Clinical Chemistry

By
Dr. Rabea Al-Sayed
2018
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Chapter 1- Endocrine
1) Hyper Calcemia
 Causes:
1) Parathyroid diseases:
* 1ry hyperparathyroidism: the commonest cause
* 3ry hyperparathyroidism * Multiple endocrine neoplasia syndrome.
2) Excess intake of vit D or Ca:
* Milk alkali syndrome * self- medication with vit D
3) Bone diseases:
* Paget’s disease. * Ectopic PTH production.
* Carcinoma with Osteolytic secondary deposits.
4) Familiar hypocalciuric hypocalcaemia.
5) Drugs: thiazide diuretics.
6) Thyrotoxicosis, cushing syndrome.
7) Malignancy:
 Multiple myeloma, Leukemia.
 Investigations:
1) S.Ca   Increased. 2) High critical value > 13 ml /dl (ICU)
3) S.Phosphatase   in parathyroid hypercalcemia.
4) PTH level  in 1ry hyperparathyroidism & ectopic.
5) 25 hydroxy vit D 6) Urinary Ca 
7) Urinary Ca AMP 
8) Alkaline Phosphatase  when bone affected
9) Renal function test.
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2) Hypocalcaemia
 Causes:
1) Hypoalbuminemia: the commonest cause in chronic hypocalcaemia.
2) Hypoparathyrodism.
a) Post- Surgical hyperparathyroidism.
b) Autoimmune disease.
c) Low Mg d) Extensive radiotherapy
3) Pseudo hypoparathyroidism: due to peripheral resistance to PTH.
4) CRF: hypocalcemia is mainly leading to hyperphosphatemia.
5) Hypo- Mg: as a mal- absorption syndrome, starvation, excess diuretics.
6) Osteomalacia & Rickets.  Vit. D   absorption of calcium
7) Acute hemorrhagic &edematous pancreatitis due to:
a) Deposition of Ca in necrotic peripancreatic fat
b) Impaired PTH secretion
 Pre- analytical preparation:
1) Specimen should be withdrawn out of tourniquet.
2) The patient is in sitting position.
3) Using a wide needle. 4) Overnight fast.
 Methods of assays:
1) Total Ca:
- Spectrophotometer Method. - Atonic absorption.
2) Free Ca++:
- Free Ca analyzer. - Ca ion selective electrode.
3) Corrected Ca (mg/dl) = S.ca +0.8 (4 – S.albumin)
4) Critical Low value < 6 mg/dl
 Investigations:
1) S.ca   (Critical Value ≤ 6 mg/dl) 2) S.albumin  in liver cirrhosis.
3) S.creatinine & blood urea in CRF. 4) Mg deficiency.
5) In Osteomalacia & Rickets:  Ca,  phosphorus,  Vit.D &  PTH & 
alk. phosphatase.
6) Pseudohypoparathyroidism: PTH  or normal.
7) Hyperparathyroidism.  PTH &  phosphorus,  Ca.
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3) Hyperprolactinemia
* Causes:
1) Hypothalamic – Pituitary disorders:
* Hypothalamic Lesion
* Parahypopituitarism.
* Traumatic or surgical section of pit. stalk
2) Pregnancy:
Decreased to the basal level after several weeks of delivery.
3) 1ry hypothyroidism:
  TRF, TSH, PRL.
4) Liver diseases: sever Cirrhosis.
5) Uremia.
6) Chest wall injury.
7) breast diseases & nipple stimulation.
8) Ectopic PRL secreting tumours
9) Drugs: Antidepressants.
Indications for PRl Measurements:
1) Galactorrhea.
2) Enlarged sella turcica.
3) Suspected pituitary tumour.
4) Hypogonadism, oligomenorrhea in women &  lipido – impotence in
men.
5) unexplained amenorrhea: (Secondary amenorrhea) FSH is essential for
proper diagnosis.
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* Interpretation of results:
1) analysis of repeated samples should be done in agreat peripheral PRL
levels.
(in suspected hyperprolactinemia)
2) In pathological cases: there are possibilities:
- 30 - 100 ng/ml
- 1ry hypothalamus.
- hypothalamus affection.
- Pit. Tumour.
3) PRl may be normal in some pituitary tumour Cases;
- If PRl 30 – 100 ng/ml  MR1 (high resolution) is recommended.
- Radiological Studies of pituitary & sella –turcica is recommended.
4) In Prolactinomas, there is
- Subnormal responsiveness to TRH stimulation.
- In 90 % in cases, the 2folds increase doesn’t occur.
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4) Laboratory investigations of thyroid gland:

1ry 2ry 1ry 2ry

Hypothyroidism Hypothyroidism Hyperthyroidism Hyperthyroidism

TT3    

FT3    

TT4    

FT4    

TSH    

Autoantibodies +ve - ve +ve - ve

CK & cholesterol  

Normocytic or
pernicious Present Absent Absent Absent
anemia

Hyperglycemia - ve - ve +ve - ve

Ca: serum &

-- --  --
urine

TRH stimulation Exaggerated Lack response to

Lack response Lack of response
test response TRH

Causes:

Hypothyroidism Hyperthyroidism

1ry Hashimoto’s disease Grave’s disease

Iodine deficiency Subacute thyroiditis
Surgical removal & irradiation Benign thyroid tumor
Post therapy of hyperthyroidism Thyroid hormone intake
Excess iodine & anti- thyroid drugs

2ry Pituitary hypopituitarism TSH pit. secreting tumor

3ry Hypothalamic causes Hypothalamic causes

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5) Investigations of hypothyroidism:
Presentation:
1- New born: (congenital hypothyroidism)
* Respiratory difficulty, cyanosis
* Jaundice, poor feeding, retarded bone maturation
2- Children:
* Retard growth &mental retardation
3- Adult:
Easy fatigue, coldness, constipation with gain, menstrual irregularity, Ms.
Cramps.
4-  TG &  cholesterol
5-  prolactin
6- Macrocytic anemia
How to investigate:
I- Tests to measure concentration of Hormones
* T3 T4, FT4, FT3, TSH
- By RIA & Elisa
* Thyroglobulin
- By RIA (N: 10- 40 ng/ml) - In hyperthyroidism with thyroiditis
* rT3 - By RIA & Eliza (N: 80- 200 ng/dl)
II - Tests to examine function of thyroid gland
* Evaluation of thyroid gland size & iodine metabolism
- Radioactive iodine uptake
- Thyroid imaging (fluorescent scanning –US- MRI)
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lll- Test to evaluate the hypothalamic - pit. axis.

