Hani Susianti

THE ROLE OF URINE

FLOWCYTOMETRY
IN HEMATURIA EVALUATION
 OUTLINES

1 Introduction

Glomerular and Non

2
Glomerular Hematuria

3 The Role of Urine Flowcytometry in

4
6 Hematuria Evaluation
 1. INTRODUCTION

• In Japan, as of the end of

Year 2008, 1 out of 450
people was a dialysis
patient
• Patients with CKD at
stages 3 and 4
accounted for nearly 10%
of the total population.

Considering that the incidence of dialysis introduction due to chronic

nephritis (IgA nephropathy), a condition often diagnosed by the
presence of asymptomatic proteinuria and hematuria, has decreased,
these urinalysis screening measures seem to be successful
(Iseki, JMAJ 54(1): 27–30, 2011)
 INTRODUCTION….
• In 2016, there were 12,673 cases of Hematuria (Ery >
3/hpf) in RSSA laboratory
• Hematuria cases are found in 1-20% of population

• 14% of cases in RSSA laboratory has

> 51% dysmorphic erythrocytes
• The morphological differences between :
Dysmorphic erythrocyte
( glomerular source of bleeding)
Eumorphic erythrocyte
(non glomerulalar source of bleeding)
Identification by Participants of EQAS of
Particles of Nephrological Importance

5
ACANTHOCYTURIA IS MORE EFFICIENT IN TO DIFFERENTIATE
GLOMERULAR FROM NON-GLOMERULAR HEMATURIA THAN
DYSMORPHIC ERYTHROCYTES

RESULTS:
• 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, Arch Esp Urol. 2002 Mar;55(2):164-6.

2. HEMATURIA GLOMERULAR AND
NON GLOMERULAR
History and Physical examination

URINALYSIS

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

Search other Check RBC Myoglobinuria or

cause of red morphology Hemoglobinuria
urine color

GLOMERULAR NON GLOMERULAR

 EXTRAGLOMERULAR
AND GLOMERULAR HEMATURIA

Extraglomerular Glomerular
(Isomorphic) (Dysmorphic)
Colour Red or pink urine Red, smoky brown, or "Coca-
Cola"

Clots May be present absent

RBC morphology Normal shape Dysmorphic

Casts Absent May be present

Proteinuria May be absent May be present

 Causes of Hematuria
Glomerular
• IgA nephropathy (Berger's
disease)
• Postinfectious
(streptococcal) GN
• Membranoproliferative GN
• Focal Segmental
Glomerulosclerosis
• SLE nephritis
• etc
Non Glomerular
• Urolithiasis
• Pyelonephritis
• Bacterial cystitis (UTI)
• Benign prostatic
hyperplasia
• Transitional cell
carcinoma
• etc
 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)
 3. The Role of Urine Flowcytometry
in Hematuria Evaluation
Core (CR) channel: Nucleic acid Surface (SF) channel: Components
containing components not 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
Material genetik

Side Fluorescent
Light System

Laser Diode
Photo Multiplier
Spectrum
Filter
Dichroic
Mirror
Side Scattered
Light System

Condenser Kompleksitas
Collimating Lens Lens
Beam Photo
Condenser
Stopper Diode
Lens

Flow cell Forward

Condenser Scattered Light
Lens Pin System
Hole
Photo
Diode Ukuran
IDENTIFICATION OF RBC ISOMORPHIC/
DYSMORPHIC/MIXED
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
• Diagnostic value ??
• Accuracy ? ?

