URINE FLOWCYTOMETRY

IN THE DIAGNOSIS OF
HEMATURIA AND UTI

Hani Susianti

PDS Patklin Malang

 OUTLINES

1.Introduction

2.Urine flowcytometry in the diagnosis of hematuria

3.Urine flowcytometry in the diagnosis of UTI

 INTRODUCTION

• Urinalysis is one of the key tests to evaluate renal, extra renal and urinary tract
disease
• Urinalysis is more susceptible to poor standardization of analytical phases and
preanalytical issues, especially for urine particle analysis

• Time • High inter • Centrifuga-

• Exhibits low
consuming observer tion problem,
reproducibility
variable etc
 INTRODUCTION

• Over the past 25 years, new automated technologies have greatly reduced the
labor intensity of urinalysis and have created new technical possibilities

The Urinalysis Flowcytometry tool offers a range of clinical added values.

Which one is the most useful for you?
1. Estimating the location of the occurrence of hematuria through RBC
morphology (47%)
2. Urinary Tract Infection Screening (40% )
3. Identify problem of drug abuse sample through urine conductivity (11%)
4. Others (2% )
 AUTOANALYZER : Urine Flowcytometry

Material
Side
Fluorescent genetik
Light System

Laser Diode Spectrum

Photo Multiplier
Filter

Dichroic
Mirror
Side
Scattered
Light
Kompleksitas
Condenser System
Collimating Lens
Lens
Condens Beam
er Lens Stopper Photo
Diode

Flow
cell Condenser
Lens Pin
Hole
Photo
Diode Forward Ukuran
Scattered Light
System
 Urine Flowcytometry
• The introduction of a new blue laser
• Staining : Core & Surface channel
Core (CR) channel: Nucleic acid Surface (SF) channel: Components not
containing components containing nucleic acids
Nucleic acid specific staining Staining of RBC, casts, etc
WBC, epithelial cells, bacteria, fungi, etc.

Diluent

Incubation

Detection Unit
Staining
 URINE
FLOWCYTOMETRY
RESULT

UTI :
RBC : WBC
dysmorphic Bacteria
and
eumorphic
 2. URINE FLOWCYTOMETRY
IN THE DIAGNOSIS OF HEMATURIA
Hematuria
Hematuria Hematuria (Glomerular) (Non Glomerular)
• Hematuria cases are • IgA nephropathy (Berger's • Urolithiasis
found in 1-20% of disease)
• Pyelonephritis
population • Postinfectious
(streptococcal) GN • Bacterial cystitis (UTI)
• in RSSA laboratory there
were 12,673 cases of • Membranoproliferative GN • Benign prostatic
Hematuria (Ery > 3/hpf) • Focal Segmental hyperplasia
in 2016 Glomerulosclerosis • Transitional cell
• SLE nephritis 1 carcinoma
• etc • etc
2
 2. URINE FLOWCYTOMETRY
IN THE DIAGNOSIS OF HEMATURIA…

• The morphological differences between the dysmorphic

erythrocyte (glomerular source of bleeding) and eumorphic
erythrocyte (non glomerular source of bleeding) have been
described
• The prominent features of dysmorphic RBCs are variously
shaped blebs or projections at the cell membrane
(acanthocytes).
• In contrast, the morphological features of isomorphic RBCs
resulting from renal pelvis, ureter, or bladder bleeding are
uniform and no more than two types
• 14% of cases in RSSA laboratory has > 51% dysmorphic
erythrocytes
 Laboratory Test for Hematuria
Distinguishing extra-glomerular from glomerular hematuria
Extraglomerular Glomerular

Colour Red or pink urine Red, smoky brown, or "Coca-

Cola"

Clots May be present absent

RBC morphology Eumorphic Dysmorphic

Casts Absent May be present

Proteinuria Absent May be present
Acanthocyturia is more efficient in to differentiate glomerular from
non-glomerular hematuria then dysmorphic erythrocytes

• Glomerular hematuria defined as dysmorphic red cell count > 35% showed a
sensitivity and specificity of 69% and 100%, respectively.
• Glomerular hematuria defined as acanthocytes > 5% showed a sensitivity and
specificity of 88% and 100%, respectively.

Lopez & Brouad, Arch Esp Urol. 2002 Mar;55(2):164-6.

