Comparison of hematologic, biochemical, and coagulation parameters in α1,3-galactosyltransferase gene-knockout pigs, wild-type pigs, and 4 primate species

Abstract

Background

The increasing availability of genetically-engineered pigs is steadily improving the results of pig organ and cell transplantation in nonhuman primates (NHPs). Current techniques offer knock-out of pig genes and/or knock-in of human genes. Knowledge of normal values of hematologic, biochemical, coagulation, and other parameters in healthy genetically-engineered pigs and NHPs is important, particularly following pig organ transplantation in NHPs. Furthermore, information on parameters in various NHP species may prove important in selecting the optimal NHP model for specific studies.

Methods

We have collected hematologic, biochemical, and coagulation data on 71 α1,3-galactosyltransferase gene-knockout (GTKO) pigs, 18 GTKO pigs additionally transgenic for human CD46 (GTKO.hCD46), 4 GTKO.hCD46 pigs additionally transgenic for human CD55 (GTKO.hCD46.hCD55), and 2 GTKO.hCD46 pigs additionally transgenic for human thrombomodulin (GTKO.hCD46.hTBM).

Results

We report these data and compare them with similar data from wild-type pigs, and the 3 major NHP species commonly used in biomedical research (baboons, cynomolgus, and rhesus monkeys) and humans, largely from previously published reports.

Conclusions

Genetic modification of the pig (e.g., deletion of the Gal antigen and/or the addition of a human transgene) (i) does not result in abnormalities in hematologic, biochemical, or coagulation parameters that might impact animal welfare, (ii) seems not to alter metabolic function of vital organs, though this needs to be confirmed after their xenotransplantation, and (iii) possibly (though by no means certainly) modifies the hematologic, biochemical, and coagulation parameters closer to human values. The present study may provide a good reference for those working with genetically-engineered pigs in xenotransplantation research and eventually in clinical xenotransplantation.

INTRODUCTION

Pigs have provided a valuable and popular large animal model for biomedical research, especially during the last 3 to 4 decades (1), and are the source-animal of choice for xenotransplantation (2). Pigs offer many similarities to humans in terms of anatomy, physiology, biochemistry, pathology, and pharmacology (2,3) and therefore provide a large animal model to bridge the gap between rodents and humans. Knowledge of normal hematologic and biochemical values in any species used in biomedical research is important. Normal hematologic, biochemical, and physiologic values in several breeds of wild-type (WT, genetically-unmodifed) pigs, e.g., Yorkshire, Yucatan, Landrace, have been reported by several groups (1, 3–5).

With increasing numbers of genetically-engineered pigs becoming available (Table 1), research experience obtained from small animal models (e.g., gene-knockout and/or knock-in technology) can be translated to large animal models. Whereas the ultimate goal is clinical application of cells, tissues, and organs from genetically-engineered pigs for human therapeutic applications (6), it will be critical from a regulatory and safety perspective to have data available on hematologic, biochemical, and physiologic parameters in the source animals.

Table 1.

Common genetically-engineered pigs currently available for biomedical research

Knock-out technology for deletion of antigen expression

- GTKO (α1,3-galactosyltransferase gene-knockout)

Knock-in technology for human complement regulation

- human CD46

- human CD55

- human CD59

Knock-in technology for human thromboregulation

- human CD39

- human thrombomodulin (TBM)

- human endothelial protein c receptor (EPCR)

Pigs with multiple gene modifications exist (e.g., GTKO.hCD46.hCD55 or GTKO.hCD46.hTBM or GTKO.hCD55.hCD59.hCD39.hTBM)

Measurement of these parameters essentially serves two aims, namely assessment of (i) the health status of the animals themselves, which includes the effect of the genetic modification (i.e., gene knockout or knock-in) on the respective parameter, and (ii) any molecular and/or physiologic incompatibilities following a xenogeneic transplant. While the first aim relates to safety, the second relates to the efficacy of a xenotransplantation “product”.

The genetic modification of pigs has been essential to progress in overcoming the barriers to xenotransplantation (7–10). Early experience in the 1990s using pigs transgenic for human decay-accelerating factor (hCD55) showed significantly extended survival of pig kidneys in NHPs (7). Expression of human complement-regulatory transgenes (e.g., CD46, CD55, CD59) is now common in pigs (7,8), as is knockout of the α1,3-galactosyltransferase gene (Table 1) (9). Islets obtained from pigs transgenic for human CD46 when transplanted into diabetic monkeys have demonstrated >1 year normalization of blood glucose and cure of diabetes (11). Casu et al. (12,13) and Graham et al. (14) have reported differences in glucose metabolism between pigs and NHPs; pigs differ from NHPs and humans by having a much lower C-peptide level, and a less rapid response to a glucose challenge and to arginine stimulation.

Extended survival was also achieved with the transplantation of organs from GTKO pigs (15,16). Recently, heart xenograft survival has been extended to 8 months using GTKO pigs expressing human CD46 (GTKO.hCD46) (17).

In our recent experience in liver xenotransplantation (18), we observed that pig alanine transaminase (ALT), but not aspartate transaminase (AST), in GTKO pigs is significantly lower than in WT pigs, but similar to human and baboon levels (19). We hypothesized that there would be other differences in hematologic, biochemical, and coagulation parameters between WT and GTKO pigs. To our knowledge, there is, hitherto, no published report of normal laboratory values of GTKO pigs in the literature.

In the present study, we report normal hematologic, biochemical, and coagulation values in healthy pigs with various genetic modifications. We compared these values with those of WT pigs and 4 primate species - (i) baboons (Papio species), (ii) cynomolgus monkeys (Macaca fascicularis), (iii) rhesus monkeys (Macaca mulatta), and (iv) humans, to identify possible differences and similarities.

MATERIALS AND METHODS

Animals

Genetically-engineered and WT pigs

Genetically-engineered pigs (on a Landrace large white WT background) were obtained from Revivicor Inc. (Blacksburg, VA, USA). There were a total of 71 GTKO pigs (49 females, 22 males), 18 GTKO pigs transgenic for human CD46 (GTKO.hCD46) (14 females, 4 males), 4 GTKO.hCD46 pigs additionally transgenic for human CD55 (GTKO.hCD46.hCD55) (2 females, 2 males), and 2 GTKO.hCD46 pigs transgenic for human thrombomodulin (GTKO.hCD46.hTBM) (2 males). The number of pigs with a GTKO or GTKO.hCD46 background was 95 and 24, respectively. Their mean ages and weights are shown in Table 2.

