The Role of Serum Lactate Dehydrogenase in Etiological Diagnosis of Macrocytic Anemia

 

Maram Jbaily¹, Firas Hussein², Nisrin Kaddar³

¹Postgraduate Student, Department of Biochemistry and Microbiology, Faculty of Pharmacy,

Tishreen University, Lattakia, Syria.

²Assistant Professor, Department of Internal Medicine and Heamatology, Faculty of Medicine,

Tishreen University, Lattakia, Syria.

 

ABSTRACT:

Background: Macrocytic anemia is common and is characterized by decreased hemoglobin levels with elevated Mean Corpuscular Volume (MCV), due to a range of diseases and divided into Megaloblastic and Non-megaloblastic anemia. Serum vitamin B12 and folic acid tests are usually performed but they are limited by their low sensitivity and specificity. To confirm diagnosis of macrocytic anemia bone marrow examination is required but it is invasive procedure. Vitamin B12 and/or B9 deficiency leads to a defect in DNA synthesis leading to ineffective erythropoiesis and intramedullary hemolysis in patients of megaloblastic anemia, this leads to increased serum  LDH (Lactate Dehydrogenase) and unconjugated bilirubin. Aims of study: This study was carried out to evaluate the role of serum LDH in the distinction between megaloblastic and non-megaloblastic anemia, and to study the correlation between serum LDH and MCV Patient and Methods: The study included 60 patients with non-regenerative macrocytic anemia (We exclude patients with regenerative macrocytic anemia because elevated reticulocytes leads us to hymolysis anemia or bleeding and it’s not a diagnostic problem(. Complete blood count, biochemical investigation, peripheral blood examination, reticulocyte count, bone marrow examination was performed in all cases Results: The most common cause of macrocytic anemia was Megaloblastic anemia (65%). The other causes were primary bone marrow disorders (35%). There was a significant difference in the mean values of serum LDH  and MCV between two groups ( megaloblastic and non-megaloblastic anemia). When LDH>2076.5 IU/L, there is more probability of having megaloblastic anemia than non-megaloblastic anemia but when LDH>2076.5  and MCV>109.45 with bilirubin> 1.2mg/dl will must more probably not have non-megaloblastic anemia (the specificity was 100%), all three parameters combined together can be used as screening test to distinguish between the 2 groups of macrocytic anemia (megaloblastic and non-megaloblastic)  without necessity of bone marrow aspiration in patients of non- regenerative macrocytic anemia. Present study had also shown that there were a positive relationship between serum LDH and MCV (r=+0.613).

 

 

INTRODUCTION:

Anemia is defined as a reduction in the red blood cell (RBC) mass or blood haemoglobin concentration^1-3, Macrocytic anemia is a condition in which the red blood

cells are larger than the normal size. In macrocytic anemia, mean corpuscular volume (MCV) of red blood cells is more than 100 fL (normal range of MCV should be 80-100 fl)^4-6. Macrocytic anemia is divided into  megaloblastic and non-megaloblastic anemia based on bone marrow examination, this category is important for determining the etiology of macrocytic anemia⁷.  Diagnosis of  macrocytic anemia depends on the study of peripheral blood smear. The presence of macro-ovalocytes, hypersegmented neutrophils (>5% of neutrophils with 5 segments, or >1% with 6 segments), anisocytosis and poikilocytosis refers to megaloblastic anemia⁸. To confirm the diagnosis of macrocytic anemia bone marrow examination should be performed, but it is invasive procedure. In megaloblastic anemia bone marrow shows abnormal proliferation and maturation of multiple myeloid cells, resulting from defect in nuclear differentiation. These abnormalities occur in the morphology of the erythrocyte and other myeloid cells (erythroblasts and giant metamyelocytes are seen in bone marrow) leading to ineffective erythropoiesis and intramedullary hemolysis causing an elevated lactate dehydrogenase and indirect bilirubin in the serum^5,9. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the interconversion of lactic and pyruvic acids with a very wide distribution throughout the body^10-13. It is released into circulation when cells of the body are damaged or destroyed. The highest levels of total LDH are seen in megaloblastic anemia and hemolytic disorders. Intramedullary destruction of erythroblasts causes elevation as a result of the high concentration of LDH in erythrocytes¹⁴. This study was done to evaluate the significance of serum LDH estimation as a diagnostic indicator of macrocytic anemia (megaloblastic or non-megaloblastic anemia).

