CHEMICAL INVESTIGATIONS
Very great assistance is afforded by chemical and histological
diagnosis in the determination of cases of lead poisoning, especially
when the case is likely to involve proceedings under the Workmen’s
Compensation Act. In addition, a large amount of information is
afforded to the certifying or appointed surgeon and the medical
practitioner by adoption of certain easily carried out methods of
diagnosis. It will be our purpose in the present chapter to describe
as far as possible methods by which chemical or other investigation of
a case of lead poisoning can be pursued, and the clinical methods of
diagnosis which are applied in ordinary routine.
The majority of the methods described, especially the chemical
investigation of material obtained from alleged fatal lead poisoning
for the purpose of determining the presence or absence of lead,
the histological examination of such tissues, and the examination
quantitatively of excretions for lead, are processes which can only be
carried out in a fully equipped laboratory, and certainly do not belong
to the ordinary routine of medical work. The medical practitioner
cannot be expected in the ordinary course of his routine work to
examine blood-films for basophile staining or to make differential
blood-counts. Especially is this the case in the routine examination of
large numbers of factory hands. Further, many of the processes involved
in either the chemical or histological examination of the tissues
require so much special apparatus, without which such work cannot be
undertaken, that the mere cost of the necessary instruments precludes
the investigation being carried on except in special laboratories. At
the same time, our purpose is to point out how additional methods of
research may be made use of in obscure cases, and how recourse should
be had to a well-equipped laboratory in doubtful cases. Further,
the coroner, when ordering a post-mortem examination, may ask for a
histological and chemical examination.
=Methods of Chemical Diagnosis.=--The presence of lead may require
to be determined qualitatively and quantitatively, and the procedure
may differ slightly as to which process it is necessary to adopt. The
quantitative determination of the amount of lead present in organs or
excretions of the body is of far more importance than the estimation
or determination of the fact of its presence. We have already referred
to the work of Gautier[1], who has found lead present in the tissue of
normal persons with such constancy that French observers, at any rate,
now speak of “normal lead,” to distinguish it from lead which may be
found in pathological conditions. There is, however, little doubt that
the quantity of lead existing in the human body is exceedingly small.
It is possible, with certain refined methods of chemical examination,
that qualitative traces of this substance might be found. On the
other hand, the methods of determination of lead are some of the most
difficult in toxicological analyses, on account of the presence of
other metals, particularly iron, which are exceedingly difficult to get
rid of, and may easily lead to errors.
1. =Qualitative Tests.=--The group reagent for lead is sulphuretted
hydrogen in acid or alkaline solution. Lead is precipitated by this
reagent as a black precipitate. Where no other metals are present, and
where no organic matter is present at the same time, there is very
little difficulty, and the usual method of the determination of lead in
water by means of sulphuretted hydrogen is exceedingly easy.
Potassium iodide gives a yellow precipitate soluble on warming, and
forms large crystals on the tube. Hydrochloric acid and chlorides give
needle-shaped crystals, soluble in heat, and crystallizing out when
cold. But the double chloride of potassium and lead is more soluble
in heat, and still more soluble in cold, than is the pure chloride.
This fact is made use of in the process to be described presently in
separating lead from organic mixtures.
The direct examination is rarely possible or satisfactory, but the
potassium cupric acetate method is one that may be applied to the
qualitative estimation of lead in the tissues.
_Method for detecting Small Quantities of Lead qualitatively._--Dry and
incinerate the material to be tested. Extract with hot dilute nitric
acid, after repeated incinerations, finally extracting with ammonium
acetate. Filter, incinerate, and take up the residue in dilute nitric
acid. Evaporate to dryness and add a few drops of dilute acetic acid,
and transfer drop to microscope slide.
To the drop on the microscope slide add one drop of dilute copper
acetate solution, and two to three drops of saturated solution of
potassium nitrate. Stir up the drops and well mix with a platinum wire,
allow to stand for a few minutes, and then examine with a two-thirds
objective. If lead is present, violet-black cubes of potassium copper
lead nitrate appear (K₂CuPb[NO₃]).
This test is said to give a reaction in the presence of 0·00003 gramme.
_Determination of Lead in the Urine._--The quantity of lead passed
by the kidneys into the urine is always small, even in acute lead
poisoning. Further, the lead is excreted in organic combination, and is
therefore difficult to detect. For absolute quantitative examination it
is essential to evaporate the whole bulk, using at least ¹⁄₂ gallon of
the fluid, and proceeding with the concentrate in the manner indicated
in the estimation of lead.
For the qualitative examination many methods have been suggested, but
all of them are more or less fallacious.
In certain instances in acute lead poisoning, or where a relatively
large quantity of lead is excreted by the kidney, acidulation of
the fluid with strong sulphuric acid direct will at times produce
a precipitate of lead sulphate, which may be filtered off and the
filtrate examined by the usual tests--namely:
A white precipitate with dilute sulphuric acid.
A yellow precipitate with potassium chromate, hardly soluble in nitric
acid, but soluble in alkalies.
