is .fqh
58
7.6.3 SAVE DATA SETTINGS

To save the analyzed data, click File Save As.
Figure 81
A folder to store the experiment files will open. Save the file by entering the
name and click Save.
Another option for saving data is click Save As in the lower tool bar.
Figure 82

7.6.4 EXPORT EXPERIMENT DATA TO TEXT AND EXCEL

Click Data Summary Export Experiment Export Experiment to Text the
exported experiment data will generate TEXT file.
Figure 83
Click Data Summary Export Experiment Export Experiment to EXCLE the

exported experiment data will generate EXCEL file.
Figure 84
7.6.5 EXPORT RAW DATA

To export raw data of the experiment click File Export Raw Data.
Figure 85
Enter the name of the file and select the folder to export the raw data. To open
the file again, start the software and click onto the file name.
60
7.7 Data Review

To review recent runs and data files click File Open and select the correspond-
ing experiment file.
Figure 86
To view file information click File File Information.
Figure 87
The experiment type, serial number of the instrument as well the use of cross-
talk are listed and if proportion parameters were measured.

RELATIVE QUANTIFICATION 61
8 RELATIVE QUANTIFICATION
8.1 EXPERIMENT DESIGN

Start

Design Experiment

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to design a relative quantitative experiment and

covers the creation of a new relative quantitative experiment, the inspection
of the item settings, the sample information, the reaction plate and program
settings.
8.1.1 CREATE A NEW RELATIVE QUANTITATIVE EXPERIMENT

a. Click Relative on the HCy 4 software interface or
b. Click New Relative on the toolbar
c. Click File New Relative on the menu bar.
Figure 88
62

1. Click Setup Detector
Figure 89
Figure 90
2. Enter the experiment properties

-- Experiment name: Is generated automatically but can be modified.
-- User name: User name can be added to refer the experiment to operator.
-- Comment: Add any comments.
3. Detector Settings
Figure 91
4. Inspection Item Setting

a. Set up the detectors, assays, dyes and colors.
b. Add detectors.
c. Delete detectors.
d. Add detectors from library.
Users can select, if the target detects the endogenous control. Add information
about Primers/Probes, Supplies and Batch Number if required.

Figure 92
5. Set up Reference Dye
Figure 93

Figure 94
2. Add sample information

a. Itemized addition: input ID in Sample ID press Enter
or
b. Batch addition: click Batch Add the Batch Add window will pop up.
64
Figure 95
Insert the start Sample ID and add the numbers of the batch samples.
3. Delete sample
a. Itemized deletion: select the sample to delete click Delete the select-
ed sample is deleted.
4. Import/Export sample information

Figure 96
5. Set up Sample information
Figure 97
Enter the sample names and modify the measuring time and submitting date, if
necessary.

8.1.4 REACTION PLATE SETTINGS

Figure 98
2. Select a plate well

Select a sample by clicking into the empty field in the corresponding row (check-
mark appears). The sample color appears in the well.
Determine the property of the sample and select the reference sample.
Figure 99
a. Users can also do a right click into one well of the plate to Copy, Paste and
Add New Detector. Adding a new detector will open the Edit Detector Li-
brary window.
66
To arrange samples automatically, click Sample Auto Arrange determine the

order of the samples.
To select the well sample, target or specific properties, click Select Wells select
a feature.
All samples and their properties are listed in the Well Table.
Figure 100

Figure 101

b. To insert new steps, select the step before or after a new step should be
inserted. Click Add Step. Click Delete to delete selected steps.
Figure 102
dow will pop up and the details of the current experiment will be dis-
played in a table.
d. To set up temperatures and times of hold stages, cycling and melting stag-
es enter temperatures and times directly or use the arrows.
Figure 103

The setup can be saved as template file. Click Save As Save as Template.
68
8.2 Preparation of PCR RUN

Start

Design Experiment

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End

layout of the plate as described in section 7.1.4.
8.3 PCR RUN

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End

This section describes how to run the relative quantitative PCR after loading the
reaction tubes and how to operate the fluorescence and temperature curves.

