REF86100 FAQs HumaCycler PDF

HumaCycler
| Frequently Asked Questions
Cat No. 86100/7

REVISION LIST OF THE MANUAL
Rev. /DATE. REVISION DESCRIPTION
01/2015-05 First edition
SYSTEM VERSION
COPYRIGHT
Copyright 2015, Human Gesellschaft für 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
1.1 INTRODUCTION 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 BASIC FREQUENTLY ASKED QUESTIONS (FAQS) 9

2.1 WHAT IS THE INSTRUMENT’S APPLICATION? 9
2.2 WHAT ARE THE SPECIFICATIONS OF THE INSTRUMENT? 9
2.3 WHAT ARE THE SPECIFICATIONS OF THE FLUORESCENT DYES? 10
2.4 WHICH MINIMUM COMPUTER CONFIGURATIONS ARE REQUIRED? 10
2.5 TO WHICH PRODUCT CATEGORIES DOES THE INSTRUMENT BELONG? 10
2.6 WHAT IS THE BASIC STRUCTURE OF THE INSTRUMENT? 11
2.7 HOW DOES THE BOTTOM DETECTION TECHNOLOGY WORK? 11
2.8 WHERE TO PLACE THE INSTRUMENT? 11
2.9 HOW DOES THE SHIPPING LOCK WORK? 12
2.10 HOW TO TURN THE INSTRUMENT ON AND OFF? 13
2.11 HOW TO CONNECT THE INSTRUMENT TO THE COMPUTER? 14
2.12 HOW TO INSTALL THE DRIVER AND USB ADAPTER BOX? 14
2.13 HOW TO CLEAN AND MAINTAIN THE INSTRUMENT? 14
2.14 WHICH CONSUMABLES ARE RECOMMENDED? 15
2.15 WHICH FUNCTION HAS THE YELLOW PANEL LAMP? 15
2.16 HOW DOES THE AUTOMATIC HOT-LID WORK? 15
2.17 HOW DOES THE XY-SCANNING SYSTEM ACT ? 16
2.18 HOW LONG DOES THE INSTRUMENT NEED FOR PLATE SCANNING? 16
2.19 CAN THE INSTRUMENT BE ADJUSTED TO SCAN SINGLE ROWS? 17
2.20 CAN TWO INSTRUMENTS RUN WITH ONE COMPUTER? 18
2.21 WHICH FUNCTION HAS THE TAG-NUMBER OF THE INSTRUMENT? 18
2.22 HOW TO IMPORT THE CALIBRATION PARAMETERS TO ANOTHER
INSTRUMENT? 19
2.23 DO THE DATA FILES EXIST AFTER UNINSTALLING THE SOFTWARE? 19
2.24 WHY DOES THE DISPLAY OF THE SYSTEM PARAMETER MENU
REQUIRE THE INPUT OF A PASSWORD? 19
3 PRODUCT CALIBRATION AND ALARMING 21
3.1 HOW TO HANDLE THE FAILURE ALARM? 21
3.2 AT WHICH TEMPERATURE RANGES DOES THE INSTRUMENT WORK? 21
3.3 WHAT ARE THE ALARMING CONDITIONS OF THE TEMPERATURE
SENSOR? 21
3.4 WHY DOES THE ACTUAL TEMPERATURE DISPLAYS 0°C OR 100°C
AFTER DETECTING THE SAMPLE POSITION? 21
3.5 WHY IS THE OPEN KEY NOT WORKING DURING THE INSTRUMENT
IS RUNNING? 21
3.6 WHY CAN’T THE MODULE NOT LOCKED IF IT’S PULLED OUT
AND PUSHED IN QUICKLY? 22
3.7 WHAT ARE THE DEFAULT TEMPERATURES OF THE HOT-LID AND
THE BLOCK IN A NON-OPERATION STATE? 22
4 USB AND BLUETOOTH INSTALLATION 23

4.1 HOW TO INSTALL THE BLUETOOTH CONNECTION? 23
4.2 HOW TO CONFIGURE THE BLUETOOTH DEVICE? 23
4.3 HOW TO CONFIGURE THE BLUETOOTH SOFTWARE? 23
5 FAQS OF THE INSTRUMENTS FAILURE CASES 25

5.1 THE PORTS ARE OKAY, BUT THE INSTRUMENT DOES NOT
COMMUNICATE 25
5.2 WHY DOES THE INSTRUMENT NOT RESPOND ALTHOUGH THE FLIP
SWITCH AT THE REAR WAS TURNED ON? 25
5.3 WHY THE MODULE TEMPERATURE RUNS NORMALLY, BUT
THE FLUORESCENCE DETECTION DOES NOT SCAN? 25
5.4 WHY DOES THE STEP MOTOR NOT WORK AND THE
COMMUNICATION FAILS AT THE DETECTING SAMPLE POSITION? 25
5.5 WHY THE POWER LAMP FAILS TO LIGHT UP AFTER TURNING
THE FLIP SWITCH ON? 26
5.6 THE YELLOW LAMP LIGHTS UP 26
5.7 THE INDICATION LAMPS ARE ALWAYS IN THE ON MODE AFTER
THE INSTRUMENT STARTED 26
5.8 THE HOT-LID DOES NOT HEAT 26
5.9 THE MODULE FAILS TO HEAT AND REFRIGERATE 26
5.10 THE MODULE TEMPERATURE HEATING OR COOLING RATE OBVI-
OUSLY DECREASE OR THE TEMPERATURE CONTROL IS INCORRECT 27
5.11 WHY IS THE CONTINOUS SCANNING ABNORMAL IF THE
CONSTANT TEMPERATURE IS SHORT? 27
5.12 THE RECORD DATA ARE ABNORMAL 27
CONTENTS
5.13 THE FLUORESCENCE DETECTION VALUES ARE ABNORMAL 27

