Upsala J Med Sci 84: 67-74, 1979 The Effect of Short-term High-dose Treatment with Methenamine Hippurate of Urinary Infection in Geriatric Patients with Indwelling Catheters I. The preparation and morphology of a quantified urine sediment Bo Norberg, Astrid Norberg, U l f Parkhede, Hans Gippert and MBns Akerman Departments of Internal Medicine, Pathology and Education, Univer.tity of Lund and the School of Nursing, Lund, Sweden. 3 A4 Riker Laboratories, Skurholmen, Swyden ABSTRACT A quantified sediment of the urine from patients with indwelling catheters was prepared by fixation of 0.1 ml urine in 0.9 ml 2% glutaraldehyde immediately after sampling. Slide preparations were then made from 0.2 ml of the glutaral- dehyde suspension by means of a cytocentrifuge. were properly contrasted by the May-Grunwald-Giemsa stain but haematoxylin- eosin and the Papanicolaou stain were superior as regards leukocyte morphology. It is suggested that g l u t a r a l d e h y d e - c y t o c e n t r i f u g e preparations of the urine cytology may be useful in the evaluation of urinary infection and in the evaluation of the therapy of urinary infection. Bacteria and epithelial cells INTRODUCTION The microscopic examination of urine sediments is a rapid and simple proce- dure which provides essential information in many cases of kidney disease or infections in the urinary tract. The conventional urinary sediment has, how- ever, serious pitfalls, e.g. low reproducibility, low precision and high vul- nerability to delay in transport and preparation (1-10). It nevertheless seemed desirable to make a quantified urine sediment from patients with indwelling catheters in order to evaluate urinary infection and the effects of therapy. Since delay in transport and preparation could not be avoided under the conditions prevailing, we chose to "freeze" the cell picture at the moment of sampling by fixation. The aim of the present study was to identify a simple and rapid method of sampling, fixing and quantifying the cytology of the urine from patients with indwelling catheters. Different staining procedures were also compared as re- gards the contrast and detail revealed in the morphology of bacteria and leu- kocytes. 67 MATERIAL AND METHODS The urine was sampled from 14 inpatients at the somatogeriatric wards at Saint Lars Hospital, Lund, twice a week during the pre-treatment control period, days 10-17 , during treatment with methenamine hippurate (MH, HiprexR, Riker Laboratories, Loughborough, Leicestershire, England), 2 g x 3 , days 18-52 and during the post-treatment control period, days 6 6 - 7 3 . During days 1-9 different cytocentrifuge preparations of leukocytes and bacteria were evaluated. All patients had an indwelling catheter with continuous flow of the urine in- to a bag with a valve preventing back flow (closed system) for months or years prior to the present study. The catheter was plugged 20-30 minutes before sam- pling. The urine, approximately 15 ml, was collected in a sterile plastic tube and thoroughly shaken in order to disperse the solid matter. The specimen (0.1 ml) was then immediately transferred to 0.9 ml 2% glutaraldehyde in phosphate buffer, 0.135 M, pH 7 . 4 . Cells and bacteria were spun down on slides 2-4 hrs later by means of a cytocentrifuge (Shandon-Elliot Cytospin), 1,000 r.p.m., 10 minutes, 0 . 