Table 2.

Normal hematologic, biochemical and coagulation values in healthy GTKO pigs

	Test	Unit	GTKO	N	GTKO.hCD46	N	GTKO.hCD46.hCD55	N	GTKO.hCD46.hTBM	N	Mean	SD	Total N
Age/Weight			154 days (range 10-647) / 79kg (range 3-247)		72 days (range 18–390) / 8kg (range 3-29)		45 days (range 40-54) / 9kg (range 8-10)		38 days (range 34-41) / 7.5kg (6.4-8.7)		132 days (range 10-647) / 69kg (range 3-247)
Hematology	WBC	/mm3	15.9	71	14.8	17	13.3	4	10.7	2	15.5	1.0	94
	RBC	×106/mm3	6.5	71	5.9	17	4.2	4	5.1	2	6.3	1.4	94
	Hemoglobin	g/dL	12.8	71	11.0	17	6.7	4	9.1	2	12.2	2.5	94
	Hematocrit	%	40.3	71	33.3	17	21.9	4	26.4	2	37.9	8.0	94
	Platelets	×103/mm3	432	71	406	17	444	4	310	2	425	210	94
	MCV	fL	63	71	56	17	53	4	52	2	61.3	9.0	94
	MCH	pg	20	71	19	17	18	4	18	2	19.7	2.3	94
	MCHC	g/dL	32	71	33	17	34	4	34	2	32.3	1.9	94
	RDW	%	18.2	2	21.4	10	22.9	4	21.2	2	21.4	3.7	18
	MPV	fL	9.7	2	9.1	7	9.1	4	9.1	2	9.1	0.8	15
	Neutrophil	%	40.0	71	48.4	17	43.5	4	46.0	2	41.8	13.8	94
	Lymphocyte	%	51.7	71	40.9	17	38.5	4	46.0	2	49.1	15.5	94
	Monocyte	%	5.1	71	4.6	17	4.3	4	2.5	2	4.9	3.1	94
	Eosinophil	%	2.6	71	1.5	17	0.8	4	1.0	2	2.3	2.5	94
	Basophil	%	0.1	71	0.0	17	0.5	4	0.0	2	0.1	0.3	94
Renal Function and Electrolytes	Sodium	mmol/L	146	61	140	18	136	4	140	2	144	7	85
	Potassium	mmol/L	5.6	61	5.1	18	3.3	4	3.8	2	5.3	1.1	85
	Chloride	mmol/L	104	61	101	18	99	4	106	2	103.0	8.0	85
	Calcium	mg/dL	11.0	61	10.4	18	10.0	4	9.7	2	10.8	0.8	85
	Phosphorus	mg/dL	8.9	61	8.5	16	9.2	4	8.5	2	8.8	1.8	83
	CO2	mmol/L	28	61	28	11	30	4	26	2	28	5	78
	Urea	mg/dL	14.6	61	8.8	17	5.8	4	5.5	2	12.8	9.9	84
	Creatinine	mg/dL	1.2	61	0.9	17	0.5	4	0.7	2	1.1	0.5	84
Liver Function	AST	IU/L	38	71	36	18	30	4	36	2	37	29	95
	ALT	IU/L	55	2	40	18	47	4	40	2	42	15	26
	ALP	IU/L	145	2	249	18	409	4	324	2	272	146	26
	LDH	IU/L	559	2	471	9	433	4	468	2	472	109	17
	GGT	IU/L	78	61	65	16	58	4	47	2	74	16	83
	Tot Bilirubin	mg/dL	0.2	61	0.1	9	0.2	4	0.2	2	0.2	0.2	76
	Dir Bilirubin	mg/dL	0.1	61	0.1	9	0.1	4	0.1	2	0.1	0.1	76
	Indir Bilirubin	mg/dL	0.1	61	0.1	9	0.1	4	0.1	2	0.1	0.1	76
	Tot Protein	g/dL	6.2	61	5.5	18	3.9	4	3.8	2	5.9	1.4	85
	Albumin	g/dL	3.8	61	2.8	18	1.3	4	1.3	2	3.4	1.1	85
	Cholesterol	mg/dL	98	2	85	16	62	3	59	2	81	22	23
	Triglyceride	mg/dL	14	2	39	16	14	3	11	2	31	22	23
	Glucose	mg/dL	90	71	93	18	75	4	66	2	89	27	95
Coaulation Profile	PT	sec	14.4	2	13.6	9	13.9	4	13.2	2	13.6	0.5	17
	PTT	sec	32.5	2	34.8	9	35.8	4	29.3	2	34.1	14.0	17
	INR		1.1	2	1.1	9	1.1	4	1.1	2	1.1	0.1	17
	D-Dimer	μg/mL	0.22	2	0.22	9	0.22	4	0.14	2	0.20	0.05	17
	FDP	μg/mL	5to20	2	<5	9	5to20	4			5to20		15
	Fibrinogen	mg/dL	265	71	197	9	156	4	143	2	250	121	86
Others	Amylase	IU/L	459	2	1292	16	2941	4	2780	2	1622	1130	24
	Lipase	IU/L			14	14	10	4	11	2	13.1	13.2	20
	Iron	μg/dL			110	9	79	3	121	2	104.0	48.0	14
	Total CPK	IU/L	1209	59	1228	8	510	4	652	2	1166	2305	73
	CPK-MB isoenzyme	ng/mL			1.2	1	5.1	4	4.8	1	4.4	4.0	6
	CPK-MB relative index				0.1	1	0.9	4	0.6	1	0.7	0.5	6
	Troponin I	ng/mL			0.10	1	0.10	4	0.10	1	0.11	0.02	6

Wild-type (Landrace large white) pigs (n=19; 9 females, 10 males) were obtained from Country View Farm, Schellsburg, PA, USA. Their mean ages and weights are shown in Table 3.

Table 3.