Figure 104
The fluorescence curves of selected or all samples are shown during the ampli-
fication.
2. Click Start Run
Figure 105
quired.
70
Figure 106
Users can select Reference Gain, if they analysed the gain setting for the corre-
4. After the PCR run started, users can:

a. Skip the Current Stage
b. Add cycles
c. Delete cycles
d. Stop the run
a. Assay item
b. Plot color

Figure 107

Figure 108
2. Click Run Start
Figure 109
72
quired.
Figure 110
4. After starting the PCR run, users can:

b. Add cycles
c. Delete cycles
d. Stop the run
Figure 111


Users can only review the program setting but cannot make modifications.
8.4 DATA ANALYSIS

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to analyze the results of the PCR run and how to
adjust the parameters for the re-analysis. In addition, it covers the analysis of
amplification curves and the analysis of the relative quantification.

Figure 112
74
2. Analysis of the Amplification Plot

of the corresponding sample. In addition, the amplification plots of the
dyes can be displayed.
rithmic format.
time.
Figure 113

a. The default setting to analyze a plate means that all wells are selected. To
view an amplification plot of a special well, select the well on the plate
(Figure 110).
b. To analyze the well table, click Well Table. All CT values are listed.
with the CT values in the wells is shown.
d. Users can zoom-in, zoom-out and reset the reaction plate.

Figure 114
Figure 115
4. Set up the Plot Settings

a. To analyze the amplification plots of a target dye, click Assay.
tomatic baseline.
Figure 116
76
8.4.2 RELATIVE QUANTIFICATION DATA ANALYSIS

1. Click Analysis Relative Quantification
Figure 117
1. Analyze the relative quantification results

a. To set up the figure type, select which results should be shown on the
X-axis and if the results should be shown as linear or logarithmic plot.
Figure 118
b. Analyze the results

On the right side of the interface, all results, e.g. Ct Mean or Ct, are
shown.

Figure 119
8.4.3 ADJUST PARAMETERS AND RE-ANALYSIS

Figure 120
Users can adapt many features to re-analyse the data:

a. Adjust the start cycle and end cycle of the baseline.
b. Adjust Ct analysis algorithm.
c. Set up the use of S fitting.
d. Set up the stage to use for Ct analysis.
e. Set up the automatic threshold value.
78
f. Adjust Advanced setting.

g. Set up the endogenous control.
Figure 121
After finalizing the adjustments, click Apply Analyse Settings.
8.5 DATA REPORT

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to design the data report and covers the design of the
report template and the print settings.


pop up.
Figure 122
The Consolidated Report includes the basic, sample and plate information, am-
plification curves, endogenous controls etc. To adapt the consolidated report to
the users needs, change the features on the right side by clicking on the feature
to insert or delete and checkmarks appear.
To print the results click Print Report.

80

Figure 123
2. Analyze the QC Summary
Figure 124
are fulfilled by using checkmark.
8.6 DATA EXPORT

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End

This section describes how to export data and covers the export to a database,
the experiment filing and exporting the experiment results to EXCEL and TXT.

Click Data Summary Export to Database the Save File dialog box will pop
up save the exported database file of the HumaCycler.
Figure 125

Click Data Summary Experimental Archive Storage Folder the experimen-
tal archive storage directory window will pop up set up the storage path of
file.
Figure 126
To change the location of the storage folder, click Change and determine, where
to archive your data.
3. Experiment filing
Click Data Summary Archived Experiment the experiment will be exported
to the archive folder. The suffix of the archived experiment file is .fqh .
82
Figure 127

Figure 128
A folder to store the experiment files will open. Save the file by entering
the name and click Save.
Figure 129


Figure 130
Click Data Summary Export Experiment Export Experiment to EXCEL the

Figure 131

To export the raw data of the experiment click File Export Raw Data.
Figure 132
the file again, start the software.
84
8.7 DATA REVIEW

To review recent runs and data files click File Open and select the correspond-
Figure 133
Figure 134
talk and proportion parameters are listed and if the proportion parameters have
been measured.