A blue flame on heating on a platinum wire, and finally, if sufficient
substance is present, the reduction to metallic form with the blowpipe
flame.
A method has been recommended of suspending a small bag of calcium
sulphide in the sample to be examined, the supposition being that, if
the calcium sulphide in the bag showed blackening, it would be due
necessarily to lead. This is highly questionable, and in the hands of
one of us (K. W. G.) has not given satisfactory results.
A further method, which is quite simple in application, and
occasionally gives confirmatory results, may be applied in the
following manner: The urine to be examined is inoculated with the
_Bacillus coli communis_. For this purpose a small quantity of fæces
may be used. The _B. coli_ in its growth makes use of the organic
substances in the urine, and at the same time sets free sulphuretted
hydrogen. The urine left is filtered, the filtrate dissolved in 10 per
cent. nitric acid (minimal quantity), and the filtrate examined by the
usual tests. This method has occasionally given quite good results in
the hands of one of us (K. W. G.), and is, moreover, an exceedingly
easy one to carry out.
Passing sulphuretted hydrogen direct through the fluid is of no value,
as it is necessary first of all to split up the organic compound before
it will react to sulphuretted hydrogen.
_Electro-Chemical Methods._--Of all the methods at present in
use for the estimation of the presence of lead in the urine, the
electro-chemical gives by far the most satisfactory results. Several
methods are described:
The first method consists in using magnesium, which is left for some
hours in the urine, which has been previously strongly acidulated. In
this manner Marsden and Abram[2] say that they have been able to detect
1 part in 50,000 in the urine without difficulty. The method adopted is
as follows:
“A strip of pure magnesium is placed in the fluid to be examined.
Ammonium oxalate in the proportion of about 1 gramme to 150 c.c. is
added. If lead is present, it is deposited on the magnesium. A deposit
is seen within half an hour, but we have usually left it twenty-four
hours. The slip is then washed with distilled water and dried.
Confirmatory tests: (1) Warm the slip with a crystal of iodine (yellow
iodide proves lead, cadmium may be ignored); (2) dissolve deposit in
HNO₃, and apply usual tests. The magnesium can be used again after
careful washing with acid and distilled water. The surface of the
magnesium, when used, must be bright and free from oxide. The delicacy
of the method has been tested with aqueous solutions containing known
quantities of lead, also with normal urine to which known quantities of
lead have been added. In all cases a control experiment was performed
to insure the freedom of the materials from lead. Lead has been
detected when present in the proportion of 1 part to 50,000, whether in
simple aqueous solution or in urine.”
Shufflebotham and Mellor[3] describe the following method as one by
which lead may be detected in organic tissue, and in each case this
necessitated a large amount of evaporation. The method has value,
but the difficulty of dealing with large quantities of fuming nitric
acid, and the addition of this acid from time to time during the
operations, render it difficult unless a good fume chamber is at hand.
Shufflebotham and Mellor state that they obtained no reaction with the
potassium chloride-hydrochloric acid method suggested by Dixon Mann.
“_On the Detection of Lead in Urine and Post-Mortem Specimens._--A
piece of kidney of 20 c.c. capacity was cut up into about a dozen
pieces. These were placed in an evaporating basin, and about 50 c.c.
of fuming nitric acid were poured into the dish. Dense brown fumes of
nitrogen oxides were evolved. When the action had subsided (in from
two to three minutes), the dish was placed upon a sheet of asbestos,
and allowed to simmer over the Bunsen flame for about an hour. If the
frothing appears in danger of running over the sides of the dish,
stirring with a glass rod or removal of the flame for a short time
may be necessary. Twenty-five c.c. of the fuming acid were added at
intervals of a quarter of an hour, and this process was repeated
three times. The destruction of the organic matter was so complete
that the whole of the piece of kidney passed into complete solution.
The solution was then evaporated down to a few c.c., neutralized
with caustic soda, filtered, and treated with hydrogen sulphide. A
dark-brown precipitate of lead sulphide was obtained. With potassium
chromate a yellow precipitate of lead chromate was obtained with
the same specimen of kidney which gave a negative result with the
KClO₃-HCl method of destroying the organic matter. Our reagents,
dishes, etc., were then examined with a blank test, but we found no
lead.
“_Urine._--We then sought the presence of lead in the urine of
Cases 2, 3, and 4. Half a gallon of urine was evaporated down to
dryness in each of two basins. In one basin the residue was heated
until it was charred. Both residues were then treated separately
with fuming nitric acid, as just described. The uncharred residue
passed into solution, and on cooling deposited a white sediment. The
mother-liquor was neutralized and tested in the usual way. A brown
precipitate of lead sulphide was obtained in Case 2, while in Case
3 a well-marked black precipitate was obtained. The urine of Case 4
gave a negative result. The charred residue did not pass completely
into solution, and the tests for lead were not so well defined as
when the residue was uncharred. This shows that care must be taken to
prevent charring of the residue during evaporation.”