5.14 HOW TO GET THE REAGENT TUBES BACK IF THE POWER FAILS
DURING THE PCR RUN? 27
6 FAQS OF REAGENT APPLICATIONS 29

6.1 WHAT IS THE BASIC PRINCIPLE OF PCR? 29
6.2 WHAT IS THE APPLICATION SCOPE OF QUANTITATIVE
REAL-TIME PCR? 29
6.3 WHAT IS A FRET PROBE? 29
6.4 WHAT IS THE PRINCIPLE OF THE TAQMAN® TECHNOLOGY? 30
6.5 WHAT ARE BASELINE, THRESHOLD AND CT VALUE? 30
6.6 WHAT IS THE PRINCIPLE OF THE ABSOLUTE QUANTIFICATION
ANALYSIS? 31
6.7 WHAT IS THE RELATION BETWEEN THE CT VALUE AND THE
INITIAL TEMPLATE AMOUNT? 31
6.8 WHAT IS THE STANDARD CURVE? 32
6.9 WHAT IS THE CORRELATION COEFFICIENT? 33
6.10 WHAT ARE THE REASONS FOR LOW AMPLIFICATION EFFICIENCIES? 33
6.11 WHAT COULD BE THE REASON FOR POOR LINEAR REGRESSION
OF THE STANDARD CURVE? 33
6.12 HOW TO MEASURE THE REFERENCE GAIN? 33
6.13 IS THE GAIN VOLUME OKAY? 34
6.14 ARE THE DENATURATION TEMPERATURE AND TIME OKAY? 34
6.15 ARE THE ANNEALING TEMPERATURE AND TIME OKAY? 34
6.16 AT WHICH STEPS SHOULD THE FLUORESCENCE BE DETECTED? 34
6.17 WHAT IS A MELTING CURVE AND WHAT DOES TM MEAN? 35
6.18 WHY IS A CONSTANT TEMPERATURE NEEDED BEFORE MELTING
CURVE DETECTION? 35
6.19 WHY THERE ARE MORE THAN ONE PEAK IN A MELTING CURVE? 35
6.20 WHICH METHODS SHOULD BE USED TO ANALYZE THE DATA? 35
6.21 WHY DOES THE CT VALUE APPEARS LATE? 36
6.22 WHY THERE ARE NO SIGNALS AND CT VALUES? 36
6.23 WHY GIVES THE NEGATIVE CONTROL A POSITIVE RESULT? 37
6.24 WHAT ARE THE REASONS FOR FALSE POSITIVE RESULTS AND
HOW TO AVOID IT? 37
6.25 WHAT ARE THE REASONS FOR FALSE NEGATIVE RESULTS AND
HOW TO AVOID IT? 37
6.26 WHY ARE TEST RESULTS NOT REPRODUCIBLE? 38
6.27 WHY ARE THE FIRST 3 CYCLES IN THE AMPLIFICATION CURVE
ALWAYS ACCOMPANIED WITH A LIFT OR FALL? 38
6.28 WHY IS THERE A STEP-SHAPE PLATFORM IN THE AMPLIFICATION
CURVE? 38
6.29 WHAT ARE THE REASONS FOR GETTING NON S SHAPED CURVES
AND HOW TO AVOID IT? 38
6.30 THE FLUORESCENCE OR TEMPERATURE CURVE ARE ABNORMAL
OR THERE IS A STRAIGHT LINE OR LOSS OF PARTIAL DATA 39
6.31 THE FLUORESCENCE VALUE INCREASES BETWEEN THE WELLS
OR THE BACKGROUND FLUORESCENCE IS VERY HIGH 39
6.32 CAN DIFFERENT REAGENTS RUN ON THE INSTRUMENT AT THE
SAME TIME? 39
6.33 CAN MULTI-CHANNEL DYES USED IN THE SAME TUBE WITH THE
HUMACYCLER? 39
6.34 WHY DO F1’S DYES RESPOND IN F2 IF DUAL CHANNELS? 40
6.35 THERE IS CROSSTALK AMONG THE CHANNELS 40
6.36 WHY DOES THE DYE CHANNEL MATCH BUT NAMES AND
DEFAULT NOT? 40
6.37 HOW TO SET UP A NEW PROGRAM DURING THE HCY4 SOFTWARE
IS RUNNING? 40
6.38 CAN PARAMETER SETTINGS BE CHANGED DURING THE
HCY4 SOFTWARE IS RUNNING? 40
6.39 HOW TO DECREASE OR INCREASE THE NUMBER OF CYCLES
DURING A PCR RUN? 41
6.40 WHICH OF THE INFORMATION CAN USERS EXTRACT FOR REPORT? 41
6.41 CAN THE SAMPLE INFORMATION BAR ON THE INTERFACE BE
MODIFIED? 41
6.42 REAGENTS EVAPORATE 41
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 manufacturer‘s 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.
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
user‘s responsibility to arrange for proper disposal of the individual components.
All parts which may contain potentially infectious materials must bedisinfect-
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 | Frequently Asked Questions

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

BASIC Frequently Asked Questions (FAQs) 9
2 BASIC FREQUENTLY ASKED QUESTIONS (FAQS)
2.1 What is the instrument’s application?

The 96-well fluorescent PCR detection system HumaCycler allows the real-time
detection of amplified DNA. Application areas for the use of the HumaCycler in-
clude the clinical diagnosis of pathogens, cancer, genetic diseases and research
of the human genome, forensics, oncology, epidemiology, zoology and bota-
ny. The HumaCycler belongs to IVD medical equipment, which uses the Poly-
merase-Chain-Reaction to perform quantitative or relative analyses of genes,
mutations or Melting Curve analyses in clinical laboratories.
2.2 What are the specifications of the instrument?
Specification/Model: HumaCycler (4 channel instrument) Table 1

96 x 0.2 ml tubes (suitable for single tubes, 8-strip
Sample capacity:
tubes, 96-well fully, half and non- skirted plates)
F1 (FAM, SYBR Green I); F2 (VIC, HEX, JOE, TET);
Applicable dyes:
F3 (Cy5); F4 (ROX, Texas Red)
Temperature range of 4…105°C (minimum division 0.1°C)
block working: Heating/cooling rate: 4°C/s (max.)
Temperature range: ≤±0.1°C (full-range), (55°C typical value ≤±0.1°C)
Temperature accuracy: ≤±0.2°C (full-range), (55°C typical value ≤±0.1°C)
Temperature uniformity: ≤±0.4°C (full-range), (55°C typical value ≤±0.3°C)
Temperature range of 30°C…110°C
hot-lid working: (adjustable, default temperature 105°C)
Repeatability of fluores-
5%
cence intensity detection:
Running mode: Continuous running
Operation system: Windows 2000/XPSP2/Windows Vista/Windows7
Power supply: 100...240V, 50/60Hz 600W
Dimensions: 430mm×395mm×352mm
Weight: 28kg
10
2.3 What are the specifications of the fluorescent dyes?
Table 2 Channel F1 F2 F3 F4
(nm range) (nm range) (nm range) (nm range)
FAM VIC Cy5 ROX
(492/515) (538/554) (552/570) (568/595)
Texas-Red
SYBR Green- I HEX (535/556)
(595/620)
Fluorescence Dye
TET
(521/536)
JOE
(525/555)
Center Excitation
470 523 550 571
Wavelength (nm)
Half Peak Range (nm) 30 20 20 20
Center Emission
525 564 584 612
Wavelength (nm)
Half Peak Range (nm) 20 20 20 20
2.4 Which minimum computer configurations are required?

Operation System: Microsoft Windows 2000/XPSP2/Vista/7
Table 3 Application Software: Excel 2000/2003/2007 or Access 200/2003/2007
Memory: 2GB
Free Hard Disc Space: 10GB
Processor: Pentium®4, Intel i3
Peripheral: CD-ROM
Interface: USB, RS232C, Bluetooth
2.5 To which product categories does the instrument belong?

Table 4 Equipment category: Category I (equipment category I with protective
grounding device and structure category II)
Insulation category: Reinforced insulation
Power supply: 100…240V, 50/60Hz
Input power: 600W
Operation: Continuous operation
Pollution class: II
Protective class: IP20
Electromagnetic Class A
compatibility:
Areas under 2000m without high temperature
Applicable region:
(>30°C) or high humidity (>80%)

2.6 What is the basic structure of the instrument?

Figure 2
Side view of the
4
HumaCycler
1 Control Unit
2 Transmission Unit
3 Computer system
4 Hot-lid Unit
2
5 Thermal Circulation
6 Unit
6 Photoelectric Unit
7 Power Supply Unit
3
7
2.7 How does the bottom detection technology work?

