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LORRCA MAXSIS

USER MANUAL Version 5.04 MRN-231-EN

Master Registration Number: MRN-231-EN

Manufacturer: RR Mechatronics Manufacturing B.V.

Phone: +31 229 291 129

Fax: +31 229 241 534

E-mail: [email protected]

Internet: http://www.rrmechatronics.com

Postal address: P.O. Box 225

1620 AE Hoorn The Netherlands

Office address: De Corantijn 13 1689 AN Zwaag The Netherlands

© Copyright RR Mechatronics Manufacturing B.V.

All rights reserved. Subject to changes without prior notice.

Issued by the Support Department of RR Mechatronics

Lorrca Maxsis User Manual Page 3 Version 5.04 MRN-231-EN

Document history overview MRN-231-EN

Published date Friday, 15 March 2019

Issue No Date Revised Section(s)

Changes Authorised

5.04 March 2019 Safety

Installation

Operational instructions

Maintenance

Mains specification changed

Oxygenscan procedure changed

Cleaning instructions changed

Document History added as appendix, only latest changes in introduction

H. Schavemaker

5.03 January 2019 Installation Program


Introduction of Oxygenscan option

H. Schavemaker

5.02 October 2017 Installation Program

Introduction Software version 5.xx

Lorrca 1.01

H. Schavemaker

Contents


CONTENTS

1. ABOUT RR MECHATRONICS .................................................................................... 11

2. INTRODUCTION .......................................................................................................... 12

2.1. About this manual .................................................................................................... 12

2.2. Typographical conventions ...................................................................................... 13

2.3. Used symbols .......................................................................................................... 14

3. INSTRUMENT DESCRIPTION ..................................................................................... 15

3.1. Instrument label Lorrca® MaxSis ............................................................................ 16

3.2. Technical specifications ........................................................................................... 17

3.3. Introduction Lorrca® MaxSis ................................................................................... 20 3.3.1. Intended use ................................................................................................. 20 3.3.2. Features of the Lorrca® MaxSis ................................................................... 21 3.3.3. Symbols ........................................................................................................ 22 3.3.4. General measurements description .............................................................. 24

3.3.4.1. Principle of operation Lorrca® MaxSis ..................................................... 24 3.3.4.2. Introduction Osmoscan ............................................................................. 26 3.3.4.3. Introduction RBC-elongation .................................................................... 26 3.3.4.4. Introduction RBC-aggregation .................................................................. 26 3.3.4.5. Introduction Oxygenscan .......................................................................... 27

4. SAFETY ........................................................................................................................ 28

4.1. General safety rules ................................................................................................ 28

4.2. Electrical safety ....................................................................................................... 30

4.3. Laser and mechanical safety ................................................................................... 31

4.4. Bio Safety bob assembly ......................................................................................... 32

4.5. Safety for working with compressed Nitrogen ......................................................... 33

4.6. Bio safety Standard Operating Procedures (S.O.P.) ............................................... 34 4.6.1. Basics of bio safety ....................................................................................... 34 4.6.2. S.O.P. for working with bio hazardous materials .......................................... 35

4.6.2.1. Facts and definitions: ................................................................................ 35 4.6.2.2. Medical requirements: .............................................................................. 35 4.6.2.3. General laboratory practices: ................................................................... 35 4.6.2.4. Specific laboratory practices and requirements: ....................................... 36

5. INSTALLATION ........................................................................................................... 37

5.1. Pre-installation requirements ................................................................................... 37

5.2. Transport ................................................................................................................. 39

5.3. Environmental conditions and setting up the instrument ......................................... 40 5.3.1. Conditions ..................................................................................................... 40 5.3.2. Main power connection ................................................................................. 41 5.3.3. Unpacking / Setting Up ................................................................................. 41

5.4. First Start-up ............................................................................................................ 45

Contents

Page 6 Lorrca Maxsis User Manual Version 5.04 MRN-231-EN

5.5. Checking Lorrca® MaxSis functionality ................................................................... 47 5.5.1. Instrument status .......................................................................................... 48 5.5.2. Lorrca IO ....................................................................................................... 49 5.5.3. Camera ......................................................................................................... 50

6. LORRCA MAXSIS PROGRAM .................................................................................... 51

6.1. Main screen ............................................................................................................. 52

6.2. Main screen settings ................................................................................................ 52 6.2.1. Screen communication settings .................................................................... 53 6.2.2. End of the day wash screen ......................................................................... 54

6.3. Hardware check status ............................................................................................ 54 6.3.1. Hardware check IO screen ........................................................................... 55 6.3.2. Hardware check Camera screen .................................................................. 56

6.3.2.1. Hardware check Camera Led on screen .................................................. 57 6.3.3. Hardware pO2 .............................................................................................. 57

6.4. Settings screen ........................................................................................................ 58 6.4.1. Files .............................................................................................................. 59 6.4.2. General options ............................................................................................ 60 6.4.3. Elongation ..................................................................................................... 60

6.4.3.1. Deformability curve Parameters ............................................................... 60 6.4.3.2. Stress curve determination Parameters ................................................... 61 6.4.3.3. Stability test parameters ........................................................................... 62

6.4.4. Aggregation .................................................................................................. 62 6.4.4.1. Disaggregation options ............................................................................. 62 6.4.4.2. Syllectogram options ................................................................................ 63 6.4.4.3. Iteration parameters setting ...................................................................... 64 6.4.4.4. FSAR options ........................................................................................... 66

6.4.5. Osmoscan options ........................................................................................ 66 6.4.6. Oxygenscan options ..................................................................................... 67

6.4.6.1. pO2 Steps................................................................................................. 68 6.4.7. Screen communication settings .................................................................... 71

6.5. Osmoscan screen .................................................................................................... 73 6.5.1. Osmoscan result screen ............................................................................... 74

6.5.1.1. Osmoscan parameters explanation .......................................................... 75 6.5.1.2. Rinse gradiënt Osmoscan screen ............................................................ 76

6.5.2. Osmoscan curve ........................................................................................... 77 6.6. Deformability curve determination screen ............................................................... 78

6.6.1. Deformability curve result screen ................................................................. 79 6.6.1.1. Deformability curve determination parameters explanation ..................... 80 6.6.1.2. Explain Rinse cup deformability screen .................................................... 80

6.7. Oxygenscan screen ................................................................................................. 81 6.7.1. Oxygenscan result screen ............................................................................ 82

6.7.1.1. Oxygenscan parameters explanation ....................................................... 83 6.7.1.2. Explain Rinse gradient Oxygen screen .................................................... 83

6.8. Stability Curve determination screen ....................................................................... 85 6.8.1. Stability curve determination results screen ................................................. 86

6.8.1.1. Stability curve determination parameters explanation .............................. 87 6.8.1.2. Open Stability curve files .......................................................................... 87

Contents


6.9. Cell Membrane stability screen ............................................................................... 88 6.9.1. Deformability results 1 screen ...................................................................... 89 6.9.2. Rinse cup cell membrane screen ................................................................. 89 6.9.3. Empty cup cell membrane screen ................................................................ 90 6.9.4. Cell membrane stability results screen ......................................................... 91 6.9.5. Deformability results 2 screen ...................................................................... 92 6.9.6. At measurement completion ......................................................................... 93

6.9.6.1. Deformability results 1 parameters explanation ....................................... 93 6.9.6.2. Stability results parameters explanation ................................................... 94 6.9.6.3. Deformability results 2 parameters explanation ....................................... 95

6.10. RBC aggregation screen ...................................................................................... 96 6.10.1. RBC aggregation result screen ..................................................................... 97

6.10.1.1. Aggregation parameters explanation ...................................................... 98 6.10.1.2. Open Aggregation files ......................................................................... 100 6.10.1.3. Rinse cup RBC Aggregation................................................................. 100

7. GENERAL OPERATION PROCEDURE .................................................................... 101

7.1. Powering up ........................................................................................................... 101

7.2. Preparations .......................................................................................................... 102 7.2.1. Lorrca reagents ........................................................................................... 102 7.2.2. RBC necessary per test/scan ..................................................................... 103 7.2.3. Estimated RBC in laser ............................................................................... 104 7.2.4. Oxygenscan whole blood volume calculation ............................................. 104

7.3. Performing Osmoscan ........................................................................................... 104 7.3.1. Preparing a blood sample ........................................................................... 105 7.3.2. Place sample tube Osmoscan screen ........................................................ 106 7.3.3. Adjust video camera Osmoscan screen ..................................................... 108 7.3.4. Osmoscan result screen ............................................................................. 109 7.3.5. Start new measurement .............................................................................. 109

7.4. Performing Deformability curve test ...................................................................... 111 7.4.1. Preparing a blood sample ........................................................................... 111 7.4.2. Insert blood sample deformability screen ................................................... 112 7.4.3. Adjust video camera deformability screen .................................................. 114 7.4.4. Deformability curve result screen ............................................................... 115 7.4.5. Start new measurement .............................................................................. 116

7.5. Performing Cell stability test .................................................................................. 117 7.5.1. Preparing a blood sample ........................................................................... 117 7.5.2. Insert blood sample stability screen ............................................................ 118 7.5.3. Adjust video camera stability screen .......................................................... 120 7.5.4. Stability curve determination results screen ............................................... 121 7.5.5. Start new measurement .............................................................................. 122

7.6. Performing Cell membrane stability test ................................................................ 123 7.6.1. Preparing a blood sample ........................................................................... 123 7.6.2. Insert blood sample membrane screen ...................................................... 124 7.6.3. Adjust video camera membrane screen ..................................................... 126 7.6.4. Cell membrane stability results screen ....................................................... 127 7.6.5. Deformability results 2 screen .................................................................... 128 7.6.6. Start new measurement .............................................................................. 128

Contents


7.7. Performing RBC Aggregation test ......................................................................... 130 7.7.1. Preparing blood sample for RBC aggregation test ..................................... 131 7.7.2. Insert blood sample RBC aggregation screen ............................................ 132 7.7.3. Adjust temperature RBC aggregation screen ............................................. 133 7.7.4. RBC aggregation result screen ................................................................... 134 7.7.5. Start new measurement .............................................................................. 135

7.8. Performing Oxygenscan ........................................................................................ 137 7.8.1. Pre-test preparations .................................................................................. 137 7.8.2. Preparing a blood sample Oxygenscan ...................................................... 138 7.8.3. Place sample Oxygenscan test .................................................................. 138 7.8.4. Oxygenscan ................................................................................................ 141 7.8.5. Discard of sample ....................................................................................... 142

7.9. End of day wash .................................................................................................... 142

7.10. Powering down .................................................................................................. 144

7.11. Decontamination procedures Routine ................................................................ 145

7.12. Waste container disposal ................................................................................... 145

8. REPORTING .............................................................................................................. 146

8.1. Deformability Report .............................................................................................. 146

8.2. Cell Membrane Stability report page 1 of 3 ........................................................... 147



8.5. Osmoscan Report .................................................................................................. 150

9. TROUBLE SHOOTING .............................................................................................. 151

9.1. Error messages ..................................................................................................... 152

9.2. Error Messages (Oxygenscan) .............................................................................. 154

9.3. Trouble shooting table ........................................................................................... 156

10. MAINTENANCE ......................................................................................................... 157

10.1. Maintenance schedule ....................................................................................... 158

10.2. Cleaning ............................................................................................................. 159

10.3. Oxygen sensor spot protocol ............................................................................. 160

10.4. Replacement sensor-spot .................................................................................. 161

10.5. Visual checks ..................................................................................................... 163

10.6. Lorrca reagents routine ...................................................................................... 163

10.7. Adjusting geometry parameters ......................................................................... 164

10.8. Back up the results file ....................................................................................... 164

10.9. Update the software ........................................................................................... 164

10.10. Adjustments and replacements ...................................................................... 165 10.10.1. Adjusting projection screen ..................................................................... 165 10.10.2. Adjusting camera for slides ..................................................................... 167 10.10.3. Replace the main fuses ........................................................................... 170 10.10.4. Waste and rinse pump tube replacements .............................................. 171

Contents


10.10.4.1. Waste and rinse pump tube replacement ........................................... 171 10.10.4.2. Pump connection tubing ..................................................................... 172

10.10.5. Tubing replacements ............................................................................... 174 10.10.5.1. Gradient pump tube replacement ....................................................... 175 10.10.5.2. Install LORC070901 tubing ................................................................ 176 10.10.5.3. Install LORC070902 tubing set 1 ........................................................ 179 10.10.5.4. Install LORC070903 tubing set 2 ........................................................ 181 10.10.5.5. Install LORC070904 tubing set 3 ........................................................ 183 10.10.5.6. After replacement of all tubing ............................................................ 183

10.10.6. Preparation for long time inactivity .......................................................... 184

11. APPENDIX FOR LORRCA® MAXSIS ....................................................................... 185

11.1. Preparation for transport. ................................................................................... 186

11.2. Instructions for packing: ..................................................................................... 187

11.3. Part 1: ................................................................................................................ 187

11.4. Part 2a: .............................................................................................................. 188

11.5. Part 2b: .............................................................................................................. 189

11.6. Appendix - Document History Extended overview ............................................. 194 11.6.1. Preface ....................................................................................................... 197 11.6.2. Principle of operation .................................................................................. 198

11.6.2.1. RBC-deformability measurements ........................................................ 199 11.6.2.2. RBC-aggregation and –relaxation measurements ............................... 205 11.6.2.3. Flow to Stasis Aggregation Ratio (FSAR) ............................................ 212

11.6.3. References ................................................................................................. 215

12. DRAWINGS AND PARTS LORRCA® MAXSIS ........................................................ 219

12.1. LORC070020 Tubing diagram ........................................................................... 219

12.2. LORC080020 Connection diagram .................................................................... 220

13. LOG BOOK (EXAMPLE) ........................................................................................... 221

14. GLOSSARY OF TERMS ............................................................................................ 223

15. INDEX ......................................................................................................................... 225

About RR Mechatronics


1. ABOUT RR MECHATRONICS

Since being founded in The Netherlands in1986, we have adhered to the belief in the exponential strength of technological advancement. RR Mechatronics develops, manufactures and markets worldwide instruments for scientific research- and medical centres. Microprocessor technology plays a vital role in every aspect. We have a track record of thinking in terms of solutions, not problems and do not have all the answers, but persistently strive to find them in close collaboration with research scientists and our other partners. These partnerships significantly contribute to our high quality work.

RR Mechatronics is ISO 13485:2016 certified by Dekra certification.

The unique collaboration between the scientific world and our research & development department enabled us to develop the Lorrca® MaxSis. This also makes it our responsibility to link and disseminate the knowledge gained in the worldwide community of researchers and clinicians.

Our people are passionate about the invention and development of highly precise analytic instruments. RR Mechatronics focuses on medical analytics laboratory instruments. We serve laboratory customers and other partners all over the world. About 90% of our total production is exported through a worldwide network of exclusive and qualified distributors.

RR Mechatronics is the world leader in automated true Westergren ESR instruments.

In addition to the haematology related instruments, RR Mechatronics develops very specific environment related high technology instruments. To complement our line of instruments, we also offer diluents, cleaning-agents, controls, and specific reagents.

Mechatronics USA is the specialized sales and service organization for RR Mechatronics’ instruments sold in the United States. We understand that service of daily used instruments must be performed on-site and we are organized to do so. Mechatronics operates one service number for any service request and is available 24/7.

Introduction


2. INTRODUCTION

2.1. About this manual

This manual describes all necessary procedures and information required for the correct and successful use of the Lorrca® MaxSis, covering:

Installation

Normal operation

User maintenance

Troubleshooting

This manual does include details about possible errors, listed in the chapter troubleshooting. Here a description can be found about known errors of the instrument. If other errors occur, which are not mentioned in this section, please contact our service department.

RR Mechatronics retains the right to make any changes to the contents without any previous warning. Check our website for the latest version of this manual http://www.rrmechatronics.com.

For information concerning settings, repair and maintenance which is not mentioned in this manual, please contact RR Mechatronics.

Any remarks or suggestions will be gratefully received. RR Mechatronics accepts no liability for any mistakes in this User Manual, nor for the consequences of any faulty interpretation of text or drawings.

Read the whole Manual through carefully, especially chapter Safety (on page 28), before using the instrument.

The section LoRRca MaxSis Program (on page 51) is interactive and most of the software functions are the same as in the program itself.

Introduction


2.2. Typographical conventions

Menu selection Items you must select, such as menu options, command buttons, or items in a list.

Emphasis Use to emphasise the importance of a point or for variable expressions such as parameters.

In Software descriptions:

Tabs like SCAN SETTING

Text buttons like "Stop", "Save"

Toggle-buttons like [p-type], [<=X]

Input parameters like Current Density, Scan Speed

Names of the keys on the keyboard like

[ALT]+[PRINT SCREEN]

Specific warning text is shown as:

Warning: Do not open during operation

Introduction


2.3. Used symbols

In this manual, the following graphical symbols are used to draw attention to important information:

This symbol indicates warnings and other danger related information. Ignoring this information could result in personal injury, damage to the instrument or property or malfunction of the instrument.

Warning sign to prevent personal injury due to electrical shocks

Warning sign to prevent personal injury due to rotating parts

Warning sign to prevent personal injury due to biohazard.

Dangerous laser radiation. Avoid direct exposure to laser beam. Laser safety class 3R

Information sign on the supplied materials intended to inform about the “USE BY” date.

This symbol indicates useful tips and other important information, that are not related to any specific danger.

This symbol indicates a reference to this or other product documentation.

Instrument description


3. INSTRUMENT DESCRIPTION

The Lorrca® MaxSis consists of the instrument and a control PC with printer. You can find detailed drawings of the main components in LORC109000 Desk arrangement.

Main components:

1. Computer and display 2. BOB 3. CUP 4. Projection screen 5. De-ionized water bottle, Waste bottle 6. Pumps 7. Valves 8. Laser 9. Osmo High, Osmo Low, Elon ISO bottles 10. Sample unit



3.1. Instrument label Lorrca® MaxSis

The CE marking and other important information can be found on the Mechatronics instrument identification plate, an illustration of this is shown below with explanation.

The instrument identification plate is located on the right-hand side of the rear panel of the instrument under the main switch panel.

Explanation:

1. Name 2. Type 3. Instrument serial number 4. Electrical information 5. Protection class 6. Date of manufacturing 7. Warning(s) 8. Marking(s) and info



3.2. Technical specifications

Technical specifications for the Lorrca® MaxSis:

Lorrca® instrument models:

Model Model name Catalogue number

Lorrca® MaxSis LORC109003

Mains voltage 100/240V 50-60Hz

Fuse (20 x 5 mm) Slow blow 220V 1.6 Amp

Slow blow 110V 3.15 Amp

Power consumption Standby 100 VA

Maximum 150 VA

Heat output Standby 100 Watt

Full operation 150 Watt

Dimensions Width 470 mm

Height with closed lid 400 mm

Height with open lid 630 mm

Depth 300 mm

Total weight 70 Kg

Min. table size Width 1000 mm

Lorrca+Laptop Depth 700 mm

Sound level < 55 ± 2 dB

Working temperature 15 - 35 °C

Relative humidity 10 - 90%

Specifications of other equipment

Camera Objective mount C-mount

Connection USB 3.0

Minimal resolution 780x582 black/white



Dimensions 40x40x40 mm

Laser Wave length 655 nm

Focus distance 170 mm

output power Max 3 mW

Blood sample Tube solution EDTA-Blood

Deformability test +/- 25 l

Osmoscan +/- 200 l

Oxygenscan +/- 50 l

Tech specs Couette assembly

CUP Inner Radius 16.01 ± 0.005 mm

Surface roughness Ra<0.026 µm

Material PMMA

BOB Outer Radius 15.65 ± 0.005 mm

Surface roughness Ra<0.026 µm

Material PMMA

Ambient temperature + 4°C

Drive Minimal cup speed 0.03 rps

Maximum cup speed 20 rps

Break time from sudden stop from 106 rpm to 0 rpm

<30 ms

Shear rate 0 - 800 s-1

Warming-up time Bob Maximum 2 minutes

Heater Heating accuracy ± 0.2 °C

Range 25 - 45 °C

Tech specs Gradient assembly

Gradient mixer range 0 - 25 mS/cm

mixer accuracy ±-0.5 mS/cm

Reagents 50 - 500 mosm/kg



Duration maximum 10 minutes

Dilution (with full blood) < 200:1 ± 20%

VC elongation measurement

<5% at 3Pa



3.3. Introduction Lorrca® MaxSis

The Lorrca® MaxSis is a new version of the earlier released Lorca (Laser-assisted Optical Rotational Cell Analyzer). The Lorrca® MaxSis (Lorrca® hereafter) combines Red Blood Cell (RBC) deformability by ektacytometry, osmoscan and aggregometry; all temperature controlled. It is capable of fully automated measurement and calculation of various phenomena of RBC’s by analysis of their rheological behaviour.