A method has recently been described by Hebert[4]--a modification of
Trillet’s. This method is based upon the fact that peroxide of lead,
when mixed with tetramethyl of diphenyl-methylen, gives in acetic acid
solution a fine blue coloration. Unfortunately, a number of other
peroxides give the same blue coloration, amongst them manganese,
potash, copper, magnesium. In addition, the sodium peroxide used to
convert the lead present into the peroxide also gives a bright blue
coloration with the reagent, even if present in minute quantities.
The test is made in the following manner: The substance is incinerated,
sulphuric acid added in the usual way, and the substance evaporated to
dryness. It is treated with a cold solution of sodium hypochlorite. The
hypochlorite is then removed partly by washing and partly by heating,
and the reagent is then added directly to the substance in the capsule,
and if a peroxide is present the blue colour results.
Unfortunately, this dissociation-point of the hypochlorite and the
temperature at which peroxide of lead is changed back again to the
oxide are very close together, being only about 25° C. In addition,
it is very difficult to remove the last traces of the substances
which give a blue coloration in addition to lead. One of us (K. W.
G.) has made extensive trials with this method, as, if it had been a
reliable process, it would have been one which would have considerably
facilitated the estimation of lead in small quantities. The method has
been adopted by certain French observers, who by drawing 2 c.c. of
blood from the median basilic vein, and estimating the lead present in
this small amount by means of the blue coloration, have sought to show
that at least 25 milligrammes of lead were circulating in the blood of
the body. In addition to other grave considerations, the fact that the
reagent itself is colourable by certain other peroxides which exist in
the blood-ash renders these figures entirely untrustworthy.
2. =Quantitative Methods of Estimation.=--Two methods may be used in
the detection of lead in organic substances, either in organic fluids
or in solids, and are generally termed the “wet” and “dry” methods,
from the original treatment of the substance.
In the dry method the substance is incinerated with or without the
addition of sulphuric and nitric acid; in the wet the material is
treated with hydrochloric acid and potassium chlorate. Subsequent
treatment in both cases is on the same lines.
_Method of Fresenius and Von Babo[5]--Moist Method._--The substance
which is suspected to contain the poison, if solid, is reduced to a
pulp, and is mixed with sufficient water until of the consistency of
thin gruel. The urine should be evaporated to one-fourth or one-sixth
of its volume. Fæces should be well stirred up with distilled water.
The substance is then placed in a large flask together with crystals
of potassium chlorate; each 100 grammes of the substance require 3 to
4 grammes of potassium chlorate. Pure HCl of the same weight as the
original substance is then added, the flask is placed on a water-bath
and gently heated. Care must be taken that the heating is not too
brisk, as otherwise the evolution of the chlorine peroxide takes place
too rapidly. If necessary, additional crystals of potassium chlorate
are added from time to time until the fluid becomes limpid and of a
slight yellow colour, or, if there is much organic matter, until it
assumes the appearance and colour of thin oatmeal gruel. On account of
more gradual evolution of chlorine, the chlorate that is present before
the flask is heated acts much more energetically, weight for weight,
than fragments added after the liquid is heated, as a great deal of
the gas then escapes without rendering any service. If the substance
contains sugar, starch, or alcohol, extra care must be taken to avoid
frothing over. When the fluid contents are clear or reduced to a thin
consistency, the liquid is transferred to an evaporating basin, and
allowed to remain on a water-bath until the smell of chlorine has
disappeared; it is then filtered while hot. The whole of the organic
matter is not destroyed by this process, fatty substances especially
being resistant; but if the organic matter is reduced to small
fragments, any mineral poison present will be liberated.
The objections raised against this process are that some important
poisons--such, for instance, as arsenic and antimony, specially the
former--are liable to escape partially in the form of vapour, and that
others, such as lead and silver, may remain as insoluble precipitates
on the filter. As regards the first objection, it is to be observed
that, when the hydrochloric acid is diluted with water (as in a moist
method of destroying organic matter), any arsenic which may be present
in the hot solution is not given off with its acid aqueous vapour,
arsenious chloride dissolved in hydrochloric acid being volatile
only when the solvent is concentrated. Any possibility of loss may
be avoided, however, by furnishing the flask, in which the organic
matter is being destroyed, with a condenser and receiver. The second
objection, as far as lead is concerned, is met by taking care to filter
the solution whilst hot; if only a limited amount of lead is present,
it then remains in solution as chloride so long as the liquid is hot,
and will consequently pass through the filter. A considerable quantity
is kept in solution in the cold, as it forms a combination with
potassium chloride, which is more soluble than lead chloride alone. If
a large amount is present, it will not all be found in the filtrate;
the substance left on the filter, therefore, must always be tested for
lead. In toxicological work, however, the amount of lead present is not
as a rule more than will remain dissolved in the cold. Silver chloride,
being insoluble either in hot or cold water, will not pass through the
filter; consequently the salts of silver require dealing with in a
special manner.
_Dry Method._--This is effected by heating the finely divided substance
to redness, so that it is either carbonized or completely incinerated.
