Specimen of choice is the first-morning, midstream and
For routine urinalysis, the specimen of choice is the
first-morning miction. This choice is a compromise. For the
chemical testing, the first-morning miction is usually the
prefered specimen because it is the most concentrated but for the
microscopical examination of cells: imagine cells incubated
overnight in a 37°C urine. Cytologists prefer the second-morning
specimen but, the aim of the tests is quite different.
A free-voided specimen is often used, this being unavoidable
for the emergency patient. The main disadvantage comes from the
dilution of the specimen. This dilution, in some cases, can be
sufficiently high to cause some false-negative results,
especially with the chemistry testing.
The ideal situation is when the specimen is analyzed shortly
after miction. But let's stay realistic! There will always be a
certain delay between collection and analysis. It is important to
set some rules for an acceptable period and conditions of
What is an acceptable delay?
This is an easier question to ask than to answer. While it is
reassuring to have clearly determined delay rules, strict
obedience to rules is undesirable, it all depends on the specific
situation. Some decisions must be left to common sense.
Before rejecting a specimen, we must consider the following:
- Specimens are unique and represent a punctual situation
in time. A second specimen is necessarily obtained; later
and perhaps in a changed clinical condition.
- The patient that comes to a lab for analyses will have to
come back in the case of a rejected specimen. We hope
that the former does not return for only a urinalysis
prescribed without conviction.
- Some substances can disappear quite rapidly on standing;
for example, glucose in the case of a urinary tract
infection. But what was the first reason of the clinician
request: to know if there's any sugar in his patient,s
urine or to know if he has a urinary tract infection?
- As for the serum, urinary substances and elements don't
have the same stability. Cells degenerate rapidly while
the characteristics of casts will be more easily
preserved. Many crystals seen in urine are absent in the
freshly voided specimen.
Specimens that no longer represent the patient at the time of
collection and those that could lead to false interpretation
should be rejected. It should be remembered that these are an
For several reasons, we think that all specimens should be
analyzed. The specimen with a delay problem should have a mention
as;"unacceptable delay: many results possibly invalid."
The quality of a specimen, that cannot be analyzed rapidly
depends on its conservation method. Several centers recommend
that specimens be refregerated if they cannot be processed
shortly after collection. A major disadvantage with this practice
is the precipitation of crystals. Amorphous urates are easily
recognizable by the pink pellet they form upon centrifugation. In
some cases, the precipitation is so abundant that it obscures all
the elements rendering microscopical analysis quite difficult. A
simple way to get rid of these urates is to place the complete
uncentrifuged specimen at 37C (once centrifuged and decanted, it
is often impossible to dissolve the amount of precipitate in the
small remaining volume). When the precipitate is formed of
phosphate, it can be dissolved by an incubation at 37C or by the
addition of 1 or 2 drops of 2% acetic acid to the sediment.
Adding a bacterial growth inhibitor to a specimen expected to
be processed after a long standing period, would be of some help.
Some of these substances (like those used with contact lens) can
be added and dried to the wall of the container.
The widely accepted urine volume is 12 mL. In a majority of
centers, specimens are received in a graduated conical tube. The
excess volume can be aspirated through a special device before
In some cases, filling to the 12 ml mark is impossible. Some
have suggested that the missing volume be completed with a
suitable solution (normally saline). In some cases, a low volume
should be used as is. Another problem with the unfilled container
is whether to correct or not the results to a 12 ml volume. We
prefer not to correct and add the mention to the report;
"Results obtained with a .... mL specimen". The main
reason for this choice is that it is not obvious that a count
made from 6 ml of urine difficultly obtained from a urinary tract
infected child would be doubled with a 12 ml specimen. This
solution leaves the clinician with the opportunity to correct the
values, if indicated.
The recommended parameter for the urine centrifugation is 5
minutes at 400 RCF. The term RCF means « relative centrifugal
force » and is dependent of the squared rotation speed (RPM) and
the head's radius. In most centrifuge instruction manuals, a
calculation nomogram is illustrated. This graphic is an easy way
to calculate the correct RPM to obtain a 400 RCF. A speed of 1200
RPM is a representative example.
Specimens must not be overspun. With compacted pellets,
resuspension is more difficult, leucocytes and some other
elements form clumps. These factors will give unevenly
The most efficient way to eliminate the
supernatant is by aspiration. It is relatively easy to construct
a constant volume aspiration device with a water siphon. The
device shown here is efficient and clean.
Some recommend to keep a residual volume of 1 ml which gives a
concentration factor of 12.