3.3.1. Intended use

The Lorrca® MaxSis is intended for the following measurements on human and veterinary blood samples:

Red Blood Cell Deformability

Laser diffraction ektacytometry

Relaxation rate

Parametrization of deformation curve

Cell Stability test

Cell Membrane Stability

Red Blood Cell Deformability under various osmotic conditions (Osmoscan)

Osmotic resistance

Red Blood Cell Aggregation

Cell aggregation (kinetics, tendency)

Syllectometry (back-scatter)Disclaimers:

Until otherwise labelled, the Lorrca® MaxSis is NOT released by the manufacturer as an in-vitro diagnostic medical device (IVD).

Until otherwise published, the Lorrca® MaxSis is NOT validated by the manufacturer for any diagnostic or therapeutic purposes.

The manufacturer does not accept liability for any consequences arising from the use of the Lorrca® MaxSis for diagnostic and therapeutic purposes.

Note:

The manufacturer encourages researches who have developed or investigated any possible diagnostic or therapeutic applications for the Lorrca® MaxSis, to contact the manufacturer in order to establish and validate the Lorrca® MaxSis as an IVD for these applications.



3.3.2. Features of the Lorrca® MaxSis

Only 25 l blood needed for each deformability measurement.

Easy sample changing.

3 mW laser-diode in shielded setup for maximum user safety.

Adjustable shear rates from 0 - 800 s-1.

On-line data analyses and storage via digital camera system.

"On screen" control of diffraction pattern.

Temperature controlled.

Automatic rinsing.

Osmoscan, the instrument is equipped for automatic addition of reagents.



3.3.3. Symbols

Quantities

Ý Shear rate (1/s)

Dynamic medium viscosity (mPas; 1 Poise = 100 mPas).

Orientation angle.

Shear stress (Pa; 1 Dyne/cm2 = 0.1 Pa).

Isc Back-scatter intensity (au = arbitrary units).

I Intensity parameter resulting from curve fit.

Units

au Arbitrary units.

s Time, seconds.

Pa Shear stress, Pascal (1 Pa = 10 Dynes/cm2).

mPa.s Dynamic viscosity (1 mPa.s = 1 cP (centi-Poise)).

mm Distance, millimeter.

°C Temperature, degrees Centigrade.

Elongation parameters

A Vertical axis of diffraction pattern (pixels)

B Horizontal axis of diffraction pattern (pixels).

EI Elongation Index (1).

R Image aspect ratio (%).

Eof Error of fit (au), average distance of the edge pixels to the best-fit ellipse.

Aggregation parameters

Isc dis Back-scatter intensity during disaggregation phase (au).

Upstroke Height of syllectogram upstroke (au).

Isc top Back-scatter intensity of syllectogram peak (au).



Isc ½ Back-scatter intensity after one syllectogram half-life (au).

Isc0 Syllectogram base level (au).

Amp Syllectogram amplitude (au).

AreaA Area above syllectogram (first 10 s) (au*s).

AreaB Area below syllectogram (first 10 s) (au*s).

AI Aggregation Index (%).

t top Time to reach syllectogram peak after cup cessation (s).

t ½ Syllectogram half-life (s)

FSAR Flow to Stasis Aggregation Ratio (1).

Eof Root of mean square sum of 300 syllectogram samples uniformly spaced on a log time scale (au).

Ir RBC-shape recovery contribution (au), result of curve fit.

Tr RBC-shape recovery time constant (s), result of curve fit.

If RBC-rouleaux formation contribution (au), result of curve fit.

Tf RBC-rouleaux formation time constant (s), result of curve fit.

Is RBC-3D aggregate formation contribution (au), result of curve fit.

Ts RBC-3D aggregate formation contribution (s), result of curve fit.

I0 Syllectogram base level (au) result of curve fit.

Isc max Highest back scatter intensity found during iteration procedure (au).

Ý at Isc max

Shear rate (1/s at Isc max.

dIsc min Lowest back scatter intensity found during iteration with intermediate disaggregation (au).

ý at min Shear rate (1/s at dIsc min.

Textual

<…> Text between < > refers to program buttons or to menu items.

NaN Not a Number (when, e.g., a parameter cannot be calculated)

RBC Red Blood Cell



SD Standard deviation

3.3.4. General measurements description

This section gives a short description of the measurements, for more information see Appendix - Additional Information.

3.3.4.1. Principle of operation Lorrca® MaxSis

In order to evaluate the flowing capacity of blood in different parts of the body it is important to have a technique available for the accurate measurement of Red Blood Cell (RBC) properties.

The LoRRca MaxSis is therefore an indispensable instrument. It uses a Couette geometry with a static BOB and a rotating cylinder (CUP) to create a simple shear flow.



Cell deformability

A thin layer of red blood cell suspension is sheared between two concentric cylinders. The rotation of the outer cylinder (CUP), causes deformation (elongation) of the red blood cells. The software instructs the operator when to insert the test suspension, shows the current temperature during warming-up and performs fully automatic RBC deformability and stability measurements. The laser beam diffraction pattern is detected with a video camera and analysed by a computer. The RBC elongation program quantifies the following:

Stress dependent (Deformability curve)

Time dependent (Stability test) deformability of red blood cells

Combination of both (Cell Membrane Stability Test)

Cell aggregation

Various aggregation indices are determined in whole blood by means of reflection measurements. The software instructs the operator when to insert the test suspension. A laser diode, mounted in the BOB, serves as light source. The reflected light is sensed by a photodiode.

The RBC-aggregation software is written for creating the syllectogram and for measuring the threshold shear rate at which RBC aggregation is just prevented.

The default setting is the tri-exponential model investigator is enabled to select a syllectogram-fitting model containing one to three exponents, which describes the whole syllectogram, including the upstroke. It is possible to change the settings:

The uni-exponential model is interesting to study, e.g., RBC-shape recovery in buffer solution

The conventional bi-exponential model describes the aggregation process while the tri-exponential model includes both RBC-shape recovery and aggregation



3.3.4.2. Introduction Osmoscan

The Lorrca® MaxSis has an optional equipment with the possibility for automatic measurement of Red Blood Cell (RBC) deformability (expressed as elongation index, EI) as a continuous function of suspending medium osmolality. Both the shape and the position along the osmolality axis of specific features of these, so called osmoscans provide information about the cell`s deformability (at the chosen shear stress) as well as their intracellular viscosity and surface-volume ratio. For characterization of each osmoscan 4 parameters are defined:

Omin (the osmolality at the EI minimum found in the hypotonic region)

EImax (the maximal EI of the total curve)

Omax (the osmolality at EI-max)

Ohyper (the osmolality in the hypertonic region corresponding to 50% of the EI-max).

Application of this approach to a variety of RBC disorders is in line with first generation ektacytometers.

Osmoscans obtained after various experimental manipulations (heat treatment, exposure to shear stress and glutaraldehyde) of normal RBC, demonstrating the expected effects.

3.3.4.3. Introduction RBC-elongation

RBC elongation program quantifies the stress dependent (Deformability curve) and time dependent (Stability test) deformability of red blood cells, or a combination of both (Cell Membrane Stability Test). The software instructs the operator when to insert the test suspension, shows the current temperature during warming-up and performs fully automatic RBC deformability and stability measurements.

3.3.4.4. Introduction RBC-aggregation

RBC-aggregation software is written for measuring the syllectogram and for measuring the threshold shear rate at which RBC aggregation is just prevented. The software instructs the operator when to insert the test suspension. The investigator is enabled to select a syllectogram-fitting model containing one to three exponents. The uni-exponential model is interesting to study, e.g., RBC-shape recovery in buffer solution. The conventional bi-exponential model describes the aggregation process while the tri-exponential model includes both RBC-shape recovery and aggregation. Unlike other models, the tri-exponential model describes the whole syllectogram, including the upstroke.



3.3.4.5. Introduction Oxygenscan

The Oxygenscan measures the relative oxygen pressure at the critical point the RBCs of a SCD patient start to sickle. This so called "point of sickling" (see Figure. 1) quantitatively and reproducibly describes the defined loss of deformability of sickle cell disease (SCD) RBCs.

Fig.1 Graphic representation of the measured loss of deformability, the point-of-sickling of SCD RBCs, as a result of oxygen-depletion in time, followed by subsequent gain of deformability of RBC’s during reoxygenation, as is visualized on the Oxygenscan. It is described by pO2: Oxygen- pressure (controlled, in mmHg), and by EI: elongation index (of the RBCs in shear rate), in SC RBCs: Sickle Cell patients Red Blood Cells.

Oxygenscan read out parameters are:

EImax, which represents RBC deformability at normoxia;

EImin represents deformability upon deoxygenation;

The point of sickling (PoS), the point at which a >5% decrease in EI is observed during deoxygenation, reflecting the patient-specific pO2 at which sickling begins (Figure 1).

This PoS is the most characteristic parameters of this measurement. It is typical for each sickle cell RBC sample (i.e. patient) and sensitive to its status; the PoS shifts due to alteration in treatment.

Supported by the (dynamics of) the change in cell deformability (Elongation index; EI), this measurement of typical RBC behaviour directly reflects the status of the patients’ susceptibility to oxygen depletion.

The Oxygenscan measurement represents the ‘whole population’ of RBCs (incl. HbS as well as HbF containing cells).

Safety


4. SAFETY

Read this paragraph carefully before using the instrument.

4.1. General safety rules

The instrument described in this manual is designed to be used by properly trained personnel only. For the correct and safe use of this instrument it is essential that both operating and servicing personnel follow generally accepted safety procedures in addition to the safety precautions specified in this manual.

Execute your work according to this manual. Read the instructions before operating the instrument. Observe all cautionary markings in the manual and on the instrument. Keep this manual for future reference.

Follow the bio-safety procedures when working with blood-contaminated parts.

Repair can only be executed by trained and qualified personnel.

Wear protective clothing.

When the instrument is running it is not allowed to open the hood.

It is not allowed to give access to the instrument to a non-authorised person at any time.

Whenever it is likely that safety-protection has been impaired, the instrument must be made inoperative and be secured against any unintended operation. The matter should then be referred to qualified technicians.

Safety protection is likely to be impaired if, for example, the instrument fails to perform the intended measurements or shows visible damage or unusual smells, smoke, liquids are flowing out.

Treat reagents according the material safety data sheets. On our web site the latest version of Leaflet on material safety data sheets of the used reagents can be found.

Do not spill fluids on to the instrument. This could cause a short-circuit. If this happens, turn the main switch OFF immediately and unplug the power cable. Contact your service representative.

Should the instrument emit unusual odours or smoke, turn the main switch OFF immediately and unplug the power cable. Using the instrument further carries the risk of fire, electric shock or personal injury. Contact your service representative.

Do not touch the electric circuits inside the instrument particularly with wet hands, as there is a risk of electric shock.

This instrument must be connected to a power outlet of the correct voltage.

Avoid damage to the power cable. Do not place any appliances on the power cable nor pull on the power cable.

Safety


Use the instrument only for measurements the way described in this manual. The vendor is not responsible for any damage or harm resulting from incompetent or undocumented use.

Inside the instrument laser light is used for both the elongation and aggregation measurements. Avoid direct exposure to the laser radiation since this may cause permanent damage to human tissue. Always close the hood before starting a measurement.

During measurements with the instrument the cup is rotating. Never touch the cup while it is rotating.

The waste must be treated as potentially infectious (biohazards) material and disposed of according to local regulations. Check your local environment rules about discharging the waste.

Inside the instrument hazardous voltages reside. Never open the Lorrca® MaxSis. Leave servicing to qualified service personnel only.

Avoid exposing the instrument to high temperatures (higher than 35°C), direct sunlight or high humidity (> 80 %).

Before starting work check the technical condition of the instrument and pay particular attention to:

Protective safety parts: Do they work properly and are they in the correct place.

Loose tubes: Connect them to the right position. See drawing LORC070020 Tubing diagram (on page 219)

Electrical connections: are they not damaged.

Light: Does it works properly.

Prepare required tools.

Never leave the instrument unattended during work. Refit the protective hood after any repair and/or maintenance work.

WARNING: Ensure the protective hood is closed during normal instrument operation!

Safety


4.2. Electrical safety

The instrument has protective covers, which prevent direct access to the inner parts of the instrument.

Use only the original delivered main power cord or exact equivalent

The protective covers have the sign:

WARNING: Ensure that covers are closed during normal use.

Always connect to grounded wall sockets. Set the main switch to the "0" position before opening and/or closing the covers! Work and/or measurements on the mains power section and other control parts may only be carried out by properly trained personnel.

Safety


4.3. Laser and mechanical safety

When the hood is open avoid direct eye exposure

The corresponding position of each label on the product indicated:

Label on the outside of the instrument

This instrument is equipped with a Class 3R laser. Avoid direct eye exposure.

Label inside of the instrument

Be aware of the laser aperture on the left side of the bob.

Safety


Laser aperture

"CAUTION – USE OF CONTROLS OR ADJUSTMENTS OR PERFORMANCE OF PROCEDURES OTHER THAN THOSE SPECIFIED HEREIN MAY RESULT IN HAZARDOUS RADIATION EXPOSURE"

A Class 3R laser is considered to be safe if handled carefully, with restricted beam viewing.

EYE INJURY HAZARD -- DIRECT AND REFLECTED BEAM

Do NOT deliberately look into or stare into the beam this can cause injury to the retina.

Be aware of beam reflections off glass and shiny surfaces. Depending on the surface, the reflected beam could be about as strong and as focused as a direct beam.

The Nominal Ocular Hazard Distance (NOHD) for the applied laser is ~15 centimeter.

The likely closest distance of approach is >20 centimeter (the minimum distance for the eye at which the laser can be clearly focused on the retina)

4.4. Bio Safety bob assembly

The bob assembly has a combined sign to for laser safety, bio hazard and temperature restrictions.

See for all bio hazard safety instructions chapter Bio safety Standard Operating Procedures (S.O.P.) (on page 34)

Safety


4.5. Safety for working with compressed Nitrogen

Applicable for Lorrca instruments with Oxygenscan

Please be aware of the danger in terms of changing atmospheric levels of nitrogen or oxygen.

The biggest danger to lab personnel is a rapid depletion of oxygen levels in the lab.

O2 (Volume %) Effects and symptoms on exposed

18-21 No noticable effect detected by the individual. Any source of gas contributing to change in atmosphere should be investigated and assessment carried out

to determine whether environment is safe for continuation of work

11-18 Reduced physical and cognitive function without the individual realising

8-11 Possibility of sudden loss of consciousness. Risk of death below 11%

6-8 Loss of consciousness after a short time period, resuscitation possible if carried out immediately

0-6 Almost immediate loss of consciousness and brain damage highly likely

An oxygen meter can be used to detect rapid increasing gas levels in the working space.

Safety


4.6. Bio safety Standard Operating Procedures (S.O.P.)

In this section the following issues can be found:

Basics of Bio safety

S.O.P. for working with bio hazardous materials

If another, more stringent, safety protocol is in place, please adhere to the strictest version.

4.6.1. Basics of bio safety

Basic rules on bio safety in a laboratory;

Wash hands after handling biological materials, after removing gloves and before leaving work area.

Don't eat, drink, etc. in the work area.

Never mouth pipette.

Take extreme precautions when sharps must be used. Dispose sharps carefully and properly.

Conduct procedures likely to create splashes, sprays, or aerosols within a biological safety cabinet that is certified annually.

Decontaminate work surfaces at least daily.

Decontaminate waste materials before disposal.

Wear a BUTTONED lab coat to protect your clothes.

Wear gloves when hands may come in contact with potentially infectious materials, contaminated surfaces, or equipment.

Wear eye/face protection if splashes or sprays are anticipated during work outside a biological safety cabinet.

Transport materials outside of the laboratory using secondary containment and a cart. Avoid public areas during transport.

Transfer materials according to federal and international regulations.

Be familiar with written instructions for laboratory procedures and proper responses to emergencies.

Report spills, exposures, illnesses, and injuries immediately.

Safety


4.6.2. S.O.P. for working with bio hazardous materials

Purpose:

To inform and educate all persons that work with biohazards

4.6.2.1. Facts and definitions:

Biological hazards are present in all human and animal tissues and body fluids.

The "normal" research activities carried out in a blood laboratory expose workers to human blood, urine, sweat, semen, saliva and muscle tissue.

For the purpose of assessing risk, we assume that all volunteers to our clinical studies are not normal healthy individuals, and take appropriate precautions.

We remain aware at all times that increased knowledge of disease transmission and occupational hazards may result in situations currently considered safe to be reclassified as having risk.

"Universal Precautions" describes a set of procedures for dealing with subjects based on the assumption that they are positive for blood borne pathogens. Other precautions are necessary to prevent exposure to potential respiratory diseases.

4.6.2.2. Medical requirements:

Routine personal medical assessments are advised at regular intervals (yearly) for all personal exposed to potential biohazard. Immunisation for Hepatitis B is recommended for everyone who is taking blood samples or dealing with human blood or body fluids.

4.6.2.3. General laboratory practices:

All users must follow all Departmental Safety Guidelines and bio safety Policy. Each user is responsible to leave a clean, disinfected and tidy work place. All biohazard waste must be properly disposed.

Safety


4.6.2.4. Specific laboratory practices and requirements:

Biohazard waste:

Dispose blood tubes into a biohazard sharps container. Dispose sharps into a biohazard sharps container. All other bio hazardous waste is to be deposited into a biohazard bag. All bio hazardous waste is deposited into the Medical Waste Management (MWM) bin for pick up.

Decontamination procedures:

Routine: At the end of each experiment, or each day, disinfect lab benches and any equipment Spills: Small spills of biohazard material should be treated by first covering them with an absorbent paper to avoid the formation of aerosols. Disinfect the spill by slowly pouring on a disinfecting solution working from the outside to the centre of the spill in a circular motion. Leave the spill long enough for disinfection to take place (check decontaminating instructions on the disinfectant container for time) and then carefully wipe up wearing gloves. Pick up any glass using forceps. Once all the material has been removed disinfect the area thoroughly. Inform the Technician in Charge of the spill.

Food:

No food or beverages will be brought into or consumed inside a blood laboratory at any time.

Accident reporting:

All accidents and injuries must be reported within 24 hours to the technician in Charge, to the Departmental Joint Health and Safety Committee and to the Department of Environmental Health and Safety using an Incident Report from the main office or the Technician in Charge.

Laboratory access:

Access to the haematology laboratory is limited to persons who are directly involved with the testing equipment. Children are not permitted in the laboratory.

Personal protective equipment:

Laboratory and maintenance personnel are expected to use a laboratory coat while working in the blood laboratory. We advise the use of non-canvas closed-toe shoes wherever there is a potential for foot injury from hazardous materials or from small physical objects. Personal outer clothing should not be stored in the blood laboratory. Lab coats worn in the blood laboratory should not be worn outside of the blood laboratory and should not be stored with personal outer clothing, to avoid transfer of contaminants. Gloves are considered contaminated after being worn. Avoid contamination of work surfaces with gloves. Dispose of gloves into a biohazard container. The use of eye protection is advised while processing samples. Remove and properly dispose of gloves and wash hands before leaving the laboratory.

Installation


5. INSTALLATION

Installation means: Transport, final assembly, fitting the connections and the starting up of the instrument. This should only be performed by trained and skilled personnel.

Starting up the instrument and giving instructions for the first (series of) measurements should always be carried out under the supervision of a Service Technician from the manufacturer or their representative.

5.1. Pre-installation requirements

Only when pO2 Scan will be used:

Laboratory Integrated Dry Nitrogen Gas Supply (NOT part of accessory set) consisting of:

Laboratory dry N2 purge system wall socket connection

Low pressure gas tube connection between N2 delivery system and pO2 Scan black tube, which is the PUN system at 4.0 mm outer diameter.

Flux requirement: gas flow minimal 6 ltr/min, optimal 9 ltr/min

Pressure requirement: pressure larger than 0.8 bar, smaller than 1.0 bar or

Stand-Alone Cylinder Dry Nitrogen Gas Supply consisting of:

Pressurized N2 gas cylinder: FOR EXAMPLE UN1066 (F10): Nitrogen 3.0 (purity > 99,9 vol %), CAS Nr.: 7727-37-9 , EG Nr. (EINICS) : 231-783-9. Max 10 ltr (mandatory for safety).

Installation


AR-CO2 gas pressure reduction valve / flow regulator: FOR EXAMPLE Alwell type EN ISO 2503 (as depicted) set to 9 ltr/min. Output pressure 1 bar max. RU3 compatible.

Low pressure gas tube connection between pressure reduction valve and pO2 Scan black tube, which is the PUN system at 4.0 mm outer diameter.

Installation


Low pressure gas tube connection

For safety purposes: Oxygen meter

5.2. Transport

When lifting the packaged instrument, take the following information into account:

The total weight of the instrument is 75 kg (including packaging).

The total weight of the reagent box is depending on the ordered amount.