 

MATERIALS AND METHODS:

The study was conducted on 60 patients newly diagnosed with macrocytic anemia (haemoglobin<12g/dl with MCV>100 fl) admitted in service of Hematology, Tishreen University hospital, during the period of August 2019 and August 2021. No patient included in the study had  regenerative macrocytic anemia because elevated reticulocytes leads us to hymolysis anemia or bleeding and it’s not a diagnostic problem.

 

Peripheral blood smear, reticulocyte count, bone marrow aspiration, bilirubin levels, serum LDH were done on all cases. Thyroid function tests, serum iron, and coombs test are done wherever necessary.

Lactate dehydrogenase was assayed by Biochemistry Department in Tishreen Hospital, using an automatic biochemical analyzer (DIRUI CS-600), normal values (207-414 IU/L).   

 

Those cases who were recently transfused and those already on hematinic therapy or had active hepatitis, consistent hepatic enzyme abnormalities, myocardial infraction, pulmonary embolism were excluded from the study.

 

Patients were divided into two groups (megaloblastic and non-maegaloblastic), based on bone marrow examination.

 

Statistical analysis: Data were analyzed using SPSS version 25 software, P values were calculated by Mann Whitney test. ROC Curve analysis was done for MCV values and LDH values to get a cut-off value which can be used as screening tests in macrocytic anemia to differentiate between megaloblastic and non-megaloblastic anemia.

 

RESULTS:

60 cases of macrocytic anemia were included in the study. Out of 60 cases, 65% had megaloblastic anemia and 35% had non-megaloblastic anemia. Non megaloblastic anemia included Aplastic anemia, Myelodysplastic syndrome, Multiple myeloma, Acute and chronic Leukemia. The various causes of macrocytic anemia in our study are detailed in Table 1.

 

The minimum age was 17 years and maximum age was 84 years with a mean of 53.1 years. Out of 60 cases studied 36 cases were males and 24 were females.

 

Table 1. Etiology of macrocytic anemia and percentage of cases in each diagnostic group.

 

MCV values ranged from 101 to 137fl with a mean of 111.48fl and standard deviation of ±8.19. Serum LDH values ranged from 272 to 8847IU/L with a mean of 2601.27IU/L and standard deviation of ±1703.99IU/L. The hematology values are listed in Table 2. Asignificant difference was seen in serum LDH values (P value=0.000), MCV (p value=0.005) and T Bili (P value=0.000) between two groups.

 

Serum LDH values were <1242 IU/L in 14 cases of non-megaloblastic anemia (66.7%). 87.3% of megaloblastic anemia group had serum LDH >2070IU/L (5 times higher than normal) and 30.8% of them had serum LDH>4141IU/L (10 times higher than normal) (Table3).

 

Table 2. Comparisons of various complete blood count parameters between megaloblastic and non-megaloblastic group.

 

Table 3. Precentage of cases in  megaloblastic and non-megaloblastic group under different serum LDH values.

 

MCV values were in the range of 100-110fl in 32 cases, out of which 18 cases had non-megaloblastic anemia (85.7%), but 61.5% of megaloblastic group had MCV>110fl (Table 4)

 

Serum bilirubin was rised in 28 cases of megaloblastic anemia (71.8%) with a mean value of 1.61mg/dl and a standard deviation of 0.63mg/dl.

 

ROC Curve analysis was done for MCV values and LDH values to get a cut-off value which can be used as screening tests in macrocytic anemia to differentiate between megaloblastic and non-megaloblastic anemia. Roc Curve analysis for serum LDH values gave a criterion value of >2076.5IU/L for megaloblastic anemia, at which  the sensitivity was 87.2%, specificity was 90.5% with a positive predictive value of 94.4%. The area under the curve was 0.902 and the p value was 0.000 (Figure 1).

 

Figure 1.ROC curve for serum LDH values.