We think that a concentration factor of 20 ( residual volume
of 0,6 ml) is a good compromise. This choice increases the
probability of finding some rare elements, like blood casts which
are usually present in a small number. If the slide is too
loaded, it is always possible to dilute it with a proper dilution
It is frequent to have an unequal distribution of elements on
the microscope slide. This is specially true for the leucocytes
that have a tendency to aggregate. An inadequate resuspension can
be the cause of an uneven distribution although, the presence of
mucus, to which elements may adhere, can cause a significant
variation in the different field counts. The resuspension
procedure has to provide the better homogeneous distribution
possible. The use of a vortex mixer at low speed is an efficient
mixing method. Vortex mixing does not seem to disrupt the
different urinary elements. With a well distributed sediment,
examination of ten fields is often sufficient to obtain a
It is highly recommended that the microscopical examination be
done on a constant volume of sediment. Some companies ( Kova ...)
propose special calibrated slides that always contain
the same examination volume. The recent version of the Kova
acrylic slide is: ready to use, has multiple wells, and allows
examination under polarized light with the 40x objective.
For those that prefer the usual slide and coverslip, an
examination volume of 20 uL, with a coverslip of 22 x 22 mm, is
well suited. With this coverslip size, 20 uL is the ideal volume
preparation being neither too thick or too thin. The sediment
volume is dispensed with a pipetting device. With an SMI™
type pipette that is used properly, carry over is not
The microscopical analysis report for routine urinalysis should
be concise and clear. A heavy listing of elements must be
avoided. We prefer the use of a grid report form designed so that
reporting is resumed to making a check in the correct square.
Checklist-style reports are easier to fill and easier to read.
Cell count should be done at high power field (40X) while the
cast count is done at low power (10X).
Statland recommends the use of a unique graduation for all the
elements. The proposed scale (0-2, 3-5, 6-10, 11-20, 21-100,
>100) is practical and has a good discriminating power. This
scale, with its few classes, is also well adapted to a computer
system like the Clinicom.
It is not easy to establish the microscopical urinalysis
reference values for a single voided specimen. Some values are
reported in Ringsrud and Linné (Urinalysis and Body Fluid). Many
can be estimated from the Addis count and the Schumann
Addis, over a 12-hour collect, has established the following:
White Blood Cells < 1 000 000, Red Blood Cells < 500 000 ,
Hyaline casts < 5 000.
From Schumann the following values were estimated:
Reference values for the urinary
||Values considered as abnormal
||>5 cells at hpf
||>5 cells at hpf
|Renal tubular cells
||>2 cells at hpf
||>5 cells at hpf
||>3 cast at lpf
||>1 cast at lpf
One of the difficulties with the routine microscopical analysis
is the number of samples to examine. Some labs use selection
criteria, based on the aspect of the urine and the reaction of
the dipstick (blood, protein, leukocytes), to eliminate some microscopical
analysis. This operation allows an elimination of 40
to 60% of the microscopy depending on the center's clients and
the activity of specialized clinics. Even after a first
selection, there are still many specimens that can be classified
as "ordinary". Real interesting specimens usually
represent less than 4% of the workload. In such a context, it is
difficult to forget the fifty undone specimens in front of a
complex sediment. In this situation, it is normal to rush, and
maybe not give enough time to the special sediment. To overcome
this situation, we have proposed a three-level organization that
we have called: phase I, phase II and phase III
Phase I is a screening method. At this level, the usual microscopical results (WBC, RBC...) are estimated and reported.
Depending of the screening results, the report is transmitted and
the test ends, or the specimen is put aside for the phase II
analysis. The selection criteria used are the following:
Phase II selection criteria
Protein............if >30mg/dl......................... *
Glucose................if > 0
Microscopical Ob. 10X
Physiological casts....if >2/lpf............*
Epithelial fragments ...........................................*
Renal tubular cells............if
***(3) and over is selected for phase II
NB: the *** selection may vary with the
The phase II step is a more sophisticated analysis. If needed,
staining methods are used. Again, we are using selection criteria
that command some specific actions. We have found that the system
is more efficient if the phase II analysis is done by a different
person or done by the sameone, but only after all the phase I
analysis has been completed.
Findings and actions of phase II
||Search for oval fat bodies
||Evaluate sediment type
|RBC dysmorphocytosis, proteinuria
||Search for blood casts
|Inflammation picture with casts
||Search for WBC casts
|Numerous renal tubular cells
||Search for casts
|Numerous transitional cells
||Search for tissu fragments, search for
The phase III level is something like the coordinated approach
proposed by Schumann. Very special stainning methods, like the
PAP stain, can be used. This level is rarely needed for specimens
issued from routine.
It is also at this level that a specimen is transferred to
specialized resources like the cytology or the parasitology