Installation


5.3. Environmental conditions and setting up the instrument

5.3.1. Conditions

The instrument should be set up on a flat table, level and with sufficient room around it. (see Table size and or Unpacking/Setting Up (on page 41) for dimensions). For good operation, the CUP, BOB, bottles, keyboard and mouse should be easily seen and easy to reach. It is necessary that the locking protection covers are fully accessible for adjustment and repairs.

WARNING: Always connect to grounded wall sockets.

In order to ensure fault-free operation of the instrument, it is essential that the room where it is located is temperature controlled.

(The temperature may not be less than 15 °C, nor exceed 35 °C). Keep the area around the instrument clean and free of dust.

Installation


5.3.2. Main power connection

The instrument is delivered with an 10A/230V rated Euro plug lead 10A with locking system for IEC inlets. Do not replace with inadequately rated cords. Also delivered is an extension socket.

For a 110V connection an IEC Lock PC 1063 120V US power cord can be delivered.

The pO2scan comes along with an additional power strip to make sure both Lorrca + pO2scan can be powered. The power strip is equipped with a safety switch, enabling the user to power down the entire Lorrca + pO2scan setup in case of calamities. The pO2scan draws power from a standard laptop charging power supply.

5.3.3. Unpacking / Setting Up

1. When setting up, take a minimum table space of 700x900 mm in account. Check for damage during transport.

Installation


2. Check the Tip N Tell indicator if the packaging has been tipped. If so, act accordingly.

3. Remove the straps from the packaging and open it.

a. Check delivery for completeness; Lorrca Reagents

b. Printer (if included in order)

c. Lorrca® MaxSis

d. Laptop

e. Accessory kit

4. Take the Laptop, printer and monitor out of the packaging and remove their boxes accordingly. Place it on the table.

5. Connect the Laptop and Printer cables according the laptop and Printer installation instructions. 6. Take the Lorrca® MaxSis box out of the packaging in upright position and place it on the floor. 7. Open the box and lift the instrument out of the box by grabbing the handles (H) on the sides

(not the hood handle) of the instrument. Place it on the table.

8. Remove the protective materials from the instrument (store them to reuse in case of future

transport);

Foam pieces.

Installation


Tie-wraps.

Safety plate on pinch valves

9. Unscrew the bolt from the pump assembly 10. Check/place the correct fuse (F). According ID-plate. Factory default is 230VAC. See section

Replace the main fuses (on page 170)

11. Fill the bottle de-ionized water. Fill the bottles for the Osmoscan. 12. Place the bottles and connect them according the labels. See drawing LORC070020 Tubing

diagram (on page 219) 13. Place tube cassettes of the gradient pump

14. Connect power cables to grounded sockets.

WARNING: Always connect to grounded wall sockets.

Installation


Connect data cables between the instrument and PC and printer.

RS232 symbol

Connect camera connection (USB 3.0) on back panel and on the USB port at the right side of the laptop

Connect the USB-Hub on USB port at the left side of the laptop

For Oxygenscan only: Connect the instrument to a N2-supply with use of the parts from the accessory set and advised additional material (see Pre-installation requirements (on page 37)).

The Instrument is now ready for first start-up.

Make sure you are familiar with the use of the Lorrca® MaxSis, detailed instructions can be found in the electronic version of the User manual which is installed on the PC, click on the Manual ICON to get access to the manual.

Installation


5.4. First Start-up

Steady state

After switching on, the Lorrca® MaxSis needs 5 minutes to warm up.

Before the instrument can be operated, you have to carry out the following checks and procedures:

Check that there are no strange objects (tools, for example) in the instrument.

Switch ON the instrument.

Start-up the PC.

Choose LoRRca software program.

Select <HARDWARE CHECK>.

Move the Bob down.

Choose <CAMERA>

Set camera gain at 250.

Close the hood.

Check the image:

OK: The instrument is now ready.

Not OK: Misaligned

Check position of the projection screen.

Installation


Alignment of the projection screen:

Use the hexagon key 2mm to loosen the projection screen holder.

Align the projection screen until the black dot on the screen is aligned with the laser spot. Note: The projection screen holder itself can also be adjusted (equipped with slotted holes)

Tighten the screws of the projection screen holder.

Not OK: Driver failure.

Update the camera driver. See Camera driver installation (on page 156)

Choose <LORRCA IO><RINSE CUP> and confirm. The tubes are being filled.

Dry BOB and CUP. Do not scratch the cup and do not touch the glass with your fingers to prevent staining! Pay attention to the hole in the BOB.

After this procedure, the instrument is in the start position.

Installation


5.5. Checking Lorrca® MaxSis functionality

Both the elongation and aggregation program feature a “check” window that can be used to test the hardware functions individually:

LoRRca status

LoRRca IO

Camera

The adjustments made with this checking feature are only temporary and will be restored to default when closing the check window.

Installation


5.5.1. Instrument status

Function Description

At home position LED is lit when CUP is at home position (= fixed position along cup circumference where back-scatter intensity is acquired)

Intensity (au) Back-scatter intensity (arbitrary units), should be within the range [0, 100] au during measurements (when blood is inserted).

BOB temperature Current BOB temperature in degrees Centigrade

Run motor at (rps) Motor control. Set to desired cup revolution speed (rps = revolutions per second).

During this test, the laser is turned on (flash) in order to enable back-scatter intensity measurements.

Installation


5.5.2. Lorrca IO


Laser state

Control to select one of the following laser states:

1. On, continuous 2. On, Flash (Modulated) 3. Off.

Motor mode

Control to select one of the following motor modes:

1. Run until in home position, 2. Run continuous. If in the home position, the motor can be re-

activated by changing any of the other motor parameters.

Run motor at (rps) Sets motor revolution speed

Valves With toggling ON/OFF the functioning of the pinch valves can be checked (each valve should give a "click")

Installation


5.5.3. Camera

With this test, the laser is turned ON (continuous) and the motor speed is maintained as set in any of the other tabs. The image shows a coloured version of the diffraction pattern (see screen). The “Use colors” check box can be cleared to view the underlying gray scale image.

Lorrca MaxSis Program


6. LORRCA MAXSIS PROGRAM

The Lorrca® MaxSis is controlled via an external computer that runs the Lorrca® MaxSis program and controlled by mouse pointer.

The following type of measurements can be selected from the main screen:

Default screen is the Main screen (on page 52)

Deformability curve determination screen (on page 78)

Stability Curve determination screen (on page 85)

Cell Membrane stability screen (on page 88)

RBC aggregation screen (on page 96)

Osmoscan screen (on page 73)

Oygenscan screen (on page 81)



6.1. Main screen

6.2. Main screen settings



6.2.1. Screen communication settings

COMMUNICATION SETTINGS: Select the output port for communication.

Lorrca Connection

1. Serial output comport.ASRL1::INSTR. By default 2. Baud rate. Selectable 1200, 4800 ,9600, 38400 and many more default is set on 115200 3. Data bits. Selectable 7 or 8 data bits 4. Party bit: Selectable none, space, mark, even and odd 5. Stop bits: Selectable 1, 1.5 or 2 stop bits 6. Flow control: Selectable for;

None

XON/XOFF

RTS/CTS

DTR/DSR

XON/XOFF & RTS/CTS

XON/XOFF & DTR/DSR

pO2 Connection



6.2.2. End of the day wash screen

When the program is shut down a pop-up screen appears with “End of day wash” message. To perform an End of day wash exchange the rinse bottle with the cleaning solution bottle and follow the software instructions.

6.3. Hardware check status



6.3.1. Hardware check IO screen


Laser state

Control to select one of the following laser states:

1. On, continuous 2. On, Flash (Modulated) 3. Off.

Motor mode

Control to select one of the following motor modes:

1. Run until in home position, 2. Run continuous. If in the home position, the motor can be re-

activated by changing any of the other motor parameters.

Run motor at (rps) Sets motor revolution speed

Valves With toggling ON/OFF the functioning of the pinch valves can be checked (each valve should give a "click")



6.3.2. Hardware check Camera screen



6.3.2.1. Hardware check Camera Led on screen

6.3.3. Hardware pO2



6.4. Settings screen

Before a measurement can be started check if the chosen settings are the appropriate ones, either the default settings or customized settings.

You can always revert to the factory default settings by choosing <FILE ->LOAD DEFAULT>



6.4.1. Files

Settings Description

Storage directory Location for storing result files (measurements, diffraction pattern images). If a "relative" path/name is given, it starts from the program directory.

Auto export method Method for storing measurement results (as Comma Separated Values (CSV) files):

<NO EXPORT> The results of the last measurement are always available from file "Auto export file".CSV". This file is overwritten after each measurement.

<CONCATENATE RESULTS TO SINGLE FILE> With this selection measurements results are always added to a file named "NameHistory.CSV". Some text editors or spreadsheet programs cannot read extremely large CSV-files. It is therefore advisable to backup and delete this file from the hard disk regularly, before its size exceeds the applicable maximum.

<ENUMERATE FILE NUMBER> With this selection, each measurement is stored in a separate file with a name being equal to the patient name (as entered in the "Insert blood sample dialog"). Additional measurements for the same patient, will be stored with an enumeration number [0..n]. For example, the first result file of patient Jones is stored as Jones_0.CSV, the next as Jones_1.CSV and so on.

Auto export file name Name of the file (with extension ".CSV") that holds the last measurement results. If the "Auto export method" is set to "Concatenate results to single file", the name is also used to create the name of the file containing the measurement history.

Store diffraction patterns

Checkbox to enable storing diffraction patterns. Only diffraction patterns are stored that include the actual measurement, i.e., not those during camera adjustment.



6.4.2. General options

Settings parameter Description Range

Fill empty speed (rps) Cup revolution speed (revolutions per second) used at the moment of inserting or removing the test suspension.

[0.1, 10] rps

Shear stress for camera adjustment (Pa)

Shear stress applied during camera adjustment (diaphragm).

[0, 100] Pa

Bob temperature (°C) Desired bob (and hence, suspension) temperature in degrees Centigrade.

[20, 45] °C

Auto start if temp. is stable within (°C)

If temperature is within ± 0.2 °C, the measurement starts after the value given at parameter ... for at least (see next)

[0.1, 10] °C

… for at least (s) Time to automatic start [5, 300] s

6.4.3. Elongation

6.4.3.1. Deformability curve Parameters

Settings parameter Description

Logarithmic shear stress axis

Checkbox Turns this option on/off



6.4.3.2. Stress curve determination Parameters


First shear stress (Pa)

Last shear stress (Pa)

First and last shear stress to apply during the determination of the deformability curve. By taking "First" higher that "Last", the curve will be completed in reverse order.

[0.01, 100] Pa

Initial image stabilisation time (ms)

Following camera adjustment at a high shear stress, the measurement starts with the "first measurement-shear stress", described above. The cells in the suspension need time to adjust to each new shear stress, especially when the shear stress for camera adjustment is high compared to the first measurement-shear stress. This parameter defines the initial cell stabilisation time following camera adjustment. If the standard deviation of the elongation measurement at the first measurement-shear stress is still quite high, the initial stabilisation time may need to be increased.

[0.1, 600] s

Intermediate image stabilisation time (ms)

Like the stabilisation time mentioned in the former parameter description, the cells in the suspension need time to adjust to each new shear stress. This parameter defines the time for stabilisation of the suspension between subsequent elongation measurements.

[0.1, 600] s

Measuring points on curve

Determines the number of shear stresses that are used to build up the deformability curve.

[2, 2000]

Valid determinations per shear stress

Prescribes the number of subsequent EI determinations (each from a newly acquired diffraction image) to find the average EI (and SD) per shear stress.

[2, 1000]



6.4.3.3. Stability test parameters


Stability shear stress (Pa)

Shear stress used during the stability test (Pa) [0.01, 100] Pa

Measurement duration (s)

Duration of stability test. [1, 100000] s

Determine deformability every (s)

Time that elapses before performing another deformability measurement (diffraction pattern analysis).

[1, 3600] s

Valid determinations per measuring point

Prescribes the number of subsequent EI determinations (each from a newly acquired diffraction image) to find the average EI (and SD) per deformability measurement.

[1, 200]

6.4.4. Aggregation

This section gives a short description for all Settings parameters that influence the way aggregation measurements are performed.

Settings parameter Description

Auto store measurements

Checkbox Turns this option on/off

6.4.4.1. Disaggregation options


Disaggregation shear rate (1/s)

This disaggregation shear rate and time apply throughout the aggregation measurement procedure:

1. Before measuring the syllectogram, 2. For intermediate disaggregation during the

iteration procedure. 3. Just before applying the "Low shear rate" during

the FSAR measurement.

[5, 2500] /s

disaggregation time (s) [0, 3600] s



6.4.4.2. Syllectogram options


Logarithmic time axis Check to obtain a logarithmic time scale for the syllectogram. Remove check mark for linear time scale.

Checkbox

Draw syllectogram fit If checked, the syllectogram is superimposed by the result of exponential fitting, using the selected "Syllectogram model (see below).

Checkbox

Sample rate (Hz) Number of samples to take per second when measuring the syllectogram curve. Must be high enough to record fastest details (the peak) of the syllectogram.

[10, 1000] Hz

Syllectogram duration (s)

Syllectogram measurement duration. [1, 3600] s

Syllectogram fitting AUTO: In auto-mode, all models are evaluated and the program will present the best fitting model. The result of other models can still be examined and printed. Mathematical model used to fit the syllectogram:

Tri-exponential (rise-fall-fall)

Bi-exponential (rise-fall)

Bi-exponential (fall-fall).

Uni-exponential (rise)

Uni-exponential (fall)

See Appendix Additional Information section Tri-exponential syllectogram representation (on page 208)

-

Max Error Of Fit (au) It is evaluated whether or not each of the fitting models was successful from the error-of-fit parameter (EOF). Since the syllectogram is sampled uniformly most data points stem from the tail of the curve. To prevent the fitting algorithm from focusing on this region, the curves were re-sampled by selecting 300 points uniformly distributed on a logarithmic time scale. The EOF is defined as the root of the mean square sum of these 300 samples.

[0.01, 100] au



6.4.4.3. Iteration parameters setting


Perform iteration procedure

Checkbox, to include the iteration procedure for the determination of the threshold shear rate (see Appendix). The iteration procedure is determined following measurement of the syllectogram. During iteration, only one sample is taken per cup revolution to avoid optical disturbances.

-

Intermediate disaggregation

Checkbox to disaggregate the blood suspension between successive measurement shear rates. Intermediate disaggregation is recommended and may be favourable as the standard procedure, since potential aggregates will break down during disaggregation, resulting in the same starting point before each back-scatter measurement at a given shear stress.

-

Samples per shear rate

Number of back-scatter-intensity measurements to perform at each shear stress. Each individual sample is taken once per revolution, at the home position. The program records both the mean back-scatter and its standard deviation.

[2, 200]

Maximum deviation (au)

When a new shear rate is adjusted, the aggregation state of the suspension will gradually become stable. The program checks the absolute error between N successive data points and keeps on sampling until the absolute error is smaller than the value entered here or until an iteration-timeout occurs.

[0.1, 0.5] au

Iteration timeout (s) When the aggregation signal does not get stable within the time entered here, the measurement is aborted and an error message appears.

[10, 3600] s

Binary search steps A binary search algorithm is used to automatically generate two more shear rates to fine-tune the rough procedure (which used shear rates obtained from the user defined "iterate.txt" file). Each binary search step results in two more shear rates to measure the back-scatter intensity. They are chosen centered between the current optimum shear rate and next higher and lower shear rates. See also Appendix.

[0, 100]

Sort results Checkbox, to sort the results: The lowest shear rate first and the highest shear rate last (default). By not

-



Settings parameter Description Range sorting the measurement points, and if intermediate disaggregation is not enabled, it is possible to study memory effects (hysteresis) of RBC aggregation vs. shear rate.

Iteration shear rates Presets values to which the iteration procedure is performed. Default values are applicable for human RBC’s.

Press <EDIT> to change the values.

[1, 7745] 1/s

6.4.4.3.1. Edit Iteration shear rates



6.4.4.4. FSAR options


Perform FSAR procedure

Checkbox, to include the FSAR procedure in the aggregation measurement. The FSAR measurement is performed immediately following the iteration procedure. Note: The acquisition of the backscatter intensity during the FSAR measurement is similar to that during the iteration procedure (one sample per cup revolution). Acquisition parameters of the iteration procedure (Samples per shear rate, Maximum deviation, Iteration timeout) also apply to the FSAR measurement.

-

Low shear rate (1/s) Low shear rate for determining FSARIscglow/I0. The low shear rate is taken 10/s by default.

[1, 2500] /s

6.4.5. Osmoscan options

This is only available when “Enable Osmoscan” is checked, and Osmoscan option is available on the instrument


Enable Osmoscan Checkbox, Turns this option on/off -

Osmoscan shear stress (Pa)

… [30, 30] (Pa)

Step size (mOsm/kg) … [5, 5] (mOsm/kg)

Pump speed (ml/s) [0.0053,0.0] (ml/s)

Total scan time (s) … 540 (s)

Total consumption (ml) … (ml)

Calc. area between: Checkbox, Min or [75,75]

…and (mOsm/kg) [450,450]



6.4.6. Oxygenscan options

This is only available when “Enable pO2 scan” is checked, and Oxygen scan module is available on the instrument


Enable pO2 axis Checkbox, Turns this option on/off

Logarithmic pO2 axis Checkbox

pO2 scan shear stress (PA)

0-100

Determine pO2 every (s)

0-3600

Moving average size 0-40

Calc area between: 0-250

...and (mmHg) 0-250

Scan steps see pO2 Steps (on page 68)

pO2 control Checkbox

pO2 control max. speed (mmHg/min)

(visible when pO2 control is activated) For adjusting deoxygenation

pO2 control P-term (visible when pO2 control is activated) For adjusting deoxygenation

pO2 control I-term (visible when pO2 control is activated) For adjusting deoxygenation



6.4.6.1. pO2 Steps

If pO2 control is disabled:



If pO2 control is enabled:



Select HARDWARE SETTINGS at the top of the SETTINGS-screen:

Screen Instrument settings appears:



Settings parameter Description Range/Setting

BOB radius (mm) Outer radius of the static bob, in millimeters.

[5, 50] mm

CUP radius (mm) Inner radius of the rotating cup, in millimeters.

[5, 50] mm

Camera cam1

pO2 Sensor batch number (used for pO2 option)

File name for calibration information sensor-spot

XXXXXX-xxx

6.4.7. Screen communication settings



COMMUNICATION SETTINGS: Select the output port for communication.

Lorrca Connection

1. Serial output comport.ASRL1::INSTR. By default 2. Baud rate. Selectable 1200, 4800 ,9600, 38400 and many more default is set on 115200 3. Data bits. Selectable 7 or 8 data bits 4. Party bit: Selectable none, space, mark, even and odd 5. Stop bits: Selectable 1, 1.5 or 2 stop bits 6. Flow control: Selectable for;

None

XON/XOFF

RTS/CTS

DTR/DSR

XON/XOFF & RTS/CTS

XON/XOFF & DTR/DSR



pO2 Connection

6.5. Osmoscan screen



This screen appears when starting a scan. See Osmoscan options (on page 66) settings for details on the Settings parameters that influence the measurement.

A measurement starts after subsequently clicking <NEW...(F1)>.

On this screen are shown:

1. Coloured diffraction pattern. 2. Curve display screen. 3. Indication of cell shape. 4. Measurement and sample details. 5. Measurement results. 6. Parameters.

6.5.1. Osmoscan result screen

At measurement completion measurement results are displayed on-screen, the software offers an option to print (yes/no). It is possible to print a measurement report by clicking the <PRINT REPORT> button. The latest results automatically overwrite the results file (Default name: Results.CSV). If enabled, the results will also be written to other files (see “Settings/Files (on page 59)”). Return to the main menu by clicking the <MAIN MENU> button.

Check Osmoscan parameters explanation (on page 75)



6.5.1.1. Osmoscan parameters explanation

The following result parameters are given in Results file and printed report:

Parameter Description

# Measurement number

Start time Actual time that was valid after setting a new shear stress and immediately following the image stabilisation time.

End time Actual time that was valid after the last EI measurement at the given shear stress was performed.

Time (s) Time, in seconds, since the beginning of the measurement.

Coordinate count Number of coordinates in the iso-intensity curve that describes the shape of the diffraction pattern.

A (pixels) Vertical axis of ellipse.

B (pixels) Horizontal axis of ellipse.

EI Elongation index

Eof (pixels) Error of fit This parameter represents the average distance (in pixel units) of the iso-intensity pixels to the best-fit ellipse that resulted. It reflects how well the iso-intensity coordinates comply with the ellipse model.

O, (mOsm/kg) Osmolality is a count of the number of particles in a fluid sample, mOsm/kg means milli-osmole per kilogram



6.5.1.2. Rinse gradiënt Osmoscan screen

Screen RINSE GRADIENT SYSTEM appears:

1. Open the hood. 2. Remove empty sample tube. 3. Place tube with demi-water (+/- 2,5 ml). 4. Press <RINSE>. to remove sample and rinse the system. Sample is lost and no longer available.

