The Hematofluorometer - Clinical Chemistry

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William E. Biumberg, Josef Elsinger, Angelo A. Lamola, and David M. Zuckerman. A portable filter fluorometer, the hematofluorometer- which utilizes “front face” ...
CLIN.CHEM.23/2, 270-274(1977)

The Hematofluorometer William E. Biumberg, Josef Elsinger, Angelo A. Lamola, and David M. Zuckerman

A portable filter fluorometer,

the hematofluorometer-

which utilizes “front face” optics, internal standards, and digital computation capabilities-has been specifically designed for the rapid (-5 s) assay of erythrocyte zinc protoporphyrin in unprocessed blood. A small (unmeasured) drop of blood, such as is obtained from a finger puncture, is placed on a disposable cover slip and inserted in the sample holder of the instrument. The operator pushes the holder, which also holds the permanent blank and standard, into the instrument. Zinc protoporphyrin concentration is automatically and instantaneously computed and the value is displayed on a digital readout as micrograms of zinc protoporphyrin per deciliter of blood for some standard hematocrit. No calibration adjustment need be performed by the operator. The hematofluorometer is designed for “field” use as well as for laboratory assays. Although lead intoxication perturbs many metabolic processes, the primary screening variables have been the concentration of Pb in blood and more recently the free erythrocyte protoporphyrin (FEP) level in blood (1). The former is subject to error caused by ubiquitous lead contamination, and both tests are performed in clinical laboratories, rather than at the screening site. Therefore they are expensive and commonly entail a delay of several days between sample collection and the availability of results. Effective intervention in screening programs for lead intoxication (and iron deficiency anemia) would be greatly aided if the results of the primary screening test could be made available simply and immediately at the screening site. We describe here an instrument, the hematofluorometer (2)1, that makes this possible. The hematofluorometer is a portable filter fluorometer specifically designed for the assay of erythrocyte zinc protoporphyrin in a small drop of whole unprocessed blood. The optical design (front-face fluorometry), internal standards, and digital computation capabilities that are incorporated in the instrument allow great simplicity in its use. Without the need to measure its volume, a small drop of oxygenated blood

(as obtained

from

a finger

puncture)

is spread

Description

of the Instrument

Optical design. Most commercial spectrofluorometers are designed to view the emitted light at right angles to the excitation direction, in order to minimize the amount of excitation light scattered into the emission monochromator. If, in this arrangement, the sample absorbs a negligible fraction of the excitation light intensity (Iex) at the excitation wavelength (Aex), the observed emission intensity (‘em) is proportional to the concentration of the emitting species (c). Considerably greater emission intensities are obtainable with use of an alternative sample geometry (commonly referred to as “front surface illumination”) in which the surface of the samples is at an acute angle to the excitation light direction and the emitted light is detected upon emerging from the same surface. If the sample is optically thick (at Aex) so that it absorbs virtually all of the excitation light within a sufficiently thin layer of the front surface to permit all of the light emitted through the surface to be collected with equal efficiency, then A ‘em

Bell Laboratories,

Murray Hill, N. J. 07974. Reprints of this complete symposium (no single papers) will be available only from the AACC National Office, 1725 K St., N.W.,

Washington, D.C. 20006. Bell Laboratories is not engaged

in the manufacture

of the

hematofluorometer, but has supplied (gratis) all interested instrument manufacturers with technical information about our prototype hematofluorometer. At this time two companies are in production: Aviv Associates, Lakewood, N.J. 08701, and Environmental Sciences Associates, Burlington, Mass. 01803.

270

CLINICALCHEMISTRY.Vol. 23, No. 2, 1977

on a

disposable glass cover-slip on a 6-mm (14-inch) target area and placed into the sample holder of the instrument. The operator pushes the holder into the instrument, stopping at three positions (total time, 5 s) that contain, respectively, a blank, a standard, and the specimen. The instrument automatically and instantaneously computes the zinc protoporphyrin (ZPP) concentration in the specimen in micrograms of ZPP per deciliter of blood2 (for some standard hematocrit) and this value appears on a digital readout. No calibration adjustment need be performed by the operator. Values of ZPP obtained by use of this hematofluorometer correlate well (r = 0.99) and linearly with erythrocyte porphyrin values obtained by accepted extraction methods. The hematofluorometer is designed for “field” use, as well as for laboratory ZPP assays. Blood specimens can be collected in capillary tubes (e.g., microhematocrit tubes) and sent to the laboratory for testing with the hematofluorometer.