Sensitivity and specificity (95% confidence interval)
of dysmorphic urinary red cells for glomerular
microhaematuria

Postgrad Med J (1992) 68, 648 - 654

ISOMORPHIC AND DYSMORPHIC RBC

Microscopic Flowcytometry (Su et al, Nature, 2017)

Identification of RBC Isomorphic/
Dysmorphic/Mixed Using Urine
Flowcytometry
Research 1: RBC Information
 RED BLOOD CELL ANALYSIS WITH
AUTOMATED URINE PARTICLE ANALYSER

• Differentiates between glomerular and non glomerular hematuria based

on flowcytometry
• Needs 20 RBC/uL or 3 /HPF
• Fast

1. Accuracy ? ? 2. Diagnostic value ?? 3. Cut off ??

16
1. Accuracy ? ?
Study of glomerular and non-glomerular hematuria 2. Diagnostic value ??
by Urinary Flow Cytometer
Paper Title Sensitivity Spesificity NPV PPV
(%) (%) (%) (%)

Hyodo T, et al Differential Diagnosis between Glomerular 90 92

and Nonglomerular Hematuria by Automated
Urinary Flow Cytometer (Nephron 1999;82:312–323)

Apeland* Flow Cytometry of Urinary Erythrocytes for Evaluating the 75 86

Source of Haematuria Scand J Urol Nephrol 29: 33-37, 1995 (93) (100)

Apeland* et al Assessment of haematuria: automated urine 83 94 95 78

flowmetry vs microscopy, ephrology Dialysis Transplantation,
Volume 16, Issue 8, August 2001

Aulia D, et al Detection Glomerular and non glomerular hematuria using 87 82 90 77

automated urine particle analyzer (ISBN 978:979-496-702-7)
18
 CONFLICTING CRITERIA REGARDING
3. Cut off ??
THE LEVEL OF GLOMERULAR HEMATURIA

• Minimal proportion of dysmorphic RBC considered to indicate

glomerular bleeding varied between 10 - 90 %.
• There are widespread conflicting criteria regarding the level of
hematuria which becomes pathologically significant (such as :
more than 3000, 8000 or 20000 dysmorphic
RBC/mL)

Cut off of dysmorphic erythrocyte is needed for our laboratory

Postgrad Med J (1992) 68, 648 - 654

Diagnostic test results for glomerular hematuria
in RSSA Malang using Sysmex UX-2000

Sensitivity 96 %
Specificity 90%
Negative
predictive value 84.3%
Positive 89.2%
predictive value
Cut off of
dysmorphic >51%
erythrocyte

90 urine samples with hematuria Area under curve (AUC) : 0.97

glomerular and non glomerular
Int J Clin Oncol (2014) 19:928–934
 The Role of Urine Flowcytometry
in Hematuria Evaluation

• Flowcytometry
Crosscheck
Eumorphic
function Dysmorphic
History and Physical examination
• Microscopic
URINALYSIS

No RBC, Hb only RBC/Hb absent RBC(+)/Hb(+/-)

Myoglobinuria or Search other Check RBC

Hemoglobinuria cause of red morphology
urine

GLOMERULAR NON GLOMERULAR

URINARY TRACT INFECTION
• UTI is a common problem in population
• In Indonesia, the prevalence of UTI as a nosocomial infection is highest at
around 39% -60% (Riskesdas, 2013)

• Urine culture : time-consuming, labor intensive, expensive and often yield a

negative results
• The use of rapid and reliable screening method may help clinician to improve UTI
patients management
 Urine Flowcytometry – UTI Screening
Constructive Differences of Bacterial Cell Wall
Gram Gram
Properties Positive Negative
(GP) (GN)

Peptidoglycan layer Thick Thin

(multilayered) (single layer)

Outer membrane - +
LPS layer - +
Teichoic acids + -
Lipid content Low High
Gram Positive Gram Negative

Detecting the differences of Bacterial Cell Wall

by combination of reagents and FCM –based technology.
26
Urine Flowcytometry – UTI Screening

Sediment Bacteria
Diluents

Sediment Bacteria
Incubation

Detection
unit
Sediment Bacteria Stains
FLOWCYTOMETRY METHOD ON URINALYSIS : Scattergram B1

• Can be used to estimate gram

type of bacteria
• Based on angle 30˚
• Can reduce inappropriate
antibiotic using

