In-vitro Dilution of Human Plasma to Reduce ABO Antibody Titer Levels While Preserving Coagulation Factors

Abstract

The objective of this study was to prepare universal plasma by mixing Group A and Group B plasma with normal saline in varying proportions. This prospective in vitro study involved mixing blood group A and B plasma in ratios of 1:1, 3:1, 1:3, and with normal saline in ratios of 1:1:1, 2:2:1, and 1:1:2. The titration of IgG and IgM levels of anti-A and anti-B antibodies, as well as fibrinogen and factor VIII levels, were performed. The titers of anti-A and anti-B antibodies of both IgG and IgM types were found to have significantly decreased in all ratios compared to the control sample. Coagulation factors were found to be higher than the allowable limit when simple plasma mixing ratios (1:1, 3:1, and 1:3) were used, while they significantly decreased upon dilution with normal saline in 1:1:1 and 1:1:2 ratios. In conclusion, the mixing of A and B blood group plasma at a fixed ratio may be a promising strategy for the preparation of universal plasma with a low titre of haemolytic antibodies, while maintaining an adequate amount of coagulation factors.

Introduction

Fresh frozen plasma (FFP), also known as plasma, is primarily indicated for patients who have excessive bleeding or abnormal coagulation tests, in order to restore the activity of blood coagulation factors [1, 2]. To avoid haemolysis of recipient red blood cells (RBCs) due to naturally occurring ABO antibodies, plasma intended for transfusion should be blood group specific or compatible [3]. These antibodies are predominantly of the IgM type and have titers ranging from 2–1024, although they can also be of the IgG type, particularly in individuals with blood group O [4]. If FFP with incompatible ABO antibodies is transfused, the higher titer levels or presence of IgG antibodies can lead to immediate haemolysis of the recipient's RBCs [3].

AB blood group plasma has been suggested as a potential "universal plasma" as it lacks both anti-A and anti-B antibodies and can be transfused to patients of any blood type in emergency situations where the patient's blood group cannot be determined [5]. However, the relatively low prevalence of AB blood group individuals (approximately 3–9% of the population) limits the utility of AB plasma in some scenarios, such as trauma and obstetrics, where larger volumes of plasma may be required [6, 7].

Previous research has explored the use of group A plasma as an alternative due to its higher availability in the donor pool [8, 9]. However, the presence of high-titer anti-B antibodies in some group A donors has made this approach unsafe [8, 9]. In the 1960s and 1970s, researchers attempted to prepare modified plasma products by pooling small volumes of plasma units with saline in different ratios, but these attempts were often unsuccessful due to incorrect ratios or the presence of high-titer antibodies [10, 11]. As a result, standard guidelines for the preparation and modification of plasma products have yet to be established.

Currently, large volumes of plasma units are pooled and processed using pathogen reduction techniques and plasma fractionation to produce specific plasma products such as albumin, immunoglobulin, and Factor VIII concentrate [12]. However, intravascular haemolysis due to the presence of anti-A antibodies in concentrated Factor VIII has been reported in some recipients [13].

In resource-limited developing countries, access to plasma pooling, pathogen reduction, and plasma fractionation facilities can be limited, making it challenging to procure universal plasma (AB group) in emergency situations. In this study, we aimed to address this issue by investigating the feasibility of preparing universal plasma by combining A and B group plasma with normal saline in varying ratios, while ensuring that the resulting product maintained acceptable levels of ABO antibodies and key coagulation factors, such as Fibrinogen and Factor-VIII.

Material and Methods

Following approval from the Ethics Committee, a prospective experimental in-vitro pilot study was conducted at a tertiary care institute in North India from January 2019 to December 2019. A sample size of 60 participants was selected, with 30 participants from each blood group.