The HumaCycler provides a unique bottom detection technology. The short-dis-
tance bottom fluorescence detection is achieved by special porous semicon-
ductor refrigerator, porous flat plate hot pipe radiator and maximum 8 channel
coaxial optic fiber.
2.8 Where to place the instrument?

The instrument must be placed in a low humidity area (≤70%), away from water
resources (e.g. water pipes) with a well indoor ventilation, free of corrosive gas
or strong magnetic fields. Don’t place it in damp or dusty areas. The work bench
to put instrument must be horizontal and steady. The openings on the instru-
ment are designed for the ventilation circulation. Don't block or cover them or
the instrument body might get over heated. The distance between ventilation
opening and the nearest object must be at least 30 cm for single instrument
operations or 50 cm for several instruments operations. The test performance
might get impacted or failure might happen, if ambient temperature is too high.
Don’t use the instrument in places with direct sunshine or strong light sources
12
to protect fluorescence detection. Keep it away from heater, stove and any other
heat source! If the instrument will not be used for a long time, the power supply
should be turned off and power plug shall be withdrawn and the instrument
should be covered with soft cloth or plastic film to avoid dust or dirt come into
the instrument.
2.9 How does the shipping lock work?

In order to prevent the inner moving parts to shift and collide during transport,
they were secured with a Fixing Pin before leaving HUMAN (Figure 3). After the
instrument is placed in the desired location, 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
and fully tightened by clockwise rotation to release the locking mechanism in-
side the instrument (Figure 4). Only after the correct installation of the Fixing
Pin, the instrument may be switched on; otherwise, the instrument’s tempera-
ture control system is able to run, but the XY-axis scanning system isn’t able to
move.
Figure 3
Fixing Pin on the right

side of the instrument

Figure 4
Fixing Pin in the

unlocker port
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.
2.10 How to turn the instrument on and off?

The flip switch is at the back on instrument to turn on or off the main power sup-
ply. The Run Switch button at the front cover works as the remote switch for an
internal power supply. After pressing the Run Switch button (it outputs 24V) the
control loop gets energy, the green lamp turns on and a beep signal sounds. It is
designed to facilitate the operation and equal to the standby mode. After reset
the button, it closes the output of the flip switch supply, but there is still hazard
voltage at wire entrance of the internal power supply in the instrument. It is
recommended to turn off the instrument completely by pressing the flip switch,
if the instrument will not be used for longer times or the Technical Support
needs to open the instrument for maintenance.
14
2.11 How to connect the instrument to the computer?
Accessory Accessory Configuration

RS232C-RS232C Standard computer
“Isolation RS232 transit RS232C interface Standard configuration
box” Two-way signal isolation
USB-RS232C Standard computer USB
Standard configuration
“USB transit box” interface
Wireless transmission
BlueTooth-RS232C distance 10m
Standard configuration
“Bluetooth transit box” Suitable for short distance
signal transmission
2.12 How to install the driver and USB adapter box?

Insert the USB adapter box into the instrument and connect it with the com-
puter. Follow the instructions after installing the driver according to the instruc-
tions shown on the computer screen.
2.13 How to clean and maintain the instrument?

In order to guarantee a full contact between the PCR tubes and the wall of
the conical cavities, good heat conduction should be ensured and contamina-
tions should be avoided! The conical cavities and hot-lid of the module shall be
regularly cleaned. Wells may be cleaned with dust free nail wipes and if
necessary, they may be soaked with 95% absolute ethyl alcohol used in medi-
cine or distilled water. Don’t scratch the optic element in the conical wells in the
instrument’s module and try to prevent the contamination of the instrument.
The stained surface of the instrument, shall be cleaned with soft cloth soaked
with mild detergents. Heat conductive oil medium shall not be used. The mod-
ule should be pushed in time to prevent dust during normal conservation or
after use.

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 damages of the optics in the wells, NEVER
use sharp or hard objects to clean the wells.
The maintenance of the HumaCycler should be performed by local Human
authorized Technical Support or well trained users depending of the fre-
quency of use.
2.14 Which consumables are recommended?

For Real-Time PCR analysis, 0.2ml single tubes, 8-tube stripes, skirted, half-skirt-
ed and non skirted 96-well plates are recommended. The bottom of the test
tubes must be transparent. In addition, filter tips to avoid contamination of aer-
osols should be used.
2.15 Which function has the yellow panel lamp?

When the instrument lock is in the right position and optic coupling doesn’t act,
the hot-lid motor can’t move downwards, the module temperature control can
work, the yellow alarm lamp on the panel turns on and the software generates
a message. When the instrument’s optic coupling is pushed in without blocking,
the hot-lid motor doesn’t move down. It goes down if it’s pushed to the right
position during operation. When the optic coupling for the test tube pressure
or for the lower limit is blocked, the motor also doesn’t move downward. The
motor doesn’t run anymore if it changes during operation.
2.16 How does the automatic hot-lid work?

The instrument has an automatic hot-lid function, which means the hot-lid
is driven up or down by a step motor. The hot-lid stops automatically, once it
reaches the upper or lower optic coupling limit. If there are PCR tubes in the
block module, when the hot-lid goes down, the spring structure generates pres-
sure to act optic coupling, the hot-lid motor perceives it and stops automatically.
The hot-lid lifts to the upper limit when the instrument was turned on. When
the instruction is running, the instrument must receive the signal that the mod-
ule has to be pushed into the right position. After that, the hot-lid moves down-
16
wards, presses and the program runs. If the plate spring pressurization structure
is used, only certain deformation can generates pressure to act optic coupling,
so it is a “constant pressure type”. Without any PCR tubes, the hot-lid still keeps
the certain gap with the module when it goes down to lower limit.
2.17 How does the XY-scanning system act ?

The self-check at the beginning could be described as follows: The XY-system
moves to the left and twice to the right, then forward and stops at the top front
of the Y-axis, moves backwards, slows down and finally, stops next to the Hall
sensor inside Y-axis. The photo-electric unit is at right side when it stops, which
facilitates the locking of moving parts at the stop and avoids the damage of
photoelectric parts during shipment. During the normal operation, the origin
can be found at left side of X-axis, scans rightwards, moves one line forward,
scans leftward and repeats like this to scan the whole plate. The photo-electric
unit waits at left side when the operation status is “waiting to scan”.
2.18 How long does the instrument need for plate scanning?
The relation between theoretical running time and recommended re-scanning
interval settings is shown in Table 6:
Total Channel Parameter 1 Time 2 Times 3 Times 4 Times
Table 5 Scanning Time
Whole Plate Time difference 5.04
Scanning between wells
(sec.)
Theoretical 6.54 13.08 19.62 26.16
cumulative time
(sec.)
Rescanning Recommended 7 14 20 27
Interval minimum setting
(sec.)
Single row scan- Time difference 0.48
ning fluorescence between wells
collection (sec.)
Theoretical cu- 1.98 3.96 5.94 7.92
mulative time
(sec.)
Rescanning Recommended 2 4 6 8
Interval minimum setting
(sec.)

