CLINICAL CASES

1. A woman with severe crystalluria and AKI

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2. A boy with crystalluria and renal stones

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3. A crystalluria appearing only once every 130 years?

4. A kidney transplant recipient with two types of crystals in his urine?

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G.B. Fogazzi, MD,�Clinical and Research Laboratory on Urinary Sediment�U.O.C. di Nefrologia, Dialisi, e Trapianto di Rene�Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico�Milano, Italy

CLINICAL CASE 1

A woman with severe crystalluria

and AKI

CLINICAL HISTORY/1

• On February 7th, 2009, a 82-year-old woman is

admitted to the Emergency Unit of our hospital for

malaise & vomitus started 1 month before, associated

with progressive reduction of food & fluid intake and

reduced urine output

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• Physical examination: BP 110/70 without treatment,

hypoelastic skin, dry mucosae, intense thirst

CLINICAL HISTORY/2

• S-creat 8.8 mg/dL (777.9 μmol/L) &

severe metabolic acidosis (pH 7.32, HCO3: 10.8 mmol/L, BE – 13.8 mmol/L)

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• By ultrasounds, the kidneys are mildly hyperechogenic without obstruction

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• Working hypothesis: AKI due to dehydration, the cause behind malaise and vomitus being unclear at the moment

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• IV hydratation is started & the patient is hospitalized in the Internal Medicine Unit

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MAIN LABORATORY TESTS

FEBRUARY 9th:

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• S-creat: 7.7 mg/dL (680.6 μmol/L)

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• S-uric acid: 28 mg/dL (nv ≤ 5.7)

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• S-LDH: 932 U/L (nv ≤ 480)

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• WBC count: 3,800/mm³

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• Hb: 7.0 g/dL

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Plt count: 44,000/mm³

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%

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URINALYSIS

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• Dipstick:

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pH: 5.0; SG: 1.015; Albumin: +; Hb: +++; LE: ++;

Nitrites: negative

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• Urine sediment:

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• RBC: 4-5/HPF
• WBCs: 1-3/HPF
• RTECs: 1 every 9-10 HPFs
• Uric acid crystals: ++++
• Bacteria: ++++

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• Urine colture: E. coli + Enterococcus sp > 10⁶/mL

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• Urine output 1,500 mL/24 hrs

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MASSIVE URIC ACID CRYSTALLURIA

MASSIVE URIC ACID CRYSTALLURIA

AT THIS POINT

• Severe AKI

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• Associated with:

- marked hyperuricemia

- increased S-LDH

- reduced blood cell counts

- marked uric acid crystalluria

- urinay tract infection

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• Hydration, alkalinization & allopurinol

started

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ON FEBRUARY 13th:

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• S-creatinine: 4.2 (371 μmol/L)

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• S-uric acid: 15.7

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• Urine sediment:

persisting & massive uric acid

crystalluria, but morphologically

different from that seen 4 days

before

%

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MASSIVE URIC ACID CRYSTALLURIA

MASSIVE URIC ACID CRYSTALLURIA

(%) ON FEBRUARY 13th

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• RASBURICASE 7.5 mg IV
• S-uric acid: 15.7 🡪 0.6 🡪 5.0 mg/dL

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• Uric acid crystalluria disappears rapidly

& completely

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• S-creatinine slowly decreases to

normal value (1.0 mg/dL on March 16th)

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• On February 17th, bone marrow biopsy:

Acute lymphoblastic leukemia

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COMMENT (1)

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• AKI associated with very severe hyperuricemia

and very severe uric acid crystalluria, both

secondary to acute lymphoblastic leukemia

(= Tumor Lysis Syndrome)

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• Regression of AKI and crystalluria after

correction of hyper-uricemia with rasburicase,

a recombinant urate oxidase, which degrades

UA to allantoin, which is 10 times more soluble

than UA and is easily eliminated by the kidneys

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COMMENT (2)

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• In TLS AKI is mainly the consequence of the

precipitation of UA crystals in the renal

collecting ducts

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• The examination of the urine sediment is a very

valuable, quick and inexpensive tool to diagnose

the renal involvement in TLS and to follow-up

its course

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CLINICAL CASE 2

A boy with crystalluria and renal stones

08.02.2017

Many unusual and birefringent crystals are found in the urine sediment of a 11-year-old boy (U-pH: 5.5)

BASED ON MORPHOLOGY: LEUCINE or 2,8-DHA?

