M. Pfeifer
Blood as a
Soil on Surgical
Instruments:
Chemical Profile,
Cleaning,
Detection
Classified as medical devices, surgical
instruments require standardised and validated reprocessing. The procedure
conventionally employed here is cleaning and disinfection in a
washer/disinfector followed by sterilisation in an autoclave. Since the organic
contamination of surgical instruments encountered in practice can be very
pronounced, cleaning constitutes the basis for the subsequent reprocessing
steps of disinfection and sterilisation. In this respect, blood represents the
most common and hence the most important form of contamination.
Keywords: Blood coagulation, fibrin fibres,
denaturation, residual soil, hydrolysis
1 Introduction
By virtue of
the exceedingly complex composition and broad spectrum of functions of blood,
enormous difficulties continue to be faced when trying to reproduce this fluid
as a substitute for donor blood (1). But if one views blood as a cleaning problem
when reprocessing surgical instruments, the majority of its biological
functions can be disregarded. The only aspect meriting attention in this
context is the process of blood coagulation with the end product fibrin. Since,
to perform their preordained tasks, all blood components are necessarily
present in a water-soluble form, the insoluble fibrin fibres formed during
coagulation are particularly relevant for cleaning.
Of
importance concomitantly are the chemical properties of the proteins, since most
of the compounds found in the blood are such proteins. Even though approx. 45%
of the blood is composed of red blood cells (erythrocytes), these need not be
dealt with separately because, during the cleaning process, they very easily
release through haemolysis the protein haemoglobin which accounts for up to 95%
of their contents (2).
1.1 Properties of the Proteins
Proteins are
generally composed of 20 different ammo acids, linked together by means of very
stable peptide bonds. Encountered here
are molecular masses ranging from 10,000 to several million. Due to this
complexity, the structure of these compounds is very complex, thus explaining
the high sensitivity to chemical and even physical influences.
Despite the
high molecular masses, the blood proteins are water-soluble compounds. The
ionizable side chains as well as the spatial arrangement (tertiary structure)
of the peptide chain itself play an important role. But it is precisely the
tertiary structure of the proteins that is amenable to change mediated by
external influences: this process is called denaturation.
Unfolding of the polypeptide chain detracts
in most cases from solubility. In the case of blood proteins on a surface, this
process results in hardening (coagulation) due to the formation of
high-molecular aggregates. Temperatures between 60-70o C suffice for
complete denaturation. An important consideration here is that denaturation is
very dependent on the presence of water. For example, in a dry state casein
forfeits its solubility only after 6 hours at 130o C, whereas blood
immediately coagulates on immersion in boiling water (3).
Other
denaturing factors are marked acidic or alkaline reactions of the solution,
organic solvents. UV light and chemicals, such as e.g. urea or guanidine.
Furthermore, aldehydes can also cause proteins to undergo direct chemical
change (4). But the agent inducing the most hardening action is glutaraldehyde
due to cross-linking of proteins (5).
1.2 Blood
Coagulation
Blood
coagulation induces solidification of the liquid blood in the blood clot, thus
sealing traumatised blood vessels. This ultimately entails the formation of
insoluble fibrin fibres from the soluble fibrinogen present in the blood. This
multi-step process, in which almost 20 different substances are involved, can
be activated by two different pathways (6):
·
The
extrinsic pathway triggers coagulation due to the vascular injury itself. This
form of activation is effected within the space of seconds.
·
The
intrinsic pathway is initiated by contact with an unphysiological surface. This
pathway is activated only within a few minutes.
During the
most important step here, thrombin cleaves two fibrin peptides from fibrinogen.
Hitherto, the latter had prevented fibrinogen aggregation thanks to their highly
negative charge and due to occupation of binding sites. The fibrin monomers
thus formed polymerise and are further stabilised by coagulation factor XIII
(cross-linking due to isopeptide bonds). A network of fibrin fibres, enclosing
the blood cells, is thus formed (6).
2 Materials
and Methods
The soil
exclusively employed for the investigations was fresh, coagulable human blood.
Dosing only during the first four minutes after withdrawal of the blood ensured
that coagulation would take place on the test bodies, thus making it possible
to take account of the important fibrin portion. The test bodies used were
stainless-steel sheets (stainless steel
material 1.4301) measuring 20 x
70 mm.
Since the
lack of standardisation of human blood precludes the procurement of exact
reproducible findings, quantitative results were largely omitted. Hence what
was investigated here was the chemical profile of blood in respect of cleaning
and of detection possibilities of any residues. Quantitative investigations, as
required for verification and optimisation of cleaning processes, must hence be
conducted with a standardised test soil.
3 Results
3.1 Solubility
Figure 1
shows protein detachment (in an immersion experiment without mechanical
cleaning component) of a dried blood soil at 18 oC in demineralised
water. Here it is demonstrated that after approx. 8 minutes no further
reduction is made in the remaining residue. The residual white layer is
composed of insoluble fibrin fibres, which account for approx. 4% of the total
protein content of this blood sample. This confirms that the red blood cells
easily release their well-soluble haemoglobin content. Translated into a
practical setting, this means that already pre-cleaning with clean water can
remove the greatest part of contamination.
Conversely,
a problem is caused by the fibrin fibres which cannot be dissolved even with
surfactants, which, not least, becomes obvious in the fact that in the
production of fibrin preparations sodium dodecyl sulphate is used for their
cleaning (7). The fastest way to dissolve fibrin is by using an alkaline
detergent at as high a temperature as possible. By means of hydrolytic cleavage
of peptide bonds this generally degrades insoluble proteins to smaller and
hence more soluble products.
