Hematology

Defining the optimal calcium repletion dosing in patients requiring activation of massive transfusion protocol

a b s t r a c t

Purpose: massive transfusion protocols (MTP) commonly result in severe hypocalcemia due to the calcium- binding affinity of citrate in blood components. The purpose of this study is to determine the optimal grams

(g) of citrate to repletion calcium (Ca) milliequivalents (mEq) (Citrate:Ca) ratio to reduce 30-day mortality.

Methods: This was a retrospective, single-centered, cohort study at a level 1 trauma center evaluating trauma and surgical patients in need of MTP activation from January 1, 2010-July 31, 2021. Patients with severe hypocalce- mia at baseline, defined as ionized calcium (iCa) <0.9 mmol/L, were compared to patients without severe hypo- calcemia. The primary endpoint was to determine the optimal ratio of grams of citrate to calcium mEq to reduce mortality in patients receiving a MTP. Secondary endpoints included mortality at 24 h and 30 days, blood com- ponents used in MTP, and type of calcium used.

Results: Overall, 501 patients were screened for inclusion. Of these patients, 193 were excluded, leaving 308 patients, of which 165 patients (53.6%) had an iCa <0.9 mmol/L within 24 h and 143 patients (46.4%) had iCa

>=0.9 mmol/L within 24 h. The ratio of Citrate:Ca for each patient was not significantly associated with mortality at 24 h (P = 0.79) or 30 days (P = 0.91) at a repletion Citrate:Ca ratio of median 1.97 (IQR 1.14-2.91). The rate of mortality was lowest at a Citrate:Ca of 2 in both <24-h mortality and 30-day mortality.

Conclusions: There were no differences in 24 h or 30 day mortality based on repletion ratios seen in this study. A Citrate:Ca ratio between 2 and 3 in patients undergoing MTP was sufficient to obtain a normalized iCa within 24 h of MTP activation regardless of baseline iCa level. Further prospective studies will be needed to determine the optimal Citrate:Ca ratio.

(C) 2023

  1. Introduction

massive transfusion protocol (MTP) provides life-saving replace- ment of blood and blood factors in patients experiencing hemorrhagic shock [1,2]. MTP is defined as the administration of >=10 units of Packed red blood cells (RBCs) in 24 h or >=4 units of RBCs in one hour [3,4]. MTP can result in hypocalcemia due to the anticoagulant citrate added to

? This research did not receive any specific grant from funding agencies in the public, commercial, or non-for-profit sectors.

* Corresponding author at: Department of Pharmacy, Community Health South, Greenwood, IN, United States of America.

E-mail address: [email protected] (A. Robinson).

RBCs, fresh frozen plasma , and platelets (PLT) to maintain stability of the blood components during ex vivo storage [5]. In normal circum- stances, citrate is cleared quickly by the liver; however, in critical illness and in the administration of multiple blood components, the clearance is slowed due to liver saturation This results in the ionized (free) calcium in the serum to chelate to citrate, which left untreated, may result in profound hypocalcemia [5].

Calcium plays an important role in many physiologic processes [6,7]. Roughly 99% of all body calcium is found in bones and 1% is found in serum; of the calcium in the serum, half is bound to albumin and half is active, or ionized calcium (iCa) [7]. In hemostasis, calcium is a cofactor that plays a role in the initiation, amplification, and propagation of the coagulation cascade [5]. Additionally, calcium aids in the contraction

https://doi.org/10.1016/j.ajem.2023.05.017

0735-6757/(C) 2023

of cardiac and smooth muscles, acting as a co-factor for vasoconstric- tion, inotropy and chronotropy; thus, calcium is essential for hemody- namic stability [6]. Calcium deficiency is one of the factors leading to coagulopathy observed in trauma patients, which in turn is associated with increased mortality [8]. This combination of hemostatic and hemo- dynamic effects make normal calcium levels crucial in critically ill patients with traumatic injury.

A retrospective cohort of patients requiring MTP activation found that 97% of patients experienced hypocalcemia, defined as an iCa

<1.12 mg/dL, and 71% of patients had severe hypocalcemia defined as an iCa <0.9 mmol/L despite calcium replacement up to 4 g of calcium chloride within the first 24 h [9]. No consensus replacement ratio for calcium in MTP has been established. Given the calcium-binding poten- tial of citrate in blood products, the purpose of this study is to determine the optimal grams (g) of citrate to Ca milliequivalents (mEq) ratio to reduce 30-day mortality.