It takes a certain amount of time for the module temperature to conduct to the
reagents in the PCR tube and to stabilize the temperature. This time for heat
conduction depends on the PCR tube type, wall thickness and the material heat
conductivity. Therefore, to develop a PCR amplification program, time for the
heat conduction should be added on the recommended re-scanning time in-
terval to consider the time for a constant temperature at the detection point.
When the program is running, the instrument will forecast before the constant
temperature period on detection point is finished and will scan in advance. If
the time for constant temperature is set shorter than the time that scanning
needs, the instrument will extend the constant temperature period automati-
cally to meet the scanning requirements. The time difference at the detection
cavity must be very small, if the reagent fluorescence detection is required. To
scan the whole plate, the time difference for single color fluorescence detection
between the wells is 5.04 sec. To scan the designated single color in a single row,
the re-scanning interval is recommended to set at 2 sec. The time difference for
the fluorescence detection between wells is 0.48 sec. To scan designated several
rows, the time difference is between 0.48 sec. and 5.04 sec.
1. The Photomultiplier (PMT) gain needs 1.5 sec. for stabilization upon the
channel bank replacement.
2. For the theoretical calculation, the entire X-axis takes 0.48 sec., Y-axis
takes 0.15 sec/line, X+Y=0.563 sec. (the total time for X and Y to complete
one row and move one column). The phase difference of total scanning
time over the whole 96-well plate is 0.63*8=5.04 sec. Since it prepares to
replace the gain during the scanning process, return plus gain stabiliza-
tion is calculated as 1.5 sec.
3. The theoretical cumulative time includes the time for zero return and the
time for data transmission.
4. The time difference between the wells refers to the time difference bet-
ween first and the last cavity. The re-scanning interval time includes the
time for the constant temperature and the time for the temperature
change at detection point.
2.19 Can the instrument be adjusted to scan single rows?

The instrument can scan single rows for the faster detection compared to scan
the whole block. The time difference for single row scanning is 0.48 sec., which
saves a lot of time compared with full plate scanning and improves the detec-
tion consistency significantly. Select Block Scan Method under Tools and select
the rows to be scanned after selecting Line Scan.
18
Figure 5
2.20 Can two instruments run with one computer?

One computer can be connected to several instruments if the instruments are
connected with different communication wires (USB, Bluetooth, RC232C) to the
computer. Click onto the instrument icons on the desktop. Click onto the same
shortcut to re-open the software of the instrument with same specifications
and click another specification icon for an instrument with other specifications.
Note, that the files of the background correction and inter-channel dye cross-
talk correction at different instrument cannot be shared. The relevant para-
meter files must be imported (probably from a different CD-ROM) for all instru-
ments to the root directory of the software installation. The software matches
the parameters automatically or the wells are shown without the fluorescence
or without background corrections. To install the new software, it generates a
message to delete the installed program. Please, select all instrument specifica-
tions to be used in software installation.
2.21 Which function has the Tag-number of the instrument?

The purpose of the Tag-number of the instrument is to name the correction and
calibration coefficient for each host machine, so that the software can identi-
fy and use the correction coefficient automatically. The Tag number is set at
ex-factory. The Tag-number of the instrument and detection channels which
are permitted, are displayed in status bar under the software interface after
connection between instrument and computer successful. The Tag-number
consists of the last 6 numbers of the serial number. When the instrument runs
normally, the Tag-number will be shown in the status bar of the software inter-
face. If users need to replace the instrument, a restart of the software is neces-
sary to refresh the contents after the success in the communications.

2.22 How to import the calibration parameters to another

instrument?
Select Import Instrument Calibration Parameters in the Tool menu and se-
lect the calibration parameter file with the suffix .icp. Note, if one computer
is connected to several instruments, be aware that the Tag-numbers of the
instruments are stored in different files. It automatically searches the proper
parameters under the root directory. There is just one operation for each com-
puter necessary. If background correction is normal and the wells are empty, the
adjusted gain and each channel show the no-background value.
2.23 Do the data files exist after uninstalling the software?

If the HCy4 software is installed correctly, the user’s experiment data and in-
strument’s correction parameter files are kept in the installation directory. Us-
ers can search files and delete them manually, if they are not needed anymore.
After uninstalling the HCy4 software, the files still exist, but cannot be opened.
2.24 Why does the display of the system parameter menu require
the input of a password?
The system parameters are for the instrument manufacture’s internal calibra-
tion and require a special accession password. The function is not required for
the end user and for calibration.
20

PRODUCT CALIBRATION AND ALARMING 21
3 PRODUCT CALIBRATION AND ALARMING
3.1 How to handle the failure alarm?

When something fails, the software shows a temperature control failure or an
abnormal ambient temperature. The red lamp turns on, the buzzer sounds with-
in an interval of 200 ms. The red lamp is locked once it is triggered. When the
failure is found, the software stops all control signals and programs. It is not
a failure if status lamp RUN and Error LED blink once at same time during the
instrument starts.
3.2 At which temperature ranges does the instrument work?

The working temperature range of the instrument is 10…30°C. The ambient
temperature detection sensor is set on the instrument, which prompts once it is
over 35°C and stops operation once it is over 40°C. Let the instrument cool down
until it meets the working temperature scope; turn it off and start again.
3.3 What are the alarming conditions of the temperature sensor?

The software reports “Temperature Control Failure” if the temperature sensor
of the temperature module detected temperatures below 0°C or above 110°C
or if the sensor is detected as open-circuit or short-circuit. The red lamp, an in-
dication of an error, turns on. Once a failure is detected, all temperature ele-
ments, the auxiliary heating system and hot-lid heating film are closed and the
run stops.
3.4 Why does the actual temperature displays 0°C or 100°C after
detecting the sample position?
The temperature module is damaged. It accompanied the panel red lamp
alarm and a message appears in the software. The instrument stops running
automatically. Please, contact the local HUMAN authorized Technical Support.
3.5 Why is the OPEN key not working during the instrument is
running?
While the instrument is running, the hot-lid presses onto the test tubes and the
module is not allowed to move. After the run is completed, the hot-lid lifts to the
right position and the button works again.
22
3.6 Why can’t the module not locked if it’s pulled out and pushed
in quickly?
According to the instrument design, the module locks 0.5 sec. to lift the slide
block to get it out. Please, wait when it is pushed in.
3.7 What are the default temperatures of the hot-lid and the block
in a non-operation state?
The default control temperature of the hot-lid is 105°C and 30°C of the block.
Start the running program, the control temperature of hot-lid will change ac-
cording to the customer’s program. After finishing the PCR run, the instrument
restores to the non-operational status.

USB AND BLUETOOTH INSTALLATION 23
4 USB AND BLUETOOTH INSTALLATION
4.1 How to install the Bluetooth connection?

