Immunoglobulin G determination for foals in the veterinary practice,
which tests are most accurate?
G.J. Zandstra
3383539
g.j.zandstra@students.uu.nl
Supervisor:
Dr. I.D. Wijnberg
i.d.wijnberg@uu.nl
Department of Equine Sciences, Section Internal Medicine, Faculty of Veterinary Medicine,
Utrecht University, Yalelaan 16, 3508 TD Utrecht, The Netherland
CONTENTS
ABSTRACT
2
INTRODUCTION
3
MATERIALS AND METHODS
Foals
5
Collection of blood
5
Test methods
6
Interpretation of data
8
Data analysis
8
Statistical analysis
9
RESULTS
10
Correlation between IgG concentration and total serum protein
13
Correlation between IgG concentration and serum globulin concentration
13
Correlation between IgG concentration and total plasma protein
14
Correlation between IgG concentration and plasma globulin concentration
14
Correlation between IgG concentration and gammaglobulin concentration
15
Formula for estimating the IgG concentration
16
SNAP Foal IgG Test Kit
17
Glutaraldehyde coagulation test
18
Correlation between IgG concentration and total plasma protein
measured by refractometer
19
DISCUSSION
20
CONCLUSSION
21
CONFLICT OF INTEREST
22
ACKNOWLEDGEMENTS
22
FOOTNOTES
22
REFERENCES
23
1
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ABSTRACT
Monitoring the success of passive transfer of immunoglobulins is important because foals
with failure of passive transfer are at increased risk for the development of infection and death
during the first month of life. The purpose of this study was to identify which test is most
suitable for determining the foal’s IgG concentration in practice. A distinction was made
between hospitalized foals and healthy low risk foals in the field. One formula and several
screening tests were compared to the IgG concentration measured by turbidimetric
immunoassay: total protein, albumin and protein spectrum measured by chemistry analyzers,
total protein measured by refractometer and IgG concentration determined by the SNAP Foal
IgG Test Kit and glutaraldehyde coagulation test. Blood was collected from 46 foals of seven
days of age or younger. The most reliable alternative test for determining the IgG
concentration appeared to be the combination of total serum protein measured by a chemistry
analyzer and gammaglobulin concentration. A total serum protein of ≥ 49 g/L and a
gammaglobulin concentration of ≥ 6 g/L was corresponding with an IgG concentration of ≥ 8
g/L. If the immunoglobulin concentration has to be known in healthy low risk foals in the
field, SNAP test can be an alternative test if its weaknesses are recognized.
Keywords: IgG; Neonatal foals; Protein; Screeningtests; Turbidimetric immunoassay
2
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INTRODUCTION
Foals are born with low concentrations of immunoglobulins because there is no significant
immunoglobulin transfer across the equine epitheliochorial placenta to the fetus 1, 2. Therefore,
transfer of passive immunity is really important for the health of the neonatal foal. They live
in an environment heavily populated by bacteria and many of them are capable of causing
disease 2. To obtain passive immunity, the neonatal foal is dependent on the ingestion and
absorption of good-quality colostrum with high levels of maternal antibodies. IgG is the
predominant immunoglobulin in equine colostrum, IgA and IgM are present in lower
quantities 2. The maternal immunoglobulins are essential to prevent infection caused by
pathogens until the foal develops his own protective immune response 2, 3.
Absorption of immunoglobulins occurs by pinocytosis through epithelial cells of the small
intestine, especially in jejunum and ileum 4. The rate of uptake is highest in the first six to
eight hours after birth and then declines1. Therefor the ingested quantity of colostrum should
be 1.8 – 2.8 liters within the first eight hours 2. When the foal is 24 – 36 hours of age, no
immunoglobulins are absorbed anymore1. The reason for the decrease in immunoglobulin
absorption is the replacement of enterocytes capable of pinocytosis by mature enterocytes, not
been able of pinocytosis 2. Most foals ingest colostrum within two hours after birth and the
immunoglobulin concentration peaks after 18 – 24 hours 2, 4. An adequate transfer of passive
immunity in an one day old foal is an IgG concentration of ≥ 8 g/L 3, 5, 6. Many normal healthy
foals have IgG concentrations that are much higher than 8 g/L 7. When the foal does not
absorb enough immunoglobulins, it results in a partial failure of passive transfer (PFPT) or a
complete failure of passive transfer (FPT) of immunoglobulins. FPT is the most common
secondary immunodeficiency disorder of foals with an incidence between 3% and 24% 5.
There has been a disagreement regarding the definitions of PFPT and FPT. Some researchers
define FPT as IgG concentrations of < 2 g/L after 24 hours of age and PFPT as IgG
concentrations of 2 to 4 g/L or 2 to 8 g/L 6, 8. Other researchers define FPT as IgG
concentrations of < 4 g/L after 24 hours of age and PFPT as an IgG concentration of 4 to 8
g/L 2, 3, 9, 10. The latter is the most widely recognized classification of FPT and PFPT 2. If a foal
is a healthy low-risk foal on a well-managed farm with minimal exposure of pathogens, IgG
concentrations of 4 to 8 g/L may be adequate and generally there is no need for IgG
supportive therapy 2, 5-7, 10.