SINGLE-NUCLEOTIDE-POLYMORPHISM (SNP) DETECTION 85
9 SINGLE-NUCLEOTIDE-POLYMORPHISM (SNP) DETECTION
Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to design a new SNP experiment and covers the in-
spection of the item settings, sample information, reaction plate and program
settings.
9.1 CREATE A SNP EXPERIMENT

a. Click SNP on the HCy 4 software interface or
b. Click New SNP on the toolbar or
c. Click File New SNP on the menu bar
Figure 135
86

Click Setup Detector
Figure 136
Figure 137
1. Enter the experiment properties:

-- Experiment name: is generated automatically but can be modified
-- User name: user name can be added to refer the experiment to operator
-- Comment: add any comments
2. Inspection of the Item Settings

-- Set up the Detectors, Alleles, Dyes and Colors.
Figure 138
If necessary, users can also:

a. Add Detectors
b. Delete Detectors
c. Add the Detector in the Detector library: click Add Detector From Library
the Detector Library window will pop up select the Detector on the win-
dow to be added.

3. Set up a Reference Dye

If it is necessary to add a reference dye to the run, add the name of the dye in the
corresponding from the list.
Figure 139

Figure 140
2. Add sample information

a. Itemized addition: input the ID in Sample ID press Enter
or
b. Batch addition: click Batch Add the Batch Add window will pop up. Insert
the start ID and the sample numbers of the batch and click Add.
Figure 141
88
3. Delete Sample
a. Itemized deletion: select the sample to delete Delete the selected
sample is deleted.
4. Import/Export Sample Information

Figure 142
5. Set up Sample Information
Figure 143
9.1.3 PLATE SETTINGS

Figure 144

2. Select a plate well

Select a sample by clicking into the empty field in the corresponding row (a
checkmark appears). The sample color appears in the well.
3. Determine the sample properties

Mark all the detectors that should be detected.
Figure 145
Figure 146
Users can also do a right click into one well of the plate to Copy, Paste and Add
New Detector. Adding a new detector will open the Edit Detector Library win-
dow.
90
To arrange samples automatically, click Sample Auto Arrange and determine

the order of the samples.
All samples and their properties are listed in the Well Table.
Figure 147

Figure 148

2. Run the Program Setup
Click Program a default temperature profile is opened.
Figure 149
b. To insert new steps, select the step before or after an existing step. Click
Add Step. Click Delete to delete selected steps.
dow will pop up and the details of the current experiment will be displayed
in a table.
d. To set up the temperatures and times of the hold stage, cycling and melt-
ing stages enter temperatures and times directly or use the arrows.
Figure 150

The setup can be saved as template file. Click Save As Save as Template.
92
9.2 PREPARATION OF PCR RUN
Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End

layout of the plate described in section 7.1.4.
9.3 PCR RUN

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End

This section describes how to run the PCR and how to operate the fluorescence
and temperature curves.
! Caution: Before starting, read the instructions to start the instrument carefully and follow
the procedure for a correct start of the instrument. Before running the program, push the
module smoothly until you hear the locking sound and the alarm lamp is switched off. If the
module is pulled out or is improperly closed, the software will produce a pop-up warning and
the alarm lamp will light up. In this case, the temperature program can be run, but the fluo-
rescence scanning data would become invalid.

Figure 151
2. Click Start Run
Figure 152
3. Operating confirmation
quired.
c. Modify Target Fluorescence value settings if required.
94
Figure 153
4. After starting the PCR run, users can:

b. Add cycles
c. Delete cycles
d. Stop the run

a. Assay item
b. Plot color

Figure 154

2. Click Start Run
Figure 155
Figure 156
The temperature curve shows the profile of the temperature during the cycling
process.

Users can only review the program settings but cannot make any modifications.
96
9.4 DATA ANALYSIS

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to analyze the results of the PCR run and how to ad-
just the parameters for the re-analysis.

