Tacrolimus (FK506) Levels in Transplants by two Methods

COMPARISON BETWEEN WHOLE BLOOD TACROLIMUS BY ABBOTT IMx AND PLASMA TACROLIMUS BY ENZYME- LINKED IMMUNOASSAY.

S. Zuckerman, T. McKaveney, S. Mehta, K. Velmer, J. Chao, R. Venkataramanan, A. Jain, V. Warty and (Depts. of Path. and Surg., Univ. of Pittsburgh School of Medicine, Pittsburgh, PA 15213)

Tacrolimus is a potent immunosuppressive agent used in preventing organ rejection. Due to its narrow therapeutic index and highly variable pharmacokinetics, routine monitoring of tacrolimus concentrations in blood/plasma is required. We compared two distinct methodologies, Abbott IMx for whole blood and enzyme-linked immunosorbent assay (ELISA) for plasma using multiple trough samples from 15 patients who underwent liver, kidney of heart transplantation. The IMx whole blood tacrolimus assay is based on micro-particle enzyme immunoassay technology . Its linearity range is 5-60 ng/mL with an inter-assay CV of 9.6% and intra-assay CV of 9.9%. The ELISA method for plasma tacrolimus requires a solid-phase extraction by Sep-pak before analysis. The assay linearity is 0.1-8.0 ng/mL with an inter-assay CV of 17% and intra assay CV of 7.1%. The therapeutic range for whole blood tacrolimus is between 10-20 ng/mL whereas for plasma the patients were maintained between 0.5-2.0 ng/mL . The comparison between these two methods resulted in following correlations. For Liver Tx patients, IMx =5.5 ELISA + 3.6, r2 =0.48 (n=101); for kidney Tx patients, IMx =8.9 ELISA +8.2, r2 =0.76 (n=48) and for heart Tx patients, IMx =9.8 ELISA + 3.2, r2 =0.72 (n=53). The average blood to plasma ratio of tacrolimus is 8.6 +/- 3.8 in liver, 15.1 +/- 5.3 in kidney and 14.7 +/- 4.9 in heart transplant patients, suggesting that this ratio is dependent on the nature of the organ transplanted. In liver patients this ratio tends to be lower perhaps due to accumulation of tacrolimus metabolites which do not appreciably partition in to red blood cells, s but tend to accumulate in plasma. Our study indicates that it is inappropriate to extrapolate blood tacrolimus concentrations based on its corresponding plasma value, due to poor correlation. IMx whole blood assay for tacrolimus is automated with a quick turn around time. The correlation between Tacrolimus whole blood levels as measured by IMx and clinical status of the patient is being currently evaluated.

Introduction

Tacrolimus is a potent immunosuppressive drug approved by FDA in 1994.
Routine therapeutic monitoring of tacrolimus is essential because of its:
- narrow therapeutic index
- large inter-individual variability in kinetics
Assay Methodologies currently used include
- IMx - Whole blood
- ELISA - Plasma

Objective

To compare the whole blood tacrolimus concentrations as measured by IMx to plasma tacrolimus concentrations as measured by ELISA in liver, kidney, and heart transplant patients during the immediate post- operative period.

Methods

Clinical protocol

Six livers, four kidney and four heart transplant patients participated in this study.
Patients received tacrolimus 0.1 mg/kg/day intravenously. for up to 3-7 days post transplant.
Once able to tolerate oral intake, patients received tacrolimus 0.1-0.24 mg/kg/day twice daily.
Daily blood samples were collected in heperinized tubes prior to morning dose of tacrolimus.

Analysis

Whole blood was maintained at 4C until IMx analysis( 1 ).
Plasma was separated from the blood that was incubated at 37C for 45 min. prior to centrifugation, and maintained at -20C until analyzed by ELISA( 2 ).
A minimum of ten blood/plasma specimens were analyzed for each of the patients.

Table I : Analytical methods and characteristics for measurement of Tacrolimus.