FPT can be caused in several ways and include the following: (1) the mare produces poorquality colostrum; colostrum with an inadequate immunoglobulin concentration, (2) the foal
does not ingest an adequate volume of colostrum early in the postnatal period because of
prematurity, dysmaturity, injury or disease, (3) failure of the foal to absorb the ingested
colostral immunoglobulins by the intestinal tract or (4) loss of colostrum by premature
lactation; leads to low IgG concentrations at the time of foaling2, 5, 6. Generally, IgG
concentration is measured when it has reached its peak at 18 to 24 hours of age. Monitoring
the success of passive transfer of immunoglobulins is important because foals with FPT are at
increased risk for the development of infection and death during the first month of life 5. Foals
younger than 12 hours of age and FPT is suspected or confirmed by a blood test, oral
3
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supplementation of colostrum or an oral immunoglobulin preparation can be given. Foals with
IgG concentrations < 4 g/L at 18 to 24 hours of age, should receive an intravenous
administration of equine plasma or a hyperimmune serum due the closure of the
gastrointestinal tract wall. This also applies to foals with IgG concentrations between 4 and 8
g/L with risk factors for sepsis 2, 3, 5, 6, 11.
The serial radial immunodiffusion (RID) is the most quantitatively accurate test available for
determination of serum immunoglobulin concentration 5-7, 10, but the technical skills, costs and
especially the time to obtain the test results (18 to 24 hours) are the disadvantages. For the
diagnosis of FPT or PFPT in a diseased foal, the RID as gold standard is an impractical test
when a rapid diagnosis and treatment are required. In addition to the RID, there are several
rapid screening tests available for the diagnosis of FPT or PFPT and they are widely used in
the equine. An optimal screening test should be accurate, inexpensive, easy to perform and the
results should be obtained in a short period 3, 5, 9, 10.
This study has investigated the diagnostic performance of one formula and six commonly
used screening tests performed at all foals. A distinction in tests was made for hospitalized
foals and healthy low risk foals in the field under practical circumstances. In the field there is
a need for a simple test, easy to carry and which primarily serves to check whether the foal
has sufficient immunoglobulins. All these six screening tests were evaluated and compared
with the turbidimetric immunoassay (TIA) that is considered the currently best available ‘gold
standard’. Studies have shown that values generated by TIA are highly correlated with data
obtained by RID assay 1, 6. The advantages of TIA is automation and a shorter turnaround time
when compared to RID 1, 5, 6, 9, 12.
4
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MATERIALS AND METHODS
Foals
Forty-six Foals (mostly Royal Dutch Sport horses (n=20), Friesian horses (n=13) and
Standardbreds (n=8)) and less than seven days of age were included in the study. Twenty
foals were male and 26 were female. The mean age was 42 hours (SD = 40 hours). Thirty-two
foals were presented to the Faculty of Veterinary Medicine Utrecht University. Twelve foals
were presented to three private practices in the Netherlands. All foals were divided into four
different groups: group one (n=16) healthy foals > 12 hours of age; group two (n=13) healthy
foals < 12 hours of age; group three (n=12) diseased foals; group four (n=5) diseased foals,
four of them had received hyperimmune serum intravenously and one diseased foal received a
blood transfusion. The seventeen diseased foals are described in table 1.
Table 1 Diagnosis of the seventeen diseased foals of group three and four.
Foal
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Diagnose
Dehydrationa
Colic of unknown origin
Meconium obstipation, urachus rupture
Meconium obstipation
Epithelial haemartoma †
Megaesophagus †
Megaesophagus †
Neonatal maladjustment syndromea
Premature, dysmature caused by a placentitis †
Premature, sepsis caused by a placentitis †
Premature, diarrhea of unknown origin
Perinatal asphyxia syndrome, aspiration pneumonia, meconium obstipationa
Perinatal asphyxia syndrome, meconium obstipation
Perinatal asphyxia syndromea
Diarrhea of unknown origin
Neonatal iso-erythrolysisb
Bladder rupture †
a
Hyperimmuunserum Vet Immunogenics (800 mL)
Plasma infusion (1500 mL)
† Dead
b
Collection of blood
Blood samples (12 mL) were collected from the vena cephalica by use of a 20-mL syringe
and 20-gauge needle. Blood was immediately distributed into one heparin blood collection
tube (4-mL) and two collection tubes (8-mL) with no additivesa. Some blood samples were
collected from a jugular vein by use of a vacutain holderb. The samples in the blood collection
tubes with no additives, were allowed to clot at room temperature, approx. 17 - 20 °C for one
hour. Immediately after clotting, the samples were centrifuged for four minutes (2800 rpm) to
avoid haemolysis. Whole blood in the heparin collection tube was centrifuged after the first
5
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test was performed. Serum and plasma were transferred into labeled cryogenic vials and
stored at 4 °C when several tests were done.
Test methods
All tests and formula were considered as screening tests for hospitalized foals. The SNAP
Foal IgG Test Kit (SNAP test), glutaraldehyde coagulation test (GCT) and refractometer were
also considered as screening tests for healthy low risk foals in the field.