When cold, the residue is drenched with nitric acid, and sufficient
heat is afterwards applied to drive off the free acid. The nitrate
of the metal is then dissolved in water, filtered, and dealt with
according to the kind of metal present.
The dry method is unsuitable in the case of the more volatile metals,
as arsenic, antimony, and, in a lesser degree, lead, tin, and zinc.
Further, it is extremely difficult and troublesome to carry out with
large masses of organic matter. It is convenient with small amounts,
and in the absence of the more volatile metals yields good results.
The following two methods are given by Glaister[6] on the one hand, and
Dixon Mann[7] on the other. Both methods are good. It will be seen that
Glaister recommends the estimation of the lead as sulphide.
When minute quantities of lead are present in combination with large
amounts of organic matter, the dry process is tedious, difficult to
carry out, and uncertain in its results. The plan adopted in the
elimination of lead by Dixon Mann is as follows:
The urine is evaporated down to the consistency of gruel, and the
fæces mixed in distilled water to a like consistency. They are then
treated by the wet method, as given above. The filtrate after cooling
is placed in a glass cell, the bottom of which consists of a sheet
of vegetable parchment; the cell is immersed to such a depth in a
deeper cell, containing distilled water acidulated with a few drops of
sulphuric acid, that the liquids in the inner and outer cells stand at
the same level. A piece of platinum-foil enclosing a surface of about
50 square centimetres, constituting the kathode, was submerged in the
liquid contained in the inner cell, a similar piece of platinum-foil,
constituting the anode, being immersed in the outer cell. The pieces
of foil are so placed as to be opposite each other, separated by the
parchment diaphragm. A current, 3 or 4 volts, is then passed through
for from six to eight hours, after which the foil is removed from the
inner cell and gently washed and dried. The metallic lead is dissolved
off the foil with dilute nitric acid aided by heat, and after driving
off most of the free acid the solution is decomposed with dilute
sulphuric acid with an equal volume of alcohol added. It is then set
aside for twenty-four hours. The precipitate of lead sulphate is washed
with water containing 12 per cent. of alcohol, until all the free acid
is removed; it is then separated by decantation, ignited, and weighed.
The amount of lead is calculated from the weight of the sulphate; 100
parts of sulphate are equal to 68·319 parts of metallic lead.
Whether the moist or the dry process is used, the residue after the
primary filtration should be tested for lead, which may be present as
sulphate and remain undissolved. If the original substance contains
lead as sulphate, the salt should be dissolved with heat in an aqueous
solution of ammonium tartrate to which a little free ammonia has been
added, then precipitated with sulphuretted hydrogen (100 parts of lead
sulphide equal 86·61 parts of metallic lead). It is better, however,
to convert the sulphide into sulphate by treating it with nitric and
subsequently sulphuric acid, after which it is ignited, weighed, and
the amount of the metal calculated by the lead sulphate factor.
Having obtained the substance by decomposing the organic matter, two
methods of estimation may be made use of:
(1) Colorimetric; (2) gravimetric.
Where the estimation is to be made gravimetrically, the substance is
always obtained as a sulphate, and the lead estimated by weighing as
a sulphate. This process is an exceedingly tedious one when a large
number of small samples have to be estimated, as in the determination
of the amount of lead dust present in the air. On the other hand, it
is probable that the use of the balance in the estimation of lead as
sulphate is more accurate where larger quantities up to 10 milligrammes
are present; but where only 2 or 3 milligrammes are present in the
amount of substance examined, the experimental error in washing is
too large to warrant the expenditure of time required in this form of
estimation, and the colorimetric method is used.
=Detection of Lead in Organic Mixtures.=--Acidulate the organic
substances, reduced to fine proportions, with nitric acid, heat for
some time, then permit to cool; filter, wash residue, and mix washings
with filtrate; concentrate filtrate; pass H₂S; place mixture in warm
place to allow precipitate to settle; after which decant supernatant
fluid, collect precipitate on tared filter, thoroughly wash, dry on
water-bath, and weigh. One part of sulphide is equivalent to 0·9331
part of lead oxide and 1·5837 parts of acetate of lead.
The electrolytic method is better adapted for the detection of
minute quantities of lead, as, for example, in the urine or fæces or
in vomited matter. The urine may be evaporated to a viscous state;
the others, finely broken up, are treated in the same way, after
HCl is added, as recommended above, the mixture heated, and pinches
of powdered chlorate of potash added, as necessary, to break down
organic matter. The heating is continued until the odour of chlorine
disappears, after which it is filtered and the filtrate allowed to
cool. The filtrate is then placed in the outer cell of a two-celled
arrangement, not unlike a dialyser, the bottom of which is formed
of vegetable parchment, the outer cell containing distilled water
acidulated with H₂SO₄. Into the inner cell is placed a piece of
platinum-foil measuring about 50 square centimetres of exposed
surface, which is connected with the kathode or negative pole of four
Grove cells, and into the outer cell is placed a like-sized piece
of platinum-foil connected with the anode or positive pole. These
pieces of foil are so placed in relation to one another that they
are only separated by the parchment. The galvanic circuit being now
closed for some hours, any lead in the filtrate will be deposited
on the platinum-foil connected with the kathode in the inner cell.