=

KIex

(1)

A0

where A and are the absorbance (at X) and quantum yield of the emitting species, is the total absorbance of the sample, and K is a geometrical factor character2

The SI unit is micrograms

grams per deciliter

per liter; multiplying

by 10 gives the value

in SI units.

values

in micro-

istic of the instrument. Since A is proportional to the concentration of the emitting species, c, ‘em is proportional to c as long as A is only a small fraction of The absorbance of oxygenated blood containing CHb g of hemoglobin per deciliter at the optimum wavelength for exciting ZPP (424 nm) is given by: =

(or about 900 cm1 sorbance is:

if cHb

AZ424

15 g/dl) while the ZPP ab-

=

i0=

(2)

10C}ThCHb424

c 2

M2

424

(3)

2

where c2 is the ZPP concentration in micrograms per deciliter of whole blood, and MHb and M2 are the molecular weights of hemoglobin and ZPP, respectively. Because CHb is typically 15 g/dl while c2 rarely exceeds 1000, even in serious cases of lead poisoning, A2 is always small compared to Ab and equation 1 may be simplified to: ‘em

=

(10_6KIex

M M2

424 C CHb424

)

Fig. 1. Schematic diagram of the optical system of the hematofluorometer

C

(4’

CHb

if ZPP is the only emitting species in blood. The ratio Cz/CHb is an extremely useful parameter for monitoring lead intoxication and may, according to equation 4, be obtained from the fluorescence intensity after proper calibrationof a frontsurface fluorometer. The instrument that was designed for this purpose is shown in Figures 1 and 2. In the prototype built by us, the excitation tight source is a 50 W, 12 V tungsten halogen lamp (Osram 64610; Opti-Quip, Highland Mills, N. Y. 10930), and its light is filtered by means of a three-cavity interference filter transmitting from 420 nm to 430 nm (Ditric optics, Marlboro, Mass. 01752), which is mounted with a blue glass filter (e.g., Hoya B-390; Hoya Optics, Menlo Park, Calif. 94025). The excitation light is focussed on the bottom of a drop of blood placed on a horizontal slide chosen to have minimum background fluorescence. One-millimeter-thick quartz slides (Esco Products, Oak Ridge, N.J. 07438) or 0.15-mm-thick glass microscope cover slips (Corning Glass Works, Corning, N. Y. 14830) have proven acceptable for this purpose. The excitation light direction makes an angle of 37#{176} with the normal of the sample slide and the light emitted from the sample is collected in a vertical direction. With this arrangement excitation light reflected from the sample slide is prevented from reaching the emission collecting lens, which, like the excitation system, is an f/2 lens. The emitted light is collimated and passes through a broad-band interference filter, which transmits between 580 and 680 nm with a peak transmission of 80% (Ditric Optics). The two interference filters were designed to have a combined rejection ratio greater than 10 outside their respective passbands below 825 nm. The photomultiplier tube was chosen for its high red sensitivity (Hamamatsu R456; Hamamatsu Corp., Middlesex, N. J. 08846). All lenses are 1 inch in diameter, 2 inch

A and B represent the blank and the fluorescence standard slides; C represents the drop of blond on a supporting slide in its measuring posItion. PM is the photomultiplier tube

Fig. 2. Photograph of the hematofluorometer (right) connected by cable to the display box, which also contains power supplies and logic circuitry

focal-length quartz lenses (Esco Products), which, unlike most glass lenses, do not fluoresce. The optical system described above is housed in an anodized aluminum box 2 inches wide and about 9 inches high (see Figures 1 and 2). The inside faces of the two side panels are provided with slots that are perpendicular to the excitation and emission beam directions and that receive and hold (by means of set screws) the plates upon which the filters and lenses are mounted. This facilitates alignment of the optical trains and ensures that all optical components are approximately perpendicular to the light propagation directions. These components are readily accessible by removal of the narrow end plates. A metal separator prevents scattering of the excitation light into the detector compartment and bisects the angle between the excitation and emission beam directions. CLINICAL CHEMISTRY.