2 ml of the cell suspension. During the present study 197 double preparations of urine sediments were ob- tained. One preparation was always stained according to May-Griinwald-Giemsa (MGG), which was chosen as reference stain to confirm with the haematological traditions of the laboratory. Other routine stains available in the laboratory were used for the remaining preparation and compared with MGG: haematoxylin- eosin (Mayer's H a e m a t o x y l i n - E r y t r o s i n ) , the Papanicolaou stain, periodic acid Schiff (PAS), methyl green-eosin or haematoxylin-eosin-methylene blue. In ad- dition, staining with acridine orange at low pH ( 4 ) was tried. The evaluation of the staining procedures was performed by means of a Zeiss Photomicroscope equipped with a planachromatic x 100 objective (cf. Figs 1-3). RESULTS The cytological picture of urine sediments occasionally shed light on the associated disease; the renal lesions in a patient with hyperparathyroidism and urinary tract infection were confirmed by the finding of numerous hyaline casts with embedded leukocytes, sugcestive of protein loss in the urine and re- nal inflammation (Fig. 1). The glutaraldehyde fixation was so rapid and effective that leukocytes were caught in different stages of the locomotory cycle (Fig. ZB), in different sta- ges of phagocytosis (Fig. 2B) and in different stages of disintegration (Figs. l B , 3). The staining of g l u t a r a l d e h y d e - c y t o c e n t r i f u g e preparations entailed several problems. The basic haematological stain, May-Grunwald-Giemsa (MGG), produced 68 F i g . 1. From p a t i e n t no. 8, an 80-year-old man w i t h c e r e b r a l a r t e r i o s c l e r o s i s , a m o d e r a t e d e g r e e of r e n a l f a i l u r e ( s - c r e a t . 130-150-140 m m o l / l ) , and hyper- p a r a t h y r o i d i s m (s-Ca 3.7-2.5-2.8 mmol/l, s-P 0.8-0.8 m o l / l , s-parathormone 690-510 p m o l / l ) . The u r i n e s e d i m e n t c o n t a i n e d numerous h y a l i n e c a s t s w i t h em- bedded l e u k o c y t e s . The l e u k o c y t e c a s t s s u g g e s t e d r e n a l l e s i o n s i n d u c e d by h y p e r c a l c e m i a o r u r i n a r y t r a c t i n f e c t i o n , o r b o t h . S t a i n i n g a c c o r d i n g t o May- Griinwald-Giemsa. F i g . 1 A . Low-power f i e l d , m a g n i f i c a t i o n x 1 3 4 . Two h y a l i n e c a s t s (Cy) w i t h em- bedded l e u k o c y t e s . E x c e p t f o r t h e c a s t s , t h e p i c t u r e i s t y p i c a l of t h e u r i n e s e d i m e n t from a p a t i e n t w i t h i n d w e l l i n g c a t h e t e r and h e a v i l y i n f e c t e d u r i n e , i . e . numerous b a c t e r i a and l e u k o c y t e s , some i n l y s i s . T h i s specimen w a s ob- t a i n e d a f t e r t r e a t m e n t w i t h methenamine h i p p u r a t e , 2 g x 3 , f o r 2 2 d a y s . F i g . 1 B . D e t a i l from F i g . l A , m a g n i f i c a t i o n x l , 3 3 6 of a p a r t of a h y a l i n e c a s t ( e n c l o s e d a r e a ) . Two l e u k o c y t e s ( L ) , s u r r o u n d e d by b r i g h t h a l o s , a r e embedded i n t h e c a s t . Two c e l l s i n l y s i s (C) are a l s o s e e n , one embedded i n t h e c a s t . B: b a c t e r i a , p r o b a b l y p r o t e u s m i r a b i l i s , which were r e p e a t e d l y c u l t u r e d from t h e u r i n e of t h i s p a t i e n t . a b r i l l i a n t c o n t r a s t i n e p i t h e l i a l c e l l s and b a c t e r i a ( F i g . 