Page 1

V National Conference Clinical Laboratory Varna, 25.09. 2009

Basic Principles of Sysmex Fluorescence Urine Flow Cytometry Sysmex Europe GmbH


The Art of Urine Particle Analysis From the birth of microscopes.... ... to modern days‘ microscopy

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Quantitative and Accurate Counting => Difficult! Concentration factor? • Initial volume? • Sedimentation efficiency? • Residual volume?

Analysed volume? • • • •

Volume of sediment suspension? Thickness of volume under cover slide? Volume/optical field ? Number of checked HPF or LPF?

Analytics • Precision for low counts? • Subjective classification?

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Comparison Method: Sediment Microscopy Source of errors

CV [%]

● Volume of sediment suspension

3

● Edge length of cover slip

1

● Error propagation to the thickness of field of view

10

● Diameter of field of view

1

● Error of the WBC count per field of view

17

● Error of residual volume

50

● Error of initial volume

3

● Error propagation for WBC count/µL in native urine

32

● Error of centrifugation efficiency

19

● Total error propagation for WBC in native urine

136%

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Standardisation of Sediment Analysis is impossible Centrifugal loss (RBC) monitored with UF-100:

native urine

after 5 min

after 20 min

1,428

1,370

123

400

191

45

17,030

318

1

45

27

19

Univ. D端sseldorf, 1996

V National Conference on Clinical Laboratory, Varna, September 25, 2009


The Art of Urine Particle Analysis in Modern Days ....

V National Conference on Clinical Laboratory, Varna, September 25, 2009


The Measurement Principle


Starting with standardised sample preparation ● Automatic mixing, aspiration and dispensing of urine ● Aspiration of 800µL ● Dilution and staining ● Defined concentration ● 1:4 for sediment analysis ● 1:8 for bacteria analysis

● Incubation at certain temperatures

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Flowcytometry with Hydrodynamic Focussing ● Stained urine is injected into a flow channel surrounded by a carrier fluid ● Sheath fluid stretches the cell suspension to an extremely thin arrangement so that cells are lined up one after the other ● The sheath fluid with centered urine sample flow enters the flow cell ● The flow velocity is in the range of meters per second which results in a high count rate (up to several thousands of particles per second)

V National Conference on Clinical Laboratory, Varna, September 25, 2009


The Art of Urine Particle Analysis ... Count of 9Âľl or 45 triple ruled squares Modified Fuchs Rosenthal for urinary cytology with double cell 2 x 1.8mm3

SYSMEX UF-Series

~ ~

V National Conference on Clinical Laboratory, Varna, September 25, 2009


The Art of Urine Particle Analysis ... Detection of Optical Properties ● A laser beam causes the tagged cells to emit a momentary pulse of fluorescence ● This pulse of fluorescence is measured by photomultipliers ● Forward scattered light tells us about the diameter ● Optical properties differ from particle to particle ● The unique “look inside”

V National Conference on Clinical Laboratory, Varna, September 25, 2009


RBC

These RBC are regular in size and are shaped normally. This means that they probably derive not from the kidney but from the lower urogenital tract. In Fluorescence Urine Flow Cytometers they are identified based on their low fluorescence signal (no DNA) and based on their low Fsc signal (small sized cells).

V National Conference on Clinical Laboratory, Varna, September 25, 2009


RBC

These are dysmorphic RBC most likely deriving from the kidney. The arrow indicates an acanthocyte. Acanthocytes are classified as (microcytic and therefore, possibly dysmorphic) RBC in the UF-1000i and UF-500i.

V National Conference on Clinical Laboratory, Varna, September 25, 2009


WBC

WBC are classified based on their medium size fluorescence signal (from nucleic acids!) and forward scatter signal (medium size around 10 Âľm)

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Epithelial cells (EC)

Squamous epithelial cells

Squamous epithelial have a very large fluorescence and a large forward scatter signal

V National Conference on Clinical Laboratory, Varna, September 25, 2009


SRC (small round cells)

Transitional epithelial cells The UF-1000i/UF-500i classifies transitional epithelial cells (from the bladder and urethra) as well as renal tubular cells (from the kidney) as SRC.

