Performance of Histamine Test Kits for Applications to Seafood

7/30/2019 Performance of Histamine Test Kits for Applications to Seafood

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Presentation on October 26,2001, 4 th World Fish Inspection & Quality Control Congress, Vancouver, B.C.

Performance of Histamine Test Kits for Applications to Seafood

Walter F. Staruszkiewicz and Patricia L. Rogers, Ph.D.

Washington Seafood Laboratory, Office of Seafood, FDA, Laurel, MD.

ABSTRACT

Analytical determinations for histamine are used as an index of wholesomeness and safety

for species of fish such as tuna and mahimahi which can support the growth of bacteriacapable of producing histamine during decomposition. Histamine determinations have

been routinely conducted for decades in traditional laboratory settings using an AOAC

fluorometric method, and data obtained with the method have been used to establish a limit

of 50 ppm for histamine in regulatory applications. An interest in portable proceduresfor field analysis that would be capable of rapidly screening fishery products dockside has

led to the development of commercial test kits which have been proposed for HACCP

applications. A number of these test kits have been compared to the AOAC method throughthe analyses of samples of tuna and mahimahi. The precision and reliability of the results

will be discussed as well as the practical technical requirements for using the test kits.

INTRODUCTION

Histamine is a chemical compound that forms postmortem in the muscle of scombroid fish,

such as tuna, and in other species, such as mahimahi by the action of certain bacteria that

are found in fish. Bacteria that have the ability to form histamine do so by the

decarboxylation of histidine, an amino acid found in fish, through the action of histidinedecarboxylase, an enzyme produced by the bacteria. The level of histamine produced in the

fish by these processes provides a measure of the decomposition that has occurred.

Histamine at high levels has been associated with incidents of scombroid poisoning andalso serves as an indicator of an unsafe food. Histamine levels are not reduced by heat

processing, freezing or long term storage. Other biogenic amines such as cadaverine and

putrescine are also formed during decomposition of seafood.

Histamine ranges from 0 to about 5 ppm in the best quality fish. The formation of the

amine during decomposition is highly temperature dependent and increases rapidly with

high temperatures (1). Based on current research in this laboratory, the level of temperatureabuse needed to reach a level of 50 ppm histamine in mahimahi, requires incubation times

of 20 hours @ 20 C, 14 hours @ 29C, and 6-8 hours @ 30/35 C. To reach a histamine

level of 500 ppm, requires approximately 30 hours @ 20 C and 10 hours @ 30 C. At 10C a week or more may be needed to achieve similar levels if at all. A complication in

larger fish is that the amine is not distributed evenly throughout the muscle and the

maximum levels of histamine are usually found in the anterior sections of the fish (2).When such fish are received in the form of frozen steaks or as the canned product, the

highest levels of histamine are randomly distributed within the lot and large sample sizes


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are required to ascertain the safety of the fish. For regulatory applications, fish containing

histamine levels of 50 ppm or more are considered to be of unacceptable quality (3).Lower reject levels of histamine may be applied to reduce sample sizes in practical

applications. For these reasons, the development of analytical procedures centers on a

histamine range of 5 to 50 ppm.

Published methods for the determination of histamine have been based on bioassays,

colorimetric, fluorometric and chromatographic procedures. In general, all of the

commonly used methods for histamine were developed for applications in standardlaboratory environments. For regulatory purposes, the AOAC fluorometric method, is

routinely applied (4). With the advent of HACCP plans which include testing for

histamine, several commercial firms have attempted to develop portable test kits that do notrequire solvents such as methanol and which can be more conveniently used in industrial

applications.

Descriptions of new procedures usually discuss sensitivity, range, linearity, stability, speedand costs of analysis. In practice, there are also reliability limits based upon the number of

required steps, measuring weights and volumes, and in the completeness of reactions which

may be involved. While some of these limits may be calculated, the uncertainties due toanalyst-sample-method interactions and lab to lab differences must be measured

experimentally.

Our laboratory conducted a two-part study of histamine test kits in which we determined

the relative performance of various test kits to each other and whether an analyst would

reach the same PASS/FAIL decision using these different procedures in a laboratory setting.Secondly, test kits were evaluated under field conditions by shipping the kits and a reader to

a remote testing site. There have been rapid changes in the development and availability of

test kits which were encountered during the study. While these changes are expected to

continue in the future, the results of this study provides a starting point for futurecomparisons.