  1. Methods

This retrospective, single center cohort study was conducted at a level 1 trauma center and evaluated surgical and trauma patients who received MTP activation from January 1, 2010 to December 31, 2021. On activation of MTP by the bedside physician, coolers were sent from the blood bank containing RBC, FFP, and PLT in a 5:5:1 ratio, respec- tively. Two units of whole blood were available prior to MTP cooler arrival in the emergency department (ED). Blood products were admin- istered using a rapid infuser device. Patients >=18 years of age undergo- ing surgery or presenting with traumatic injury were eligible for inclusion if MTP was initiated in the ED, hospital or during pre- hospital transport. MTP was defined as receipt of four units of RBCs within the first hour of activation or 10 or more units of blood products in 24 h [3]. The following exclusion criteria were used and applied dur- ing data collection: pregnancy, no iCa level within 24 h of MTP initiation, and missing blood bank records. Blood components assessed included RBC, FFP, PLT, cryoprecipitate, and whole blood. The content of citrate varies among blood components with 3 g per unit in RBCs, 10 g per unit in FFP, 0.22 g per unit in platelets, and 2 g per unit in whole blood [10]. The content of elemental calcium varies among calcium formula- tions with 4.65 mEq in 1 g of Calcium gluconate and 13.6 mEq in 1 g of calcium chloride [7].

The primary outcome was the optimal ratio of administered citrate (grams) from MTP blood products to calcium (mEq) dose given (Citrate:Ca) in order to reduce 30-day mortality. The aforementioned citrate content of each blood component was used in Citrate:Ca calcula- tions. Secondary outcomes included patients who had an iCa

<0.9 mmol/L at any time within 24 h of MTP initiation, 24 h and 30 day mortality, hospital and ICU length of stay for survivors at 30 days, total blood products received, time to first iCa, iCa at 24 h, amount of elemental calcium received, and amount of citrate

(g) received. For secondary outcomes, patients who had iCa

<0.9 mmol/L at any time within 24 h of MTP activation were compared to patients who had iCa >=0.9 mmol/L at all times within the same time frame. Groups were dichotomized at this level based on previous litera- ture used to describe severe hypocalcemia and its relationship to mortality [7,9].

Descriptive statistics were used to characterize baseline demograph- ics. Parametric data were reported utilizing mean and standard devia- tion and analyzed via independent student t-test. Non-parametric data were reported using median and interquartile range and analyzed by Mann-Whitney U test. Normality of continuous variables was assesses using the Shapiro-Wilk test. Categorical data were evaluating using Chi-square tests. The primary efficacy endpoint was assessed via multi- variable, mixed-effect logistic regression. The independent variable was set as the ratio of grams (g) of citrate to calcium milliequivalents (mEq), and mortality in 30 days was set as the outcome variable. The analysis was done via cubic spline regression to flexibly model the relationship

between the optimal ratio and mortality. To find the association of the optimal ratio and mortality, a spline curve was plotted. A separate mul- tivariable, mixed-effect logistic regression was performed with mortal- ity in <24 h set as the outcome variable. These variables were chosen for analysis in the primary endpoint as citrate administration has been identified as the major mechanism of hypocalcemia in MTP populations [9,11]. Further multivariable logistic regressions were performed to as- sess iCa nadir, SOFA score, and total citrate given on 30-day mortality and the use of RBCs on the incidence of hypocalcemia. The covariates for these analyses were chosen a priori based on previous literature [7,9,12].

  1. Results

Overall, 501 patients were screened for inclusion. Of these patients, 193 were excluded with 118 patients (61%) not meeting MTP criteria, 63 patients (32%) having a lack of documentation, and 12 patients (7%) being <18 years old (Fig. 1). As a result, 308 patients were included, of which 165 patients (53.6%) were grouped with an iCa

<0.9 mmol/L within 24 h and 143 patients (46.4%) were grouped with an iCa >=0.9 mmol/L within 24 h. The baseline characteristics were well balanced between groups (Table 1). Patients who had iCa

