NBSIR 76-1024

Statistical Analysis of Blood

Lead Levels of Children

Surveyed in Pittsburgh,

Pennsylvania: Analytical

Methodology and Summary

Results

Walter D. Urban

Institute of Applied Technology

National Bureau of Standards

Washington, D. C. 20234

April, 1976

Final

Prepared for Office of Policy Development and Research

The Department of Housing and Urban Development

Washington, D. C. 20410

NBSIR 76-1024

STATISTICAL ANALYSIS OF BLOOD

LEAD LEVELS OF CHILDREN

SURVEYED IN PITTSBURGH,

PENNSYLVANIA: ANALYTICAL

METHODOLOGY AND SUMMARY

RESULTS

Walter D. Urban

Institute of Applied Technology

National Bureau of Standards

Washington, D. C. 20234

April, 1976

Final

Prepared for Office of Policy Development and Research

The Department of Housing and Urban Development

Washington, D. C. 20410

U.S. DEPARTMENT OF COMMERCE, Elliot L. Richardson, SeerBtary

Dr. Betsy Ancker-Johnson, Assistant Sacretary for Seianca and Tachnoiogy

NATIONAL BUREAU OF STANDARDS, Ernest Ambler, Acting Diractor

Table o£ Contents

Page

List of Tables iii

List of Figures iv

Abstract v

1. Introduction 1

2. The Survey Approach 4

2.1 Selection of Children 4

3. Blood Sampling 15

3.1 Blood Sample Size . 15

3.2 Blood Sample Contamination 16

3.3 Blood Sample Alteration 17

4. ACHD Blood Lead Analysis Procedure 18

4.1 General Procedures 18

4.2 Validation of Analytical Results 19

4.2.1 Duplicate Children's Blood Samples 20

4.2.2 Porcine Blood Standard Samples 20

5. Results, Conclusions and Summary 26

6 . References 39

7. Acknowledgement 40

8 . Appendices 41

Appendix lA - Micro Blood Sample Collection Procedure 41 as Used by Allegheny County Health Department Lead Poisoning Control Program..

Appendix; IBl - Pittsburgh Laboratory Determination of the 43

Lead Content of Blood Sampling Equipment

and Supplies

Appendix 1B2 - I'^IBS Determination of the Lead Content of 45

Blood Sampling Equipment and Supplies

Appendix IC - Dust and Soil Survey 49
Table of Contents (con't)

Page

Appendix ID -

Appendix II -

Appendix III

Appendix IV

Weight Loss of Frozen Porcine

Blood Stored in Spinco Tubes

Analytical Procedure Employed

by the Clinical Laboratory

Serving ACHD

- The NBS Method for the Deternination

of Sub-Microgram Samples of Lead

Employing Mass Spectrometry.

Comments on Lower Values of Blood

Lead Levels After Retest

53

54

59

64

ii
List of Tables

Page

1. Room by Room Lead Content Data for V/alls 12

2. Room by Room Lead Content Data for Windows 13

3. Room by Room Lead Content Data for Doors 14

4. Cumulative Distribution of the Tested Children's 27

Blood Lead Levels

5. Children's Elevated Blood Lead Levels at Screening 28

and Results After Retest.

6. Distribution of Blood Lead Levels by Age and Sex of 32

Child and by the Age of the Dwelling.

7. Distribution of Children's Blood Lead Levels by Age 34

of Dwelling

lB-1.1 Pittsburgh Laboratory Determination of the Lead 44

Content of Supplies Used in Blood Collection

lB-2.1 NBS Determination of the Lead Content of Supplies 47

Used in Blood Collection.

iii
List of Figures

Page

1. Room by Room Comparison of Lead Content of Walls 7

2. Room by Room Comparison of Lead Content of Windows 8

3. Room by Room Comparison of Lead Content of Doors 9

4. Frequency Distribution of the Lead Content of Kitchen

Walls in the Dwellings Occupied by the Tested Children 10

5. Comparative Analysis of Duplicate Samples of Children's

Blood by NBS and the Pittsburgh Laboratory 21

6. Analyses of Porcine Reference Blood Samples by the

Pittsburgh Laboratory 23

7. Analyses of Porcine Reference Blood Samples by NBS 24

8. Frequency Distribution of the Tested Children's Blood

Lead Levels 29

9. Correlation of Children's Blood Lead Levels with the

Fractions of Surfaces Within a Dwelling that are Contaminated with Lead at Least at 2mg/sq. cm. 23

IC-1.1 Lead Content of Pittsburgh House Dust Samples 50

IC-2.1 Lead Content of Pittsburgh Dirt Samples 51

III-l Isotope Composition of Natural and Spike Lead 60

Samples

iv
Analysis o£ Blood Lead Levels o£ Children Surveyed in Pittsburgh

Pennsylvania: Analytical Methodologies and Summary Report

Abstract

A survey was conducted in Pittsburgh, Pennsylvania to estimate the

incidence of lead paint in housing and to develop a survey methodology

that could be used in other metropolitan communities. A secondary

objective of the survey was to determine whether a causal relationship

could be found between blood lead levels of children aged 7 years or

less, living in the surveyed dwellings, and the presence of lead paint.

This report deals with the latter objective. For the children tested

in Pittsburgh the incidence of elevated blood lead levels, defined as

40 micrograms of lead per 100 milliliters of blood or greater, was

found to be less than one percent, which is too low to permit the

establishment of a causal relationship. Since more than 801 of the

tested children lived in dwellings containing lead, the low incidence

of elevated blood lead levels makes it appear that the presence of lead

paint does not necessarily result in children having elevated blood

lead levels. There was a low correlation (6-15%) between the blood

lead levels of the children and the fraction of lead bearing surfaces

within their dwellings. There was a stronger correlation (40%) between

the blood lead levels and the age of the dwellings in which the children

resided. This correlation appeared to be independent of the lead paint

levels in the dwellings.

This report presents a summary of the survey procedures, the blood

lead measurement process and associated problems and the more significant results of the analysis of the housing/blood lead data obtained in

V
Pittsburgh.

Key Words: Blood; blood lead; elevated blood lead; children; housing;

lead paint; lead poisoning; surveys.

vi
I. INTRODUCTION

Lead poisoning of children is a serious national health problem.

It has been estimated that as many as 500,000 children [11 >* below the

age of seven years, may have excessive body burdens of lead resulting

from a variety of environmental exposures including lead in air, water,

food, and dust.

It is generally believed that one important source of lead poisoning

of children is lead paint in housing. In addition to the natural tendencies of young children to chew and teethe on non-food items, there is an

abnormal tendency among some children, called "pica", which is an active

consumption of non-food items. Dried, peeling paint on walls, woodwork

and other accessible surfaces represents a health hazard to children who

exhibit an interest or craving for such chewable materials especially if

the paints contain lead compounds.

Although the health effects of low level body burdens of lead in

children are not known, high body burdens result in severe neurological

damage including mental retardation and in some cases, death. A primary

diagnostic tool in determining the body burden of lead is the measurement

of lead in blood.

The Department of Housing and Urban Development (HUD) is required

by Title III of PL 91-695, the Lead Based Paint Poisoning Prevention Act of 1971,

to detemine the nature and extent of the lead paint poisoning hazard to

children throughout the nation. As one of several related research

efforts, HUD undertook a program of investigation into the numbers and

*NLimbers in brackets refer to references

1
geographical distribution of children under the age of seven vdth abnormally high levels of lead in their bodies and the nature and numbers

of dwellings which constitute potential hazards by virtue of their lead

paint content and physical condition.

The National Bureau of Standards (NBS) has provided technical

assistance to HUD in this research area because of its capabilities in:

housing related research; chemistry; mathematics; statistics and computer

science. Under HUD sponsorship, NBS had previously estimated the national

incidence of elevated blood lead levels among children age seven or

less. The estimate, reported in NBS TN 746 [1], was based primarily on

very limited child screening data, obtained from two community administered surveys, which provided too small a sampling to serve as a sound

basis for an adequately valid estimate. In 1973, HUD requested NBS to

develop a program to update and refine its previous estimates of the

number of dwelling units constituting lead paint hazards. NBS responded

by developing a housing survey methodology which included both the

operational aspects and statistical and analytical functions of such

a survey. The procedures were evaluated and refined by means of a field

test in Washington, DC which was reported in NBSIR 74-426 [2],

V Subsequent to the development of the NBS survey plan, HUD reviewed

its own definitions of the lead paint hazard in housing and recognized

the need for additional data which might prove the correlation between

the lead paint content of housing and blood lead levels in children.

NBS was therefore requested to modify its survey plan to include the

screening of children for blood lead levels as well as surveying housing

for lead paint. NBS was also requested to select a city in which to

2
carry out the survey and formalize the arrangements for its implementation.