Data from the first part of the study was published last year and will be summarized today(5 ) in addition to new results from a continuation of the study conducted during the past

summer. That part of the study included the 2 most commonly available test kits available

in the U.S. compared in a laboratory setting and in field experiments.

EXPERIMENTAL

In the first part of the study, several histamine test kits were selected to provide a range oftypes available at that point in time. The kits tested included the Histamarine (Coulter

Corp., Opalocke, FL), K1-HTM and K3-HTM (Immuno Diagnostic Reagents, Vista, CA),

and the Alert, Veratox, & Agrimeter II kits (Neogen Corp., Lansing, MI) With the exceptionof the Agrimeter II, all of the kits are based on a form of ELISA (enzyme-linked

immunosorbant assay) assay. Extracted histamine competes with a histamine-conjugate for

binding to antibodies coated on the walls of microtiter wells and the amount of histamine isdetermined by the development of a color based on the bound conjugate. For example, in

the Veratox test kit, a color is developed by the action of the bound conjugate on an


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enzyme substrate and histamine determined as an inverse function of the color. Other

details of the test kits may be are found in the previously cited reference (5). A Micro-Reader III (Hyperion, Miami, FL) was used to quantitate the developed color.

There were significant differences in the procedural steps of the different test kits. The

basic outline of the steps are given in Table 1. For the purposes of this study, none of theprocedures were modified and all were used as the manufacturers instructions directed.

Some of the procedures used a PASS/FAIL approach and some allowed estimates of the

amount of histamine to be calculated.To provide a standard reference against which to compare the test kits, the AOAC

fluorometric method was used to analyze the same samples. The fluorometric method has

supplied consistent results over the years and is generally considered the standard methodfor measuring histamine in seafood. Eight laboratories participated in the original

collaborative study ( 6 ), and the coefficient of variation (repeatability for a individual

analyst) was found to be 4%. In a 1999 collaborative study by 16 laboratories (7) the

repeatability was again calculated as 4%.All of the testing in the current study was conducted by a single analyst who had experience

using such procedures. The test kits were evaluated for their ability to detect defective

samples without giving false positive results and for quantitative measures of histaminewhere possible. The ease of use was also considered since analyst behavior and

performance can be significantly affected by the construct of a procedure.

Samples for testing (Table 2) were selected to provide a range of histamine levels as formed

during the decomposition of the fish. Tuna and mahimahi were chosen because they

represent the 2 species most commonly associated with incidents of scombroid poisoning.

In the second part of the study, attention was focused on the two kits most readily available

at that time for application in a field trial. These kits were the Veratox kit and the

Histaquant test kit (Biomedics, Vista, CA). The two test kits were first evaluated in thelaboratory on prepared samples of cooked tuna loins containing incurred histamine levels

and then in a field trial on tuna canned in aqueous media. In the field trial, the analyses

were conducted on aliquots of the aqueous liquid in the cans which provide a good estimateof histamine content without blending the entire contents of the cans.

RESULTS

Table 3 shows the procedures which were tested and the analytical results on acceptable

quality samples In general, the test kits were in agreement with the AOAC values except for

a few elevated levels for the IDR-K1, the Histamarine and the Agrimeter results. Howeverall values were less than 50 ppm.

In Table 4 are results for 2 intermediate level samples which contained 20 and 58 ppm

histamine. These samples are in the range most difficult for some procedures to handle.With 1 exception, all values agreed with a decision to Pass or Fail the materials.

For samples prepared from decomposed fish, the kits worked well except for one decision(Table 5). A summary of the Pass/Fail decisions for the test kits is given in Table 6. Good


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agreement in rendering decisions on acceptability of product. In only 2 instances, were

different decisions reached between the official method result and that from a test kit.

In developing rapid procedures, compromises must be made. Highly precise histamine

values are not necessary for screening purposes except at the 50 ppm threshold and proper

sampling can minimize even that problem. The important features to retain are

1. Identical decisions to PASS or FAIL a sample between the screening procedure and the

AOAC method;2. Minimal variability for replicate analyses as that will determine the operational limit of a

procedure in the hands of a particular analyst; and

3. Small lab to lab variations if the results are to be compared directly between processingfacilities or laboratories.

Estimates of the capability to quantitate levels of histamine were obtained for 3 procedures,

using replicate analyses to measure the variability of data at 30, 63 and 72 ppm histamine(Table 7). While the overall agreement is again quite good, note that the variability for the

Histamarine procedure portends problems for analyses at the 50 ppm level especially if

limited to single determinations.