09.02

Through hospital intranet it is found that:

1. Liver enzymes are normal (which weakens leucine hypothesis).
2. The boy is under observation and treatment in the Pediatric EU for a colicky pain associated with left kidney stones.

Thus: severe and unusual crystalluria associated with kidney stones in an 11-year-old boy.

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Working hypothesis: hereditary disease, ?due to congenital deficiency of adenin phosphorybosil transferase

(APRT)?

After the reversion of the colic, the boy is discharged and is enrolled in the “Urolithiasis Clinic” of the Pediatric Nephrology Unit of our hospital, where a new check is planned in two-week time.

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27.02

A new urine sample is supplied:

1. The urine sediment examination confirms the presence of a severe crystalluria, identical with the previous one.

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2. An aliquote of urine is filtered, dried, and shipped to Prof. Michel Daudon (Hôpital Tenon, Paris) for infrared spectroscopy (IRS) investigation.

The urine sediment of 27.02

Many crystals mostly in aggregates…

…of variable size, colour, shape, and birefringence

… some of which with a rather pretty arrangement…

Diameter of crystals (number measured = 50):

6.3 ± 1.7 μm, median 6; range: 4.3-11.7

28.02

Measurement of APRT in the peripheral RBC lysate is performed (by Prof V. Rizzo, Fondazione IRCCS Policlinico San Matteo, Pavia):

APRT 1.1 U (mg of AMP/min, nv: 6.5-44.6)

23.03 RESULT OF INFRARED SPECTROSCOPY

The typical spectrum of 2,8-DHA showing stretching vibration of the NH₂ group of adenine at 3358 and 3258 cm^-1 and the stretching vibration of the two lactame groups (O=C-NH) of 2,8-DHA at 1655 cm^-1. Other typical vibrations of 2,8-DHA are observed at 1446, 980, 797 and 764 cm^-1.

28.03

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Surgical removal of stones from the left kidney.

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During surgery a kidney biopsy is performed:

only very few 2,8-DHA crystals within the distal tubular lumens are found (= no signs of crystalline nephropathy)

29.03

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Infrared investigation of the removed stone:

(central laboratory of our hospital): 2,8-DHA.

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Final diagnosis: Left kidney stones and crystalluria due to the inherited APRT deficiency in an

11-year-old boy.

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Treatment with allopurinol, 100 mg/day,

is started.

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Due to the persistence of crystalluria, the

dosage of the drug is gradually increased to 300

mg/day, with complete reversion of crystalluria.

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SUMMARY

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An accurate and complete diagnostic & therapeutic roadmap (opened and closed in

50 days) for a rare and

severe kidney disease

started and triggered

by a motivated & skillful U-sed examination

WHAT IS

APRT DEFICIENCY?

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�2,8 DHA CRYSTALS�(APRT DEFICIENCY)�

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APRT

Adenine

Adenosin monophosphate

2,8 -DHA

(highy insoluble at any U-pH)

XANTIN DEHYDROGENASE

X

APRT DEFICIENCY

Autosomic recessive transmission

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• Type I : Enzyme activity in RBC lysate: virtually absent.

Exposed subjects: Caucasians, hemizygous and compound heterozygous.

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• Type II : Enzyme activity in RBC lysate: 10

-25%.

Exposed subjects : Japanese (especially

hemizygous).

WHAT CLINICAL EVENTS

HAVE WE PREVENTED

TO OUR YOUNG PATIENT?

• Recurrent radiolucent urolithiasis (65%)

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• AKI from intra-renal precipitation (tubular lumen and interstitium) of 2,8-DHA crystals (26%)

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• CKD from possible chronic interstitial

nephritis (17%)

(Edvarsson V et al. Am J Kidney Dis 2001; 38: 473-80)

23 Pts (M11/F 12, age 0.5-62 yrs (29.0 ± 19.8)

SOME IMAGES OF INTRARENAL PRECIPITATION OF 2,8-DHA CRYSTALS (associated with severe AKI in a kidney transplant recipient with APRT deficiency)

Courtesy of Dr. Elisabetta Margiotta

H&E

POL

H&E

POL

H&E

POL

A virtuous diagnostic and therapeutic roadmapd triggered by a motivated and skillful urinary sediment examination

Garigali G, Marra G, Rizzo V, de Liso F, Berrettini A, Daudon M, Fogazzi GB

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Clin Chim Acta 2019; 492: 23-25

CRYSTALLINE AKI

• Acute urate nephropathy

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• Ethylene glycol intoxication

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• Drugs (e.g, amoxycillin, cyprofloxacin, indinavir

acyclovir, orlistat, felbamate, etc.)