Screen EMPTY CUP appears.

Press <CLEAN>

5. Remove empty (rinse) tube.

6. Press OK and the Osmoscan screen appears. 7. Press <F1> or click on NEW...F1 to perform another Osmoscan.

or Press <MAIN MENU> to perform other tests (perform in that case also an “END OF DAY WASH (on page 142)”). or Exit the software and perform an “END OF DAY WASH (on page 142)”.



6.5.2. Osmoscan curve

The Osmoscan window appears by clicking the <Osmoscan>-button from the introduction window. A measurement starts after clicking "NEW…(F1)". The figure shows the window that appears before starting a scan. See "Settings" for details on the Settings parameters that influence the measurement.



1. Coloured diffraction pattern. 2. Curve display screen. 3. Indication of cell shape. 4. Measurement and sample details. 5. Measurement results. 6. Parameters



6.6. Deformability curve determination screen

This screen appears when starting a deformability measurement. See Deformability curve (on page 60) for details on the Settings parameters that influence the measurement.



1. Coloured diffraction pattern. 2. Curve display screen. 3. Indication of cell shape. 4. Measurement and sample details. 5. Measurement results.



6.6.1. Deformability curve result screen


Check Deformability curve determination parameters explanation (on page 80)



6.6.1.1. Deformability curve determination parameters explanation





Shear stress (Pa) Applied shear stress.

Coordinate count or Crd cnt

Number of coordinates in the iso-intensity curve that describes the shape of the diffraction pattern.



EI Elongation index.

Eof (pixels) Error of fit. This parameter represents the average distance (in pixel units) of the iso-intensity pixels to the best-fit ellipse that resulted. It reflects how well the iso-intensity coordinates comply with the ellipse model.

#Tries Number of diffraction patterns that were analysed at the current shear stress.

#Valid The number of diffraction patterns that were within the ±2SD confidence interval. This number agrees with the desired count as given in the Settings menu (<Settings<Determinations per shear stress).

6.6.1.2. Explain Rinse cup deformability screen



After a measurement has completed, the program sets the fill/empty speed (<SETTINGS> <GENERAL OPTIONS FILL/EMPTY SPEED (RPS)>) and asks the operator to remove the blood sample.

Press <CLEAN>. Sample is removed and system is rinsed. Sample is lost and no longer available.

A new measurement can now be started or return to the main menu by pressing <MAIN MENU> to perform other tests. Or exit the software and perform an “END OF DAY WASH (on page 142)”.

6.7. Oxygenscan screen

This screen appears when starting a scan. See Oxyscan options (on page 67) settings for details on the Settings parameters that influence the measurement.



1. Coloured diffraction pattern. 2. Curve display screen. 3. Indication of cell shape. 4. Measurement and sample details. 5. Measurement results. 6. Parameters.



6.7.1. Oxygenscan result screen


Check Oxygenscan parameters explanation (on page 83)



6.7.1.1. Oxygenscan parameters explanation










EI Elongation index

P02 (mm/Hg)

N2 (valve)

6.7.1.2. Explain Rinse gradient Oxygen screen


1. Open the hood. 2. Remove empty sample tube. 3. Place tube with demi-water (+/- 2,5 ml).



4. Press <RINSE>. to remove sample and rinse the system. Sample is lost and no longer available. Screen EMPTY CUP appears.

Press <CLEAN>


6. Press OK and the Oxygenscan screen appears. 7. Press <F1> or click on NEW...F1 to perform another Oxygenscan.




6.8. Stability Curve determination screen

This screen appears when starting a stability test. See Stress curve determination (on page 61) for details on the Settings parameters that influence the measurement.






6.8.1. Stability curve determination results screen


Check Stability curve determination parameters explanation (on page 87)



6.8.1.1. Stability curve determination parameters explanation










EI Elongation index


6.8.1.2. Open Stability curve files

A .SMP or .CSV result file can be shown again. Default folder C://LoRRca MaxSis/Results/Elongation.



6.9. Cell Membrane stability screen

This screen appears when starting a stability test. See Stability test (on page 62) Settings for details on the Settings parameters that influence the measurement.

The cell membrane stability test consists of 3 stages.






6.9.1. Deformability results 1 screen


Check Deformability results 1 parameters explanation (on page 93)

6.9.2. Rinse cup cell membrane screen



After a measurement has completed, the program sets the fill/empty speed (<SETTINGS> <GENERAL OPTIONS FILL/EMPTY SPEED (RPS)>) and asks the operator to remove the blood sample.

Press <CLEAN>. Sample is removed and system is rinsed. Sample is lost and no longer available.


Return to Cell Membrane stability screen (on page 88) or Deformability results screen (on page 89).

6.9.3. Empty cup cell membrane screen



6.9.4. Cell membrane stability results screen


Check Stability results parameters explanation (on page 94)



6.9.6. At measurement completion

6.9.6.1. Deformability results 1 parameters explanation

















6.9.6.2. Stability results parameters explanation










EI Elongation index





6.9.6.3. Deformability results 2 parameters explanation
















6.10. RBC aggregation screen

This screen appears when starting RBC aggregation test. See Aggregation (on page 62) settings and the following option selections for details on the Settings parameters that influence the measurement.

1. Disaggregation level. 2. Syllectogram, following cup cessation.

3. Iteration curve (I()) retrieving the threshold shear rate. 4. Results area. 5. Syllectogram model selection, control for displaying the curve fit and an indication of the best-

fitting model



6.10.1. RBC aggregation result screen


Check RBC parameters explanation (on page 98)



6.10.1.1. Aggregation parameters explanation


Result parameter Description

Patient and sample data Contains patient and sample data:

Patient name, Blood donation time, Remarks, Date and time, Sample start/end temperature.

Conventional parameters

Iscdis Average back-scatter intensity measured during disaggregation (prior to syllectogram measurement)

Upstroke Change in back-scatter intensity due to RBC relaxation. Isctop – Isc dis

Isc top Maximum back-scatter intensity recorded throughout the syllectogram => Peak intensity (usually the top of the syllectogram).

Isc½ Back-scatter intensity at t ½

(Isc½ = Isc top – (Amp/2)).

Isc0 Syllectogram base level (average of last 40 samples). Note that the syllectogram duration must be long enough to obtain a valid base level, i.e., at full aggregation.

Amp Syllectogram amplitude (Isc top – Isc0) representing the extent of aggregation.

Area A, Area B, AI See Appendix E.

t top Time at which the peak occurs within the syllectogram (cup cessation is taken as t=0).

t ½ Time that elapses until the peak intensity is reduced by half the amplitude (hence, at Isc ½).

FSAR Flow to Stasis Aggregation Ratio See Appendix E

Results of fit These results correspond to the selected syllectogram fitting model.

Fit Indicates whether the fit was successful (Good) or not (Bad). The fit is considered "Bad" if the Eof exceeds the



Result parameter Description limit given in <Settings<Max Error Of Fit>.

Eof Error Of Fit. Root of mean square sum of 300 syllectogram samples uniformly spaced on a logarithmic time scale. This parameter gives an indication of the goodness of fit.

Ir RBC-shape recovery contribution

(r = recovery).

Tr RBC-shape recovery time constant.

If RBC-rouleaux formation contribution

(f = fast).

Tf RBC-rouleaux formation time constant.

Is RBC-3D aggregate formation contribution

(s = slow).

Ts RBC-3D aggregate formation time constant.

I0 Syllectogram base level.

Iteration

Isc max Highest back-scatter intensity found during the iteration procedure. N.B. This is not the same as "Isc top" obtained from the syllectogram!

g at Isc max Shear rate at which "Isc max" was found.

dIsc min Minimum change in back-scatter intensity found during the iteration procedure (when intermediate disaggregation is enabled). The change in back-scatter intensity is determined with respect to the intensity during the preceding disaggregation period.

g at dIsc min Shear rate at which "dIsc min" was found



6.10.1.2. Open Aggregation files

A .SMP or .CSV result file can be shown again. Default folder C://LoRRca MaxSis/Results/Aggregation.

6.10.1.3. Rinse cup RBC Aggregation

After a measurement has completed, the program sets the fill/empty speed (<SETTINGS> <FILL->EMPTY SPEED>) and asks the operator to remove the blood sample.

Press <RINSE> Sample is removed and system is rinsed. Sample is lost and no longer available.


General operation procedure


7. GENERAL OPERATION PROCEDURE

7.1. Powering up

Switch ON the Lorrca.

Switch ON the PC and printer.

Start Lorrca software.

Steady state After switching on, the Lorrca MaxSis needs 5 minutes to warm up.

For pO2 scan, the Lorrca Maxsis needs at least 30 minutes to warm up.



7.2. Preparations

Check de-ionized water bottle, should be filled

Check Waste bottle, should be empty

Place reagent bottles

Check applicable viscosity of reagents

Check the gradient pump tubes by pushing cassettes downwards (no click if correctly fixed and in parallel position)

In case of a period of inactivity: Check the functioning of the valves with HARDWARE CHECK/LORRCA IO/VALVES. During toggling ON/OFF each valve. should give a "click". If a pinch valve sticks, perform a "PRIME ALL"

Always check consumables for “USE-BY” date before starting preparation.

Discard if “USE-BY” date has passed.

Start each day with performing a control sample (containing blood with normal values) before a sample measurement. This control sample gives reference values for the patient samples.

7.2.1. Lorrca reagents

The Lorrca reagents are delivered in 5 ml vials (Elon ISO and Oxy ISO) or in storage bottles of 0.5 l (Osmo HIGH, Osmo LOW)

In all cases:

Store the reagent as indicated at the packaging. Lorrca reagents have a shelf life of one year, stored at 4-30 °C.

Prior for use the reagents must be at room temperature. Do not heat!

Lorrca reagents are strongly adhesive after drying up! Therefore, we recommend to clean the instrument by means of the automated rinse cycle and perform an end of day wash!

Once blood is added to the reagent, the sample tube may be used during one hour.

Invert for use, do not shake.

Do not forget to change the default viscosity value* in case of a new reagent batch at SETTINGS SCREEN/GENERAL OPTIONS.

Batch information can be found on the Lorrca website.

1. Go to www.lorrca.com . 2. Select "PRODUCTS". 3. Click on the link for "BATCH INFORMATION". 4. On this page all batch information can be found for each Lorrca reagent 5. Click on "CERTIFICATE OF ANALYSIS" of the applicable reagent batch number which is mentioned on

the label.



6. Check or change (if needed) the default medium viscosity before the first test sequence in the 'VISCOSITY' field in the Sample data screen

* For Osmoscan measurements the mean of the viscosity of the Osmo HIGH and Osmo LOW has to be set as default medium viscosity. For the other measurements the viscosity of the Elon ISO and Oxy ISO (used for blood preparation) has to be set as default medium viscosity.

Correct use of storage bottles:

Close the storage bottles immediately after filling the instrument bottles.

Transfer the Lorrca reagent directly from storage bottles in instrument bottles.

Check for turbidity and discard if turbidity develops.

Do not return reagents from the instrument bottles back in storage bottles.

After opening, the reagents can be used during one week, stored at 4-7°C (in instrument bottles as well as the remaining reagents in the storage bottle).

Use only the reagent bottles as supplied with the Lorrca® MaxSis.

7.2.2. RBC necessary per test/scan

For a good quality and stable RBC diffraction pattern a minimum number of cells is required.

Whole healthy blood contains ~4 million cells per µl

Add 25 microliter whole blood to 5 milliliter ELON ISO for a Deformability measurement

25 * 4* 106 results in 100 * 106 RBC per test (Deformability measurement)

25µl Deformability 1 min.

200µl Osmoscan 6 min.

50µl Oxygenscan 20 min.

For a Deformability test 100 * 106 RBC are needed

For an Osmoscan 1000 * 106 RBC are needed

For an Oxygenscan 200 * 106 RBC are needed



7.2.3. Estimated RBC in laser

The couette volume uses 1 milliliter resulting in 100 / 5 = 20 * 106 RBC in the couette system

The suspension volume in the laser beam = 0,5 microliter

The diffraction pattern uses (20 * 106 / 1000) * 0,5 = 20.000 RBC

7.2.4. Oxygenscan whole blood volume calculation

Measure RBC level CBC on a Cell Counter.

250/RBC = xx µL blood in 5 ml Oxy ISO

RBC in 106/µl or RBC in 1012/l

this should result in a value between 50 and 250 µl depending on the level of anemia

7.3. Performing Osmoscan

Start software

In case of starting Osmoscan measurements after other tests: Perform a prime step with corresponding Lorrca reagent first:

1. Check that all tubes are connected correctly to the Osmo HIGH and Osmo LOW, water and waste bottles



2. Select "HARDWARE CHECK" and "LORRCA IO".

3. Place a sample tube with basic Elon ISO (QRR 030901) on needle position.

4. Move the sample needle to the bottom of the tube.

5. Select the "PRIME ALL" button. Check that both Osmo LOW and HIGH are pumped, indicated by different click-noises, moving air-fluid meniscus (eventually after the introduction of an air bubble). Check the sample feeding through the needle after introduction of an air bubble. In case of a problem check whether all tubing and connections have a free passage and are not folded or clogged.

6. Remove (priming) tube from the needle position.

7. Return to main screen

In case of starting the day with Osmoscan: Prepare a blood sample Osmoscan (on page 105).

7.3.1. Preparing a blood sample

Use the dedicated Lorrca reagents (see Appendix - Parts list (on page 191))

A blood sample can be stored at room temperature during a limited time. After that the sample cannot be used anymore.

1. Mix 5 ml ready to use Elon ISO with 200 μl of normal EDTA-anticoagulated blood (shortly before the measurement). Gentle mix the blood by immediately inverting the tube 10-12 times (don’t shake) until it colours homogeneous red.

2. Click on OSMOSCAN on the Main screen. The Osmoscan start screen appears:

3. Press NEW...(F1)



7.3.2. Place sample tube Osmoscan screen



Lower the BOB if necessary.

Start a new measurement by clicking the <NEW…(F1)> button in the screen.

Enter measurement ID.

The measurement ID is not allowed to contain characters other than digits or letters. It is used to store measurement data (if enabled in the Settings menu). The “REMARKS” and “DONATION TIME” fields may be ignored but serve to include sample details. The "VISCOSITY" field is the viscosity parameter of the test sample, which is mainly determined by the reagent. The indicated value differs per batch and have to be entered at the start of each test run. See chapter Lorrca reagents (on page 102) for obtaining batch information. For Osmoscan measurements the mean of the viscosity of the Osmo HIGH and Osmo LOW has to be set as default medium viscosity.

1. Always check whether CUP and BOB are clean before taking a blood sample. When CUP and/or BOB are dirty or some cleaning fluid is left, the measurement results may be influenced. reagent bottles are in place.

2. Place the prepared sample tube at sampling position.

3. Close the hood 4. Press <OK>



7.3.3. Adjust video camera Osmoscan screen

It displays a coloured version of the diffraction pattern:

It is optional to adjust the camera gain (bar on the right side) until the diffraction pattern image fills the entire viewing field (green light upper right of screen).

In case the "auto start" is enabled the measurement starts automatically when the osmolality has reached a value less than 75 mOsm/kg and the image is OK, otherwise you can standarise by counting to 30. If needed this can be extended by pressing '+ 10 sec', the measurement is postponed with 10 seconds. This can be done repeatedly if needed.

Press <Start now>.

The measurement starts and the curve starts to appear.



7.3.4. Osmoscan result screen


Check Osmoscan parameters explanation (on page 75)

7.3.5. Start new measurement

After a measurement has completed and the report is printed (yes/no)

Press the <YES> for printed report.

Press the <NO> if no report is required.




1. Open the hood. 2. Remove empty sample tube. 3. Place tube with demi-water (+/- 2,5 ml). 4. Press <RINSE>. to remove sample and rinse the system. Sample is lost and no longer available.


Press <CLEAN>


6. Press OK and the Osmoscan screen appears. 7. Press <F1> or click on NEW...F1 to perform another Osmoscan.




7.4. Performing Deformability curve test

Start software




1. Mix 5 ml vial ready to use Elon ISO solution with 25 μl of normal EDTA-anticoagulated blood (shortly before the measurement).



2. Gentle mix the blood by immediately inverting the tube 5-10 times (don’t shake) until it colours homogeneous red.

3. Click on DEFORMABILITY CURVE on the Main screen. The Stability start screen appears:

4. Press <NEW...(F1)>

7.4.2. Insert blood sample deformability screen



Before insert blood Preparing a blood sample Deformability (on page 111) test first.




The measurement ID is not allowed to contain characters other than digits or letters. It is used to store measurement data (if enabled in the Settings menu). The “REMARKS” and “DONATION TIME” fields may be ignored but serve to include sample details. The "VISCOSITY" field is the viscosity parameter of the test sample, which is mainly determined by the reagent. The indicated value differs per batch and have to be entered at the start of each test run. See chapter Lorrca reagents (on page 102) for obtaining batch information.

Always check whether CUP and BOB are clean and dry before entering a blood sample. When CUP and/or BOB are dirty or some cleaning fluid is left, the measurement results may be influenced.



Insert 600-900 µl of the blood suspension in the gap between BOB and CUP using a pipette (see above Figure.). Press Aspirate and check if the suspension level is high enough and without air bubbles. Capillary forces usually keep the suspension within the gap. However, if capillary forces are insufficient, more of test suspension is required to fill the whole geometry (including the bottom). After inserting more blood suspension, aspirate again.

Close the hood.

Press <OK>

7.4.3. Adjust video camera deformability screen





In case the "auto start" is enabled the measurement starts automatically when the temperature has reached the correct value.

Press <START NOW>.


7.4.4. Deformability curve result screen


Check Deformability curve determination parameters explanation (on page 80)








1. Press <CLEAN> 2. Open the hood. 3. After a measurement on whole blood use the automated rinse cycle and simultaneous rinse the

funnel with a spray bottle with de-ionized water to clean and empty the CUP.



4. Press <F1> or click on NEW...F1 to perform another measurement. or Press <MAIN MENU> to perform other tests (perform in that case also an “END OF DAY WASH (on page 142)”). or Exit the software and perform an “END OF DAY WASH (on page 142)”.

7.5. Performing Cell stability test

Start software








3. Click on CELL STABILITY TEST on the Main screen. The Stability curve start screen appears:


7.5.2. Insert blood sample stability screen



Before inserting blood, read Preparing a blood sample Stability (on page 117) test first.








Insert 600-900 µl of the blood suspension in the gap between BOB and CUP using a pipette (see above Figure.). Press Aspirate and check if the suspension level is high enough and without air bubbles. Capillary forces usually keep the suspension within the gap. However, if capillary forces are insufficient, more of test suspension is required to fill the whole geometry (including the bottom). After inserting more blood suspension, aspirate again.

Close the hood.

Press <OK>

7.5.3. Adjust video camera stability screen






Press <START NOW>.


7.5.4. Stability curve determination results screen


Check Stability curve determination parameters explanation (on page 87)




7.6. Performing Cell membrane stability test

Start software








3. Click on CELL MEMBRANE STABILITY TEST on the Main screen. The Cell membrane stability start screen appears:


7.6.2. Insert blood sample membrane screen



Before insert blood Preparing a blood sample Stability (on page 117) for RBC test first.






Insert 600-900 µl of the blood suspension in the gap between BOB and CUP using a pipette

(see above Figure.). Press Aspirate and check if the suspension level is high enough and without air bubbles. Capillary forces usually keep the suspension within the gap. However, if capillary forces are insufficient, more of test suspension is required to fill the whole geometry (including the bottom). After inserting more blood suspension, aspirate again.

Close the hood.

Press <OK>



7.6.3. Adjust video camera membrane screen




Press <START NOW>.




7.6.4. Cell membrane stability results screen


Check Stability results parameters explanation (on page 94)



7.7. Performing RBC Aggregation test

Start software



7.7.1. Preparing blood sample for RBC aggregation test

A blood sample can be stored at a maximum of one hour (at room temperature). After that the sample cannot be used anymore.

Since aggregation is determined through light back-scatter measurement and indicated as Isc-values are dependent on the amount of oxygenated Hb present, it is important to use fully oxygenated whole blood. When the degree of oxygenation is different in two samples of the same blood, all absolute Isc-values, derived from the Syllectogram, will be different!

1. Incubate 4 ml of whole blood, with at least 40 ml of air (1:10), in a tube preferably on a roller-bank for 15 minutes, minimum. About 600-900 µl of whole blood is required to perform an aggregation measurement.

2. Click on AGGREGATION on the Main screen. The RBC Aggregation start screen appears:




7.7.2. Insert blood sample RBC aggregation screen

Before inserting blood, read Preparing blood sample for RBC test (on page 131) first.




The measurement ID is not allowed to contain characters other than digits or letters. It is used to store measurement data (if enabled in the Settings menu). The “REMARKS” and “DONATION TIME” fields may be ignored but serve to include sample details.