<0.9 mmol/L within 24 h had higher sequential organ failure assess- ment (SOFA) scores on ICU admission (Table 1). On average, patients in the iCa <0.9 mmol/L group received significantly more units of RBC, median 12 vs 7 (P < 0.001), and units of FFP, median 9 vs 4 (P < 0.001), within 24 h of MTP activation. Between groups there was no difference in mEq of elemental calcium administered (55.8 vs 36.3). The primary outcome of the ratio of grams of citrate to calcium re- pletion (mEq) (Fig. 2) was not significantly associated with a mortality difference at 30 days (P = 0.91). The repletion ratio of grams of citrate to calcium repletion was evaluated at a median of 1.97 (IQR 1.14-2.91) in this population. Additionally, the ratio of grams of citrate to calcium mEq for each patient (Fig. 3) was not significantly associated with 24- h mortality (P = 0.79). Observing the cubic spline regression, the rate of mortality was lowest at a Citrate:Ca between 2 and 3 in both <24-h

Fig. 1. Inclusion/Exclusion.

Fig. 3. The dark blue line is the predicted probability of mortality in <24 hours based on the ratio of grams of citrate to calcium milliequivalents (mEq).The light blue shaded area is the standard deviation of the predicted probability

Baseline characteristics.

iCa < 0.9 mmol/L (n = 165)

iCa >= 0.9 mmol/L (n = 143)

Age, year; mean(SD)

39.7 (24.7-57.9)

44.7 (28.5-66.9)

BMI; mean(SD)

27.7 (23.9-32.5)

27.4 (23.5-31.5)

Male sex, n(%)

115 (69.7)

106 (74.1)

SOFA score, median(IQR)

8 (4-11)

6 (3-8)

Race/Ethnicity*, n(%) Caucasian

64 (38.7)

68 (47.2)

Black

49 (29.7)

36 (25)

Hispanic

15 (9.1)

4 (6.9)

Asian

3 (1.8)

6 (4.2)

Pacific Islander

1 (0.6)

0 (0)

Mixed/Other

33 (20)

30 (20.8)

Trauma, n(%)

91 (55.1)

80 (55.9)

Penetrating

56 (61.5)

33 (41.3)

Blunt

31 (34)

46 (57.5)

Penetrating and blunt

4 (4.5)

1 (1.2)

Baseline Medications, n(%) Calcium

6 (3.6)

13 (9.1)

vitamin D

14 (8.4)

18 (12.6)

Aspirin

20 (12.1)

28 (19.6)

Clopidogrel or

5 (3.0)

5 (3.5)

Ticagrelor

Apixaban or

5 (3.0)

8 (5.6)

Rivaroxaban

Warfarin

7 (4.2)

7 (4.9)

Dipyridamole

0 (0)

0 (0)

Reversal Agents within 24 h of MTP activation, n(%)

Tranexamic acid 22 (13.3) 17 (11.9)

vitamin K 19 (11.5) 13 (9.1)

Desmopressin 6 (3.6) 2 (1.4)

between patients who had iCa <0.9 mmol/L and patients who had iCa

>=0.9 mmol/L (Table 2). At 24 h, patients in both groups had a normalized iCa level.

A logistic regression (Table 3) was performed to ascertain the effects of iCa nadir, SOFA score, and total citrate given on 30-day mortality and found both iCa nadir (1.139 odds ratio [OR] 95% Confidence Interval [95% CI] 1.023-1.267) and SOFA score (1.173 OR [95%CI]

(1.1.03-1.247)) to be significant. Furthermore, based on the finding of

4-Factor prothrombin complex concentrate

4 (2.4) 2 (1.4)

a preferential RBC administration, a logistic regression was preformed to assess the administered quantity of RBCs on the development of iCa

Image of Fig. 3Factor VIIa 15 (9.0) 7 (4.9)

Comorbidities, n(%)

end stage renal disease

5 (3.0)

4 (2.8)

Renal replacement

4 (2.4)

2 (1.4)

Cirrhosis

12 (7.3)

15 (10.5)

Cancer

11 (6.7)

16 (11.2)

mortality and 30-day mortality. Patients in the iCa <0.9 mmol/L group received significantly more RBC units (median 12 vs 7; p < 0.001), FFP units (median 9 vs 4; p < 0.001), and platelet units (median 2 vs 1; p < 0.001) within 24 h of MTP activation. There was no difference in the amount of calcium received or duration of ICU and hospital LOS

Image of Fig. 2

Fig. 2. The dark blue line is the predicted probability of mortality in 30 days based on the ratio of grams of citrate to calcium milliequivalents (mEq).The light blue shaded area is the standard deviation of the predicted probability

<0.9 mmol/L (Table 3), which was found to be significant (1.044 OR [95%CI] (1.019-1.069)).