Normal hematologic, biochemical and coagulation values in healthy WT pigs

	Test	Unit	Ekser (2012)	N	Klem* (2010)	N	Egeli* (1998)	N	Rispat* (1993)	N	Hannon* (1990)	N	Mean	SD	Total N
Type /Weight			Landrace		Landrace		Landrace		Yucatan		Yorkshire
			12.7 kg		30-50 kg		12.2 kg		13.8 kg		21.3 kg
Hematology	WBC	/mm3	16.0	19	27.3	99	13.4	60	14.9	135			18.6	6.4	313
	RBC	×106/mm3	5.1	19	7.4	99	5.8	60	7.34	135			6.9	1.1	313
	Hemoglobin	g/dL	9.3	19	12.0	99	10.1	60	8.9	135	8.5	26	10.0	1.4	339
	Hematocrit	%	26.3	19	39.0	99	32.0	60	47.0	135	27.0	69	37.9	8.7	382
	Platelets	×103/mm3	261	19	549	99			510	135			507	156	253
	MCV	fL	51	19	54	99	57	60	64	135			59	6	313
	MCH	pg	18	19			18	60	17	135			17	1	214
	MCHC	g/dL			31	99	31	60	19	135			25	7	294
	RDW	%			18	99	20.6	60					19.0	1.8	159
	MPV	fL
	Neutrophil	%			37	99	41	60	54	135			45.6	8.9	294
	Lymphocyte	%			51	99	51	60	39	135			45.5	6.9	294
	Monocyte	%			6.5	99	1.6	60	4.7	135			4.7	2.5	294
	Eosinophil	%			2.6	99	1.6	60	1.6	135			1.9	0.6	294
	Basophil	%			0.7	99	0.9	60	0.06	135			0.4	0.4	294
Renal Function and Electrolytes	Sodium	mmol/L	139	19	149	101	143	60	140	127	138	35	143	4	342
	Potassium	mmol/L	3.9	19	6.3	101	4.3	60	5.5	127	4.4	35	5.3	1.0	342
	Chloride	mmol/L	103	19	106	101	107	60	103	127	106	17	104.8	1.9	324
	Calcium	mg/dL			11.2	101	11.2	60	10.9	127	9.6	15	11.0	0.8	303
	Phosphorus	mg/dL			10.8	101	10.2	60	6.7	127	4.0	17	8.6	3.2	305
	CO2	mmol/L	29	19									29	4	19
	Urea	mg/dL	10	19	8.7	101	5.9	60	19.2	127	8.9	17	12.4	5.1	324
	Creatinine	mg/dL	1.0	19	1.2	101	1.0	60	0.9	127	1.0	17	1.0	0.1	324
Liver Function	AST	IU/L	41	19	46	101	32	60	39	127			40	6	307
	ALT	IU/L	79	19	68	101	39	60	57	127			58	17	307
	ALP	IU/L	140	19	211	101	824	60	58	127			263	349	307
	LDH	IU/L	140	19	795	101	1207	60	773	127			826	440	307
	GGT	IU/L	50	19	50	101	29	60					43	12	180
	Tot Bilirubin	mg/dL	0.1	19	1.0	101	0.1	60	0.3	127			0.5	0.4	307
	Dir Bilirubin	mg/dL	0.1	19									0.1	0.0	19
	Indir Bilirubin	mg/dL	0.1	19									0.1	0.0	19
	Tot Protein	g/dL	3.7	19	5.8	101	4.8	60					5.2	1.1	180
	Albumin	g/dL	1.6	19	2.5	101	3.0	60			2.5	40	2.6	0.6	220
	Cholesterol	mg/dL			120	101	116	60	78	127			101	23	288
	Triglyceride	mg/dL			80	101	80	60	29	127			58	29	288
	Glucose	mg/dL	62	19	115	101	121	60	67	127	83	33	92	27	340
Coaulation Profile	PT	sec							11.7	135			11.7	0.6	135
	PTT	sec							15.5	135			15.5	1.2	135
	INR
	D-Dimer	μg/mL
	FDP	μg/mL
	Fibrinogen	mg/dL
Others	Amylase	IU/L
	Lipase	IU/L
	Iron	μg/dL			173	101	179	60					175.2	4.2	161

^*Klem et al (4), Egeli et al (5), Rispat et al (3), Hannon et al (1).

Baboons

All baboons used in our own studies (n=45; 13 females and 32 males) were obtained from the University of Oklahoma Health Sciences Center (Oklahoma City, OK, USA). Their mean age was 2.7±0.5 (range 1.8–3.6) years and mean weight was 8.5±2.0 (range 5.6–15.9) kg, respectively.

All animal care was in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). Protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Blood collection and tests

Blood was collected when the animals were surgically and immunologically naïve. Animals were sedated by an intramuscular injection of 5–10mg/kg of ketamine hydrochloride (Fort Dodge, IA USA). Blood samples were collected by venepuncture for hematologic (EDTA tube), biochemical (plain tube), and coagulation (sodium citrate tube) analysis using standard methods either in the Central Laboratory of Presbyterian Hospital of the University of Pittsburgh Medical Center, Pittsburgh, PA, USA or of Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA.

Equipment used at the University of Pittsburgh and Virginia-Maryland Regional College were, respectively, Beckman LH750 (Fullerton, CA) and Siemens ADVIA 2120 (Tarrytown, NY) for hematologic values, Diagnostic Stago STAR Evolution (Parsippany, NJ) for coagulation parameters, and Beckman DXC 800 (Fullerton, CA) and Olympus America AU400 (Melville, NY) for biochemical parameters.

Literature search and collection of data

A literature search was carried out to identify significant reports on normal values of various parameters in healthy WT pigs, baboons, cynomolgus monkeys, and rhesus monkeys. Published reports detailed normal values in different species and considered factors such as (i) gender, (ii) age, (iii) weight, and (iv) diet. We have not subdivided the data by age, etc., as we wished to compare our data with a large number of animals from each species, as this is how normal human ranges are reported. We have included data from the literature on various parameters from a large number of WT pigs or NHPs. Normal human values and ranges were obtained from the Central Laboratory of Presbyterian Hospital of the University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Data and statistical analyses

Data analyses were conducted with GraphPad Prism v5.01 (La Jolla, CA, USA). Mean values of sample subsets were calculated and compared using the Student t-test, with a p value of <0.05 being considered statistically significant.