The city of Pittsburgh, Pennsylvania was selected as the site for

the survey, and the Allegheny County Health Department (ACHD) was chosen

as the agent to conduct the survey. The ACHD is responsible for administering both public health and public housing functions in the city of

Pittsburgh. The administrative control of the two major parts of the

survey activity (housing and health measurements) by this one agency

was a major selective factor. In addition, the ACHD was actively engaged

in screening children by blood lead measurement as part of a Federal

grant program to identify and treat children with lead poisoning. The

ACHD had experienced staff, administrative control of a large city area

and an enthusiastic desire to carry out the survey, all of which contributed to make Pittsburgh uniquely appropriate for the program.

In a contract arrangement between NBS and the ACHD a housing survey

was conducted with the areas of responsibility defined as follows: NFS

furnished the survey methodology, lead detection equipment, and training in

the use of that equipment; monitored the inspection procedure; and

prepared and analyzed the data base. The ACHD provided the staff required

to administer the program and conducted the various survey operations

including: inspection of dwellings for lead; collection of blood samples

of children aged 7 years or less, and the analysis of those samples for

lead

.

This report describes those aspects of the survey which pertain to

the collection and analysis of the blood samples and presents an analysis

of the data. Other reports, being prepared by NBS, are directed to the

housing aspects of the survey [3]

.

3
2 . THE SURVEY APPROACH

The MBS/HUD sponsored "Pittsburgh Survey" is the only major statistically designed housing survey for lead paint hazards that has been

carried out to date. The Center for Disease Control (CDC) of the

Department of Health, Education and Welfare issues grants to Public Health

agencies throughout the country for child screening programs that are

basically limited to areas where high lead poisoning incidence is expected.

Local programs identify so-called "lead-belts" by accumulating lead

poisoning data submitted by neighborhood clinics and public health

offices

.

A goal of the survey was to obtain a representative sampling of the

entire Pittsburgh area and not just identifiable "lead -belts". This was

achieved by a housing selection strategy. Four thousand dwellings were

selected randomly from a listing of the entire housing population of

the city. This process was accomplished using a computer generated

list of random numbers which were then used to select pages, columns,

and lines from a Polk [4] directory of Pittsburgh that listed all the

buildings by their addresses.

The ACHD contacted the occupants of the selected dwellings by means

of direct mailings and follow-up telephone calls. Initial visits were

limited to housing inspections for measuring lead content of interior

and exterior surfaces and identifying other pertinent housing conditions

and construction characteristics.

2.1 SELECTION OF CHILDREN

At the time of the first visit by the ACHD, occupancy by children

4
under seven years of age was determined and parents or guardians were

requested to allow their children to be included in the blood testing

program. Following this procedure, 800 children were identified as

being eligible to participate in this program.

Full participation in the blood lead testing program could not be

obtained since the participation was on a voluntary basis. The large

number of refusals (about 40% of the eligible children) introduced a

bias to the survey sample. Fventually 456 children were tested. The

reasons for refusal were noted in order to assess their impact upon the

randomness of the sample and the nature of the bias. Refusals by parents

or guardians to allow their children to be tested were based on: the

absence of lead paint in their homes as determined by the ACHD inspection

team and the parent's reluctance to allow the blood collectors to take

the child's blood because of unfamiliarity with the public health departm.ent.

Of the reasons given, those based upon the absence of lead paint

in the dwellings were of the type that could bias the survey. Therefore the

lead paint profiles of the dwellings of the tested children, the children

who were not tested and of the entire surveyed population of Pittsburgh

were compared on a room by room basis to determine what the differences

were.

To date there is no specific index by which dwellings may be intercompared \\rith regard to the extent and degree of lead contamination.

The decoration and painting of dwellings is a highly individual matter.

The only direct way that dvrel lings may be compared is on a room bv room

basis and by the amount of lead paint on each surface type. Th.e results

of the comparisons of the 550 dwellings occupied by the 800 children are

5
shoMi for walls, windows and doors in Figures 1, 2, and 3 respectively,

and the data from which these results v/ere compiled are shown in Tables

1, 2, and 3. The results for baseboards obtained in the survey are so

similar to the results for walls, windows, and doors that they are not

included in this report.

The data shown in Tables 1-3 were obtained in the following manner.

Readings were taken from each room in a dwelling with a portable x-ray

fluorescence lead detector [5] at various accessible locations on

different surface types such as walls, windows, doors, and baseboards.

The readings were recorded and for each room a high value was established

for each type of surface. A reading of 2 milligrams of lead per sauare

centimeter is taken by convention to be an indication of significant

lead contamination.

The high readings for each type of room for their respective surfaces

were aggregated and averaged according to dwelling population, such as

those of the tested children. These averaged readings are referred to as

mean high readings. The distributions of such readings were found to be

strongly skewed toward high values. The consequence of this is that the

standard deviations may exceed their associated mean high values. One such

distribution is shown in Figure 4 in the form of a histogram. In this

figure the frequency distribution of the lead content of walls in 429

kitchens is shown for the dwellings of the tested children. The values

range from zero to more than 20 milligrams of lead per sauare centimeter

of wall surface. The mean value for this distribution is 3.3 and the

standard deviation is 5.7;a value that exceeds the mean. Nevertheless

the standard deviation may be used as a r.seful measure of the deviation of

6


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10
the lead values from the mean.

In the same distribution of 429 kitchens, the lead contents of 53,

24, and 12 rooms (12, 5 and 3?; of the total respectively) exceeded the

mean by 1, 2 and 3 standard deviations respectively. In Tables 1, 2, and

3 the standard deviations have been provided to serve as indicators of the

broadness of the frequency distribution curves.

Mean readings were computed by using an aggregation process for each

type of room. All of the readings for the surface of a specified type

were used, not just the high readings. Because of placement of cabinets,

heavy appliances and furniture some surfaces were inaccessible, so that

not all rooms had an equal number of readings taken for each surface.

In some rooms more high readings were taken than low readings , consequently

some mean readings exceeded their associated mean high readings.

The room by room comparisons in Figures 1, 2, and 3 show that the

tested children lived in dwellings that contained more lead than did

the dwellings of both the total surveyed population and the dwellings

of children who were not tested. The untested children lived in dwellings that contained less lead than did those of the surveyed population

as a whole. By implication, the non-tested children (by living in

dwellings that contain less lead than those of the tested children) are

likely to have no higher, if not actually lower, blood lead levels than

those of the tested children. Thus, while the sample of children may

be biased because of refusals, the bias is in the direction of finding

higher blood lead levels if, in fact, there is a relationship between

lead levels in housing and children's blood lead levels.

As noted above, the housing and consequently the households were

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selected on a randor basis. Children in households having only one

child were therefore also selected on a randoip, basis. Povever, any number

o£ eligible children in each household were allowed to participate in

the program and since siblings were not individually selected on a

random basis, their inclusion introduces a small statistical bias. On

the average in this program there were 1.5 children tested per household.

3. BLOOD SAMPLING

The blood collection procedure employed by the ACHD was comprised

of the following essential steps. The child's finger was cleansed with

a cotton swab to both disinfect the finger tip and to remove dirt and

grime which might contain lead particles or other interfering contaminents. The finger tip (usually the index finger) \\^s then punctured with

a sterile needle and blood was forced from it by gentle pressure on the

finger on both sides of the puncture. Several drops of blood were

elicited and wiped away with sterile swabs. Approximately 0.25 milliliters were then drawn into a sterile capillary tube. A clean swab -vvras

finally pressed to the puncture to stop the bleeding and a simple bandage was applied. This procedure is described in greater detail in

Appendix LA. Various factors comhine to make this procedure of critical

importance in obtaining valid blood lead data. The principle factors

are: the small size of the blood samples; contamination of the blood

sanples during collection; and alteration or contamination of the samples

after collection and prior to chemical analysis for lead.

3.1 BLOOD SAMPLE SIZE

Large blood samples of 5 to 10 milliliters are dra-KTi from children

only when absolutely necessary and only by personnel who are specifically

15
qualified and licensed to carry out such procedures. The sampling of

blood by finger puncture is essentially hazard free; requires a minimal

level of experience and qualification of the clinician; is much less

expensive in terms of the cost of disposable equipment; is much more

readily performed in the patient's home; and is therefore the method of

choice for blood screening programs or surveys.

The blood sample size used in this survey was limited to 0.25

milliliters (250 microliters* or about 1/120 fluid ounce). This small

sample size, along with the low level of lead being sought (80-100 nanograms**) greatly magnifies the effects of sample contamination and strains

the limits of the analytical procedure's accuracy and precision.

