Dr. Amany Ragab

TUMOR MARKERSTUMOR MARKERSSOME LABORATORY ASPECTS OF SOME LABORATORY ASPECTS OF

PREGNANCYPREGNANCYCEREBROSPINAL FLUID ANALYSISCEREBROSPINAL FLUID ANALYSIS

ByByDr.Dr. Amany RagabAmany Ragab

Lecturer of Clinical PathologyLecturer of Clinical PathologyMansoura Faculty of MedicineMansoura Faculty of Medicine

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Tumor markersTumor markers

• Tumor markers are macromolecules mostly protein with carbohydrate or lipid component whose appearance in blood or body fluid is related to the presence or progress of the neoplasm.

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• Cell (tissue) tumor markers

These include antigens located on cell membranes such as cell markers for leukemia, hormone receptors, genetic intracellular components

• Humoral markers

Substances which are present in high concentration in blood, urine, other body fluid. They are synthesized and excreted by tumor tissue.

• The ideal tumor markers should have the following characters:

• Specificity for cancer:Number of true –ve cases (low number of false

+ve cases)• Tumor markers should produced only by the

tumor• Should be absent or present in low

concentration in normal individuals• Shows no elevation in benign diseases• Sensitivity:Number of true +ve cases (low number of false

–ve cases)• Means that a very small growth of tumor

produces measurable amounts of markers

• The amount of the marker should be correlated with the tumor load

• the assay for it should be inexpensive, easy to do and sensitive

• the half life of it should be short enough so that when production the level rapidly.


Clinical application of tumor markers:• Screening in general population• Differential diagnosis in symptomatic

patients• Clinical staging of cancer• Estimating tumor volume• prognostic indicator for disease

progression• Monitoring the response to therapy• Detecting the recurrence of cancer

tumor markers may be:

• Enzymes

• Hormones

• Oncofetal antigens

• Carbohydrate markers

• Blood group antigens

• Proliferation markers (mitotic index)

• Hormone receptor markers (estrogen & progesterone receptors)

ENZYMES:ENZYMES:

elevated levels can serve as tumor markers, examples are:

• Alkaline phosphatase (ALP): primary or secondary liver cancer, metastatic cancer with bone or liver involvement, specially with osteoblastic lesions.

• Lactate dehydrogenase (LDH): liver cancer, non-Hodgkin’s lymphoma, acute leukemia, non-Seminomatous germ-cell testicular cancer, seminoma, neuroblastoma, etc ……

• Prostatic acid phosphatase (PAP): prostatic cancer, osteogenic sarcoma, multiple myeloma, and some benign conditions such as benign prostatic hyperplasia, osteoporosis and hyperparathyroidism.

• Prostate-specific antigen (PSA): it is extremely useful tumor marker to detect, stage, and monitor treatment of prostate cancer. Serum PSA rises also with age, benign prostatic hypertrophy and lower urinary tract infection

Hormones:Hormones:

• production of hormones in cancer involves either production of hormones in excessive amounts by its original gland or production at a distant site by a non endocrine tissue (ectopic syndrome), e.g production of ACTH by the small cell carcinoma of the lung.


• Calcitonin: medullary carcinoma of the thyroid

Carcinoma of the lung pancreas

prostate and ovary

• B-HCG: Vesicular mole choriocarcinoma,

Carcinoma of the liver, lung.

• ACTH: Carcinoma of the lung, colon, prostate and ovary

• Prolactin: Pituitary tumors, carcinoma of the lung

• Parathyroid hormone (PTH): Parathyroid tumors, carcinoma of the lung, kidney, liver and breast.

• ADH: Carcinoma of the lung.

• Oncofetal antigens:

• are proteins produced during fetal life. These proteins are present in high concentration in the sera of fetuses and decrease to low levels or disappear after birth. These proteins reappear in cancer patients. The production of these proteins demonstrates that certain genes are reactivated as a result of malignant transformation of cells.