RESULTS

Normal values obtained from healthy GTKO pigs with or without added transgenes from our own study are shown in Table 2. Table 3 shows normal values in different breeds of WT pigs, such as Landrace, Yucatan, and Yorkshire, from our own center and from published studies. Healthy naïve baboon normal values from our own center and from the literature are shown in Table 4. Normal values for healthy cynomolgus and rhesus monkeys from the literature are shown in Tables 5 and 6, respectively. Table 7 compares data on GTKO and WT pigs and from baboons and monkeys with normal human values.

Table 4.

Normal hematologic, biochemical and coagulation values in healthy baboons (Papio species)

	Test	Unit	Ekser (2012)	N	Schuurman* (2004)	N	Havill* (2003)	N	Harewood* (1999)	N	Hainsey* (1993)	N	Mean	SD	Total N
Age / Weight			2.7 years (8.5 kg)		7.7-8.9 kg		0-12 Months		not available		4.4 years
Hematology	WBC	/mm3	7.5	45	7.9	85	11.6	109	10.2	1024	9.6	89	10.0	1.7	1352
	RBC	×106/mm3	5.0	45	5.1	85	5.2	110	5.1	1024	5.0	90	5.1	0.1	1354
	Hemoglobin	g/dL	12.4	45	13.0	85	13.2	109	12.6	1023	12.6	89	12.7	0.3	1351
	Hematocrit	%	37.7	45	40.2	85	40.8	109	40	1024	38.2	89	39.9	1.3	1352
	Platelets	×103/mm3	295	45	368	85	410	110	439	1020	316	89	419	61	1349
	MCV	fL	76	44	79	85	78	110	77	1024	77	90	77	1	1353
	MCH	pg	25	44	26	85	25	110	25	1023	25	90	25	0	1352
	MCHC	g/dL	33	44	32	85	33	110			33	90	33	0	329
	RDW	%	12.7	37			13.5	110			12.8	90	13.1	0.4	237
	MPV	fL	8.6	37			8.7	60			8.3	89	8.5	0.2	186
	Neutrophil	%	54	45	40	85	52	91			62	90	52.0	9.1	311
	Lymphocyte	%	38	45	56	85	45	110			36	90	44.4	9.0	330
	Monocyte	%	5	45	3	85	3	103			2	90	3.2	1.1	323
	Eosinophil	%	1	44	1	85	1	97			1	90	0.9	0.1	316
	Basophil	%	1	44	0	85	1	88			0	90	0.3	0.5	307
Renal Function and Electrolytes	Sodium	mmol/L	146	40	150	67	144	103	146	1044	149	25	146	2	1279
	Potassium	mmol/L	3.7	40	5.0	67	4.3	104	3.7	1045	3.9	25	3.8	0.5	1281
	Chloride	mmol/L	107	40	108	67	110	104	108	1042	99	24	108	4	1277
	Calcium	mg/dL	10.0	39	10.2	67			9.5	1034	9.0	25	9.5	0.5	1165
	Phosphorus	mg/dL	5.2	35	7.2	67			4.1	1034	2.9	25	4.3	1.8	1161
	CO2	mmol/L	27	35			20	103	23	1040	24	25	23	3	1203
	Urea	mg/dL	15	40	22	67	12	103	14	1042	14	25	14	4	1277
	Creatinine	mg/dL	0.7	40	0.93	67	0.7	104	0.7	1046	1	25	0.7	0.1	1282
Liver Function	AST	IU/L	37	40	31	67	46	106	36	1037	39	25	37	6	1275
	ALT	IU/L	31	40	39	67	33	106	40	1042	45	25	39	6	1280
	ALP	IU/L	832	40			1221	104	581	1036	248	25	638	411	1205
	LDH	IU/L	287	35	287	37					276	25	284	6	97
	GGT	IU/L	59	38	74	67			36	1043	39	25	39	18	1173
	Tot Bilirubin	mg/dL	0.2	40	0.23	67			0.11	1023	0.2	25	0.1	0.1	1155
	Dir Bilirubin	mg/dL	0.1	36									0.1	0.1	36
	Indir Bilirubin	mg/dL	0.1	36									0.1	0.1	36
	Tot Protein	g/dL	6.5	40	7.02	67	6.3	106	6.9	1043	7.1	25	6.8	0.3	1281
	Albumin	g/dL	3.8	40	4.39	67	3.8	103	4.1	1042	3.5	25	4.1	0.3	1277
	Cholesterol	mg/dL	122	36	133	67	134	106	97	948	99	25	103	18	1182
	Triglyceride	mg/dL	50	35	51	67			61	933	66	25	60	8	1060
	Glucose	mg/dL	86	40	88	67	83	106	99	1046	83	24	96	7	1283
Coaulation Profile	PT	sec	16	34	12	6					13	32	14.3	1.9	72
	PTT	sec	34	34	28	6					32	32	32.6	3.1	72
	INR		1.3	34									1.3	0.1	34
	D-Dimer	μg/mL	0.7	22									0.7	0.8	22
	FDP	μg/mL	<5	16									<5	n.a	16
	Fibrinogen	mg/dL	213	32							166	32	190	33	64
Others	Amylase	IU/L	153	35	178	37					243	25	186	46	97
	Lipase	IU/L	38	13							25	22	30	9	35
	Iron	μg/dL	122	13					162	849	68	24	159	47	886

^*Schuurman et al (30), Havill et al (31), Harewood et al (32), Hainsey et al (33).

Table 5.