Figure 157

2. Analysis of the Amplification Plot

of the corresponding sample. In addition, the amplification plots of the dyes
can be displayed.
rithmic format.
time.
Figure 158
3. Analysis of the reaction plate
b. To analyze the well table, click Well Table. All CT values and calculated con-
c. To analyze the summary of the results click Result Summary. The plate with
the CT values of the samples are shown (Figure 155).
d. Users can zoom-in, zoom-out and reset the reaction plate.
98
Figure 159
Figure 160

4. Set up Plot Settings

a. To analyze the amplification plots of a target, dye click Assay:
tomatic baseline.
Figure 161
9.4.1.2 SNP Result Analysis

1. Click Analysis SNP
Figure 162
2. SNP Analysis
a. Select signals
Users can select the signals of the corresponding wells by dragging a rectan-
gle with the mouse around the wells of interest or select wells one by one.
b. Set up targets if there are more than 1.
c. Define properties of the signals (manual calls). The signals and their proper-
ties can be distinguished due to different colors they have.
100
Figure 163
3. Analysis of the reaction plate

a. The default setting to analyze a plate means, all wells are selected.
b. To analyze the well table, click Well Table. All allele dyes and calls are listed.
c. Users can zoom-in, zoom-out and reset the reaction plate.
9.4.2 ADJUST THE PARAMETER FOR RE-ANALYSIS

a. Adjust the analysis data.
b. Adjust, whether the inspection item will retain manual recognition geno-
type.
Figure 164

9.5 DATA REPORT

Start

Experiment Design

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End
This section describes how to print an experiment report and covers the design
of a report template and the print settings.
9.5.1 DESIGN THE REPORT TEMPLATE

1. Click Report Report Template Editor the Report Designer window will
pop up.
Figure 165
The report consists of controls and users can add, modify and delete controls.
Available controls include Static Text, Dynamic Text, Line, Static Image and the
SNP Typing Curve.
102
.
Figure 166
To insert the required information into the report, click Used Controls mark
the field to insert the information go to the appearance interface and add the
information on the right side in the corresponding field.
Figure 167
The inserted information appears in the report. To change the appearance, de-
sign and layout, click on the feature to change, a scroll bar appears on the right
side. Select the design by clicking on it. In addition, to delete selected controls
either click Delete Selected Controls on the toolbar or click right on the corre-
sponding field.
To preview the adapted report click Preview on the toolbar.
Figure 168

9.5.2 PRINT SETTINGS

1. Click Report Print Template Setting the Print Template Setting window
will open.
Figure 169
Users can set up the report name, operator, investigator, the amplification plot
color, the default report template and paper size.
Figure 170

pop up.
The Consolidated Report includes the basic information, sample information,

amplification curve, SNP, plate information, etc.
104
.
Figure 171
9.5.4 REPORT PRINTING

1. Click Report Report Print
Figure 172

2. Report the print settings.

a. Set up report template.
b. Print setting.
c. Select print items.
d. Print preview.
e. Print the report.
Figure 173

Figure 174
106
2. Analyze the Quality Control Summary
Figure 175
are fulfilled by using checkmarks.
9.6 DATA EXPORT

Start

Design Experiment

PCR Preparation

PCR Run

Data Analysis

Data Report

Data Export

End

This section describes how to export data and covers exporting to a database,
experiment filing and exporting the experiment data to EXCEL and TXT.

Click Data Summary Export to Database the Save File dialog box will
pop up save the exported database file of the HumaCycler.
Figure 176

Click Data Summary Experimental Archive Storage Folder the Experimen-
tal archive storage directory window will pop up set up the storage path of
file.
Figure 177
Figure 178
To change the location of the storage folder, click Change and determine, where
to archive your data.
108
3. Experiment filing
Click Data Summary Archived Experiment the experiment will exported to
the archive folder. The suffix of the archived experiment file is .fqh

Figure 179
A folder to store the experiment files will open. Save the file by entering the
name and click Save.
Figure 180


Figure 181
Click Data Summary Export Experiment Export Experiment to EXCEL the

Figure 182

To export the raw data of the experiment click File Export Raw Data.
110
Figure 183
the file again, start the software and click onto the file name.
9.7 DATA REVIEW

To review recent runs and data files, click File Open and select the correspond-
Figure 184

Figure 185
talk and proportion parameters are listed and if the proportion parameters were
measured.
112

GENE STUDY 113
10 GENE STUDY
Start

Create Gene Study

Gene Study Analysis

End
This section describes how to create gene studies and covers how to change
settings. Gene studies can be performed with relative quantification assay to
detect and measure comparable gene expression.
10.1 Create Gene Study