Low Angle Pattern High Angle Pattern Board Angle Pattern

Gram negative bacteria Gram positive bacteria Mixed, or

Unclassified clearly
28
UTI STUDIES BASED ON URINE FLOWCYTOMETRY
Paper Location Year Instru- Number Cut Off Sensitivity Spesificity PPV NPV
ment of (%) (%) (%) (%)
Samples Bact WBC
(/uL) (/uL)
Manoni Italy 2019 UF-1000i 1,463 125 40 99 77 82 98
De Rossa Italy 2010 UF-1000i 1,394 170 150 98.8 76.5 59.2 99.5
Van der Zwet Netherlands 2010 UF-1000i 358 50 110 100 54 43.7 98.6
Wang China 2010 UF-1000i 313 100 56 97 79 70 99
Jollkonen Finland 2010 UF-500i 10,596 405 16 93.4 82.3 - 98.3
Prof Lisyani Indonesia 2012 UF-1000i 111 105 21 93.7 83.3 88.1 90.0
Verle Phillippine 2015 UX-2000 293 55 27 95.2 82.3 81.2 95.5
Prof Ida Indonesia 2015 UX-2000 203 572 19 95.07 58.3 96.4 50.0
Schuh Switzerland 2019 UX-2000 613 125 17 M: 100 M:54.8
F : 97.3 F : 45.9
 Reduction of Unnecessary Urine Culture

Paper Location Year Instrument Number of Reduction

Samples Culture(%)

De Rossa Italy 2010 UF-1000i 1,394 57.1

Van der Zwet Netherlands 2010 UF-1000i 358 42.0
Pierretti Italia 2010 UF-1000i 703 43.0
Jollkonen Finland 2010 UF-500i 10,596 69.0
Verle Phillippine 2015 UX-2000 293 43.6
Prof Ida Indonesia 2015 UX-2000 203 58.0
According to the European urinalysis guidelines, the parameters that should be counted
most accurately are RBC, WBC and bacteria

Bakan et al, Biochem Med (Zagreb) 2018;28(2):020712

Copyright PT Sysmex Indonesia 32
Precision Tests on urine flowcytometry
and manual microscopy

Xiang et al, Clin. Lab. 2012;58:1-5

 Case 1

• Male, 47 years old

• Since 2 weeks before
admission the patient
complained of an open
wound on the right foot.
• Since one week before the
hospital admitted the
patient was feverish and
weak.
• History of diabetes since 10
years ago, uncontrolled,
sometimes drinking
Gkibenclamid.
 Scatter diagram

Culture test result

Organism isolated :
Escherichia coli

Catatan :
• Hasil scatergram bakteri mengesankan kemungkinan suatu bakteri gram negatif
35
 Case 2

• Male, 62 year old

• Patient is referred from
public health with BPH.
• Patients complain of pain
during urination and back
pain
 Scatter diagram

Culture test result

Organism isolated :
Staph. aureus

Catatan :
• Hasil scatergram bakteri mengesankan kemungkinan suatu bakteri gram positif

37
38
 Hasil

Makroskopis
Parameter
Kekeruhan
2-11-2016
Jernih
CAPD analysis
Warna Kuning

Kimia pH 8,0
Berat jenis 1,020
Ny AP
Glukosa 2+ Nyeri perut
Protein 2+ sejak 3 hr SMRS hilang
Nitrit Neg timbul
Lekosit 3+ Dugaan : Infected CAPD
Darah 1+
Mikroskopik Eritrosit 22,78/L (4,10/LPB)
Leukosit 2262,78/L (407,30/LPB)
PMN Sel 94 %
MN Sel 6%
Bakteri 760,8/L
Bakteri (mL) 760800/mL
Lain-lain Gram Negatif
Pewarnaan Gram Batang Gram Negatif
I : Batang Gram (-),
Klebsiella pneumoniae
Kultur Cairan CAPD II : Batang Gram (-),
Klebsiella pneumonia
 CONCLUSION
• Hematuria management using urine flowcytometry provides important data for
glomerular and non glomerular hematuria, so management of hematuria becomes
more efficient and faster and Reduces the microscopic examination review rate and
provides more time for microscopic observation

• Urine flowcytometry gives precise data for UTI, reliable method for screening out of
major part of UTI-negative sample, help clinician to improve patients management

• Combining the automated and traditional analyses of urinary formed elements in

general laboratories—starting with automated cell counting followed by microscopic
analysis,—may be a time-sparing policy, that allowing the operators to dedicate more
time to the morphologic definitions.