Alpha-fetoprotein (AFP):Alpha-fetoprotein (AFP):

a marker for hepatocellular and germ-a marker for hepatocellular and germ-cell carcinoma (non-seminoma) its cell carcinoma (non-seminoma) its measurement can also used to measurement can also used to monitor treatment. Level in healthy monitor treatment. Level in healthy adults is less than 10 ug/L. [During adults is less than 10 ug/L. [During pregnancy, maternal AFP levels start pregnancy, maternal AFP levels start to show progressive increase]. Levels to show progressive increase]. Levels can also be increased in non-can also be increased in non-cancerous liver diseases such as cancerous liver diseases such as hepatitis and cirrhosis, but the hepatitis and cirrhosis, but the increase is mild.increase is mild.

• Carcinoembryonic antigen (CEA): this is a marker for colorectal, gastrointestinal, lung and breast carcinoma. The upper limit in the healthy population is about 3 ug/L for non-smokers and 5 ug/L for smokers.


• Because of the false-positive and false-negative results, CEA testing is better used as an adjunct to clinical staging and together with other markers. CEA levels decline after successful therapy. Rising values may indicate recurrence.

• Carbohydrate markers: These are either antigens on the tumor cell surface or secreted by the tumor cells. They tend to be more specific than enzymes or hormones and are abbreviated as CA. Examples are CA 15-3, CA 549 (for breast and ovary), and CA 125 (for ovary and endometrium).

• Blood group antigens: These include CA 19-9 (colorectal and pancreatic cancer) and CA 72-4 (cancer of GIT and ovary).

Some laboratory aspects of Some laboratory aspects of pregnancypregnancy

The clinical laboratory has an important role in diagnosis, management and evaluation of pregnancy, both normal and abnormal.

• Diagnosis of pregnancy and ectopic pregnancy:

The diagnosis of pregnancy is based on the detection of hCG in serum about 10 days after conception, with a marked and gradual increase thereafter.

Typical intervals for serum hCG in Typical intervals for serum hCG in pregnancypregnancy

After fertilization (wk)

2-----------------------------------------------------

3-----------------------------------------------------

4-----------------------------------------------------

5-12-------------------------------------------------

13-24-----------------------------------------------

26-38------------------------------------------------

IU/L

• 5-100

• 200-3000

• 10 000-80 000

• 90 000-500 000

• 5000-80 000

• 3000-15 000

In ectopic pregnancy plasma Serial measurement of hCG fails to rise at the normal rate.

The use of serum progesterone together with hCG is more predictive of outcome than a single hCG measurement

Diagnosis of gestational diabetes mellitus:

I- Screening

II- Diagnosis

I-Screening:Performed between 24 and 28 wk of

gestation on all pregnant women not identified as having glucose intolerance

50 g oral glucose load is given without regard to time of day or time of last meal.

Venous plasma glucose is measured at 1 h.

If glucose is > or equal 140 mg/dl, perform glucose tolerance test.

II-Diagnosis:OGTTPerformed in the morning after a 8-14 h fastThe fasting venous plasma glucose is

measured100 g glucose is given orallythe plasma glucose is measured orally for 3

hoursAt least two values must exeed the following:Fasting 105 mg/dl1 h 190 mg/dl2 h 165 mg/dl3 h 145 mg/dl


Maternal serum screening for Maternal serum screening for fetal defectsfetal defects • diagnosis of fetal abnormalities is advised

for pregnancies at risk because of either advanced maternal age or family history.

• Maternal serum -fetoprotein (AFP) is used to screen for neural tube defects (anencephaly and spina bifida) at 18-20 wk gestation. If elevated levels are found on two occasions, ultrasound and amniocentesis may be indicated.

• Screening for the presence of a fetus with Down’s syndrome can be performed by measurement of the following analytes in maternal serum at 16 wk gestation:

AFP: values are approximately 30% lower in mothers having trisomy 21 or trisomy 18.

Chorionic gonadotropin: values are approximalety 2 times higher when fetal Down syndrome is present.

Unconjugated Estriol: values are approximately 0.7 time lower when fetal Down syndrome is present.

Values of these analytes (triple markers) should be compared to median values reported for women of matched age, body weight and gestational age in weeks.

Cerebrospinal fluidCerebrospinal fluid

It is the fluid that occupies the spaces of the C.N.S, it circulates upward over the cerebral hemispheres and downward over the spinal cord and nerve roots.