Use the Bluetooth manager of the Microsoft Windows XP Service Pack 2 (SP2)
to install and configure Bluetooth with a virtual serial port or use a commercial
available Bluetooth management software, e.g. BLUESOLEIL. Usually, the USB
Bluetooth adapter connected to Windows XPSP2 supports the common soft-
ware. If common software doesn’t work, install the driver attached on Bluetooth
adapter that provided with the commercial available software. To install the
Bluetooth adapter, just connect the device. It doesn’t need any configurations
before connection. Once the USB Bluetooth adapter is inserted, Windows XP will
install the common Bluetooth driver automatically. Installed Bluetooth adapter
can be seen in the Windows XP’s device manager.
4.2 How to configure the Bluetooth device?

The Bluetooth settings can be configured by selecting the Bluetooth device in
the Control or System Panel. Open the Bluetooth device software and follow
the instructions. The software will guide the user to connect, match and install
virtual serial port.
4.3 How to configure the Bluetooth software?

Use the Bluetooth interface, if the Bluetooth device is not available in the com-
puter (e.g., Z-TEK Class II V2.0 Bluetooth interface application software). Follow
the basic steps according to manufacturer’s Instructions for Use.
After connecting with the computer:
1. Start the HCy4 software on the computer and press Run.

2. Wait a few seconds.
3. Once the link succeeds, the hot-lid on host machine goes down and data
starts to be transferred.
24

FAQS OF THE INSTRUMENTS FAILURE CASES 25
5 FAQS OF THE INSTRUMENTS FAILURE CASES
5.1 The ports are okay, but the instrument does not communicate
If the software’s running communication fails, use the serial port communica-
tion assistant. A message is shown if COM1 or COM2 are occupied. Another rea-
son could be that software can occupy ports, so that the instrument does not
communicate.
It might be also helpful to install the driver software. Base upon your computer
configurations, use either the CP210xVCPInstaller_64.exe (64 bit) or CP210x-
VCPInstaller_86.exe (32 bit) for driver installation. These files can be found in
the HCy4 software. Restart the HCy4 software program and connect the instru-
ment again.
5.2 Why does the instrument not respond although the flip switch
at the rear was turned on?
The Run Switch at the front of the instrument is not turned on. The internal
switching power has not voltage output. For working with the instrument, the
Run Switch must be pressed and the green indicator lamp should be lit green to
indicate that the control system is energized.
5.3 Why the module temperature runs normally, but the fluore-
scence detection does not scan?
The Fixing Pin is not removed or not inserted and tightened into the rear un-
locker port. Remove the Fixing Pin and tighten it into the unlocker port.
5.4 Why does the step motor not work and the communication
fails at the detecting sample position?
1. The reason could be a poor contact or damage of the interface wire. Please
verify, connect or replace the interface wire.
2. The flip switch is not turned on or it’s turned on only after the program starts
running. Please, turn the flip switch on and restart the program.
3. The step motor or the drive is damaged. Please, contact the local HUMAN
authorized Technical Support.
4. The Fixing Pin is not fully inserted. Tighten it into the unlocker port and
switch the power button on after shutdown.
26
5.5 Why the power lamp fails to light up after turning the flip
switch on?
The Run Switch is not pressed. This switch is for the temporary turning the
ON/OFF of the output power and its equivalent to the standby mode. For a
prolonged shut down, the rear flip switch should be switched off. Anoth-
er reason could be that the power button is not switched on. Please, turn the
flip switch on. In addition, check if the fuses are blown. Therefore, replace the
fuses included in the shipment. In case of one of the switches are damaged,
contact the manufacturer or the HUMAN authorized Technical Support or the
local distributor.
5.6 The yellow lamp lights up

The module is not fully pushed in and the optical coupler fails to detect the mod-
ule. Push the module in again. If the light is still on, contact a Human authorized
local Technical Support.
5.7 The indication lamps are always in the ON mode after the
instrument started
If the switch button on communication connection box is at “Update” position,
3 lamps on small panel are always on, which means the CPU program is not run-
ning. This status is for CPU program upgrading. Please keep the switch button at
the “Normal” position for use.
5.8 The hot-lid does not heat

The thermal-sensitive fuse is damaged or the plug pieces are loose, the heating
element of the hot-lid is damaged or the temperature sensor of the hot-lid is
damaged. Contact the local Human authorized Technical Support.
5.9 The module fails to heat and refrigerate

The refrigeration sheet is damaged. Contact the local Human authorized Tech-
nical Support. If the module fails to heat and refrigerate during the hot-lid is
heating up, wait until the hot-lid temperature comes to the target value. When
it stops running, the module temperature holds down to 30°C automatically.

FAQS OF THE INSTRUMENTS FAILURE CASES 27
5.10 The module temperature heating or cooling rate obviously

decrease or the temperature control is incorrect
The ventilation opening is blocked or there is a loose connection wire. Clear the
ventilation opening and fix the power cord connection.
The refrigerating sheet or temperature sensors are damaged. In these cases,
contact the local Human authorized Technical Support.
5.11 Why is the continous scanning abnormal if the constant

temperature is short?
Data transmission can’t catch up if the continuous scanning interval is short.
Please, consider the time for XY-scanning and multi-color scanning or it might
suffer the computer buffer overflow, data block, signal missing etc. in commu-
nication, which results in abnormal curve data.
5.12 The record data are abnormal

During the amplification, the curve is normal, but the well position data for cer-
tain wells is all zero, once the data were saved onto a memory stick. Reflected on
the analysis curve, suddenly the fluorescence data of all wells decrease to 0 syn-
chronously and sustain. These cases could happen, when data files are infected
by virus files. Please, clean all hard and software components, delete infected
files and use virus protection.
5.13 The fluorescence detection values are abnormal

There could be an irradiation from an external light source during the program
runs and the hot-lid is open. Close the hot-lid and delete the external light source.
The photo-electric system is damaged. Please, contact the local Human author-
ized Technical Support.
5.14 How to get the reagent tubes back if the power fails during
the PCR run?
If the power supply fails during a PCR run, the instrument cannot be opened and
the reagent tubes cannot taken out. Please act as below:
1. Turn the plastic gear at the instrument is top continuously as instructed; lift
the internal hot-lid and stop when you think it reaches the position.
2. Insert a flat screwdriver (diameter <5mm, length approx. 150 mm) from the
hole at the top behind the instrument until an unlocking sound is heard, the
28
module is bounced out slightly, pull the module out manually, take the rea-
gent tubes out.
3. Push the module in a normal position.

FAQS OF REAGENT APPLICATIONS 29
6 FAQS OF REAGENT APPLICATIONS
6.1 What is the basic principle of PCR?

PCR is a laboratory method to amplify DNA. Usually, a PCR cycle consists of 3
steps. Double-strand DNA melts at 95°C to single-strand DNA. In the second
step, target primers (forward and reverse) bind sequence specific to the sin-
gle-strand DNA (annealing) to determine the target sequence that should be
amplified (amplicon). The annealing temperature depends on the composition
of the primers. In the second step, the DNA polymerase extends the strand with
Nucleotide-triphosphates at 72°C. These cycles were repeated 40-50 times. The
amplicon can be detected by agarose-gel electrophoresis, Melting Curve analy-
sis or with fluorescence dyes by Real-Time PCR analysis.
6.2 What is the application scope of quantitative Real-Time PCR?