1) Serum TSH:
- By RIA or IRMA.
- Normal 0.5 - 5 µu/ml.
- In primary hypothyroidism TSH is markedly increased.
- Corticosteroids and dopamine inhibits TSH.
2) TRH test:
- I.V. injection of 500 µg TRH.
- Measurement of TSH before and 15', 30' after injection.
- Normally there is 2- 3 folds increase in TSH level or the TSH should be
increased more than 6 µu/ml.
- TRH is the only test available to distinguish between 2ry (pit.) and tertiary
(Hypothalamice) hypothyroidism.
- In Pit hypothyroidism  TSH response is decreased or absent.
In hypothalamic TSH response is adequate but delayed.
1- Thyroglobulin and thyroid microsomal autoantibodies
2- Thyroid - stimulating immunoglobulin: TSI autoantibody against TSH
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6) Mention Value of TSH

1) Plasma
- TSH circulates unbound, with half- life 50- 60 min
- Normal level differ according to the method used:
o By RIA technique from 1- 10u/ml
o By IRMA technique from 0.4- 4.5 µu/ml
Evaluation of TSH:
I- Basal measurement:
1. T3; T4 and Free T4 by RIA should be done
2. TSH level is estimated by RIA or IRMA
 Elevated TSH with low T3&T4  Primary hypothyroidism
 Low or normal TSH with low T3&T4  Secondary hypothyroidism due
to Hypoth-pit dysfunction
 In hyperthyroidism TSH is low with high T3&T4
 In Low T3 syndrome TSH is Normal with low T3
II- TSH Dynamic test is recommended (TRH test)
I.V. injection of 500 µg TRH
o Normally produce TSH of at least 6 µIU/ml  within 15- 30 min
o In lry hypothyroidism with normal basal TSH  Exaggerated
o Impaired responses in:
 Delayed TSH response (60- 120 min.) means hypothalamic disease
(Tertiary hypothyroidism).
 Absent or subnormal response: means pituitary disorder. (Secondary
hypothyroidism).
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7) Low T3 Syndrome
* Def.: A state of normal or slightly  T4, low T3, T3 & normal TSH.
* Causes:
- Activate of type II di-iodine enzyme and inhibition of type I.
- accelerate conversion of T4 into T3.
* Occurs:
- Physiologically in fetus
- Pathologically in:
1) CHO restricted:
* Malnutrition, Starvation
* D.M & Anorexia nervosa
2) Acute illness
* M.I
* Liver diseases.
* CRF.
* Cancer in advanced stage.
* D.D of low T3 Syndrome.
* Low T3 syndrome   rT3 & normal TSH
Hyperthyroidism   T3,  T4 and  TSH
Hypothyroidism   T3,  T4,  rT3 and  TSH
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8) Demonstrate Reversed T3:

• rT3 measured by RIA Elisa.
• Norma l adult range 25 - 75 ng/dl (1/3 total T3)
• It can be used to differentiate hypothyroidism from low T3 syndrome &
starvation, where rT3 is reduced in hypothyroidism & elevated in chronic
illness as rT3 & TSH normal in low T3 syndrome
• Hypothyroidism: in cases with elevated T4 (to compensate low T3) Those
• Can be differentiated from hyperthyroid by low T3, normal TSH and
elevated rT3,
Direct estimation of free T4 is essential
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9) Primary Hyperparathyroidism
 General characteristics:
1. One or more glands produce inappropriately high amounts of PTH
relative to the serum calcium level.
2. Most common cause of hypercalcemia in the outpatient setting
 Causes:
1. Adenoma (80% of cases) - majority involve only one gland
2. Hyperplasia (15% to 20%) of cases
3. Carcinoma
 Clinical features
- Stones: nephrolithiasis, nephrocalcinosis
- Bones: bone aches, pains, osteitisfibrosa cystica (brown tumors)
- Muscle pain and weakness Pancreatitis, peptic ulcer disease, Gout and
Constipation
- Psychiatric overtones: depression, fatigue, anorexia, sleep
disturbances, anxiety and lethargy
- Polydipsia, polyuria, HTN, shortened QT interval and weight loss
 Laboratory diagnosis
a. Calcium levels: hypercalcemia and ionized calcium level
b. PTH levels: Should be elevated relative to serum calcium level
c. Hypophosphatemia
d. Hypercalciuria e. Urine cAMP is elevated
f. Chloride /phosphorus ratio > 33 is diagnostic of primary
hyperparathyroidism
 Radiographs
a. Subperiosteal bone resorption b. Osteopenia E.
Treatment. Surgery is the only definitive
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10) Secondary hypercholesterolemia

* Causes:
1) Diet rich in cholesterol, total & saturated fat.
2) Hypothyroidism.
3) D.M.
4) Hepatic disease & Obstructive Jaundice.
5) CRF & Nephritic syndrome.
6) Pregnancy, Alcohol, Obesity, Smoking.
7) Anorexia nervosa.
8) Lipid Storage disorders.
9) Drugs: BB, estrogen, thiaziade.
Total cholesterol levels:
• Desirable: Below 200 mg/dL
• Border line high: 200 to 239
• High: Above 240
LDL cholesterol levels:
• Optimal for people with heart disease or who are at high risk: Below 70
mg/dL
• Optimal for people at risk of heart disease: Below 100
• Optimal: 100 to 129
• Borderline high: 130 to 159
• High: 160 to 189
HDL cholesterol levels:
• Poor: Below 40 mg/dL • Acceptable: 40 to 5,9
• Optimal: 60 or above
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Triglyceride levels:
• Optimal: Below 150 mg/dL • Borderline high: 150 to 199
• High: Above 200
If you have high cholesterol, you should be checked every 2 to 6 months.
• Lab. investigations:
• Serum lipid profile (measured total cholesterol, TG, and HDL cholesterol
and calculated LDL cholesterol and VLDL)
• Dyslipidemia is suspected in patients with characteristic physical findings
or complications of dyslipidemia (e.g., atherosclerotic disease).
A family history of atherosclerotic: disease, or serum cholesterol > 240
mg/dL.
• Prof iles can vary for about 30 days after an acute MI; however, results
obtained within 24 h after MI are usually reliable enough to guide initial
lipid- lowering therapy.
VLDL is estimated by TG ÷ 5.
𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒𝑠
LDL cholesterol = Total cholesterol - [𝐻𝐷𝐿 𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 + ( )]
5

• Other tests for Secondary causes

Fasting glucose, liver enzymes, creatinine, TSH and urinary protein
Screening by:
A fasting lipid profile should be done in all children between age 9 and 11 or
at age 2 if children have a family history of severe hyperlipidemia or
premature CAD. Adults are screened at age 20 yr and every 5 yr thereafter.
Assessment of other cardiovascular risk factors, defined as:
• Diabetes mellitus
• Cigarette use
• Hypertension
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11) 2ry hyperlipidemia

* Causes:
1) Exogenous:
* Obesity * Drugs: thiazide, cortisone
2) Endocrine:
* D.M * Hypothyroidism * Pregnancy * Hypopituitarism.
3) Storage D:
* Glycogen storage disease. * Gaucher disease.
* Neiman pick disease.
4) Renal diseases:
* NS   LDL- C
* CRF  HUS, dialysis: lipoptn.
5) Liver diseases:
Congenital biliary atresia
6) Acute & transient:
* MI * Bacterial & viral infection * Burns * Hepatitis.
* Investigations:
1) S. cholesterol:
(Total, HDL, LDL)
2) Serum TG.
3) Lipoprotein electrophoresis.
(Done in unknown disorders) as:
* Familial type * If TG > 200 mg/dl. * Lipemic Sample.
* Significant hyperglycemia. * CHD in a patient < 40 y old.
* Strong family hereditary sex with CHD.
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12) Familial hypercholesterolemia

