INTRODUCTION:

Cancer has become the second ranking cause of death there, led only by heart disease. Cancer calls can be viewed as altered self cells that have escaped normal growth regulating mechanism. These cells give rise to clones of cells that expand to a considerable size, producing a tumor, or neoplasm. A tumor that is not capable of indefinite growth and does not invade the healthy surrounding tissues extensively is benign. A tumor that continues to grow and becomes progressively invasive is malignant; the term cancer refers specifically to a malignant tumor.

Lymphoma refers to malignant tumor of the lymph system includes lymph nodes and related organs that are part of the body’s immune and blood forming system. There are two types of lymphoma include Hodgkin’s lymphoma and Non-Hodgkin’s lymphoma. Histologically all forms Hodgkin lymphoma is characterized by the presence of reed Sternberg cells and their variant forms. Reed Sternberg cells are varying large cells with two or more nuclei or nuclear lobes, each which contains a single large eosinophilic nucleolus. Non-Hodgkin Lymphoma is characterized by absence of Reed Sternberg cells. About 85% of Non-Hodgkin arises in B cells, the rest occurs in T cells.

When a diagnosis of lymphoma is suspected, the cancer specialist will do several blood tests, a thorough physical examination (including manual examination of lymph node sites) and possibly, depending on his or her suspicion of the type of lymphoma, a biopsy (removal and examination) of bone marrow. The technique used in the diagnosis are morphology (examination of cell appearance), phenotype (specific cell sub-type) which includes Bone Marrow Aspiration, finding the parameters of Lactate dehydrogenase, Microglobulin and then analyzing through Flowcytometry.

MATERIALS AND METHODS:

Different diagnostic methods and materials are followed in the analysis of neoplastic cells. The taken out blood from patients is then stored in special type of tubes called vaccutainers. The walls of these tubes are coated with anti-coagulant such as EDTA, sodium fluoride, etc.

The study group was of Indian origin; of a particular region (Andhra Pradesh). The cases are in the age group of (35-75). The diagnostic criteria for lymphoma (both HL and NHL) were strictly followed .Blood, Bone Marrow, Immuno-Histocompatability tests were carried out at the pathology department for clinical evaluation of diagnosis and prognosis. Clinical, Biochemical, and Hematological characteristics are analyzed for the entire diagnosed patient. The parameters recorded are age, sex, LDH level, β2m I and IHC markers. Three of the patients in the group complained of general lymphoma symptoms like lymph node swelling and weight loss. In case of HL patients, the common complaint was severe itching.

CBC - Complete Blood Count: This is a collection of tests including hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, and count. Blood is collected from the patient in a vaccutainer containing EDTA as an anticoagulant, and then the sample is mixed well using rotary shaker for one minute. The sample is aspirated into the instrument.

Bone Marrow Aspiration: Bone marrow is usually located in the centre of many bones and is responsible for producing blood cells. This test is used for the diagnosis and staging of cancers involving the blood cells. Bone marrow biopsies are also used to look for metastatic diseases.

Beta 2-Microglobulin: β2microglobulin is synthesized by most nucleated correspond to a production of approximately 150mg per 24hours. Half the plasma β2microglobulin, which is renewed daily, originate from lymphocytes cells. Circulating β2 microglobulin is filtered through renal glomeruli and then reabsorbed and catabolised by the proximal tubule. Elevated plasma β2 microglobulin levels indicate either decreased glomerular filtration or increased synthesis. Plasma beta2 microglobulin levels can therefore be elevated in patient with diseases other than renal disorder and in particular, those which affect the immune system.

The assay principle combines a two step enzyme immuno assay sandwich method with a final fluorescent detection (ELFA).The solid phase receptacle (SPR) serves as the solid phase as well as pipetting device for the assay. After diluting the sample, the β2 microglobulin in the sample binds with the specific monoclonal antibody coating the interior of the SPR. Unbound components are eliminated during the washing steps. The beta2 microglobulin retained is revealed by an alkaline phosphates-labeled polyclonal anti-human beta2 microglobulin antibody (sheep). Unbound conjugate is eliminated during the washing phase.