The principle of a quantitative Real-Time PCR can be used for gene detection and
comparisons of gene expression levels. It can be used for the quantification and
detection of Infectious Diseases and it is also used widely in criminal investiga-
tion, veterinary, agriculture, biochemistry etc.
6.3 What is a FRET probe?

FRET is the abbreviation of Fluorescence Resonance Energy Transfer. A FRET
probe is a small piece of DNA similar to a primer which is linked to a fluores-
cence dye (F) on the one hand and on the other hand to a quenching molecule
(Q). Usually, stimulated by exciting light, fluorescence generates emitting light
that can be detected. Due to the close localization of the quencher to the report-
er, the original emitting light is absorbed by the quencher which converts to
emitting light with another wavelength or thermal energy, which is called FRET.
The principle of fluorescence probes: Figure 6
R: Reporter
Q: Quencher
30
6.4 What is the principle of the TaqMan® technology?

The TaqMan® technology uses FRET probes for the detection of amplified DNA.
Beside forward and reverse primer pairs, FRET probes bind close to the primers
within the sequence to be amplified. During the primer extension process by the
DNA polymerase, the enzyme cuts the FRET probe completely out of the target
sequence. The reporter and quencher are not longer in a close distance to each
other and the emitted light of the reporter can be measured. So, the fluores-
cence signal increases with each further PCR cycle and the amount of amplified
DNA. Due to the fluorescence detection system of the HumaCycler, the amplifi-
cation of the target sequence can be observed in real time. That’s why it’s called
Real-Time PCR.
Figure 7
6.5 What are baseline, threshold and Ct value?

The Ct value or threshold cycle is the cycle number at which the fluorescence
generated within a PCR run crosses the fluorescence threshold, a fluorescent
signal significantly above the background fluorescence. At the threshold cycle, a
detectable amount of the amplicon has been generated during the early expo-
nential phase of the reaction. The Ct value correlates inversely with the original
amount of the template DNA or RNA.
Threshold: The horizontal line to obtain Ct value. It is the value of the fluores-
cence background noise during the PCR run. The threshold line must be high-
er than baseline. In addition, it’s a numerical value, which reflects a significant
point above the baseline, which is assigned for each run.
Baseline: The minimum horizontal line that is not affected by the fluorescence
background noise. The baseline value can be determined by testing the dyes that
customers use. Adjust it properly, so that the threshold horizontal line crosses

the linear line of fluorescence S-shaped curve. In general, to start with Real-Time
PCR, customers can calculate 10 times of the standard deviation of the fluores-
cence background signal before amplification. For further information, please
see the Instruction for Use of the HumaCycler DYE CALIBRATION KIT (REF 86150).
Figure 8
To set up these parameters manually, try to select the early stage that comes to
exponential phase and make sure the regression coefficient is bigger than 0.95.
6.6 What is the principle of the absolute quantification analysis?

The principle of an absolute quantification is the quantification of the target
DNA in a sample with a standard curve. The standard curve is generated with 3
or 4 Quantification Standards (QS) of known concentrations during the PCR run.
Each of the standards generates specific Ct values. Due to the standard curve
and the Ct value of unknown samples, the HCy4 software calculates the concen-
tration of the unknown sample. Usually, concentrations are determined as IU/
ml or copies/ml. The slope of a standard curve is -3.32 or 3.32, if the PCR reaction
has an efficiency of 100%.
6.7 What is the relation between the Ct value and the initial
template amount?
The more template amount is in the sample, the less cycles are needed for the
specific fluorescence signal to reach the threshold.
32
Figure 9
6.8 What is the standard curve?

The log concentrations and cycles are in linear relation. A standard curve can
be obtained from quantification standards with a known initial copy amount.
The template amount contained in the sample can be calculated according to
Ct value of sample.
Figure 10

6.9 What is the correlation coefficient?

The correlation coefficient reflects whether a standard sample is linear on log-
arithmic concentration. Usually, the closer the absolute value of a correlation
coefficient is to - 1 or 1, the better is the concentration gradient precision.
During optimum conditions, the correlation coefficient should be over 1± 0,05
and ideally, the difference of Ct values between 1 log is 3.32. That means, that
the efficiency is close to 100%. These features depend on the quality of sample,
primers/probes and pipetting mistakes.
6.10 What are the reasons for low amplification efficiencies?

1. Some components (especially fluorescence dyes) in the reaction reagent are
degraded.
2. There are PCR reaction inhibitors in reaction system that usually come with
the template. To reduce the impact of inhibitors, dilute the template proper-
ly before add to the Master-Mix.
3. The reaction conditions are not optimized. Decrease the annealing tempera-
ture properly or change to a three-step amplification method.
6.11 What could be the reason for poor linear regression of the
standard curve?
1. Quantification Standards show no linear regression due to errors of the sam-
ple application.
2. Quantification Standards are degraded. Avoid multiple freezing and thaw-
ing cycles, aliquot the Quantification Standards in convenient portions.
3. Primers or/and probes are not working. Please, check the storage conditions,
expiry dates, aliquot them in convenient sizes and avoid multiple freezing
and thawing cycles.
4. Template contains inhibitors or suffers too high concentration. Dilute the
templates with TE, pH 8.
6.12 How to measure the reference gain?

Users can measure reference gains with the amplification outcome and save
the parameter as Referengain.icp. When experimenting with dyes, users may
use the“best gain” , which can get more reasonable fluorescence signals than
that get from the optical system gain settings manually. For further informa-
tion, please contact the manufacturer of the dyes and/or see the Instruction for
Use of the HumaCycler Dye Calibration Kit (REF 86150) if the assay contains the
corresponding dyes.
34
6.13 Is the gain volume okay?

Usually, the curve is completely or partly beyond the window extents or fluores-
cence intensity zero area is in straight line. The possible reason is an improper
gain setting. The software has a maximum display and self-adaptive range dis-
play. The normal analysis is available for data within 150% of the normal range.
The detection gain might need to be increased if the curve flat zone is too low
or amplification is not obvious. The gain can be set in the Tool menu before
operation.
6.14 Are the denaturation temperature and time okay?

Most double-strand DNA melts at 95°C. Lower temperatures (90-94°C) de-
crease the melting capability of double-strand DNA, which lead to decrease the
efficiency of the PCR reaction. Usually, 15...20 sec. are enough to melt double-
strand DNA to single-strand DNA. However, longer products might take 30 sec.;
a 60 sec. denaturation time is not necessary.
6.15 Are the annealing temperature and time okay?

Verify the Tm (Metling Temperature) value of primers and probes if using home-
brew assays. The annealing time for SYBRGreenI is about 20…35 sec. Double-la-
beling probes usually take a two-step method that combines annealing and
extending, which takes 45…60 sec. and temperature is usually 60°C. The anneal-
ing of FRET probe takes around 20…30 sec. For commercial available CE-marked
assay the annealing temperature is optimized to the validated instruments and
can be found in the Instruction for Use of the assay.
6.16 At which steps should the fluorescence be detected?