Vol.

23, No. 2, 1977 271

>.

I-. 2 LU 2 U

z

LU U

0 -J U-

SENSE SENSE p0 WAVELENGTH

mm)

Fig. 3. Uncorrected emission spectra of ZPP-containing whole blood and other liquids, obtained by front-face Illumination in a Perkin-Elmer MPF-2 spectrofluorometer. The undiluted samples were in quartz cuvettes and the excitation and emission slit widths were 20 nm. Hoya B-390 and 0-54 filters were used in the excitation and emission beams, respectively (a) Blood obtained from a man occupationally exposed to lead and showing a ZPP burden of 370 ig/dl of blood. (b) Blood ofa male city-dweller (50 g ZPP/100 ml blood). (c) Rhodamine B dissolved In K,ylon No. 1602 Ultra-fist Black Enamel and deposited on a glass slide. The spectrum, taken after drying of the sample, resembles spectrum a sufficiently to make this substance a useful calibration standard, with which blood fluorescence is compared In the course of each ZPP determination by means of the hematofluorometer. (c “Mock blood’ containing ZPP. Note that this spectrum is slltly shifted compared to spectrum a. A very similar spectrum is obtained when Ammonyx-LO detergent is added to whole blood. (e) Transmission characteristics of the 0-54 filter used on the emission side of the fluorometer

Because the hematofluorometer is intended for use in the field, where line voltage and temperature fluctuations as well as aging of the light source and of the phcomultiplier could alter the calibration of the instrument profoundly, we thought it highly desirable to standardize each fluorescence measurement of a blood sample by comparing it to a measurement of the fluorescence from a stable dye, when exposed to the same excitation light. Ideally, this fluorescence standard should have the same emission spectrum as ZPP in blood, because the photomultiplier response is wavelength-dependent. Rhodamine B was found to have a suitable fluorescence spectrum and is so stable that it was used for this purpose (Figure 3). A drop of blood is placed on a sample slide and inserted into the sample slide well of a metal carriage, which is then pushed into the instrument. Springloaded plungers and detents in the carriage permit it to come to rest in three positions consecutively. In the first detented position (P0) the excitation beam falls on a blank slide, which matches the standard and sample slide, an optical sensor senses the position of the carriage, the light source is turned on for 1 s and a background photocurrent (Is) is registered by the photomultiplier. In the next detented position (P5) the excitation light falls on the standard sample, the light goes on for 1 s and an emission intensity reading I, is recorded. In the third detent position (Pb), the light again goes on for 1 s and a reading of ‘b is recorded for the blood (Figure 4) A dedicated digital microprocessor, described in greater detail below, computes the value of: 272

CLINICAL CHEMISTRY,

Vol. 23, No. 2, 1977

SENSE

Fig. 4. Block diagram of the logic circuit, showing how the carriage position-sensing signals govern data handling and display

R

=

R

: ‘:

(5)

which is a standardized value for the blood fluorescence, corrected for background fluorescence from the supporting slide as well as other causes (such as scattering within the instrument). The value of R2 may be adjusted so that R will be computed in any desired units. The reading, R appears as a digital display in units of micrograms of ZPP per deciliter of blood of a standard hematocrit. If there are significant contributions from spurious fluorescences (i.e., from blood components other than

ZPP), this data analysis is, of course, not valid. For the filter combinations described here, the background fluorescence from the blood is negligible. Data Handling In each of the three positions Po, P, and Pb, the lamp timer is enabled (E) for 1 s as indicated schematically in Figure 4. During that time the current from the photomultiplier (PM) is converted to a digital value (by the analog-to-digital converter), and that value is stored in the appropriate register 1o, L, or ‘b. Analog voltages corresponding to the stored values of these registers are differenced (by differential amplifiers) and are entered as the numerator (N) and denominator (D) of an analog divider. The factor R8 of equation 5 can be set by adjusting the gain of either of the differential amplifiers. The quotient (Q) is converted to digital form and presented on a 2.5-digit display, which is enabled (E) by sensing that the carriage is positioned (Pb) to measure the blood sample. It is convenient to have two ranges, 0-199 and 0-1990 ig of ZPP per deciliter of blood, and this is arranged by dividing the numerator signal by a switchable factor of 10 before the divider. In the prototype version of the hematofluorometer described here, the duty cycle of the lamp is prevented from exceeding 10% so that no cooling of the lamp or its housing is necessary. The position of the carriage is sensed by three LED-phototransistor sensors (Spec-

a.