TURBIDIMETRIC IMMUNOASSAYc. TIA determined plasma IgG concentrations and is
based on the immunologic agglutination between specific antigens (IgG) and antibody’s
(sheep anti-equine IgG). The resulting turbidity caused by these immune complexes, is
proportional to the immunoglobulin concentration of the plasma sample 1. Plasma (0.5 mL)
was sent to the laboratory, once a week. In three to eight days, plasma samples arrived at
Beaufort Cottage Laboratories in Newmarket. The used materials were a dilution buffer
(Phosphate Buffered Saline (PBS) pH 7.4), an assay buffer (PBS pH 7.4 with 40 g/L
Polyethylene Glycol (PEG)), an anti-equine IgG antisera raised in the sheepd and an equine
IgG standarde, assayed by RID. Plasma samples were diluted 1:20 with PBS pH 7.4.
Antiserum was diluted 1:30 with PBS-PEG pH 7.4 and mixed well. Before the diluted
antiserum was centrifuged at 2,5000 rpm, it was left to stand for approximately 30 minutes at
4 °C. After that, the supernatant was used for the assay. Diluted plasma sample was added to
the diluted antisera (1:125) and antigen-antibody complexes were formed. The resulting
turbidity caused by these immune complexes, is proportional to the immunoglobulin
concentration of the plasma sample. The change in optical density, the nonscattered light, is
measured by a spectrophotometer. First absorbance measured at 340nm, final absorbance
measured after ten minutes at 37 °C. The coefficients of variation for between-run and withinrun precision for the IL650 automated biochemistry analyzer were 4.4 and 3.6%, respectively.
UNICEL DXC 600f
AND HYDRASYS® 2SCANg. Serum (0.5 mL) was brought to UVDL
(University Veterinary Diagnostic Laboratory), Faculty of Veterinary Medicine Utrecht
University. The samples were kept refrigerated in the laboratory until total serum protein
(TSP), serum albumin and serum protein spectrum were measured. Serum globulin
concentration was determined by TSP minus albumin. Both TSP and albumin were measured
by UniCel DxC 600. The used methods were the Biuret and the homemade Broomcresol
Green respectively. Serum protein spectrum was determined by HYDRASYS® 2 SCAN method
amido black. The serum proteins in the sample were separated into five distinct fractions: α1,
α2, β1, β2 and γ. The laboratory results were obtained within two working days. Quality
control for the automated biochemical analyzers was performed daily.
FUJI DRI-CHEM 4000Ih. This method is an automated clinical chemistry analyzer and
determined total plasma protein (TPP) and plasma albumin in less than ten minutes. The used
methods were the Cupric sulfate pentahydrate and Bromscresol green respectively. Plasma
globulin concentration was determined by TPP minus albumin. One TPP test slide and one
albumin test slide were placed in the analyzer. At least 20 µL plasma was transferred into a
6
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sample tube and both the sample tube and an Auto TIP were placed in the specified sample
rack. Setting the correct species ‘horse’ and measurements were started.
SNAP FOAL IGG TEST KITi. The SNAP test is a semi-quantitative enzyme immunoassay
and determines IgG concentration. In this study the test was performed with whole blood,
anti-coagulated with heparin. After blood collection, the SNAP device and reagents were
removed from the fridge. They must be at 7 ° - 29 °C when used. Whole blood was mixed
well by inverting the tube eight to ten times. Only the loop tips of two separate loops were
immersed in the sample that remains in the cap of the evacuated tube. The two filled loops
were transferred into the bottle of sample diluent and the sample diluent bottle was mixed
thoroughly by inverting five times. The first five to ten drops were disposed from the bottle,
as described in the manual. One drop of diluted sample was placed on the sample spot in the
result window of the SNAP device. The contents of the conjugate bottle was poured into the
sample well of the SNAP device. The diluted sample flowed across the result window and
reached the activation circle in about 30 seconds. When the color appeared in the activation
circle, the activator was pushed firmly so the reagents stored in the SNAP device were
released automatically. Seven to ten minutes later the IgG concentration was visible. For the
determination of the IgG, the color of the two calibrator spots with known calibrated IgG
levels were compared with the color of the sample spot. When the color intensity of the
sample spot was lighter than the 400 mg/dL calibrator spot or darker than the 800 mg/dL
calibrator spot, the foal had a blood IgG concentration less than 400 or more than 800 mg/dL,
respectively. When the sample spot was the same as the 400 mg/dL or the 800 mg/dL
calibrator spot, the foal had a blood IgG concentration of 400 or 800 mg/dL, respectively. If
the color intensity of the sample spot was darker than the 400 mg/dL calibrator spot, but
lighter than the 800 mg/dL calibrator spot, the blood IgG concentration was between 400 and
800 mg/dL.
GLUTARALDEHYDE COAGULATION TEST. The GCT is a semi-quantitative test to
determine the serum gammaglobulin concentration. The test is based on the coagulation
between glutaraldehyde and the free amino groups of proteins. Lysine and arginine are amino
acids with free amino groups and gammaglobulins have the highest proportion of these amino
acids. A serum sample with a high gammaglobulin concentration will form cross-links and
this will be visible as a solid gel. The GCT is only feasible with serum. Plasma contains
fibrinogen, another blood protein, what also makes an irreversible binding with
glutaraldehyde13. The 5% glutaraldehyde solution was stored at 4 °C. For the performance of
the test, 50 µl glutaraldehyde solution was pipetted in a clean plastic tube. After approx. 20
minutes, 0.5 mL serum was added and the tube was swirled a number of times. After 30
minutes the tube was turned upside down. The formation of a gel in 30 minutes which stuck
in the reverse tube, indicated a gammaglobulin concentration of ≥ 8 g/L. A gammaglobulin
concentration of < 8 g/L when the gel did not stick in the reverse tube after 30 minutes. The
tube was turned upside down again after another 20 minutes. When the gel remained in the
reverse tube after a total of 50 minutes, the gammaglobulin concentration would be 5.5 – 8
g/L. A gammaglobulin concentration of < 5.5 g/L when the solution was still flowing out of
the tube after these 50 minutes14.