SYBRGreenI should be detected at 72°C, at which temperature most DNA is
double-stranded. As mentioned above, double-labeling probe usually detects
by two steps, so signal collection must be in the combining step of annealing
and extending. If the signal collection points are not determined, several points
can be collected for comparison. If no signal is detected on software interface,
please verify, whether there is at least one signal collection point in program
setting. For commercial available CE-marked assay the step to measure the fluo-
rescence is optimized to the uses technology and can be found in the Instruction
for Use of the assay.

6.17 What is a melting curve and what does Tm mean?

A melting curve is a physical characteristic curve that reflects the DNA compo-
nent characteristics. When temperature exceeds a certain value, the fluores-
cence intensity decreases quickly, the fluorescence mutant point is Tm peak
(Melting Point) if presented by derivative. Tm point is same, if amplification
product is single (most difference in actual test are caused by difference of sys-
tem module temperature uniformity), several Tm peaks appear, if nonspecific
amplification happens.
6.18 Why is a constant temperature needed before melting curve

detection?
No thermal insulation process before the melting curve is generated, makes the
real melting peak almost invisible. Therefore, a thermal insulation process with
same initial temperature of melting program is added in melting curve.
6.19 Why there are more than one peak in a melting curve?
1. The primer design is not optimized. Avoid primer dimers and pin structures.
2. The primer concentration is too high. Decrease the primer concentration pro-
perly and pay attention to the concentration ratio of the forward and reverse
primers.
3. The concentration of magnesium ions is too high. Decrease the magnesium
ion concentration properly.
4. The template is contaminated. Avoid DNA contamination during the RNA ex-
traction process or avoid non-specific amplification by an optimum primer
design.
5. Some multi-probe reagents may have many Tm peaks.
If using commercial available CE marked assays, just the point 4) and 5) might
be relevant.
6.20 Which methods should be used to analyze the data?

Usually, the 2nd derivative method is used for amplification curve with very
good effect and very obvious linear section. It gets high Ct value and users don’t
have to adjust baseline and thresholds and a fast analysis can be done. The
Fit-point method has wider applications and stronger adaptability. Users have
to set a sample point number, baseline and thresholds and users need to be
experienced in analyzing data. Fit point works well on amplification curve that
36
is not very ideal. If users are doing an absolute or relative quantification exper-
iment with the HCy4 software, the algorithms to analyze the data are prein-
stalled.
6.21 Why does the Ct value appears late?

There are several reasons for the late appearance of Ct values. The Ct values of
commercial available CE marked assays strongly depend on the quality of the
sample and the extracted nucleic acids or pipetting mistakes. These mistakes
could be related to low amplification efficiencies. For home-brew assays, late
Ct values could base on poor reaction conditions. It might be useful to decrease
the annealing temperature or design other primers and probes. In case, the PCR
product is too long, the product length should be between 80-150 bp.
If customers use commercial available tests, the reaction conditions for validat-
ed instruments are not necessary to optimized. In addition, the expiry date of
the reagents and the numbers of thawing and freezing cycle should be deter-
mined.
6.22 Why there are no signals and Ct values?

1. The number of reaction cycles is too low. Usually, 35 cycles are the minimum
to select. Users should add cycles up to 45 or 50 cycles in total. The back-
ground signal will increase if more than 45 cycles are selected.
2. The step to measure the fluorescence signal is wrong. Usually, the fluore-
scence is detected at 72°C, depending on primer/probe technology that is
used. This information should be get from the Instruction for Use of the used
assay.
3. Primers or probes are degraded. Use PAGE electrophoresis to analyze them
or use a new assay. Please, avoid multiple freezing and thawing cycles.
4. Primers or probes have a general design problem. For example, the tempera-
ture of the probe is higher than the temperature of the primer. That leads to
product extension without probe cross-breed.
5. The template amount is not sufficient. For sample with unknown concentra-
tion, use non-diluted, nucleic acids.
6. The template is degraded. Avoid multiple freezing and thawing cycles, ali-
quot the isolated material into convenient portions and analyze the sample
storage.

6.23 Why gives the negative control a positive result?

Either the negative control or Non-Template-Control is contaminated with
positive template material or the water is polluted. Another reason could be
the presence of primer dimers, which can be analyzed by performing a melting
curve.
6.24 What are the reasons for false positive results and how to
avoid it?
If a positive result appears in negative controls, the detection result of other
samples in this test might be false positive. The reasons of false positive include
the contamination of samples, amplification reagents or amplification products.
The common contamination source includes lab environment, sample injector,
aerosols generated during operation, reagent or anything in contact with am-
plification product. It is recommended to isolate the working area and to decon-
taminate all items that are used (pipettes, centrifuges, vortex mixers, benches
with fresh prepared 10% bleaching solution and or UV light. Dispose all reagents
of the assay and templates and use a new assay for re-testing. Users should
improve the lab operation, e.g. to avoid the reagent splash during the sample
application, take high-pressure treatment on sucker and centrifugal tube. Don’t
open the lid of the PCR tube after amplification and use filter tips for transfer
the reagent and templates to the PCR tubes, etc. Use 3 or 4 working areas and
wear separate gloves, coats and shoes or shoe covers.
6.25 What are the reasons for false negative results and how to
avoid it?
If a positive control set is not amplified into positive result, the test might have
a false negative result. Besides that, if there are problems with the extraction
steps of specimen nucleic acid purification, the specimen detection might find
false negative even though positive control is positive. So, the reason might be
that there is no DNA or RNA in the sample or after extraction. Standardize the
operation of DNA/RNA extraction, use commercial available extraction assays
and pay attention to clean working. Store the nucleic acids at -20°C or lower in
aliquots. Avoid multiple freezing and thawing cycles.
38
6.26 Why are test results not reproducible?

1. The template concentration is low. The lower the initial sample concentrati-
on is, the worse is the reproducibility. Do not dilute samples or concentrate
the sample.
2. The sample application to the Master-Mix is not accurate. Practice accurate
pipetting!
3. The temperature uniformity of the instrument is bad. Contact the local
Human authorized Technical support.
6.27 Why are the first 3 cycles in the amplification curve always
accompanied with a lift or fall?
When the amplification begins, the temperature transfer in the test tube takes
time. If the constant temperature cycle time set in PCR reaction program is short,
the temperature of the block is not transferred to reagent completely, the re-
agent fluorescence is affected by temperature and the PCR tube material and
structure cause thermal conductive coefficient difference. As a result, an in-
creasingly steady process is shown. PCR tubes are recommended, which have a
higher thermal conductive coefficient.
6.28 Why is there a step-shape platform in the amplification

curve?
The phenomenon appears during the same-tube double-color reagent amplifi-
cation, means: the fluorescence amplification curve of one channel is normal,
but the curve for another channel shows a step-shape platform and then an
S-shape change. For example, F1 has a high fluorescence value, while F2 has low
one. For the same-tube double-color amplification, F1 rises first, its fluorescence
is detected by F2 channel, the F1 ray filter signal contains micro residual of F1
and then a platform appears on F2 first. When F2 also amplifies, as fluorescence
intensity increases, the F1 impact is hard to detect. The HCy4 software has a
function of fluorescence crosstalk correction among channels, which is able to
minimize this effect.
6.29 What are the reasons for getting non S shaped curves and
how to avoid it?
If a positive control curve is not S-shaped, the reason could be that the reagents
are not fully thawed or not mixed uniformly before the sample was added.
Besides, many unknown components in the sample might interrupt the PCR