Blood donors were selected in accordance with the departmental standard operating procedure, which adhered to the Drugs & Cosmetic Acts (DCA), 1940, and rules therein, 1945 of India [14]. Initial blood grouping was performed using the "slide method" to determine if the prospective donor met the selection criteria. After collection of the whole blood (350 ml or 450 ml), the plasma component was separated and frozen for 24 h, followed by storage at -30 °C for 6 months.

Baseline Laboratory Testing

The collected samples were utilized for confirmatory blood grouping and antibody titration, which were referred to as control plasma samples. Antibody titration was performed for both IgM and IgG types of anti-A and anti-B antibodies using the conventional tube technique (monoclonal AHG antisera by Tulip diagnostics Pvt. Ltd.) and column agglutination (gel) technique (AHG Gel cards of Biorad Pvt. Ltd., California, USA), respectively.

An aseptic aliquot of the plasma was separated from the fresh frozen plasma (FFP) unit and was utilized for assessment of the Fibrinogen and Factor VIII levels using a semi-automated coagulation analyzer (Start 4 coagulation analyzer, STAGO Pvt. Ltd., USA).

Preparation of Mixing Ratios of Plasma in Varying Combinations

After 6 months of storage, FFP was thawed and an aliquot of 1–3 ml plasma was separated and mixed in pre-fixed combinations as shown below:

1. Ratio 1:1 – 1 ml of both group A and B plasma each.

2. Ratio 1:3 – 1 ml of group A plasma and 3 ml of group B plasma.

3. Ratio 3:1 – 3 ml of group A plasma and 1 ml of group B plasma.

4. Ratio 1:1:1 – 1 ml of both group A and B plasma each with 1 ml of normal saline (0.9% w/v).

5. Ratio 2:2:1—2 ml of both group A and B plasma each with 1 ml of normal saline(0.9% w/v).

6. Ratio 1:1:2—1 ml of both group A and B plasma each with 2 ml of normal saline(0.9% w/v).

The antibody titration and coagulation factor analysis of each mixture were performed using the same methodology as the unmixed samples. The titration value at which Fibrinogen (mg/bag) with minimum of 150 mg/bag and Factor VIII levels (IU/bag) with minimum of 80 IU/bag were in accordance with the standard criteria of the quality control for Fresh Frozen Plasma (FFP) as per the Drugs & Cosmetic Acts (DCA) (14) was considered the optimum ratio for the preparation and use of alternate plasma.

Statistical Analysis

The study utilized rigorous statistical methods to analyse the data and draw meaningful conclusions, ensuring the validity and reliability of the findings. Data with a normal distribution were presented as mean and standard deviation (SD), while skewed data were presented as median and range. To compare the medians of ABO antibody titers in different test groups, the Kruskal–Wallis equality-of-populations rank test was employed, followed by the Dunn-Bonferroni test. Nonparametric variables were analysed using the Chi-square test, and statistical significance was determined by a p-value less than or equal to 0.05.

Results

In the study, a total of 60 male plasma donors were recruited, 30 from blood group A and 30 from blood group B. The median age of the donors was 28 years, ranging from 18 to 55 years, with the largest proportion (46.7%) in the 26–35 age group. Out of the 60 blood collections, 21 were obtained in 350 ml blood bags while 39 were obtained in 450 ml blood bags.

Antibody Titers

In the control plasma samples, the median titers of both IgM and IgG types of anti-B antibodies in blood group A plasma were 32 (range 8–256 and 4–128, respectively). In blood group B plasma, the median titers of IgM and IgG types of anti-A antibodies were 48 (range 4–256) and 32 (range 1–128), respectively.

Upon mixing plasma in various ratios (individually or with normal saline), the titers of both IgM and IgG antibodies of were significantly reduced (p < 0.05) compared to the control plasma sample (Table 2). The IgG type anti-A antibody titer was reduced by 97%, 100%, 94%, 98%, 98%, and 100%, while the IgM type was reduced by 96%, 98%, 92%, 98%, 97%, and 98% in the 1:1, 3:1, 1:3, 1:1:1, 2:2:1, and 1:1:2 mixing ratios, respectively. The IgG and IgM types of anti-B antibodies were reduced by 97%, 94%, 100%, 97%, 97%, and 100% in the 1:1, 3:1, 1:3, 1:1:1, 2:2:1, and 1:1:2 mixing ratios, respectively (Table 1 and Fig. 1).

