Abstract
Background:
Urine cytology is useful to diagnose urinary neoplasms, whereas its role in the study of microhematuria is debatable. Usually, standard urinalysis (dipstick test and sediment examination with bright field microscope) detects the presence of microhematuria, but only urinalysis with phase-contrast microscopy (PCM) (dipstick test and sediment examination with PCM) allows the observation of red blood cell (RBC) morphology and identify their source. Usually glomerular diseases show RBCs with morphological alterations in high percentages, whereas on urologic bleeding, RBCs are rather homogeneous without morphological alterations.
Aims:
We compare, for the first time, RBC morphology observed in urine cytology and in urinalysis with PCM, to verify whether urinary cytology allows the recognition of the source of bleeding.
Methods and Material:
A total of 60 patients who had performed both urine cytology and urinalysis with PCM for microhematuria, detected with standard urinalysis, were investigated. Urine cytology showed RBCs and were negative for neoplastic cells or for inflammatory events. Urine samples were processed with the automated method ThinPrep®. RBCs with abnormal and variable shapes were defined as deformed. RBCs of the same spherical shape were defined as non-deformed.
Results:
Fifty-six urine cytology with RBCs deformed were confirmed in 55 urinalysis with PCM. One case showed RBCs non deformed in urine cytology and in urine sediment. Overall, agreement, between RBC morphology in urine cytology and urinalysis with PCM, was found in 56/60 cases (93%).
Conclusions:
Therefore, since sediment examination with PCM is available in only few laboratories, we propose that cytopathologist always reports, in urine cytology, any morphological abnormalities of RBCs in order to provide information of the hematuria origin and correctly refer the patient to a nephrologist rather than a urologist.
Keywords: Deformed red blood cells, dysmorphic red blood cells microhematuria, urine cytology
INTRODUCTION
The main role of urine cytology is the detection of cancer cells exfoliated in urine. Currently, urine cytology is the best available test for diagnosis, screening and monitoring urinary tract malignancies. It is known to have high sensitivity and specificity for high grade urothelial carcinoma but sensitivity decreases significantly for low-grade urothelial carcinoma.[1,2] Pathologists, who focus on the identification of neoplastic cells, do not normally examine for other elements such as erythrocytes, crystals, or casts. In the literature, the value of urine cytology in the study of microhematuria is debatable.[3-6] Usually, standard urinalysis (dipstick test and microscopic sediment examination in bright field) is needed to detect the presence of microhematuria. However, only urinalysis with phase-contrast microscopy (PCM) (dipstick test and microscopic sediment examination with PCM) allows us to study RBC morphology to identify whether the source of bleeding is urological or nephrological.[7-11] Usually, in fact, patients with glomerular disease show high percentages of RBCs with morphological alterations. These erythrocytes are defined as dysmorphic. Various types of dysmorphic erythrocytes have been described according to the red blood cells morphology in hemathology: echinocytes, codocytes, anulocytes, schizocytes, discocytes, stomatocytes, knizocytes, and erythrocytes with loss of hemoglobin (ghost cells). Particularly, acanthocytes are doughtnut-shaped dysmorphic erythrocytes with one or more vescicle-like protrusions. A percentage of acanthocytes ≥5% of erythrocytes observed is enough to define a glomerular hematuria. The alterations of RBC morphology are the consequence of RBC egression through the damaged glomerular filtration barrier and subsequent exposure to a hostile tubular environment (osmotic changes and acid urine). Conversely, on urologic bleeding, RBCs are normocytic, rather homogeneous without morphological alterations. These erythrocytes are defined as normoconformed and isomorphic.[1,8,9,11-14] To the best of our knowledge, there is no data in the literature about using urine cytology to evaluate RBC morphology but only brief descriptions in urinary cytology texts.[1,5,15] Since urinalysis with PCM is only rarely available in medical laboratories, the aim of our paper is to verify whether urinary cytology, detecting the morphological alterations of erythrocytes, could be a diagnostic tool to recognize their origin. In fact, the identification of glomerular RBCs can lead to patients being referred to a nephrologist rather than a urologist, thus avoiding invasive and costly diagnostic investigations such as cystoscopy, computerized tomography (CT), or magnetic resonance imaging (MRI). To achieve our goal, we compared, for the first time, PCM sediment examination with urine cytology, to confirm the proper morphology evaluation of erythrocytes in cytology.
