Laboratory Challenges in Screening and Monitoring of...

Ch ll i L b tChallenges in Laboratory Tests for CKD

Hassan Argani

Professor of NephrologyProfessor of Nephrology,

Shahid Beheshti University of Medical Sciences

ddIntroductionIntroduction

ChallengeChallenge‐‐11: Who should be screened for CKD: Who should be screened for CKD

ChallengeChallenge‐‐22: Which method is better for : Which method is better for ggscreening and follow up for CKDscreening and follow up for CKD

ChallengeChallenge‐‐33: The biases of each method: The biases of each method

Revised chronic kidney disease classification based upon glomerular filtration rate and albuminuria

GFR stages GFR(mL/min/1.73 m2) Terms

G1 >90 Normal or highgG2 60 to 89 Mildly decreasedG3a 45 to 59 Mildly to moderately decreasedG3b 30 44 M d l l d dG3b 30 to 44 Moderately to severely decreasedG4 15 to 29 Severely decreased

G5 <15 Kidney failure (add D if treated by dialysis)G5 <15 Kidney failure (add D if treated by dialysis)

Albuminuria stages

AER(mg/day) Terms

A1 <30 Normal to mildly increased (may be subdivided for risk prediction)

A2 30 to 300 Moderately increased

A3 >300Severely increased (may be subdivided into nephrotic and non‐nephrotic for differential diagnosis, management, and risk prediction)g , g , p )

Who needs screening for CKD?The ACP recommendations,

October 2013The ASN in response to the ACP recommendations

Asymptomatic adults without risk f f CKD h ld b

CKD screening even in patients i h i k f f CKDfactors for CKD should not be

screened for the disease (Grade: weak recommendation, low‐quality

without risk factors for CKD

weak recommendation, low quality evidence

Adults with or without diabetes Disagreed. Present or absent who are currently taking an angiotensin‐converting enzyme (ACE) inhibitor or an angiotensin II

Diabetes in adults taking an ACE inhibitor or an ARB, should be tested for proteinuria (G d k(ACE) inhibitor or an angiotensin II‐

receptor blocker (ARB) should not be tested for proteinuria (Grade:

tested for proteinuria (Grade: weak recommendation, low‐quality evidence)

be tested for proteinuria (Grade: weak recommendation, low‐quality evidence)

Flowchart for diagnosing CKD

How we should screen patients with CKD?

M t f BPAll PatientsMeasurement of BP Serum Cr to estimate GFR Protein/Cr Ratio or Albumin/Cr Ratio in the first morning urine sample Protein/Cr Ratio or Albumin/Cr Ratio in the first morning urine sample Examination of Urine Sediment or Test Tape for RBC and WBC

Selected Patients Depending of Risk Factor

Sonography S l t l t Serum electrolytes Urinary Concentration Urinar pH Urinary pH

Diagnostic Evaluation in Chronic Kidney Disease-1

CLINICAL URINE PROTEIN/CRDISORDER

CLINICAL CLUES

URINE SEDIMENT

PROTEIN/CR RATIO ADDITIONAL TESTS

Diabetes mellitus Diabetes for > 15 years,

Benign > 30 to > 3,500 mg of protein

Fasting blood sugar, A1C

retinopathy per g of creatinine

Essential hypertension

LVH, retinopathy

Benign > 30 to 3,000 mg of protein per

No additional testshypertension retinopathy of protein per

gram of creatinine

Glomerulonephritis History and Dysmorphic > 30 to > 3,500 C3 and C4 for all physical examination: infections; rash arthritis;

RBCs or RBC casts

mg of protein per g of creatinine

patientsTests for infections: anti-ASO, ASK, HIV, HBsAg, HCV RPR bloodrash, arthritis;

Old ageHCV, RPR, blood culturesTests if there is rash or arthritis: ANA, ANCA, cryoglobulin, anti-GBMTests if patient is older than 40 years: SPEP, UPEPUPEP

Diagnostic Evaluation in Chronic Kidney Disease-2URINE PROTEIN/CR

DISORDER CLINICAL CLUES SEDIMENT RATIO ADDITIONAL TESTSLow flow

statesVolume depletion,

hypotension, ti h t

Hyaline casts, eosinophils

< 200 mg of protein per g of creatinine

FENa: < 1 percent;

congestive heart failure, cirrhosis, atherosclerosis

Urinary tract Urinary symptoms Benign, or None KUB radiography,Urinary tract obstruction