3.2 BLOOD SAMPLE CONTAMINATION

Extensive experience of public health agencies, including the ACLD,

has shown that failure to rigorously follow a prescribed method for

collecting blood samples results in false positives, that is, lead

analyses that erroneously indicate that the subject child has an elevated

blood lead level. In most cases, such children, when retested using

approved and carefully implemented sampling procedures, are found to

have blood lead levels in the normal range. The cause of false positives

is usually assumed to be contamination of the blood sample rather than

systematic errors in the laboratory chemical analysis of the collected

samples

.

The ACHD tested its supplies and equipment (see Appendix TBI) and

*1 microliter = one-millionth liter or 34 millionths fluid ounce

**1 nanogram = one-billionth gram or 35 million millionths ounce avoirdupois

16
did not find serious lead contamination potential from these sources.

Ffowever, NBS tested the same types of supplies and equipment and found

them to contain trace levels of lead (see Appendix IB-2) . Thus, a potential

may exist for contamination by the supplies and equipment used during

the blood collection, but it is not possible to estimate how much actual

•contamination may occur in practice.

As a part of the ACHD survey effort, thirty-seven samples of household dust and outdoor soil were taken in Pittsburgh (see Appendix IC)

.

From the lead values obtained it is evident that the environments in which

the blood samples are taken can contribute enough lead to seriously contaminate the blood samples. The households dust samples ranged from less

than 1 to as much as 230 micrograms* of lead per gram of dust while the

dirt samples ranged from 200 to more than 4000 micrograms of lead per

gram of dirt. From these values, an estimate can be made of the amount of

lead that may be found in average dust and dirt films. For convenience 1

gram of dirt is assumed to occupy a volume of 1 cubic centimeter.

If a dust film is assumed to be 1/100 millimeter (.0004 inch) in thick

ness, it could contain 10 or more nanograms of lead per square centimeter

while a dirt film of similar dimensions could contain over 200 nanograms

These amounts represent a serious risk of contamination since they are of

the same order of magnitude as the amounts being sought in the blood sample

Elevated blood lead levels (EBL) were designated by ACHD to be 40 micrograms

lead or more per 100 milliliters of blood, which amounts to 100 nanograms le

or more per blood sample of 250 microliters.

3.3 BLOOD SAMPLE ALTERNATION

NBS has found that even when blood samples are frozen, at about

*1 microgram = 1 millionth gram = 1000 nanograms.

17
-15°C, significant dehydration occurs i£ the samples are stored for

several weeks (see Appendix ID) . The storage containers used by the

ACHD in the blood collection process were tubes made of polyethylene or

polypropylene. Their caps do not close by friction and a partial

vacuum must be induced during capping, to insure that the closure will

be hermetic.

Dehydration of the blood samples increases the blood density. Since

the blood lead levels are defined in terms of weight of lead per volume

of blood the interpretation of such measurements are affected by a

positive error. The apparent levels of lead will increase as the sample

is dehydrated.

Dehydration is reduced but not eliminated by freezing the blood

samples prior to analysis.

Another potential source of error is the analysis of clotted blood.

Clotting occasionally occurs and makes the sample non-uniform, in density.

As it is customary to subdivide the blood in the collection tube, into

smaller samples for duplicate analyses, it is unlikely that the subdivided

samples are comparable to the bulk composition. The clotted portions

and fluid portions are likely to contain different fractions of lead.

The ACHD laboratory requested new samples when this occurred.

4. ACHD BLOOD LEAD ANALYSIS PROCEDLIRE

4.1 GENERAL PROCEDURES

The ACHD blood samples were analyzed by a comm.ercial clinical

laboratory in Pittsburgh (Pittsburgh Laboratory) . This laboratory

employed a modified Delves Cup Atomic Absorption Spectrophotom.etric

procedure which is a micro analytical technique. Macro (large scale)

18
procedures were precluded by the small size of the blood samples. This

procedure was originally developed by H. T. Delves and modified

and improved upon by others including D. Olson and I. Jatlow [5] and is

described in detail in the literature.

Appendix II presents the procedure employed by the Pittsburgh

Laboratory. The method essentially consists of partially oxidizing the

sample with hydrogen peroxide, volatilizing the sample with an airacetylene flame and aspirating the decomposition products into a nickel

absorption tube situated in the flame. By noting an absorption peak

compared with peaks obtained by using reference standards, the amount

of lead that was in the sample may be determined.

4.2 VALIDATION OF ANALYTICAL RESULTS

Due to the low level of lead present in the blood samples, accurate

analyses are often difficult to obtain. Yet, in screening programs,

accurate analyses are necessary if the results are to be trusted and

interpreted meaningfully,

Two strategies for validation were adopted to substantiate the

findings of the Pittsburgh Laboratory. The first strategy involved the

collection of duplicate samples of blood from about 10% of all the

children tested, and having these samples analyzed by both Pittsburgh

and NBS chemists respectively. The analytical methods employed differed

greatly in methodology and in realizable absolute accuracy and precision.

The other strategy was to have porcine blood laden with various

but very accurately known amounts of lead analyzed by both ACW and NBS

These strategies are described below.

19
4.2.1 DUPLICATE CHILDREN'S BLOOD SAMPLES

Duplicate blood samples of 0.25 milliliters each were drawi from

54 children. One sample was sent, as usual, to the Pittsburgh Laboratory. No indication was provided to the laboratory that the sample was

unusual in any way. The other sample was frozen in dry ice and sent

to NBS for analysis. NBS analyzed these samples by an isotopic dilution

mass-spectrometric method, which is described in Appendix III.

The results of the duplicate analyses of children's blood are shown

in Figure 5. The NBS results are indicated along the abscissa, while

the Pittsburgh laboratory values are indicated along the ordinate. A

"line of equality" is shown in this figure. The data points that fall

upon this line indicate agreement between the results obtained by the

Pittsburgh laboratory and by NBS. The Pittsburgh laboratory results were

higher or lower than the NBS results according to whether the data points

shown are above or below this line. The less the data points deviate

from this line the better the agreement. The vertical deviations of the

data points from the "line of equality" provide a measure of the precision

of the Pittsburgh laboratory's analyses. Another line labeled "least

squares fit" is shown as the best straight line to fit the data. It may

be noted that this line does not deviate much from the "line of equality".

4.2.2 PORCINE BLOOD STANDARD SA^'(PLES

Porcine blood lead reference standards being prepared and evaluated

by NBS as part of a program relating to clinical standards were made

available for use in the Pittsburgh survey. The lead in the blood was

obtained from pigs which had been fed lead bearing foods. Blood sarrples

with differing levels of lead were prepared by blending the blood samples

20
Figure 5

Cranparative Analyses of Duplicate Samples of Children's

Blood by NBS and the Pittsburgh Laboratory
from different pigs in varying proportions. The resulting levels of lead

were determined by exhaustive macro -analytical procedures. These samples

viere used as porcine blood lead standards. Standard samples containing

lead in the range of 17 to 34 and some at 105 micrograms per 100 milliliters of blood were used. Unfortunately blood samples having lead

between 40 and 70 micrograms per 100 milliliters were not available.

Six to ten standard specimens at each concentration were prepared

by subdividing the porcine blood standard samples and were made up to

resemble children's blood samples. They were provided with fictitious

names and residence addresses, and were included along with children's

blood sent to the Pittsburgh laboratory for analysis.

Similarly, six standard samples of porcine blood, at each of three

concentrations, were anlayzed at NBS in the same manner as the children's

blood.

Figures 6 and 7 show the comparisons of these results. Figure 6

shows the Pittsburgh results of the analyses of porcine blood samples

compared with NBS porcine standard sample values. Figure 7 shows the

NBS results of the analyses of the porcine blood samples by the NBS

micro -isotopic dilution method as compared with the known values of the

NBS porcine standard samples.

The analytical determ.inations of the subdivided portions of the

porcine blood lead standard samples were averaged and the mean values

of the determinations of the Pittsburgh laboratory and NBS respectively

are shown in Figures 6 and 7. These lines are referred to as "mean

lines". For the subdivided portions of each standard sample, the amount

that the individual determinations deviate from the mean is a measure of

22

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24
the reproducibility or precision of the analyses. The mean value is the

basis of the measure of accuracy. Specifically the amount that the mean

deviates from the true value, in this instance, from the "line of equality",

is a measure of the accuracy of the analysis. In Figures 6 and 7 the

"line of equality" is implicit and is the line that connects equal values

on both the horizontal and vertical axes. For the standard sample having

the lead level of 34 micrograms per 100 milliliters, the mean value in

Figure 6 is 5% low and the range of values is 20^. It is reasonable to

assume that in the region of 40 micrograms approximately the same conditions

apply. The corresponding NBS accuracy and range of values is 2% high and

41 respectively.