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Cluster Analysis

V National Conference on Clinical Laboratory, Varna, September 25, 2009


The analytical Performance


Demands on automated Urine Particle Analysis ● Method with focus on ● Sorting out negative samples ● Better indicating most of the pathological samples ● Urinary tract infections ● Haematuria Kidney diseases Haematuria/ urinary tract infections

Negative samples

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Detection of Urinary Tract Infections


Bacteria specific staining by a dedicated interplay of reagents

Diluent and polymethine dye together bring out an effective staining of bacteria only.

Cell debris, often seen in urine, is not stained

V National Conference on Clinical Laboratory, Varna, September 25, 2009


UF-1000i: The evaluation of superior bacteria detection

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Results of a correlation study between UF1000i-technology and culture Bacteria in culture (Level 3, ECLM)

UF-1000itechnology

Bacteria in culture (Level 3, ECLM)

pos

neg

pos

44

6

50

neg

3

51

54

47

57

104

Sensitivity = 94 % Specificity= 89 %

V National Conference on Clinical Laboratory, Varna, September 25, 2009

Bacteria in routine culture (Level 2, ECLM)

pos

neg

pos

36

11

47

neg

3

54

57

39

65

104

Sensitivity = 92 % Specificity = 83 %


Better results in the clinically relevant low concentration range for bacteria E.coli 1E+09 1E+08

1E+07

1E+07

1E+06

1E+06

1E+05

1E+05 BACSYS

BACSYS

Ps.aeruginosa 1E+08

1E+04

1E+04

1E+03

1E+03

R=0.98012

R=0.954414

1E+02

1E+02

1E+01

1E+01

1E+00

1E+00

1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09

culture

V National Conference on Clinical Laboratory, Varna, September 25, 2009

1E+00

1E+01

1E+02

1E+03

1E+04

culture

1E+05

1E+06

1E+07

1E+08


Decisive results within short time All relevant indicators in respect to urinary tract infection available after approx. 1 minute

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Detection of Haematuria


Frequency of Haematuria The prevalence of asymptomatic microscopic hematuria in adult men and postmenopausal women*: â—? from 10 % to 20 percent

* Mohr DN, Offord KP, Owen RA, Melton LJ 3d. Asymptomatic microhematuria and urologic disease. A population-based study. JAMA 1986;256: 224-9. * Messing EM, Young TB, Hunt VB, Emoto SE, Wehbie JM. The significance of asymptomatic microhematuria in men 50 or more years old: findings of a home screening study using urinary dipsticks. J Urol 1987;137:919-22. * Britton JP, Dowell AC, Whelan P, Harris CM. A community study of bladder cancer screening by detection of occult urinary bleeding. J Urol 1992; 148:788-90. V National Conference on Clinical Laboratory, Varna, September 25, 2009


UF-1000i and the detection of haematuria ● Early detection of haematuria by keeping the proven high sensitivity of UFSeries ● Supplying the known additional RBC information essential for further investigation ● Improve the positive predictive value (PPV) and carry on to avoid unnecessary invasive diagnostic procedures such as cystoscopy and radiological investigations

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Glomerular Haematuria Tthe urinary sediment of patients with glomerular haematuria is characterised by* ● the presence of a large variety of erythrocyte shapes ● heterogeneity in shape and size of the erythrocytes ● also cell fragments can be found

*J. Huussen*, R.A.P. Koene, L.B. Hilbrands *The (fixed) urinary sediment, a simple and useful diagnostic tool in patients with haematuria

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Detection of Haematuria and Display of first Information

V National Conference on Clinical Laboratory, Varna, September 25, 2009


A Strong Combination: RBC, RBC Size and RBC Size Distribution ● Sensitivity is 100% (laser microscopy 96%) ● Specificity is 93% (laser microscopy 93%) to detect a renal bleeding Hyodo et al; Jpn J Nephrol 37: 35-43 (1995)

● Sensitivity is 83% with NPV 0.78 (microscopy 79% with NPV 0.74 ) ● Specificity 94% (PPV 0.95) ● (microscopy 90% with PPV 0.79) Apeland et al: Nephr. Dial Transplant (2001) V National Conference on Clinical Laboratory, Varna, September 25, 2009


UF-1000i: modern technology and its advantages

● UF-1000i is conveniently operated via its IPU ● The fast screening enables ● to quickly sort out negative samples ● to save unnecessary tests ● to cut down on unnecessary, blindly started administration of antibiotics

V National Conference on Clinical Laboratory, Varna, September 25, 2009


Thank you for your attention

V National Conference on Clinical Laboratory, Varna, September 25, 2009

5c_05  
5c_05  
Advertisement