7
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SERUM PROTEIN REFRACTOMETER SPR-NEk. Total serum protein (TSPr) and total
plasma protein (TPPr) were also determined by a refractometer. After calibration, a few
sample drops were placed on the surface of the prism. The daylight plate was closed gently
and the protein scale was read.
FORMULA FOR ESTIMATING THE IGG CONCENTRATION. Since IgG concentrations
are not immediately available, according to the University Clinic a foal is considered to have
a successful passive transfer of immunoglobulins when beta- and gammaglobulin
concentration measured by HYDRASYS® 2 SCAN are at least 26% of total serum protein
measured by UniCel DxC 600. The use of this formula is only reliable when the foal has a
total protein falling within the reference range which is 40 – 66 g/L according to Paradis 15.
Interpretation of data
A positive test result was defined as an IgG concentration of < 4 g/L for healthy low risk foals
in the field 2, 5-7, 10 and < 8 g/L for hospitalized foals 10, 16. A negative test result for healthy low
risk foals in the field and for hospitalized foals was defined as an IgG concentration of ≥ 4 g/L
and ≥ 8 g/L respectively. According to GCT, a positive test result for a healthy low risk foal
in the field is an IgG concentration of < 5.5 g/L instead of < 4 g/L 14.
Data analysis
First, it was determined whether if significant differences exist (1) between the mean total
protein concentration (TSP and TPP) in foals with an IgG concentration of < 4 g/L, 4 – 8 g/L
and those with a successful passive transfer of immunoglobulins and (2) between the four
different foal groups as described above in TSP, TPP and IgG concentration measured by
TIA. Then the correlation was determined between the IgG concentration measured by TIA
and (1) TSP and serum globulin concentration, (2) TPP and plasma globulin concentration,
(3) gammaglobulin concentration and (4) TPPr. In addition the correlation between TSPr and
TPPr, both measured by the same refractometer, was determined. With the aid of a linear
regression line, a formula was determined for calculating (1) what TSP and serum globulin
concentration, (2) what TPP and plasma globulin concentration and (3) what gammaglobulin
concentration was corresponding to an IgG concentration of ≥ 8 g/L measured by TIA. Also
what TPPr corresponded to an IgG concentration of ≥ 4 and ≥ 8 g/L. The reliability of these
calculated values for determining the IgG concentration of a foal was determined using
sensitivity and specificity calculations. Sensitivity and specificity calculations were also used
for testing the reliability of the formula as described above, the SNAP test and GCT.
8
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Statistical Analysis
All results were compiled and analyzed by IBM SPSS Statistics 20. The one way ANOVA with
a 95% confidence interval (CI) was used for determining whether if significant differences
exist between (1) the mean TSP and TPP concentration in foals with an IgG concentration of
< 4 g/L, 4 – 8 g/L and those with a successful passive transfer of immunoglobulins and (2)
between the four different foal groups in IgG and total protein as described above. The level
of significance was set at P <0.05. The correlation between the reference method and the
screening tests as described above and between TSPr and TPPr were analyzed by lineair
regression with a 95% CI. The level of significance was set at P < 0.05. Multiple 2X2 tables
are used to illustrate how many foals should be supplemented with IgG according to TIA and
how many according to the formula and the six screening tests as described above. Then, the
reliability of the formula and experimental screening tests for determining the IgG
concentration of a foal were determined by calculating sensitivity and specificity manually
(table 2).
Table 2 Calculating sensitivity and specificity. Sensitivity: number of animals which are
positive tested by the experimental test of the actual positive animals tested by the reference
method (A/A+C). Specificity: number of animals which are negative tested by the
experimental test of the actual negative animals tested by the reference method (D/B+D).
Turbidimetric
Immunoassay
<4 g/L or
< 8 g/L
Turbidimetric
Immunoassay
≥ 4 g/L or
≥ 8 g/L
Total
A
B
A+B
Negative experimental
test resultsb
C
≥ 4 g/L or ≥ 8 g/L
D
C+D
Total
B+D
A+B+C+D
Positive experimental
test resultsa
< 4 g/L or < 8 g/L
a
b
A+C
IgG support.
No IgG support.
9
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RESULTS
Nine foals (19.6%) had an IgG concentration of < 4 g/L, whereas ten foals (21.7%) an IgG
concentration between 4 and 8 g/L. There were significant differences between the mean TSP
and TPP in foals with an IgG concentration of < 4 g/L, 4 – 8 g/L and those with a successful
passive transfer of immunoglobulins (P <0.001, table 3). There were no significant
differences in IgG concentration measured by TIA (P = 0.422), TSP (P = 0.377) and TPP (P
= 0.412) between the four foal groups, so the 46 foals can be regarded as one group (table 4).