Figure 2
shows the hydrolytic kinetics (detachment from a stainless steel surface) of
fibrin, in one instance at 100 oC in 1 molar sodium hydroxide
solution (procedure for elution of residual soils) and once at 65 oC
in 0.1 molar sodium hydroxide solution (similar conditions as in automated
reprocessing). In neither case are the proteins completely decomposed to amino
acids. This would require at least 5 hours of heating with reflux in 5 molar
sodium hydroxide solution (8). But the hydrolysis products of the proteins are
in solution.
3.2
Repercussions of Denaturation or of Chemical Change
Is has
already been mentioned that heat in conjunction with water results in
coagulation of blood proteins. Figure 3 illustrates the quantity of blood
protein remaining on the stainless-steel surfaces after a 10-minute immersion
in demineralised water at 50-80 oC. Noticeable here is that even at
80 oC. approx. one third of the proteins are detached from the
surface. The remaining residues, however, were in all cases hardened due to
coagulation and evidence increased adhesion to the surface. Hence, in automated
reprocessing the temperature employed for pre-cleaning should never exceed 40 oC.
In a dry
state proteins are largely insensitive to thermal denaturation. Accordingly,
dried blood can be easily heated for 10 minutes up to 100 oC,
without the proteins forfeiting their water solubility. Much more resistant
soils are obtained due to the effects of aldehydes contained in disinfectants,
which easily react with the amino groups of the proteins (9). Glutaraldehyde
hardens blood proteins so much that, during hydrolysis at 65 oC in
0.1 molar sodium hydroxide solution, even after two hours no solution can be
observed. The elution procedure (In NaOH/100 oC/lh) used here for
determination of residual proteins leads, depending on the layer thickness, to
quantitative elution only after approx. 50 minutes. Conversely, iodine-and
alcohol-based disinfectants generate only a slight change, which does not
essentially hamper reprocessing with alkaline detergents.
3.3
Detection Methods
Myriad
detection methods are described in the literature (10). In all these
determination methods it is, however, essential to quantitatively bring the
residual soil to solution. Since in the case of a residual soil in
washer/disinfectors, water-soluble, surfactant-soluble and easily hydrolysed
proteins have been removed and the remaining proteins have been hardened due to
the thermal final disinfection and heat denaturation, simple elution, for
example with a surfactant solution or with a sodium hydroxide at room
temperature, does not suffice. Moreover, methods used to determine the free
amino groups of proteins are too imprecise, since for example after chemical
changes induced by aldehydes, no amino groups can be detected any more.
Concomitantly, the number of free amino groups changes depending on the
hydrolytic cleavage of a protein.
UV
absorption has proved to the method of choice, which is based on the light
absorption of aromatic amino acids. This method has the advantage of enabling
hydrolysis to be employed as an elution method, since absorption at 240 nm is
not dependent on the degree of hydrolysis. Accordingly fibrin fibres, denatured
proteins and even proteins that have been cross-linked by glutaraldehyde can be
quantitatively acquisitioned. In addition, haemoglobin as the chief constituent
of the blood can be separately acquisitioned due to the maximum absorption of
its hydrolysis products which is 388 nm.
Residual
proteins can also be well recognised visually. As illustrated in figure 4, even
0.001 mg (lug) of haemoglobin can be easily seen on a stainless-steel surface.
The haemoglobin had been distributed by dosing aliquots of 10 ul of a
corresponding standard solution, and thus takes up a surface of approx. 15 mm2.
Of course, visual inspection does not permit either quantitative or qualitative
classification of a residual soil.
4 Discussion
The
experiments conducted here give an insight into the manifold investigation
methods, attributable to the ultra complexity of the chemistry of the proteins.
For example, denaturation is also largely a function of the pH value, while in
practice various pH values can be encountered due to neutral, alkaline and acidic
detergents. More thorough, quantitative investigations should be conducted in all these domains, using a
standardised test soil, since only in this manner exact, reproducible and
comparable results can be obtained.
It has been concomitantly
revealed that elution with sodium dodecyl sulphate (SDS) does not
suffice for quantitative
determination of residual proteins in prac-tice. Conversely, in laboratory
experiments a high recovery rate can be obtained by mechanical fragmentation of
test bodies. Determination of free amino groups with o-phtaldialdehyde (OPA)
also entails problems, since their number in the case of chemically transformed
and partially hydrolysed proteins is no longer proportional to the baseline
protein quantity. Using hydrolysis and measurement of the UV absorption,
residual proteins can, conversely, be reliably and quantitatively
acquisitioned.
References/Literatur
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Spektrum der
Wissenschaft 1998 (4): 56-61.
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Mutschler E:
Arzneimitteiwirkunyen, Lehrbuch der Pharniakoloigie und Toxikologie, 6.. vollig
neu bearbeitete und erweiterte Auflaye: 353-354.
3.
Joly M: A
Physio-chemical Approach to the Denaturation of Proteins 1965: 162.
4.
Fraenkel-Conratu
H. Oclott HS: J. Amer. Chem. Soc. 1948:70:2673.
5.
Rompp Chemie
Lexikon, 9.. erweiterte und neubearbeitete Auflaye: 1607.
6.
Voet D.
Voet JG: Biochemie. 1
Korr. Nachdruck, 1994:1100-1109.
7.
Duhamel RC.
Meezan E. Brendel K: A simple procedure for the purification of bovine fibrin
from clottet blood by the use of detergents. Prep. Biochem. 1980; 10 (1):43-58.
8.
Neurath H:
The Proteins. Composition. Structure and Funktion. Second Edition. Volume 1: 35.
9.
Fraenkel-Conratu
H. Oclott HS: J. Amer. Chem. Soc. 1948:70: 2673.
10.
Rompp Chemie
Lexikon. 9., erweiterte und neubearbeitete Auflage: 3655.