During the final detection step, the substrate (4-methyl-umbelliferyl phosphate) is cycled in and out of the SPR. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-methyl-umbelliferone), the fluorescence of which is measured at 450nm. The intensity of the fluorescence is proportional to the concentration of beta2 microglobulin present in the sample.

Lactate Dehydrogenase: The estimation of lactate dehydrogenase enzyme involves the following reaction, Pyuvate + NADH + H + LDH à lactate + NADNADH = nicotinamide adenine dinucleotide (reduced form), LDH = lactate dehydrogenase

In the presence of coenzyme NADH, lactate dehydrogenase catalyzes the conversion of pyruvate to lactate. LDH activity is measured as the amount of pyruvate consumed by the continuous monitoring of the decrease in absorbance due to the oxidation of NADH at 340nm.

Flow Cytometry: Immunphenotyping of cells is a method of detecting specific markers on cell populations and there by characterizing them. These markers are generally the clusters of differentiation (CD), a molecule that is present on cell surface and differentiates populations of cells from each other. The CD molecules can be recognized by monoclonal antibodies, and thus populations of cells expressing CD molecules can be identified.

RESULTS AND DISCUSSION:

Patient profile and disease characteristics:

The clinical characteristics of 10 patients diagnosed with lymphoma were analyzed. The Diagnosis was confirmed by hematology test and bone marrow tests. The serum samples of 10 patients of whom 7 were males and 3were females are analyzed for LDH, β2 Microglobulin and also tumor markers. Of the ten patients, 8 patients (80%) showed increased levels of serum LDH and remaining (only 20%) had shown normal LDH level.

Complete blood count:

The RBC, WBC and platelet count for each patient was found out. The results of this test are shown in the following tabular column:

Patient	RBC 10¹²/litre	WBC 10⁹/litre	Platelets 10⁹/litre
A	4.0	11.5	210
B	4.2	12.8	215
C	3.5	11.9	140
D	4.5	12.4	310
E	4.9	12.9	408
F	3.2	14.8	121
G	5.7	12.4	287
H	4.8	11.7	356
I	5.5	13.5	335
J	3.9	14.2	375

From this table, WBC counts were found to be increased in all the ten patients. The normal range of WBC is 4-11 x 10⁹/litres. In all the patients WBC values were found to be above 11x 10⁹/litres. Elevated WBC counts may occur in case of infection, allergy, systematic illness, inflammation, tissue injury and cancer.

RBC counts were found to be normal in 8 patients; the other two patients had decreased RBC counts (patient C and F). The normal/ range of RBC cells are 4-6 x 10¹²/ litres. Decreased RBC counts may occur in case of anemia, polycythemia and cancer. When the cancer cell spread to the marrow, it suppresses the production of all other blood cells.

Platelets counts are usually measured before the bone marrow biopsy procedure. It is useful to find clotting time. The patients C and F showed decreased platelets count (normal range 150-400 10⁹/litre). Decreased platelet count may occur in case of aplastic anemia thrombocytopenia and cancer.

Significance of LDH in diagnosis of lymphoma:

The analysis of ten patients with tumor symptoms, showed increased levels of LDH. These LDH values are shown in the following tabular column.

Name	LDH	Biopsy Diagnosis
A	340	NHL ( T-cell)
B	562	NHL ( T-cell)
C	399	NHL ( B-cell)
D	278	NHL ( T-cell)
E	414	HL
F	263	NHL ( B-cell)
G	528	NHL ( T-cell)
H	151	HL
I	490	NHL ( B-cell)
J	452	NHL ( T-cell)

In the cases analyzed, LDH concentration in blood was found to be abnormal for both Hodgkin’s and Non-Hodgkin’s lymphoma patients. In any cancer, tissue damage is the major factor during the tumor evasion. Tissue damage is estimated by measuring the concentration of LDH in blood, as many cells have this enzyme including RBC. In patient B, LDH level was found to be higher (562 IU/L) than all other patients. This suggest that the patient is subjected to extensive tissue damage and also of stage 3 or stage 4 type lymphoma. In patient H, LDH level is less elevated than the others. This indicates that the centre is in the early stages (stage 1 or stage 2 type of lymphoma). Lower aberrated results suggest of lesser tumor burden.