The foil is then removed, carefully washed, and the metallic lead
dissolved by dilute nitric acid aided by heat, after which the solution
is concentrated until most of the free acid is driven off; dilute
sulphuric acid is added to throw down the sulphate, alcohol being also
added to expedite precipitation. The precipitate is allowed to settle
for twenty-four to thirty-six hours, filtered on a tared filter, washed
with water containing 12 per cent. of alcohol, dried, ignited, and
weighed. One part of sulphate is equivalent to 0·68319 part of metallic
lead and to 1·25 parts of acetate of lead.
The estimation of the lead, especially if the amount be small, may
be more accurately made by the volumetric colorimetric method. The
metallic lead deposited on the platinum-foil is dissolved in nitric
acid, and distilled water added, and an aliquot portion placed in a
Nessler glass. A few drops of freshly-prepared H₂S water, or H₂S gas
itself, may be added to or bubbled through the contents of the glass so
as to form lead sulphide. The colour formed is now matched in a similar
glass, using a standard solution of lead nitrate, and forming the lead
sulphide as before.
Some have advocated the magnesium wire deposition test, originally
devised by von Jaksch, and modified by Hill Abram, for the detection
of lead in the urine of persons who are suspected to be suffering from
chronic lead poisoning (see _ante_).
The colorimetric method of estimating lead has been made use of with
very great success by Duckering in the estimation of lead in the air
of potteries. The method is that devised by Mr. Vernon Harcourt[8],
with a few modifications. The whole of the method is given, as
observation on the quantity of lead present in the air of lead
factories helps greatly in suggesting rational methods of precaution.
“After drying and weighing the filters, the following is the method.
Solutions required:
“_Nitric Acid._--One part of pure concentrated nitric acid to three
parts of water.
“_Caustic Soda._--One hundred grammes of pure caustic soda dissolved
in 250 c.c. of water.
“_Sugar._--A saturated solution of sugar in water.
“_Sulphuretted Hydrogen._--A saturated solution of sulphuretted
hydrogen in water.
“_Coloured Solution._--Cotton-wool dissolved in concentrated nitric
acid and evaporated to dryness, and the residue dissolved in a little
water and filtered; the solution is deep yellow in colour.
“_Standard Lead._--A solution of lead acetate or lead nitrate made up
to contain exactly 0·0001 gramme of lead per c.c. of solution.
“The bulk of the dust in the filter was removed to a beaker (No.
1), by gently tapping the inverted funnel. The cotton-wool was then
removed from the funnel, and the upper one-third, containing the
remainder of the dust, was cut off and added to the dust in beaker
No. 1. The remainder of the cotton-wool was placed in a second beaker
(No. 2); 2¹⁄₂ c.c. of hot nitric acid was dropped on the dust in
beaker No. 1 from a pipette, a little water added, and the whole
heated. The solution was filtered into a 50 c.c. Nessler glass, and
the liquid remaining in the cotton-wool also removed by squeezing the
wool with a glass rod against the side of the beaker. The cotton-wool
in beaker No. 2 was similarly extracted with 2 c.c. of nitric acid,
and the solution added to that remaining in beaker No. 1. The
liquid was heated, the cotton-wool macerated in it and filtered as
before. The wool was then washed with hot water about three or four
times, and the liquid filtered into the Nessler glass. A number of
standards were then made up by running into Nessler glasses, from a
burette, varying amounts of standard lead solution covering a fair
range. Usually five standards were made up, containing 0·5, 0·8,
1·0, 1·2, and 1·4 c.c. lead solution, depending on the volume of
the air aspirated and the quantity of lead expected in the known
weight of dust found. To each standard 4¹⁄₂ c.c. nitric acid was
added, and 5 c.c. of the caustic soda solution and 4 c.c. of the
sugar solution were run into all the six solutions--_i.e._, one test
and five standards--from pipettes. It was invariably found that the
test was coloured faintly yellow, and if this is not allowed for in
the standards high results are obtained. Hence a drop or two of the
coloured solution (see solutions required) was added to the standards
placed on white paper till they matched the test. Lastly, to the
contents of each of the six glasses was added 4 c.c. of sulphuretted
hydrogen solution, and the liquid in each made up to the 50 c.c.
mark, and the whole well stirred. Usually it was found that the
colour of the test came somewhat deeper than that of one standard,
and a drop or two of lead solution was added to the standard till its
colour matched that of the test. The elaborate method of making up a
number of standards was adopted because it was found that any other
way gave high results. In the way described many trial experiments
were made, and they were invariably correct within half a drop of the
standard solution.”
The estimation of the quantity of lead present in organic fluids,
with the disturbing influence of the presence of other metals, is a
factor which always complicates the use of the sulphuretted hydrogen
colorimetric estimation. It is almost impossible, when dealing with
fæces, with blood, or, on the other hand, with artificial digests
containing bread and milk, to eliminate the disturbing influence of
iron. Further, if steps are taken to remove the iron and other metals,
so much loss takes place in the manipulations necessary that the
results arrived at are not satisfactory. Whenever dealing with organic
matter, such as urine and fæces, it is best to make a blank test with a
similar quantity of the substance under examination obtained from other
sources, and to subtract the error found due to iron, when a rather
closer approximation may be arrived at.