Insert 600-900 µl of the blood suspension in the gap between BOB and CUP using a pipette

(see above Figure.). Press Aspirate and check if the suspension level is high enough and without air bubbles. Capillary forces usually keep the suspension within the gap. However, if capillary forces are insufficient, more of test suspension is required to fill the whole geometry (including the bottom). After inserting more blood suspension, aspirate again.

Close the hood.

Press <OK>

7.7.3. Adjust temperature RBC aggregation screen



It subsequently displays a “Wait until temperature is stable” window.

• Press <START NOW> if you want to start the measurement immediately.


7.7.4. RBC aggregation result screen


Check RBC parameters explanation (on page 98)



7.8. Performing Oxygenscan

This manufacturer’s standard operating protocol is to be validated in each lab.

Prior to use, samples should be stored at 2-8 ⁰C; test the sample after a fixed (cold) storage time.

Prior to use, the Oxy ISO reagent should be stored at room temperature for at least 24 hours (to accommodate to the level of dissolved oxygen corresponding to ambient temperature)

7.8.1. Pre-test preparations

Start-up

1. Switch pc and Lorrca on 2. Open the nitrogen cylinder 3. Lower de Bob into de Cup (to start accommodating to a 37⁰C temperature) 4. Start software

5. Run the 'Self Check' (if it fails, re-run Self check in Hardware --> pO2) 6. If the self-check consistently fails because the measured value of ambient oxygen level is

below 130 mmHg it is time to change the oxygen sensor spot1). 7. Perform a 'test run' with any (old) blood sample 8. Rinse



9. Dry Bob, Cup and Luer syringe-connector with soft cloth, flush the connector and the tube connecting to the bottom of the Bob with air from the big syringe to make it dry.

7.8.2. Preparing a blood sample Oxygenscan


Sample preparation:

1. Measure RBC level CBC on a Cell Counter (RBC in 106/µL or RBC in 1012/L). 2. Oxygenscan whole blood volume calculation: 250/RBC = xx µL blood in 5 mL Oxy ISO (this

should result in a value between 50 and 250 µL depending on the level of anaemia) 3. Gently mix the sample 10 to 15 times in the primary tube. 4. Gently pipet calculated standardized sample volume into the Oxy ISO 5. Gently mix the Oxy ISO vial 40x, use the blood/Oxy ISO solution within 30 minutes. (Oxy ISO is

not a storage medium) 6. Slowly draw 2,0 mL of the blood/Oxy ISO solution in a 3 mL syringe


7.8.3. Place sample Oxygenscan test

1. Remove big air bubble(s) from the syringe, 2. Connect the syringe to the Luer-connector and inject the blood/Oxy ISO solution slowly and

continuously via syringe into the Bob through the Luer-connector, inject till above the “black ring” on the Bob and near the level of the suction port in the Bob

Overview of syringe on the Luer-connector, ready to inject the blood/Oxy ISO solution into the cup. The blue arrow indicates the level that needs to be surpassed by the injected solution



3. Initiate test by starting new measurement. Click on NEW <F1>

4. Add sample data (do not click any button) and viscosity data (only for the first time from each

test run).



5. Click OK 6. Check for possible rising air bubbles and wait until these have passed 7. Click Aspirate, to accurately levelling the solution in the cup. This will take 60 seconds. If the

edges are not sharp enough click Aspirate again. 8. Verify that the edges of the aspiration patch in the cup are sharp

9. Close the hood 10. Click Continue and Start now

11. Press Continue 12. Adjust gain, if necessary, to nicely fit within the image screen and press Start now



After the Run:

Remove the syringe and replace by a syringe filled with demi-water. Press ‘clean’ and slowly flush the connector during rinsing, make sure you flush during the 2 directions the cleaning fluid flows.

Dry the bob and the cup and connector, flush the Luer-connector with air using a large syringe. If everything is dry, lower the Bob in the cup. Ready to run the next sample.

The Oxygen sensor spot is tested during the self-check before every measurement. In case the self-check is not passed, see our Protocol (on page 160) how to correct the oxygen measurement.

7.8.4. Oxygenscan

Test is performed automatically, according to test-settings.

Test-time consists of:

- Deoxygenation: 20 min

- Re-oxygenation: 8 min.

At measurement completion, result-data is displayed on-screen, the software offers an option to print (yes/no), and the data is stored (see further in text).



7.8.5. Discard of sample

1. Open the hood.

2. Sample is automatically removed by clicking: <remove sample>

3. Remove syringe from the Luer and replace it with a syringe filled with de-ionized water.

4. Press <CLEAN> and slowly flush the connector during rinsing

5. Dry the Bob, Cup and the connector with cotton / paper towel and air (to assure the tube is free of water)

6. Place Bob in Cup for next measurement

7.9. End of day wash

When the program is shut down a pop-up screen appears with <END OF DAY WASH> message. To perform an End of day wash:

End-of-day wash procedure:

1. Remove both tubes on the reagent bottles.



2. Mount the reagent tubes to the cleaning tube LORC070905 from the cleaning solution bottle.

3. Place a tube with cleaning solution at sample position. 4. Follow the software instructions. 5. Remove after finishing of the End-of-day wash the cleaning tube from the reagent tubes. 6. Remove the reagent-bottles and store them at cool conditions.

7. Empty waste bottle. 8. Clean and dry bob and cup, drip plate and sample unit plate. Press OK



7.10. Powering down

CAUTION: Never turn off the main switch when the instrument is running. Always wait until the instrument has completed its cycle.

Go to HARDWARE CHECK ->LORRCA IO

Press <RINSE CUP>.

Press <RINSE GRADIENT PUMP>.(If available)

Dry CUP and BOB. Do not scratch the CUP!

Exit software.

End of day wash (on page 142) (see further in this chapter)

Switch OFF the LoRRca.

Switch OFF the PC and printer.

Empty the waste bottle.



7.11. Decontamination procedures Routine

Routine:

At the end of each test, or at least each day, disinfect lab benches and any equipment.

Spills:

Small spills of biohazard material should be treated by first covering them with an absorbent paper to avoid the formation of aerosols. Disinfect the spill by slowly pouring on a disinfecting solution working from the outside to the centre of the spill in a circular motion. Leave the spill long enough for disinfection to take place (check decontaminating instructions on the disinfectant container for time) and then carefully wipe up, use protection gloves.

Pick up any glass using forceps.

Once all the material has been removed disinfect the area thoroughly.

Inform the Technician in Charge of the spill.

7.12. Waste container disposal

The waste container has a level sensor and as soon as the level sensor generates a <WASTE FULL> signal, the waste container must be emptied.

Preferably, remove the complete waste container and replace it with a cleaned one, discard waste according the regulations.

Biohazard waste:

Dispose blood tubes into a biohazard sharps container.

Dispose sharps into a biohazard sharps container.

All other bio-hazardous waste is to be deposited into a biohazard bag.

All bio-hazardous waste is deposited into the Medical Waste Management (MWM) bin for pick up.

Disclaimer: Check your local environment rules about discharging the waste.

Reporting


8. REPORTING

Reporting examples.

8.1. Deformability Report

See Deformability curve determination parameters explanation (on page 80)

Reporting


8.2. Cell Membrane Stability report page 1 of 3

See Deformability results 1 parameters explanation (on page 93)

Reporting



See Stability results parameters explanation (on page 94)

Reporting



See Deformability results 2 parameters explanation (on page 95)

Reporting


8.5. Osmoscan Report

See Osmoscan parameters explanation (on page 75)

Trouble shooting


9. TROUBLE SHOOTING

General

This paragraph is intended for trained personal. They should be familiar with at least the operation and various basic settings.

Do not hesitate to contact RR Mechatronics if you are confronted by a fault which is not described in this paragraph.

WARNING! Ensure that no one can operate the control keys accidentally and never leave the instrument unattended during this work.

Trouble shooting


9.1. Error messages

Error Description/Check Remedy

Error 1 Error in ambient temperature measuring circuit. NTC transmits values that are rejected (<10 °C or >45 °C).

Exit software and switch off the LoRRca.

Check communication cables to computer.

Switch on the LoRRca and start the software.

Error 7

Error in conductivity measuring circuit. Measured calibration factor too high or too low. This happens during initiation and is not depending on attached measuring cell or conductivity NTC.




Error 8 Home sensor is not sensed during initiation of the servomotor.




Error 9 BOB heating wrong. Heating takes too much time to reach temperatures above 25 °C.




Temperature BOB > 440C

Error 25

I2C error. The I2C communication is not handled well.




Error 26

Error in braking system. During initiation the brake did not activate (Servo is activated and there are encoder pulses)




Trouble shooting


Error 27

Encoder error detected in servo motor (no pulses during initiation).




Error 30

Encoder error detected of the gradient mixer (no pulses during initiation).




Check in Settings screen (on page 58) that the Osmoscan option Checkbox is switched OFF.

Error 31

BOB overflow. Overflow sensor did not give pulses while the gradient mixer or rinse pump runs.




Note: For Error codes > 25: Restart the LoRRca MaxSis, if still NOT OK: Call RR Mechatronics see Service request protocol

Trouble shooting


9.2. Error Messages (Oxygenscan)

Message Description Solution

No PT100 connected! No PT100 connected to the device or PT100 not correctly connected.

Check the PT100 presence and its soldered connection to the PCB.

No sensor connected! No sensor is connected (Amplitude < 1000).

Check the optical coupling to the sensor, for example the fiber head2sensor alignment

Signal amplitude too low! Signal amplitude < 3000. Check the optical coupling to the sensor, for example the fiber head2sensor alignment

SD card error! SD card not inserted or defect. Check the SD card presence and functionality

Reference amplitude out of range!

Contact service team Device has to be sent to RRM for service

Photo diode saturated! Ambient light too high or signal strength too high

Send measurement sensor data (Phase, Amplitude Temperature and Calibration data, ideally the repo) to RRM

Reference overflow! Reference strength too high. Contact service team. Device has to be sent to RRM for service

Signal overflow! Signal strength too high. Decrease signal current.

PME timeout Contact service team. Device has to be sent to RRM for service

Pressure sensor defect! Pressure sensor out of function. Contact service team. Device has to be sent to RRM for service

SD card full! SD card is full. Backup and wipe SD card

Pulse counter overflow! The pulse counter has reached the pulse counter overflow limit.

Device has to be sent to RRM for service

Temperature sensor not available!

The temperature sensor was activated but it is not present on this device.


Trouble shooting


Pressure sensor not available! The pressure sensor was activated but it is not present on this device.


Unknown calibration parameter! Unknown calibration parameter in calibration file.

Wrong calibration file installed or file is corrupted. Check calibration file

Unknown calibration mode! Unknown calibration mode in calibration file.


Unknown measurement mode! Unknown measurement mode in calibration file.


Unknown 2nd calibration type! Unknown second calibration type in calibration file.

Check calibration file

Trouble shooting


9.3. Trouble shooting table

Field Description Check/Remedy

Waste container

Container indicates waste full when it is not. Waste liquid is clinging to the inside of the bottle and gives a false reading.

Flush bottle thoroughly with hot water or replace the bottle.

Power No power

Check the fuses at the main power inlet.

Check cables

Replace if necessary.

Check the fuse on the PC main board

Camera No camera image

Check connection cable. Reconnect if necessary.

Check Camera driver (on page 156) is not defect.

Camera Disturbed image

Check position projection screen

Adjust projection screen if necessary (see Adjusting projection screen (on page 165))

Fluid flow There is no fluid flow or not enough fluid flow in one or more tubes

Check the liquid levels in the bottles

Check the functioning of the pinch valve, in case of stitching: perform a Prime all

In case of an incorrect image, not sharp and/or black dot is not in the middle, the laser has to be adjusted or even be replaced.

To check the connection on the camera, the handle on the right side has to come off to check if the connection is good.

Check the operation of the Digital Camera driver in the window driver manager.

When the driver does not work correct re-install the driver.

Disconnect the USB cable.

Reconnect the cable again.

Re-install the Digital Camera driver.

Select in the option screen <DON'T SEARCH I WILL CHOOSE>.

Maintenance


10. MAINTENANCE

Always use disinfectant solution supplied by RR Mechatronics for cleaning purposes.

The schedule shows which maintenance procedures are allowed to be executed by an operator.

The maintenance recommendations are based on an 8-hour working day. The frequency must be increased with shift work schedules.

Ensure that others cannot accidentally operate the control keys, and never leave the instrument unattended during this work. Always switch the mains power off.

Maintenance


10.1. Maintenance schedule

Description

Daily

Weekly

Monthly

6 monthly

yearlly Clean X

Reagent handling X

Visual check X

Pump tube replacement X

Periodical maintenance* X

Report irregularities in the logbook.

*The yearly maintenance has to be performed by a trained engineer. Please contact RR Mechatronics for more information.

Maintenance


10.2. Cleaning

Clean the Instrument once a day. Note the following points:

Never use compressed air for cleaning, as this may cause swarf and fragments to be blown into the instrument. The use of flammable fluids is not allowed. Do not use medical alcohol, bleach or any solvent for cleaning, use only DI water or Starrsed X-clean.

Clean the casing with a soft dust-free cloth moistened with a few drops of subscribed disinfectant.

Clean the area around the instrument and remove dust from the floor with the vacuum cleaner.

Clean the projection screen with a clean soft cloth or lens paper.

Clean the CUP with a soft dust free cloth moistened with DI water or Starrsed X-Clean.

Maintenance


10.3. Oxygen sensor spot protocol

For Oxygenscan option only

Background

In the Oxygenscan, the oxygen level in the sample is measured through the luminescence quenching of a luminophore spot. When the luminophore spot is illuminated by a green/blue light, it reacts by returning orange light. When the green/blue light is switched off, there is a decay time of the returning orange light. The decay time is dependent on the temperature and oxygen level in contact with the luminophore spot, referred to here as the partial oxygen pressure (pO2) sensor spot.

The pO2 at sea level is approximately 149.3 mmHg at 37°C. In the Lorrca, a new pO2 sensor spot will show an equilibrium pO2 of about 149 mmHg. For improved accuracy, each batch of sensor spots is accompanied by calibration data. The response of the sensor from ambient oxygen to no oxygen is not all linear along this trajectory and can vary between different production batches of the sensor spot. The calibration data is the input for the formula to correct for this non-linearity. Beside this correction, over time, the pO2 measured by the spot will decline due to the influence of light and mechanical wear, resulting in an inaccurate reading of the real ambient pO2.

Correcting the decline in the pO2 measurement

The pO2 measured by a new sensor spot and the decline in subsequent pO2 measurements are proportional and can therefore be corrected using a proportional correction factor:

Correction factor=(pO2 of the new sensor spot)/(pO2 measured during self-check)

For example, if the pO2 measured by the new sensor spot is 143 mmHg, and the self-check measures 135 mmHg, then the correction factor is 1.06.

To then correct pO2 measurements from the Oxygenscan, the pO2 value is multiplied by the correction factor:

Corrected pO2=(measured pO2 )*(correction factor)

For example, if the Oxygenscan measures the Point of Sickling (PoS) as 31 mmHg, then the corrected pO2 would be 32.9 mmHg.

We recommend correcting the Oxygenscan pO2 results when the start-up pO2 measures less than 140 mmHg.

Checking and replacing the pO2 spot

The user should perform a pO2 self-test during each start-up of the instrument to check the pO2 measurement before using the Oxygenscan. If the pO2 sensor spot measures an ambient pO2 of less than 130 mmHg, the wear of the spot is considered too high and should be replaced with a new one. See Replacement sensor-spot (on page 161).

Maintenance


10.4. Replacement sensor-spot

For Oxygenscan option only

The sensor-spot has to be replaced regularly due to wear.

To replace the pO2 sensor spot and update the calibration file:

1. Gently, but forcefully push the sensor spot with your finger out from the inside of the cup.

2. Replace the spot by a new one provided in a black plastic pouch by pressing it into the outside

of the cup 3. A new sensor spot is accompanied by a data file containing the calibration data of the new

spot. Download the calibration file from the Lorrca website. This file must be placed in the main directory C:/Lorrca MaxSis/. The old data file should be moved to a separate directory where the old data files of the used spots are stored (this might be useful in the future)

4. Start software and select tab Lorrca Settings - and in Settings Instrument settings

Maintenance


5. Select at PO2 Sensor batch number the correct calibration file, which is given with the sensor-spot. If the new calibration file cannot be found after replacement of the sensor-spot, the following warning is given

Press OK

6. When the Lorrca program is restarted it will notice that the original sensor spot datafile is

missing. The new file will need to be linked through the settings screen and then selecting the tab instrument settings and from there hardware settings. There you can select the current sensor spot data file to be linked.

The self-check may be skipped at any time.

If Step 2 of the self-check reads 0 mmHg, the pO2 sensor spot is likely misaligned with the fiber-optic cable and must be adjusted. Adjustment is done by rotating the cup relative to its holder.

Maintenance


With the pO2 sensor spot removed and the cup sent to the home position, the fiber optic cable is visible through the sensor spot hole. This alignment is critical to accurately measure the pO2 sensor spot.

10.5. Visual checks

Report irregularities in the logbook.

Check for scratches on surface projection screen or CUP:

No scratches: Continue

Scratches: Contact RR Mechatronics

Check tube attachments.

When loose: re-attach

When damaged: replace

10.6. Lorrca reagents routine

Remove the reagent-bottles after use and store at a cool and dry place (4-6 °C.)

The instrument bottles have to be cleaned after use. Rinse with hot tap water and purge with Ultra pure water. Drain the bottles as much as possible and let it dry sufficiently.

Do not forget to change the default viscosity value* in case of a new reagent batch at SETTINGS SCREEN/GENERAL OPTIONS.

Batch information can be found on the Lorrca website.

1. Go to www.lorrca.com . 2. Select "PRODUCTS". 3. Click on the link for "BATCH INFORMATION". 4. On this page all batch information can be found for each Lorrca reagent 5. Click on "CERTIFICATE OF ANALYSIS" of the applicable reagent batch number which is mentioned on

the label. 6. Check or change (if needed) the default medium viscosity before the first test sequence in the

'VISCOSITY' field in the Sample data screen

* For Osmoscan measurements the mean of the viscosity of the Osmo HIGH and Osmo LOW has to be set as default medium viscosity. For the other measurements the viscosity of the Elon ISO and Oxy ISO (used for blood preparation) has to be set as default medium viscosity.

Maintenance


10.7. Adjusting geometry parameters

Use the supplied accessories.

To guarantee accurate shear rates/stresses, the BOB/CUP radii parameters of both the elongation and the aggregation program need to be set according to the true radii (Tab SETTINGS LORRCA

SETTINGS GENERAL OPTIONS).

10.8. Back up the results file

The memory stick supplied by Mechatronics is required here (or rename any available memory stick to LoRRca-backup).

Place the memory stick in the USB slot of the computer (or the USB extension cable).

Drag the file C:\LoRRca Maxsis\Results onto the memory stick.

If you have a previously stored backup files on the memory stick, you should rename it first.

10.9. Update the software

New software versions (if available) can be downloaded from http://www.rrmechatronics.com.

1. Click on LOGIN SUPPORT LORRCA MAXSIS 2. Log in with user name: "lorrcauser" and password: "maxsis". 3. Select DOWNLOAD and scroll to Software and download the latest version of the system software

and copy it to a memory stick.

Maintenance


10.10. Adjustments and replacements

Replacement parts list can be found in Appendix - Parts list (on page 191)

10.10.1. Adjusting projection screen

Switch ON the instrument.

Start-up the PC.

Choose LoRRca software program.

Select <HARDWARE CHECK>.

Move the Bob down.

Choose <CAMERA>

Set camera gain at 250.

Close the hood.

Check the image:

OK: The instrument is now ready.

Not OK: Misaligned

Check position of the projection screen.

Maintenance


Alignment of the projection screen:

Use the hexagon key 2mm to loosen the projection screen holder.

Align the projection screen until the black dot on the screen is aligned with the laser spot. Note: The projection screen holder itself can also be adjusted (equipped with slotted holes)

Tighten the screws of the projection screen holder.

Not OK: Driver failure.

Update the camera driver. See Camera driver installation (on page 156)

Choose <LORRCA IO><RINSE CUP> and confirm. The tubes are being filled.

Dry BOB and CUP. Do not scratch the cup and do not touch the glass with your fingers to prevent staining! Pay attention to the hole in the BOB.

Maintenance


10.10.2. Adjusting camera for slides

Maintenance


Place the EI 0 slide.

Adjust the camera gain according the value on the slide.

Close the hood.

Turn on the led.

Place the EI 0.333 slide.


Close the hood.

Turn on the led.

Place the EI 0.6 slide.

Maintenance



Close the hood.

Turn on the led.

The pattern must be uniform red. The measured value should match within +/-0.01

Maintenance


10.10.3. Replace the main fuses

The instrument is standard equipped for 230VAC , if the power voltage is 115VAC another fuse has to be placed (see ID-plate)

Pull the fuse holder in the main power entrance module open with a screwdriver or other flat tool.