Table 2.

Statistical significance of antibody titers between the control plasma sample and different mixing ratios

Variables		P Values for titres
		a vs b	a vs c	a vs d	a vs e	a vs f	a vs g	b vs c	b vs d	b vs e	b vs f	b vs g	c vs d	c vs e	c vs f	c vs g	d vs e	d vs f	d vs g	e vs f	e vs g	f vs g
Anti A	IgG	S						NS					S	NS		S	NS	NS	NS	NS
	IgM													S	S
Anti B	IgG													NS		NS			S
	IgM

^*a = Control plasma sample, b = 1:1 sample ratio, c = 3:1 sample ratio, d = 1:3 sample ratio, e = 1:1:1 sample ratio, f = 2:2:1 sample ratio, g = 1:1:2 sample ratio S = Statistically Significant (p < 0.05), NS = Statistically Non-significant (p > 0.05)

Table 1.

Quantification of antibody titers in study samples, both individually and upon mixing with varying ratios

Variables	Control plasma samplea	Mixing ratios
		1:1b	1:3c	3:1d	1:1:1e	2:2:1f	1:1:2 g
Anti A (IgG)	32 (1–128)	1 (0–8)	2 (0–16)	0 (0–4)	0.5 (0–8)	0.5 (0–8)	0 (0–4)
Anti A (IgM)	48 (4–256)	2 (0–8)	4 (0–16)	1 (0–4)	1 (0–8)	1.5 (0–8)	1 (0–4)
Anti B (IgG)	32 (4–128)	1 (0–8)	0 (0–4)	2 (0–16)	1 (0–8)	1 (0–8)	0 (0–8)
Anti B (IgM)	32 (8–256)	1 (0–8)	0 (0–2)	2 (0–32)	1 (0–8)	1 (0–16)	0 (0–8)

Fig. 1.

Trend of antibody titre on the different dilution combinations

The intergroup comparison between the 1:3 and 3:1 mixing ratios for all antibodies and the 1:1:1 and 1:3 mixing ratios for IgM type of anti-A antibodies showed statistically significant differences (p < 0.05) (Table 2). Comparison of antibody titers was statistically non-significant for other ratios (Table 2).

Coagulation Factors

In the control plasma samples of blood group A, the median value of Fibrinogen and Factor VIII were 509.5 (range 187–1700) mg/bag and 321 (range 92–728) IU/bag, respectively, while in blood group B, the median of Fibrinogen and Factor VIII were 321 (range 92–728) mg/bag and 230.5 (range 76–485) IU/bag, respectively. After mixing plasma in various ratios, a non-significant decline in fibrinogen was observed in 1:1, 3:1, and 1:3 mixing ratios, while it was significantly declined (p < 0.05) in 1:1:1, 1:1:2, and 2:2:1 ratios (Tables 3 and 4). The intergroup comparison between the mixing ratios of 1:1:1 vs 1:1, 3:1, and 1:3, and 3:1 vs 2:2:1 was statistically significant (p < 0.05). Factor VIII level was only significantly declined in the 1:1:2 mixing ratio (Tables 3 and 4). The fibrinogen level declined in 1:1, 1:3, 1:1:1, 2:2:1, and 1:1:2 mixing ratios compared to baseline blood group A and B plasma, with a range of 2%-75%. However, it increased by 8% in the 3:1 mixing ratio compared to both baseline plasma values. Factor VIII level increased by 5% and 17% in the 3:1 and 1:3 mixing ratios, respectively, compared to baseline blood group A plasma value, while it increased by 37%, 47%, 63%, and 31% in 1:1, 3:1, 1:3, and 2:2:1 mixing ratios, respectively, compared to baseline blood group B plasma value. In contrast, Factor VIII level declined by 2%, 28%, 6%, and 39% in 1:1, 1:1:1, 2:2:1, and 1:1:2 mixing ratios, respectively, compared to baseline blood group A plasma value, while it declined by 15% in the 1:1:2 mixing ratio compared to baseline blood group B plasma value. In the 1:1:1 mixing ratio, the value was similar to baseline blood group B plasma value (Table 3).