An overview of all experimental screening tests is given in table 5.
Table 3. Mean total serum protein (TSP) and mean total plasma protein (TPP) of foals (N)
with an IgG concentration of < 4 g/L, 4 – 8 g/L and those with a successful passive transfer of
immunoglobulins (≥ 8 g/L).
Foals
N
TSP (g/L)
TPP (g/L)
< 4 g/L
9
37.33
(SD = 4.03)
38.44
(SD = 6.93)
4 – 8 g/L
10
42.90
(SD = 3,48)
44.80
(SD = 3.55)
≥ 8 g/L
27
55.37
(SD = 5,41)
53.74
(SD = 4.38)
Total Mean
46
49.13
Mean
48.80
SD = standard deviation
10
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Table 4. Mean IgG concentration measured by turbidimetric immunoassay and mean total
serum protein (TSP) and total plasma protein (TPP) per foal group measured by chemistry
analyzers.
Foals
N
IgG (g/L)
TSP (g/L)
TPP (g/L)
Healthy foals
> 12 hours of age
16
9,2375
(SD = 3.35)
50,8125
(SD = 7.4)
50,1250
(SD = 5.7)
Healthy foals
< 12 hours of age
13
6,8385
(SD = 4.28)
46,1538
(SD = 8.5)
45,8462
(SD = 6.7)
Diseased foals
12
7,8917
(SD = 5.01)
48,3333
(SD = 11.5)
49,0833
(SD = 11.2
Diseased foals (infusion)
5
9,0400
(SD = 1.42)
53,4000
(SD = 8.7)
51,6000
(SD = 7.2)
Total
46
8,1870
49,1304
48,8043
N = number of foals
SD = standard deviation
11
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Table 3. An overview of all experimental test results.
Experimental test
UniCel
DxC 600
Measuring
TSP1
SGC2
R
0.90
0.84
Cut-of value (g/L)
8 g/L IgG = < 49
8 g/L IgG = < 20
Se (%)
100
84
Sp (%)
96
81
FUJI DRI-CHEM
4000i
TPP3
PGC4
0.83
0.84
8 g/L IgG = < 48
8 g/L IgG = < 20
79
84
100
89
HYDRASYS® 2 SCAN
GGC5
0.94
8 g/L IgG = < 6
95
93
78
89
Formulaa
SNAP Foal IgG
Test Kit
IgG
<8
<4
89
78
93
100
Glutaraldehyde
Coagulation test
IgG
<8
< 5.5
100
100
65
66
SERUM PROTEIN
REFRACTOMETER
SPR-NE
TPP3
8 g/L IgG = 72
4 g/L IgG = 60
74
44
85
100
0.75
For an IgG concentration of ≥ 8 g/L, beta- and gammaglobulin concentration together must be at least 26% of a
total serum protein of 40 – 66 g/L.
a
1
total serum protein
serum globulin concentration
3
total plasma protein
4
plasma globulin concentration
5
gamma-globulin concentration
2
R= correlation coefficient between experimental screening test and IgG concentration measured by TIA as the
reference method.
Se= sensitivity
Sp= specificity
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CORRELATION BETWEEN IGG CONCENTRATION AND TSP
The correlation coefficient between the IgG concentration and TSP was 0.90 (p < 0.001). The
IgG concentration can be estimated by using the following formula: IgG = -11.325 +
(0.397·TSP) with a 95% CI of 0.339-0.456. According to the formula, a TSP of ≥ 48.7 g/L (≈
49 g/L) corresponds to an IgG concentration of ≥ 8 g/L. Sensitivity and specificity would
have been 100 and 96% respectively (table 4).
Table 4. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 8 g/L and according to the
concentration of total serum protein (TSP) with a cut-of value of < 49 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
TSP
< 49 g/L
≥ 49 g/L
19
0
19
Total
CORRELATION BETWEEN
CONCENTRATION
IGG
1
26
27
CONCENTRATION
Total
20
26
46
AND
SERUM
GLOBULIN
The correlation coefficient between the IgG concentration and serum globulin concentration
(SGC) was 0.84 (p < 0.001). The IgG concentration can be estimated by using the following
formula: IgG = -1.142 + (0.465·SGC) with a 95% CI of 0.375-0.555. According to the
formula, a SGC of ≥ 19.7 g/L (≈ 20 g/L) corresponds to an IgG concentration of ≥ 8 g/L.
Sensitivity and specificity would have been 84 and 81% respectively (table 5).
Table 5. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 8 g/L and according to the serum
globulin concentration (SGC) with a cut-of value of < 20 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
SGC
Total
< 20 g/L
≥ 20 g/L
16
3
19
5
22
27
Total
21
25
46
13
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CORRELATION BETWEEN IGG CONCENTRATION AND TPP
The correlation coefficient between the IgG concentration and TPP was 0.83 (p < 0.001). The
IgG concentration can be estimated by using the following formula: IgG = -12.407 +
(0.422·TPP) with a 95% CI of 0.336-0.508. According to the formula, a TPP of ≥ 48.4 g/L (≈
48 g/L) corresponds to an IgG concentration of ≥ 8 g/L. Sensitivity and specificity would
have been 79 and 100% respectively (table 6).