MATERIALS AND METHODS
We selected retrospectively 60 cases of patients who had performed, in 2021, urine cytology for microhematuria, detected with standard urinalysis. All patients had medical history negative for nephropathy and urological neoplasm. All urine cytology tests were negative for neoplastic cells or for inflammatory events, but they showed the presence of RBCs. Therefore, all patients, to recognize the RBCs origin, performed sediment examination with PCM, according to diagram 1.
Diagram 1.
Diagnostic pathway to evaluated red blood cell morphology in patients with microhematuria
The median age of the patients was 61 (range 23-84), 24 female and 36 male. All cases were voided urine (VU) samples processed within two hours of arrival to laboratory, to avoid degenerative cellular changes. The samples were processed with ThinPrep® method, a proprietary automated liquid-based monolayer cell preparation system.[16,17] The VU collection for three days consecutive was mixed with a Cytolyt solution containing methanol, mucolytic, and hemolytic agents and was than centrifuged at 600 G for 10 minutes. The cell pellet of three samples, after discarding the supernatant, was mixed with PreservCyt solution in a single vial and treated using the ThinPrep5000 processor (Hologic, Marlbourgh, Massachusetts). ThinPrep slide was stained with Papanicolau. The cytology slides were revaluated by two pathologists (R.DS and C.C) at light microscopy. RBCs were quantified at low magnification (LPF, 20x) according to a semi-quantitative scale: rare (<4/field), several (5-15/field), moderate (16-25/field), and numerous (>26/field). RBCs were observed at HPF (40x). The comparison between two pathologists led to define the various morphological forms also referring to the red blood cell morphology in hematology. RBCs were defined as deformed when showed the following various forms: speculated, ring form also with one or more cytoplasmic blebs, bell-shaped, triconcave [Figure 1a-b]. Differences in size were considered as another morphological alteration [Figure 1a]. RBCs were defined as having normal appearance when almost all had the same spherical shape [Figure 2a], sometimes “crumpled”. After reading 10 fields (40X), the cases were defined with erythrocytes: a) deformed when over half of RBCs showed various forms; b) non-deformed when over half of RBCs showed spherical shape or “crumpled”; c) doubtful when deformed and no deformed erythrocytes were presented in the same quantity.
Figure 1.
(a) Red blood cells deformed showing various forms and differences in size (Papanicolau stain 400x). (b) Doughtnut –shaped dysmorphic red blood cells with one or more vescicle-like protursions defined acanthocytes in sediment examination with phase-contrast microscopy (arrows) (Papanicolau stain 400x)
Figure 2.
(a) Red blood cells non deformed showing same spherical shape (Papanicolau stain 400x). (b) Red blood cell cast (Papanicolau stain 400x)
Casts, particularly RBC casts [Figure 2b], were sought and when identified, they were reported.
We compared the reports of sediment examination with PCM with data of urine cytology revaluation.
Urinalysis with PCM was performed in a properly collected early morning midstream urine sample: dipstick test, for chemical-physical parameters, and urine sediment examination with PCM for study of pathological elements (erythrocytes, leucocytes, tubular cells, casts, crystals, or other) and erythrocyte morphology. Standardized procedures were used for the following steps: 10 ml urine volume, centrifugation at 550 G for 6 minutes, 0.5 ml sediment concentration, 40 μl volume of urine resuspended to an analyzer in the slide, and quantification of the observed elements at LPF and HPF. Particularly, RBCs were observed and quantified at HPF in the form of numeric range. After a description of various types of erythrocytes, a percentage of dysmorphism was calculated, reading 10 fields (40X). In urine sediment examination with PCM, hematuria was defined as consistent with a glomerular origin when the percentage of dysmorphic cells was higher than 70% of erythrocytes observed or when the percentage of acanthocytes was higher than 5% of erythrocytes observed. Instead, erythrocytes with normal form or however isomorphic in percentage higher than 70% of erythrocytes observed, identified a non-glomerular hematuria. Observation of other pathological elements such as proteinuria, leucocytes, tubular cells, and casts, helped us to confirm a glomerular origin of hematuria.
