M. Pfeifer
Standardised
Test Soil Blood 1:
Composition,
Preparation, Application
In addition to the protein content, the behaviour
evinced by a blood soil is essentially determined by the blood coagulation
process. This process can be monitored in a controlled manner due to the
reaction of fibrinogen with thrombin, with the high protein content being
simulated by a matrix of albumin and haemoglobin. A two-component system based
on freeze-dried protein fractions and solvents provides, before application,
for separation of the coagulation factor fibrinogen from thrombin. This setup
permits a stable test soil of standardised composition with reproducible
behaviour and correlation with human blood.
Keywords: test soil, blood coagulation,
cleaning process
1
Introduction
Test soils
are an important fundament for the development of detergents and
washer/disinfectors as well as for optimisation of existing cleaning processes.
While simple starch and fatty soils are adequate for many domains, the
requirements addressed to a test soil for verifying the reprocessing action of
surgical instruments are infinitely more stringent: since blood is the most
prevalent contaminant encountered in this domain, in addition to the general
requirements - such as standardised composition, reproducible dissolution
action or facilities for elucidation of residues - there are special
requirements dictated by the process of blood coagulation.
The extremely complex sequence of blood coagulation gives rise to the formation of insoluble fibrin fibres thanks to an enzyme cascade (1). This process is initiated only by injury to blood vessels or by contact with unphysiologic surfaces, which explains why fibrin formation also takes place directly on the surface of the instruments. This means that in the case of a practice-oriented test soil which is correlated with human blood. not only the fibrin component, which is of special relevance to cleaning, but also its formation is to be implemented only at the time of application.
2 Materials and Methods
2.1 Composition of the Test Soil
To effect fibrin formation in a protein-based soil. a major part of the enzyme cascade, which is required in the blood circulation for assured wound closure, can be omitted. Of decisive importance is the enzymatic cleavage of fibrinogen to fibrin monomers, which automatically merge to thread-shaped polymers (1).
To integrate the important step of blood coagulation into the test soil. a two-component system, which containes fibrinogen separately from thrombin and calcium ions. was developed. Hence coagulation takes place only after mixing both components, thus permitting the formation of fibrin fibres on the surfaces of challenge devices being used for test purposes.
Stability of the sensitive protein fractions is assured by employing lyophilised products. By dissolving the two components in the respective solvents one obtains a coagulable test soil that is always of a similarly high quality. The composition of a test kit for preparation of 10 ml standardised test soil is described in the following:
- Component A: 400 mg albumin
400 mg haemoglobin
60 mg fibrinogen
- Solvent A: 5.0 ml 0.4% NaCI solution
- Component B: 400 mg albumin
400 mg haemoglobin
12.5 NIH units thrombin
- Solvent B: 5.0 ml 0.4% NaCI solution
+ 8.0 mmol./l CaC12
2.2 Preparation of the Test Soil
Before application, the two solid components A and B are dissolved in the respective solvents A and B. To bring all protein fractions completely to solution, dissolution is effected for 1 hour at 36o C while shaking or agitating. Here temperatures above 40o C must absolutely be avoided. Temperatures that are 5-0 oC lower reduce the speed of dissolution, but do not adversely affect the test soil. After dissolution, components A and B are stable for four hours when stored separately. After mixing both components, coagulation immediately begins with formation of insoluble fibrin fibres and is complete after 30 min. Analogous to this process, blood escaping from a blood vessel - and in this case the test soil - is transformed into a gelatinous mass.
2.3 Application of the Test Soil
If both components are separately pipetted onto
challenge devices and immediately mixed a reproducible quantity can be spread
out. coagulating only on the desired surface (Fig. 1). By adding test microbes
to component A before use, microbiological investigations can also be conducted
while having recourse to a standardised soil.
Of special importance for reproducible control
of cleaning processes - in particular on recording the cleaning kinetics - is
not only a standardised composition but also a reproducible layer thickness of
the test soil. In the present case, this is achieved with a robot dosing system
(Fig. 2). In this manner, e.g. soils can also be applied for minimally invasive
surgery instruments.
By
treating challenge devices for instance with heat, alcoholic or aldehyde
solutions, cleaning problems emanating from denaturation or chemical changes
can be investigated. After drying, challenge devices with the standardised test
soil can be preserved for 6 months, when stored protectedly against humidity,
light and temperature fluctuations.
3 Results
A blood soil can be compared with the
standardized test soil by simply immersing it in water. Employed for this
comparison were a high-grade steel challenge device with 20 mg (protein
content) of a standardised test soil on 430 mm2 and a challenge
device with 75 uL human blood, spread on the same surface. After 10 minutes, in
both cases only a white layer composed of fibrin can still be seen. The
remaining components, in both cases primarily haemoglobin and albumin, are
easily dissolved by water. If the detached protein content is continually
recorded at 240 nm for this dissolution experiment by using a flow cuvette, the
detachment kinetics can be concurrently recorded (Fig. 3). The curves rapidly
assume a flat shape within 5 min. and in both cases there remains a residue,
since the fibrin layer cannot be removed by water alone without a mechanical
cleaning action.
Since the fibrinogen content in human blood is
subject to fluctuations between 0.2-0.4% (2), this has been set at a higher
value for the test soil compared with the illustrated blood sample. Thanks to
standardisation, the cleaning performance of an automated reprocessing
procedure as well as of detergents can be investigated in the immersion
experiment (Fig. 4).
4 Discussion
The employment of easily soluble plasma proteins
in conjunction with the enzymatic reaction for formation of fibrin fibres, and
hence of coagulation, gives rise to a standardised test soil endowed with a
very good correlation with a human blood soil. The high protein content of
blood is simulated by the haemoglobin content in addition to the albumin. Hence
investigations can also be conducted in respect of the chemical or thermal
denaturation action.
In conjunction with standardised challenge
devices and a reproducible layer formation, the test soil constitutes the
fundament for scientific as well as practice oriented investigation and
examination of the cleaning step for
reprocessing surgical instruments. Concomitantly, microbiological tests
can be conducted by virtue of the suitability or the suspension medium.
Reagents'
Appendix
Albumin. Bovine: Clinical Reagent Grade. Purity 98- 99%. 1CN Cat.
No. 105033
Calcium chloride hexahydrate, purity DAB (German Pharmacopoeia),
Merck No. 102072
Fibrinogen. From Bovine Plasma. Purity 75%. ICN Cat. No 820212
Hemoglobin. Freeze Dried. Purity 98%. MW 64.5 kDa. ICN Cat. No
151234
Sodium chloride, purity DAB. Merck No. 106400
Thrombin. From Bovine Plasma. High Purity Grade.
Activity > 2000 NIH Units/mg Protein ICN Cat No. 154163