Urinary symptoms Benign, or RBCs

None KUB radiography, intravenous

pyelography, spiral CT scanning, renal lt hultrasonography

Chronic urinary tract

infection

Urinary symptoms WBCs, RBCs < 2,000 mg of protein per g of creatinine

Pelvic examination, urine culture, voiding cystourethrography,infection cystourethrography,

renal ultrasonography, CT scanning

Neoplasm, Old ages, RBCs, RBC False-negative result SPEP, UPEP, calcium Sparaproteine

miaconstitutional

symptoms, anemiacasts,

granular castsor > 30 to > 3,500 mg

of protein per g of creatinine

level, ESR

Interstitial Medications fever WBCs WBC 30 to 3 000 mg of ACE level; SS–A SS–BInterstitial nephritis

Medications, fever, rash, eosinophilia

WBCs, WBC casts,

eosinophils

30 to 3,000 mg of protein per g of

creatinine

ACE level; SS A, SS Beosinophilia

Diagnostic Evaluation in Chronic Kidney Disease-3URINE

SEDIME PROTEIN/CRDISORDER CLINICAL CLUES

SEDIMENT

PROTEIN/CRRATIO ADDITIONAL TESTS

Cystic kidney disease

Palpable kidneys with or without

RBCs 30 to 3,000 mg of protein per g of

Renal ultrasonography or CT scanning if there isdisease with or without

family history of cystic kidney disease, flank pain

protein per g of creatinine

or CT scanning if there is a complex kidney cyst or mass

Renovascular disease

Late-onset or refractory hypertension, sudden onset of

Benign < 200 mg of protein per g of creatinine

Renal Doppler ultrasonography, radioisotope renal scanning MRA renalsudden onset of

hypertension in young woman, smoking history,

scanning, MRA, renal angiography

abdominal bruitVasculitis Constitutionalsympt

oms, peripheral neuropathy rash

RBCs; granular casts

> 30 to > 3,500 mg of protein per g of creatinine

C3, C4, ANA, ANCA; HBsAg, HCV, cryoglobulins ESR RFneuropathy, rash,

respiratory symptoms

casts creatinine cryoglobulins, ESR, RF, SS–A, SS–B, HIV

Screening for Complications in CKD(Stages 3 and 4*)

TEST COMPLICATIONS DETECTEDHemoglobin concentration Anemia

Red blood cell indexes, reticulocyte count, iron studies, fecal occult

blood test

For ruling out other causes of anemia before erythropoietin

therapy is startedblood test therapy is started

Serum electrolyte levels Hyperkalemia, hyponatremia, id iacidosis

Calcium, phosphorus, and parathyroid hormone levels

Hypocalcemia, hyperphosphatemia secondaryparathyroid hormone levels hyperphosphatemia, secondary

hyperparathyroidism

Serum albumin and total protein Hypoalbuminemia decreasedSerum albumin and total protein levels

Hypoalbuminemia, decreased levels of immunoglobulins in

patients with nephritic levels of p pproteinuria or signs of

malnutrition

Evaluation of Intrinsic Renal Failure

Glomerular Vascular Interstitial

RBC castUrinalysis RBC cast,OFB, fatty cast RBC cast None

24 h protein24‐h protein excretion

(g/d/1.73 m2)>3.5 1‐5 <2

Hypertension 50% 75% RareHypertension 50% 75% Rare

Minimal initial diagnostic tests for patients with CKD

• Estimation of GFRMinimal initial diagnostic tests for patients with CKD

• Urinalysis• Quantification of proteinuria Quantification of proteinuria• Renal ultrasound

Additional diagnostic tests, depending on the clinical situation

• Serologies for autoimmune diseases• Serologies for chronic infections (hepatitis B and C, HIV, and others)g f f ( p , , )• Serum and urine protein electrophoresis and immunofixation• Blood and urine cultures

d f l• Imaging studies for malignancy• Kidney biopsy??

Sensitivity and specificity of each equation to Diagnosis and classify patients with CKDDiagnosis and classify patients with CKD

‐EPI

CG: Cockcroft‐Gault equation; CGi: Cockcroft‐Gault equation (calculated with ideal weight); CKD‐EPI: the Chronic Kidney Disease Epidemiology Collaboration equation; CrCl: Creatinine l MCQ M Cli i Q d i E i MDRD i lifi d MDRD d iclearance; MCQ: Mayo Clinic Quadratic Equation; sMDRD: simplified MDRD study equation.