:1

a. a.

r.J 0

z a Ui

Ui I-.

Ui 0 0 -J

U0

Ui I

1200 FEP (PIOMELLI)

(g/d1)

PEP (SASSA)(gid)

Fig.6.Hematofluorometerreadingvs.theFEP valuesobtained by an extraction method for a population of city children who Fig.5.Hematofluorometerreadingvs.the FEP values obtained by an extraction method for a population ofmen occupationally were being screened exposed to lead

tronics SPX1396; Spectronics Corp., Westbury, N. Y. 11590), which sense reflective spots on the underside of the carriage.

Calibration To calibrate the hematofluorometer absolutely, one places a drop of oxygenated blood with a known hematocrit and ZPP content into the sample compartment and the gain of the photocurrent amplifier, which R5 in equation 5 is proportional to, is adjusted until the digital display corresponds to the ZPP concentration (c2) in g ZPP/dl blood.3 c was determined by three different methods, which gave results in excellent agreement with each other. Although this absolute calibration is necessary in order to be able to use established ZPP (or equivalent FEP) cut-off values in screening programs, it was deemed desirable to have available a secondary calibration liquid, the fluorescence of which could be used to check on possible calibration changes. A suitable liquid for this purpose must mimic the absorption characteristics of blood in the vicinity of the Soret band As was shown in a previous section, method gives directly a measure of the matocrit-independent figure. It would flects this fact; for example, tg ZPP/g

the front surface fluorometric ZPP/hemoglobin ratio, a hebe best to use a unit that re-

Hb would be an appropriate unit. However, zg ZPP/dl blood is the unit upon which federal standards for childhood lead poisoning screening are based because it is the simplest unit with respect to extraction methods for assaying ZPP. The hematocrit

must be taken into account in order to relate ZPP/ hemoglobin to ZPP/volume. It is simplest to calibrate the hematofluorometer based on the average hematocrit for the population being tested-e.g., 35 for young children-since any deviation within the normal range would lead to a minor error. An anemic state would lead to an artificially large value of for the concentration, but this would only serve to accentuate the presence of an abnormal condition.

and must be relatively stable, nonvolatile, orescent. When dissolved in this “mock

and nonflublood,” ZPP

must, moreover, have absorption and emission spectra similar to those of ZPP in blood of a lead-poisoned individual, although the ZPP fluorescence quantum yield and the absorbance of the mock blood at the excitation wavelength (424 nm) need not be the same. A mock blood that satisfies these requirements consists of hemin dissolved in a 300 ml/liter detergent solution (“Ammonyx-LO”; Onyx Chem. Co., Jersey City, N. J. 07302). When ZPP is added and its fluorescence spectrum is examined under front surface illumination it is found to have a spectrum that is very similar to that of blood from an individual with lead intoxication and a ZPP qu.antum yield that is about half of that in blood (see Figure 3). However, this quenching can be compensated for by preparing mock blood with a lower hemin concentration than obtains in blood. With a hemin concentration of 10 g/liter the effective ZPP concentration in mock blood is about 0.7 of the actual ZPP concentration. The fluorescence signal of this mock blood as determined in the hematofluorometer or from the 595 nm peak of the fluorescence spectrum obtained by front surface illumination is linear with the ZPP concentration. This mock blood can be prepared quickly and conveniently by dissolving the ZPP and hemin in the detergent, and it remains stable at room temperature in the dark for several days. Testing Procedure The hematofluorometer is designed to be used in the field, with fresh whole blood obtained from a finger puncture.4 The following methods are recommended. For infants, a heel stick may be done more easily. CLINICALCHEMISTRY,Vol. 23, No. 2, 1977 273