Def.: Genetic disorder characterized by  cholesterol level esp. LDL “bad
cholesterol”
C/P:
- Xanthomata, Atherosclerosis, CHD.
- Angioma.
* Investigations:
1)  total cholesterol.
2)  LDL.
3) Normal HDL.
4) Genetic study to establish defective gene
5) Test for fibroblasts to detect Ab
IIa   total cholesterol + LDL
IIb   TG + Cholesterol + LDl
IV   TG
* In all  Apolipoprotein.
* In lipoprotein electrophoresis.
IIa   B region only.
IIb   both B & pre B.
Type III   both B & pre B with broad band.
Type IV   Pre B only.
Type V   Pre B only & Chylomicrone
Type I   B & Pre B & Chylomicrone
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13) Apolipoprotein
* Def: the proteins components of lipoprotein.
* Function:
1) Maintain the integrity & Stability of lipoproteins particles and promote
the Solubility of Lipids in plasma.
2) Activate Key Regulatory enzymes in lipid metabolism.
3) Facilitate the uptake of Lipoprotein into cells.
* Types:
1) A  synthesized in liver, intestine
- It’s the major ptn in HDL
- Apo A1  cofactor for LCAT
- Apo A II  inhibit LCAT & activate hepatic lipase.
2) B  in plasma in 2 forms:
- Apo B – 100
- Apo B - 48  made in intestine.
- It’s the only found in Chylomicrons
- Play an important role in secretion of chylomicrons.
3) C  3 types apo C- 1, C- 2, C- 3.
* made by liver & incorporated into HDL.
* transferred from HDL  chylomicrons & VLDL.
4) D  transfer cholesterol esterase from HDL  LDL, VLDL
5) E  Synthesed in the liver & incorporated in HDL & play central role in
metabolism of chylomicrons remnants & VLDL.
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Apolipoprotein B100
It is a protein that plays a role in moving cholesterol around body. It is a
form of low density lipoprotein (LDL). Changes in apoB 100 can cause
familial hypercholesterolemia.
Apolipoprotein B (apoB) levels are used to evaluate the risk for
cardiovascular disease. The reference range of apoB levels in adults is less
than 130 mg/dl.
High apoB100 levels associated with:
• Familial combined hyperlipidemia
• Diabetes
• Hypothyroidism
• Kidney disease
• The use of certain drugs, such as diuretics, androgens, or beta- blockers
Low apoB100 levels indicate:
• Hyperthyroidism
• Reye’s syndrome
• Abetalipoproteinemia
• Cirrhosis or severe scarring of the liver
• Malnutrition
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14) Hyperadrenalism (Cushing syndrome):

- Elevated cortisol and loss of diurnal variation
- Causes: Iatrogenic, Cushing disease & Cushing syndrome.
o Iatrogenic e.g. cortisone therapy.
o ACTH dependent causes (85%)  Cushing disease.
  ACTH by anterior pituitary gland adenoma.
 Ectopic (extra- pituitary source): Oat cell carcinoma of the lung,
epithelial thymoma, pancreatic islet cell tumour, medullary carcinoma of
thyroid or bronchial carcinoid tumour.
o ACTH independent causes (15%)  Cushing syndrome.
 Adrenal gland tumours: malignant more common than benign:
- Scheme for investigation of hyperadrenalism:
I. Screening test:
1. Urine cortisol
2. Serum cortisol
3. Overnight dexamethasone suppression (suppression to ACTH) test:
II. Confirmatory test: Low dose dexamethasone suppression test:
III. To know the etiology:
1. High dose dexamethasone suppression test:
2. plasma ACTH
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15) Dexamethasone suppression tests:

One of the most powerful synthetic glucocorticoids,
A- Low dose tests:-
1- These procedures are used to establish the presence of Cushing’s
syndrome whatever its cause.
2- Dexamethasone suppress pit.
ACTH release with a resulting fall in plasma and urine corticosteroids
3- In Cushing's syndrome this mechanism is abnormal & failure of
suppression occur.
4- Dexamethasone does not interfere with the measurement of plasma or
urine cortisol.
2) Two- day low- dose test: [suppression tets of Liddle]
• Dexamethasone administered 0.5 mg every 6 hours for 2 days. (8 doses)
• 24 hrs. urine collection are obtained before and on the 2nd day of Dex
administration.
• Urine 17- hydroxy corticosteroids fall to less than 4mg/24hs.
• Failure to suppress exclude pituitary adenoma & indicating either adrenal
neoplasma or ectopic ACTH secretion.
• ACTH> 200 pg/mlEctopic tumour
• The same false positive & negative results as in overnight test.
• Less useful than overnight test.
B- High- Dose tests:-
High dose of dexam. Differentiat Cushing's disease (Pit. ACTH
hypersecretion) from Ectopic ACTH syndrome and adrenal tumors.
1) Overnight high dose Dexamethasone supp.test.
Faster & Simpler than the 2 days test.
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• After a base- line cortisol sample at 8- 9AM is obtained a single dose of

Dexamethazone 8mg orally is administered at 11 PM
• At 8- 9 AM next morning measure cortisol:
a- In Cushing's disease plasma cortisol depress to 50%S of basa.
b- Ectopic or adrenal adenoma there is no depression.
This test is more reliable than 2 days test.
2) Two days higher dose test:
• Oral administration of 2 mg dexameth. Every 6hrs for 2day (8 doses) (2mg
x 4)
• 24hrs urine collected before & on 2nd day of Dexameth Administration.
• Reduction of 17- hydroxycorticosteroid of urine to about 50% of baseal
level is diagnostic for Cushing disease while no response in Ectopic or
adrenal tumours.
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16) Clinical significance of β- HCG:

Normal range of β- HCG in early detection of pregnancy
- < 5 IU/L  negative - 5 – 25 IU/L  equivocal -  25 IU/L  positive
We must repeat the test twice to notify the duplication mechanism of the
level.
Clinical application
1. Diagnosis of early pregnancy in acute care setting:
- Detect early pregnancy in cases of in- vitro fertilization.
- Early detection of pregnancy is made if:
 Β- HCG  25 IU/L
 Β- HCG shows a 2 fold increase at 2 days interval (100%  in 48 hours).
2. Diagnosis of ectopic pregnancy: It is made if
- β- HCG shows β- HCG < 66% β- HCG  over 48 hours
- β- HCG is low for that gestational age especially after 4 weeks of
gestation: actually.
- β- HCG never exceeds 6500 IU/L: if β- HCG  6500 IU/L, this denotes that
the intrauterine pregnancy and presence of gestational sac should be visible
by ultrasound. Therefore, if β- HCG  6500 and the sac not seen by
ultrasound  ectopic pregnancy.
3. Diagnosis of pregnancies at increased risk of down syndrome:
- The triple test for down syndrome:
 β- HCG: , α- fetoprotein:  & Estriol:  than the expected for
gestational age.
 The test done in 15th to 17th week of gestation.
4. Predicting of inevitable (obligatory) abortion:
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- In the 1st 30 days of gestation: if the β- HCG is < 66%  0ver 48 hours 
abortion is likely.
- At 8th week of gestation: if the serum β- HCG β- HCG < 10,000 IU/L β-
HCG  abortion generally takes place, but if it is greater than 18,000 IU/L
abortion doesn’t occur.
5. In missed abortion:
- β- HCG levels are low and repeated analysis are necessary to verify the
diagnosis.
6. Diagnosis and follow up of non- trophoblastic tumours: Malignant
ovary teratoma
- Where low levels of β- HCG are detected < 10,000 IU/L.
7. Diagnosis and follow up of benign trophoblastic disease: vesicular mole
- The activity of syncitio- trophoblastic cells  resulting in excessive β- HCG
production (200,000 – 1,000,000 IU/L).
- Follow up program of vesicular mole:
 successive assays.
 Repeat β- HCG every month for at least 1 year before the patients
is released.
- After evacuation:
 β- HCG rising for 2 successive or constant for 3 successive weeks.
 β- HCG is elevated at 15 weeks post evacuation.
 Rising β- HCG, after reaching normal level.
Note:
 50% of patients  vesicular mole.
 50% of patients develop choriocarcinoma.
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8. Diagnosis and follow up malignant trophoblastic disease:

High serum β- HCG (200,000 – 1,000,000) observed in
- β- HCG in non- metastatic malignancy:
  40,000 IU/L  indicate poor prognosis.
9. detects late postpartum hemorrhage:
- Detectable β- HCG concentration may indicate residual placental tissue in
utero.
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17) Osteoporosis & Osteomalacia

1- Osteomalacia is the softening of the bones while osteoporosis is the
decreasing of the mass of the bones.
2- Osteomalacia is caused by a deficiency in calcium and phosphorus due to
inadequate intake of Vitamin D while osteoporosis has many causes
3- Osteomalacia is characterized by a deceased ratio of bone mineral to
matrix.
Osteomalacia when it appears in children is called rickets.
4- Diagnosed by:
* An elevated alkaline phosphatase, low phosphate, low or normal calcium,
and low 25(0H) D levels. Plain radiographic films may show osteopenia or
characteristic pseudofractures
5- Clinical features of osteoporosis include:
a- fractures from minimal trauma, particularly in the thoracic and lumbar
spine, wrist and hip
b- Lab values of serum calcium, phosphorus and alkaline phosphatase are
not diagnostic.
c- Plain x-rays, show decreased bone density. A DEXA > 2.5 is diagnostic of
osteoporosis.
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Chapter 2 - D.M
1) Juvenile diabetes
Causes
1- Autoimmune destruction of B cells of pancreas autoantibodies.
2- Environmental factors: Viral (mumps, Rubella).
3- Genetic Factors.
Symptoms & Signs:
1- Heavy thirst.
2- Increased hunger.
3- Nausea & Vomiting.
4- Frequent urination
5- Blurred Vision.
6- Heavy breathing.
7- Fruity Smell of breath.
8- Loss of Consciousness
9- Frequent infection of skin, vagina & urinary tract.
Laboratory diagnosis:
RBS ≥ 200 mg/dl
FBS ≥ 126 mg/dl
HbA1C: provide an estimate of plasma glucose levels during the preceding 1
– 3 Months.
If > 6.9 on 2 Separate tests suggests D.M
Fructosamine Levels: Measure glucose levels & reflects glucose control in
the previous 1 – 3 Weeks.
TLC & blood and urine culture to rule out infection.
Plasma acetone level: specifically & more reliable indicator of DKA than
urine ketones.
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2) Oral glucose tolerance test (OGTT) :

* (OGTT) measures the body's ability to use glucose,
* Used to diagnose prediabetes and diabetes.
*Indications of OGTT:
1) Diagnosis of gestational D.M.
2) Diagnosis of impaired glucose tolerance & impaired fasting glucose.
3) For epidemiological studies.
4) Evaluation of unexplained microangiopathic Complications of D.M with
Random
Blood sugar < 140 mg/dl
5) Diagnosis of:
- Renal glucosuria.
- Alimentary glucosuria.
- Reactive hypoglycemia.
- After response.
*Advantages:
More sensitive > FBS
*Disadvantages:
- Imprecise (affected by large No of variants)
- Should be repeated > one occasion
- Vigorous exercise
- Several drugs may cause glucose intolerance.
*Precautions before test:
1) Stop drugs  BB, OCPS.
2) Not done in  bed ridden, hospitalized, sever infection, trauma.
3) Unrestricted  diet (150gm CHO / day) at Least: 3days before test
exercise.
4) When to be form
* 7 – 9 AM
* fasting for (8- 14hrs)
*No smoking or Caffeine in the day of Test.
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5) Mental & Physical rest.

6) Sitting or lying Rt side.
How to proceed
1) Eat a balanced diet that contains at least 150 grams(g) of carbohydrate
per day for 3 days before the test
2) Do not eat, drink, smoke, or exercise for at least 8 hours before your first
sample is taken
3) Stop taking certain medicines before the test (BB, OCPS – thiazides)
4) Do not eat during the test. You may drink only water during this time.
On the day of testing, the following steps will be done:
5) A % sample for fasting blood glucose will be collected when you arrive.
- For the standard glucose tolerance test, you will drink 75 grams or 100
grams glucose.
- Blood samples will be collected at timed intervals of 1, 2, and sometimes 3
hours after you drink the glucose.
*Normal glucose tolerance test values
Fasting: Less than or equal to 100 mg/dL
1 hr: Less than 184 mg/dL
2- hour: Less than 140 mg/dL
*Values that indicate gestational diabetes
Fasting: More than or equal to 92 mg/dL
1 hr: More than or equal to 180 mg/dL
2 hr: More than or equal to 153 mg/dL
3 hr More than or equal to 140 mg/dL
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3) I.V glucose tolerance test:

*Indications:
1) Assessment of 1st phase insulin response to glucose.
2) Patient can’t tolerate Oral glucose load (repeated Vomiting).
3) Malabsorption $, Post gastrectomy.
4) Hypothyroidism.
5) Patients showing a flat tolerance curve after administration of the oral
glucose load
*Glucose load: 25 gm / dl Solution given over 3 min (I. V infusion)
*Sampling:
- Every 10 min, over 60 min. (6 Samples).
- Start from mid injection time.
Disadvantage:
Poor reproducibility
Data presentation:
1- Blood glucose decreases algorithmic manner
2- So, blood glucose levels are blotted as algorithmic scale as the Y- axis
versus time in minutes as the X- axis.
On Log paper
Blood
glucose K= 70 / t ½

Time (min)
10 20

K  rate of disappearance of glucose.

