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, Ulf 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. Bacteria and epithelial cells
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.

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 moderate 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 mmol/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 sediment 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 induced by
hypercalcemia 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 . Except 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
sediment 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 22 days.

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 ) , surrounded 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 sediment 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 faeca-
l
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 .
2
The upper p a r t of t h e microphotograph 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 arrows. N: 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 .
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- cytoplasm 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 . 2B. Urine sediment 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 caught 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 have 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 locomotion, 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 lamellipodium ( 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 surrounded 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-
nification ~1,336.

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 ) . The PAS s t a i n v7as n e a r l y as bac! as t h e

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 .

70
3A 3B

F i g . 3. The hematoxylin-eosin 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 cytoplasm

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). I t 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 polymorphonuclears. 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 shape s u g g e s t i v e of locomotion 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 ) .
Urine sediment from an 81-year-old m a l e , p a t i e n t no. 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 moderate 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 days. 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 were
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 . Epi-
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 . Bac-
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 . Other s t a i n i n g

71
p r o c e d u r e s w i t h methyl g r e e n and e o s i n , HE combined w i t h methylene b l u e and ac-
r i d i n e orange 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 .
Treatment 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 morphological 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 were, 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 persisted. 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 be 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 secondary 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 sediment un-
l e s s microscope, 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-
side. Leukocytes d i s i n t e g r a t e ( c f . 2 , 5, 10) 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 cloudy 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 major s o u r c e of v a r i a t i o n between double p r e p a r a t i o n s from t h e same sam-
ple. 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 adherence
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.) This finding
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 sediment, 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 . T h i s phenomen i s n o t
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
slowness 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 evidenced by t h e f i n -
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 shape 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 . 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 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 due 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 ) . T h i s f a c t r e n d e r e d t h e assessment
of b a c t e r i a more d i f f i c u l t .
The glutaraldehyde-cytocentrifuge method had some obvious advantages 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 sediment and t h e c o n t r a s t i n g proce-
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 sampling.
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-
scope 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 sampling, p r e p a r a t i o n and assessment
of t h e inflammatory 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 sediment 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

T h i s work w a s s u p p o r t e d by t h e Swedish Medical Research C o u n c i l p r o j e c t

no * 2296 and no. 5362 and k r s n t s frorii t h e H e d i c a l Facillty of Lund.

REFERENCES

1. 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.
2. Gadeholt, 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.
3. Helgason, S . & L i n d q v i s t , B.: Eosinophiluria. Scand J Urol Nephrol 6:257-
259, 1972.
4. 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 orange 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.
5. L i n d q v i s t , B. & Wahlin, A.: D i f f e r e n t i a l count 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 phase c o n t r a s t microscopy. Acta Med Scand
198:505-509, 1975.
6. 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.
7. L i t t l e , P.J.: 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
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 Urol 36:360-363, 1964.
8. P r e s c o t t , L.F. & B r o d i e , D . E . : A simple 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
sediment. Lancet I I : 9 4 0 , 1964.
9. 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.
10. 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 microscopy 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 specimens. Uppsala J Med
S c i 82:43-47, 1977.

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