Table 3.

Quantitative analysis of coagulation factors in control plasma samples and following mixing with varying ratios

Variables		Control plasma samplea	Mixing ratios
			1:1b	3:1c	1:3d	1:1:1e	2:2:1f	1:1:2 g
Fibrinogen (mg/bag)	Blood Group A	509.5 (187–1700)	473.5 (84–963)	550 (170–1503)	499 (119–1968)	253 (48–728)	336.5 (158–869)	128.5 (63–638)
	Blood Group B	506.5 (67–951)
Factor VIII (IU/bag)	Blood Group A	321 (97–728)	315.5 (102–844)	338.5 (91–853)	375 (106–854)	231.5 (93–683)	303 (130–713)	196.5 (82–582)
	Blood Group B	230.5 (76–485)

Table 4.

Statistical significance of coagulation factors between control plasma sample and on mixing with different ratios

Variables		P Values
		a vs b	a vs c	a vs d	a vs e	a vs f	a vs g	b vs c	b vs d	b vs e	b vs f	b vs g	c vs d	c vs e	c vs f	c vs g	d vs e	d vs f	d vs g	e vs f	e vs g	f vs g
Fib (mg/bag)	BG A	NS			S		NS			S		NS		S						NS		S
	BG B
F VIII (IU/bag)	BG A	NS			S		NS			S		NS		S		NS		S		NS		S
	BG B

^*a = Control plasma sample, b = 1:1 sample ratio, c = 3:1 sample ratio, d = 1:3 sample ratio, e = 1:1:1 sample ratio, f = 2:2:1 sample ratio, g = 1:1:2 sample ratio, S = Statistically Significant (p < 0.05), NS = Statistically Non-significant (p > 0.05)

Discussion

In transfusion practice, blood group-specific or compatible plasma is usually transfused. However, during emergencies or shortages, ABO non-identical plasma transfusion is also in practice [15]. Universal plasma with low titers of antibodies and adequate amounts of coagulation factors is required to prevent hemolytic reactions and achieve the desired response. In large settings, universal plasma is prepared by pooling several thousand liters of A, B, AB, and O plasma in order to neutralize the anti-A and anti-B IgM antibodies by soluble A and B antigens and possible anti-idiotypic antibodies in plasma [16, 17]. However, this requires a significant infrastructure and finance, which is quite challenging for low-resource settings. Therefore, in this study, we pooled small amounts of plasma of A and B blood group donors individually and along with normal saline in different ratios to obtain universal plasma.

We found that in approximately ≥ 75% of donors, IgG and IgM types of anti-B and anti-A antibody titers were less than 64. Our findings were in concordance with previous studies [18, 19], but in contradiction with others [20], which could be due to differences in environmental and genetic factors. Strongly haemolytic anti-A and anti-B antibodies has been reported in higher frequency in Asian and Black populations [21].The hemolysis risk due to passive transfusion of anti-A and/or anti-B ranges widely from 1 in 2000 to 1 in 46,176, which depends on the antibody titers and the number of transfusion events [22]. The critical titers level of ABO antibodies varies in different studies like Cooling et al. [23] proposed the critical titer of 128–200 while Quillen et al. [24] mentioned 250 as a critical titer.