t ½  is the time in minutes needed for blood glucose to decrease to ½ of
the 10 min level.
Normally K > 1.5% / min.
In D. M: K < 1% / min.
 (29)

4) HbA1C: Glycated Hb
 Def.:
Glycation is non – enzymatic addition of sugar residue to N terminal of
Valine of each  chain of HbA.
 Concentration of HbA1C depend on:
- Life span of RBCs - Blood glucose level last 6 – 8 weeks
 Reference range: 3 – 6 % of HBA
Good glycemic control of HbA1C  5 – 8 %
 Cl. Significance:
- Routine in 3 months in type I diabetes.
- Every month in diabetic pregnancy.
 Advantages:
1) No need for fasting sample.
2) Not affected by exercise or recent eating.
3) Not affected by glucose mutation from day to day.
 Specimen: Venous blood sample on EDTA.
 Methods:
2) Change differences:
- HPLC - electrophoresis - Isoelectric focusing
3) Chemical analysis:
- Colorimetric. - Spectrophotometer.
4) Structural differences:
- Chromatography. - Immunoassay.
 Causes of False results:
False  False 
Hemolytic anemia Lead Poisoning
 Life span of RBCs. Alcoholism
Hypersplenism Splenectomy
Hbs HbF
Recent blood loss.
Ineffective erythropoiesis.
 (30)

5) How to investigate Gestational D.M

Lifestyle interventions or metformin for diabetes prevention

• Women with diabetes in the first trimester have type 2 diabetes
*Def:
- CHO intolerance within 1st onset during Preg  due to Failure of insulin
secretion to overcome insulin resistance.
*Complications:
- Mother: - Preeclampsia.
- D.M in Subsequent Pregnancy.
- Fetus:  death or still birth.
 Congenital malformations.
*Factors  risk of G D.M:
1) +Ve family history.
2) Advanced maternal age.
3) History of 1UFD & Congenital malformations.
4) Fetus > 4 Kg.
5) Obesity.
6) Recurrent monilial infection.
7) Polyhydramnios, glucosuria.
Diagnosis of Gestational Diabetes (GDM)
Pregnant women with risk factors
Test for undiagnosed type 2 at first prenatal visit using standard diagnostic
criteria
Pregnant women without known prior diabetes
Test for GDM.at 24- 28weeks
Women with GDM
Screen for or persistent diabetes 6- 12 wks postpartum using OGTT and
standard diagnostic criteria
 (31)

Women with a history of GDM

Lifestyle screening for diabetes or prediabetes every ≥3 yrs
Women with a history of GDM and pre- diabetes
• GDM if diagnosed in the second or third trimester and not clearly
associated with type 1 or type 2 diabetes
Procedures
• Perform 75- g OGTT with plasma glucose measurement
• Test in the morning after the patient has fasted for ≥ 8 hours
• Repeat test at 1 and 2 hours after initial measurement
Diagnosis is confirmed when PG levels meet or exceed:
• Fasting 92mg/.dL
• 1 hr: 180 mg/dL
• 2 hr: 153 mg/dL
 (32)

6) Diagnosis of Diabetic ketoacidosis:

Biochemically, DKA is defined as an increase in the serum concentration of
ketones greater than 5 mEq/L, a blood glucose level greater than 250
mg/dL, and a blood arterial pH less than 7.3
Symptoms
- Flushed, hot, dry skin. - Blurred vision.
- Feeling thirsty and urinating a lot.
- Drowsiness or difficulty waking up.
- Rapid, deep breathing.
- A strong, fruity breath odor.
- Loss of appetite, belly pain, and vomiting.
- Confusion.
Laboratory findings:
- Urine dipstick highly positive for glucose, ketones
- Serum ketones present
- ABG: low bicarbonate, low PCO2, pH < 7. 35
- Anion gap > 16
- Serum K normal, or high.
- Serum Na low
- Serum phosphate: normal or high
- Serum glucose usually > 300 mg/dl but below 800mg/dl
- Leukocytosis
serum urea Elevated
S. creatinine Elevated
serum chloride usually low
serum magnesiumusually low
serum calcium usually low
serum lactate elevated in lactic acidosis
serum amylase usually elevated
serum lipase usually normal
 (33)

7) How to investigate of Fasting hypoglycemia :

1) Whipple Triad:
1) FBS: adult < 45 mg/dl.
Children < 40 mg/dl.
Neonates < 30 mg/dl.
Premature < 20 mg/dl.
2) Signs & Symptoms of hypoglycemia precipitated by Fasting
3) Signs & Symptoms of hypoglycemia Improved by glucose administration
2) Fasting Insulin: (N: 10 – 20 µ/ml)
*  In
– isulinoma.
- Non pancreatic tumours.
*  In most hypoglycemic disorders.
3) C-Peptide: (N: 0.78 – 1.8 ng/dl)
 In insulinoma.
 In insulin exogenous.
4) Insulin / fasting glucose ratio: (N : < 0.3 )
If > 0.3  insulinoma.
5) 72 hrs Fasting test:
-If FBS insulin Level on borderline.
- Using non –glucose containing Fluids for 72 hrs then:
* Measure glucose, Insulin, proinsulin, C-Peptide, Ketone bodies every 6
hours.
*Stop the test if bl.glucose < 45mg/dl.
6) Suppressive test:
- Measure insulin, C-Peptide.
- Give exogenous insulin, then remeasure insulin & C- Peptide.
- Insulin: * In insulinoma. *  In normal cell.
 (34)

8) Hypoglycemia
*Causes :
-In Neonates:
- Prematurity, RDS, Maternal DM.
- Preeclampsia of mother.
-In infants:
- ketotic hypoglycemia, glycogen storage D, galactosemia.
-In adults :
- Alcohol, liver Cirrhosis, insulinoma.
- Hormone deficiency.
-Lab diagnosis:
Whipple Triad:
1) FBS: adult < 45 mg/dl.
Children < 40 mg/dl.
Neonates < 30 mg/dl.
Premature < 20 mg/dl.
2) Signs & Symptoms of hypoglycemia precipitated by Fasting
3) Signs & Symptoms of hypoglycemia Improved by glucose administration
 (35)

9) C-peptide:
C-peptide is a peptide composed of 31 amino acids. It is released from the
pancreatic beta-cells. It is mainly excreted by the kidney.
The reference range of C-peptide is 0.8-3.1 ng/mL.
C-peptide is measured the difference between insulin as a body produces
and insulin injected into the body.
C-peptide levels are elevated in the following:
- Sulfonylurea intoxication. - Chronic kidney disease

- Noninsulinoma pancreatogenous hypoglycemia syndrome (NIPHS)

C-peptide levels are suppressed in the following:

- Exogenous insulin injection
- Due to insulin-like growth factor secreting tumor
- Insulin-independent hypoglycemia
Indications
• C-peptide should be measured in the combination of insulin and
proinsulin to differentiate between insulin-dependent hypoglycemia (high
C-peptide levels) versus insulin-independent hypoglycemia (low C-peptide
levels).
• In combination with serum and/or urine sulfonylurea screening, C-
peptide testing can help differentiate between factitious hypoglycemia due
to exogenous insulin use (low C-peptide level, high insulin level) and
sulfonylurea intoxication (high C-peptide level, high insulin level).
• To monitor pancreatic function after a pancreatic transplantation or
pancreatectomy
• To monitor beta-cell function in a patient with early-stage type 1
diabetes mellitus.
• To differentiate between type 2 diabetes mellitus and latent
autoimmune diabetes of adults.
 (36)

10) Insulin- induced hypoglycemia stress Test

The most reliable stimulus for GH secretion. Normally GH increase to more
than l0 ng/ml after adequate hypoglycemia is reached.
*Principle:
- Rapid reduction of blood glucose by I.V injection of insulin  Stimulation
of adrenals (measured by plasma cortisol &GH secretion).
- Contraindicated in elder Patients with epilepsy or heart diseases.
Procedure:
- 0.1 Iu/kg I.V. of insulin then plasma GH, PRI, ACTH & glucose are
estimated. In suspected insulin resistance 0.3m/kg is used. Then plasma GH
estimated 30, 1H, 1.5Hs & 2Hs after injection:
- Normally peak at 1H is reached > 7ng/mI.
- GH deficiency is excluded by the attainment of GH concentration in excess
of 7ng/ml at any time. But cannot be diagnosed unless the patient fails to
respond to at least 2 different provocative tests. Since 10- 15% of cases fail
to respond in normal individuals.
N.B:
* When induction of hypoglycaemia is contraindication glucagons may be
used to stimulate cortisol and GH, secretion instead of insulin.
* At the end of test give the Patients Glucose containing drink & meat.
* Avoid stress of Patients on testing.
*Terminate the test if Signs of marked hypoglycemia appears (by giving I.V
5% glucose immediately).
*Interpretation:
* In Hypopit:
- GH not respond - No  Cortisol
- ACTH  low or normal.
*Normal response:
-  GH > 10 ng/ml - Cortisol up to 18 mg/dl in 60- 90 min
- PRL  in two folds
 (37)

Chapter 3 - GITE & Liver

1) Hyperbilirubinemia:

*Causes:

1) Pre- hepatic ( un conjugated) :

*Hemolytic anemia. * Bleeding into the tissues.

*In effective erythropoiesis.

2) Hepatic (hepatocellular) :

*defect in uptake:

( unconjugated bil)

- Gilbert’s syndrome. - Hepatitis. - Cirrhosis.

*defect in conjugation:

( Unconjugated)

- Gilbert’s syndrome. - Crigler Najjar syndrome.

- Premature babies.

*defect in excretion:

( Conj > unconjugated bil)

- Generalized hepatocellular damage. - Rotor and Dubin Johnson.

3) Post- hepatic (Obstructive) :

( Conjugate)

*Intrahepatic:

HCC – drugs – Cirrhosis

 (38)

*Extra hepatic:

- Gall stones in CBD. - Cancer head of Pancreas. - Biliary atresia.

*HOW to investigate a case of hyperbilirubinemia

1) Liver function test:

Hymolytic HC Obstructive
Unconjugated   Ormal
bilirubin
Conjugated Normal  
Bilirubin
SGPT, SGOT Normal  Normal or 
Alk.Ph Normal mild 
GGT
stercobilinogen   
Urobilinogen   
Cholesterol Normal Normal or  
S.amylase ------- ----- in cancer head
pancrease
PT Normal Normal or  Prolonged due to
vit.k
2) CBC

- Features of Hemolytic anemia

3) ESR: 

4) Serological tests: Hepatitis markers & autoantibodies.

5) Therapeutic tests:

a) Vit.k injection to differentiate between Obstructive & HC jaundice

- Vit.K Corrects the prothrombin time in obstructine jaundice, not in HC

b) Cortisone Level to differentiate between intrahepatic & extrahepatic.

 (39)

2) Serum albumin & significance method:

a. It’s the most abundant Ptn in human plasma

b. Total ptn 6 – 8 gm/dl, serum albumin 4 – 5 gm/dl
c. Synthesized in liver.
A. Causes of hypoalbuminoma = Significance:
1) Impaired Synthesis  chronic liver disease
  Ptn intake
2) Catabolism:  tissue damage

 Inflammation

3)  Absorption of a.a  malabsorption syndrome

4) Protein Loss in Urine  nephrotic Syndrome, D.M, SLE, and
chronic glomerulonephritis
5) Protein Loss in Faeces  protein Losing enteropathy due to
inflammation.
6) Protein Loss from skin  burns 7) Altered distribution:
ascites
B. Causes of hyperalbuminemia  Dehydration

Specimen:

 Serum is of Choice, may be heparinized plasma

 Avoid Venous stain   conc. of albumin  Urine
sample
C. Methods

* Electrophoresis

* Immunochemical Methods (Turbidimetry + nephelometry)

* Dye binding Method (most widely used) * Urine Strips.

 (40)

3) Obstructive Jaundice

Causes:

Intrahepatic

* Viral hepatitis.

* Malignancy.

* Alcohol.

* Drugs (OCP, testosterone, penicillin)

Extrahepatic

* Stone in CBD

* Cancer head of pancrease

* Cancer of biliary tract.

* Biliary atresia or stricture

Pathophysiology

1-  Excretion of conjugated Bilirubin into intestine due to

obstruction   sterocobilinogen (pale stool).
2- Conjugated Bilirubin regurgitation into blood   serum
bilirubin Jaundice.
3- Conjugated bilirubin is water souble, so appear in Urine (dark
urine).
4-  Bile excretion into intestine (steatorrhea)
Bile salts regurgitate into blood appears in urine.
 (41)

C/P:

1- Jaundice
2- Pruritis
3- Steatorrhea
4-  Bleeding tendency
5- Xanthomata  due to Cholesterol.
6- Bradycandia
7- +Ve Urobilinogen.

Laboratory Tests for obstructive Jaundice

(1) Serum bilirubin level

Serum bilirubin values (especially direct) are usually elevated. However, the
degree of hyperbilirubinemia.

 Extrahepatic obstruction

This is typically associated with considerable direct and indirect bilirubin

elevation.

 Intrahepatic obstruction

Both conjugated and unconjugated bilirubin fractions may increase in

varying proportions.

(2) Alkaline phosphatase (ALP) level

ALP is markedly elevated in persons with biliary obstruction. However, high

levels of this enzyme are not specific to cholestasis.

 Extrahepatic obstruction

ALP levels are elevated in nearly 100% of patients, except in some cases of
incomplete or intermittent obstruction.

 Intrahepatic obstruction
 (42)

ALP levels are usually elevated, and they often are less than 3 times the
upper limit of the normal reference range.
(3) Levels of serum transaminases

Moderately elevated in patients with cholestasis but may be markedly

increased, especially if cholangitis is present.