Figure 3(a-b) shows the various erythrocyte morphology in urine cytology and in sediment using oil immersion (100X).
Figure 3.
(a) Red blood cells deformed in urine cytology at light microscopy (LM) and red blood cells dysmorphic in sediment examination with phase-contrast microscopy (PCM) (oil immersion 100x). (b) – Red blood cells non deformed in urine cytology at LM and red blood cells non dysmorphic in sediment examination with PCM (oil immersion 400x)
To exclude that morphological changes of RBCs, in urine cytology, were due to any mechanic or chemical artifacts of ThinPrep® method, we added a control group. We selected 15 patients with non-glomerular hematuria studied with urinalysis with PCM. They performed successively urine cytology that confirmed the absence of deformed RBCs.
RESULTS
Figure 4 summarizes the results, comparing RBC morphology observed in urine cytology and in sediment examination with PCM.
Figure 4.
Comparison of red blood cell morphology in urine cytology and in sediment
Urine cytology of 56/60 cases showed deformed RBCs. Urinary sediment confirmed the presence of dysmorphic RBCs in 55/56 (98%) cases: 11 cases showed acanthocytes higher than 5% of erythrocytes observed and 44 cases showed dysmorphism higher than 70-80%. One case (1/56) with rare deformed RBCs in cytology showed non-RBCs in urinary sediment and negative dipstick test for blood. Furthermore, in 10/56 urinary cytology with deformed RBCs, RBC casts were identified.
One case (1/60) which was defined as doubtful in cytology, due to the presence of deformed and non-deformed RBCs, showed non-dysmorphic RBCs in urinary sediment. One case (1/60) with numerous and non-deformed RBCs in cytology had non dysmorphic erythrocytes in urinary sediment. Two cases (2/60) with numerous RBCs with non-deformed morphology in cytology did not have RBCs in urinary sediment and dipstick test for blood were negative.
Overall, agreement, between RBC morphology in urine cytology and sediment, was found in 56/60 cases (93%): 55 deformed and 1 non deformed.
DISCUSSION
Currently, cytologic urine examination is used in screening and diagnosis of high-grade carcinoma, in follow-up of urothelial tumor regardless of grade, in follow-up of patients with atypical cells without documented urinary tract neoplasm, and in patients with microscopic or macroscopic hematuria. The Paris System Reporting allows the definition, in adequate samples, of the various morphological categories in urinary tract cytopathology for the identification of negative cases, low-grade urothelial neoplasms, atypical urothelial cells, suspected high-grade urothelial carcinoma, and high-grade urothelial carcinoma.[18,19] This classification is useful to standardize a universally usable reporting system.
However, an experienced cytopathologist should recognize in urine examination the presence of other non-neoplastic pathological elements, indicative of kidney disease (erythrocytes, renal tubular epithelial cells, inflammatory cells, crystals, and casts).