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• Metabolic disorders (e.g, hyperoxaluria,

2,8-DHA)

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• Myeloma kidney with crystalline light chains

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• Foods (e.g, Averroha carambola or star fruit)

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CLINICAL CASE 3

A crystalluria appearing only once every 130 years?

CASE REPORT/1

• On March 25, 2003, very unusual crystals are found in the urine of a 30-year-old woman in the 8^th week of her first pregnancy

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• The patient is under medication with folic acid (5 mg/day), iron sulphate (525 mg/day), and levotyroxin (100 μg/day)

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CASE REPORT/2

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• The day before urinalysis, the patient has eaten large amounts of vegetables, especially spinach and salad

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URINALYSIS BY DIPSTICK

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• pH: 7.5, SG: 1.025

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• Albumin, Glucose, Haemoglobin,

Leukocyte esterase, Nitrites:

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All negative

�URINARY SEDIMENT �

• A ++ crystalluria without any other particles is found

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• The crystals have a very unusual morphology, never seen before, which we define as «daisy-like» crystals

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BRIGHT FIELD

PHASE CONTRAST

WHAT CRYSTALS ARE THEY?

Two steps are undertaken to identify their nature:

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1. REVIEW OF THE IMAGES OF CRYSTALS CONTAINED IN BOOKS OF OUR SPECIALIZED LIBRARY

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2. INFRARED SPECTROSCOPY

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REVIEW OF THE IMAGES CONTAINED IN OUR SPECIALIZED LIBRARY�

58 books devoted to urinalysis, urine sediment or clinical microscopy were consulted:

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43: historical books covering the period

1844-1974

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15: books covering the period 1981-1999

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Altogether, more than

1530 images of crystals were examined

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RESULTS

In only two works crystals

similar to ours were found

Calcium carbonate

crystals

found in the saliva of a dog

ROBIN C, VERDEIL F�“Traité de Chimie anatomique et physiologique normale et pathologique” (1853)

ULTZMAN R, HOFMANN KB�“Atlas der physiologischen und �pathologischen Harnsedimente” (1871)

Creatin & zinc chloride

crytals found in the

human urine,

zinc chloride being

a reagent

used in the laboratory

to extract creatinine

OUR PATIENT (2003)

unknown

(urine)

ROBIN-VERDEIL 1853

calcium carbonate

(saliva)

ULTZMAN-HOFMANN 1871

creatine + zinc chloride

(urine)

RESULTS AT THAT POINT

�2.INFRARED SPECTROSCOPY�

• The crystals contain calcium carbonate

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• However, the infrared spectrum does not correspond to calcium carbonate commonly found in biological fluids

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• Therefore, our crystals are made up of an atypical form of calcium carbonate

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WHAT DO WE KNOW ABOUT CALCIUM CARBONATE CRYSTALS?

• Rare in humans

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• A frequent finding in the urine of horses and other

herbivores

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• With a pleomorphic morphology (dumbels, four-leaved

clovers,daisies, pumpkins,etc) and birefringence

under polarized light

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• UpH: alkaline

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• Effervescence of the urine by adding acetic acid

due to the production of carbon dioxide

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• Clinical significance in humans: ?just due to ingestion of large quantities of vegetables ?

CALCIUM CARBONATE CRYSTALS IN HORSES’ URINE

Bright field

Phase contrast

Polarized light

A spinach load given to a member of our urine group:

No crystals found in the urine!!

THE ROLE OF SPINACH

However, in the following years we were contacted by 7 colleagues from 5 different countries who also had found daisy-like crystals in the urine, so that in 2017…

“Daisy-like” crystals: A rare and unknown type of urinary crystal

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G.B. Fogazzi, R. Anderlini, S. Canovi, C. Covarelli, J. Gras, J. Kučera, A. Proietti, D. Rogic, R. Teboul, C. Ferraris Fusarini, F. de Liso, G. Garigali, M. Daudon

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Clinica Chimica Acta 2017; 471: 153-157

The 10th case was a 1-year-old male crossbred dog, whose diet was urine showed a pH of 7.0 and a specific gravity of 1.018, without abnormalities. After centrifugation and removal of the supernatant, the sample revealed the presence of 5 to 15 DLcr/high power field (400×) intermingled with tiny rhombohedral thin plates. A new urine sample, examined 5 days later, confirmed the presence of DLcr in the same amount

PATIENT 1

PATIENT 2

PATIENT 4

But the story is not yet finished…� …in 2021�

Three types of crystals, including daisy-like ones (2 🡪) are found in the urine of a 64-year-old woman. With infrared spectroscopy and other sophisticated techniques, it is found that each type of crystal is made of a variant of calcium carbonate : 1 calcite; 2: vaterite; 3: aragonite.