 Extrahepatic obstruction
(AST) levels mild to moderate  (< 10 times the upper reference limit)
when extrahepatic obstruction occurs acutely.
 Intrahepatic obstruction

(ALT) levels mild to moderate 

(4) GGT levels

These levels are elevated in patients with diseases of the liver, biliary tract,
and pancreas.

(5) Prothromhin time (PT)

Prolonged because of malabsorption of vitamin K. Correction of the PT by

parenteral administration of vitamin K may help distinguish hepatocellular
failure from cholestasis.

(6) Hepatitis serology

Serologic assays for acute viral hepatitis for all patients with cholestasis

(7) Antimitochondrial antibody levels: High titers

(8) Urine bilirubin levels

When it is present, only conjugated bilirubin is passed into the urine.

 (43)

4) Hepatocellular jaundice

*Causes:

1)  Uptake

a) Acute hepatitis. b) Gilbert’s syndrome.

c) Liver cirrhosis. d) Drugs: rifampicin.

2)  Conjugation

a) Gilbert’s syndrome & Crigler Najjar syndrome.

b) Drugs: Novobiocin. c) Premature: Physiologically Jaundice.

3) excreation:

a) Rotor dubbin Johnson. b) Drugs: Halothane.

*Pathogenesis:

 Up take of Bilirubin  unconjugated Bilirubin.

 Conjugation  uncongugated

 Excretion  congugated Bilirubin  dark urine.

 Excretion stercobilinogen  Pale stools.

*C/P:

1) Orange yellow jaundice. 2) Dark urine.

3) Pale stool. 4) Features of liver disease.

*Investigation:

1) In serum: both conjugated & unconjugated

2) In urine  urobilinogen & Bilirubin present.

3) In stool  stercobilinogen.
 (44)

5) Liver cell failure

A. Def.: A condition in which the liver cells failed to function

adequately.
B. Predisposing Factors:
1) Viral hepatitis B&C
2) Drugs: alcohol, COP, Anasthesia, Chemotherapy.
3) Metabolic disorders: α1 antitrypsin, Wilson disease.
C. Etiology:
1) Acute LCF: due to toxins, fulminant viral hepatitis, crude injury.
2) Chronic LCF: due to chronic liver D.
D. CL. manifestations:
 Hypoglycemia, hyponatremia, hypokalemia
 Oedema, ascitis, jaundice.
 ↑ Viability of infection → septicemia
 Bleeding, the most common cause of death.
 Portal hypertension, esophageal varies.
 Hepatorenal syndrome
 Coma: the accumulation of toxic substances → encephalopathy
 Pancreatitis: may be a further complication
E. Lab diagnosis: depend on the degree of lab abnormalities &
reflect degree of liver failure.
1) S. bilirubin:
- Is the 1st persisting feature.
-  Unconjugated then  conj.
2) Urine Bilirubin:  +ve before jaundice.
3) Urobilinogen:   then normal then 
4) ALT, AST:   in early cause then normal or  when massive
cell failure.
5) Alk.ph:   in obstruction & HCC.
6) LDH isoenzyme 4, 5  
 (45)

7) Cholesterol   (< 90 mg/dl)

8) S. albumin
9)  GGT
10) hypoglycemia
11)  Na,  K (dilutional hyponatremia + 2ry hyperaldosteronemia).
12)  PT.
 (46)

6) Liver Carcinoma

A. Laboratory diagnosis:

No Single Lab test is useful for early diagnosis & Monitoring of Cancer

1. Hepatic tumor Markers:

a. αFP:(n<20ng/ml)

Moderate  in Benign Liver disease (Cirrhosis)

 early 1ry Cancer Liver.

 Cancer Liver Metastasis

Marked  in  advanced Liver Cancer.

***AFP is Sensitive indicator in Recurrence Cases.

b. CEA (Carcino embryonic Ag)

 In Cancer Colon & Liver Cancer

If > 400 ng/ml in old man  highly Suggestive of 1ry Cancer Liver

IF  αFP +  CEA  2ry Liver Metastasis from Cancer Colon

2. Liver Enzymes:

a. ALP   in Liver metastasis, obstructive Jaundice

b. GGT:  in – Hepatocellular Cancer & Liver metastasis.

3- 5-Nucleotide

4- Total LDH

5- Hypocalcemia & Hypoglycemia.

6-  Vit B12 & dysfibrinogen.

 (47)

7) Liver Cirrhosis

*Causes:

1) Post necrotic (most common type)

- Viral hepatitis (B, C).

2) Biliary cirrhosis:

A) 1ry

*Causes:

- Autoimmune

- Endocrine origin

- Drug hyper sensitivity.

B) 2ry: due to obstruction of CBD

3) Alcohol Cirrhosis (more common type)

*Laboratory diagnosis:

1) Post necrotic.

a)  S. bilirubin

b) ALT, AST.

c)  Urine bilirubin.

d)  Urine Urobilinogen.

e) Mild  in Alk.Ph&GGT.

f) Pt.

g)  Albumin.

h) NH3.
 (48)

i) Na, K  2ry hyperaldosteronemia.

2) Biliary cirrhosis

a) S.bilirubin.

b) alk. ph (2 – 10 folds)

c) Bile salts.

d) Lipoprotein α +ve

e) Mild  AlT, AST

f) Urine Bilirubin

g) Liver copper

h) IgM

i) AMA, ANA +ve by IF

3) Alcoholic cirrhosis:

a) alk. ph &  GGT

b) Mild  in ALT, AST, Bilirubin.

c)  NH3, Na, K.

d)  Pt & albumin

e) B- d binding (IgA)

f) Respiratory alkalosis.

g) AST/ALT ratio >2.0

 (49)

8) Mention Value of TG. & Amylase in acute pancreatitis

 Laboratory findings
- Serum amylase: o > 3 x ULN 0 Typically return to normal in 48- 72
hours
- Serum lipase: o > 3 X ULN o Increases in parallel with Serum
amylase level
- Hypocalcemia (25% of patients)
- Hyperglycemia
- Hypoalbuminemia
- Leukocytosis: 15, 000~20, 000/uL
- Hypertriglyceridemia (15- 20% of patients)
- Transient elevations: serum bilirubin, alkaline phosphatase,
aspartate aminotransferase
- Hypoxemia (25% of patient)

The most common causes of high triglycerides are obesity and poorly
controlled diabetes.

In a healthy individual, a normal blood amylase level is 23- 85 units per liter
(U/L) , although some lab results for amylase go up to 140 U/L.

A normal lipase level is 0- 160 U/L.

If the pancreas is damaged, these digestive enzymes can be found in the

blood at higher levels than normal. Amylase levels more than four times
normal levels, or greater than 450 U/L, and lipase levels greater than 400
U/L, are likely to mean there's damage to your pancreas or pancreatitis.
 (50)

9) Compare: Amylase and lipase in Pancreatitis

* If the pancreas is damaged, these digestive enzymes can be found in the

blood at higher levels than normal. Amylase levels more than four times
normal levels, or greater than 450 U/L, and lipase levels greater than 400
U/L.

* Serum lipase is more specific and will stay elevated for a longer period of
time, as hyperlipasemia persists for 7 days and amylase should normalize
within 4 days.

