Differentiation of Yin, Yang and Stasis Syndromes in Severe Aplastic Anemia Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation and Their Correlation with Iron Metabolism, cAMP/cGMP, 17-OH-CS and Thyroxine

Figures & data

Table 1 Scoring System for Basic TCM Syndrome

Symptom		Scoring
		None (0)	Mild (2 for Major Syndromes, and 1 for Minor)	Moderate (4 for Major Syndromes, and 2 for Minor)	Severe (6 for Major Syndromes, and 3 for Minor)
Major syndromes	Pale face		Slightly	Moderate, pale without vitality	Severe, pale with a white paper looking
	Dizziness		Occasionally	Frequently	Persistently and hard to relieve
	Fatigue		Slightly, can still work on daily life	Moderate, hard to work	Severe, can only stay in bed
	Palpitations and shortness of breath		Occasionally	Frequently	Recurrently and hard to relieve
Minor syndromes	Feverish palms and soles		Slightly feverish in night	Moderate feverish with vexation	Feel burning, and away from clothing
	Hectic fever and night sweating		Occasionally, mostly on head	Recurrently, on chest and waist	Frequently, showing as wet body
	Dipsia		Occasionally	Frequently, tolerable	Frequently, intolerable
	Yellow urine		Primrose yellow	Medium yellow	Dark yellow
	Cold body		Cold in hands and feet	Cold limbs	Clod body, cannot relieve with warming
	Anorexia		Slightly, appetite decreases 1/3 to 2/3	Bad, appetite decreases above 2/3	Severe anorexia, even no diet
	Diarrhea		Once daily	Two to three times daily	More than three times daily

Table 2 Scoring System for Stasis Syndrome

	Grading and Scoring
	Normal (0)	Mild (1)	Medium (2)	Severe (3)
Inspection of the tongue	Pink tongue, without ecchymosis and sublingual varices	Dark red tongue, with petechiae	Dark purple tongue, with petechiae and ecchymosis	Bluish purple tongue, or with sublingual varices
Inspection of the facial	No purplish black at face, lips, gums or eyes	Dark red less than two sites	Dark red or bluish purple more than two sites	Purple black more than two sites
Skin manifestation	No rough skin nor scales	Rough hand and foot, but without scales	Rough hand and foot, with scales	Rough skin throughout the body with scales
Pain	No fixed pain, prickling or cramping	Occasionally pain, and always relieved within half an hour	Pain sometimes, always relieved within three hours, and no need for drug	Persistent pain, and painkillers needed
Bleeding	No bleeding, hypodermic ecchymosis, nor menstrual disorders	Small petechiae in one site, or with occasionally dysmenorrhea and dark menstruation	Petechiaes more than two sites, or always with dysmenorrhea and dark menstruation	Petechiaes more than three sites, or suffer from abdominal pain during menstruation, and even amenorrhea
Conscious	No vertigo or forgetful	Occasionally happen	Happened sometimes, and more frequently after physical work	Always happen, and even during rest
Mentality	No mental abnormality	Occasionally happen, and relieved without intervene	Happened sometimes, and can be stopped by persuading	Always happen, and can only stopped by constraint or medication
Physical sensation	No numbness or hemiplegia	Occasionally happen, and relieved without intervene	Numbness cannot be alleviate by self, with mild hemiplegia	Numbness Limbs and with hemiplegia
Pulse manifestation	Normal pulse	Hesitant pulse	Thread and hesitant pulse	No pulse or irregularly pulse
Lump in abdomen	No organ enlargement, mass, tissue hyperplasia, new organisms, etc.	Slightly stiff and painful with pressing	Moderate stiff and painful	Painful and reject pressing

Table 3 The Distribution of TCM Syndrome in AA Patients Undergoing HSCT

Time Points (HSCT Following Up)	Yang Deficiency (n)			Yin Deficiency (n)
	Kidney Yang Deficiency	Spleen–Kidney Yang Deficiency	Kidney Yin and Yang Deficiency	Kidney Yin Deficiency	Liver–Kidney Yin Deficiency	Kidney Yin and Yang Deficiency
10 days before stem cell reinfusion (d-10)	1	6	2	4	5	2
The day before stem cell reinfusion (d-1)	2	1	11	4	0	11
7 days after stem cell reinfusion (d+7)	0	7	8	2	1	8
14 days after stem cell reinfusion (d+14)	2	5	5	1	5	5
30 days after stem cell reinfusion (d+30)	1	11	3	1	2	3
60 days after stem cell reinfusion (d+60)	5	10	0	1	2	0
90 days after stem cell reinfusion (d+90)	5	4	1	3	1	1

Figure 1 Evolution of TCM syndromes in severe aplastic anemia (SAA) patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) based on “Yin–Yang” and “Zang–Fu viscera” syndrome differentiation.