2 A ) . The l e u k o c y t e s were, however, o v e r l o a d e d w i t h s t a i n which v e i l e d i n t r a c e l l u l a r d e t a i l s ( F i g s . 69 F i g . 2A. The u r i n e s e d i m e n t from an 82-year-old f e m a l e , p a t i e n t no. 5 , p r i o r t o t r e a t m e n t w i t h methenamine h i p p u r a t e . The c y t o l o g i c a l p i c t u r e w a s dominated by b a c t e r i a of which two s t r a i n s a r e shown, B a p p a r e n t l y s t r e p t o c o c c u s f a e c a - l i s , B a p p a r e n t l y E c o l i , b o t h of which were found i n h e r u r i n e c u l t u r e s . The u p p e r p a r t of t h e m i c r o p h o t o g r a p h i s dominated by an e p i t h e l i a l c e l l , t h e b o r d e r s of which a r e i n d i c a t e d by a r r o w s . N : L: l e u k o c y t e s , one p r o b a b l y l o c a t e d w i t h i n tho- c y t o p l a s m of t h e e p i t h e l i a l c e l l . S t a i n i n g a c c o r d i n g t o May-Grunwald-Giemsa. M a g n i f i c a t i o n ~ 1 , 3 3 6 . F i g . 2 B . U r i n e s e d i m e n t from an 82-year-old f e m a l e , p a t . no. 7 , p r i o r t o t r e a t - ment w i t h methenamine h i p p u r a t e . Leukocytes c a u g h t i n t h e h u n t and phagocy- t o s i s of b a c t e r i a . Many of t h e l e u k o c y t e s h a v e t h e e l o n g a t e d s h a p e t y p i c a l of c e l l s i n l o c o m o t i o n , amoeboid movement c o n f i g u r a t i o n . P h a g o c y t o s i s i s p e r f o r - med by e x t e n s i o n of t h e t h i n g r a n e l a - f r e e c y t o p l a s m i c v e i l i n t h e f r o n t p a r t of t h e l e u k o c y t e s , t h e l a m e l l i p o d i u m ( I p ) towards and around t h e b a c t e r i a (B). S e v e r a l b a c t e r i a are found w i t h i n l e u k o c y t e s and s u r r o u n d e d by p h a g o c y t i c v a c u o l e s ( s e e e . g . a r r o w ) . S t a i n i n g a c c o r d i n g t o May-Grunwald-Giemsa. Mag- n i f i c a t i o n ~ 1 , 3 3 6 . l 2 n u c l e u s of t h e e p i t h e l i a l c e l l . 1, 2A) w i t h r a r e e x c e p t i o n s ( F i g . 2 B ) . MGG s t a i n a s r e g a r d s l e u k o c y t e morphology and c l e a r l y i n f e r i o r t o VGC i v t h e s t a i n i n g of h a c t e r i a . The PAS s t a i n v7as n e a r l y as bac! as t h e 70 3A 3B F i g . 3 . The h e m a t o x y l i n - e o s i n s t a i n p r o v i d e d b e t t e r t r a n s p a r e n c y of c y t o p l a s m and n u c l e u s t h a n t h e May-Griinwald-Giemsa s t a i n b u t b a c t e r i a was h a r d l y v i s i b l e ( B ) . It i s e v i d e n t t h a t t h e v a s t m a j o r i t y of l e u k o c y t e s i n t h e u r i n e of p a t i - e n t s w i t h i n d w e l l i n g c a t h e t e r s are p o l y m o r p h o n u c l e a r s . AMC: amoeboid movement c o n f i g u r a t i o n , t h e e l o n g a t e d c e l l s h a p e s u g g e s t i v e of l o c o m o t i o n a t t h e moment of f i x a t i o n . Some l e u k o c y t e s were f i x e d i n t h e s t a t e of p y c n o s i s ( P ) , k a r y o r - r h e x i s ( K ) o r c y t o l y s i s ( C ) . U r i n e s e d i m e n t from an 81-year-old m a l e , p a t i e n t n o . 