Preferred Methods for Assessing Kidney FunctionMETHOD SITUATIONS FOR USE

MDRD study equation for estimating GFR

Patients with diabetic kidney disease

Patients with chronic kidney disease in middle age (average age: 51 years)middle-age (average age: 51 years)

Black patients with hypertensive chronic kidney diseasekidney disease

Patients with a kidney transplant

Cockcroft Gault equation for Older patients (performs better than theCockcroft-Gault equation for estimating creatinine clearance

Older patients (performs better than the MDRD study equation)

24-hour urine collection for Pregnant womencreatinine clearance Patients with extremes of age and weight

Patients with malnutritionPatients with skeletal muscle diseases

Patients with paraplegia or quadriplegia

Patients with a vegetarian diet and rapidly changing kidney function

Formulas to Estimate Creatinine Clearance as an Estimate of GFR

Cockroft‐Gault formula (mL/minute) (Cockroft, 1976):

([140−Age] × [IBW])/(72×SCr)× 0.85 if female.the CG formula overestimates the measured GFR at levels lower 60 mL/min/1.73 m2.

The modified MDRD formula , 2000

GFR=175×Cr−1.154×Age−0.203×1.212(for black)×0.742(for women)the MDRD underestimates the measured GFR at levels above 60 mL/min/1.73 m2.

Males: IBW= 50kg+0.9 kg/Cm over 150 cm

F l IBW 45 5k 9 k /C 150Females: IBW= 45.5kg+o.9 kg/Cm over 150 cm

The formulas for estimating creatinine clearance for children

Schwartz Formula 1976:GFR 0 55 h i h ( )/ i i ( /dL)GFR=0.55× height(cm)/serum creatinine(mg/dL)

Counahan‐Barrett Formula 1976:Counahan‐Barrett Formula 1976:GFR=40× height(cm)/serum creatinine(μmol/L)

Modified Schwartz Formula in 2009:GFR(mL/min/1.73m2)=39.1[height(m)/Cr(mg/dL)]0.516×[1.8/cystatin C( /L)] 0 294[30/BUN( /dL)]0 169[1 099] l [h i ht( )/1 4]0 188C(mg/L)] 0.294[30/BUN(mg/dL)]0.169[1.099]male[height(m)/1.4]0.188

Most recently, a new formula, known as the CKD‐EPI (Chronic Kidney Disease Epidemiology Collaboration) equation, was reported to give improved performance over the widely used MDRD equation.

TionsInfection,D i t i tiDrug intoxicationDehydrationObstructionSevere hypertensionSevere hypertension

1.5mg/dL

Cr production in male (mg/Kg)=Cr production in male (mg/Kg)=2828‐‐00..2 2 ageage

Cr production in Cr production in female female (mg/Kg)=(mg/Kg)=2323..88‐‐00..17 17 ageage

Common causes of false estimates of elevated creatinine

Urea as Measure of Renal Function

Urea is the main waste product of nitrogen‐containing chemicals in the body.the body.

It has a molecular weight of 60 Da.

The concentration of urea is expressed only by the nitrogen t t fcontent of urea.

Each molecule of urea contains 2 nitrogen atoms, the molecular Each molecule of urea contains 2 nitrogen atoms, the molecular weight of urea nitrogen is 28 Da.

Serum urea is widely used as a measure of renal dysfunction, but its value as a measure of GFR is not very good for several reasons.

Causes of Prerenal Azotemia

Low cardiac output• Acute myocardial infarction• Chronic heart failure• Valvular heart disease• Valvular heart disease• Diarrhea• Vomiting

Decreasedplasma volume

• Sweating• Nasogastric suction• Burns• Diuretics

Decreasedhemoglobin levels • Bleedinghemoglobin levels

Endocrinedysfunction

• Uncontrolled diabetes mellitus(polyuria) • Diabetes insipidusdysfunction • Addison disease

Renal dysfunction • Salt‐wasting nephropathy• S i dVasodilation • Sepsis syndrome• Endotoxemia

Cystatin CCystatin C is a 122 amino acid with a molecular weight of 13,000 Da.Is produced by all nucleated cells, and its production is constant. The rate of production is not affected by muscle mass, sex, or race.It is freely filtered and completely reabsorbed by the proximal tubule it is destroyed rather than reentering the circulationtubule it is destroyed rather than reentering the circulation.

It has no extrarenal elimination, so its plasma concentration is inversely related to GFR.inversely related to GFR.

Cystatin C is better than creatinine for estimation of GFR. However, cystatin C is not widely used clinically because measurements are difficult and expensive.

The results of cystatin C equation were better than those achieved by the MDRD equation and the Schwartz formula for estimation of GFRthe MDRD equation and the Schwartz formula for estimation of GFR.