17
 Study of glomerular and non-glomerular
hematuria by Urinary Flow Cytometer
Author Publication Sensiti Spesifi NPV PPV
vity (%) city (%) (%) (%)

Hyodo T, Differential Diagnosis between Glomerular 90 92

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

Apeland* Flow Cytometry of Urinary Erythrocytes for 75 86

Evaluating the Source of Haematuria Scand J (93) (100)
Urol Nephrol 29: 33-37, 1995
Apeland* Assessment of haematuria: automated urine 83 94 95 78
et al flowmetry vs microscopy, ephrology Dialysis
Transplantation, Volume 16, Issue 8, August
2001

Aulia D, Detection Glomerular and non glomerular 87 82 90 77

et al hematuria using automated urine particle
analyzer (ISBN 978:979-496-702-7)
 QUESTIONNAIRE

The Urinalysis Flowcytometry tool offers a

range of clinical added values.
Which one is the most useful for you?
• Estimating the location of the
occurrence of hematuria through RBC
morphology (47%)
• Urinary Tract Infection Screening (40% )
• Identify problem of drug abuse sample
through urine conductivity (11%)
• Others (2% )
 CONFLICTING CRITERIA REGARDING
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 renal 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
glomerular and non glomerular Area under curve (AUC) : 0,92
 Case 1

ARP ( 6 y.o.)
• The patient
came to the
hospital
(diagnosed with
nephrotic
syndrome).
• The patient had
cough but there
were no edema,
shortness of
breath, fever, or
flu.
 Laboratory Examination

Urine Microscopic
27/10/17 02/11/17 08/11/17 Reference
Parameter

Erythrocyte 23.0 21.0 90.0 ≤3 /HPF

Eumorphic 5% 10% 11%
Dysmorphic 95% 90% 89%
Acanthocytes
+

Leukocyte 5.8 5.8 8.2 ≤5 /HPF

Cast Positive Positive Positive
Hyaline 4–5 4–5 0–1 ≤2 /LPF
Granular 7–8 - 6–8 Negative

Erythrocyte 1–2 - -
 Urine
microscopics
examination

The patient
had
glomerular
hematuria
Case 2
Mrs. SS (19 y.o.)
• The patient was
referred with G1P0A0
and eclampsia.
• She had seizures 3
times at private
hospital and history
of hypertension since
she got pregnant.
 Laboratory Examination

Urine Microscopic
06/11/2017 09/11/2017 10/11/2017 Reference
Parameter

Erythrocyte 148.4 92.1 311.1 ≤3 /HPF

Eumorphic 96% 72% 75%
Dysmorphic 4% 28% 25%
Leukocyte 6.2 8.0 35.0 ≤5 /HPF
Crystal - Uric amorphous Uric amorphous /HPF
+ +
Bacteria 180 40.2 651.6 ≤ 93
.103/mL
Cast Negative Negative Negative
Urine microscopic
(Isomorphic)

The patient had

non glomerular
hematuria
Int J Clin Oncol (2014) 19:928–934
 The causes of discrepancies between blood dipstick and
erythrocyte sediment results
Blood
Negative (-) Positive (+)
Negative (-) • Normal • Old urine
• Alkaline urine/urine
hypotonicity
• Urine Hemoglobin
Urinary • Urine Myoglobin
Sediment • Mixture of oxides
RBC • Bacteria POD
Positive (+) • High SG urine (high protein)
• When lower than sensitivity
measurement figures Bloody urine
• Large quantities of ascorbic
acid contained in urine
• False recognition (yeast,
white blood cell, sperm
head)
 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

 Microscopic Urinalysis and Automated
Flow Cytometry in a Nephrology
Laboratory

• In conclusion, combining the automated and

traditional analyses of urinary formed elements in
general laboratories—starting with automated cell
counting followed by microscopic analysis, which is more
specific in revealing morphologic aspects—may be a
time-sparing policy.
• Nonetheless, the use of the automated procedure may
help to save time on red and white cell counts, thus
allowing the operators to dedicate more time to the
morphologic definitions.
Gai et al, Clinical chemistry, 2003
 CONCLUSION
Hematuria management using flowcytometry :
• Provides important data for glomerular and non
glomerular hematuria, so management of hematuria
becomes more efficient and faster

• Reduces the microscopic examination review rate

and provides more time for microscopic
observation.

• Improving urinalysis technology gives precise data

& speeds up workflow in routine laboratory work
Thank You ….
I WILL NOT GIVE UP TO GET
ABNORMALITIES OF YOUR URINE