Table 6. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 8 g/L and according to the
concentration of total plasma protein (TPP) with a cut-of value of < 48 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
TPP
< 48 g/L
≥ 48 g/L
15
4
19
Total
CORRELATION BETWEEN
CONCENTRATION
IGG
Total
0
27
27
CONCENTRATION
15
31
46
AND
PLASMA
GLOBULIN
The correlation coefficient between the IgG concentration and plasma globulin concentration
(PGC) was 0.84 (p < 0.001). The IgG concentration can be estimated by using the following
formula: IgG = -2.655 + (0.528·PGC) with a 95% CI of 0.423-0.632. According to the
formula, a PGC of ≥ 20.2 g/L (≈ 20 g/L) corresponds to an IgG concentration of ≥ 8 g/L.
Sensitivity and specificity would have been 84 and 89% respectively (table 7).
Table 7. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 8 g/L and according to the plasma
globulin concentration (PGC) with a cut-of value of < 20 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
PGC
Total
< 20 g/L
≥ 20 g/L
16
3
19
3
24
27
Total
19
27
46
14
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CORRELATION
BETWEEN
CONCENTRATION
IGG
CONCENTRATION
AND
GAMMAGLOBULIN
The correlation coefficient between the IgG concentration and gammaglobulin concentration
(GGC) was 0.94 (p < 0.001). The IgG concentration can be estimated by using the following
formula: IgG = 2.965 + (0.907·GGC) with a 95% CI of 0.806-1.007. According to the
formula, a gammaglobulin concentration of ≥ 5.55 g/L (≈ 6 g/L) corresponds to an IgG
concentration of ≥ 8 g/L. Sensitivity and specificity would have been 95 and 93% respectively
(table 8). Figure 1 illustrates the comparison between the IgG concentration and the
gammaglobulin concentration. In 45 of 46 foals the IgG concentration was higher as the
gammaglobulin concentration (figure 1).
Table 8. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 8 g/L and according to the
gammaglobulin concentration (GGC) with a cut-of value of < 6 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
GGC
Total
< 6 g/L
≥ 6 g/L
18
1
19
2
25
27
Total
20
26
46
Figure 1. Difference between IgG concentration in g/L measured by turbidimetric
immunoassay (TIA) and gammaglobulin concentration (GGC) in g/L (y-axis). In 45 of 46
foals (x-axis), IgG concentrations were higher as the gammaglobulin concentration.
18
16
14
12
10
TIA
8
GGC
6
4
2
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
15
-
FORMULA FOR ESTIMATING THE IGG CONCENTRATION
Thirty-six of 46 foals had a total serum protein of 40 – 66 g/L. Sensitivity and specificity of
this formula were 78 and 89% respectively (table 9). Meaning that 78% of the foals who had
an IgG concentration of < 8 g/L had a beta- and gammaglobulin concentration of < 26% of
the total protein and 89% of the foals who had an IgG concentration of ≥ 8 g/L, had a betaand gammaglobulin concentration of ≥ 26% of the total protein. This formula estimated the
right IgG concentration in 86% of the foals.
Table 9. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 8 g/L and according to the
following formula: IgG support is needed when beta- and gammaglobulin concentration
(BGGC) together are less than 26% of a total serum protein (TSP) of 40 – 66 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
BGGC
Total
< 26% of TSP
≥ 26% of TSP
7
2
9
3
24
27
Total
10
26
36
16
-
SNAP FOAL IGG TEST KIT
The results of the SNAP test were similar to those obtained by TIA in 87% of the foals.
According the SNAP test, 12 foals had an IgG concentration between ≥ 4 and < 8 g/L. TIA
measured an IgG concentration between ≥ 4 and < 8 g/L in eight of these 12 foals (67%). Two
foals had an IgG concentration < 4 g/L and two foals ≥ 8 g/L (table 10). For an IgG cut-off
value of 8 g/L, sensitivity and specificity of the SNAP test were 89 and 93% respectively
(table 11). For an IgG cut-off value of 4 g/L, sensitivity and specificity of the SNAP test were
78 and 100% respectively (table 12).
Table 10. Illustrates number of foals divided into three categories of IgG concentrations
measured by turbidimetric immunoassay and SNAP Foal IgG Test Kit (SNAP).
Turbidimetric immunoassay
< 4 g/L
SNAP 4 – 8 g/L
≥ 8 g/L
Total
< 4 g/L
7
2
0
9
4 – 8 g/L
0
8
2
10
≥ 8 g/L
0
2
25
27
Total
7
12
27
46
Table 11. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 8 g/L and according to the SNAP
Foal IgG Test Kit (SNAP) with a cut-of value of < 8 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
SNAP
< 8 g/L
≥ 8 g/L
Total
17
2
19
2
25
27
Total
19
27
46
Table 12. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with a IgG cut-of value of < 4 g/L and according to the SNAP
Foal IgG Test Kit (SNAP) with a cut-of value of < 4 g/L.
Turbidimetric immunoassay
< 4 g/L
≥ 4 g/L
SNAP
Total
< 4 g/L
≥ 4 g/L
7
2
9
2
37
37
Total
7
39
46
17
-
GLUTARALDEHYDE COAGULATION TEST
The results of the GCT were similar to those obtained by TIA in 66% of the foals (table 13).