Click File New Gene Study on the menu bar.
Figure 186
114
10.2 Gene Study Setup

1. Click Gene Study Study Setup
Figure 187
The following window will be opened.
Figure 188
Please, enter study and user name or comments in the study properties column
if required. To add a new relative experiments click Add Experiments and select
the relative quantification experiments that should be added. If files should be
removed, click Remove Selected Experiments.
10.3 Gene Study Analysis

Start

Create Gene Study

Gene Study Analysis

End
This section describes how to view Gene Study analyses results after entering
the data and adjusting parameters for re-analysis.

GENE STUDY 115
10.3.1 VIEW ANALYSIS RESULTS

Click Gene Study Study Analysis
Figure 189
The relative quantification study results will be opened.
Figure 190
10.3.2 ANALYSIS SETTINGS
Click Gene Study Study Analysis Analysis Settings.

116
Change the settings of Gene Study analysis: analysis (e.g method, reference
sample, endogenous control, RQ min/max calculations) as required.
Figure 191

TOOL USAGES 117
11 TOOL USAGES
11.1 GAIN SETTINGS

The Gain Setting tool is used to set up gain modes. Determine the Gain Settings
before starting the PCR run.
Click Tools Gain Setting the following window will pop up.
Figure 192
Gain settings can be set up as Reference Gain, Custom Gain and Auto Gain.
In Custom Gain mode, users can modify the gain value.
11.2 BLOCK SCAN METHOD

Click Tools Block Scan Method the following window will pop up.
Users can select either the Whole Block Scan or a Line Scan depending on the
experiment set up. The lines to scan should be marked by clicking on the empty
field (checkmarks appear).
Figure 193
118
11.3 DETECTOR LIBARY

The Detector Library tool is used to set up the inspection libraries for absolute
and relative quantification and SNP analyses.
Click Tools Detector Library (Absolute /Relative/SNP) the following win-
dow will be opened.
Figure 194
Users can:
a. Add Detectors
b. Modify Detectors
c. Delete Detectors
11.4 CUSTOMIZED DYES

The Customized Dyes tool is used to set up existing dyes and newly added dyes.
Click Tools Customize Dyes the following window will be opened.
Figure 195

TOOL USAGES 119
Users can:
a. Create new dyes.
b. Modify dye names and channels.
c. Delete dyes.
d. Move dyes upward.
e. Move dyes downward.
After adding new dyes or modifying dyes the operator should conduct the cross-
talk parameter measurements.
11.5 CUSTOMIZED COLUMNS
Click Tools Customize Columns the following window will pop up.
Users can:
a. Add columns
b. Delete columns
c. Modify column name
Figure 196
11.6 COLUMN SELECTION

The Select Columns tool is used to add the new columns into current existing
columns or remove existing columns in current column.
Click Tools Select Columns the following window will pop up.
120
Figure 197
1. Current existing column items include sample, report, report setting, query
and query condition.
2. Double click onto the column can add or remove a column.
3. Column with (*) indicates it cannot be removed.
11.7 SAMPLE COLUMN LIBRARY

The Sample Column Library tool is used in the experiment design phase. The
operator can select the definition of contents in the drop-down box, when set-
ting up sample information.
Click Tools Sample Column Library the following window will pop up.

TOOL USAGES 121
Figure 198
Users can:
a. Add columns
b. Delete columns
c. Edit the columns content
11.8 INSTRUMENT CALIBRATION PARAMETERS

The Instrument Calibration Parameters tool is used to calibrate the instrument
parameters.
Click Tools Instrument Calibration Parameters the following window will

pop up.
122
Figure 199
11.9 CROSSTALK
11.9.1 MEASURE CROSSTALK CALIBRATION PARAMETERS

The Measure Crosstalk Calibration Parameters tool is used to measure the cross-
talk correction parameters.
Click Tools Measure Crosstalk Calibration Parameters the following

window will pop up.
Figure 200
Users can add and modify the channels to be tested and dyes according to his
needs, upload corresponding reaction plates and operate the experiment. When
the experiment is completed, the system will automatically save the crosstalk.

