detection of glomerular and non glomerular hematuria using...

Detection of glomerular and non

glomerular hematuria using

flowcytometry

IRA PUSPITAWATIDEPARTEMEN PATOLOGI KLINIK DAN KEDOKTERAN LABORATORIUMFKKMK UGM-RSUP DR SARDJITO YOGYAKARTA

Introduction

Hematuria

Very important feature in kidney disease

Variation in hematuria prevalence

Introduction

Woolhander, et al. JAMA. 1989;262(9):1214-1219

• Prevalence of asymptomatic hematuria was 0,19-16,1%.

Woolhander, et al 1989

• Prevalence of asymptomatic hematuria was 13% among >35 years old man and postmenopousal woman.

Mohr, et al, 1986

• Prevalence of occult haematuriawas 20.1% among men over 60 years of age screening for bladder cancer using dipstick.

Britton, et al

1989

Hematuria4

• Gross hematuriaMacroscopic

• More than 5 RBC/ high-power field (hpf) on microscopic analysis of 2 or 3 properly collected urine specimens.

Microscopic

Karnath, et al. Hospital Physician 2007;62: 20-26; Grossted, et al. American Family Physician. 2007. 63

5Hyodo et al.and Pellet et al.showed that more than 80-90% of the microhematurias were rated as glomerular hematuria

Microscopic hematuria is thus predominantly associated with glomerular disease.

Identification of the type RBC in urine diagnose the etiology

Hyudo, et al. Nephron 1999;82:312-323

6Etiology of Hematuria

RENAL

Glomerular

Thin basement membrane

disease (benign familial hematuria)

IgA nephropathy

Alport’s syndrome

Other glomerulonephritides

Nonglomerular

Polycystic kidney disease

Medullary sponge kidney

Papillary necrosis

Pyelonephritis Sickle cell disease

Renal cell carcinoma

EXTRARENAL

Upper urinary tract

Nephrolithiasis

Ureteral cancer

Lower urinary tract

CystitisBladder cancer

Bladder stones

Prostate cancer

Schistosomiasis

otherVigorous exercise

Coagulation related

Factitious

False hematuria

Karnath, et al. Hospital Physician 2007;62: 20-26

7

Early detection of hematuria and differentiaton between glomerular and non glomerular type.

Essential for determination of correct line of investigation and management

Avoid unnecessary invasive diagnostic procedures such as cystoscopy and radiologicalinvestigations.

Dinda, Indian J Nephrol 2001;11: 37-38

8Glomerular vs Non Glomerular

GlomerularNon Glomerular

Evaluation of Urine RBC morphology

Glomerular vs Non Glomerular

1.Glomerular erythrocytes are smaller than

nonglomerular erythrocytes

2.The smaller glomerular erythrocytes vary greatly

in both shape and size whereas nonglomerular

erythrocytes are uniform in size and shape.

3.Glomerular erythrocytes usually have lost a large

amount of their hemoglobin pigment and are pale.

4.Erythrophagocytes and RBC cast commonly

accompany glomerular hematuria.

Smith dan Fairley. Semin Nephrol 2005. 25:127-135

Glomerular vs Non Glomerular

Glomerular Non Glomerular

Non Glomerular HematuriaGlomerular Hematuria

Glomerular

ErythrophaghocyteRBC cast

Glomerular bleeding

Glomerular

Glomerular Hematuria

13Glomerular hematuria Dysmorphic Erythrocyte

14Glomerular bleeding

Glomerular bleeding is associated with more than 80% dysmorphic RBC.

Kohler, et al. Kidney International, Vol. 40 (1991), pp. 115—120

No clear cut definition of dysmorphic erythrocyte

15Dysmorphic vs Isomorphic RBC

• Variously shaped blebs or

projections at the cell

membrane. Classify as

acanthocytes, G1 cells, or

D cells based.

DysmorphicRBC

•Uniform and no more than two types

Isomorphic RBC

Kohler, et al. Kidney International, Vol. 40 (1991), pp. 115—120; Kidney International, Vol. 21(1982),

pp. 105—108; Letgen, et al. Pediatric Nephrology 1995: 9(4); 435–437|

https://link.springer.com/journal/467

17Dysmorphic vs Isomorphic


Yu Chu Su. 2017. Scientific report 7:40521, 1-10


The shapes of isomorphic RBCs can change in response to the

osmolarity of urine.

The isomorphic RBCs swell to spheres (Fig. 2B-07) in urine

with a low specific gravity, and they shrink to the shape of a

spiked disk (Fig. 2B-11 and B-12) or a spiked sphere (Fig. 2B-

13) in urine with a high specific gravity.


Some “dysmorphic” cells could be easily and reversibly induced by

changes of osmolality, pH or coverslip (Kohler, et al, 1991)

20Achantocyturia

Sumber: Kohler, et al. Kidney International 1991: 40; pp. 115—120

One irreversible cell type, a ringform with vesicle-shaped

protrusions (acanthocyte), was found almost in glomerular disease

and was not induced by journey of the renal tubules (changes in

osmolality, pH or the investigation procedur.

21

Achanthocyte in urine ≥ 5% (specificity 98%, sensitivity 52%) was found almost in glomerular disease.