For an IgG cut-off value of 8 g/L, sensitivity and specificity of the GCT were 100 and 65%
respectively (table 14). For an IgG cut-off value of 5.5 g/L, sensitivity and specificity of the
GCT were 100 and 66% respectively (table 15).
Table 13. Illustrates number of foals divided into three categories of IgG concentrations
measured by turbidimetric immunoassay and glutaraldehyde coagulation test (GCT).
Turbidimetric immunoassay
GCT
< 5.5 g/L
5.5 – 8 g/L
≥ 8 g/L
Total
< 5.5 g/L
12
0
0
12
5.5 – 8 g/L
6
0
0
6
≥ 8 g/L
5
4
17
26
Total
23
4
17
44
Table 14. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with a IgG cut-of value of < 8 g/L and according to the
glutaraldehyde coagulation test(GCT) with a cut-of value of < 8 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
GCT
< 8 g/L
≥ 8 g/L
Total
18
0
18
9
17
26
Total
27
17
44
Table 15. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with a IgG cut-of value of < 5.5 g/L and according to the
glutaraldehyde coagulation test (GCT) with a cut-of value of < 5.5 g/L.
Turbidimetric immunoassay
< 5.5 g/L
≥ 5.5 g/L
GCT
Total
< 5.5 g/L
≥ 5.5 g/L
12
0
12
11
21
32
Total
23
21
44
18
-
CORRELATION BETWEEN IGG CONCENTRATION AND TPPR
The correlation coefficient between TSPr and TPPr was 0.97 (p < 0.001). Indicating that the
refractometer could also be performed with plasma. The correlation coefficient between the
IgG concentration and TPPr was 0.75 (p < 0.001). The IgG concentration can be estimated by
using the following formula: IgG = -16.217 + (0.338·TPPr), with a 95% CI of 0.250-0.427.
According to the formula, a TPPr of ≥ 71.6 g/L (≈ 72 g/L) corresponded to an IgG
concentration of ≥ 8 g/L. Sensitivity and specificity would have been 74 and 85% respectively
(table 16). A TPPr of ≥ 59.8 g/L (≈ 60 g/L) corresponded to an IgG concentration of ≥ 4 g/L
and sensitivity and specificity would have been 44 and 100% respectively (table 17).
Table 16. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 8 g/L and according to the
refractometer with a total plasma protein (TPPr) cut-of value of < 72 g/L.
Turbidimetric immunoassay
< 8 g/L
≥ 8 g/L
TPPr
< 72 g/L
≥ 72 g/L
Total
14
5
19
4
23
27
Total
18
28
46
Table 17. Illustrates number of foals who should receive IgG support according to the
turbidimetric immunoassay with an IgG cut-of value of < 4 g/L and according to the
refractometer with a total plasma protein (TPPr) cut-of value of < 60 g/L.
Turbidimetric immunoassay
< 4 g/L
≥ 4 g/L
TPPr
Total
< 60 g/L
≥ 60 g/L
4
5
9
0
37
37
Total
4
42
46
19
-
DISCUSSION
In the veterinary practice, several quantitative and semi-quantitative diagnostic tests are used
for the assessment of passive immunity in foals 3, 7, 8, 14, 17. It seemed useful to know what
screening test is the most accurate for use in the field and which one for hospitalized foals.
Because RID was not available, TIA has been used as the standard laboratory method for
determination of plasma IgG concentration. TIA is reliable as the RID for measuring plasma
IgG concentration and is acceptable for use as a standard reference laboratory method 1, 9. TIA
can be used with samples of either serum or plasma. The coagulation factors in plasma do not
affect the measured IgG concentration 6. In this study, TIA was performed with plasma
because this can be obtained more quickly than serum when results are needed as soon as
possible. The non-refrigerated transport and the fact that some plasma samples were older
than other plasma samples when arrived in Newmarket, do not influence the results of TIA 6.
TIA correlated well with TSP, but less with TPP. The fact that UniCel DxC 600 was
performed using serum and FUJI DRI-CHEM4000i was performed using plasma could
explain the lower correlation coefficient between TIA and TPP. Based on our study, TSP
seems to be an useful alternative for the TIA with a cut-off value of < 49 g/L for hospitalized
foals since sensitivity and specificity were high. TSP concentration was significantly
different between foals with a IgG concentration < 4 g/L, 4 – 8 g/L and those with a
successful transfer of passive immunity. TSP has been found to be an unreliable indicator of
hypogammaglobulinemia in foals 18-20. Dehydration and variable serum albumin
concentrations in hypogammaglobulinemic foals can produce a TSP within the reference
range for normal foals. TSP is a poor sole indicator of FPT in foals, but may be useful as an
adjunctive test according other authors 7, 17. TSP might not be able to accurately determine the
IgG-status of the foal, but according to our results any foal after 18 hours of age with a TSP of
< 49 g/L is highly likely to have (P)FPT. These findings corresponds largely with TylerMcGowan et al. 21. TSP can be performed in combination with a more reliable test such as the
protein spectrum for the gammaglobulin concentration, in our study the most reliable
screening test for determining the IgG status of the foal. Almost all IgG concentrations
measured by TIA were higher as the gamma-globulin concentrations. Probably, γ-fraction
does not contain all IgG’s and part of the IgG concentration must be somewhere else as taken
into consideration in the clinical emergency calculations for FPT as described above as the
formula. Most but not all immunoglobulins are found in the γ-fraction which can be
differentiated into у1 and у2 13. IgG is primarily found in the у2-fraction. IgA, IgM and IgE
are primarily found in the у1-fraction and to some extent in the β2-fraction, indicating that a
part of the IgG concentration might also be in the β-fraction.