 

Figure 2.ROC curve for MCV values.

 

Roc Curve analysis for MCV values gave a criterion value of >109.45fl for megaloblastic anemia, at which the sensitivity was 69.2% specificity was 81% with a positive predictive value of 87.1%. The area under the curve was 0.719 and the p value was 0.005 (Figure 2).

 

When LDH>2076.5 IU/L, there is more probability of having megaloblastic anemia than non-megaloblastic anemia but when LDH>2076.5  and MCV>109.45 with bilirubin> 1.2mg/dl will must more probably not have non-megaloblastic anemia, all three parameters combined together can be used as screening test to distinguish between the 2 groups of macrocytic anemia (megaloblastic and non-megaloblastic) and further evaluation can be done depending upon the categorization (Table 5).

 

Table 5. Sensitivity, specificity, positive predictive value and negative predictive value for diagnosing megaloblastic anemia at ROC criterion values

 

DISCUSSION:

Macrocytic anemia describes an anemic state characterized by elevated mean corpuscular volume (MCV>100 femtoliters) and divided into  megaloblastic and  non-megaloblastic anemia based on bone marrow examination^5,15. In present study megaloblastic anemia was the most common cause of macrocytic anemia, this may be due to several reasons such as deficient intake¹⁶, chronic Helicobacter pylori gastritis¹⁷, chronic alcoholism, excessive use of proton pump inhibitors¹⁸.

 

The difference between means of serum LDH between megaloblastic and non- megaloblastic group was significant with those of megaloblastic anemia group having higher serum LDH than the non-megaloblastic group , the maximum value of serum LDH was 8847 IU/L which was 21 times the normal value, the significant increase in serum LDH concentrations in the megaloblastic group can be explained by the fact that vitamins B12 and B9 are important cofactors for normal maturation of all cells including Marrow erythroblasts. Deficiency in either of these two vitamins leads to a defect in DNA synthesis and abnormal RBC maturation (nuclear/cytoplasmic asynchrony). The erythroblasts become large, oval shaped. These bone marrow abnormalities are called “megaloblastic” and lead to ineffective erythropoiesis and intramedullary hemolysis in patients of megaloblastic anemia, this leads to increased serum  LDH and unconjugated bilirubin^5,8,19,20. In addition to increased intramedullary turnover of megaloblastic, higher LDH content of these cells is also responsible for high LDH plasma levels²¹.

 

This result is in agreement with a number of previous studies conducted in india and confirmed the great diagnostic important of LDH enzyme in the casual diagnosis of macrocytic anemia^22-25. The study conducted by Aarthi Kannan et al on 100 macrocytic anemia patients stated that when serum LDH>1345.2 IU/L or MCV>121fl with reticulocyte count <2% was taken as criteria,  the sensitivity was 92.1%, specificity was 93.5%  for diagnosis megaloblastic anemia²³, another Indian study conducted by  Thimmappa et al on 100 macrocytic anemia patients stated that when serum LDH>1124.5 IU/L or MCV>120.5fl with reticulocyte count <2% was taken as criteria,  the sensitivity was 94.4%, specificity was 93%  for diagnosis megaloblastic anemia²⁴. In present study, we found that when serum LDH >2076.5 IU/L (criterion value of ROC curve) there is more probability of having megaloblastic anemia than non-megaloblastic anemia, the sensitivity was 87.2%, specificity was 90.5% with positive predictive value of 94.4% and negative predictive value of 79.2%. but when serum LDH>2076.5 IU/L (5 times higher than normal) and MCV>109.45fl with bilirubin> 1.2mg/dl will must more probably not have non-megaloblastic anemia (the specificity was 100%), Present study had also shown that there were a positive relationship between serum LDH and MCV (r=+0.613).

 

CONCLUSION:

Depending on the previous data and the result of our study, we can diagnose megaloblastic anemia in a patient with non- regenerative macrocytic anemia when three criteria are combined: serum LDH>2076.5 IU/L  (5 times higher than normal) and MCV>109.45fl with bilirubin> 1.2mg/dl without the need for invasive procedures such as bone marrow aspiration.

 

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Accepted on 16.04.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5421-5424.