FIELD EVALUATIONS

An evaluation of the performance of the test kits in field experiments was undertaken to see

if their routine use for the determination of histamine would lead to new difficulties. Two

test kits, the Veratox test kit (Neogen) and the Histaquant kit (Biomedics), were selectedbased on their availability to the U.S. industry. Initially they were compared under

laboratory conditions similar to the first part of this study against the AOAC fluorometric

method on a series of samples prepared from cooked tuna loins prior to canning. As shown

in Table 8, results from the test kits on samples of acceptable quality tuna were all less than50 ppm. The results of the Veratox kit most closely resembled those of the AOAC method.

Values obtained with the Histaquant kit averaged 21.9 ppm, significantly higher than the

other procedure. For decomposed tuna containing approximately 42 ppm histamine and forsamples of acceptable tuna spiked with 50 ppm histamine, both test kits agreed quite well

with the AOAC method.

The 2 test kits were then used in a field study to evaluate their ruggedness. At a workshop

for the sensory training of analysts on canned tuna at a marine research station, portions of

aqueous packing media were taken from 34 cans prepared from a range of qualities of

authentic tuna of known composition. The aqueous media was used to permit a rapidanalysis of the can contents while sensory decisions were being made. It has been found in

our laboratory that the histamine content in the aqueous layer of stored canned aqueous

packed tuna is very similar to that determined on the entire contents of the can. Histaminewas determined with the 2 test kits as the cans were opened for the sensory analysts.

Portions of the liquids were also frozen & shipped to the main laboratory for later analysis

by the AOAC method. The results are given in Table 9. Nineteen cans containingscombrotoxic fish were analyzed and results from both test kits showed histamine levels in

excess of 1000 ppm. For 9 samples of acceptable quality tuna, the AOAC method reported


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an average of 13.1 ppm histamine while an average of 14.2 ppm histamine was found with

the Veratox kit. The results from the Histaquant kit averaged 53.4 ppm histamine for thesame samples which would cause the product to be rejected. Pass/Fail decisions by the

AOAC method and the Veratox kit were identical for all quality levels of tuna. The

Histaquant kit gave more variable results. Importantly, both test kits correctly identified the

cans which contained scombrotoxic fish. The concern with the Histaquant kit was the falsepositive results found for some samples and one decomposed sample which was passed.

An important consideration would be the need for replicate analyses at the threshold of

rejection or a check analysis with the AOAC method.

The analysts comments on the ease of use and performance of the test kits in the field were

positive. Both test kits were relatively easy to transport and use. A Micro-Reader wastransported with the kits for quantitation.

SUMMARY

In general, the test kits had the capability to provide usable histamine results for tuna and

mahimahi. When such tests are applied by a trained analyst to a sample which is

representative of a lot under evaluation, reliable decisions on histamine content may beachieved. With few exceptions, all procedures yielded identical PASS/FAIL decisions

when a single analyst in 1 lab conducted the analyses. Data are not available to calculate

analyst to analyst variability for these test kits. The quantitative data show adequateprecision for screening samples except for a large coefficient of variation in the Histaquant

data. Errors would be expected near the threshold of 50 ppm for some analyses and

replicate determinations would probably be required for reliable decisions.

Are the test kits portable? Yes. Fast? Varies with the analyst. The end result of the trials

with histamine test kits is that they are convenient but do require training and an

appreciation for their limitations. Periodic cross checking with the AOAC method isadvisable using spiked samples and fish with naturally incurred histamine levels.

Notes:

Kits no longer available include the Agrimeter, IDR-K1 and IDR-K3 test kits.

REFERENCES

1. Baranowski, J.D., Frank, H.A., Brust, H.A. & Premarathe, R.J. Decomposition and

histamine content in mahimahi (Coryphaena hippurus). J of Food Protection 1990, 53:217-222.


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2. Frank, H.A., Yoshinaga, D.H., & Nip, W.K. Histamine formation and honeycombing

during decomposition of skipjack tuna. Marine Fisheries Review 1981, 43(10):9-14.3. Federal Register Aug 3, 1995. Decomposition and histamine in raw, frozen, & canned

tuna, mahimahi, & related species. 60(149):39754-30956.

