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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 12 - 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 - 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 - 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 - REFERENCES 1. 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