  1. Discussion

This is the first study to evaluate dosing strategies of calcium reple- tion in relation to citrate content of blood components in patients un- dergoing surgery or presenting with traumatic injuries necessitating MTP activation. This study sought to examine optimal calcium dosing strategies by creating a novel ratio of grams of citrate to calcium mEq (Citrate:Ca) in order to account for the different citrate concentrations in different components. Data for citrate content in blood components was extrapolated from the blood bank technical formula manual [10]. This study did not find a mortality difference in Citrate:Ca ratio median of 1.97 (IQR 1.14-2.91). To explain this ratio, a quantity of 3 units of RBC for 2 g of calcium gluconate would be equivalent to a Citrate:Ca ratio of 1, and 9 units of RBC for 2 g of calcium gluconate would be equivalent to a Citrate:Ca ratio of 3 [7,10]. Additionally, patients treated with this re- pletion ratio of median of 1.97 (IQR 1.14-2.91) achieved normalized iCa levels at 24 h. However, the predicted probability of mortality was the lowest at a Citrate:Ca ratio between 2 and 3 in both 30 days and 24 h mortality. This suggests that supplementing 1 g of calcium gluconate for every 3 units of RBC given is appropriate.

To date there is little evidence for the appropriate or optimal dosing of calcium in MTP [11]. A previous study characterized hypocalcemia in patients needing MTP and found that those with iCa <0.9 mmol/L re- ceived a mean of 54.4 mEq of elemental calcium or 4 g of Calcium chlo- ride, however the patient population utilized a higher quantity of blood components compared to our study [9]. Another repletion strategy has been described as administering 2 g of calcium gluconate for every 2-4 units of RBCs administered, but this has not yet been validated by research.511 In our trial, patients with iCa < 0.9 mmol/L had increased mortality [9]. Further, for every decrease in iCa nadir by 0.1 mmol/L, there was an increase in mortality rate by 13.9%. This mortality

Table 2

Secondary outcomes.

iCa <0.9 mmol/L (n = 165)

iCa >=0.9 mmol/L (n = 143)

P- Value

Baseline labs

iCal on initiation of MTP, mmol/L; Median (IQR)

0.89 (0.61-1.07)

1.13 (0.99-1.29)

<0.001

Hemoglobin on initiation of MTP, g/dL; Median (IQR)

8.9 (7.4-12.6)

9.6 (7.5-12.8)

0.281

pH on admission; mean (SD)

7.17 (0.18)

7.27 (0.16)

0.01

Lactate on initiation of MTP, mmol/L; Median (IQR)

6.2 (2.9-9.9)

4.5 (3.4–7.7)

0.011

Time to 1st iCal, hr; Median (IQR)

0.67 (0.25-1.25)

1.38 (0.27-4.00)

<0.001

24 h labs

iCal at 24 h after initiation of MTP, mmol/L; Median (IQR)

1.11 (1.07-1.16)

1.12 (1.09-1.17)

0.249

Hemoglobin at 24 h after MTP initiation, g/dL; Median (IQR)

8.8 (7.6-10.6)

9.6 (8.2-11.1)

0.008

Lactate level 24 h after initiation of MTP, mmol/L; Median (IQR)

2.9 (1.7-6.4)

3.0 (1.8-4.4)

0.882

Blood Product and Calcium Administration

Total RBCs given in 24 h after initiation of MTP, units; Median (IQR)

12 (7-20)

7 (4-12.25)

<0.001

Total FFPs given in 24 h after initiation of MTP, units; Median (IQR)

9 (4-15)

4 (2-8)

<0.001

Total Platelets given in 24 h after initiation of MTP, units; Median (IQR)

2 (1-3)

1 (0-2)

<0.001

Total Cryoprecipitate given in 24 h after initiation of MTP, units; Median (IQR)

0 (0-1)

0 (0-1)

0.773

Total citrate 24 h after initiation of MTP, g; Median (IQR)

126.2 (66.9-204.5)

65.2 (35.5-108.7)

<0.001

Total elemental Calcium over 24 h, mEq; Median (IQR)

55.8 (32.1-103.5

36.3 (13.5-70.05)

0.076

Outcome Data

Mortality within 24 h, n (%)

71 (43.0)

23 (16.1)

0.023

Mortality at 30 days, n (%)

85 (51.5)

43 (30.1)

<0.001

ICU length of stay, days, median (IQR)

7.5 (4-21.75)

7 (3-17)

0.348

Hospital length of stay, days, median (IQR)

16 (9-33.25)

17 (8-26)

0.626

difference further illustrates the need to develop an evidence based strategy to maintain iCa >0.9 in MTP patients.