The solid lines shown in Figures 6 and 7 connect the mean values

obtained for the respective subdivided samples. The dashed lines connect

the respective limits which are twice the sample standard deviation above

and below the means.

Based upon the determinations made in the comparative analysis of

childrens blood and the porcine blood lead standard samples, the accuracy

of the Pittsburgh laboratory is apparently adequate to determine whether

blood lead levels are above or below the 40 yg/100 ml. level.

25
5. RESULTS, CONCLUSIONS AND SIMIARY

In the discussion below, the blood lead levels are expressed in

units of micrograms per 100 milliliters and the definition of an Elevated

Blood Lead Level (EBL) is 40 micrograms per 100 milliliters of whole

blood or more. A frequency distribution of the blood lead levels after retest o£ all

the children who were tested in- this survey, is shown in Figure 8. In this

histogram the vertical bar represents the number of children (and fraction

of all the children) whose blood lead levels were contained in the

interval between the value posted below the bar and the next higher

value, 5 micrograms per 100 milliliters higher. For example, 135

children had blood lead levels in the range of 15 to 19.9. It should

be noted that the distribution is skewed toward the higher values after retest,

only three children had lead levels exceeding 40 micrograms.

The same data are shown in Table 4 in the form of a cumulative

distribution. In each percentile, the percent and number of children

is shown whose lead levels do not exceed the stated value.

Of the 456 children tested all but 15 of them had blood lead

levels that were found to be below the ERL defined above. Fourteen

of the fifteen children (Table 5) x;ere retested and all but two of these

were then found to be below the ERL level . One of these two children

with elevated blood lead levels was placed under surveillance to determine the source of lead. The other child, aged 8 years and retarded

since an early age, had a blood lead level of 75 as confirmed by a macro

analysis procedure and was treated for lead poisoning. The fifteenth

child could not be located and thus was unavailable for retest. The

26
Table 4

Cumulative Distribution of the Tested

Children's Blood Lead Levels

Percent of Samples

within Blood Lead Blood Lead Number of Samples

Range Range (yg/100 ml) in Range

10 0-13.6 46

20 0-15.5 91

30 0-17.3 137

40 0-18.8 182

SO* 0-20.7 228

60 0-22.4 274

70 0-24.3 319

80 0-26.9 365

90 0-30.6 410

95 0-33.6 433

99 0-38.0 451

99.3 0-39.9 453

100 0-73.0 456

*Median

27
Table 5

Children's Elevated Blood Lead Levels at Screening

and Results after Retest

Child Screening

Micro

yg/100 naRepeat

Micro

yg/100 nd

Macro*

yg/100 ml

Case

Sl"a1'ii<;

1 45 26 nosed

2 73 73 75

3 42 35 nosed

4 43 11 n osed

5 45 38 nosed

6 41 28 nosed

7 46 34 nosed

8 42 38 nosed

9 47 35 Closed

10 59 28 Closed

11 42 19 Closed

12 43 Can't be located

13 40 21 Closed

14 50 48 .43

15 41 32 Closed

*Macro blood sample size is 10 milliliters

28
>

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29
child's blood data is retained in this report as a case o£ EBL.

In Appendix TV various possible causes are discussed for the predominantly lower blood lead levels of these children after retest, with

the conclusion that most of the initial values that were high were due

to the inherent limitations of accuracy for the analytical method.

Some of the statistics for the Pittsburgh survey are summarized as

follows: Of the 4000 randomly selected dwellings, 3300 were fully

inspected for the presence of lead paint, the remainder were at least

partially inspected. Of the fully inspected dwellings, 500 had 800

children in the target age range. The 456 children ultimately tested

for elevated blood lead levels occupied 300 of the these dwellings.

Although 391 of the tested children (85.71) lived in dwellings that

contained at least one surface containing lead at a level of 2 mg/square

centimeter or more (see Figure 7) , the incidence of confirmed blood lead

levels was less than II. Nevertheless, meaningful statistical analyses

of the data were performed. The results of those analyses are discussed

below.

Most of the blood lead levels (991) were found to be below the EBL

threshold, and since experience has shown that the analytical precision

is significantly lower at the lower lead levels (see Figure 6) , the

individual blood lead values are of limited accuracy. Nevertheless,

the number of blood lead samples is sufficiently large to permit trend

analysis and the exploration of interrelationships between various

parameters by statistical techniques. Extensive statistical analyses

were performed by computer on the blood lead data and the most significant findings are reported below.

30
The mean and median blood lead levels o£ the tested children as

categorized by their age and sex in combination with the age of the

dwelling in which they live in shown in Table 6. From this table it

may be noted that children under 1 had lower lead levels than those above

1. The median value for children under 1 in all dwellings was 16 while

for those above 1 was 22. In the housing constructed after 1959 no

significant differences in lead levels was found according to children's

age. No significant difference in blood lead levels were found between

males and females; the median values being 22 and 21 respectively.

The blood lead levels of the children, according to the fraction

of surfaces that exceeded a value of 2 milligrams of lead per scpoare

centimeter is shown in Figure 9. The solid line shows blood lead levels

as a function of fraction of lead contaminated surfaces which were in good

condition. The dashed line shows the blood lead levels as a function

of the fraction of the surfaces which were in poor condition, namely

surfaces with loose, peeling or chalking paint. The relation between

blood lead levels and the fractions of lead contaminated surfaces is not

strong; the correlation coefficient is less than 15%. The blood lead levels

increase only slightly with an increasing fraction of surfaces contaminated

with lead and are a bit higher in dwellings whose surfaces are in poor

condition. This finding applies only to the older homes since the newer

homes have, at most, 201 of their surfaces contaminated with lead.

The distribution of the children's blood lead levels according to

the year of construction of the dwelling in which the children live and

the highest lead reading in the dwelling is shown in Table 7. From the

data in this table and Table 6 above, the conclusion may be drawn that

31
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OO <U -P « « « « «

34
regardless of the lead level, for houses in the same age category, the

median blood lead of the children is essentially the same. Only the age

of the dwelling and the median blood lead levels seem to be related.

The older the dwelling the higher the median blood lead levels. This

finding is more significant than the one relating the blood lead levels

and increasing fraction of surfaces contaminated with lead. The correlation coefficient is about 40%. Even this coefficient is low, but it is

the highest one that was found in all the statistical analyses that were

done on the children's blood data.

An example of a plausible relationship which was not proven by analysis

is the relation between the blood lead levels of the children and the

amount of lead in the house dust and exterior dirt of the samples

described in Attendix C. No correlation was apparent; the correlation

coefficients were essentially zero. Although the number of test samples

was only 32, any trend should have been discernible.

Two conjectures as to why occupancy of older homes may affect

children's blood lead levels are that:

a) The plumbing systems of many older homes in Pittsburgh, which

are still occupied, were constructed of lead piping. Thus

drinking water may bear significant quantities of dissolved

lead.

b) Older homes are often located in the more congested areas within

a city and are likely to be exposed to heavy vehicular traffic

with consequent heavier pollution by lead particulates from such

traffic and from industry.

Even though the blood lead levels of children, (living in dwellings

35
having lead contamination levels of 2 milligrams per square centimeter

of surface and higher) do not differ significantly, the possibility that

the presence of lead paint does affect the children's blood lead levels

cannot be completely ruled out.

All of the indicators of lead levels in a dwelling (such as, high

median or mean reading of lead) for a specific surface type or room,

are highly correlated with each other. The best correlation that was

obtained was between the blood lead levels and the high readings.

The failure to find high correlations between blood lead levels and

lead paint in housing is probably due to the data being accurately representative of nonrial urban background lead levels.

One of the principle objectives of this survey was to determine the

blood lead levels of children living in housing which contains lead paint

and to establish causal relationships between lead paint in housing and

EBL's. This survey objective was achieved. The low incidence of EBL's

in Pittsburgh (less than 1%) precludes any attempt to establish a causal

relationship between EBL's and the presence of lead paint in housing.

However, the absence of EBL*s in Pittsburgh is a valid and highly significant finding v^ich will have ramifications on future research on lead

poisoning and the total lead environment.

36
SlffWlY

In this survey the incidence o£ elevated blood lead levels ainon^

the tested children was less than 1?;. Since inore than 80?^ of the

children lived in the dwellings contaminated to some degree with

lead paint (Table 7), it appears that in Pittsburgh at least, the

presence of lead paint in housing does not per se cause elevated blood

lead levels. No explanation may be advanced at this time as to v^hy this

is so. Nevertheless, the reason for this apparent anomaly is of great

interest and potential value. If a causative agent can be identified

as being a limiting factor in lead poisoning it might he applied to

other communities vrhere lead poisoning is a widespread and serious

problem. NBS is funding continuing research in Pittsburgh, which is

intended to develop and evaluate hypotheses which might explain the

low incidence of lead poisoning in that city.