TOOL USAGES 123
11.9.2 MEASURE CROSSTALK GAIN

The Crosstalk Gain Parameters Measurement tool is used to measure the cross-
talk between gain parameters.
Click Tools Measure Crosstalk Gain Parameters the following window will
pop up.
Figure 201
Users can add and modify the channels to be tested and dyes according to his
needs, upload corresponding reaction plates and operate the experiment. When
the experiment is completed, the system will automatically save the crosstalk.
Please review the Instructions for Use of the assay to set up the corresponding
program.
124

SERVICE USAGE 125
12 SERVICE USAGE
To manage users and their permissions click User Management (see chapter 6.3).
Figure 202
The following window will pop up.
Figure 203
Users can be added, deleted and their permissions can be determined. Click
Add and enter User Name and password and determine the users permission.
Figure 204
126
To change password click Change Password.
To manage and search for experiments click Experiment Management Exper-

iment Management.
Figure 205
Enter the query conditions, like experiment name, operator, start and end time
to define the experiment features.
Click Query, the corresponding experiments files are listed. Click Download to
download the file.
.
Figure 206
If templates are stored in the data base, click Template Management, select the
template and click Download.
Figure 207

OTHER FUNCTIONS 127
13 OTHER FUNCTIONS
13.1 INSTRUMENT OPERATION

The instruments operations include Connect, Disconnect and Instrument Infor-
mation.
Figure 208
13.1.1 CONNECT
Click Instrument Connect select Automatically Connect or Search Instru-
ments.
Figure 209
13.1.2 DISCONNECT
Click Instrument Disconnect disconnect the currently connected instru-
ment.
13.2 INSTRUMENT INFORMATION

When the instrument is connected, users can review the instrument informa-
tion.
Click Instrument Instrument Information the following dialog box will pop
up.
128
Figure 210
Instrument information includes instrument serial number, run time, currently

connected ports and whether an experiment is in operation.
13.3 INSTRUMENT ALARM

To adapt the duration of the alarm signal, click Instrument Alarm Options.
Figure 211
Adapt the signal duration by changing the seconds.

OTHER FUNCTIONS 129
Figure 212
13.4 SYSTEM MAINTENANCE

The maintenance of the HumaCycler should performed only by local Human au-
thorized Technical Support or well trained users depending of the frequency of
use.
13.5 DATA QUERY

Data Query is used to query the data already exported to the database.
Click Data Summary Data Query the following window will pop up
Figure 213
Users can:
a. Select database files
b. Set up query condition
c. Query
d. Clear all query conditions
130
13.6 SYSTEM HELP

Click Help Help Topics
Click about Help About the following window will pop up.
Figure 214

MAINTENANCE 131
14 MAINTENANCE
14.1 REGULAR CLEANING

In order to ensure the normal operation, detection and use, the instrument
needs to be cleaned regularly. To clean the outer surface: Clean only with a soft
cloth and if necessary, the cloth may be soaked with alcohol, distilled water or
a mild detergent. To clean the module wells: Wells may be cleaned with nail
wipes, which are dust-free and if necessary, they may be soaked with 95% abso-
lute ethyl alcohol used in medicine or distilled water.
Warning!
1. Before cleaning the instrument, the power supply must be switched off.
2. When cleaning the conical wells of the module, prevent any cleaning agents
dropping into the wells.
!
3. The surface of the instrument MUST NOT be cleaned with corrosive cleaning
agents.
4. In order to avoid scratches or damage to the optics in the wells, NEVER use
sharp or hard objects to clean the wells.
The maintenance of the HumaCycler should performed only by local Human

authorized Technical Support depending of the frequency of use.
132

ANALYSIS AND TROUBLESHOOTING 133
15 ANALYSIS AND TROUBLESHOOTING
Problem Possible Cause Correction Table 1