Normal hematologic, biochemical and coagulation values in healthy Cynomolgus monkeys (Macaca fascicularis)

	Test	Unit	Bonfanti* (2009)	N	Schuurman* (2005)	N	Koga* (2005)	N	Mean	SD	Total N
Type / Age			Mauritius		not available		Chinese
			not available		3.5 years		3-7 years
Hematology	WBC	/mm3	8.6	60	11.9	106	12.4	440	11.9	2.1	606
	RBC	×106/mm3	6.9	60	6.5	106	5.5	440	5.8	0.7	606
	Hemoglobin	g/dL	13.8	60	12.2	106	13.1	440	13.0	0.8	606
	Hematocrit	%	49	60	40	106	43	440	43	5	606
	Platelets	×103/mm3	359	60	430	106	442	440	432	45	606
	MCV	fL	72	60	62	106	78	440	75	8	606
	MCH	pg	20	60	19	106	24	440	23	3	606
	MCHC	g/dL	29	60	31	106	31	440	31	1	606
	RDW	%	13.7	60					13.7	0.9	60
	MPV	fL	9	60					9.0	1.1	60
	Neutrophil	%	27	60	42	106	32	440	33.2	7.8	606
	Lymphocyte	%	65	60	48	106	60	440	58.4	8.7	606
	Monocyte	%	5.7	60	6.8	106	3.6	440	4.4	1.6	606
	Eosinophil	%	1.6	60	2.8	106	2.7	440	2.6	0.7	606
	Basophil	%	0.4	60	0.3	106	0.8	440	0.7	0.3	606
Renal Function and Electrolytes	Sodium	mmol/L	158	60	159	106			158	0	166
	Potassium	mmol/L	6.3	60	5.5	106			5.8	0.6	166
	Chloride	mmol/L			113	106			113	4	106
	Calcium	mg/dL	12.3	60	10.5	106			11.1	1.2	166
	Phosphorus	mg/dL	6.3	60	5.4	106	5.6	304	5.6	0.5	470
	CO2	mmol/L
	Urea	mg/dL	44.5	60	19.1	106	20.5	328	23.1	14.3	494
	Creatinine	mg/dL	0.92	60	1.1	106	0.6	328	0.8	0.3	494
Liver Function	AST	IU/L	43	60	31	106	32	328	33.1	6.5	494
	ALT	IU/L	45	60	61	106	43	328	47.1	9.9	494
	ALP	IU/L	2046	60			964	312	1139	765	372
	LDH	IU/L	789	60	340	106	653	296	599	230	462
	GGT	IU/L	153	60	108	106			124.1	31.5	166
	Tot Bilirubin	mg/dL	0.3	60	0.4	106	0.2	328	0.3	0.1	494
	Dir Bilirubin	mg/dL	0.1	60					0.1	0.0	60
	Indir Bilirubin	mg/dL
	Tot Protein	g/dL	8.1	60	8.9	106	7.5	304	7.9	0.7	470
	Albumin	g/dL	4.9	60	4.7	106	4.0	288	4.3	0.5	454
	Cholesterol	mg/dL	127	60	144	106	128	304	132	9	470
	Triglyceride	mg/dL	64	60	56	106	37	304	45	14	470
	Glucose	mg/dL	79	60	79	106	82	304	81	2	470
Coaulation Profile	PT	sec					9.4	616	9.4	0.5	616
	PTT	sec					21.6	616	21.6	2.2	616
	INR
	D-Dimer	μg/mL
	FDP	μg/mL
	Fibrinogen	mg/dL	236	60					236	28	60
Others	Amylase	IU/L			440	106			440	150	106
	Lipase	IU/L
	Iron	μg/dL	156	60					156	36	60

^*Bonfanti et al (34), Schuurman et al (35), Koga et al (36).

Table 6.

Normal hematologic, biochemical and coagulation values in healthy Rhesus monkeys (Macaca mulatta)

	Test	Unit	Chen* (2009)	N	Smucny* (2004)	N	Buchl* (1997)	N	Mean	SD	Total N
Age / Weight			3.6 years		8.6 ± 2.6 kg		3.6 years
Hematology	WBC	/mm3	15.7	35	7.6	3086	10.1	476	8.0	4.1	3597
	RBC	×106/mm3	5.3	35	5.6	3093	5.8	476	5.6	0.3	3604
	Hemoglobin	g/dL	12.8	35	13.3	2974	12.9	476	13.2	0.3	3485
	Hematocrit	%	41.8	35	41.0	3023	40.0	476	40.9	0.9	3534
	Platelets	×103/mm3	359	35					359	71	35
	MCV	fL	79	35	75	2952	70	476	74	5	3463
	MCH	pg	24	35	24	3068	22	476	24	1	3579
	MCHC	g/dL	31	35	32	3062	32	476	32	1	3573
	RDW	%	13	35					13.0	0.7	35
	MPV	fL
	Neutrophil	%	39	35	52	1611	63	476	54.3	12.0	2122
	Lymphocyte	%	57	35	39	2977	32	476	38.2	12.9	3488
	Monocyte	%	2.3	35	3.7	1436	3	476	3.5	0.7	1947
	Eosinophil	%	0.1	35	3.9	1419	0.8	476	3.1	2.0	1930
	Basophil	%	0.0	35			0.2	476	0.2	0.1	511
Renal Function and Electrolytes	Sodium	mmol/L	151	36			147	476	147	3	512
	Potassium	mmol/L	4.8	36	4.0	1558	4.0	476	4.0	0.5	2070
	Chloride	mmol/L	107	36			113	476	113	4	512
	Calcium	mg/dL	10.7	36	9.2	2476	10.3	476	9.4	0.8	2988
	Phosphorus	mg/dL			4.0	2465	4.9	476	4.1	0.6	2941
	CO2	mmol/L	13.9	36					13.9	3.9	36
	Urea	mg/dL	21.9	36	16.9	2594	19.0	476	17.3	2.5	3106
	Creatinine	mg/dL	0.8	36	1.1	2492	0.8	476	1.0	0.2	3004
Liver Function	AST	IU/L	39	36	33	2464	36	476	33.6	3.0	2976
	ALT	IU/L	53	36	45	2259	42	476	44.6	5.7	2771
	ALP	IU/L	546	36	131	2474	430	476	184	214	2986
	LDH	IU/L	514	36	276	1553	410	476	311	119	2065
	GGT	IU/L	74	36			60	476	61.0	9.9	512
	Tot Bilirubin	mg/dL	2.8	36			0.2	476	0.4	1.8	512
	Dir Bilirubin	mg/dL	0.7	36					0.7	0.2	36
	Indir Bilirubin	mg/dL	2.1	36					2.1	0.7	36
	Tot Protein	g/dL	7.8	36	7.1	2773	7.3	476	7.1	0.4	3285
	Albumin	g/dL	5.4	36	3.9	1864	4.4	476	4.0	0.8	2376
	Cholesterol	mg/dL	114	36	142	2482	142	476	142	16	2994
	Triglyceride	mg/dL	74	36	93	818	45	476	75	24	1330
	Glucose	mg/dL	103	36	66	2516	70	476	67	20	3028
Coaulation Profile	PT	sec	14.2	24					14.2	0.9	24
	PTT	sec	43	24					43.0	5.4	24
	INR
	D-Dimer	μg/mL
	FDP	μg/mL
	Fibrinogen	mg/dL
Others	Amylase	IU/L
	Lipase	IU/L
	Iron	μg/dL	156	36					156	30	36

^*Chen et al (37), Smucny et al (38), Buchl et al (39).