reaction. Ineffective reagents also causes bad amplification curve. In addition,

baseline settings influence the shape of curves. Make sure, that all reagents are
thawed completely and mix the components thoroughly before the preparation
of the PCR reaction. In addition, verify the baseline settings.
6.30 The fluorescence or temperature curve are abnormal or there

is a straight line or loss of partial data
The running program is infected by a virus, the computer configuration fails to
meet the requirements or the set up of the communication port is not appro-
priate. After deleting the virus, re-install the HCy4 software or configure the re-
quired features.
6.31 The fluorescence value increases between the wells or the

background fluorescence is very high
The test tube well or the hot-lid is contaminated. In addition, the baseline back-
ground parameters are set incorrectly. Eliminate the contamination. After per-
ennial use, the offset would occur in the optical elements. In this case, contact
the manufacturer to re-calibrate the background value.
6.32 Can different reagents run on the instrument at the same

time?
As long as the amplification temperature profile is same, they can be run at
same time. A maximum of 4 groups of standard samples can be analyzed due to
the 4 channels of the HumaCycler. Flexible grouping for analysis can be done by
setting different name for channel dyes.
6.33 Can multi-channel dyes used in the same tube with the
HumaCycler?
Yes, but pay attention to certain situations, the amplification of a kind of probe
component might inhibit the amplification of other component or generation
of fluorescence. Separate dyes to different test tubes, if there are doubts on the
test result. If using CE marked multiplex assays, the reaction systems are opti-
mized to the different targets and dyes.
40
6.34 Why do F1’s dyes respond in F2 if dual channels?

Each dye has a wide light spectrum. The ray filter length of F2 and F1 are close
to each other, that’s why it is normal that they have overlapping areas. Usually,
the HCy4 software can correct the fluorescence among channels. It records the
F1’s impact on channel F2 to deduct this impact later, minimize the “interfer-
ence” among channels and avoid the wrong judgment in same-tube multi-color
synchronous detection. The correction time is long and takes usually 45 min.
The instrument uses genuine multi-color LED excitation to make the fully use
of light intensity of the LED main peak. The exciting light is narrow band, which
facilitates the classification of different dye probes and multi-color analysis.
6.35 There is crosstalk among the channels

The dye signal crosstalk among channels can happen. Use Crosstalk Measure-
ments and save the parameters to modify.
6.36 Why does the dye channel match but names and default not?
User can name the new dye by most suitable ray filter channel wavelength. Se-
lect Customize Dyes from Tools on the HCy4 software, input channel number
and dye name.
6.37 How to set up a new program during the HCy4 software is

running?
To ensure that the data collection is reliable and effective, new settings or pro-
grams on the software should not be entered while the software program runs.
But users can click the icon on desktop, start the second process; re-open the
software to set a new program. It won’t affect other processes.
6.38 Can parameter settings be changed during the HCy4 software

is running?
It is possible to view the parameter settings while software program runs, but
modification cannot be make. It is “read only”.

6.39 How to decrease or increase the number of cycles during a

PCR run?
To change settings is not allowed during a PCR run. But users can increase/de-
crease the cycle numbers temporarily by Plus Cycle/Minus Cycle on the HCy4
software interface. For Melting Curve analyses, users can increase or decrease
temperature steps.
6.40 Which of the information can users extract for report?

Report Template Editor and Print Template Setting are available in the HCy4
software. Settings and analyzed results can be output to Excel, Text and data
base. All functional modules have a data query interface to query previous test
records. It is especially good for technical reports. Diagrams for serveral curves
(e.g. concentration, fluorescence, temperature, derivative-temperature) can be
extracted and saved.
6.41 Can the sample information bar on the interface be modified?

The HCy4 software allows users to custom the sample item. Select Customize
Columns and select Columns on the Tools menu and customize the information
bar.
6.42 Reagents evaporate

The PCR tube cap is not sealed tightly enough. Change the consumables to an-
other with a tighter fitting cap.
42

HUMAN
Gesellschaft für 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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HumaCycler

| Frequently Asked Questions

Cat No. 86100/7

SERVICE UND SUPPORT

2 BASIC FREQUENTLY ASKED QUESTIONS (FAQS) 9

4 USB AND BLUETOOTH INSTALLATION 23

5 FAQS OF THE INSTRUMENTS FAILURE CASES 25

5.13 THE FLUORESCENCE DETECTION VALUES ARE ABNORMAL 27

6 FAQS OF REAGENT APPLICATIONS 29

1.2 User Warranty

1.3 Intended Use of the Instrument

1.4 General Safety Warnings

1.5 Disposal Management Concept

HumaCycler | Frequently Asked Questions

1.6 Biohazard Warning

1.7 Instrument Disinfection

HumaCycler | Frequently Asked Questions

2 BASIC FREQUENTLY ASKED QUESTIONS (FAQS)

2.1 What is the instrument’s application?

2.2 What are the specifications of the instrument?

Specification/Model: HumaCycler (4 channel instrument) Table 1

2.3 What are the specifications of the fluorescent dyes?

2.4 Which minimum computer configurations are required?

2.5 To which product categories does the instrument belong?

HumaCycler | Frequently Asked Questions

2.6 What is the basic structure of the instrument?

2.7 How does the bottom detection technology work?

2.8 Where to place the instrument?

2.9 How does the shipping lock work?

Fixing Pin on the right

HumaCycler | Frequently Asked Questions

Fixing Pin in the

2.10 How to turn the instrument on and off?

2.11 How to connect the instrument to the computer?

Accessory Accessory Configuration

2.12 How to install the driver and USB adapter box?

2.13 How to clean and maintain the instrument?

HumaCycler | Frequently Asked Questions

2.14 Which consumables are recommended?

2.15 Which function has the yellow panel lamp?

2.16 How does the automatic hot-lid work?

2.17 How does the XY-scanning system act ?

HumaCycler | Frequently Asked Questions

2.19 Can the instrument be adjusted to scan single rows?

2.20 Can two instruments run with one computer?

2.21 Which function has the Tag-number of the instrument?

HumaCycler | Frequently Asked Questions

2.22 How to import the calibration parameters to another

2.23 Do the data files exist after uninstalling the software?

HumaCycler | Frequently Asked Questions

3 PRODUCT CALIBRATION AND ALARMING

3.1 How to handle the failure alarm?

3.2 At which temperature ranges does the instrument work?

3.3 What are the alarming conditions of the temperature sensor?

HumaCycler | Frequently Asked Questions

4 USB AND BLUETOOTH INSTALLATION

4.1 How to install the Bluetooth connection?

4.2 How to configure the Bluetooth device?

4.3 How to configure the Bluetooth software?

After connecting with the computer:

1. Start the HCy4 software on the computer and press Run.

HumaCycler | Frequently Asked Questions

5 FAQS OF THE INSTRUMENTS FAILURE CASES