Formation:-70% by the ventricular choroid plexus and 30% by epindymal lining of the ventricles as well as cerebral and arachinoid space. Its normal adult volume is 100-200 ml. It functions as an excretory vehicle, circulate nutrients and lubricates CNS.

C.S.F. examination:-

C.S.F is obtained by lumbar puncture, lateral cervical puncture or through ventricular cannula or shunts under aseptic conditions. Indications for C.S.F examination are meningeal infection, subarachinoid haemorrhage, CNS malignancy and demylinating diseases .

Appearance: Physical examination -

Normal C.S.F is:

• Clear

• Colourless

• Clot free. Clot formation may be due to its traumatic tap, complete spinal block (Froin`s syndrome) and suppurative or T.B meningitis.

• Has the viscosity of water.

Turbidity in fresh CSF may be due to: erythrocytes, leukocytes or bacteria. Coloured CSF is due to oxy- hemoglobin, bilirubin or drugs.

Bloody CSF sample may be due to:• A traumatic tap (clear supernatant after

centrifugation) • Hemorrhage (yellow supernatant, after

centrifugation;this is called xanthochromia).

Chemical examination:-Chemical examination:-

Total protein (normal range 20 – 40 mg/dl): -

• CSF proteins:

More than 80% of CSF protein content originate from plasma by ultrafiltration (forced diuresis) through walls of the capillaries in meninges and choroid plexuses.

The reminder 20% from intrathecal synthesis.

Causes of Causes of protein in CSF protein in CSF

I- Increase permeability of blood brain barrier to plasma proteins:

• High intracranial pressure due to tumor or hemorrhage.

• Inflammation: bacterial meningitis

• Obstruction of spinal cord by tumor

• The degree of permeability of blood brain barrier can be evaluated by

measurement of albumin in CSF and serum simultaneausly:

The CSF/ Serum albumin index is calculated = albumin in CSF(mg/dl)/Albumin in serum (g/dl)

• An index value < 9 is normal

• 9-14 slight impairment of the barrier

• 14-30 moderate impairment of the barrier

• 30-100 severe impairment of the barrier

• > 100 complete breakdown of the barrier

II- II- increase intrathecal synthesis:increase intrathecal synthesis:

• Demyelinating disease of CNS; multiple sclerosis, where immunoglobulin production is increased


To identify the intrathecal production of immunoglobulin one of the following ratios are used

1. IgG (CSF) mg/dl / Alb |(CSF) mg/dlValue > 0.27 indicates synthesis of Ig

G2. CSF IgG index= IgG CSF x Albumin

serum / IgG serum x Albumin CSFReference range for index: 0.3-0.77Value >0.77 = increase synthesis in 90 %

of cases of multiple sclerosis

Glucose (normal CSF level 40-80 mg/dl):-

Increased level (relative to plasma glucose) is an evidence of hyperglycemia 2-4 hour prior to lumbar puncture. Decreased level of CSF glucose < 40 mg/dl in fasting patients with normal blood glucose may be due to bacterial meningitis, T.B meningitis, fungal meningitis, amaebic meningo-encephalitis and subarachinoid hoemorrhge. In viral meningitis CSF glucose is usually normal.

In Rhinorrhea : The fluid is tested for glucose, if glucose is present the fluid is CSF.

Chloride :- (Normal 120 – 130 mmol/L,is higher than plasma level).

Its level is low in pyogenic meningitis and much lower in T.B meningitis.

Increased level of lactic acid occurs in case of traumatic brain injury and bacterial meningitis.


Enzymes:

Lactic dehydrogenase (LDH): Increased in bacterial meningitis, viral meningitis, leukaemia, lymphoma, metastatic and subarachinoid haemorrhage.

Creatine kinase (CK):-

Increased CK is sensitive but not specific for CNS diseases as haemorrhage, thrombosis, multiple sclerosis.

Aspartate amino transferases (AST):- Increased in neurological disease with bad prognosis.

Microscopic examination:Microscopic examination:

• Without centrifugation of the sample for total leucocytic counting (cells/ ml)

• With sample centrifugation for RBCS counting / HPF from the precipitant.

• Stained smear by Leishman stain for determination of the type of leucocyte

• Gram's stain for microbiological examination. a


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