Table 7.

Comparison of normal GTKO and WT pig mean values with 4 primate species

	Test	Unit	GTKO*	WT*	Baboon	Cyno*	Rhesus*	Human
Hematology	WBC	/mm3	15.5	18.6	10.0	11.9	8.0	3.8 – 10.6
	RBC	×106/mm3	6.3	6.9	5.1	5.8	5.6	3.73 – 4.89
	Hemoglobin	g/dL	12.2	10.0	12.7	13.0	13.2	11.6 – 14.6
	Hematocrit	%	37.9	37.9	39.9	43.1	40.9	34.1 – 43.3
	Platelets	×103/mm3	425.0	506.6	419.2	431.7	359.0	156 – 369
	MCV	fL	61.3	58.7	77.2	74.6	74.4	82.6 – 97.4
	MCH	pg	19.7	17.4	25.1	22.7	23.7	27.8 – 33.4
	MCHC	g/dL	32.3	25.5	32.6	30.7	32.0	32.7 – 35.5
	RDW	%	21.4	19.0	13.1	13.7	13.0	11.8 – 15.2
	MPV	fL	9.1	n.a.	8.5	9.0	n.a.	6.8 – 10.4
	Neutrophil	%	41.8	45.6	52.0	33.2	54.3	44 – 77
	Lymphocyte	%	49.1	45.5	44.4	58.4	38.2	13 – 44
	Monocyte	%	4.9	4.7	3.2	4.4	3.5	4 – 13*
	Eosinophil	%	2.3	1.9	0.9	2.6	3.1	0 – 6
	Basophil	%	0.1	0.4	0.3	0.7	0.2	0 – 1
Renal Function and Electrolytes	Sodium	mmol/L	144.0	142.9	146.1	158.4	147.3	136 – 146
	Potassium	mmol/L	5.3	5.3	3.8	5.8	4.0	3.5 – 5.0
	Chloride	mmol/L	103.0	104.8	108.0	113.4	112.6	95 – 110
	Calcium	mg/dL	10.8	11.0	9.5	11.1	9.4	8.4 – 10.2
	Phosphorus	mg/dL	8.8	8.6	4.3	5.6	4.1	2.5 – 4.5
	CO2	mmol/L	28.1	29.0	22.9	n.a.	13.9	21–32
	Urea	mg/dL	12.8	12.4	14.3	23.1	17.3	5.0 – 20.0
	Creatinine	mg/dL	1.1	1.0	0.7	0.8	1.0	0.6 – 1.1
Liver Function	AST	IU/L	37.0	40.1	36.6	33.1	33.6	< 40
	ALT	IU/L	42.0	58.5	39.2	47.1	44.6	< 40
	ALP	IU/L	272.0	263.1	637.7	1138.6	183.7	38 – 126
	LDH	IU/L	472.0	825.9	284.2	598.8	311.0	< 170
	GGT	IU/L	74.0	43.0	39.0	124.1	61.0	< 40
	Tot Bilirubin	mg/dL	0.2	0.5	0.1	0.3	0.4	0.3 – 1.5
	Dir Bilirubin	mg/dL	0.1	0.1	0.1	0.1	0.7	0.1 – 0.4
	Indir Bilirubin	mg/dL	0.1	0.1	0.1	n.a.	2.1	0.2 – 1.1
	Tot Protein	g/dL	5.9	5.2	6.8	7.9	7.1	6.3 – 7.7
	Albumin	g/dL	3.4	2.6	4.1	4.3	4.0	3.4 – 5.0
	Cholesterol	mg/dL	81.0	100.6	103.2	131.5	141.7	<200
	Triglyceride	mg/dL	31.0	57.5	60.1	44.8	75.3	<150
	Glucose	mg/dL	89.0	92.1	96.4	81.0	67.1	70 – 99
Coaulation Profile	PT	sec	13.6	11.7	14.3	9.4	14.2	11.3 – 14.5
	PTT	sec	34.1	15.5	32.6	21.6	43.0	22.7 – 35.6
	INR		1.1	n.a.	1.3	n.a.	n.a.	0.8 – 1.2
	D-Dimer	μg/mL	0.2	n.a.	0.7	n.a.	n.a.	<0.45
	FDP	μg/mL	5to20	n.a.	<5	n.a.	n.a.	<5 neg, >20 pos
	Fibrinogen	mg/dL	250.0	n.a.	189.5	235.8	n.a.	200 – 400
Others	Amylase	IU/L	1622.0	n.a.	185.7	440.0	n.a.	35 – 118
	Lipase	IU/L	13.1	n.a.	29.8	n.a.	n.a.	22 – 51
	Iron	μg/dL	104.0	175.2	158.9	156.3	156.0	28 – 170
	Total CPK	IU/L	1166.0	n.a.	n.a.	n.a.	n.a.	60 – 400
	CPK-MB isoenzyme	ng/mL	4.4	n.a.	n.a.	n.a.	n.a.	0 – 3
	CPK-MB relative index		0.7	n.a.	n.a.	n.a.	n.a.	0 – 3
	Troponin I	ng/mL	0.1	n.a.	n.a.	n.a.	n.a.	< 0.4

^*GTKO = α1,3-galactosyltransferase gene-knockout pigs; WT = wild-type pigs; Cyno = cynomolgus monkeys; Rhesus = rhesus monkeys. n.a. = not available. GTKO values indicated in bold are statistically different from human values.

Hematologic parameters

White blood cell (WBC) count

GTKO pigs had a significantly lower mean WBC than WT pigs (p<0.01) (Table 7). Pigs with a GTKO background appeared to have a lower WBC when young (Table 2). Mean WBC count was significantly higher in GTKO and WT pigs than in humans or NHPs (p<0.01) (Table 7). All NHP species tested showed a similar WBC to humans, except in cynomolgus monkeys where the WBC count was significantly higher (p<0.01), though cynomolgus monkeys from Mauritius exhibited similar WBC counts to humans (Tables 5 and 7).

