26/10/2022

EFLM WEBINAR

The EFLM Update of the

European Urinalysis
Guidelines
Dr. Timo Kouri, MD, Docent
Dept Clinical Chemistry, University of
Helsinki,
and
HUSLAB, HUS Diagnostic Center, Helsinki,
FINLAND

Chair, EFLM TfG Urinalysis

25th October 2022

Members of the EFLM Task and finish Group Urinalysis

Chair
Timo KOURI Finland term: 2018-2022
Chemistry, Particles
Jan BERG GERTSEN Member, Bacteriology Denmark term: 2018-2022
Rosanna FALBO Member, Particles Italy term: 2018-2022

Walter HOFMANN Member, Chemistry Germany term: 2018-2022

Audrey MERENS Member, Bacteriology France term: 2018-2022

Matthijs OYAERT Member, Particles Belgium term: 2021-2022

Martine PESTEL-CARON Member, Bacteriology France term: 2021-2022

Sören SCHUBERT Member, Bacteriology Germany term: 2022

The members of the EFLM TfG Urinalysis have no personal conficts of interest interfering with this project

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From the ECLM to EFLM

in the URINALYSIS GUIDELINES
The document to be updated
ECLM. European Urinalysis Guidelines. (Kouri T, Fogazzi G, Gant V,
Hallander H, Hofmann W, Guder W, editors). Scand J Clin Lab Invest 2000;
60 (Suppl 231): 1-96.

Sponsors from the

IVD diagnostic industry

eLearning, Timo Kouri 3

Contents being Updated

Gyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y,
Format of GRADE:
Alonso-Coello P, Schünemann HJ, for the GRADE Working Group.
RECOMMENDATIONS GRADE: an emerging consensus on rating quality of evidence and
BASED ON EVIDENCE strength of recommendations. Brit Med J 2008; 336:924-6.

1. Section: Medical needs and Preanalytics (55 pages)

Medical needs, Patient preparation and Specimen collection
Hierarchy of Measurement Procedures
2. Section: Chemistry (56 pages)
3. Section: Particles (30 pages)
4. Section: Bacteriology (67 pages) Total 207 pages

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Provisional Recommendations: Medical Needs and Requisition

Recommendations (1-2) Gyatt GH, et al, for the GRADE Working Group. Level of
1 = Strong, 2 = Weak recommendation Brit Med J 2008; 336:924-6. Evidence
*
INDICATIONS: Urinalysis tests should be requested based on assessment of risk to B
severe disease. The specific tests should be planned between laboratories and
clinics, to balance benefits against resource. (1)
Asymptomatic bacteriuria shall not generally be sought to avoid unnecessary A
antimicrobials and multiresistant strains of uropathogens. (1)
STRATEGY: General screening for low-risk and routine patients is to be separated B
from targeted diagnostics for high-risk patients. (1)
Requisition and reporting of urinalysis tests using electronic interfaces is B
encouraged, with local diagnostic algorithms. Electronic transfer improves exchange
of systematic information between clinicians and laboratories. (1)
*Levels of Evidence: A = high, B= moderate, C= low quality of evidence, D = consensus by the experts
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TARGETED WORKLOAD: Bacteriuria screening

(1) Low-risk patients: non-pregnant females with typical symptoms of cystitis diagnosed with a validated ACSS
questionnaire (Acute Cystitis Symptoms Score, from: http://www.acss.world/downloads.html )
(NO LABORATORY TESTS) (note the exceptions, such as Chlamydia, yeast and Trichomonas diseases)

(2) Other routine symptomatic patients: WORKLOAD OPTIMISATION

Mid-stream samples may be screened for bacteriuria by using WBC and Bacteria detection
by rapid examination (strip test), to detect bacteriuria at 105 CFU/mL (= 108 CFB/L) in emergencies,
noting the false negative cases with symptomatic UTI
by automated counting to rule out bacteriuria at 104 CFU/mL (= 107 CFB/L) in the laboratories
at a sensitivity of 90-95 % (considering clinical symptoms, way of urine collection, and leukocyturia)
Workflow to be organised locally/regionally based on patient populations, laboratory organisation & resource.