2 , w i t h c e r e b r a l a r t e - r i o s c l e r o s i s and a m o d e r a t e d e g r e e of c a r d i a c f a i l u r e , a f t e r t r e a t m e n t w i t h methenamine h i p p u r a t e f o r 1 7 d a y s . M a g n i f i c a t i o n 1 , 3 3 6 . Haematoxylin-eosin and t h e P a p a n i c o l a o u s t a i n p r o v i d e d i n t e r e s t i n g a l t e r n a - t i v e s t o t h e MGG s t a i n . The n u c l e a r and c y t o p l a s m i c d e t a i l s o f l e u k o c y t e s w e r e b e t t e r v i s u a l i z e d t h a n i n MGG-stained p r e p a r a t i o n s ( F i g . 3) w i t h o u t l i t t l e d i f - f e r e n c e between t h e HE s t a i n and t h e P a p a n i c o l a o u s t a i n . A t l e a s t 90-99% of t h e l e u k o c y t e s i n t h e u r i n e of t h e s e c a t h e t e r i z e d p a t i e n t s were g r a n u l o c y t e s . t h e l i a l c e l l s were s t a i n e d p r o p e r l y by b o t h HE and t h e P a p a n i c o l a o u s t a i n . t e r i a were o f t e n h a r d l y v i s i b l e i n p r e p a r a t i o n s s t a i n e d by HE and o n l y s l i g h t l y e a s i e r t o s e e i n p r e p a r a t i o n s s t a i n e d by t h e P a p a n i c o l a o u s t a i n . Epi- Bac- O t h e r s t a i n i n g 71 p r o c e d u r e s w i t h m e t h y l g r e e n and e o s i n , HE combined w i t h m e t h y l e n e b l u e and ac- r i d i n e o r a n g e a t low pH d i d n o t improve t h e c y t o l o g i c a l p i c t u r e . T r e a t m e n t of t h e p a t i e n t w i t h MH, 2 g x 3 d a i l y , d i d n o t i n h i b i t l e u k o c y t e a c t i v i t y , a s r e f l e c t e d by m o r p h o l o g i c a l s i g n s of locomotion and p h a g o c y t o s i s . Both c l i n i c a l and c y t o l o g i c a l e f f e c t s of MH t r e a t m e n t w e r e , however, ambiguous. I n some p a t i e n t s t h e u r i n e c l a r i f i e d , i n o t h e r p a t i e n t s i t d i d n o t . I n some pa- t i e n t s , t h e p r e s e n c e of e p i t h e l i a l c e l l s w i t h o u t l e u k o c y t e s and b a c t e r i a sug- g e s t e d a b e n e f i c i a l e f f e c t of MH t r e a t m e n t , i n o t h e r p a t i e n t s p y u r i a and bac- t e r i u r i a p e r s i s t e d . I t w a s e v i d e n t t h a t a p o s s i b l e r e d u c t i o n i n p y u r i a , bac- t e r i u r i a and o t h e r c o m p l i c a t i o n s of c a t h e t e r i z a t i o n had t o b e e v a l u a t e d by more q u a n t i t a t i v e methods amenable t o s t a t i s t i c a l t e s t s . DISCUSSION The u r i n e from p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s i s u s u a l l y h e a v i l y con- t a m i n a t e d by b a c t e r i a w i t h s e c o n d a r y i n v a s i o n o f l e u k o c y t e s . The c o n t a m i n a t i o n o f t h e u r i n e cannot b e e v a l u a t e d by c o n v e n t i o n a l microscopy of t h e s e d i m e n t un- l e s s m i c r o s c o p e , c e n t r i f u g e and m i c r o s c o p i s t are v i r t u a l l y a t t h e p a t i e n t ’ s bed- s i d e . Leukocytes d i s i n t e g r a t e ( c f . 