With regard to WBC subsets, GTKO pigs had significantly fewer neutrophils than WT pigs, humans, baboons, and rhesus monkeys (p<0.01). Cynomolgus monkeys had the lowest neutrophil counts among all species tested (p<0.01 vs all other species) (Table 7), but had the highest lymphocyte counts (p<0.01 vs all other species). Monocyte counts were significantly higher in pigs (GTKO and WT) in comparison to other species (p<0.01). Eosinophil and basophil counts were similar in all species (Table 7).

Red blood cell (RBC) parameters

RBC counts were significantly higher in GTKO and WT pigs than in humans and NHP species (p<0.01). Hemoglobin values were comparable in all species, except WT pigs in which the hemoglobin was significantly lower (p<0.01 vs all other species). GTKO and WT pig hematocrits were significantly lower than in NHPs (p<0.01), but were within the human range (Table 7). GTKO and WT pigs exhibited a significantly lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) than seen in human and NHP species (p<0.01). Mean corpuscular hemoglobin concentration (MCHC) in all species, except WT pigs, was comparable to that in humans. Percentage RBC distribution width (RDW) was significantly higher in pigs (GTKO and WT) than in primate species (p<0.01) (Table 7).

Renal function and electrolytes

Pigs (GTKO and WT) exhibited higher potassium, calcium, and phosphorus values than humans, baboons, and rhesus monkeys (p<0.01 in all comparisons) (Table 7). Cynomolgus monkeys showed the highest potassium, calcium, and chloride values in comparison to other species (p<0.01) (Table 7). Sodium values were comparable in all species, except in cynomolgus monkeys, which had significantly higher values (p<0.01) (Table 7). Cynomolgus monkeys showed significantly higher urea values than other species (p<0.01) (Table 7). Serum creatinine values were comparable in all species. Carbon dioxide (CO₂)levels were significantly lower in rhesus monkeys (p<0.01). GTKO and WT pigs and baboons exhibited CO₂ levels within the human range (Table 7).

Hepatic function

AST and ALT values were comparable in all species, except that WT pig ALT was significantly higher than in other species (p<0.01) (Table 7). Alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were higher in pigs and NHPs than in humans (Table 7). ALP was highest in cynomolgus monkeys (10-fold more than in humans) and baboons (5-fold more than in humans). WT pigs exhibited the highest LDH values (5-fold higher than in humans, and 2-fold higher than in GTKO pigs) (Table 7). Total, direct, and indirect bilirubin values were comparable in all species. Total protein and albumin levels were significantly lower in pigs than in NHPs and humans (p<0.01) (Table 7). Levels of total protein and albumin appeared to be lower in younger than in older GTKO pigs (p<0.01) (Table 2). In contrast, WT pigs did not show significantly different total protein and albumin levels between low (young) and high (older) weight pigs (Table 3). Total cholesterol, triglyceride, and glucose levels were comparable in all species (Table 7), except in rhesus monkeys, which exhibited higher cholesterol and triglyceride and lower glucose levels (p<0.01) (Table 7).

Coagulation profiles

WT pigs and cynomolgus monkeys had significantly lower prothrombin times (PT) and partial thromboplastin times (PTT) than GTKO pigs, baboons, rhesus monkeys, and humans (p<0.01 in all comparisons). GTKO pigs exhibited similar PT and PTT to humans. Rhesus monkeys had significantly prolonged PTT compared with other species (p<0.01) (Table 7). While GTKO pigs had international normalized ratio (INR) and d-dimer comparable to humans, baboons showed significantly increased INR and d-dimer (p<0.01). GTKO pigs showed positive fibrinogen degradation products (FDP). Fibrinogen levels were comparable in GTKO pigs, cynomolgus monkeys, and humans, but baboons had significantly lower fibrinogen levels than other species (p<0.01) (Table 7).

Other parameters

Although lipase levels were comparable in humans, GTKO pigs and baboons, amylase levels were significantly lower in humans than in other species (p<0.01). GTKO pigs showed a 13-to-45-fold increase in amylase in comparison to humans and baboons, but only a 4-fold increase in comparison to cynomolgus monkeys (Table 7). Younger GTKO pigs showed significantly higher levels of amylase in comparison to older GTKO pigs (p<0.01) (Table 2). In contrast, amylase levels were higher in older baboons (p<0.01) (Table 4). Iron levels were comparable in all species.

The cardiac enzymes, total creatine kinase (CPK), myocardial band of CPK (CPK-MB) and its relative index, and troponin I were measured only in pigs of GTKO.hCD46 background (Table 2). Total CPK and CPK-MB were significantly higher in GTKO.hCD46 pigs than in humans (p<0.01). However, the CPK-MB relative index and troponin I values were comparable in GTKO.hCD46 pigs to humans (Table 7).

DISCUSSION

In biomedical research, it is essential to compare pre-treatment values (i.e., in a surgically and immunologically naïve animal) with post-treatment values. Therefore, knowledge of normal hematologic, biochemical, and coagulation parameters is important. The present study reports, for the first time, the mean values in genetically-engineered pigs important to xenotransplantation research, all on a GTKO background. Moreover, the study compares these values with those in WT pigs and 4 species of primate, including humans.

We report differences in certain parameters between GTKO and WT pigs and/or pigs between primates and/or between NHPs and humans, which may prove important in xenotransplantation research and, ultimately, in clinical xenotransplantation. It should be kept in mind that the health status of the animals may affect a specific parameter. For example, designated pathogen-free pigs may have lower white blood cell counts than pigs housed under routine circumstances. Differences in normal levels of potassium or other electrolyte may be problematic after pig kidney xenotransplantation. After the transplantation of a pig kidney or liver into a NHP, the level of a parameter may reflect the normal level in the NHP (e.g., WBC count), or the normal level in the pig (e.g., serum potassium or albumin). In fact, prominent proteinuria has been underlined by several groups after pig-to-NHP kidney xenotransplantation (16,20). The loss of protein may reflect the physiologic ability of the pig kidney to reduce the albumin levels of the NHP to the normal pig albumin level, which is significantly lower (Table 7). Alternatively, it could reflect an inability of pig kidneys to retain NHP albumin, or reduced synthesis of albumin in the pig liver. Soin et al previously reported severe hypophosphatemia and persistent hypoalbuminemia due to increased proteinuria after pig-to-NHP renal xenotransplantation (21). Whether this was related to a physiologic incompatibility between pig and primate or was the result of a low-grade immune response remains unknown. In our experience, healthy pigs do not have proteinuria (Hara H, personal observation). The topic of physiologic incompatibilities has been reviewed elsewhere (22).