The main power entrance is located on the rear side.

Replace both fuses with the appropriate type, as indicated on the type label

Push the fuse holder back into the power entrance module.

Maintenance


10.10.4. Waste and rinse pump tube replacements

10.10.4.1. Waste and rinse pump tube replacement

Important: The rinse- and waste peristaltic pumps use different tubing, the waste pump uses QWLV090011 (3,2mm tube) and the rinse pump uses QWLV090012 (1,6mm tube) Always replace the correct tube!

The pumps are in the foot of the Lorrca® MaxSis, pull on the lid to open the pump drawer.

The pump tube needs to be replaced every 6 months.

Switch the instrument OFF.

Remove the old tube from the pump as shown:

Pull the tube from the connectors and install the new tube in the reverse order.

Check that all connecting tubes and the tube cover are attached.

Maintenance


10.10.4.2. Pump connection tubing

Pumps to Needle assembly (LORC070904)

Note: Start to install with the tube end with label 6 which is shown GREEN in the picture. Next picture: The installed tube is now BLUE continues with the next GREEN tube. -

1. Connect tube with label 6 to the output port of the Rinse pump

2. Connect tube with label 1 to the Input port of the Rinse pump.

3. Connect tube with label 7 to the output port of the Waste pump.

4. Connect tube with label 4 to the Input port of the Waste pump.

Maintenance


Tube to needle assembly (LORC070901)

Connect tube with label 5 onto connector 5



Maintenance


10.10.5. Tubing replacements

Tubing as used for Osmoscan

Maintenance


10.10.5.1. Gradient pump tube replacement

After 500 measurements/ half year.

Disconnect water and reagent tubing and pull up the sample needle

Lower the BOB and perform two times a PRIME ALL function

HARDWARE CHECK [ LoRRca IO ] [ Prime all ] [ Start ]

Open the pump drawer and pull the drawer out.

Maintenance


Release the tube cassette.

Replace the tube assembly with new tube assembly following Install LORC070901 tubing (on page 177) (see also Appendix - Parts list (on page 191) spare- and replacement parts)

10.10.5.2. Install LORC070901 tubing

Put the waste tubing labeled with 3 and 5 into the first tube bracket

Assure that the tubing is not twisted and the locking ridge is pointing up

Maintenance


Continue the with the four other tube brackets

Hook the first tube bracket onto the pump frame. (Waste bracket)

Continue the with the rest of the tube brackets

This how it looks when all the tube brackets are in place.

Tube to needle assembly

Maintenance


Connect tube with label 5 onto connector 5 Connect tube with label 3 onto connector 3

Connect small (sample) tube onto the first cassette.


Maintenance


10.10.5.3. Install LORC070902 tubing set 1

Note: Start to install with the tube end with label 2 which is shown GREEN in the picture. Next picture: The installed tube is now BLUE, continue with the next GREEN tube. -

1. Put both small tubes into the pinch valve V4 (short end at the front)

2. Connect tube with label 2 on the connector 2

3. Put both small tubes into the pinch valve V3: The tube with label Osmo HIGH is at the back of V3, the tube with label Osmo LOW is at the front of V3

4. Install both long tubes to the PVP bottles. Connect tube "Osmo LOW" onto the bottle Osmo LOW bottle

5. Connect tube "Osmo HIGH" onto the Osmo HIGH bottle

6. Take connection tube from V4 and connect the tube "H2O" onto the H2O bottle.

Maintenance


7. Connect the tube "1" onto connector 1.

Tube to needle assembly

1. Connect tube with label 3 on the connector 3 2. Connect the other end to the needle assembly

Maintenance



Note: Start to install with the tube end with label 5 which is shown GREEN in the picture. Next picture: The installed blue tube is now BLUE, continue with the next GREEN tube. -

Start with tube end with label 5.

1. Put the short tube into the pinch valve V2 in the back port.

2. Connect tube with "5" on the connector 5.

3. Put the other short tube into the pinch valve V2 at the front port.

4. Connect tube with label 6 on the connector 6.

5. Take the long end of the tube and run this through the back hole

6. Take connection tube and put the short tube in V1 through the back port.

7. Connect the tube with label 4 to connector 4

Maintenance


8. Take the long end of the tube and run this through the front hole

9. Connect tube "4" on the bob, connect tube "6" on the EC-sensor

Tubing to waste connector

Connect tube with label 7 on the connector 7 Connect tube labelled with waste to the waste

bottle

Maintenance



Note: Start to install with the tube end with label 6 which is shown GREEN in the picture. Next picture: The installed tube is now BLUE continues with the next GREEN tube. -

1. Connect tube with label 6 to the output port of the Rinse pump

2. Connect tube with label 1 to the Input port of the Rinse pump.

3. Connect tube with label 7 to the output port of the Waste pump.

4. Connect tube with label 4 to the Input port of the Waste pump.

10.10.5.6. After replacement of all tubing

Close the drawer.

Place the sample needle in sample tube.

Perform two times a PRIME ALL function.

HARDWARE CHECK [ LoRRca IO ] [ Prime all ] [ Start ]

Maintenance


Check the pump function and exclude leakage, check the tubes are filled correctly without air bubbles.

10.10.6. Preparation for long time inactivity

In case of long-time inactivity or temporary storage:

1. Perform a End-of-day-wash to clean all tubing. 2. Disconnect tubing from bottles except the waste line. 3. Perform a prime all and a cup rinse step to empty all tubing. 4. Clean the outside of the instrument carefully as prescribed. 5. Remove all liquid bottles, discard or store the contents. 6. Remove all tubes from the pinch valves, the tubes could stick (due to reagents). Place shim in

the normally closed section of the valves.

7. Unlock the tube cassettes of the gradient pump to relief the the tubing.

Appendix for Lorrca® MaxSis


11. APPENDIX FOR LORRCA® MAXSIS

Appendix section


Work instruction Number 516

Page 1 of 4 Purpose: Preparation for transport

Safety Bio hazard area

Instrument:Lorrca Version:1, June 2018

RR Mechatronics shall not in any event be liable for any direct, indirect, special, incidental, or consequential damages, including, without limitation, lost revenues, or lost profits, arising out of or in any way connected with the use or misuse of the information or lack of information in this Instruction.

11.1. Preparation for transport.

Perform the preparation for long time inactivity or temporary storage:

In case of long-time inactivity or temporary storage:

1. Perform an End-of-day-wash to clean all tubing.

(User Manual/General operation procedure/End of Day Wash)

2. Disconnect tubing from bottles except the waste line.

(User Manual/General operation procedure/End of Day Wash)

3. Perform a prime all and a cup rinse step to empty all tubing. (to empty and clean all tubes)

4. Clean the outside of the instrument carefully as prescribed.

(User Manual/Maintenance/Cleaning)

5. Remove all liquid bottles, discard or store the contents.

6. Remove all tubes from the pinch valves, the tubes could stick (due to reagents).

(User Manual/Maintenance/Install LORC070902 tubing set 1)

7. Unlock the tube cassettes of the gradient pump to relief the tubing.

(User Manual/Maintenance/Gradient pump tube replacement)


11.2. Instructions for packing:

11.3. Part 1:

(applicable for all types)

Use the packing foam to secure the cup as shown in the picture.

Lorrca Maxsis User Manual Version 5.04 MRN-231-EN

This picture is from a Lorrca without a cup. Please do not remove the cup!

11.4. Part 2a:

(applicable for Lorrca 109001)

Use another piece of foam and cut two blocks in the following size with a cut in the side to put it over the pump plate.

L= in mm


Put block 1 on the left and block 2 on the right over the side of the pump plate to secure it.

11.5. Part 2b:

(applicable for Lorrca109003)

Use safety plate (yellow/green in the picture) to secure the pinch valves

Tighten the bolt on the pump assembly to secure the peristaltic pumps.



Appendix - Ordering parts

You will find drawings in the Appendices showing the components without a description but with an alphanumerical code.

In your order, first state the part number followed by the quantity required.

For example: PART NUMBER QUANTITY

QWLV090011 1x

Make a clear distinction between the capital letters "O" and "D" and the number "0". (When reading certain numbers on the drawings, mistakes may be made due to the letter and number typeface chosen. The first four characters are letters, the remaining characters are numbers).

www.lorrca.com http://www.lorrca.com

www.rrmechatronics.com

[email protected] mailto:[email protected]

+31 229 291 129



Appendix - Parts list

Spare parts

A standard package of spare parts is supplied with number and description. When using spare parts, we recommend you order new spare parts immediately, to prevent delays in use.

In order to prevent confusion, there is a summary below of the contents of the spares package.

Part No Description Quantity

QWLV090011 Peristaltic Pump Tube bore 3.2 mm 1

QWLV090012 Peristaltic Pump tube bore 1.6 mm 1

QEDV130017 Fuse 1.6A Slow 230VAC 2

QEDV130020 Fuse 3.15A Slow 115VAC 2

Replacement parts

Part No Description

LORC110900 Rinse bottle

LORC110901 Waste bottle

LORC110902 Osmo Low bottle

LORC110904 Osmo High bottle

LORC110905 Lorrca Cleaning solution bottle

LORC070901* Tubing set Peristaltic pump

QWLV090011 Peristaltic pump tube 3,2mm (waste pump)

QWLV090012 Peristaltic pump tube 1,6mm (rinse pump)

LORC070902* Tubing set 1



LORC070905* Cleaning solution tube


LORC070025* EC Cell tube

*Part of tubing set LORC079200



LORC020905 Cup assembly (standard)

PO2S020900 Cup assembly (Po2S version)

Reagents

Part No Description Quantity

QRR 030901 Elon ISO 100 tubes of 5 ml in

tray

QRR 030905 Oxy ISO 100 tubes of 5 ml in

tray

QRR 030910 Osmo LOW 0.5 Ltr

QRR 030930 Osmo HIGH 0.5 Ltr

QRR 010946 Starrsed X-Clean 5 Ltr

Accessories

Part No Description

LORC060902 Set EI test screens

LORC110100 Manual Lorrca MaxSis

QEDK100017 Euro plug lead 10A with locking system for IEC inlets

QEDK130501 Cable SUBD9 male-female

QWBM540005 Hex key 2 mm



Periodical Maintenance Kit LORC110985

Part No Description

LORC110900 Rinse bottle

LORC110901 Waste bottle

LORC110902 Osmo LOW bottle

LORC110904 Osmo HIGH bottle

LORC110905 Cleaning solution bottle

LORC070901 Tubing set Gradient Peristaltic pump

LORC070902 Tubing set 1



LORC070905 Cleaning solution tube


QEDV130018 Fuse 2 A Slow

QEDV130010 Fuse 315 mA Slow

QEDV130017 Fuse 1.6 A Slow

QEDV130020 Fuse 3.15 A Slow

LORC110018 Laser Radiation label

LORC110114 LORC Reagent label

LORC110115 LORC Reagent label

QWLV090011 Pump tube assembly waste pump

QWLV090012 Pump tube assembly rinse pump

QWBM910504 Nail fastener

QWBM910302 Rubber buffer

LORC070025 EC Cell tube



11.6. Appendix - Document History Extended overview MRN-231-EN

Published date Friday, 15 March 2019

Issue No Date Revised Section(s)

Changes Authorised

5.04 March 2019 Safety

Installation


Maintenance

Mains specification changed

Oxygenscan procedure changed

Cleaning instructions changed

Document History added as appendix, only latest changes in introduction

H. Schavemaker

5.03 January 2018 Installation Program


Introduction of Oxygenscan option

H. Schavemaker

5.02 October 2017 Installation Program

Introduction Software version 5.xx

Lorrca 1.01

H. Schavemaker

Extended change history



5.01 August 2014 Instrument description

Intended use changed

H. Schavemaker

5.00 September 2013

All Addition of chapter operation, contents taken out from chapter Program

H. Schavemaker

4.03 May 2013 Maintenance

Adjustments and replacements

Pump tube replacement

Labels changed

H. Schavemaker

4.02 February 2012 Technical specs

Reagents

Shear rate is changed from 3500 to 800

PVP handling items

Calibration is not applicable for the LoRRca and all issues are removed out of the manual.

H. Schavemaker

4.01 January 2012 Yearly update and publishing

H. Schavemaker

4.00 November 2011 Introduction

Safety

General chapter structure

Review of safety items

H. Schavemaker

3.01 January 2010 Trouble shooting Add service request protocol in trouble shooting.

H.E. van Dijk

3.00 January 2010 All This version of the Manual is an extension and adaptation of the Lorrca manual version 2.1 written by J.G.G. Dobbe (AMC).

H.E. van Dijk

Preliminary November 2009 All Start of he manual H.E. van Dijk



Appendix - Additional Information

This is additional information about the basic principle of the performed test with the Lorrca.



11.6.1. Preface

The Laser-assisted Optical Rotational Cell Analyzer (LORCA) was developed at the Academic Medical Center (AMC) in Amsterdam by Dr. M.R.Hardeman and P.T.Goedhart and has been used for many years at the Laboratory for Clinical Hemorheology at the Dept. of Internal Medicine Reference 261, 312.

Later models where enhanced and the instrument was extended for aggregation measurements. The software packages and documentation were implemented by Dr. J.G.G. Dobbe from the Dept. of Medical Technological Development. 15,16 The instrument was made commercially available by R&R Mechatronics38 up to 2008 under the name LoRRca.

In 2009 the original hardware was improved and extended with the facility to measure red cell deformability under a gradient of medium osmolalities (REF). The corresponding new software was written by Mechatronics Instruments B.V. The new version of this instrument is named: LoRRca MaxSis.

1 Hardeman M.R., De Rie M.A., Schut N.H., Wilmink J.H., Impaired erythrocyte deformability in psoriasis patients after treatment with cyclosporin, Clin. Hemorheol., vol. 11:(6), pp. 784, 1991. 2 Hardeman M.R., Goedhart P.T., Dobbe J.G.G., Lettinga K.P., Laser-assisted Optical Rotational Cell Analyser (LORCA); A new instrument for measurement of various structural hemorheological parameters, Clin. Hemorheol., vol. 14:(4), pp. 605-618, 1994.



11.6.2. Principle of operation

Introduction

Tissue is provided with oxygen, nutrients etc. by virtue of the blood circulation, which also transports metabolic waste products from these tissues to e.g. the kidney and lungs. The amount of oxygen transported through the capillaries is directly proportional to the amount of oxygenated hemoglobin present in the blood and inversely proportional to the flowing capacity i.e. the viscosity of the blood. In the normal situation the latter is mainly determined by the concentration of red blood cells (RBCs) (hematocrit), their property to deform in order to pass through capillaries with diameters smaller than that of the RBCs and their potential to form aggregates (rouleaux) in low flow regions, which could disturb the flow even more. Disturbances of either one or both of these RBC properties have been described in a wide variety of diseases (References (on page 215).2-4,7,8,14,21,26,32,42,43,61,63,64)

In order to evaluate the flowing capacity of blood in different parts of the body it is therefore important to have a technique available for the accurate measurement of these RBC properties. The LORCA is therefore an indispensable instrument for hemorheological laboratories in research institutes, clinical departments of hematology, intensive care units etc. It uses a Couette geometry with a static BOB and a rotating cylinder (CUP) to create a simple shear flow. The mean geometric shear rate for small gaps, can be estimated as:(References 203, 624)

(Equation 1)

With:

Shear rate (1/s)

rb Outer radius of the BOB (mm) (engraved on BOB)

rc Inner radius of the CUP (mm) (engraved on the inside of the metal CUP holder)

N Revolution speed of the CUP (revolutions/min)

Red blood cells cause the blood to be non-Newtonian, which means that the viscosity changes with the applied shear rate. (References 125, 136) During the deformability measurements, blood is diluted in a high viscous medium (dilution 1:200) causing the suspension to behave nearly Newtonian. The shear stress is calculated as:

(Equation 2)

3 Dognon A., Loeper J., Housset E., Etude optique de l'aggregation reversible des hematies, Comptes Rendus Soc. Biol., vol. 143, pp. 769-771, 1949. 4 White F.M., Flow between long concentric cylinders, in: Fluid mechanics, McGraw-Hill, Fourth edition, pp. 261-263, 1999 (ISBN: 0-07-069716-7). 5 Chien S., Sung L.A., Physicochemical basis and clinical implications of red cell aggregation, Clin. Hemorheol., vol. 7, pp. 71-91, 1987. 6 Chien S., Usami S., Dellenback R.J., Gregersen M.I., Nanninga L.B., Guest M.M., Blood viscosity: Influence of erytrocyte aggregation, Science, vol. 157, pp. 829-831, 1967.



With:

Shear stress (Pa)

Medium viscosity (mPas)

Shear rate (1/s), as given above equation. (Equation 2)

11.6.2.1. RBC-deformability measurements

The red blood cells in EDTA-anticoagulated blood, diluted 1:200 in a viscous reagent (detailed in section 2.1.2), are exposed to a user-defined shear stress (Figure 1). The deformation of the cells, present in the gap between CUP and BOB, is measured.



11.6.2.1.1. Deformability curve

When measuring a deformability curve (EI vs. shear stress), the RBC suspension is kept at an adjustable temperature (usually 37 ºC). The applied shear stress is computer controlled and is adjustable in the range 0.01 - 100 Pa. The laser light traverses the blood suspension and will partially be diffracted by the RBCs. Under the influence of shear stress, RBCs elongate accompanied by a shape change of the diffraction pattern. The following drawing shows how the diffraction pattern is made visible on a small projection screen:

Figure 1: Principle of ektacytometry

Figure 1. Principle of ektacytometry. A laser beam is scattered by a suspension of red blood cells stressed between a static inner cylinder (BOB) and a rotating outer cylinder (CUP). The shape of the diffraction pattern, projected on a small screen, is a measure for the average RBC deformability and changes from circular at rest to elliptical at a high shear stress. The major (a) and minor (b) axis of the ellipse serve to calculate the elongation index, EI=(a-b)/(a+b). The photo diodes are used for aggregation measurements using further the same geometry.

A video camera acquires the diffraction pattern from the projection screen and transmits it to the image acquisition board of the PC. Finally, the computer calculates the best matching ellipse (curve fit) and determines the Elongation Index (EI) from the horizontal (B) and vertical (A) axes (Figure 1 and Figure 2) according to:

(Equation 3)

When deforming under increasing shear stress, RBCs change gradually from a biconcave towards a prolate ellipsoid morphology and orient themselves along the flow vector in the gap, i.e., tangential to the axis. This is accompanied by a transition from a circular into an elliptic diffraction pattern, which is oriented perpendicular to the orientation of the elongated cells. The elongation program measures the EI at shear stresses in an optional range and draws EI vs. shear stress, known as the deformability curve. Figure 2 shows this deformability curve and the shape of the accompanying diffraction patterns.



Figure 2: The RBC-deformability curve

Figure 2. The RBC-deformability curve shows the change in the diffraction pattern during application of increased shear stress; calculation of elongation index (EI).

11.6.2.1.2. Stability test

RBC stability

To perform a stability test for RBC, the cells are considerably deformed by a, user defined, high shear stress for a certain time period. The effect on the cells can be followed, either during the stress application period by the intermittent measurements of their deformability at a fixed shear rate, or by the measurement of a complete deformation curve before and after stress application. Thus, the same LORCA geometry as given in Figure 1 is used for both applying the shear stress and analysing the result of it.

Monitoring the decrease of RBC deformability is, compared to hemolysis, an early indication of shear induced cell damage (REF Sung Sik Lee).



RBC membrane stability

Using the Ektacytometer, Chasis et al. found that the continuous high shear stress slowly changes the mechanical properties of resealed erythrocyte membranes. (Reference 117) This causes the cell membranes to fragment progressively. The process can be accelerated by further increasing the applied shear stress. The diffraction pattern gradually looses its ellipticity, and hence EI decreases, under continued high shear stress (see Figure 3). The decrease in EI can be explained by the appearance of randomly oriented cell fragments which add a circular component to the –initially- elliptical diffraction pattern. A similar effect was observed, using the LORCA, in mixtures of rigid and normal deforming RBC, both in the experimental (REF) and clinical situation, e.g. in the blood from patients with sickle cell disease (REF). However, for comparison of results obtained by the Ektacytometer with those of the LORCA it should be kept in mind that the Ektacytometer uses a different method to analyse the obtained diffraction pattern, i.e. 4 diodes detecting light intensity, in contrast to LORCA`s modern digital image analysis.

Figure 3: Fixed high shear stress

Figure 3. A stability test (References (on page 215) 11) is measured as the elongation index (EI) vs. time while resealed erythrocyte membranes are subject to a continuous high shear stress.

7 Chasis J.A., Mohandas N., Erythocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations, J. Cell Biol., vol. 103:(2), pp. 343-350, 1986.



11.6.2.1.3. Ellipse fitting algorithm

The ellipticity of the diffraction pattern is determined from an iso-intensity curve. A slight misalignment of the camera with respect to the orthogonal diffraction pattern is compensated for with the new tilted ellipse fit.