Type of assay	IMx	ELISA
Technology	Semi automated	Manual
Biological fluid	blood	plasma
Extraction	precipitation	solid phase/methanol
Detection	colorimetric	colorimetric
Sample volume (ul)	100	100
Performance time (hrs.)	0.6	6
Dynamic range (ng/ml)	5-60	0.1-8.0
Intra-day variation (% )	9.9	7.1
Inter-day variation (% )	9.6	17.0
Therapeutic range(ng/mL)	10-20	0.5-2.0

Table II Biochemical profiles in kidney, liver and heart transplant patients.

	Creatinine(mg/dL)		Bilirubin(mg/dL)		ASTa(IU/L)		ALTb(IU/L)
Patient	I	II	I	II	I	II	I	II
Kidney
cw	3.6	1.7	0.4	0.3	66	20	38	66
kw	1.2	1.2	0.9	0.7	712	33	476	89
jg	0.8	2.9	0.5	0.8	46	18	34	42
pc	2.3	2.2	0.7	0.6	20	44	27	140
Liver
rc	4.1	3.3	8.0	2.9	101	90	93	96
gb	2.7	4.4	8.5	1.6	6605	24	996	31
iz	2.4	1.4	13.4	14.3	1247	91	644	127
lt	1.6	1.3	14.5	26.3	863	136	725	495
hh	1.7	1.5	15.6	8.5	5767	96	5463	198
mm	2.7	2.8	3.9	3.0	2075	87	885	121
Heart
hj	1.4	1.6	0.5	0.6	25	14	15	16
gs	2.0	1.8	0.4	0.5	19	16	25	15
kr	2.4	4.3	3.3	1.7	35	57	14	11
kg	4.0	4.0	0.3	0.5	28	20	18	25

Discussion

The blood concentration of tacrolimus is consistently higher than the plasma concentration. The average slope of blood versus plasma in kidney and heart transplant patients is similar (8.9 and 9.8 respectively). However in liver transplant patients the slope appears to be lower (5.5) . This is consistent with our previous study ( 1 ) where we noted that the slope of blood versus plasma using the solid phase extraction with detection by the ELISA method was 11.3 in kidney transplant patients and 4.0 in liver transplant patients. Similar observations have been made by Winkler et.al.( 3 ). The lower blood to plasma ratio in liver transplant patients may be due to preferential accumulation of tacrolimus metabolites in the plasma or impaired red blood cell uptake of tacrolimus in patients with liver dysfunction. Our earlier study (1 ) suggested that the parent tacrolimus is the primary species in the red blood cell and that tacrolimus metabolites do not appreciably partition into the red blood cells, but rather accumulate in the plasma. We have observed an overall poor correlation between the trough blood and trough plasma concentrations (Figures 4-6). Therefore it is inappropriate to extrapolate blood tacrolimus concentrations based on corresponding plasma values.

Conclusions

Whole blood Concentrations are on average 4 to 10 times higher than plasma concentrations
Plasma concentrations measured by ELISA cannot be extrapolated to whole blood concentrations as determined by IMx
IMx technology for tacrolimus measurement is reliable and practical
Further improvement in sensitivity is necessary to monitor transplant patients on an ongoing basis

References

1. Warty VS, Zuckerman S, Venkataramanan R, Lever J, Chao J, Mckaveney T, Fung J and Starzl T. Tacrolimus Analysis: A Comparison of Different Methods and Matrices. Ther. Drug Monitoring (1995) Vol. 17, p159-167.

2. Warty VS, Zuckerman S, Venkataramanan R, Lever J, Fung J and Starzl T. FK506 Measurement: Comparison of Different Analytical Methods. Ther. Drug Monitoring (1993) Vol. 15, p 204-208.

3. Winkler M, Christians U, Stoll K, Baumann J and Pichlmayr R. Comparison of Different Assays for the Quantitation of FK506 Levels in Blood or Plasma. Ther. Drug Monitoring. (1994) Vol.16, p 281-286.

Figures

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