After pig liver xenotransplantation, a great number of parameters may reflect those in the pig, since the liver is the major site of production of many proteins. After pig heart xenotransplantation, the knowledge of normal values of CPK and troponin I is important to monitor damage to the transplanted heart.

GTKO pigs had significantly lower WBC counts than WT pigs, which may be related to the cleanliness of the housing in which they are reared (though GTKO pig values fell within the range of published normal values for WT pigs). Pigs (both GTKO and WT) have higher WBC counts than the primates we tested (Table 7). As important as high WBC count could be, the higher percentage of lymphocytes in the recipient NHPs may also be important with regard to successful lymphocyte depletion. Cynomolgus monkeys have the highest lymphocyte count among four primate species and pigs (Table 7). This high lymphocyte count may result in an increased need for of lymphocyte-depleting agents to achieve the desired outcome.

It should be kept in mind that the health status of the animals may affect a specific parameter. For example, designated pathogen-free pigs may have lower white blood cell counts than pigs housed under routine circumstances.

Cynomolgus monkeys are also special in respect to RBC. It is well known that CD52 is expressed on erythrocytes of most NHP species. As a result, alemtuzumab (anti-CD52 monoclonal antibody) can be used only in cynomolgus monkeys of Indonesian origin, which do not express CD52 on their RBCs (23). The RBC MCV in humans is almost 30–50% greater than that in GTKO pigs, and the MCV of baboon RBC is 30–40% greater than in pigs. Theoretically, this discrepancy could well adversely impact the perfusion of a pig organ after transplantation into a primate. However, evidence from numerous pig-to-NHP organ transplantation studies suggests that this is not the case, and that organ perfusion is satisfactory (unless affected by rejection, etc.). Furthermore, biopsies obtained after pig-to-NHP kidney, heart, and liver xenotransplantation have not shown unequivocal defects in the microcirculation (except when thrombosis occurs following fibrin and platelet aggregation) (15–18,20).

These observations illustrate how baseline (pre-treatment) knowledge of parameters is key to success in biomedical research. Attention has been drawn to the importance of knowing normal parameters in NHP by recent publications by the Emory Group (24,25) with particular regard to MHC typing as a key to successful outcome.

Although our study did not detect any significant difference in total, direct, or indirect bilirubin levels among the species tested (Table 7), the relevance of these data should be interpreted cautiously. Kobayashi et al reported that hepatic bile was significantly less viscous in baboons compared with that in humans and pigs, with pig and human hepatic bile viscosity being similar (26). In our experience of GTKO pig-to-baboon liver xenotransplantation, we observed cholestatic damage on liver histopathology without structural obstruction of the bile ducts, but with the presence of viscous bile (18,27). However, bile stasis may not be a significant problem after pig liver Tx into humans (25).

The lower values for PT and PTT in WT pigs and in cynomolgus monkeys need particular attention as they may impact a coagulopathic state and related complications. A significantly shorter PTT in WT pigs could be related to intrinsic pathway coagulation factors, such as FXII, FXI, and FIX. We have previously reported data suggesting that FXII (initiator of the intrinsic coagulation pathway) in WT pigs is significantly higher (2-fold) than in GTKO pigs (27,28). We have also documented the production of pig coagulation factors after GTKO pig liver xenotransplantation in baboons (27). Knowledge of baseline coagulation values in both organ-source pig and recipient NHP is of importance when monitoring post-transplantation changes. We previously reported the baseline extended coagulation profile in nine healthy baboons (29).

There are other observations from our data that cannot be explained. For example, in GTKO pigs with added transgenes, serum amylase was high when compared with GTKO pigs (Table 2). However, at necropsy, no features suggestive of pancreatitis were observed in these pigs. Similarly, serum cholesterol was particularly low in these pigs. Larger numbers of pigs will need to be studied to confirm, and possibly explain, these observations.

While differences have been observed in various parameters between WT and genetically-engineered pigs, there is no evidence that such differences would lead to an increased risk profile, as compared to the significant benefits that genetically-engineered pigs may provide in overcoming the challenges for human clinical application.

A minor weakness of our comparative study is that data from different centers may have been obtained using different laboratory equipment. However, our own and most other studies have been carried out in hospital laboratories in which standard equipment is used. We suggest there are unlikely to be wide or significant differences in the data obtained. Furthermore, our own data, and we strongly suspect the vast majority of data in the literature, were obtained using equipment equilibrated and validated with respect to human material, not to nonhuman primate material. We do not see this as a major problem. If data from various centers are to be compared, it could be argued that this provides some uniformity to the data as they will all have been obtained on equipment validated to human material.

In conclusion, it appears that genetic modification of the pig (e.g., deletion of the Gal antigen and/or the addition of a human transgene) (i) does not result in abnormalities in hematologic, biochemical, or coagulation parameters that might impact animal welfare, (ii) seems not to alter metabolic function of vital organs, though this needs to be confirmed after their xenotransplantation, and (iii) possibly (though by no means certainly) modifies the hematologic, biochemical, and coagulation parameters closer to human values. The present study may provide a good reference for those working with genetically-engineered pigs in xenotransplantation research and eventually in clinical xenotransplantation.

Abbreviations (in text and tables)

ALP

alkaline phosphatase

ALT

alanine transaminase

AST

aspartate transaminase

GGT

gamma-glutamyl transferase

GTKO

α1,3-galactosyltransferase gene-knockout

INR

international normalized ratio

LDH

lactate dehydrogenase

MCH

mean corpuscular hemoglobin

MCV

mean corpuscular volume

MCHC

mean corpuscular hemoglobin concentration

MPV

mean platelet volume

NHP

nonhuman primate

PT

prothrombin time

PTT

partial thromboplastin time

RBC

red blood cells

RDW

red blood cell distribution width

WBC

white blood cells

WT

wild-type