Ellipse fit.

The orientation of the diffraction pattern in the video image depends on the orientation of the camera with respect to the measuring geometry. The diffraction pattern is therefore described by the equation of a tilted ellipse. (References 18, 339) The ellipse parameters (see Figure 4 section a). namely location (x0,y0), orientation () and axes (A,B) are obtained by least squares fitting of the data points to the edge of the ellipse.(References 110, 3311)

Outlier rejection.

The ellipse parameters are heavily biased if outliers exist in the data points, or when part of a neighbouring cell coexists in the rectangular analysis region. The fitting algorithm is therefore encapsulated by an iterative procedure that skips the most-distant data point until all data points are within 10 pixels from the fit. The procedure rejects patterns if the remaining number of pixels drops below a specified number (50).

Distance to ellipse.

8 1. Ahn S.J., Rauh W., Geometric least squares fitting of circle and ellipse, Int. J. Pattern Recognit. Artif. Intell., vol. 13:(7), pp. 987-996, 1999. 2. Ballas S.K., Smith E.D., Red blood cell changes during the evolution of the sickle cell painful crisis, Blood, vol. 79:(8), pp. 2154-2163, Apr. 1992. 3. Banerjee R., Nageshwari K., Puniyani R.R., The diagnostic relevance of red cell rigidity, Clin. Hemorheol. Microcirc., vol. 19, pp. 21-24, 1998. 4. Bareford D., Stone P.C.W., Caldwell N... 9 Hart D., Rudman A.J., Least-squares fit of an ellipse to anisotropic polar data: Application to azimuthal resistivity surveys in karst regions, Computers & Geosciences, vol. 23:(2), pp. 189-194, 1997. 10 1. Ahn S.J., Rauh W., Geometric least squares fitting of circle and ellipse, Int. J. Pattern Recognit. Artif. Intell., vol. 13:(7), pp. 987-996, 1999. 2. Ballas S.K., Smith E.D., Red blood cell changes during the evolution of the sickle cell painful crisis, Blood, vol. 79:(8), pp. 2154-2163, Apr. 1992. 3. Banerjee R., Nageshwari K., Puniyani R.R., The diagnostic relevance of red cell rigidity, Clin. Hemorheol. Microcirc., vol. 19, pp. 21-24, 1998. 4. Bareford D., Stone P.C.W., Caldwell N... 11 Hart D., Rudman A.J., Least-squares fit of an ellipse to anisotropic polar data: Application to azimuthal resistivity surveys in karst regions, Computers & Geosciences, vol. 23:(2), pp. 189-194, 1997.



The distance of a data point to the obtained ellipse has to be calculated in order to reject outliers. Calculating this distance requires solving a quadratic equation, which is an inefficient procedure. A good approximation can be obtained using orthogonal hyperbola. (References 5012, 5113) Confocal ellipses and hyperbola intersect orthogonally (see Figure 4 section b). This property is exploited to find the confocal hyperbola that crosses a specific data point. The distance from the data point to the point of intersection between the ellipse and the hyperbola provides a good approximation of the shortest distance to the ellipse. (References 5014, 5115) The average distance of the edge pixels to the best-fit ellipse is defined as the Error Of Fit (Eof) parameter.

Figure 4: Tilted ellipse with its five ellipse parameters

Figure 4.

A. Tilted ellipse with its five ellipse parameters indicated: location (x0, y0), orientation () and axes (a,b),

B. the closest distance of a data point to the best-fit ellipse is estimated using the confocal hyperbola that goes through the data points and intersects the ellipse orthogonally.

Accuracy of ellipse parameters.

The accuracy of the ellipse parameters is determined in one of two possible ways.

1. If one configures the software to analyse a certain number of diffraction patterns in order to reduce measurement inaccuracy, the programs displays the mean and standard deviation for each of the obtained ellipse parameters, and of the derived parameters (such as the EI).

12 Rosin P.L., Ellipse fitting using orthogonal hyperbolae and Stirling's oval, Graphical Models and Image Processing, vol. 60:(3), pp. 209-213, May 1998. 13 Rosin P.L., Analysing error of fit functions for ellipses, Pattern Recognit. Lett., vol. 17, pp. 1461-1470, 1996. 14 Rosin P.L., Ellipse fitting using orthogonal hyperbolae and Stirling's oval, Graphical Models and Image Processing, vol. 60:(3), pp. 209-213, May 1998. 15 Rosin P.L., Analysing error of fit functions for ellipses, Pattern Recognit. Lett., vol. 17, pp. 1461-1470, 1996.



2. If only one diffraction pattern is analysed, which can be configured, e.g., for RBC-stability testing, parameter accuracy will be estimated using the Bootstrap method. (References 4716, 6017) This method takes M random data points from the set of N that remained after outlier rejection. M is normally taken equal to N resulting in a new hypothetical data set in which some data points appear more than once while others are omitted. By repeatedly (300x) generating these hypothetical data sets from the original set, and by fitting each set to the equation of a tilted ellipse, a distribution is obtained for each of the five ellipse parameters. The average value is a good estimate of the true parameter while the standard error serves to indicate the accuracy of the estimate.

Normally, the diffraction pattern is displayed on an orthogonal base and the orientation angle () will not be of value in ektacytometry. Therefore, A is taken the axis closest to the vertical and B is taken the axis closest to the horizontal. With this definition EI can be negative when the diffraction pattern is rotated over 90º, which happens when the cells orient differently in shear flow, e.g., in sickle cell anemia (Reference 918) or elliptocytosis. (References 1819, 1520)

Note that the accuracy of the fit also depends on the number of edge coordinates that are extracted from the image. The number of edge coordinates can be increased by fitting more than one intensity level, at the cost of computational speed.

11.6.2.2. RBC-aggregation and –relaxation measurements

The state of RBC (dis-)aggregation is shear rate dependent: at high shear rates, all red cells in normal human blood are dispersed and deformed. At very low shear rates or at stasis, aggregates (rouleaux) will form. RBC aggregation behaviour can be studied from light intensity measurements; both changes in light absorption and in light back scatter can be used. The LoRRca applies a light back scatter system.

Various aggregation indices can be determined in whole blood by means of back scatter measurements. A diode laser, which is mounted inside the BOB, serves as light source. Light that is back scattered by the layer of blood in the gap is collected by two photodiodes inside the static BOB and is converted into an electrical signal (Figure 5).

16 Press W.H., Teukolsky A.A., Vetterling W.T., Flannery B.P., Quick-and-dirty monte carlo: The bootstrap method, in: Numercal recipes in C, The art of scientific computing, Cambridge University Press, 2nd ed., pp. 691-694, 1992 (ISBN: 0-521-43108-5). 17 Thomas G.E., Use of the bootstrap in robust estimation of location, The Statistician, vol. 49:(1), pp. 63-77, 2000. 18 Bessis M., Mohandas, N., Feo C., Automated ektacytometry: A new method of measuring red cell deformability and red cell indices, Blood cells, vol. 6, pp. 315-327, 1980. 19 Dobbe J.G.G., Hardeman M.R., Streekstra G.J., Strackee J., Ince C., Grimbergen C.A., Analyzing red blood-cell deformability distributions, Blood Cells, Molecules, and Diseases, vol. 28:(3), pp. 373-384, May/Jun 2002. 20 Dobbe J.G.G., Engineering developments in hemorheology, PhD Thesis, University of Amsterdam, Sept. 2002.



11.6.2.2.1. Syllectometry

Syllectometry as originally described by Zijlstra (References (on page 215) 10,65) is commonly employed to measure RBC aggregability. (References (on page 215) 6,22,23,31,36,44,48,54,57) In this method blood is illuminated and subjected to shear stress, causing the cells to deform and align in the direction of the flow (Figure.5). The change in back scattered light is measured after the driving mechanism stops. (The time needed for the suspension to come to a stop influences the syllectogram and its derived parameters for large gaps (> 0.6 mm), see References (on page 215) 15 or 16 for details.) The resultant time-dependent intensity plot is called a syllectogram. A high intensity peak shows up at the beginning of the plot as cells return to their original randomly-oriented biconcave shape and lose their alignment. The intensity of the back scattered light decreases as aggregation proceeds. A mathematical representation of the syllectogram yields aggregation parameters that are used to assess RBC aggregability. (References (on page 215) 6,23,31)

Figure 5: LORCA aggregometer setup

Figure 5. LORCA aggregometer setup. A laser beam is incident on a layer of blood between a static inner cylinder (BOB) and a rotating outer cylinder (CUP). Back-scattered light is collected by two photo diodes. Electrical voltage that is represented by the intensity scale of the syllectogram (range: 0 – 100 arbitrary units).

In a syllectogram, four RBC-behavioural stages are distinguished (Figure 6).

These are:

1. The initial plateau during the disaggregation stage originates from the light that is back scattered by the elongated RBCs that are aligned in the direction of the flow. The intensity of the light relates to the state of elongation and thus depends on the shear rate.

2. In the shape-recovery stage that follows immediately after cup cessation, cells collectively lose their alignment and return to their biconcave resting shape. The recovery stage is accompanied by an exponential increase in light back-scatter (References (on page 215) 5,34,59) leading to a high intensity peak in the syllectogram.



3. Aggregation starts during the shape-recovery stage when external shear forces fail to keep the RBCs dissociated. RBCs start to aggregate side-to-side as stacks of coins, called rouleaux, causing the back-scatter of light to decrease exponentially (Reference 2521) with a time-constant of about 1 -3 s in normal human blood.

5. Rouleaux formation is immediately followed by so-called 3D aggregate formation during which rouleaux connect end-to-end as well as side-to-end, creating larger 3D aggregates. In normal human blood, the formation of 3D aggregates is a slower process (Reference 622) with a time-constant of about 10 - 25 s.

21 Groner W., Mohandas N., Bessis M., New optical technique for measuring erythrocyte deformability with the Ektacytometer, Clin. Chem., vol. 26:(10), pp. 1435-1442, 1980. 22 Bauersachs R.M., Wenby R.B., Meiselman H.J., Determination of specific red blood cell aggregation indices via an automated system, Clin. Hemorheol., vol. 9, pp. 1-25, 1989.

Figure 6: The syllectogram distinguishes four behavioural stages

Figure.6. The syllectogram distinguishes four behavioural stages:

1. Disaggregation,

2. RBC-shape recovery,

3. Rouleaux formation immediately followed by

4. 3D aggregate formation.

The peak duration is exaggerated. au = arbitrary units.



11.6.2.2.2. Tri-exponential syllectogram representation

The conventional mathematical representation of the syllectogram contains two time-constants and describes the curve from the peak onward. (References 623, 2324, 3125) The tri-exponential representation (References 1526, 1627) introduces a third time-constant to include the upstroke caused by RBC shape recovery. (Reference 528) Thus, the intensity curve I(t) was fitted using a tri-exponential function containing three time-constants associated with RBC-shape recovery (Tr), rouleaux formation (Tf) and 3D aggregate formation (Ts):

(Equation 4)

where Ir, If, and Is denote the contribution of shape recovery, (fast) rouleaux formation and (slow) 3D aggregate formation, respectively.

The curve fit is performed using the Levenberg-Marquardt algorithm (Reference 4629) for fitting non-linear functions. Since the syllectogram is sampled uniformly most data points stem from the tail of the curve. To prevent the fitting algorithm from focusing on this region, the curves were re-sampled by selecting 300 points uniformly distributed on a logarithmic time scale.

Several aggregation parameters are derived from the syllectogram as indicated in Figure 6. To be able to distinguish “fitted” intensity parameters from “conventional” parameters (not resulting from the curve fit), the latter gives the symbol Isc.

23 Bauersachs R.M., Wenby R.B., Meiselman H.J., Determination of specific red blood cell aggregation indices via an automated system, Clin. Hemorheol., vol. 9, pp. 1-25, 1989. 24 Firsov N.N., Bjelle A., Korotaeva T.V., Priezzhev A.V., Ryaboshapka O.M., Clinical application of the measurement of spontaneous erythrocyte aggregation and disaggregation. A pilot study, Clin. Hemorheol. Microcirc., vol. 18, pp. 87-97, 1998. 25 Hardeman M.R., Goedhart P.T., Dobbe J.G.G., Lettinga K.P., Laser-assisted Optical Rotational Cell Analyser (LORCA); A new instrument for measurement of various structural hemorheological parameters, Clin. Hemorheol., vol. 14:(4), pp. 605-618, 1994. 26 Dobbe J.G.G., Engineering developments in hemorheology, PhD Thesis, University of Amsterdam, Sept. 2002. 27 Dobbe J.G.G., Streekstra G.J., Strackee J., Rutten M.C.M., Stijnen J.M.A., Grimbergen C.A., Syllectometry: Effect of aggregometer geometry in the assessment of red blood-cell shape recovery and aggregation, IEEE-Transactions on Biomedical Engineering, vol. 50:(1), pp. 97-106, 2003. 28 Baskurt O.K., Meiselman H.J., Determination of red blood cell shape recovery time constant in a couette system by the analysis of light reflectance and ektacytometry, Biorheology, vol. 33:(6), pp. 489-503, 1996. 29 Press W.H., Teukolsky A.A., Vetterling W.T., Flannery B.P., Levenberg-Marquardt method, in: Numercal recipes in C, The art of scientific computing, Cambridge University Press, 2nd ed., pp. 683-688, 1992 (ISBN: 0-521-43108-5).



The amplitude (Amp=Isctop – Isc0) is used to describe the extent of aggregation. (References 630 ,3131) Aggregation kinetics are described by the time-constants Tf and Ts but also by t½. The latter is the time that elapses until the peak intensity is reduced by half the amplitude (to Isc½). The overall aggregation behaviour of the suspension in described by a single parameter: the aggregation index. This aggregation index (AI) is a value between 0 and 100% and depends on both the extent and kinetics of aggregation. (References (on page 215) 632, 2233, 3134, 5735) It is determined from the areas A and B bounded by t = ttop and t = ttop + 10 as AI = 100% * A/(A+B) (see Figure 6).

The time elapsed until the occurrence of the peak is sometimes used as an indication of the RBC-shape recovery time. The peak occurs when the first derivative of equation 4 equals zero. The first derivative depends on all parameters of equation 4 including the aggregation parameters. The fact that ttop is influenced by the aggregation process makes it an unsuitable candidate for representing the RBC-shape recovery time.

The aggregation software allows the user to display the results of a fixed syllectogram model or to display the best performing one (auto-mode):

Mode Equation Relevant program parameters

Tri-exp (rise-fall-fall)

Ir, Tr : RBC relaxation

If, Tf : RBC rouleaux formation

Is, Ts : 3D aggregate formation

Bi-exp (rise-fall)


If, Tf : Mix of RBC rouleaux and 3D aggregate formation

30 Bauersachs R.M., Wenby R.B., Meiselman H.J., Determination of specific red blood cell aggregation indices via an automated system, Clin. Hemorheol., vol. 9, pp. 1-25, 1989. 31 Hardeman M.R., Goedhart P.T., Dobbe J.G.G., Lettinga K.P., Laser-assisted Optical Rotational Cell Analyser (LORCA); A new instrument for measurement of various structural hemorheological parameters, Clin. Hemorheol., vol. 14:(4), pp. 605-618, 1994. 32 Bauersachs R.M., Wenby R.B., Meiselman H.J., Determination of specific red blood cell aggregation indices via an automated system, Clin. Hemorheol., vol. 9, pp. 1-25, 1989. 33 Donner M., Siadat M., Stoltz J.F., Erythrocyte aggregation: Approach by light scattering determination, Biorheology, vol. 25:(1/2), pp. 367-375, 1988. 34 Hardeman M.R., Goedhart P.T., Dobbe J.G.G., Lettinga K.P., Laser-assisted Optical Rotational Cell Analyser (LORCA); A new instrument for measurement of various structural hemorheological parameters, Clin. Hemorheol., vol. 14:(4), pp. 605-618, 1994. 35 Stoltz J.F., Donner M., Erythrocyte aggregation: Experimental approaches and clinical implications, Int. Angiol., vol. 6, pp. 193-201, 1987.



Bi-exp (fall-fall)

Conventional syllectogram model: (References 636, 3137)


Is, Ts : 3D aggregate formation

Uni-exp (rise)


May be interesting to study pure relaxation behaviour, e.g., RBC in buffer solution

Uni-exp (fall)


Describes curve as if only one exponent would describe RBC aggregation.

36 Bauersachs R.M., Wenby R.B., Meiselman H.J., Determination of specific red blood cell aggregation indices via an automated system, Clin. Hemorheol., vol. 9, pp. 1-25, 1989. 37 Hardeman M.R., Goedhart P.T., Dobbe J.G.G., Lettinga K.P., Laser-assisted Optical Rotational Cell Analyser (LORCA); A new instrument for measurement of various structural hemorheological parameters, Clin. Hemorheol., vol. 14:(4), pp. 605-618, 1994.



11.6.2.2.3. Iteration procedure

For diagnostic reasons it is often desirable to know the threshold shear rate. (Reference 638) This threshold is found from the back scatter vs. shear rate curve as the shear rate where a peak is detected. At this point, the RBCs balance between aggregating and disaggregating. The threshold shear rate is a sensitive parameter to demonstrate hyperaggregation and cannot be found from the syllectogram. With the LORCA it is possible to search for the threshold shear rate in two ways:

With intermediate disaggregation (recommended). In this mode (see Figure 7) the program first disaggregates the RBC suspension before measuring at each user defined (lower) shear rate. This may be favourable as the standard procedure since potential aggregates will break down during disaggregation resulting in the same starting point before each back scatter measurement at a given shear stress. A user-defined list of shear rates is first processed to get a rough impression of the threshold shear rate. When intermediate disaggregation is enabled, which is preferable, the difference between the back scatter during disaggregation and that at each subsequent user defined shear rate (dIsc) is determined vs. user defined shear rate. Next, a binary search algorithm is used to automatically generate two more shear rates to fine-tune the rough procedure. Each binary search step results in two more shear rates to measure the back scatter intensity. They are chosen centered between the current optimum shear rate and next higher and lower shear rates. The number of binary search steps that is used for optimization is also user defined. From this so-called iteration procedure, the shear rate with the lowest dIsc is pointed out as the threshold shear rate.

Figure 7: Iteration procedure with intermediate disaggregation

Figure 7. Iteration procedure with intermediate disaggregation. The back scatter level during each disaggregation and that at each user defined shear rate is measured (a) and the difference in Intensity of each shear rate with that of the disaggregation shear rate is plotted vs. shear rate (b).

38 Bauersachs R.M., Wenby R.B., Meiselman H.J., Determination of specific red blood cell aggregation indices via an automated system, Clin. Hemorheol., vol. 9, pp. 1-25, 1989.



Without intermediate disaggregation. This mode (Figure 8) is very similar to the former but without intermediate disaggregation. The absolute value of the back scatter intensity at each shear rate serves to determine Isc vs. shear rate. The threshold shear rate is taken where the back scatter intensity is the highest. Note that some aggregates might still remain at the threshold shear rate. Therefore, during the iteration procedure, the back scatter level (aggregation level) at the threshold shear rate may differ from the peak value in the Syllectogram.

Figure 8: Iteration procedure without intermediate disaggregation

Figure 8. Iteration procedure without intermediate disaggregation. After disaggregating the sample once, the program determines the back-scatter intensity (Isc) vs. shear rate.

11.6.2.3. Flow to Stasis Aggregation Ratio (FSAR)

To get an indication whether there is any additional (secondary) aggregation at low flow, the ratio of the back scatter intensity at an optional low shear rate, e.g. 10 s-¹ (Isc10) and Isc at stasis (Isc0) can be calculated. In case of additional flow-induced aggregation (lower Isc), a Flow-to-Stasis Aggregation Ratio (FSAR) value of <1 can be expected.

(Equation 5)

Usually, however, FSAR values are > 1, meaning that there is rather breakdown of aggregates due to flow than hyper aggregate formation.



11.6.2.3.1. Diaphragm adjustment and sample thermostation

The measurement procedure can be cancelled by clicking “Cancel”. If the procedure is continued, by clicking the “Ok” button, a new window appears in which the operator is asked to adjust the camera diaphragm to scale the diffraction pattern for maximum size within the screen. The applied shear stress (<SETTINGS> <SHEAR> stress for camera adjustment>) is taken high enough to fully elongate the cells in order to get a maximum elliptical diffraction pattern.

Figure 9. Optimum contrast is obtained if the fit level excludes higher order diffraction patterns but is low enough to get a large image.

The original image is a gray scale image containing an intense zero order diffraction pattern and some weak higher order patterns. Figure. 9 shows the intensity distribution of a single image line. The intensity drop in the middle of the zero-order peak is caused by the laser absorber dot, which prevents camera overexposure. The smaller peaks at both sides of the zero-order peak represent the higher orders. When opening the diaphragm, light intensity increases, hereby increasing also the higher order patterns. As soon as the intensity of the higher order patterns exceed the "Fit gray level" (see <Settings><Fit gray level>), they participate in the iso-intensity curve and the pattern ellipse is detected incorrectly. When the diaphragm is closed too far, the diffraction pattern can hardly be distinguished from the laser spot, which also results in a wrong detection of the iso-intensity curve. Thus, the diaphragm should be opened to scale the diffraction pattern for maximum size within the screen, yet without including higher order diffraction patterns and without introducing secondary reflections.