Modified cystatin C equation: GFR[mL⋅min(1.73m2)]=84.69×cystatin C (mg/L) −1.680×1.384 (if a child<14years)(mg/ ) .680 .384 (if a child 4years)

Level of Cystatin C is alteredIncreased Level Decreased Level

Hyperthyroidism HypothyroidismHyperthyroidism Hypothyroidism

Peripheral arterial diseases Glucocorticostroidsb l d h lMetabolic syndrome Atherosclerosis

Alzheimer Aorta AneurysmCigarette smoking

CHF MI StrokeCHF, MI, Stroke

Malignancy

HIV infection

Increased CRPIncreased CRP

Methods for GFR Estimation

Equations developed by the MDRD Study Group and CKD‐EPI, based on serum Cr.

Filtration Marker

eGFR Research Group

Number of

Assays Equation

Advantages References

CKD EPI, based on serum Cr.

subjectsMDRD Study 1628

CKDNon-

standardizedMDRD Study

ti i

Recommended by NKF-KDOQI

2002

Levey et al. Ann

I t M dcreatinine 1999

2002 Intern Med 1999; 130:

461-70;MDRD Study Same as Re-expressed MDRD Appropriate for Levey et y

abovepfor

standardized assay

Study creatinin

e 2006

pp puse with

standardized assays

yal. Ann

Intern Med 2006; 145:

247-54

CKD-EPI 12,150 Standardized CKD-EPI creatinin

Lesser bias ateGFR >60.

Levey et al. Ann Int

e 2009 Recommended by KDIGO 2013

Med 2009; 150: 604-

12

Equations developed by the MDRD Study Group and CKD‐EPI, based on serum Cystatin‐C

Filtration Marker

eGFR Research Group

Number of

subjects

Assays Equation Advantages References

CKD EPI, based on serum Cystatin C

subjects

CKD-EPI CKD3418

Non-standardized

CKD-EPI cystatin C

2008

eGFRcr-cys more precise

Stevens et al. Am J

Kidney Dis CKD-EPI

creatinine-cystatin C

2008

than eGFRcroreGFR cys

2008; 51:395-406

CKD-EPI Same as above

Re-expressed for standardized

assay

CKD-EPI cystatin C

2011CKD-EPI

Appropriate for use with standardize

d assays

Inker et al. Am J Kidney Dis 2011; 58:

682-684CKD-EPI creatinine-cystatin C

2011

d assays 682-684

CKD-EPI Diverse6471

Standardized CKD-EPI cystatin C

2012CKD-EPI

Lesser bias ateGFR >60.Recommend

ed by

Inker et al. N Engl J

Med 2012; 367: 20-9

creatinine-cystatin C

2012

yKDIGO 2013

β‐2‐Microglobulin

β ‐2‐microglobulin, a polypeptide with molecular weight of 11.6 kDaand length of 99 amino acidsand length of 99 amino acids.

Is a component of the MHA class I molecule.It is present in all nucleated cells, and is needed for production ofIt is present in all nucleated cells, and is needed for production of CD8 cells.

Its production is increased in multiple myeloma and lymphoma. β‐2 microglobulin is freely filtered at the glomerulus, and then is reabsorbed and metabolized completely by the proximal tubule.

Similar to cystatin C the plasma level increases in renal failureSimilar to cystatin C the plasma level increases in renal failure. The protein appears in the urine when reabsorption is incomplete because of proximal tubular damage, as in acute kidney injury. p g , y j y

β Trace Protein

BTP is a low molecular weight glycoprotein with 168 amino acids. The molecular weight varies between 23 000 and 29 000 DaThe molecular weight varies between 23 000 and 29 000 Da, depending on the degree of glycosylation.

BTP belongs to the lipocalin protein family and functions as prostaglandin D synthase.

Plasma BTP originates from the brain and is freely filtered at the l l th i b b d l t l b th i l t b lglomerulus, then is reabsorbed completely by the proximal tubule and is catabolized there.

The plasma level is increased in patients with renal disease because ofThe plasma level is increased in patients with renal disease because of reduced filtration in the presence of constant production.

Estimated GFR by B Trace Protein is better than those obtained by the MDRD equation and serum cystatin C measurements. However, others showed that BTP was less sensitive than cystatin C.

GFR=112.1×BTP−0.662×urea−0.280×(0.880 if female)

Tryptophan Glycoconjugate

Is a substance normally produced in the body by glycoconjugation of tryptophan.

It is filtered at the glomerulus freely and is not reabsorbed. A strong linear correlation exists between clearances of TG and inulin. TG increases progressively with declining renal function but unlike TG increases progressively with declining renal function, but unlike

creatinine, it is not affected by muscle mass. The current limitation: it can be measured only by the HPLC. The current limitation: it can be measured only by the HPLC. It is not known whether dietary intake of tryptophan affects the

serum concentration.