(3) High-risk patients: Sensitive cultures of special specimens and examination procedures

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Provisional Recommendations: Patient Preparation

Recommendations (1-2) Level of
1 = Strong, 2 = Weak recommendation Evidence
*
PATIENT Interaction to reduce Contamination: Invite patients to become C
subjects in decision-making on their disease. This would encourage them to
learn how to collect a mid-stream urine (MSU) specimen in a best achievable
way, in order to minimise contamination.
Quality specification: desirable rate < 10%, maximum rate < 15% of MSU
specimens against 104 CFU/mL (or 107 CFB/L) in culture, calculated at a
laboratory level. (1)
Laboratories shall maintain educational material bank and to enforce routine co- B
operation with their clinical units in order to improve preanalytical processes,
including preparation of patients for delivering their urine specimens. (1)

eLearning, Timo Kouri 7

Native or second
language?

Visual + Human
presentation councelling
Culturally
adapted
videos

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Recommendations (1-2) Level of

1 = Strong, 2 = Weak recommendation Evidence *
CONCENTRATION of single-voided urine specimens shall be measured together with the B
primary measurand, expressed as measurand-to-reference ratio, e.g., albumin-to-creatinine
ratio, or reported separately as urine osmolality, relative density, or conductivity, e.g., with
Identical protein/particle excretion rate with different concentrations
urine particle counts. (1)

Dense urine (low diuresis) Dilute urine (high diuresis)

 Identical excretion rates of particles / proteins, but different amount of

water excretion (diuresis, volume rate)

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Provisional Recommendations: Specimen Collection

Recommendations (1-2) Level of
Evidence*
Measurand-to-reference ratios in single-voided urine specimens, similar to A
albumin-to-creatinine ratios, are recommended to replace timed (24-hour)
urine collections for other measurands as well
- in order to reduce incidence of non-conformities.
Verification of an intended measurand to any new patient group is needed
before clinical application. (1)

Preservation of urine specimens is obligatory if the sample is not analysed B

within 2-6 hours. Consider refrigeration if applicable. The preservation shall be
verified against the given specification of the measurement procedure. (1)
*Levels of Evidence: A = high, B= moderate, C= low quality of evidence, D = consensus by the experts

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Suggested criteria for urine preservation

The preservation should fulfill the following criteria:
(a) For urine components with exponential changes in disease, a
maximum of a two-fold change is acceptable, i.e., a maximum loss
down to -50 %, or increase to +100% from the original concentration.
(b) For urine components with linear changes in disease, an 80%
(minimum) to 90% (desirable) preservation of original concentration is
recommended.

Preservatives were reviewed for urinalysis, bacterial culture, and quantitative

chemical measurements - also required by the IVDR and MDR regulations.

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Provisional Recommendations: Chemistry

Recommendations (1-2) Level of
Evidence
Multiple (multiproperty) test strips are still recommended as screening tools for A
low-risk patient populations because of their cost-efficiency. They are NOT
recommended for urine diagnostics of high-risk patients if insensitive. (1)
Performance of strip tests must be verified against quantitative measurements and B
monitored with control solutions at the limit of positivity (1+/2+ range). (2)
Sensitive detection of renal disease in high-risk groups requires measurements of B
both albumin, and a tubular marker in urine, such as α1-microglobulin, expressed as
measurand-to-creatinine ratio. Measurement of urine total protein remains
important in screening for free light chains in urine (Bence Jones proteinuria). (1)

Levels of Evidence: A = high, B= moderate, C= low quality of evidence, D = consensus by the experts

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Internal Quality Control of test strip reading using

continuous reflectance values Focus on the dynamic
measuring interval (1+ to 2+)
LEUKOCYTES, MIDITRON
and the Limit of Detection,
NOT at 3+ category
80