Table 4 Comparison of Stasis Score Among Different Time Point After During HSCT (Wilcoxon), P value

	Day 14	Day+30	Day+60	Day+90
Day-10	NS	0.011	0.001	0.002
Day-1	NS	0.005	0.001	0.001
Day+7	0.013	0.003	0.001	0.000
Day 14	/	0.05	0.015	0.007
Day+30	0.05	/	NS	0.047
Day+60	0.015	NS	/	NS
Day+90	0.007	0.047	NS	/

Figure 2 Evolution of “blood stasis” syndrome in SAA patients during HSCT. The stasis syndrome was differentiated, scoring and compared during different stage of HSCT. A total of 61% (11/18) patients were complicated with stasis syndrome before HSCT, and increased after conditioning. After the stem cell engraftment (around +14d), the stasis score decreasing and 88.9% (16/18) had no symptoms of stasis at +90d.

Figure 3 Typical evolution of tongue manifestation during HSCT. Type I: went from the firstly “spleen–kidney Yang deficiency” stage to “liver–kidney Yin deficiency with blood stasis” stage, “spleen–kidney Yang deficiency with blood stasis” stage, and finally of relatively stable “kidney Yang deficiency” stage (2A-2E); Type II: went from “kidney Yang deficiency”, to a syndrome of “Yin deficiency with blood stasis”, to “spleen–kidney Yang deficiency” and finally “kidney Yang deficiency” stage (2F-2J); and Type III: went from “kidney Yin deficiency”, to “Yin deficiency fire hyperactivity”, to “Yin and Yang deficiency”, and finally “kidney Yang deficiency” stage (2K-2O).

Figure 4 Changes of serum cAMP, cGMP and urine 17-OH-CS during the HSCT process. Data are presented as mean ± SD; *P < 0.01 (as compared with the normal population);⁺P < 0.05, and ⁺⁺P < 0.01 (as compared with −10d); ^#P < 0.01 (as compared with −1d); ^@P < 0.05, ^@@P < 0.01 (as compared with +7d); ^&P < 0.05, and ^&&P < 0.01 (as compared with +14d).

Figure 5 Changes of serum thyroxine during HSCT process. The serum level of total triiodothyronine (TT3), free T3 (FT3) (A) and tetraiodothyronine (TT4), free T4 (FT4) (B) were detected during the HSCT. Data are presented as mean ± SD; *P < 0.01 (as compared with the normal population);⁺P < 0.05, and ⁺⁺P < 0.01 (as compared with −10d); ^#P < 0.05, ^##P < 0.01 (as compared with −1d); ^@P < 0.05, ^@@P < 0.01 (as compared with +7d); ^&P < 0.01 (as compared with +14d); ▲P < 0.01 (as compared with +30d).

Figure 6 Comparison of iron load and its regulator during HSCT process. The serum level of iron load indexes, ferritin and labile plasma iron (LPI) (A), as well as iron-absorbing regulators, hepcidin and soluble transferrin receptor (sTfR) (B) were detected. Data are presented as mean ± SD; *P < 0.05, **P < 0.01 (as compared with the normal population);⁺P < 0.05, and ⁺⁺P < 0.01 (as compared with −10d); ^#P < 0.05, ^##P < 0.01 (as compared with −1d); ^@P < 0.05, ^@@P < 0.01 (as compared with +7d); ^&P < 0.05, and ^&&P < 0.01 (as compared with +14d).

Figure 7 Changes of bone marrow VEGF, NOS, and ROS during HSCT process. The marrow serum level of VEGF, NOS, and ROS were detected. Data are presented as mean ± SD; *P < 0.05, **P < 0.01 (as compared with the normal population); ⁺⁺P < 0.01 (as compared with −10d); ^@P < 0.05, and ^@@P < 0.01 (as compared with +7d).