Kohler, et al. Kidney International, Vol. 40 (1991), pp. 115—120

Glomerular Bleeding

22PROBLEMS

Dysmorphic RBC

Isomorphic RBC

Classification and Interpretation

sometimes not easy

Underinterpretation of dysmorphic

RBC

Dysmorphic erythrocyte

23

Phase-contrast microscopy is considered the gold

standard for the differentiation between

glomerular and non-glomerular hematuria.

Limited availability

Supra vital stain using brightfield

microscope

Cutoff ≥20% dysmorphic erythrocyte for diagnosing glmerular heusing supravital stain and brightfield microscope specificity

82%, sensitivity 100%.

Pemeriksaan yang sederhana ini memiliki efisiensi yang setara

dengan mikroskop fase kontras.

Prinsip pengecatan supravital dilakukan dengan menggunakan

cat kristal violet 1%, safranin 0,5% dan normal saline, selanjutnya

cat akan dicampurkan dengan sedimen urin hasil sentrifugasi

dengan perbandingan 1:1

25PROBLEMS

Laboratory interpretation

Nephrologist

- Nephrologist found a higher number of Renal tubular epithelial (RTE), granular

cast, hyaline cast and RTE cast compared to medical technologist.

- Nephrologist reported the presence of acanthocytes in 7 samples that were

not detected by laboratory personels.

Discrepancies

Tsai, et al (2005). Am. J. Kidney Dis. 46: 820-829

26

Urinalysis report usually

lack a description of

dysmorphic RBCs,

Tools that can give information related with the dysmorphic RBC

Urine Flowcytometry

PROBLEMS

The drawbacks of microscopic urinalysis high inter-observer variability, low sensitivity, and time consuming

Automation of Urine analyzer

Principle of Urine Flowcytometry

Classify by 3 level information

Forward scatter light (Fsc)

Side scatter light (Ssc)

Fluorescence light (Fl)

Describe particle size

Describe complexity of particle

Describe material genetic of particle

Classifying

type of

particle

32Principle of UF in detecting dysmorphic RBC

• The majority size of the RBC population

RBC-P70Fsc

• The width of the RBC size distribution

RBC-Fsc-DW

RBC Information from UF

33Principle of UF in detecting dysmorphic RBC

34Flag message Microcytic,

normocytic and non classified

Dysmorphic, Isomorphic, Mixed RBC

Principle of UF in detecting dysmorphic RBC35

RBC Morphology from UF

36

37Glomerular Hematuria

38Non Glomerular Hematuria

Study of Dysmorphic RBC using Urine

Flowcytometry

39

Shayanfar, et al 2013

UF 100i had sensitivity 76%, specificity 93%, 92% NPV, 78% PPV in detecting RBC

Some sample with dysmorphic RBC flag normal RBC but small manual reviewed

40Study of Dysmorphic RBC using Urine

Flowcytometry

Hyodo, et al 1999

Sensitivity and specificity of UF100 in detecting glomerular hematuria were 90,3% and 92,5% respectively (Study 1)

Sensitivity and specificity of UF100 in detecting glomerular for hematuria were 100% and 86,6% respectively (Study 2)

Hyodo, et al. Nephron 1999;82:312-323

41Study of Dysmorphic RBC using Urine

Flowcytometry

Yu, Chu Su, et al. 2017

Modified urinalysis protocol with an increased

relative centrifuge force and concentration factor

↑recovery ratio of dysmorphic erythrocyte 34,7%

42% p=0,001

Correlation between dysmorphic RBC counts by the modified urinary protocol and Sysmex UF-1000i urinary flow cytometer (r ≥ 0.898, P < 0.001).

42Dysmorphic RBC Detection

Chu Su, et al (2016) proposed modified urinalysis protocol increases the

detection rate of dysmorphic red blood cells:

a. Relative Centrifuge Force (RCF) with higher recovery ratios for formed

elements in urine sediment is preferable to increase the cell count

accuracy.

The Recovery ratio of RBCs from the sediment suspension at an RCF of

500×g (49.8%) was significantly higher (P <0.001) than that at an RCF

of 400x g (44.2%).

b. Use optimal RCF setting (500xg) for the recovery of dysmorphic RBCs

in cases of haematuria. The increased concentration factor (20-fold)

for urine sedimen.

c. Apply Stenheimer malbin stainning.


43 Nephrologist

Nephrologists apply high

concentration protocols

for urine sediment

preparation. For

example, 10 mL urine

was centrifuged at 1,500–

3,000 revolutions per

minute (RPM) for 3–10

min, and the pellet was

resuspended with a

minimal volume of

supernatant (0.3–0.5 mL).

Then, a drop of the

suspension was placed or

observation

Medical Technologist

Based on Clinical and

Laboratory Standards

Institute (CLSI), GP-16A3

Under this protocol, 12

mL of urine was

centrifuged at an RCF of

400× g for 5 min, and the

pellet was resuspended

with 1 mL of supernatant.

Therefore, the

concntration factor was

only 12-foled,


1. Differentiating glomerular and non glomerular hematuria is an

important steps in urinalysis.

2. Urine flowcytometry can help us in differentiating glomerular

and non glomerular hematuria based on RBC P70 FSc that give

information related with the majority size of RBC population

and RBC FSc-DW that give information related with the width of

RBC variation.

3. Manual review is needed to confirm the Dysmorphic RBC.

Conclusions

4

4

detection of glomerular and non glomerular hematuria using...

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