2

2

2

…and 3 months ago one new piece of information…�

Clin Chim Acta 2021; 523: 169-171

Daisy-like crystals were found in the urine of a 5-year-old girl whose urine had been collected in a glass recipient which had been sterilized by the patient's family the night before sample collection by boiling water with high calcium and magnesium content (hard water), and letting the recipient cool overnight with the water in it.

Hypothesis: Sampling artifacts are therefore a possible explanation for at least some of the previously described "daisy-like" urinary crystals. 

Extremely rare «daisy-like» crystals in urinary sediment can be due to a sampling artifact.

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G HudáK, G Farkas, B Vajik, et al.

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Clin Chim Acta 2021; 523:395-396

Letter to the editor

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Daisy-like crystals: Not just the result of sampling artifact and not only in urine

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S Baroni, S Kučera, R Anderlini, et al.

In order to verify the hypothesis proposed by Hudák et al, the urine collection procedures used for the 11 subjects described previously were retraced.

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The results demonstrates that 9 out of 11 subjects had used appropriate sterile plastic containers (tubes or jars), while for the 2 other subjects no information could be retrieved.

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In addition, one of the authors (S.B.) found DLcr in the seminal fluid of two male patients investigated for infertility.

Also in these patients the sperm had been collected in a plastic sterile jar.

At this point… We expect some new interesting information in the future… which one will it be?

CLINICAL CASE 4

A kidney transplant recipient with two types of crystals in his urine?

CLINICAL HISTORY (1)

• On July 15, 2014, a 50-year-old man of Moroccan origin is submitted to a periodical control in the outpatient clinic (two months before kidney transplant from cadaveric donor for uremia due to pANCA+ systemic vasculitis):

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- S-creat 1.77 mg/dL (nv 0.5.1.20)

- S-Uric acid 6.8 mg/dL (nv 2.4-7.0)

- Urine dipstick: pH 5.0, SG 1.030 Hb, LE, albumin: negative

URINARY SEDIMENT FINDINGS

• Hyaline and hyaline-granular casts, 1/lpf (160x)

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• Monohydrate calcium-oxalate crystals, 1-3/HPF (400x)

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• Hexagonal crystals, 1-3/HPF

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HEXAGONAL CRYSTALS

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Monohydrate calcium-oxalate crystals

POLARIZED LIGHT

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One or two types of crystals???

And which one(s)???

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At this point….

Infrared spectroscopy analysis

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After IRS result, we went back to the patient clinical notes and found that he was under a daily treatment with:

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• Prednisone 10 mg
• Tacrolimus 4 + 3 mg
• Mycophenolate mofetil 1 g bid
• Omeprazole 20 mg
• Co-trimoxazole (= sulfamethoxazole 800 mg + trimethoprim 160 mg)

initially 1 tablet on alternate day to prevent

Pneumocistis carinii infection, but recently increased to

1 tablet/day in view of the removal of a ureteric stent

CLINICAL HISTORY (2)

WHAT DO WE KNOW ABOUT SMX CRYSTALS?

* M. Daudon (personal communication) on 14 pts investigated:

lozenges 6; triangles: 4; globules 2; hexagons 2.

TAKE HOME MESSAGE

1. We would not have identified these crystals if we

our microscope had not been equipped with filters

for polarized light!!!

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2. SMX can cause the precipitation of crystals which may have a wide spectrum of morphologies and may be associated with AKI

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3. SMX crystalluria may occur in the presence of:

- High dosage of the drug

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- Hypoalbuminemia

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- Hypovolemia/dehydration

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- Urine pH 5.0-6.5

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- Already reduced renal function

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How to identify sulfamethoxazole crystalluria

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de Liso F, Garigali G, Ferraris Fusarini C,

Daudon M, Fogazzi GB

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Clin Chim Acta 2016; 452: 106-108

THANK YOU FOR YOUR ATTENTION