A weak correlation (1S%) was found between the fraction of surfaces

within dwellings contaminated by lead paint and the children's blood

lead levels. A stronger correlation (40?^) was found betv^een the age of

the child's dwelling and that child's blood lead level. Both of these

trends, though statistically weak, were the strongest relationships that

were found in this survey. No correlation was found between the levels

of lead on the surface and the blood lead levels.

Although the blood lead levels encountered in this survey may appear

to be low, the findings appear to be reliable since the analytical accuracy

of the Pittsburgh laboratory has been tested and has been found to be

satisfactory.

37
It may be necessary to match the results of the Pittsburgh survey

with those of other cities and have any differences reconciled by other

studies

.

38
6 . REFERENCES

[1] Gilsin, Judith F., "Estimates o£ the Nature and Extent of Lead

Paint Poisoning in the United States," NBS Technical Note 746,

December 1972.

[2] Hall, William, Ayers, Tyrone, Doxey, Dwight, "Survey Plans and

Data Collection and Analysis Methodologies: Results of a PreSurvey for the Magnitude and Extent of the Lead Based Paint

Hazard in Housing," NBSIR 74-426, January 1974.

[3] Untitled NBS Report on the Pittsburgh Housing Survey in process

of preparation.

[4] Polk Directory citation.

[5] Spurgeon, Joe C, "Response Characteristics of a Portable X-Ray

Fluorescence Lead Detector: Detection of Lead in Paint,"

NBSIR 73-231, June 1973.

[6] Olsen, Eugene, D., Jatlow, Peter I., "An Improved Pelves Cup

Atomic Absorption Procedure for Determination of Lead in Blood

and Urine," Clinical Chemistry, Volume 18, No. 11, 1972.

[7] Hessel, D.W. , "A Simple and Rapid Quantitative Determination of

Lead in Blood, Atomic Absorption," Newsletter, Volume 17, Page 55,

1968.

[8] Barnes, I. L. , Murphy, T. J., Gramlich, J. W. , Shields, W. R. , "Lead

Separation by Anodic Deposition and Isotope Ratio Mass Spectrometry

of Microgram and Smaller Samples," Anal. Chem. pp. 1881-1884,

September 1973.

[9] Kuehner, E.C., Alvarez, R., Paulsen, P.J., Murphy, T.J. , "Production

and Analysis of Special High Purity Acids Purified by Sub-Boiling

Distillation," Anal. Chem. 44, pp. 2050-2056, October 1972.

39
7. ACKNOWLEDGEMENT

The author is indebted to William Hall for his technical assistance

with the computer analysis of the data and the many stimulating recommendations and suggestions with regard to possible interrelationships of

the various data parameters, and to James Filliben for his assistance

with statistical analyses, particularly the Pittsburgh and NBS laboratory's

chemical analysis data. Further, the author is indebted to Janet Bonk

and Cindy Treser, formerly with the Allegheny County Health Department

(ACHD) , for their assistance in resolving data inconsistencies and

errors

.

The author thanks the staff of the National Bureau of Standards

Analytical Chemistry Division for its assistance in providing porcine

blood lead standards and carrying out analyses of blood samples.

40
APPENDIX lA

MICRO BLOOD SAMPLE COLLECTION PROCEDURE AS USED BY ALLEGHENY

COUNTY HEALTH DEPARTMENT LEAD POISONING CONTROL PROGRAM

The method of drawing blood samples by finger pricking involves

several steps. After the child is secure or even before, his hand may be

vashed to clean it of all noticeable dirt, grime and anything else, such

as food stains. After that, the finger to be stuck is selected and held.

The finger is initially washed with 0.1 molar hydrochloric acid. The

acid is put on a sterile gauze pad and the finger is wiped vigorously. The

acid is thought to mobilize the surface lead thus rendering the finger lead

contamination free. The finger is then wiped with a dry gauze pad.

Next, the finger is wiped with an individually wrapped sterile alcohol

prep pad. After this, the finger is massaged by applying pressure from

the palm of the hand towards the fingertip, pushing the blood up the

fingertip. When the fingertip appears somewhat red and engorged with

blood, the finger is then stuck with a blue monolet. The monolets are

individually packaged finger stick needles. The first drop of blood is

then wiped off with an individually wrapped sterile gauze pad. The blood

is then collected with a Natelson* capillary blood collection tube of

approximately 280 microliters in volume. During the blood collection

procedure, care is taken to see that the tip of the Natelson* tube

is held right on the point of puncture and the blood collected there

so that the blood does not run down the finger and then into the tube,

thereby opening possibilities for contamination. The Natelson tube

fills itself through capillary action; when it is full, the blood

collector picks up a Beckman Spinco* tube. The Spinco tube is a micro

*Commercially available supplies used during blood collection by the ACHD.

Use of trade names in this report does not constitute an endorsement of

those products. ,^
centrifuge tube of approxipately 400 microliters in volume. The blood

that is in the Natelson tube is put into the Spinco tube. The child's

finger is then wiped with a gauze pad and a bandage is put on the

puncture. The Spinco tube is capped and labelled with the name, address

and identification number for the child.

The Spinco tubes are carried in a small plastic bag back to the

office where they are placed in the refrigerator. At the end of the

day all blood collected that day is removed from the refrigerator and

taken to the laboratory. During the transport from the office to the

laboratory, the blood is not refrigerated. At the laboratory the blood

is turned over to the technicians.

NOTE: The ACHD requested that the clinical laboratory provide

them with lead free supplies for use during blood collection. The laboratory does not routinely test the supplies as they receive than but

has tested seme supplies for the presence of lead, (see Appendix lB-1).

42
APPENDIX IB-1

PITTSBURGH LABORATORY DETER>nNATinN OF THE LEJ\D CnNTEOT

OF BLOOD SAMPLING EOUII^IENT Am SUPPLIES

The clinical laboratory selected by ACHD to analyze the blood

samples for lead, tested some of the supplies used in the blood collection procedure for the presence of lead using macro as v/ell as micro

procedures.

First the lead content of a pool of ant i- coagulated human whole

blood was assayed. The blood ivas then drawn into twenty (20) Natelson

tubes and subsequently transferred to Spinco tubes and stored overnight.

Lead assays were then determined on each tube. The alcohol in the wipes

and V/ashkins -was mixed with the control blood and assays were also made

on the resultant mixture.

The results are listed below in Table IP-1.1

DISCUSSION OF RESULTS

It appears that even though NBS has found the sterile gauze, PrePads and Washkins to contain significant quantities of lead, the Pittsburgh laboratory has not found that serious contamination results when

these items come in contact vnth blood.

43
Table IB- 1.1

Pittsburgh Laboratory Determination of the Lead Content

o£ Supplies Used in Blood Collection

BASELINE

BLOOD

NATELSON/SPINCO

LOT # 04441

NATELSON/SPINCO

LOT # 03741

WIPES WASHKII

1. 7* 7 9 3 6

2. 9 8 8 5 7

3. 9 8 8 4 5

4. 7 7 10 6 5

5. 11 7 10 7 7

6. 7 8 11 6 7

7. 7 7 8 6 8

8. 8 9 7 6 6

9. 10 9 7 8 6

10. 9 8 7 8 8

11. 10 7 10 7 8

12. 11 7 11 6 7

13. 11 8 11 6 8

14. 9 7 10 7 6

15. 10 9 11 7 7

16. 10 7 9 5 8

17. 11 9 10 8 10

18. 11 11 10 7 9

19. 11 11 10 6 7

20. 11 9 11 8 8

*Units in micrograms/ 100 milliliters

44
APPENDIX IB2

NBS DETERMINATION OF THE LEAD CONTENT OF BLOOD SAMPLING

EQUIPMENT AND SUPPLIES

The lead content of the supplies used during blood sampling was

deteimined by the following procedures:

1) O.IN HCl. - Approximately 21 g of the test solution was transferred

to a 30 ml Teflon beaker.

2) Gauze Steri-Pad* - The pad was transferred to a 50 ml Teflon* beaker,

10 g of O.IN HCl was added. The pad and solution were stirred

with a Teflon stirring rod for two minutes and the pad was removed

after most of the solution was saueezed out with the Teflon rod.

Approximately two -thirds of the solution was recovered.

3) Prep-Pad* - The pad was transferred to a 50 ml Teflon beaker, 5 g

of H2O was added, and after stirring for two minutes the squeezed

pad was removed. Approximately 4 g of solution was recovered.

4) Washkin* - The pad was treated as the Prep- Pad except 10 g of H2O

were used for four minutes of stirring. Approximately 9 g of solution was recovered.