In NHL, of the 8 patients, 7 had shown abnormally increased levels of serum LDH. Of those 8 patients,5 are males and 3 patients were females. The elevated serum LDH levels ranged from151-562 IU/L. The patients were in the age group of 35-75 years. In the study period, only two patients (male) were diagnosed with Hodgkin’s lymphoma. Of them, one showed an abnormal LDH value and the other had the normal.

Significance of β2 microglobulin in diagnosis of lymphoma:

In the cases of analyzed, β2 microglobulin concentration in blood was found to be abnormal for both Hodgkin’s lymphoma and Non-Hodgkin lymphoma patients. This protein β2 microglobulin is associated with MHC class I molecules. In the presence of foreign invader, class I MHC molecules are synthesized by granulocytic cells. So elevated β2 microglobulin indicate the presence of antigens in the blood. In patient J β2 microglobulin level is highly aberrated than all other patients. This elevated level indicates the presence of cancer antigen in the blood, encountered by the granulocytic cells such as macrophages.β2 Microglobulin is released into blood due to the cancer cell properties such lurking the antigenic peptide from T_c cell, the extensive tissue damage and also the over synthesis of the β2 microglobulin by the cells. Malignant transformation of cells is associated with class I MHC Molecules, and the tumors have been shown to express decreased levels of class I MHC molecules. Due to this tumor property, β2 microglobulin in the blood gets elevated. So the elevation indicates the malignant transformation of lymphoma (staging).The lower value of β2 microglobulin for patient-I can be attributed to lesser extent of tissue damage, which in turn suggests of lesser tumor burden. In case of β2 microglobulin 6 of 8 NHL patients showed elevated levels ranging from 1.68-12.84 gm/ml, where has in HL one patient showed elevated level of β2 microglobulin and the other had normal.

Lymphoma markers:

Phenotypic markers on the surface of the lymphocytes are identified by flow cytometry analysis. The patients in the study are analyzed for these markers. Four of them showed positivity for either of the CD2, CD3 or CD5 marker, confirming B-cell NHL. Similarly four of them showed positivity for either of the CD19, CD20 or CD22 marker, confirming T-cell NHL. Two patients were tested positive for either of the CD15 or CD30 markers and hence the HL.

CONCLUSIONS:

β2 microglobulin and LDH have been already established as an indicator of tumor burden. Our observations have more or less been similar. All the patients in the study group showed some sort of deviation in their LDH and β2 microglobulin levels. This reveals the importance of these parameters in the diagnosis of lymphoma. These results are of greater importance in the planning of prognosis. Also the morphological features are essential for the lymphoma confirmation. However, numbers of cases are required to establish the role of serum lactate dehydrogenase and β2 microglobulin in accessing the tumor burden. These results play an important role in planning the treatment and understanding the patient response to chemotherapy and other radiation treatments.

ACKNOWLEDGEMENT:

The authors thanked Dr. S. Sudha Murthy, Head, Department of Laboratory Medicine, Indo American Cancer Institute and Research Centre, Hyderabad, for providing all the facilities to carry out the work.

Declaration: The authors do not have conflict of interest and this work was not financed by any organization. This wok was a nonstipendiary work.

Name

Age

Sex

Clinical picture

CD 19

CD 20

CD 22

CD 2

CD 3

CD 5

CD 15

CD 30

Biopsy Diagnosis

Three weeks of right shoulder and swelling in arms.

NHL

(T-cell)

Bilateral masses in groin present for 5 to 6 weeks

NHL

(T-cell)

4 day history of right groin pain.

NHL

(B-cell)

Non tender, firm, slight moveable mass over the right submandbicular area

NHL

(B-cell)

Swelling in groin, fever and red patches on the skin.

Enlarged lymph node in armpit

NHL

(B-cell)

Tonsls enlargement and lump iin abdomen

NHL

(T-cell0

Lymph node swelling in armpit area

Swollen, painless lymph nodes in the neck.

NHL

(B-cell)

Multiple nodules,liver mild enlargement of liver and moderate enlargement of spleen

NHL

(T-cell)