The soluble urokinase‐type plasminogen activator receptor

The soluble urokinase‐type plasminogen activator receptor (suPAR) may be a marker of CKDmay be a marker of CKD.

suPAR is a membrane protein that has been implicated in the p ppathogenesis of glomerular diseases including focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy.

Minimal initial diagnostic tests for patients with CKD

• Estimation of GFRMinimal initial diagnostic tests for patients with CKD

• Urinalysis• Quantification of proteinuria Quantification of proteinuria• Renal ultrasound

Additional diagnostic tests, depending on the clinical situation

• Serologies for autoimmune diseases• Serologies for chronic infections (hepatitis B and C, HIV, and others)g f f ( p , , )• Serum and urine protein electrophoresis and immunofixation• Blood and urine cultures

d f l• Imaging studies for malignancy• Kidney biopsy??

C l it f U i t tC l it f U i t tComplexity of Urinary testComplexity of Urinary test

CollectionCollectionStorage AnalysisAnalysis

APPEARANCE CAUSEMilky Acid urine: urate crystals

Alk li i i l bl

APPEARANCE CAUSEOrange Drugs: anthraquinones

(l ti ) if i iAlkaline urine: insoluble phosphatesInfection: pus

(laxatives), rifampicin

UrobilinogenuriaYellow Mepacrine

SpermatozoaChyluria

Smoky pink Hematuria (>0.54 mL blood/L

pConjugated bilirubinPhenacetinRib fl iurine)

Foamy ProteinuriaBlue or green Pseudomonas urinary tract

RiboflavinBrown or black Melanin (on standing)

Myoglobin (on standing)g yinfectionBilirubinMethylene blue

y g ( g)Alkaptonuria

Green or black PhenolL lMethylene blue

Pink or red Aniline dyes in sweetsPorphyrins (on standing)Blood hemoglobin myoglobin

LysolBrown Drugs: phenazopyridine,

furazolidone, l ‐dopa, Blood, hemoglobin, myoglobinDrugs: phenindione, phenolphthaleinA th i i (b t t

niridazole

Hemoglobin and myoglobin (on standing)Anthocyaninuria (beetroot,

“beeturia”)

( g)

Bilirubin

The main causes of false negative and positive testing from use of urine dipsticksp

Discounting contamination from menstrual – or other – bleeding, and exercise induced haematuria and proteinuriaexercise‐induced haematuria and proteinuria

Urinary protein Urinary protein excretion of < 150 mg/day is normal (~30

mg of this is albumin and about 70 100 mg is Tamm

y pmg of this is albumin and about 70–100 mg is Tamm‐Horsfall (muco)protein, derived from the proximal renal tubule)tubule).

Protein excretion can rise transiently with fever, acute illness UTI and orthostaticallyillness, UTI and orthostatically.

In pregnancy, the upper limit of normal protein excretion is around 300 mg/dayaround 300 mg/day.

Persistent elevation of albumin excretion (microalbuminuria) and other proteins can indicate renal(microalbuminuria) and other proteins can indicate renal or systemic illness.

Microalbuminuria is an early sign of renal and cardiovascularMicroalbuminuria is an early sign of renal and cardiovasculardysfunction with adverse prognostic significance.

AMAM

PM

Albuminuria in the Detection of Renal Lesions

• Method of measurement (of choice): albumin/creatinine ratio (mg/g creatinine)

• Definition of albuminuria: >30 mg/g in spot urine sample

Gi t i bilit 2 t f 3 iti d d 3 6• Given measurement variability, 2 out of 3 positive measures are needed over 3‐6 months to consider it pathologic

• Valid samples: first morning, mid‐morning and mid‐afternoon urine samples

• Situations that increase albuminuria: intense physical exercise, fever, infection, Situations that increase albuminuria: intense physical exercise, fever, infection, heart failure, hyperglycemic decompensation

• False positives: hematuria pyuria highly concentrated urine• False positives: hematuria, pyuria, highly concentrated urine

• Less accurate albumin/creatinine ratio in extreme values of creatinine: overestimated in reduced muscle mass and underestimated in muscular patients

The main causes of differently colored urine

Microscopic haematuria isMicroscopic haematuria is present in around 4% of

the adultl ti f h tpopulation – of whom at

least 50% have glomerular disease

ConclusionConclusion

Summary of screening Summary of screening recommendationsrecommendations for CKDfor CKD

Screen four groups of Use two tests for g ppatients with screening

Diabetes Urine albumin to creatinine ratio in 2 to 3 spot urine

Hypertension p

samples (mg/g)

Cardiovascular disease Serum creatinine and eGFR

Age over 55 yr

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