70
Neg
60
1+ (25*E6/L)
50
REMISSION %

40
3+ range
30

20 QM Pos

10 Pos 1:10 PBS

PU
0
0 2 4 6 8 10 12 14 16 18 20 QM Neg

CONSECUTIVE DAY

eLearning, Timo Kouri

Tubulo-interstitial
Quantitative nephropathy
detection of
kidney disease
with proteinuria Glomerulopathy
markers
(Hofmann W et al, 1998)
Healthy
excretion
GROUPS rates
1. Glomerulopathy
2. Secondary glomerulo-
pathy, e.g., in diabetes
3. Tubulo-interstitial
nephropathy

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Provisional Recommendations: Particles

Level of
Recommendations
Evidence *
Laboratories shall clearly describe their basic and advanced differentiation A
and reporting of urinary particles. (1)
Automated particle analysers need to be verified before implemented into A
routine, against published performance specifications (Level 3 procedure).
Appropriate review rules for visual microscopy need to be defined. (1)
Laboratories shall decide and verify one of the (Level 2) procedures of visual A
microscopy for their routine particle analysis. Application of phase contrast,
in addition to bright field optics is strongly recommended for visual
microscopy of urine particles. (1)
The recommended standard unit to particle counts is particles/L (litre), C
as decided at national level to avoid confusion and risks to patient safety. (1)

*Levels of Evidence: A = high, B= moderate, C= low quality of evidence, D = consensus by the experts
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Urinalysis Guideline: Two levels of particle identification

Basic level Advanced level
Emergency cases / Automation (24/7) Renal disease

Erythrocytes, RBC Subgroups: isomorphic and dysmorphic RBC

Leukocytes, WBC / granulocytes WBC differentiation: lymphocytes, macrophages, granulocytes

Squamous epithelial cells Renal tubular cells, transitional epithelial cells

Small (non-squamous) epithelial cells (atypical cells / cytopathologists), intestinal epith cells, others
Casts: Differentiation of pathological casts:
Hyaline casts RTC, RBC, WBC, Bacteria, Yeast casts,
Pathological = non-hyaline casts Granular, waxy, fatty, bilirubin, haemoglobin casts
Bacteria Gram staining (MICROB laboratories)
Yeasts, trichomonas, Spermatozoa Schistosoma haematobium; tropical diseases
Artefacts (hair, fibre, talc, etc) Artefacts (hair, fibre, talc, etc)

Precipitate, crystals Differentiation of crystals

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Hierarchy of Procedures (= Levels of Accuracy) in Urine Particle Analysis

Level 3: Advanced comparison level
◦ Kouri T, Györy A, Rowan RM, and the ISLH Task Force. ISLH Recommended Reference
Procedure for the Enumeration of Particles in Urine. Lab Hematol 2003; 9:58-63
Level 2: Routine quantitative level
Automated quantitative counting with different technologies
Visual quantitative counting and differentiation
◦ Urine sediment counted in a chamber (concentration 20:1?, a precise volume)
◦ Chamber counting of uncentrifuged specimens (no concentration, a precise volume)
◦ Standardised urine sediment under a coverslip (concentration factor? -> volume?)
Level 1: Ordinal scale level
◦ Non-standardized urine sediment, reporting at ordinal scale (0, 1+, 2+, 3+)

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Provisional Recommendations: Bacteriology

Recommendations (1-2) Level of
Evidence
*
Chromogenic agar is strongly recommended as primary agar medium to identify Escherichia coli B
(most frequent uropathogen) easily, quickly, and inexpensively (no need for a panel of tests to
define the species). A second agar (such as blood agar) is needed in clinically defined cases and
for fastidious organisms. (1)
Bacterial identification using matrix-assisted laser desorption ionization time-of-flight mass A
spectrometry (MALDI-TOF MS) is strongly recommended in medium-sized and large laboratories
(> 100 specimens/day), to improve patient prognosis with
(i) Accuracy and reliability of identification to the species level, and
(ii) Shortened delay of reporting (from 36-48 h to 8-24 h). (1)
New species Aerococcus spp., Actinotignum schaalii and Corynebacterium urealyticum are B
proposed into the list of Type II uropathogens. (2)
No recommendation to the unit volume (mL or L) for reporting urine bacterial cultures.
This is to be harmonised nationally to avoid confusion among professionals and patient risks. (1)
*Levels of Evidence: A = high, B= moderate, C= low quality of evidence, D = consensus by the experts
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Significance of bacteriuria and laboratory workflow