5) Natelson Tube* - Two grams of HNO^ (1:49), were transferred to a 30

ml Teflon beaker and this solution was used in filling and draining

the tube five times by capillary action.

6) Spinco Tube* - The tube was filled with HNO^ (1:49), allowed to

stand two hours, and the solution and one water rinse were transferred to a 30 ml Teflon beaker.

7) Mono let Needle* - One gram of HNO^ (1:49) was transferred to a 30 ml

beaker, the cap was removed from the needle and the needle was held

in the solution for 30 seconds.

* Trade Names
A weighed aliquot of Pb solution, 1 g o£ HCIO^ and 0.5 g of HNO^

were added to each sample. After evaporating the sample to dryness,

the lead was dissolved in 0.025N HCIO^ and electro -deposited on a platinum wire anode as Pb02. The Pb02 was dissolved in a dilute solution

of H2O2 and HNO^ and the solution was evaporated to dryness.

The sample was dissolved in enough HNO^ (1:49) to give a concentration of about 100 micrograms/milliliter . One drop of the solution

was used for isotopic analysis by mass spectrometry (see Appendix m)

using the silica gel procedure described in Appendix III. The total

amount of natural lead in the sample was then calculated from the

measured isotopic ratio of the sample and the known isotopic composition

2 06

and amount of Pb spike that was added.

The results have been corrected for a blank of 0.8 ng of lead.

The individual blank values are 0.7, 0.8, and 0.9 ng.

DISCUSSION OF RESULTS

The values in the last column of Table IB-2.I labeled Equivalent

Blood Lead requires some explanation. I'he values represent the amount

by which the blood lead level results would rise if all the lead in the

material was added to the blood sample of 0.25 milliliters.

The lead content of the Natelson tubes, Spinco tubes, and Monolets

is not significantly increased when these materials are taken into the

field and are opened for use.

It should be noted that while the particular test procedures used

do not represent a sampling blank as such, they do indicate that some of

the materials used include significant quantities of lead and that the

potential for severe contamination is high. In particular the Steri46
Table IB-2.1

NBS Determination of the Lead Content o£ Supplies Used in Blood Collection

Material or SAMPLE 1 SAMPLE 2 AVERAGE EQUIVALENT*

Device Nanograms Nanograms Nanograms BLOOD LEAD

Lead Lead Lead Micrograms/

100 Milliliters

O.IN HCl (1 gram) 2.7 2.5 2.6 1.3

Gauze Steri-Pad 81 85 83 41.5

Prep-Pad 32 28 30 15

Washkin 20 25 22 11

Natelson Tube

(unopened pkg)

** 4.4 4.4 2.2

Natelson Tube

(opened pkg;

4.6 3.9 4.2 2.1

spin CO Tube

(unopened pkg)

0 0 0

Spinco Tube

(opened pkg)

0 0 0 0

Monolet Needle

(unopened pkg)

1.7 0.9 1.3 0.6

Monolet Needle

(opened pkg)

0.0 0.3 0.1 0.05

* Potential increase in blood lead level due to contamination.

** Sample was lost during analysis.

47
pads, Prep -Pads and Washkins contain very large amounts o£ lead. However, it is not deemed possible to transfer all of the lead to the

blood sample since only a small area of one surface ever comes into

contact with the site of the blood extraction. How much of this lead

may actually contaminate a sample is not known.

Further, the acid used in the analytical procedure to leach out

the blood from the Natelson tube and Monolet, is a better solvent for

this purpose than human blood is; therefore smaller amounts are likely

to be leached out than the figures indicated.

On the other hand, the hydrochloric acid intended to mobilize lead

on the surface of the finger, also mobilizes lead in the gauze pad

which is used to apply the acid to the fingertip. Consequently wiping

of the fingertip with a dry gauze pad, absorbs not only the lead in

the acid but also may mechanically transfer lead from this pad onto the

fingertip again. Alcohol used with Prep-Pads*, can only transfer lead

from the Prep-Pad to the fingertip, since it is left there to dry

before the sample of blood is taken. The amount of lead transferred by

these means can be only a matter of conjecture.

*Commercial pads used by the ACHD during blood collection.

48
APPE^DIX IC

DUST AM) SOIL SURVEY

In order to identify alternative lead sources within the home environment, thirty- eight dust and soil surveys were made of those hom.es

having children under seven years of age. As in the housing survey for

lead paint, the children's blood lead data was included for possible

correlation with the lead content of the dust and dirt samples. The

samples were collected as follows:

Using an adhesive sivatch, interior floor dust was collected from the

following locations:

1) Five feet inside main entry

2) At the head of the child's bed

3) Child's play area

4) Kitchen

5) Dining area or front of TV

At the Pittsburgh laboratory, the samples v/ere analyzed bv macro

and micro procedures, to determine the average level of lead content.

Exterior soil was sampled using a similar procedure. Soil samples

were taken from seven and one -half feet in front of the main entry and

five feet diagonally from, each of the structure's four comers. If these

areas were paved, such as sidewalk or driveway, then samples v/ere taken

from the side or back soil at the approximate center-line of the structure.

In order to validate the laboratory procedures, duplicate interior

and exterior samples were taken from, arbitrarily selected hom.es of some

tested children. Tvro samples were taken at each of the locations listed

49
Figure lC-1.1

Lead Content of Pittsburgh House Dust Samples
Figure K:-1.2

Lead Cont^Snt of Pittsburgh Dirt Samples
above. Although the basic data was not inade available to W>S for analysis

the average results for the duplicate analyses are shown in Figures IC-1.1

and IC-2.1

The parameter 'cumulative percent' used in these figures is defined

as the percent of the values, of the whole sample population, that are

equal to or less than the indicated value at the specified cumulative

percent

.

Thus, for dirt at the cumulative percent of 40, 40 percent of all

the dirt samples had a content of 310 micrograms of lead per gram of

dirt or less or, conversely, 60 percent of the dirt samples had a lead

content of 310 micrograms per gram or more.

By definition, at the cumulative 50 percent level, the value indicated

is the median.

As can be seen, the lead content of dust varies widely with a

median value of 1.8 micrograms/gram.

For dirt the values range from 30 micrograms of lead per gram, of

dirt to a high value of 4000. This latter amount is equivalent to

about 9 lbs. of lead per ton of dirt. The median value is 380 micrograms

per gram.

No correlation was found between the levels of lead in the house

dust and exterior dirt of the dwellings sampled and the blood lead levels

of the children living there.

52
APPENDIX ID

WEIGHT LOSS OF FROZEN PORCINE BLOOD STORED IN SPINCO TUBES

Six sanples o£ porcine blood in Spinco Tubes were checked for weight

loss after three weeks and after four months of storage in a freezer.

These samples were weighed after allowing them to warm to room temperature and three empty tubes were carried along for tare correction. These

six samples (0.12-0.37 g) lost an average of 0.17 percent after storage

for four months.

It had been observed by visual inspection that some other blood

samples had dehydrated when stored in a freezer for a period of several

months. Those samples, showing visual dehydration, were stored with only

one or two tubes within a 30 ml or 50 ml Teflon beaker. The six

samples in this study were all stored together in a 30 ml Teflon beaker

covered with Parafilm.

The Spinco Tubes appear to be suitable containers for storage of

blood in a freezer for periods of several weeks if several are stored

together to conserve the moisture loss, in a plastic bag or a covered beaker

Weight Loss of Porcine Blood in Spinco Tubes

Weight Loss

Tube # Weight of Blood Sample Three Weeks Four Months

g %

11

12

14

15

16

17

0.29

0.12

0.23

0.37

0.24

0.25

0.07

0.06

0.01

0.01

0.03

0.03

U.17

0.21

0.13

0.11

0.19

0.14

53
APPENDIX II

ANALYTICAL PROCEDURE EMPLOYED BY THE CLINICAL

LABORATORY SERVING ACHD

This laboratory is certified by the Center for Disease Control (CDC)

as performing satisfactory analyses of lead in blood. Periodically

they analyze standard reference samples sent them by CDC for validation.

This laboratory employs a variation of the Delves Cup procedure

as modified by Eugene D. Olson and Peter J. Jatlow [6], to determine

the lead levels in blood.

54
LEAD IN BIOLOCICAl miERIALS

TYPE C PROCEHTRE [7]

APPARATUS

1. Vacutainer* tubes, heperanized (Becton- Dickinson* No. L3200XE 313)

2. Polyethylene bottles, screw capped, 4 oz.

3. Homogenizer (Sorvall Co. Omni-Mixer-Om* Model 17105) equipped with

a 50 ml stainless steel chamber assembly (Model 17077)

4. Shaker (Eberbach Corp.*)

5. Centrifuge tubes (Fisher Scientific* Cat. No. 5-558 or 5-558-5),

50 ml.