2 , 5 , 1 0 ) and b a c t e r i a m u l t i p l y e v e r y 20- 60 m i n u t e s . The c l o u d y u r i n e of c a t h e t e r i z e d p a t i e n t s - due t o t h e p r e s e n c e of mucus, s a l t p r e c i p i t a t e s and a g g r e g a t e s of b a c t e r i a , l e u k o c y t e s and e p i t h e l i a l c e l l s - w a s a m a j o r s o u r c e of v a r i a t i o n between d o u b l e p r e p a r a t i o n s from t h e same sam- p l e . O t h e r p i t f a l l s were l o s s of c e l l s d u r i n g c e n t r i f u g a t i o n due t o a d h e r e n c e t o t h e w a l l s of t h e p l a s t i c chamber of t h e c e n t r i f u g e , l o s s of c e l l s i n t o t h e f i l t e r p a p e r d u r i n g c e n t r i f u g a t i o n , and l o s s of c e l l s from t h e s l i d e s d u r i n g t h e d r y i n g and s t a i n i n g . The l e u k o c y t e s p r e s e n t i n t h e glutaraldehyde-cytocentrifuge s e d i m e n t s of ca- t h e t e r u r i n e c o n s i s t e d t o 90-99% of g r a n u l o c y t e s . ( F i g s . 1-3.) T h i s f i n d i n g i s i n agreement w i t h o b s e r v a t i o n s of p r e v i o u s a u t h o r s on p a t i e n t s w i t h bac- t e r i u r i a ( 5 ) . The morphology of t h e b a c t e r i a w a s sometimes s u f f i c i e n t l y d i s - t i n c t t o p e r m i t a t e n t a t i v e i d e n t i f i c a t i o n of b a c t e r i a l s p e c i e s ( F i g . 2 A ) . I n c o n v e n t i o n a l c o v e r s l i p p r e p a r a t i o n s o f u r i n e s e d i m e n t , v i t a l l e u k o c y t e s are sometimes s e e n h u n t i n g and p h a g o c y t i s i n g b a c t e r i a . s e e n i n a i r - d r i e d p r e p a r a t i o n s of l e u k o c y t e s u s p e n s i o n s , a p p a r e n t l y due t o t h e s l o w n e s s of t h e method; t h e l e u k o c y t e s s t o p h u n t i n g and t a k e up a s t a t i o n a r y s p h e r i c a l form d u r i n g p r e p a r a t i o n . The g l u t a r a l d e h y d e f i x a t i o n was r a p i d and e f f e c t i v e , as e v i d e n c e d by t h e f i n - T h i s phenomen i s n o t d i n g of l e u k o c y t e s w i t h t h e e l o n g a t e d s h a p e of moving c e l l s and l e u k o c y t e s i n d i f f e r e n t s t a g e s of d i s i n t e g r a t i o n . The l e u k o c y t e s were f i x e d i n t h e rounded s h a p e of suspended c e l l s , n o t i n t h e 72 f l a t t e n e d s h a p e of smeared and a i r - d r i e d c e l l s . t h u s became l o c a t e d i n d i f f e r e n t o p t i c a l p l a n e s on t h e s l i d e d u e t o t h e r e s t r i c - t e d d e p t h of t h e v i s u a l f i e l d ( 0 . 2 - 0 . 4 ~ m ) . of b a c t e r i a more d i f f i c u l t . The l e u k o c y t e s and t h e b a c t e r i a T h i s f a c t r e n d e r e d t h e a s s e s s m e n t The glutaraldehyde-cytocentrifuge method had some o b v i o u s a d v a n t a g e s o v e r t h e c o n v e n t i o n a l c o v e r s l i p p r e p a r a t i o n of u r i n e s e d i m e n t and t h e c o n t r a s t i n g p r o c e - d u r e s d e s c r i b e d by p r e v i o u s a u t h o r s (1-10). The c y t o l o g i c a l and b a c t e r i o l o g i c a l p i c t u r e of t h e u r i n e w a s f r o z e n i n t h e s t a t e e x i s t i n g a t t h e moment of s a m p l i n g . Cells and b a c t e r i