After mixing A and B blood group plasma in different ratios and diluting with saline, the IgG titer values for both anti-A and anti-B were ≤ 16, while the IgM titer was ≤ 16 and ≤ 32, respectively. Therefore, the titer values after mixing and diluting were below the critical titers reported in previous studies [24–26]. We found that the decrease value of antibody titer was directly proportional to the ratio of normal saline added, i.e., more in the 1:1:2 mixing ratio compared to the 2:2:1 mixing ratio. Our mixing ratio of 2:2:1 was more similar to the mixing ratio of 6:2.5:1.5 of a previous study [19]. In our study we also found statistically significant reduction in IgG type of anti-A antibody titers in mixing ratio of 1:1 and 1:3, which was similar to Sikora et al. [11]. The levels of these antibodies were also lower than the recommended product specifications for Uniplas(IgM < 8, IgG < 32) and Bio plasma FDP(IgM < 32) [27, 28].

The baseline fibrinogen and factor VIII levels were lower in blood group B plasma compared to blood group A plasma. In almost 75% of donors, the baseline fibrinogen and factor VIII levels were 300–900 mg/bag and 200–400 IU/bag, respectively, for both blood group A and B plasma. We found non-significant changes in fibrinogen and factor VIII values after mixing group A and B plasma in different ratios (1:1, 3:1, 1:3). In our study, we found an increase value of factor VIII levels after mixing plasma in some ratios. Previous study also pooled FFP or Cryoprecipitates in different numbers and they also did not find any significant change in fibrinogen and Factor VIII after pooling [29].

Upon mixing plasma with normal saline in varying dilutions, a notable reduction in fibrinogen and factor VIII was observed, indicating the occurrence of a dilutional effect. This reduction was statistically significant. Furthermore, the findings of Darlington et al. [30] support these results, as they demonstrated a linear decrease in fibrinogen and factor VIII levels with increasing levels of haemodilution, ranging from 60 to 80%. It was also concluded that the type of fluid used did not have an effect on the decline of coagulation factors, rather the extent of dilution played a crucial role.

As universal plasma is not available in many lower-middle-income countries, this kind of small volume plasma pooling can be useful there. The advantage of this plasma over transfusion of A blood group plasma in emergent situations is that it can be used for extended resuscitations. In some ratios, we found increase effect in fibrinogen and factor VIII level, which is also helpful for rapid recovery of coagulation factors in patients.

The major limitation of this study is its in-vitro nature. We did not transfuse our mixing ratio plasma, so we cannot comment on the beneficial or deleterious effects of this plasma after transfusion. Another limitation was that we mixed only a small amount of the plasma samples. Furthermore, it should be noted that the majority of donors in our study exhibited a baseline antibody titre of ≤ 64. Therefore, it is possible that our findings may differ when high titre plasma is utilized. Additionally, the use of normal saline for dilution presents a limitation to our study, as normal saline lacks coagulation factors, which may not provide optimal benefit for patients suffering from bleeding disorders.

In conclusion, the mixing of A and B blood group plasma at a fixed ratio may be a promising strategy for the preparation of universal plasma with a low titre of haemolytic antibodies, while maintaining an adequate amount of coagulation factors. Our study demonstrated that the optimal mixing ratio was 1:3 of A and B blood group plasma. However, the addition of normal saline was found to be unhelpful due to the significant reduction in coagulation factors resulting from dilutional effects. So, within the reference of the pilot study in vitro, it is proposed that mixing of A and B plasma with ration of 1:3 is an option, the scrutiny of the results in larger studies and in vivo clinical results and drug authorities vouching needs before any such practice can be envisaged.

Authors Contributions

SKM collected study data and wrote initial draft of the manuscript, GKP designed the study concept, performed data analysis, wrote and reviewed the manuscript, and prepared the final manuscript, RC collected patient data and reviewed the final manuscript with RL and AH.

Funding

No financial support was taken.

Declarations

Conflict of Interest

Authors have no conflict of Interest.