Rapid measurements (test strip, counting)
Clinical picture Urine specimen • Leukocyturia
• Patient population • Bacteriuria
• Symptoms and signs Rapid screening
• Background diseases as locally applied Colony counts (CFU/mL or CFB/L?)
• Special samples > 102 CFU/mL (limit of positivity)
Way of urine collection Urine • Routine samples
• Midstream specimens bacterial • Borderline 103 CFU/mL or 106 CFB/L?
• Paediatric bags and pads • Positive growth > 104 CFU/mL
• Catheterization
culture
Polymicrobial growth > 3 species
• Special samples

Identification of species Type of grown species in culture

• Chromogenic agar 60% • Type I uropathogens (E. coli; S. saprophyticus)
• MALDI-TOF MS 40% • Type II uropathogens
• Potential uropathogens
• Biochemistry tests occasionally
• Contaminants
Antimicrobial Susceptibility Test

Report 19

Workflow of primary urine cultures from routine specimens

no
GROWTH Finding: no growth
ID =
yes 103 CFU/mL b ≥104 CFU/mL
Leukocyturia a Identification
to species
1 - 2 species Interpretation d Interpretation level
Class I + ID + AST c 2 ID + AST 2
uropathogens - ID 1 ID + AST 1 AST =
antimicrobial
Class II + ID + AST 2 ID + AST 2 susceptibility
uropathogens - ID 0 ID 1 test
Class III, doubtful + ID + AST 1 ID + AST 1
- ID 0 ID 0 CFU =
uropathogens
colony-
Contaminants, + ID 3 ID 3 forming units
no uropathogens - ID 0 ID 0

≥ 3 species No No
(Interpretation: contamination)

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Take home messages

Preanalytics: True medical indications and patient guidance create the backbone of
urinalysis tests.
Hiearchy of measurements: A reference procedure of a higher level of accuracy is
needed for all clinical laboratory examinations.

Chemistry: Quantitative quality control is needed also for strip tests. Kidney disease is
to be screened both for albuminuria and α1-microglobulinuria (tubular proteins).

Particles: Basic level of particle identification is for 24/7 service. Verify your automated
and visual procedures, using phase contrast microscopy.

Bacteriology: Consider improving your workflow, to be able to move from specimens

of low-risk patients to those of high-risk patients.

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Thank you for

Your attention!

Appendix
in the following
slides

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Males Females

Available since
2000

Timo Kouri 23

Diagnostic performance of urine particles in renal diseases

Perazella MA, Am J Kidney Dis 2015
ROC analysis
Acute kidney injury (AKI) TP
• RTC and casts in visual microscopy; AUC = 0.66 0.66
(Useless AUC = 0.50, maximum AUC = 1.00)
• Prediction of a severe disease in AKI, AUC = 0.85 0.50 line
• Urine NGAL, AUC = 0.74
• Sensitivity and specificity depend on tested populations:
Se 22 % with Sp 91% in separating AKI from non-AKI among patients at emergency room FP

• Urine microscopy is more specific (but less sensitive) than NephroCheck (combination of IGFBP7 and TIMP-2)
Chronic kidney disease
• Particles present more often in proliferative GN than in non-proliferative glomerulonephritis (GN)
(Fogazzi GB et al, J Nephrol 2005)

•Urine microscopy is cheaper than specific immunochemical tests from urine

 selection of the test reflects availability and national economy

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ISLH REFERENCE MEASUREMENT PROCEDURE (Level 3)

Kouri T, Rowan RM, Györy A & ISLH Task force. Lab Hematol, 2003; 9:58-63
Chamber counting, no centrifugation
o 200 (RBC, WBC) or 50 counted particles (Epithelian Cells, Casts)
(Poisson statistics)
Counting volume: Fuchs-Rosenthal chamber: 0.2 µm  2x 3.2
µL;
o Bürker chamber: 0.1 µm height with 2x 0.9 µL volume, is applicable
to Sternheimer staining with a background colour