The power switch at the rear The Run Switch at the front of For working with the instru-
of the instrument is set ON, the instrument is not turned ment, the Run Switch must be
but the instrument does not on. The internal switching pressed and the green indicator
response. power has no voltage output. lamp should be lit green to in-
dicate energizing of the control
system.
The module temperature runs The Fixing Pin is not removed Remove Fixing Pin and tight-
normally, but the fluorescence or is not inserted and en into the unlocker port. (For
detection does not scan. tightened into the rear transport, the instrument will
unlocker port. normally be shutdown and the
Fixing Pin should be removed
and then screwed into the side
locking port).
The display of system The system parameters are The function is not required for
parameters menu requires for instrument manufacturers end users and calibration. Con-
the input of Password. internal calibration and tact the manufacturer or sup-
require special accession pliers service personnel.
password.
After turning the power The Run Switch is not pressed. This switch is for temporary
switch on, the power lamp turning the ON/OFF of the
fails to light up. output power and is equiva-
lent to the standby mode.
For prolonged shutdown, the
flip switch should be switched
OFF.
Power is not switched on. Turn the flip switch on.
Blown fuses. Replace the fuses.
(250V 8A, 5x20).
Damaged switch flip Contact the local Human au-
thorized Technical Support.
At detecting sample position, Poor contact or damage Check, connect or replace the
the step motor fails to work of the interface wire. interface wire.
and the communication fails.
The flip switch is not Turn the flip switch on and
turned on or is turned on restart the program.
only after the program
starts running.
The step motor or the drive Contact the local Human au-
is damaged. thorized Technical Support.
134
Problem Possible Cause Correction

The Fixing Pin is not Tighten it into the unlocker port
fully inserted. and switch the power button on
after shutdown.
After detecting the The module temperature Contact the local Human
sample position, the sensor is damaged. It authorized Technical Support.
actual temperature accompanies the panel red
displays 0C or 100C. lamp alarm and a software
occurrence. The instrument
automatically stops running.
The flip switch is turned on Switch the power on and restart
only after the program starts the program.
running.
The program is searching the Contact the local Human
communication port. During authorized Technical Support.
the process, data will not be
sent.
Module temperature The ventilation opening is Clear the ventilation opening.
heating or cooling rate blocked.
obviously decreases or
temperature control is
incorrect.
Loose connection wire. Contact the local Human au-
thorized Technical Support
The refrigerating sheet Contact the local Human au-
is damaged. thorized Technical Support.
Fan is damaged or fails to run. Contact the local Human
authorized Technical Support.
The temperature sensor is Contact the local Human
damaged. authorized Technical Support.
The module fails to heat The refrigeration sheet is Contact the local Human
and refrigerate damaged. authorized Technical Support.
during the hot-lid Waiting until the hot-lid tem-

heating up. perature comes to the target
value. When it stops running,
the module temperature holds
down to 30C automatically.
Abnormal temperature or The running program is After deleting the virus, re-
fluorescence curve; straight infected by a virus. install the HCy4 software.
line or loss of partial data.

ANALYSIS AND TROUBLESHOOTING 135
Problem Possible Cause Correction

Computer configuration fails Configure the required
to meet the requirements or features.
the set up of the communica-
tion port is not appropriate.
Yellow lamp on the panel The module is not fully pushed Push the module in again. If
lights on. in and the optical coupler fails the light is still on, contact a
to detect the module. Human authorized local Tech-
nical Support.
The hot-lid does not heat-up. The thermal-sensitive fuse is Contact the local Human au-
damaged. thorized Technical Support.
Loose plug pieces. Contact the local Human au-
thorized Technical Support.
The heating element of the Contact the Human authorized
hot-lid Is damaged. Technical Support
The temperature sensor of Contact the Human authorized
the hot-lid is damaged. Technical Support.
Under no test tube state, the The test tube well or hot-lid is Eliminate the contamination.
fluorescence value difference contaminated or the baseline
between the wells increases background parameters are
or the background fluore- set incorrectly.
scence is very high.
After perennial use, offset
would occur in the optical ele-
ments. In this case, contact the
manufacturer to re-calibrate
the background value.
Reagent evaporation. The PCR tube cap is not Change the consumables to an-
sealed tightly enough. other with a tighter fitting cap.
Single Crosstalk among the The dye signal crosstalk Using Crosstalk Measure-
channels. among channels can ments and save parameters to
happen. modify.
Fluorescence detection value Irradiation from an external Switch off the external light or
is abnormal. light source. remove the instrument from
the light source.
During a program run, the hot- Close the hot-lid (detection
lid is open. result unreliable).
The photo-electric system is Contact the local Human au-
damaged. thorized Technical Support.
! During the warranty period, opening the instrument casing to inspect the internal workings will
invalidate the warranty. If any problems should arise, please contact a Human authorized local
Technical Support in the first instance.
136