Since it is difficult to distinguish the zero-order diffraction pattern from higher order patterns within the gray-scale image, each gray level (range 0..255) is assigned a colour. The on-screen diffraction pattern is created using this colour look up table (see table Colour look-up table).

The “User colours” checkbox can be used to switch between gray images and colour images. Click the “Ok” button if the camera diaphragm is well adjusted and the sample has reached the desired temperature.

Gray level Colour



0 Black

1..Fit gray level Dark..Light blue

Fit gray level..Fit gray level + higher levels Black

Rest..254 Dark..Light red

255 White

Table, Colour look-up table definition for converting gray scale images into colour images.



11.6.3. References

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17. Dobbe J.G.G., Streekstra G.J., Hardeman M.R., Ince C., Grimbergen C.A., The measurement of the distribution of red blood-cell deformability using an automated rheoscope, Clinical Cytometry, vol. 50, pp. 313-325, Dec. 2002.

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19. Deng L.H., Barbenel J.C, Lowe G.D.O., Influence of hematocrit on erythrocyte aggregation kinetics for suspensions of red blood cells in autologous plasma, Biorheology, vol. 31, pp. 193-205, 1994.

20. Dognon A., Loeper J., Housset E., Etude optique de l'aggregation reversible des hematies, Comptes Rendus Soc. Biol., vol. 143, pp. 769-771, 1949.

21. Dondorp A.M., Kager P.A., Vreeken J., White N.J., On the pathophysiology of servere falciparum malaria (Chapter 2, The rheology of severe falciparum malaria), in: MD Thesis, University of Amsterdam, pp. 25-44, 1999 (ISBN: 90-90-9012968-5).

22. Donner M., Siadat M., Stoltz J.F., Erythrocyte aggregation: Approach by light scattering determination, Biorheology, vol. 25:(1/2), pp. 367-375, 1988.

23. Firsov N.N., Bjelle A., Korotaeva T.V., Priezzhev A.V., Ryaboshapka O.M., Clinical application of the measurement of spontaneous erythrocyte aggregation and disaggregation. A pilot study, Clin. Hemorheol. Microcirc., vol. 18, pp. 87-97, 1998.

24. Fisher T., Schmid-Schönbein H., Tank tread motion of red cell membranes in viscometric flow: Behavior of intracellular and extracellular markers (with film), in: Red Cell Rheology, Springer-Verlag Berlin, pp. 347-361, 1978 (ISBN: 3-540-09001-0).

25. Groner W., Mohandas N., Bessis M., New optical technique for measuring erythrocyte deformability with the Ektacytometer, Clin. Chem., vol. 26:(10), pp. 1435-1442, 1980.

26. Hardeman M.R., De Rie M.A., Schut N.H., Wilmink J.H., Impaired erythrocyte deformability in psoriasis patients after treatment with cyclosporin, Clin. Hemorheol., vol. 11:(6), pp. 784, 1991.

27. Hardeman M.R., Dobbe J.G.G., Ince C., The laser-assisted optical rotational cell analyzer (LORCA) as red blood cell aggregometer, Clin. Hemorheol. Microcirc., vol. 25, pp. 1-11, 2001.

28. Hardeman M.R., Goedhart P.T., Interrelationship of changes in hemorheological parameters induced by low osmolar radio-contrast media, Clin. Hemorheol., vol. 12, pp. 381-392, 1992.

29. Hardeman M.R., Goedhart P.T., Laser-assisted Optical Rotational Cell Analyser (LORCA); Red blood cell aggregometry, in: Editions Medicales Internationales, Paris, 1994.

30. Hardeman M.R., Goedhart P.T., Schut N.H., Laser-assisted Optical Rotational Cell Analyser (LORCA); II Red blood cell deformability: Elongation Index versus Cell Transit Time, Clin. Hemorheol., vol. 14:(4), pp. 619-630, 1994.

31. Hardeman M.R., Goedhart P.T., Dobbe J.G.G., Lettinga K.P., Laser-assisted Optical Rotational Cell Analyser (LORCA); A new instrument for measurement of various structural hemorheological parameters, Clin. Hemorheol., vol. 14:(4), pp. 605-618, 1994.

32. Hardeman M.R., Ince C., Clinical potential of in vitro measured red cell deformability, a myth?, Clin. Hemorheol. Microcirc., vol. 21, pp. 277-284, 1999.

32. Hart D., Rudman A.J., Least-squares fit of an ellipse to anisotropic polar data: Application to azimuthal resistivity surveys in karst regions, Computers & Geosciences, vol. 23:(2), pp. 189-194, 1997.



34. Hochmuth R.M., Worthy P.R., Evans E.A., Red cell extensional recovery and the determination of membrane viscosity, Biophys. J., vol. 26:(4), pp. 101-114, Apr. 1979.

35. Kubota K., Kurabayashi H., Tamura J., The behavior of diabetic red cells in microcirculation examined by a new microcapillary method, Ann. Hematol., vol. 78, pp. 571-572, 1999.

36. Lademann J., Weigmann H.G., Sterry W., Roggan A., Muller G., Priezzhev A.V., Firsov N.N., Investigation of the aggregation and disaggregation properties of erythrocytes by light scattering measurements, Laser Phys., vol. 9:(1), pp. 357-362, 1999.

37. Maeda N., Seike M., Shiga T., Effect of temperature on the velocity of erythrocyte aggregation, Biochem. Biophys. Acta, vol. 904, pp. 319-329, 1987.

38. Mechatronics Instruments B.V., P.O.Box 225, 1620 AE Hoorn, De Corantijn 13, 1689 AN Zwaag, The Netherlands, Phone +31(0)229–291129, Fax +31(0)229 241534.

39. Meiselman H.J., Red blood cell role in RBC aggregation: 1963-1993 and beyond, Clin. Hemorheol., vol. 13:(5), pp. 575-592, 1993.

40. Mohandas N., Measurement of cellular deformability and membrane properties of red cells by ektacytometry, in: Red Cell Membranes. Shohet, S., Mohandas, N. (Eds). New York: Churchill Livingstone, pp. 299, 1988.

41. Mohandas N., Clark M.R., Jacobs M.S., Groner W., Shohet S.B., Ektacytometric analysis of factors regulating red cell deformability, Blood cells, vol. 6, pp. 329-334, 1980.

42. Mohandas N., Phillips W.M., Bessis M., Red blood cell deformability and hemolytic anemias, Semin. Hematol., vol. 16:(2), pp. 95-114, 1979.

43. Mokken F.Ch., Kedaria M., Henny Ch.P., Hardeman M.R., Gelb A.W., The clinical importance of erythrocyte deformability, a hemorheological parameter, Ann. Hematol., vol. 64, pp. 113-122, 1992.

44. Neumann F.J., Schmid-Schönbein H., Ohlenbusch H., Temperature-dependence of red cell aggregation, Eur. J. Physiol., vol. 408, pp. 524-530, 1987.

45. Nguyen F., Drouet L., Boisseau M., Leger P., Juchet H., Bierme R., Cambus J.P., Erythrocyte hyper-aggregation and thrombogenic dysfibrinogenemia, Clin. Hemorheol. Microcirc., vol. 18:(4), pp. 235-243, 1998.

46. Press W.H., Teukolsky A.A., Vetterling W.T., Flannery B.P., Levenberg-Marquardt method, in: Numercal recipes in C, The art of scientific computing, Cambridge University Press, 2nd ed., pp. 683-688, 1992 (ISBN: 0-521-43108-5).

47. Press W.H., Teukolsky A.A., Vetterling W.T., Flannery B.P., Quick-and-dirty monte carlo: The bootstrap method, in: Numercal recipes in C, The art of scientific computing, Cambridge University Press, 2nd ed., pp. 691-694, 1992 (ISBN: 0-521-43108-5).

48. Priezzhev A.V., Ryaboshapka O.M., Firsov N.N., Sirko I.V., Aggregation and disaggregation of erythrocytes in whole blood: Study by backscattering technique, J. Biomed. Opt., vol. 4:(1), pp. 76-84, Jan. 1999.

49. Reid H.L., Barnes A.J., Lock P.J., Dormandy J.A., Dormandy T.L., A simple method for measuring erythrocyte deformability, J. Clin. Pathol., vol. 29, pp. 855-858, 1976.

50. Rosin P.L., Ellipse fitting using orthogonal hyperbolae and Stirling's oval, Graphical Models and Image Processing, vol. 60:(3), pp. 209-213, May 1998.

51. Rosin P.L., Analysing error of fit functions for ellipses, Pattern Recognit. Lett., vol. 17, pp. 1461-1470, 1996.



52. Schmid-Schönbein H., Letter to the editors-in-chief, Clin. Hemeorheol., vol. 13, pp. 803-805, 1993.

53. Schmid-Schönbein H., Malotta H., Striesow F., Erythrocyte aggregation: causes, consequences and methods of assessment, Tijdschr. NVKC, vol. 15, pp. 88-97, 1990.

54. Schmid-Schönbein H., Van Gosen J., Heinrich L., Klose H.J., Volger E., A counter-rotating “rheoscope chamber” for the study of the microrheology of blood cell aggregation by microscopic observation and microphotometry, Microvascular Research, vol. 6, pp. 366-376, 1973.

55. Schmid-Schönbein H., Wells R., Schildkraut R., Microscopy and viscometry of blood flowing under uniform shear rate (rheoscopy), J. Appl. Physiol., vol. 26:(5), pp. 674-678, May 1969.

56. Schut N.H., Hardeman M.R., Wilmink J.M., Decrease of erythrocyte deformability in cyclosporin-treated renal transplant patients: correction with fish oil as well as corn oil, Transpl. Int., vol. 5:(1), pp. S536-S538, 1992.

57. Stoltz J.F., Donner M., Erythrocyte aggregation: Experimental approaches and clinical implications, Int. Angiol., vol. 6, pp. 193-201, 1987.

58. Streekstra G.J., Hoekstra A.G., Nijhof E.J., Heethaar R.M., Light scattering by red blood cells in ektacytometry: Fraunhofer versus anomalous diffraction, Appl. Opt., vol. 32, pp. 2266-2272, 1995.

59. Sutera S.P., Mueller E.R., Zahalak G.I., Extensional recovery of an intact erythrocyte from a tank-treading motion, J. Biomech. Eng., vol. 112, pp. 250-256, 1990.

60. Thomas G.E., Use of the bootstrap in robust estimation of location, The Statistician, vol. 49:(1), pp. 63-77, 2000.

61. Vekasi J., Marton Z.S., Kesmarky G., Cser A., Russai R., Horvath B., Hemorheological alterations in patients with diabetic retinopathy, Clin. Hemorheol. Microcirc., vol. 24:(1), pp. 59-64, 2001.

62. White F.M., Flow between long concentric cylinders, in: Fluid mechanics, McGraw-Hill, Fourth edition, pp. 261-263, 1999 (ISBN: 0-07-069716-7).

63. Williamson J.R., Gardner R.A., Boyland C.W., Carroll, G.L., Chang K., Marvel J.S., Gonen B., Kilo C., Tran-son-tay R., Sutera S.P., Microrheologic investigation of erythrocyte deformability in diabetes mellitus, Blood, vol. 65:(2), pp. 283-288, Feb. 1985.

64. Zhao H., Wang X., Stoltz J.F., Comparison of three optical methods to study erytrhocyte aggregation, Clin. Hemorheol. Microcirc., vol. 21, pp. 297-302, 1999.

65. Zijlstra W.G., Syllectometry, a new method for studying rouleaux formation of red blood cells, Netherlands society for physiology and pharmacology, vol. 8, pp. 153-154, Oct. 1957.

Drawings and parts Lorrca® MaxSis


12. DRAWINGS AND PARTS LORRCA® MAXSIS

Drawing section.

12.1. LORC070020 Tubing diagram

Parts LORC070020 Tubing diagram

Drawings and parts Lorrca® MaxSis


12.2. LORC080020 Connection diagram

Log book (Example)


13. LOG BOOK (EXAMPLE)

Name:

Date:

Description visual inspection OK NOT OK REMARKS

Scratches on glass surface of cup

Check tube attachments

Check tube damage

Description functional test OK NOT OK REMARKS

Hardware check

For accordance Manager:

Remarks:

Glossary of Terms


14. GLOSSARY OF TERMS A 14.1.1.1.1. AMC

AMC stands for the Academic Medical Center in Amsterdam, Netherlands.

E 14.1.1.1.2. EI

EI = expressed as elongation index

H 14.1.1.1.3. HE

HE stands for Hereditary Elliptocytosis.

L 14.1.1.1.4. Lorca

Lorca stands for Laser-assisted Optical Rotational Deformability Cell Analyzer

M 14.1.1.1.5. MWM

MWM stands for Medical Waste Management

R 14.1.1.1.6. RBC

RBC is used as a short for Red Blood Cell

Index


15. INDEX

A

ABOUT RR MECHATRONICS • 11 About this manual • 12 Adjust temperature RBC aggregation screen

• 133 Adjust video camera deformability screen •

114 Adjust video camera membrane screen • 126 Adjust video camera Osmoscan screen • 108 Adjust video camera stability screen • 120 Adjusting camera for slides • 167 Adjusting geometry parameters • 164 Adjusting projection screen • 156, 165 Adjustments and replacements • 165 After replacement of all tubing • 183 Aggregation • 62, 96 Aggregation parameters explanation • 97, 98,

134 AMC • 223 Appendix - Additional Information • 196 Appendix - Document History Extended

overview • 194 Appendix - Ordering parts • 190 Appendix - Parts list • 105, 111, 117, 123,

138, 165, 176, 191 APPENDIX FOR LORRCA® MAXSIS • 185 At measurement completion • 93

B

Back up the results file • 164 Basics of bio safety • 34 Bio Safety bob assembly • 32 Bio safety Standard Operating Procedures

(S.O.P.) • 32, 34

C

Camera • 50 Cell Membrane Stability report page 1 of 3 •

147 Cell Membrane Stability report page 2 of 3 •

148 Cell Membrane Stability report page 3 of 3 •

149 Cell membrane stability results screen • 91,

127

Cell Membrane stability screen • 51, 88, 90, 93, 94

Checking Lorrca® MaxSis functionality • 47 Cleaning • 159 Conditions • 40

D

Decontamination procedures Routine • 145 Deformability curve • 200 Deformability curve determination parameters

explanation • 79, 80, 115, 146 Deformability curve determination screen •

51, 78 Deformability curve Parameters • 60, 78 Deformability curve result screen • 79, 115 Deformability Report • 146 Deformability results 1 parameters

explanation • 89, 93, 147 Deformability results 1 screen • 89, 90, 93, 94 Deformability results 2 parameters

explanation • 92, 95, 128, 149 Deformability results 2 screen • 92, 128 Diaphragm adjustment and sample

thermostation • 213 Disaggregation options • 62 Discard of sample • 142 Document history overview • 3 DRAWINGS AND PARTS LORRCA®

MAXSIS • 219

E

Edit Iteration shear rates • 65 EI • 223 Electrical safety • 30 Ellipse fitting algorithm • 203 Elongation • 60 Empty cup cell membrane screen • 90 End of day wash • 76, 81, 84, 90, 100, 110,

117, 123, 129, 136, 142, 144 End of the day wash screen • 54 Environmental conditions and setting up the

instrument • 40 Error messages • 152 Error Messages (Oxygenscan) • 154 Estimated RBC in laser • 104 Explain Rinse cup deformability screen • 80 Explain Rinse gradient Oxygen screen • 83

F

Facts and definitions: • 35 Features of the Lorrca® MaxSis • 21

Index


Files • 59, 74, 79, 82, 86, 89, 91, 92, 97, 109, 115, 121, 127, 128, 134

First Start-up • 45 Flow to Stasis Aggregation Ratio (FSAR) •

212 FSAR options • 66

G

General laboratory practices: • 35 General measurements description • 24 GENERAL OPERATION PROCEDURE • 101 General options • 60 General safety rules • 28 Gradient pump tube replacement • 175

H

Hardware check Camera Led on screen • 57 Hardware check Camera screen • 56 Hardware check IO screen • 55 Hardware check status • 54 Hardware pO2 • 57 HE • 223 hematocrit • 198

I

Insert blood sample deformability screen • 112

Insert blood sample membrane screen • 124 Insert blood sample RBC aggregation screen

• 132 Insert blood sample stability screen • 118 Install LORC070901 tubing • 176 Install LORC070902 tubing set 1 • 179 Install LORC070903 tubing set 2 • 181 Install LORC070904 tubing set 3 • 183 INSTALLATION • 37 INSTRUMENT DESCRIPTION • 15 Instrument label Lorrca® MaxSis • 16 Instrument status • 48 Intended use • 20 INTRODUCTION • 12 Introduction Lorrca® MaxSis • 20 Introduction Osmoscan • 26 Introduction Oxygenscan • 27 Introduction RBC-aggregation • 26 Introduction RBC-elongation • 26 Iteration parameters setting • 64 Iteration procedure • 211

L

Laser and mechanical safety • 31

LOG BOOK (EXAMPLE) • 221 LORC070020 Tubing diagram • 29, 43, 219 LORC080020 Connection diagram • 220 Lorca • 223 Lorrca IO • 49 Lorrca MaxSis Program • 12 LORRCA MAXSIS PROGRAM • 51 Lorrca reagents • 102, 107, 113, 119, 125 Lorrca reagents routine • 163

M

Main power connection • 41 Main screen • 51, 52 Main screen settings • 52 MAINTENANCE • 157 Maintenance schedule • 158 Medical requirements: • 35 MWM • 223

O

Open Aggregation files • 100 Open Stability curve files • 87 Osmoscan curve • 77 Osmoscan options • 66, 74 Osmoscan parameters explanation • 74, 75,

109, 150 Osmoscan Report • 150 Osmoscan result screen • 74, 109 Osmoscan screen • 51, 73 Oxygen sensor spot protocol • 141, 160 Oxygenscan • 141 Oxygenscan options • 67, 81 Oxygenscan parameters explanation • 82, 83 Oxygenscan result screen • 82 Oxygenscan screen • 51, 81 Oxygenscan whole blood volume calculation

• 104

P

Performing Cell membrane stability test • 123 Performing Cell stability test • 117 Performing Deformability curve test • 111 Performing Osmoscan • 104 Performing Oxygenscan • 137 Performing RBC Aggregation test • 130 Place sample Oxygenscan test • 138 Place sample tube Osmoscan screen • 106 pO2 Steps • 67, 68 Powering down • 144 Powering up • 101 Preface • 197

Index


Pre-installation requirements • 37, 44 Preparation for long time inactivity • 184 Preparations • 102 Preparing a blood sample • 105, 111, 113,

117, 119, 123, 125 Preparing a blood sample Oxygenscan • 138 Preparing blood sample for RBC aggregation

test • 131, 132 Pre-test preparations • 137 Principle of operation • 198 Principle of operation Lorrca® MaxSis • 24 Pump connection tubing • 172

R

RBC • 223 RBC aggregation result screen • 97, 134 RBC aggregation screen • 51, 96 RBC necessary per test/scan • 103 RBC-aggregation and –relaxation

measurements • 205 RBC-deformability measurements • 199 References • 198, 202, 206, 209, 215 Replace the main fuses • 43, 170 Replacement sensor-spot • 160, 161 REPORTING • 146 Rinse cup cell membrane screen • 89 Rinse cup RBC Aggregation • 100 Rinse gradiënt Osmoscan screen • 76

S

S.O.P. for working with bio hazardous materials • 35

Safety • 12 SAFETY • 28 Safety for working with compressed Nitrogen

• 33 Screen communication settings • 53, 71 Settings screen • 58, 153 Specific laboratory practices and

requirements: • 36 Stability curve determination parameters

explanation • 86, 87, 121 Stability curve determination results screen •

86, 121 Stability Curve determination screen • 51, 85 Stability results parameters explanation • 91,

94, 127, 148 Stability test • 201 Stability test parameters • 62, 88 Start new measurement • 109, 116, 122, 128,

135

Stress curve determination Parameters • 61, 85

Syllectogram options • 63 Syllectometry • 206 Symbols • 22

T

Technical specifications • 17 Transport • 39 Tri-exponential syllectogram representation •

63, 208 TROUBLE SHOOTING • 151 Trouble shooting table • 156 Tubing replacements • 174 Typographical conventions • 13

U

Unpacking / Setting Up • 40, 41 Update the software • 164 Used symbols • 14

V

Visual checks • 163

W

Waste and rinse pump tube replacement • 171

Waste and rinse pump tube replacements • 171

Waste container disposal • 145

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