6. Centrifuge (International* Model IN)

7. Spectrophotometer, atomic -absorption (Perkin- Elmer* Model 303)

equipped with a lead Intensitron lamp and digital reader,

(Perkin-Elm.er* Model DCR-1)

P^.GENTS

All chemicals used should be analytical reagent grade. All aaueous

solutions are prepared with double distilled water.

1. Ammonium pyrrolidine dithiocarbamate -- Triton* XI 00 reagent

Mix equal volumes of a 2% solution of APDC and a S% solution of

Triton XI 00.

2. MIBK (Methyl isobutylketone)

, water- saturated

Presaturate the solvent with distilled water prior to use

3. Lead stock solution, 100 mg/liter

Dissolve 0.1599 g of dried lead nitrate in 1 liter of distilled

water containing 1.0 ml of concentrated nitric acid.

4. Lead reference solution, 1 m.g/lOOml

Dilute 10.00 ml of the lead stock solution to 100 ml with

distilled water.

PROCEDURE

Preparation of the Specimen

1 . Mark the exact level of the blood on the Vacutainer* tube in which

the specimen has been received. A sample size of 2.0 ml is

required.

2. Pour the entire blood specimen into a 4 oz. plastic bottle. Note

whether the specimen is clotted. If the sample is clotted request

new sample.

3. Rinse the collection tube by adding 1.0 ml of APDC -- Triton*

X-100 reagent and shake its.

4. Mix and let stand for 10 minutes.

5 . Add 2 . 0 ml of vTater saturated ^^IBK and shake for 5 minutes on

Eberbach* shaker.

*Trade names or manufacturers of scientific instruments.

55
6. Centrifuge the tube @3000 rpm for 10 minutes to separate the

layers.

7. Pipette upper layer into acid washed Vacutainer* and a stopper.

8. Aspirate the upper MIBK into the atomic absorption spectrophotometer .

PREPARATION OF REFERENCE SAMPLES

1. To 4 oz. plastic bottles containing solutions with known lead

concentrations add the following reagents in the order listed

below:

Reagent volumes (ml) required for reference solutions

Reagent Reference Lead Concentration yg/lOO ml

0 30 60 120

Lead reference solution

Water

APDC- Triton* XI 00 Reagent

Water saturated MIBK

0.0 .06 .12 .24

1.94 1.88 1.76

1.0 1.0 1.0 1.0

2.0 2.0 2.0 2.0

2. Shake each solution for 5 to 10 minutes.

3. Transfer each solution to a 50 ml centrifuge tube.

4. Centrifuge the solution for 10 to 15 minutes.

5. Remove the upper layer, which contains the extracted lead-APDC

chelate from each sample and aspirate it into the flame of the

spectrometer

.

56
MEASURBIENT OF UNKNOW AND REFERENCE SAMPLES

1. Set the following controls of the atomic -absorption spectrophotometer as appropriate:

Slit

Range

Gas mixture

Wavelength

Aspiration rate

Air pressure

Averaging switch

Noise suppression

Scale

Allow a warm up time of 1 hour for the instrument and 30 minutes

for the flame. Adjust the amount of acetylene in the flame while

aspirating a water saturated solution of MIBK, so that there is a slightly visible orange tint to the flame.

2. Determine the absorbance of the specimen. After performing 25

analyses check the absorbance with reference samples, make the

necessary corrections for instrumental drift or change in the

flame characteristics.

3. Plot the absorbance of the reference samples against their

respective lead concentrations, then draw standard curve from

the data obtained.

CALCULATION

Determine the lead concentration in the specim.en by comparing its absorbance with that of a suitabel reference sample or a standard curve prepared from the reference sample suggested above.

ACCURACY AND PRECISION

In the range from 25yg to 120yg per 100 ml, the relative error is on the

order of 51, with a standard deviation of approximately 4yg per 100 ml.

Results obtained by this method and those obtained by the dithiozone

procedure yield comparable data.

INTERFERING SUBSTANCES

At a wavelength of 283.3 nm there is no interference from, other metal

ions. Therapeutic blood concentrations of EDTA, used in the treatment

of lead poisoning, also do not interfere v/ith lead determinations.

57
COMMEOT

The method requires no pipetting or aliquot ing of blood, since all

volumes used are based on the total volume o£ blood collected. A techjiician can analyze up to 50 samples per day by this method including

preparation of solutions, calculations and reporting data.

QUALITY COMTROL PROCEDURES

Stock standard from Lead Nitrate per procedure requirem.ents

.

Working standards made from stock solution: 20, 40, 60, 80, 120, 160

micrograms/100 milliliters.

Standard curve run before each group of patients.

Eight (8) controls used with every six (6) patients.

a) Four aqueous controls

b) Four whole blood controls loaded with lead

LEAD LOADED IVHOLE BLOOD QUALITY CONTROL POOL

POOL PREPARATION

Lead loading standard - 100 micrograms/milliliter

Collect 500 ml pooled whole blood or use outdated blood bank blood

(RECCMIENDED - if phosphate used as additive make sure citrate also

used.) Analyze pool. Add sufficient lead standard dropwise with

stirring and continue to stir for 30 minutes to give value between

40-50 micrograms/100 milliliters.

Blind Control analysed as submitted by CDC and CAP.

All Delves Cups pre- standardized by Perkin- Elmer* , then restandardized

at the laboratory.

ALL SAMPLES RUN IN DUPLICATE

The equipment is adjusted so that the values found for the blanks

automatically are nulled out.

*Manufacturer

58
APPENDIX III

The NBS Method for the Determination of Sub ->Ticrogram

Samples o£ Lead Employing Mass Spectrometry

DISCUSSION

Samples in \vhich the amount of lead is one microgram or less, such

as in the blood samples of the Pittsburgh survey (0.1 yg or less), require

extraordinary care in handling, in cleanliness, in reagent selection

and attention to details not important in good analytical work with

larger sized samples.

A method has been developed at NBS [8] involving the dilution of

the lead in the blood sample with a specific lead isotope; separation

of the total lead from the sample by electro deposition and the subsequent determination of the lead in the orginal sample after anlaysis

by isotope ratio mass spectrometry. The combined procedure is applicable

to samples containing from 10 micrograms to less than 10 nanograms

(0.01 yg). The electro deposition is more than 9S% efficient at these

levels while the mass spectrometric procedure premits a precision of

0.1% for the measured isotope ratios.

Lead as found in nature, and in blood, is largely composed of three

isotopes of atomic weights 208, 207 and 206 respectively and a trace isotope

of atomic weight 204. The isotope compoistion of a typical lead sample is

shown in Figure III-l. Lead of atomic weight 208 (Pb208) is the major

constituent.

It appears that the fractions of these isotopes of lead in blood

does not vary much from person to person. For analyses permitting

accuracies to within a few percent, the fraction of Pb208 and Pb206 may be

assumed to be 0.5209 and 0.2519 respectively. NBS selected these values

59
V)

oooC>sJ

CO

CNI

OO ^ r-~. Osl ^ oo

(X> ^ Csj ^ to

CNI S S S S a:

0)3

00

•H

'CI

M-lOo

•H

+J •Ho

ooo O

CO

C3 CVI

OO to

UO

oo

CO

CD o

C>J LOCVJ ^ CVI CvJ

<—1 CO r-. <x> ^ „ 1 1

[i

60
as being representative on the basis of determinations of the actual

fractions of these isotopes in the blood of several persons. Only

when greater analytical accuracy is required must the actual isotope

composition be determined for each sample prior to the addition of the

lead isotope dilutant.

The 'spike' lead isotope, was largely composed of PB206*. The

actual composition of one such sample is also shown in Figure III-l as

well as the result of a one to one mixture of the typical lead sample

and the 'spike' lead.

Critical to the analytical procedure is the lead extraction process

where the lead in the sample after dilution is separated from the bulk

of the blood sample. It is critical in that unless 95% or more of the

lead is extracted the analytical accuracy would be reduced accordingly.

The preparation of the analytically ultra pure reagents used on this

procedure are described on the above citation and in more detail in

reference [9]

.

The lead extraction procedure is performed in two steps. In the

first step the lead is removed from the bulk of the blood by cathodic

deposition, redesolved and then in the second step anodically deposited

as Pb02.

EXTRACTION OF LEAD FROM THE BLOOD SAMPLE

The blood samples as received by NBS were frozen in Spinco Tubes.

After thawing, all the samples contained blood clots, making the samples

non-uniform in composition: precluding sample subdivision. Thus each

"Obtained from Isotopes Division, Oak Ridge National Laboratory of Union

Carbide Nuclear Company

61
sample had to be analyzed as a unit.

Each tube v^as weighed before and after the blood was transferred

to a 30 milliliter Teflon beaker. About 10 milliliters of redistilled

water was used to promote the transfer.