So far as can be estimated, the minimal quantity of lead required to
produce poisoning is 0·005 gramme per kilogramme of body weight; but,
on the other hand, persons who have swallowed much larger doses than
this have exhibited no symptoms of poisoning. There is every reason to
suppose that lead absorbed through the lung produces a maximum toxic
effect, and, from the estimation of the quantity of lead found in
the body after death, it is highly probable that exceedingly minute
quantities of lead have, when absorbed over long periods, produced
changes not only by their actual presence in the tissues, but also have
set up degenerative changes which progress even after the elimination
of the metal from its local position.
=Histological Examination.=--In addition to the chemical examination
of tissues from a person who has died of suspected lead poisoning,
it is of the highest importance to make histological examinations,
as the naked-eye appearance of post-mortem examination is frequently
insufficient to give any clue to the cause of the poisoning. Moreover,
in a large number of instances the necropsy may exhibit a number of
signs of disease, such, for instance, as granular kidney, cirrhosis of
the liver, and so forth, which are associated with diseases other than
lead poisoning, and, in the absence of any present or past evidence of
definite hæmorrhages found associated with the other lesions already
mentioned, an ordinary autopsy must be inconclusive. It is true that
such pathological conditions are consistent with poisoning by lead; and
if the individual has been a lead-worker, it is easy, but frequently
erroneous, to conclude that the symptoms owe their origin to the
worker’s occupation. We are entirely in sympathy with the remarks of
King Alcock[9], who says:
“I plead none the less for an impartial investigation of the symptoms
presented by a lead-worker, before assigning full or even partial
responsibility of the disease to the occupation. If any and every
departure from the normal in a lead-worker is at once assigned--the
occupation being known--to plumbism, early diagnosis naturally presents
very few difficulties to the exponents of such methods. And however
severely we may condemn in the abstract such a careless, unscientific
attitude, the tendency has, in practice, to be reckoned with and
combated. The balance of probabilities would possibly suggest that
the occupation is, after all, responsible, in one sense or another,
for the more usual illnesses classically associated with the poison;
nevertheless, the attending practitioner is in duty bound to take into
consideration, and to estimate the relation of, all the concurrent
causes of such symptoms.”
On the extremely unsatisfactory position the certifying surgeon may
find himself in at a coroner’s inquest King Alcock says:
“The problem, from a strictly scientific point of view, is
complicated--one might almost say that the truth is stifled--by the
fact that the ætiological relations of the symptoms of any suspended
worker are swamped by the insistent legal relations under which he
claims and receives compensation.
“When once a formal certificate of suspension has been issued, which
has embodied a recognition of lead as a cause of certain existing
symptoms, then it becomes almost hopeless ever to reopen the question
of causation of these or other supervening troubles, be their origin
independent of lead or not. The doctor, in a legal cross-examination,
is, in scientific honesty, bound to admit at last the bare possibility
of any fantastic chain of remote sequelæ; and his protests against the
probabilities of such sequelæ are of no avail, as opposed to his own
admission. The _post, ergo propter_, appeal carries the day easily.”
For the purpose of histological examination it is essential that
portions of the brain, spinal cord, liver, kidney, and intestine,
should be examined microscopically. The nervous tissue should be placed
in formalin and Müller’s fluid, and a portion in alcohol for the
examination of the fibres. The liver, kidney, etc., should be placed in
5 per cent. formaldehyde. The tissues are then treated by the ordinary
histological methods, and sections prepared. With nervous tissue it
is essential that those prepared for the examination of the cells
should be made by the celloidin method; the others may be treated by
imbedding in paraffin. The points to be sought for in the tissues are
sufficiently indicated in the chapter on Pathology and Symptomatology,
but may be briefly recapitulated:
In the _brain_, as well as in all the tissues, careful search should
be made for minute microscopical hæmorrhages, and for evidences of
old hæmorrhages in the form of small masses of fibrous tissue, etc.
Parenchymatous degeneration, chromatolysis of nuclei, etc., nerve
degeneration.
The _arteries_ and _veins_ should also receive close scrutiny, as the
presence or absence of arteritis should be noted.
In the _kidney_ particularly, search should be made for both
interstitial and parenchymatous nephritis.
The _liver_ frequently shows signs of microscopic hæmorrhage, and it is
as well, in taking a portion of tissue for examination, to choose those
portions which appear to be specially congested.
In the _brain_ and _spinal cord_ and _nervous tissue_, search is to
be made for the same hæmorrhages as already noted. In addition, the
condition of the nerve fibres should be noted, the presence or absence
of periaxial neuritis, as well as degeneration of the axis cells, and
the various ganglion cells both in the brain and spinal cord should be
closely examined for chromatolysis and nuclear atrophy.