a on t h e s l i d e were l i n e a r l y c o r r e l a t e d t o t h e c e l l u l a r i t y of t h e o r i g i n a l sample and were p r o p e r l y c o n t r a s t e d by r o u t i n e c y t o l o g i c a l s t a i n s . The p r e p a r a t i o n of t h e u r i n e d u r i n g i t s t r a n s f e r from t h e c a t h e t e r t o micro- s c o p e w a s r e l a t i v e l y s i m p l e and r a p i d . I n s p i t e o f t h e p i t f a l l s a s s o c i a t e d w i t h s a m p l i n g , p r e p a r a t i o n and a s s e s s m e n t of t h e i n f l a m m a t o r y c y t o l o g y of t h e u r i n e from p a t i e n t s w i t h i n d w e l l i n g c a t h e - t e r s , t h e glutaraldehyde-cytocentrifuge s e d i m e n t p r o v i d e d a rough q u a n t i t a t i v e measure of u r i n e c o n t a m i n a t i o n . B a c t e r i a were b e s t v i s u a l i z e d by t h e MGG s t a i n , l e u k o c y t e s by HE o r by t h e P a p a n i c o l a o u s t a i n . ACKNOWLEDGEMENTS no * 1. 2. 3 . 4 . 5. 6 . 7 . 8 . 9. 1 0 . T h i s work w a s s u p p o r t e d by t h e Swedish Medical R e s e a r c h C o u n c i l p r o j e c t 2296 and no. 5 3 6 2 and k r s n t s frorii t h e H e d i c a l F a c i l l t y of Lund. REFERENCES B r a d l e y , J . M . & L i t t l e , M . B . : Q u a n t i t a t i v e u r i n e c u l t u r e s . B r i t Med J 11:361-363, 1963. G a d e h o l t , H . : P e r s i s t e n c e o f b l o o d c e l l s i n u r i n e . A c t a Med Scand 183:49- 54, 1968. H e l g a s o n , S . & L i n d q v i s t , B.: E o s i n o p h i l u r i a . Scand J U r o l Nephrol 6:257- 259, 1972. K r o n v a l l , G . & Myhre, E . : D i f f e r e n t i a l s t a i n i n g of b a c t e r i a i n c l i n i c a l specimens u s i n g a c r i d i n e o r a n g e b u f f e r e d a t low pH. Acta P a t h M i c r o b i o l Scand S e c t B 85:249-254, 1977. L i n d q v i s t , B. & Wahlin, A . : D i f f e r e n t i a l c o u n t of u r i n a r y l e u c o c y t e s and r e n a l e p i t h e l i a l c e l l s by p h a s e c o n t r a s t microscopy. Acta Med Scand 198:505-509, 1975. L i t t l e , P . J . : U r i n a r y w h i t e - c e l l e x c r e t i o n . L a n c e t I:1149-1151, 1962. L i t t l e , P . J . : t h e w h i t e c e l l e x c r e t i o n r a t e . B r J U r o l 36:360-363, 1964. P r e s c o t t , L.F. & B r o d i e , D . E . : A s i m p l e d i f f e r e n t i a l s t a i n f o r u r i n a r y s e d i m e n t . L a n c e t I I : 9 4 0 , 1964. S t e r n h e i m e r , R . & Malbin, B.: C l i n i c a l r e c o g n i t i o n of p y e l o n e p h r i t i s , w i t h a new s t a i n f o r u r i n a r y s e d i m e n t s . Am J Med 11:312-323, 1951. Wahlin, A . : D i f f e r e n t i a l c o u n t of u r i n a r y l e u k o c y t e s and r e n a l e p i t h e l i a l c e l l s . A comparison between p h a s e c o n t r a s t m i c r o s c o p y of u n s t a i n e d s e d i - ments and l i g h t microscopy of f i x e d and s t a i n e d s p e c i m e n s . Uppsala J Med S c i 82:43-47, 1977. A comparison of t h e u r i n a r y w h i t e c e l l c o n c e n t r a t i o n w i t h 73 Received July 1978. Accepted October 1978. Address for reprints: Bo Norberg, M . D . Department of Internal Medicine University Hospital of Lund S-221 85 Lund, Sweden 74