Serum and plasma globulin concentrations alone seems not the best methods for predicting
the foal’s IgG concentration, which is in agreement with the study of Metzger et al. 7. The
same applies for the formula used by the Faculty of Utrecht University based on unpublished
data. They all can be used for making a rough estimation of the IgG concentrations, but a
definitive diagnose should be made with the aid of a more reliable test such as the
electrophoresis for the gammaglobulin concentration.
20
-
The SNAP test seems accurate in identifying foals with a successful passive transfer of
immunoglobulins and foals with FPT. This stall-side screening test seems not reliable when
measuring an IgG concentration between ≥ 4 and < 8 g/L. A large deviation was seen from
the actual IgG concentration measured by the reference method. So the SNAP test fails in the
category in which it is hard to estimate the status of the foals clinically. An explanation of this
unreliability could be the interpretation of the color of the sample spot. These SNAP test
results were similar as the SNAP test results of other authors and makes the test less useful for
hospitalized foals 3, 8
The GCT is only reliable when a gel is formed within 30 minutes for hospitalized foals and
within 50 minutes for healthy low risk foals in the field. According to C.M. de Bruijn et al.,
the GCT is a suitable test for determining the serum gammaglobulin concentration in foals
when using the 5% glutaraldehyde solution14. In our study, many foals were tested falsepositive when no gel was formed within 30 or 50 minutes. The difference in specificity could
be explained by the used reference method. C.M. de Bruijn et al. compared the GCT results
with serum gammaglobulin concentrations obtained by electrophoresis. Our study indicated
that a gammaglobulin concentration of ≥ 6 g/L was equivalent to an IgG concentration of ≥ 8
g/L and part of the IgG concentration must be in the β-fraction as well 22. If the tests results
were compared with the gammaglobulin concentration instead of the IgG concentration
measured by TIA, 82% of the foals had similar values instead of 66%. Disadvantages of the
GCT is the use of serum and the IgG cut-off value of 5.5 g/L instead of 4 g/L. When 4 g/L
was used as IgG cut-off value, getting the test result would have been more than one hour 14.
This study indicated that some healthy foals with an IgG concentration of < 5.5 g/L but ≥ 4
g/L would have been treated, while there was no need for an IgG supportive therapy.
In previous articles, refractometry is performed with serum and the conclusions are therefore
based on serum 3, 18, 23. We showed a good correlation between TPPr and TSPr, both measured
by the same refractometry. But the correlation between TIA and TPPr whereas others consider
refractometry an unreliable indicator of FPT 3, 18.
CONCLUSIONS
According to our study, the most reliable clinical alternative test for determining the IgG
concentration would be measuring TSP by a chemistry analyzer in combination with
gammaglobulin concentration. A TSP of ≥ 49 g/L and a gammaglobulin concentration of ≥ 6
g/L indicates an IgG concentration of ≥ 8 g/L. If the immunoglobulin concentration has to be
known in healthy foals in the field, SNAP test can be an alternative test if its weaknesses are
recognized.
21
-
CONFLICT OF INTEREST
None of the authors of this paper has a financial or personal relationship with other people or
organizations that could inappropriately influence or bias the content of the paper.
ACKNOWLEDGMENTS
Dr. J. van der Broek for the advice on statistical analysis.
J.P.H.M. Vossen for the advice on the laboratory analysis.
Veterinary Center ‘De Vallei’ (Woudenberg), Veterinary Practice ‘Gaasterland’ (Balk) and
Veterinary Equine Center ‘Kootwijkerbroek’ for contributing in sample collection.
FOOTNOTES
a
BD Vacutainer® tubes, BD Biosciences, postbus 2130, 4800 CC Breda, The Netherlands.
b
BD Vacutainer® Blood Collection Accessories, BD Biosciences, postbus 2130, 4800 CC
Breda, The Netherlands.
c
Turbidimetric immunoassay; ILab IL650 automated biochemistry analyser, Beaufort Cottage
Laboratories, Suffolk CB8 8JS, Newmarket.
d
Anti-equine IgG antisera, Polyclonal Antibodies Blauenwaun Farm. Ffostrasol, Near
Llandysul, Dyfed SA44 5 JT.
e
Equine IgG Standard, Beaufort Cottage Laboratories, Suffolk CB8 8JS, Newmarket.
f
UniCel DxC 600, Beckman Coulter Nederland B.V., Pelmolenlaan 3447 GW, Woerden.
g
HYDRASYS® 2 SCAN, Sebia Electrophoresis, Corporate Drive GA 30093, Norcross.
h
FUJI DRI-CHEM 4000i, Fujifilm Corporation, Tokyo.
i
SNAP Foal IgG Test Kit, IDEXX Laboratories Inc, Maine 04092, Westbrook.
j
Glutaraldehyde coagulation test
k
SERUM PROTEIN REFRACTOMETER SPR-NE, Atago Co. LTD., Tokyo.
22
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