A 0.1 gram aliquot containing 14.7 micro grams of the 206 isotope

of lead per gram of solution was added to the blood samples.

One gram each of ultrapure nitric and perchloric acid was added

and the samples were decomposed by heating, and evaporated to dryness.

The samples were redissolved in 20 grams of 0.025N perchloric acid,

after which the lead was removed from solution by electro -deposition for

4 to 5 hours at 3.5 volts onto platinum electrodes.

The deposited lead was redissolved in 0.025N perchloric acid and

lead deposited as lead dioxide was dissolved in a few drops of hydrogen

peroxide and nitric acid solution and was finally evaporated to dryness.

The isotope dilution measurements were made on this deposit.

The average total analysis blanks were 0.67 nanograms ranging over

less than 0.2 nanograms. The blank was about 1.5 percent of the total

lead in the samples.

The reagents used are of the ultrapure variety that test at less

than 0.1 nanograms per gram of reagent.

COMPUTATION OF THE AMOUNT OF LEAD IN BLOOD SAMPLE

After the lead extraction procedure the ratio of Pb208 and Pb206

is measured by the mass spectrometric procedure described in reference

[8] . Knowing this ratio and the amount of Pb206 added as a dilutant,

it is possible to compute the amount of lead in the original blood

sample.

62
The following formula \vas used:

(^208) (R) - S206 _N

N206 - CN208) (R) • T

L = Weight of lead in original sainple

W = Weight of added spike lead

^208" Fraction of 208 Pb in spike

S206" Fraction of 206 Pb in spike

N208" Average fraction of 208 Pb in blood

^206" Average fraction of 206 Pb in blood

R = Measured ratio 208 Pb to 207 Pb after adding

spike lead

N = Atomic weight of natural lead in blood

T = Atomic weight of spike lead added to blood

For the quantities above the following values were used:

S208= .000287

S206= .9974

N208= .5209

N206= .2519

Atomic weight for N = 207.2

Atomic weight for T = 205.9

Substituting these values in the formula results in the following:

L = W (.000287)R - .9974 . 207.2

.2519 - (.5209)R 206.98

63
APPENDIX IV

COMMENTS ON LOWER VALUES OF BLOOD LEAD LEVELS AFTER RETEST

Several reasons may be advanced for the predominantly lower values

that were obtained after retest. Four possibilities are advanced and

the merits of each are reviewed below.

(a) The Pittsburgh laboratory exercised greater care during the

repeat analyses.

This argument is easily disposed of since the repeat blood samples

were not distinguished from the routine screening samples. The laboratory

was not informed as to which samples were repeat ones.

(b) Better care was exercised during blood collection.

It does not appear that the contamination of blood samples with

lead was a problem during this survey, However, it is possible that the

exercising of greater care could lower contamination and consequently

result in lower blood lead values.

' (c) Children's blood lead levels decreased in the interval between

the first screening and the subsequent retest.

Generally less than a month elapsed between the blood samples. It

does not appear likely that the blood lead levels would change greatly

in this relatively short time interval, or that if they changed they

would uniformly tend to fall.

Since the parents of these children had no a priori reason to

suspect that their children seemingly had elevated blood lead levels,

it does not appear to be likely that either the children's behavior or

activity differed significantly from usual or that the parents supervised them more closely.

• 64
(d) The bulk of the initial high readings may be attributed to

inherent limitations of analytical accuracy.

This last conjecture is the one most likely to be correct and the

rationale for this conclusion may be supported by statistical arguments.

To simplify the discussion, the blood lead levels of all the children are assumed to be the same. Later the arguments may be broadened to

include a range of blood lead levels.

If one performs an unbiased analysis of the total population of

identical blood lead composition, preserving the identity and the quantity

of each sample for possible retest, one vrould obtain a set of values

normally distributed about a mean value, m, and vrould have a standard

deviation value, s. One-sixth of the values vrould exceed m by one

standard deviation and an equal number vrould fall below m by the same

amount, s. The mean value vrould approximate the true blood lead value.

The extreme values obtained vrould not be correct but vrould be the

unavoidable results of limited analytical accuracy.

Now if only the high valued blood samples were to be retested and

since the blood samples vrould be in no way different from the original

population of sanples, the results of the retest should approximate the

results for the population as a whole. Namely, the retest values should

be normally distributed about the same mean value, m, and should have the

same standard deviation, s. Now only one-sixth of the retest values

vrould now exceed m by as much as one standard deviation.

This reasoning applies to the low values as well. The retest of

extreme valued samples in general should tend toward the mean value.

Thus if high values are due to analytical inaccuracy the retest values

65
should tend to drop. This tendency also applies to a mixture of populations.

As long as the high values are higher than actual and exceed the mean of

their respective populations, the tendency vrould be for the retest values

to drop. On the contrary, the retest values of the samples that initially

tested below their respective means vrould tend to rise. In short, there

is a tendency for the retest values to converge toward their respective

means.

The mean blood lead level of the tested children was 21.9 micrograms

per 100 milliliters with a standard deviation of 7.0. Initially, 15

children or 3.31 of the tested children were identified tentatively as

having blood lead levels in excess of 40.0. This number is so small

that most or all of the high values could be in reality antifacts of the

analytical process.

While the tested children certainly did not have identical blood

lead levels, the above arguments apply since the aggregate results may

be considered to be a superposition of the results of a range of populations of tested children. Each such population would consist of children

actually having the same blood lead level, and where the values for each

population would have their own mean and standard deviation. It is not

possible to isolate each such population from the aggregated results for

all the tested children.

In summary, based upon statistical arguments, it is reasonable to

accept the proposition that, due to the small number and the small

magnitude of the high values obtained on initial testing, the high values

were probable analytical artifacts.

66
NBS-114A (REV. 7-73)

U S DEPT OF COMM. 1 oirni ii^ATir\M rw Rf-'PORX \^f'\ 9 *f A ^ ^ o c- i /^r> BIBLIOGRAPHIC DATA NBSIR 76-1024

Sri c fe T

3. Rec ipicnt s Accession No.

4. TITLE AND SUBTITLE

Analysis of Blood Lead Levels of Children Surveyed in

Pittsburgh, Pennsylvania: Analytical Methodologies and

Summary Results

5. Publication Date April 1976

6. Performing Organization Code

7. AUTHOR(S) , ,r T Walter D. Urban

8. Performing Organ. Report No.

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

10. Project/Task/Work Unit No. 4608400

11. Contract/Grant No.

IAA-H-35-75

12. Sponsoring Organization Name and Complete Address (Street, City, State, ZIP)

Division of Energy, Building Technology and Standards

Office of Policy Development and Research

Department of Housing and Urban Development

Washington, D.C. 20410

13. Type of Report & Period Covered Interim FY' 75

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.) A survey was conducted in Pittsburgh, Pennsylvania to estimate the incidence of lead

paint in housing and to develop a survey methodology that could be used in other

metropolitan communities for that purpose. A secondary objective of the survey was to

determine whether a causal relationship could be found between blood lead levels of

children aged 7 years or less, living in the surveyed dwellings and the presence of

lead paint in those dwellings. This report deals with the latter objective. For the

children tested in Pittsburgh, the incidence of elevated blood lead levels defined as

40 micrograms of lead per 100 milliliters of blood or greater, was found to be less

than 1%, too low to permit the establishment of a causal relationship. There was a significant correlation between the blood lead levels of the children living in

the older homes and the fraction of contaminated surfaces within the dwellings. In

addition, there was a significant correlation between the blood lead levels and the

age of the dwellings in which the children resided. This correlation appeared to be

independent of the lead paint levels in the dwellings. This report presents a summary

of the survey procedures, the blood lead measurement process and associated problems

and the more significant results of the analysis of the housing/blood lead data

obtained in Pittsburgh.

17. KEY WORDS (six to twelve entries; alphabetical order; capitalize only the first letter of the first key word unless a proper name; separated by semicolons)

Blood; blood lead; children; housing; lead paint; lead poisoning; surveys.

18. AVAILABILITY pQt Unlimited

1 For Official Distribution. Do Not Release to NTIS

1

' Order From Sup. of Doc, U.S. Government Printing Office Washington. D.C. 20402. SD Cat. No. CH

IX ! Order From National Technical Information Service (NTIS)

Springfield, Virginia 22151

19. SECURITY CLASS

(THIS REPORT)

UNCL ASSIFIED

21. NO. OF PAGES

74

20. SECURITY CLASS

(THIS PAGE)

UNCLASSIFIED

22. Price $5.00

USCOMM-DC 29042-P74

MJ.S. GOVERNMENT PRINTING OFFICE; 1976 210-801/311 1-3