4. AOAC Method 977.13, Book of Official Methods, 16th edition.

5. Rogers, P.L. & Staruszkiewicz, W.F. Histamine Test Kit Comparison. J Aq Fd ProdTech. 2000, 9(2): 5-17.

6. Staruszkiewicz, W.F. Fluorometric determination of histamine in tuna: Collaborative

Study. JAOAC 1977, 60(5): 1131-1136.7. Rogers, P.L. & Staruszkiewicz, W.F. Gas chromatographic method for putrescine &

cadaverine in canned tuna and mahimahi and fluorometric method for histamine:

collaborative study. JAOAC Int 1997, 80(3): 591-602.

Table 1. Procedural Steps of Test Kits.

Test Kit Extraction Filtration Reagents

Agrimeter water easy included

Alert water easy includedVeratox water easy included

Histamarine water easy reagent preparation

IDR-K3 0.03% HCl difficult many transfersIDR-K1 0.03% HCl difficult many transfers

Table 2. Composition of Test Samples

Acceptable Quality Fish

# Samples Type Histamine, ppm

2 Yellowfin Tuna, in water 3, 3

1 Skipjack Tuna, in water 36 Mahimahi, frozen 0.2, 1, 2, 2, 9, 20

Decomposed Fish4 Yellowfin Tuna, frozen 58, 68, 190, 300

2 Mahimahi, frozen 158, 300

Table 3. Results of Test Kits on Acceptable Quality Samples.

(Histamine, ppm)

Sample AOAC Agrimeter Alert Veratox Histamarine IDR-K3 IDR-K1a 3 10 P 3 1 P


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2

2b 3 0 P 3 tr P

2

4c 3 4 P 8 1 P 6

d 1 0 P 6 1 P 0

e 2 0 P 3 1 P 2f 9 20 P 9 17 P

17

g 2 10 P 4 2 P 5

h 1 20 P 3 1 P 2

Table 4. Results of Test Kits on Intermediate Samples(Histamine, ppm)

Sample AOAC Agri Alert Veratox Histamarine IDR-K3 IDR-K1

I 20 30 P 31 44 P 36j 58 75 P 66 73 F 96

Table 5. Results of Test Kits on Samples of Decomposed Fish.

(Histamine, ppm)

AOAC Agrimeter Alert Veratox Histamarine IDR-K3 IDR-K168 45 F 64 72 F 61

190 >100 F >100 309 F 328

300 >100 F >100 372 F 435300 >100 F >100 366 F 455

158 >100 F >100 191 F 323

Table 6. Decisions to Pass or Fail Samples

Samples Results

Acceptable Histamine Levels Test Kits Passed All Samples

Intermediate Histamine Levels Eleven of Twelve Results CorrectHigh Histamine Levels Twenty-nine of Thirty Results Correct


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Table 7. Replication of Analytical Results

(Histamine, ppm)

Content Veratox Histamarine IDR-K1

30 34 10 33

Coef. Var. 11% 24% 5%

63 69 70 62

Coef. Var. 2% 17% 8%

72 70 86 64

Coef. Var. 7% 20% 5%

Table 8. COMPARISON OF 3 METHODS FOR THE DETERMINATION OF HISTAMINE IN COOKED

TUNA LOINS

Sample Type AOAC 977.13 , ppm VERATOX, ppm HISTAQUANT, ppm

Acceptable Quality Tuna,

n=5

Range 1.0 - 10.0

Av 2.8

Range 0.2 - 1.6

Av 0.9

Range 17.3 - 26.9

Av 21.9

Decomposed Tuna 42.0 49.3 41.4

Acceptable Quality Tuna +

50 ppm

51.0 50.9 49.6


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Table 9. FIELD DETERMINATION OF HISTAMINE IN AQUEOUS LIQUID FROM CANNED TUNA

Sample Type AOAC 977.13 , ppm VERATOX, ppm HISTAQUANT, ppm

Acceptable Quality Tuna, n=

9

Range 9.5 - 13.0

Av 13.1

Range 6.6 - 29.0

Av 14.2

Range 28.0 - 116.0

Av 53.4

Decomposed Tuna 40 39 58

58 98 39

75 99 56

132 156 250

305 424 380

342 535 454

Decomposed Tuna, n= 19 --------------------------- >1000 >1000

Documents

Performance of Histamine Test Kits for Applications to Seafood