APPENDIX 137
16 APPENDIX
16.1 Packing List
No. Title Model and Specification Unit Amount Table 2

1 Instrument HumaCycler Set 1
2 Power Cord 250V 10A Piece 1
3 Power Cord 125V 12A Piece 1
4 RS232C Converter Box RS232C-RS232C Piece 1
5 USB Converter Box USB-RS232C Piece 1
6 Bluetooth Converter Box Bluetooth-RS232C Piece 1
7 Rubber typhoon Piece 1
8 PVC socket Piece 1
9 Fuse 250V 8A Piece 2
10 Anti-Condensate Water Piece 1
Seal Ring
11 HCy4 Software For HumaCycler Piece 1
12 User Manual Copy 1
13 Frequently Asked Questions Copy 1
14 Quick User Guide Copy 1
15 Instructions for Anti- Copy 1
Condensate Water Seal Ring
138
16.2 HumaCycler wiring
Figure 216

APPENDIX 139
Figure 215
140
Figure 217

APPENDIX 141
142

HUMAN
Gesellschaft fr Biochemica und Diagnostica mbH
Max-Planck-Ring 21 65205 Wiesbaden Germany
Tel.: +49 6122/9988 0 Fax: +49 6122/9988 100
eMail: human@human.de www.human.de

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Copyright 2015, Human Gesellschaft fr Biochemica und Diagnostica mbH, Wiesbaden,

SERVICE UND SUPPORT

5 SYSTEM INSTALLATION AND UNINSTALLING 17

8.4.1 Result Analysis 73

9 SINGLE-NUCLEOTIDE-POLYMORPHISM (SNP) DETECTION 85

11 TOOL USAGES 117

12 SERVICE USAGE 125

13 OTHER FUNCTIONS 127

15 ANALYSIS AND TROUBLESHOOTING 133

1.2 User Warranty

1.3 Intended Use of the Instrument

1.4 General Safety Warnings

1.5 Disposal Management Concept

HumaCycler | User Manual

1.6 Biohazard Warning

1.7 Instrument Disinfection

HumaCycler | User Manual

2.1 Intended Use

2.3 Features of the HCy4 Software

HumaCycler | User Manual

Heating/cooling rate 4C/s (max.)

HumaCycler | User Manual

4.1 Transport and Storage Conditions

4.2 Normal Working Conditions

4.4 Removal of the Fixing Pin

1 Fixing Pin on the right side

4.5 Power cord connection

! If the supplied power cord

HumaCycler | User Manual

4.6 Connection of the communication wire

HumaCycler | User Manual

5 SYSTEM INSTALLATION AND UNINSTALLING

5.1 System Environment

5.2 System Installation

a. Choose the location of the HumaServer.

b. Select the start menu folder.

c. Start the installation process of the HumaServer.

HumaCycler | User Manual

b. Select the start menu folder.

c. Start the installation process of the HCy4 software.

5.3 System Uninstallation

HumaCycler | User Manual

6.1 Inspection before Starting

6.2 Starting the Instrument

1st step: Start the computer

6.3 Starting the Software

HumaCycler | User Manual

The following window will pop up:

Another option to log in is click Service User Login.

To change your password click Change Password.

Change your password and confirm the new one.

HumaCycler | User Manual

7.1 Absolute Quantification

This section describes how to design a new absolute quantification experiment

7.1.1 CREATE A NEW ABSOLUTE QUANTIFICATION EXPERIMENT

7.1.2 DETECTOR SETTINGS

2. Enter the experiment properties

-- Experiment name: Is generated automatically but can be modified.

Enter the name of the target into the corresponding field.

HumaCycler | User Manual

b. Add/Delete Assay: A further assay can be added or deleted.

4. Set up a Reference Dye