A logistic regression performed to ascertain the effects of iCa nadir, SOFA score, and total citrate given on 30-day mortality found that iCa nadir was significantly associated with increased mortality, which was suspected based on findings of previous trials [9,13]. Between groups, patients with iCa <0.9 mmol/L received more blood components, spe- cifically, significantly more RBCs, FFPs, and PLTs received, which corre- lated with significantly more citrate received as well. Interestingly, our study found that the total amount of citrate received in 24 h was not as- sociated with mortality, which is contrary to the proposed mechanism for increased mortality in hypocalcemia. This may be explained by var- iances in how rapidly blood components and their associated citrate were transfused [14]. Faster rates and quantities of citrate infusions may result in a more potent reduction of iCa through more rapid accu- mulation of citrate and overloading of the liver’s ability to clear it [15]. This potentially could have contributed to our lack of correlation between citrate administration and iCa repletion.

Blood component administration in this study population tended to initially or preferentially favor RBC use as the primary repletion strategy over FFP and platelets. A separate logistic regression found that RBCs given alone are associated with hypocalcemia (Table 3). While not rec- ommended in severe traumas [2], this may reflect a more real world ap- proach to MTP, as it may take more time for blood to arrive from the blood bank and RBCs may be more readily available in an emergency de- partment. MTP coolers may also contain varying amounts of each com- ponent, varying among institutions that would result in differences of blood factor administration. The age of RBCs utilized in this study was not assessed, as the data linking age of RBCs to increased mortality is controversial [16,17]. There was a low incidence of anticoagulant use within this study population, which corresponds to a low usage of anti- coagulation reversal, this was similar between iCa groups however, as

Table 3

Logistic Regression.

Mortality in 30 days OR (95%CI)

iCa Nadir, 0.1 mmol/L 1.139 (1.023-1.267)

SOFA Score 1.173 (1.1.03-1.247)

Total citrate 24 h after initiation of MTP, 1 g 1.001 (0.999-1.003) Incidence of Hypocalcemia

Total RBC given in 24 h, units 1.044 (1.019-1.069)

very few patients were on anticoagulants overall, we were unable to determine the impact of calcium repletion in this cohort.

This study was limited in the fact that it was single centered with a relatively small sample, which limits its external validity when applying these findings to other institutions with different MTP protocols or pop- ulations. Additionally, this study was retrospective and could not ac- count for all confounding variables including the incidence of hypothermia and acidosis trends in this patient population, which is linked with coagulopathy in MTP populations [18]. Another limitation was the timing of the iCa levels was at the discretion of the treating phy- sician, therefore levels obtained may not reflect true calcium peaks and troughs on all patients. Finally, we were unable to explore the hypothesis that citrate infusion rate affected the incidence of hypocalcemia due to the lack of documentation. Based on the limitations noted, a larger mul- ticenter prospective study is needed to validate the results of this study.

  1. Conclusion

There were no significant differences in 24 h or 30 day mortality based on repletion ratios seen in this study. A Citrate:Ca ratio between 2 and 3 in patients undergoing MTP was sufficient to obtain a normal- ized iCa within 24 h of MTP activation regardless of baseline iCa level. Further prospective studies will be needed to determine the optimal Citrate:Ca ratio.

CRediT authorship contribution statement

Andrew Robinson: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Megan A. Rech: Writing – review & editing, Supervision, Software, Resources, Project administration, Inves- tigation, Formal analysis, Data curation, Conceptualization. Phillip J. DeChristopher: Writing – review & editing, Validation, Supervision, Re- sources, Project administration, Formal analysis. Alex Vaughn: Soft- ware, Investigation, Formal analysis, Data curation. John Rubino: Software, Investigation, Formal analysis, Data curation. Erika Bannister: Software, Investigation, Formal analysis, Data curation. Mary E. Moore: Software, Resources, Methodology, Formal analysis, Data curation. Kevin Chang: Writing – review & editing, Supervision, Resources, Pro- ject administration, Investigation, Formal analysis, Conceptualization.

Declaration of Competing Interest

None.

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