The literature has few details regarding the utility of urinary cytology to identify elements specific of nephropathy particularly RBCs, assuming that they can only be evaluated on unfixed urine. Usually, standard urinalysis (dipstick test and sediment examination with bright field microscopy) is needed to detect the presence of microhematuria, also associated with proteinuria or of other pathological elements. Various automated urinalysis systems have become more widespread, but urinalysis with PCM is invaluable because it is reliable, unlike automated tests or bright field microscopy, in order to diagnose various kidney diseases such as glomerulonephritis, vasculitis, and tubular acute necrosis.[7-11] In particular, observing RBC morphology, at HPF, it is more reliable to identify whether the source of bleeding is urologic or nephrological. In fact, usually, patients with glomerular disease show a high percentage of deformed (dysmorphic) RBCs. These morphological alterations are due to the passage of RBCs through the damaged glomerular membrane and subsequent exposure to osmotic and pH changes along tubules. Instead, on urologic bleeding, RBCs are normocytic, rather homogeneous and normal shape (isomorphic and non-dysmorphic).[1,8,9,11-14] In this study, we compared, for the first time, RBC morphology observed in urine cytology and in sediment examination with PCM and we found excellent agreement. Overall, agreement, between RBC morphology in urine cytology and sediment, was found in 93% of case (56/60). Urinalysis with PCM shows the presence of dysmorphic erythrocytes, a condition consistent with a glomerular hematuria, in 55 urine samples out of 56 patients with deformed RBCs in urine cytology, with an agreement of 98%. Only one case with rare deformed erythrocytes in urine cytology did not show erythrocytes in sediment and dipstick was negative for blood. Evidently, RBCs were too few for confirmed hematuria with dipstick test. The normal RBC morphology in urine cytology, found in three cases, was confirmed in urine sediment in one case, indicating a condition consistent with a non-glomerular hematuria. Instead, the other two cases with numerous normal morphology RBCs in cytology did not show erythrocytes in sediment and dipstick was negative for blood. It is likely that the hematuria was non-glomerular. In fact, the hematuria disappearance on urinalysis can indicate urological hematuria, often discontinuous. In this perspective, the RBC morphology had also been correctly analyzed in urine cytology in these three cases. The doubtful case with deformed and non-deformed erythrocytes showed non-dysmorphic erythrocytes in sediment examination with PCM. This can be explained by possible physical and chemical alteration in urine sample (too high pH, too high or low density) inducing morphological modification of RBCs. These results indicate that ThinPrep® method,[16,17] despite the use of hemolytic agent in the liquid medium, preserves RBC morphology well enough. Moreover, the variable number of RBCs, in analyzed cases, did not affect the study of erythrocytes morphology. The evaluation of the slides stained with Papanicolau, at HPF, consents the clear observation of the morphology of red cells present in the sample. The deformed RBCs show contour irregularities of membranes and appear speculated, ring form with one or more cytoplasmic blebs, bell-shaped, triconcave. Instead, RBCs with normal morphology or non-deformed have almost all the same spherical shape, sometimes “crumpled”. Normal and deformed RBCs can also appear as colorless “ghost cells” due to lost hemoglobin content. Ultimately, in urine cytology, the variability in the form of erythrocytes, in the same slide, is a very useful clue to identifying the glomerular origin of RBCs. Furthermore, observation in urine cytology of RBC casts, usually a hall marker of glomerular hematuria, helps us to confirm the glomerular origin of deformed erythrocytes.
In conclusion, the pathologists when observe urine cytology slides can recognize not only neoplastic cells but also erythrocytes deformed or non-deformed providing information of the hematuria origin. Our results are very interesting if you consider that hematuria affects from 1% to 18% of population[20] and that many physicians have checked patients with hematuria, for many years, by prescribing urine cytology looking for neoplastic cells. On the contrary, other physicians refer patients to urologists that often prescribe invasive and costly diagnostic investigations such as cystoscopy, CT, or MRI. Over time, patients with unrecognized glomerular microhematuria can develop other nephrological injuries (proteinuria and kidney failure) causing rising health care costs. Therefore, since urinalysis with PCM is rarely available in few laboratories, we propose urine cytology as a new diagnostic tool to recognize the origin of hematuria and to direct the patients to the nephrologist rather than the urologist.
Statement of ethics
The study was conducted in accordance with the World Medical Association Declaration of Helsinki and the Harmonized Tripartite Guideline for Good Clinical Practice from the International Conference on Harmonization. All patient’s information, data, and images were properly anonymized, to respect confidentiality and protection of patients’ data, in compliance with the processing of data covered and protected by the Italian Privacy Law and by the GDPR (General Data Protection Regulation, EU regulation 2016/679), and it is impossible for the patient to be identified. Patients involved have given their written informed consent for the routinary diagnostic procedures, and no further biological patients’ materials were depleted or collected for the purposes of the present study (see material and methods section).
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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