No evidence is afforded by micro-chemical tests of any of the sections
thus obtained, except those of the lung. It may be possible in the case
of the lung to determine the presence of lead granules in the alveolar
cells, and attention should be paid to this. It is possible also that
some evidence may be afforded by examination microscopically of the red
bone-marrow.
The intestinal walls should be examined for evidence of lead particles.
If any dark staining, deep or superficial, be found in the intestine,
it should be removed for chemical analysis. Necrotic areas of the
intestinal wall should be sought for.
=Hæmatology.=--For all practical purposes, the best stain for detection
of basophile granules in the erythrocytes is Wright’s modification of
Romanowski’s stain. This stain may be obtained in appropriate tablets,
and may be prepared immediately before use, although a stain which has
been standing for ten days or a fortnight gives much better results
than a quite new stain. The stain consists of a solution of polychrome
methylene blue, together with eosin in methyl alcohol, and the method
of procedure is as follows:
Blood is obtained by a small puncture, and slides smeared and allowed
to dry. Immediately on drying the slip is flooded with the stain,
and allowed to remain for two minutes. This causes fixation. At the
end of the two minutes the stain is diluted with an equal volume of
distilled water, and allowed to remain on for a further three minutes.
At the end of this time the stain is poured off, and the slip washed in
distilled water for another three minutes, or until the characteristic
purple-violet appearance is produced. It is better to examine such
films with an oil-immersion lens, the oil being placed directly upon
the films, and not covered with a cover-slip, as the action of Canada
balsam tends to decolorize the blue. If such specimens are required
to be kept, the oil may be washed off with xylol. It is possible to
observe basophile staining with a good sixth, but an oil-immersion
lens gives much the best result. The typical staining produced by this
means gives darkish bodies scattered about the red corpuscles, staining
sometimes deeply as the nuclei of the white corpuscles. In other cases
the appearance is like that of fine dust scattered throughout the cell.
In addition to these two forms, the whole red cell may take on a slight
generalized lilac tint, the normal cells remaining free from granules,
and stained red by the eosin. Search of 100 fields of the microscope
should be made, and if no basophile granules are found in such fields
it is unlikely that they will be found.
Basophile staining is not more pathognomonic of lead poisoning than of
any other form of anæmia, but may be regarded as a highly important
confirmatory diagnostic sign.
A differential count of the leucocytes present may be also made on
the same film in which basophile staining is observed; 300 should be
counted at least. In a typical case of lead poisoning it is found that
diminution in the polymorphonuclear leucocytes has taken place with
a corresponding increase of the lymphocytes, and possibly also the
large mononuclears, and probably a slight increase in the number of
eosinophiles.
This hæmatological method of diagnosis is of the utmost importance
in lead poisoning. A differential count such as is given on p. 137,
showing a large diminution in the polymorphonuclears, an increase in
the lymphocytes, evidence of changes in the red cells, consisting
of basophile staining, alteration in the shape of individual cells,
poikilocytosis, with vacuolation, is strong presumptive evidence of
lead absorption.
To complete the hæmatological examination, the hæmoglobin should
be estimated. This is best performed with Haldane’s instrument--an
exceedingly simple one to use. The estimation of the number of red
cells and white cells present is useful, but does not by any means give
such valuable information as does the differential count and search for
basophile granules.
=Blood-Pressure.=--Several methods are available for the estimation
of the blood-pressure. The pressure may be roughly estimated as too
high or too low by means of the finger. The presence of thickening of
the arteries may be also estimated in this way, but for determining
the absolute blood-pressure it is necessary to use one or other of
the instruments on the market. The estimation of blood-pressure
is an important point in relation to the suspected presence of
arterio-sclerosis, and should be performed wherever possible.
Sphygmographic tracings may also be taken. Such a tracing in a case of
typical poisoning gives a peculiar form of curve, which, however, may
be present in alcoholism and heavy work, and arterio-sclerosis of many
types.
=Urine Examination.=--In suspected cases of lead poisoning the
examination of the urine may reveal the presence of lead. In addition,
albumin is frequently present, especially in the early stages of
kidney inflammation. The ordinary tests for albumin should be carried
out, and it is also advisable to examine the urine spectroscopically,
as at times hæmoglobin, methæmoglobin, hæmatoporphyrin, may be
present in small quantities, each of which can be detected by means
of spectroscopic examination. Blood is not common in the urine of
lead-poisoned persons, although microscopically hæmorrhages undoubtedly
take place in the kidney. These hæmorrhages are interstitial, and as a
rule do not cause any blood-pigment to be passed in a quantity that can
be determined. It is as well, however, to centrifugalize the urine, and
examine the deposit for red blood-cells.
The presence of hæmatoporphyrin, as suggested by Steinberg[10], is
probably due to hæmorrhages in the intestine, and its presence in the
urine should be regarded with suspicion in a lead-worker.
Where a lead-worker is suffering from continued absorption of lead,
even without the manifestation of other symptoms, a change has been
noted in the acidity of the blood--namely, a loss of normal alkalinity.