The subject’s finger is cleaned and punctured and with his hand held palm down a drop of blood on his fingertip is touched to the coverslip held by its edges. The blood should entirely cover the central 6-mm diameter circle but, as explained above, the thickness of the blood spot is unimportant. After the cover-slip is inserted into the instrument, a corrected ZPP value is displayed. The reading is reproducible to within 5%; it is unaffected by the presence of tissue fluids and is the same for successive drops from the same finger puncture. Readings are only slightly lower if the blood is allowed to dry, unless the dry drop cracks severely. If the hematofluorometer is not at hand, blood is drawn from the finger puncture into a heparinized capillary tube and is transferred to a slide after shipping the tube to the hematofluorometer site. Treatment of the blood with heparin or oxalate anticoagulants does not interfere. Venous blood should be agitated gently in air until fully oxygenated before use in the hematofluorometer. Severely deoxygenated blood gives a reading on the hematofluorometer that is about half the true value, an effect that is in part a consequence of the different absorption spectra of deoxyhemoglobin and oxyhemoglobin. Whole blood with an anticoagulant can be stored in a dark refrigerator for at least one week with no change in hematofluorometer reading. Even extensive hemol-

ysis of the erythrocytes in the specimen has little effect. However, the reading becomes erroneous as methemoglobin or other oxidized hemoglobin species build up in aged blood. Blood can be kept at room temperature in room light for at least a day with no change in reading. The fluorescent porphyrins in diluted blood or extracts, in contrast, are rapidly destroyed in room light. Another feature of the hematofluorometer is the linearity of the reading with even drastic dilution of the blood. To demonstrate this feature, several blood samples of subjects with various lead burdens were diluted up to three-fold with phosphate-buffered saline and their ZPP concentrations measured with the hematofluorometer. The readings for the diluted samples differed negligibly from those for the undiluted ones. This feature is helpful in the case where too-small a blood sample is obtained, because it can be diluted up to three-fold with saline before being spread over the target area of the sample slide.

274 CLINICALCHEMISTRY,Vol. 23, No. 2, 1977

Performance

of the Hematofluorometer

The hematofluorometer has been tested in several laboratories and under “field” conditions in pediatrics clinics, at industrial sites, and in neighborhood screening programs. Results of these trials will soon be reported [a preliminary report has been made (3)]. Of chief concern here is the relationship between values of ZPP determined with the hematofluorometer and the values of erythrocyte porphyrin levels measured by use of currently acceptable laboratory extraction procedures. Figure 5 shows some data we obtained on blood specimens from men occupationally exposed to lead. Hematofluorometer readings were obtained from single drops of blood from finger punctures, and were performed “on site.” Venous blood was also drawn and these specimens transported to a laboratory where erythrocyte protoporphyrin was assayed according to the method of Granick et al. (4). The comparison shown in Figure 5 had a correlation coefficient (r) of 0.98. In an independent study, the hematofluorometer was used with blood specimens obtained from children by finger puncture. These tests were performed in a pediatrics clinic where venous blood was also drawn for lead assay. Portions of the venous blood specimens were assayed later in a specialized laboratory for erythrocyte protoporphyrin by the method of Piomelli (5). The excellent linear correlation of these data (Figure 6) is evident: r = 0.99. The hematofluorometer readings were linearly related to erythrocyte ZPP concentration in blood over the entire range from normal to severely above-normal values (from 30 to 1100 ig ZPP/dl blood), showing that interfering fluorescences from other normal blood constituents are negligibly small.

References 1. Houk, V. N., Chisoim, J. J., Curran, A. S., et at., Increased lead absorption and lead poisoning in young children: A statement by the Center for Disease Control. J. Pediatr. 87, 824 (l975. 2. Blumberg, W. E., Eisinger, J., Lamola, A. A., and Zuckerman, D. M., J. Lab. Clin. Med., in press. 3. Report of the Environmental Sciences Laboratory, Mount Sinai School of Medicine, New York, to the Natural Institute of Environmental

Health

Sciences,

May 15, 1976, ES00928.

4. Granick, S., Sassa, S., Granick, J. L., et al., Assays Proc. Nat. Acad. Sci. U.S.A. 69, 2381 (1972).

for porphyrins.

for free erythrocyte porphyrins. Clin. micromethod for free erythrocyte porphyrins: The FEP test. J. Lab. Clin. Med. 81, 932 (1972). [See also this symposium.J 5. Piomelli, S., (a) Micromethod

Res.

70,497 (1972); (b) A