Detection: phase contrast optics

Identification: phase contrast (+ Sternheimer stain if necessary)

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AUTOMATED PARTICLE DIFFERENTIATION*

*Performance depends on precise cut-off limits of low counts, and patient populations
Flow cytometry (Sysmex UF-5000) Image analysis in cuvette (UriSed3PRO)
Previtali G et al, Clin Chim Acta 2017 Falbo R et al, Clin Chim Acta 2019

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Imprecision of low particle count (x/µL) and counted volume (p):

Poisson coefficients of variation for total count, CV(n), where n= p*x
Kouri T et al, Clin Chim Acta 2021; 515: 96-103

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Sensitivity to detect bacteria by automated counting depends on

clinical sample mix and definition of positive growth
Detection limit at 80 BAC x 106/L
Categories in culture (CFB/L)
Se 93-97 % 108 CFB/L
Negat growth

Se 69-75 % M6 – M8, mixed growth at CFB

Limit of 106 – 108/L
sensitivity 107 CFB/L
(50% of negat Se 50-56 % S6 – S8, identified species at
cultures 106 CFB/L CFB 106 – 108/L
become posit
in counting)
CFB 106 – 108/L is equal to
CFU 103 – 105/mL

Kouri T, et al. Clin Chim Acta

2021; 516: 149-156.

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Automation in bacteriology
Mechanisation of preanalytics
- opening specimen containers, sample preparation, streaking, conveyors between different units
Incubators
- aerobic and CO2 incubators / standardised temperature and atmosphere, and plate readers
 increased sensitivity and shortened incubation time
Detection and identification
- high-resolution digital imaging of chromogenic agar plates, identification algorithms of colonies
- automated colony pickers allow inocula preparation for MALDI-TOF MS and Antimicrobial
Susceptibility Testing
Turn-around time (TAT), from specimen arrival to result reporting
- reduction of TAT to 5 hours (negative), or 14 hours (positive for E.coli), others longer

Typical portions of species identified in a routine workflow: Chromogenic agar 60% (role of E.coli),
MALDI-TOF Mass Spectrometry 40%, Biochemistry rarely needed after the two other procedures

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Steps of Progress in 2022

The preliminary version is now on scientific review
Professionals within EFLM societies
Other medical professionals (laboratory specialists, and clinicians)
Representatives of IVD Sponsors

The official review is taken by the Chair of Committee of Science (Prof Michel Langlois)
according to the EFLM procedures, and to a Public Consultation by ESCMID for endorsement.

Reviews and voting for acceptance by the EFLM national member societies, after which
Official publication (Guideline is a Type 1a document) in the CCLM (probably in 2023).

XVI BALTIC CONGRESS of LABORATORY MEDICINE, Tallinn, 2022 30

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Density of urine
Measurand Hypotonic Isotonic Hypertonic
Osmolality, mOsm/kgH2O 100 300 1000
Relative density 1.003 1.010 1.030
(ratio to water)
Conductivity, mS/cm 2 5-7 30

Regression equation empirically:

y (urinary osmolality; mOsm/kg) = 23.83 × (urinary conductivity, μS/cm) + 181.25 (r2 = 0.539)

Oyaert M et al, CCLM 2019;57:1169-57.

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Interpretation of urine cultures

0 = Detected microorganisms probably do not cause a UTI (even with
corresponding symptoms).
1 = Detected microorganisms possibly cause UTI in selected clinical
presentations (immunocompromised patients, early infection…) with
appropriate clinical picture.
2 = Detected microorganisms with significant colony counts. UTI is
probable with appropriate clinical picture.
3 = No microorganisms detected with the used culture procedure. In
presence of appropriate clinical picture, consider tests specific for other
microbes, e.g., Chlamydia, Mycoplasma, Ureaplasma, M. tuberculosis, N.
gonorrhoeae.”

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