The estimation of the alkalinity or acidity of the blood direct is an
exceedingly difficult process, but much information may be obtained by
careful estimation of the acidity of the urine, and of the acidity of
the urine in relation particularly to the phosphates.
Joulie[11] has pointed out the extreme value which may be obtained from
a knowledge of the urinary constituents by the means of estimation of
the acidity with suchrate of chalk. The reagent is made by slaking
lime in such a way that the resulting compound is practically dry.
A quantity of this--about 25 grammes--is then thoroughly shaken up
with 10 per cent. solution of cane-sugar, allowed to stand, and the
solution titrated against decinormal acid until it is of one-twentieth
normal. The urine is then estimated directly, the suchrate is run
into the 25 c.c. of urine until a faint white flocculent precipitate
appears. The number of c.c. of the solution of suchrate is then noted,
and multiplied by the factor of the solution. This gives the acidity
related to the phosphate and other organic acid contents, and is
similar to the method used to determine the acidity of wines.
The second estimation consists of estimating the phosphates present by
means of a standard solution of uranium nitrate, using either potassium
ferrocyanide or cochineal as an indicator. The specific gravity of the
urine is also determined. The result is then expressed in terms of
this specific gravity, or, rather, in the terms of the density of the
urine in relation to distilled water, and the whole answer returned per
litre. By this method it is not necessary to obtain a twenty-four hours
sample of the urine, the urine passed first thing in the morning being
taken for examination.
By using this density figure the quantity of acid and phosphate is
expressed in relation to the density, the equation being--
The observed acidity
--------------------- = Acidity per litre.
The density per litre
The phosphate content is expressed in the same manner, while the ratio
of phosphate to acidity gives the ratio of excretion of phosphate to
acidity.
There is in lead-workers a considerable diminution in the amount of
phosphate excreted, and, as has been pointed out by Garrod and others,
lead apparently produces alteration in the solubility of the uric acid
content of the blood, and may therefore allow of its decomposition.
Probably lead as a urate is stored up in the tissues. For further
particulars of this method of the estimation of the urine, the reader
is referred to “Urologie Pratique et Thérapeutique Nouvelle,” by H.
Joulie.
An examination of the fæces of persons suspected of lead poisoning
may often give definite results both of the presence of lead and
hæmatoporphyrin. If small hæmorrhages have occurred high up in the
intestine, the presence of hæmatoporphyrin in the fæces may result.
The substance may be easily determined by means of the characteristic
absorption bands. A quantity of fæces is taken and extracted with acid
alcohol, and the filtrate examined spectroscopically. Urobilin bands
are commonly present, and, particularly, where much constipation exists
these bands are very well marked. There is no difficulty whatever,
however, in distinguishing them from the characteristic bands of acid
hæmatoporphyrin.
=Examination of the Fæces for Lead.=--The moist method or chemical
examination given above is the best one to apply for the determination
of lead in the fæces. As has already been pointed out, lead is commonly
excreted in the fæces, and, if only about 2 milligrammes per diem are
being excreted by the fæces in a lead-worker, the quantity cannot be
regarded as indicative of poisoning. One of us (K. W. G.) has at times
found as much as 8 to 10 milligrammes of lead excreted in the fæces of
persons engaged in a lead factory, and exhibiting no signs or symptoms
whatever of lead poisoning. If, however, the quantity of lead in
the fæces rises to anything above 6 milligrammes per diem, there is
definite evidence of an increased absorption of lead, and if at the
same time clinical symptoms be present, suggesting lead poisoning, such
a chemical determination is of the first importance.
In estimating the presence of lead in fæces, it may be necessary
to deal with the separation of iron, which may be precipitated as
phosphate and filtered off, the quantitative estimation being proceeded
with in the filtrate.
Lead is much more commonly present in the fæces of lead-workers than in
the urine, and it is better to examine the fæces rather than the urine
in suspected cases.
REFERENCES.
[1] GAUTIER: Meillère’s Le Saturnisme, p. 74.
[2] MARSDEN AND ABRAM: The Lancet, vol. i., p. 164, 1897.
[3] SHUFFLEBOTHAM AND MELLOR: _Ibid._, vol. ii., p. 746, 1903.
[4] HEBERT: Comptes Rendus, tome cxxxvi., p. 1205, 1903.
[5] FRESENIUS AND VON BABO: Liebig’s Annalen, vol. xlix., p. 287,
1884.
[6] GLAISTER: Medical Jurisprudence and Toxicology. 1910.
[7] DIXON MANN: Forensic Medicine and Toxicology, p. 496.
[8] VERNON HARCOURT, A.: A Method for the Approximate Estimation of
Small Quantities of Lead--Transactions of the Chemical Society, vol.
cxvii., 1910.
[9] KING ALCOCK, S.: Brit. Med. Journ., vol. i., p. 1371, June 24,
1905.
[10] STEINBERG: International Congress Industrial Hygiene. Brussels,
1910.
[11] JOULIE, H.: Urologie Pratique et Thérapeutique Nouvelle.