Table 5 Correlation Analysis Between “Yin” and “Yang” Deficiency Syndrome and Serum Indicators (I)

	Yang Deficiency (Ratio)	Yin Deficiency (Ratio)	cAMP	cGMP	17-OH	TT3	FT3	TT4	FT4	Ferritin	LPI	Hepcidin	sTfR	FPN
Normal	0.00	0.00	33.076	3.601	29.579	1.683	4.552	6.485	1.000	9.17	0.25	73.23	73.23	2.68
Day-10	0.50	0.61	30.765	6.682	85.623	0.965	2.523	5.685	0.963	4.69	0.80	93.60	93.60	2.32
Day-1	0.78	0.83	27.949	6.164	78.7402	0.602	1.658	5.708	0.991	4.59	0.75	92.72	92.72	2.34
Day+7	0.83	0.61	26.775	5.751	75.249	0.723	1.938	5.889	0.982	4.80	0.73	92.84	92.84	2.43
Day+14	0.67	0.61	24.763	5.240	73.226	0.663	1.791	5.087	0.892	4.76	0.68	90.74	90.74	2.50
Day+30	0.83	0.33	23.012	5.042	68.391	0.665	1.881	4.814	0.844	4.63	0.66	88.02	88.02	2.56
Day+60	0.77	0.23	21.744	5.195	63.044	0.829	2.235	5.203	0.890	4.21	0.63	87.04	87.04	2.62
Day+90	0.56	0.56	22.879	4.983	61.222	0.949	2.428	5.996	0.944	4.61	0.68	87.79	87.79	2.62

Table 6 Correlation Analysis Between “Yin” and “Yang” Deficiency Syndrome and Serum Indicators (II)

		cAMP	cGMP	17-OH	TT3	FT3	TT4	FT4	Ferritin	LPI	Hepcidin	sTfR	FPN
Yang Deficiency	Coefficient of association	−0.407	0.252	0.311	−0.731*	−0.731*	−0.467	−0.371	−0.275	0.156	0.299	0.299	−0.311
	Sig	0.158	0.274	0.453	0.020	0.020	0.122	0.183	0.255	0.356	0.236	0.236	0.226
Yin Deficiency	Coefficient of association	0.220	0.830**	0.854**	−0.610	−0.610	−0.122	0.220	−0.098	0.927**	0.854**	0.854**	−0.854**
	Sig	0.301	0.005	0.007	0.054	0.054	0.387	0.301	0.409	0.000	0.003	0.003	0.003

Notes: Binary variable regression models were used to analyze the correlation among objective factors and “Yin” and “Yang” deficiency syndromes. *P<0.05; **P<0.01.

Table 7 Correlation Analysis Between “Blood Stasis” Syndrome and Serum Indicators (I)

	Average Score of “Stasis Syndrome”	cAMP	cGMP	17-OH	TT3	FT3	TT4	FT4	Ferritin	LPI	Hepcidin	sTfR	FPN
Day-10	4.111	30.765	6.682	85.623	0.965	2.523	5.685	0.963	4.69	0.80	93.60	93.60	2.32
Day-1	4.389	27.949	6.164	78.7402	0.602	1.658	5.708	0.991	4.59	0.75	92.72	92.72	2.34
Day+7	4.500	26.775	5.751	75.249	0.723	1.938	5.889	0.982	4.80	0.73	92.84	92.84	2.43
Day+14	3.389	24.763	5.240	73.226	0.663	1.791	5.087	0.892	4.76	0.68	90.74	90.74	2.50
Day+30	2.500	23.012	5.042	68.391	0.665	1.881	4.814	0.844	4.63	0.66	88.02	88.02	2.56
Day+60	1.944	21.744	5.195	63.044	0.829	2.235	5.203	0.890	4.21	0.63	87.04	87.04	2.62
Day+90	1.611	22.879	4.983	61.222	0.949	2.428	5.996	0.944	4.61	0.68	87.79	87.79	2.62

Table 8 Correlation Analysis Between “Blood Stasis” Syndrome and Serum Indicators (II)

		cAMP	cGMP	17-OH	TT3	FT3	TT4	FT4	Ferritin	LPI	Hepcidin	sTfR	FPN
Stasis syndrome	Coefficient of association	0.821*	0.821*	0.857*	−0.357	−0.357	0.143	0.714	0.536	0.750	0.857*	0.857*	−0.857*
	Sig	0.023	0.023	0.014	0.432	0.432	0.760	0.071	0.215	0.052	0.014	0.014	0.014

Notes: Binary variable regression models were used to analyze the correlation among objective factors and “blood stasis” deficiency syndromes. *P<0.05.