* Serum lipase is not a good marker of disease regression due to, its, longer
half -life. There is no benefit in following lipase as a marker of resolution.

* Lipase is produced primarily in the pancreas, with a small amount in the

liver, intestine, tongue, and stomach. Amylase is derived primarily from the
pancreas and salivary glands; it is also, present in the ovaries, small and
large intestine, and skeletal muscle.
 (51)

Chapter 4 - Kidney

1) Chronic Renal Failure

 Causes:

1. DM: 25% of cases → Diabetic Nephropathy.

2. GN: Chronic Glomeruli Nephritis.

3. Hypertension

4. 4. Stones (obstructive uropathy)

5. Others: SLE – PN – Drugs.

 Laboratory diagnosis:

I. Blood chemistry:

NPN: ↑ urea, Creatinine and uric acid. Uric acid ↑ in CFR only.

Metabolic acidosis: ↓ pH, HCO3 & total CO2.

Electrolytes disturbance:

- Hyponatremia: high blood urea causes ↑ plasma osmolality → stimulate

osmoreceptors in hypothalamus → stimulate ADH secretion → water
retention + dilutional hyponatremia

- Hyperkalemia

- Hyperphosphatemia

- Hypocalcemia & osteomalacia

- Hypermagnesemia: due to Mg retention.

 (52)

II. Urine changes:

- Polyuria with low fixed specific gravity at 1010 & osmolality at 300
mosmol/kg

- Proteinuria with casts (granular)

III. Others:

- Anemia of chronic disorders due to ↓ EPG.

- CHO intolerance: insulin resistance.

- Lipids: ↓ HDL & ↑ TG.

- Altered thyroid metabolism

- Gonadal dysfunction: ↑ prolactin, ↑ LH & ↓ testesterone.

- ↑ Aluminum due to fluids in dialysis machine → bone diseases.

- Hypertension due to disturbance in renin angiotensin system.

 (53)

2) Nephrotic syndrome

 Definition:

- A protein loosing kidney due to increase glomerular permeability. By

definition more than 5 gm protein/day in urine (may reach 30 gm) &
consequent hypoalbuminemia, edema & hyperlipoproteinemia or
hypercholesternemia.

 Causes

- Primary: (lipoid nephrosis) → either minimal lesion (selective, best

prognosis & respond to steroid), membranous & proliferative lesions.

- Secondary:

1. Acute GN 5. SLE 9. amyloidosis

2. Malaria 6. Bilhariaziasis 10. Hanoch-schonlein
purpura

3. IVC or renal vein thrombosis 7. Drugs

4. DM 8. PAN

 Laboratory investigations

I. Serum:

2. Sever hypoproteinemia/serum albumin < 2 gm/dL.

3. ↑ ESR & anemia
4. Glomerular permeability index: normally < 0.001. ↑ in nephrotic
syndrome e.g. 0.005
5. Results of patient changes include:
- Oedema
 (54)

- Decreased protein bound substances (↓ total Ca, thyroxin, cortisol &

Iron)

5. Lipoprotein pattern:

- ↑ Beta-lipoprotein (LDL) → ↑ cholesterol

- In severe cases → ↑ VLDL → ↑ TG → turbid plasma
- N.B. any hypoalbuminemia can cause hypercholerterolemia.
6. Renal function:

- Early stages & in minimal lesion → normal GFR, urea & creatinine.

- In late cases & proliferative lesions → ↓ GFR, ↑ urea and creatinine &
↓ proteinuria, plasma protein & lipid return to normal.

II. Urine:

Physical:

- Aspect: opalescent - Sp Gravity: > 1030

Chemical:

- Protein: +++ or ++++ - 24 hours protein: > 3 – 5 g/24hours.

Microscopic:

- Hyaline, lipids, waxy & granular casts → absent if alkaline urine

- Normal WBCs & RBCs in minimal lesion, but ↑ in proliferative type (> 30
RBCs /HPF), absence of RBCs exclude proliferative lesion).
III. Other:

- C3 is normal in idiopathic but ↓ in underlying GN.

- ↑ ESR, ↑ cholesterol, ↑ fibrinogen, ↓ albumin.
 (55)

3) Clinical significance of uric acid

The reference ranges for uric acid are as follows:

Men: 2. 5- 8 mg/dL

Women: 1. 9- 7. 5 mg/dL

Clinical significance used to:

Diagnose and monitor people with gout

Monitor people who are undergoing chemotherapy or radiation treatment

Check kidney function after an injury

Find the cause of kidney stones

Diagnose kidney disorders

The following may interfere with your uric acid test results:

- Alcohol

- Certain medications, such as aspirin and ibuprofen (motrin)

- High levels of vitamin c

- Dyes used in X- ray tests

High uric acid can indicate of a variety of conditions, including:

- Diabetes & metastasized cancer

- gout, which involves recurring attacks of acute arthritis

- chemotherapy & hypoparathyroidism

- bone marrow disorders, such as leukemia

 (56)

- a diet high in purines

- kidney disorders, such as acute kidney failure

- kidney stones & multiple myeloma

Low levels of uric acid in the blood may suggest:

- Wilson's disease

- Fanconi syndrome

- Alcoholism
 (57)

4) Proteinuria

 Definition: it is increase protein loss in urine more than these 100 mg. Not
all clinically significant

 Causes:

1. Glomerular leakage of plasma protein: nephrotic syndrome

- The amount of protein excreted is less importance than the degree of

selectivity

- The extent to which the glomerulus can still distinguish between large and
small molecules.

 Investigations (Selective test):

 Done to differentiate between

selective and non selective type.

 Is becoming a routine test that can

replace renal biopsy.

 Proteins with selective leakage are

more likely to respond to steroid therapy than nonselective.

 If it is high = large molecules can't

pass = selective type (vice versa).

2. Over flow filtered protein of low MW:

- BJP in myelomatosis (Bence jones proteins)

- Lysozyme in monocytic leukemia

- Hemoglobinuria in sever IV hemolysis.

- Myoglobinuria in sever muscle damage.

 (58)

- Amylase in acute pancreatitis.

3. Post renal:

- Infection

- Inflammation: proteinuria + cells (pus & epithelial) but without casts

4. Orthostatic:

- It is benign condition protein occurs when patient walks or stands erect for
any period of time, while it absent when lying down prone.

- Morning urine sample after a night rest is essential for diagnosis, it gives –
ve results for proteins.

- Evidence of renal disease may occur after some years.

5. Physiological

- Exercise - Fever - Pregnancy

6. Failure of tubular reabsorption of filtered protein:

- In chronic pyelonephritis: tubular damage accompany with glomerular

damage, so urine electrophoresis can't detect tubular affection.

- Occurs in:

* Chronic hypokalemia * Cadmium poisoning

* Fanconi syndrome.

7. Protein derived from kidney tissues:

- Glomerular basement membrane fragments in nephritis

- Tam-Horse mucoproteins

- Secretory Ig-A
 (59)

5) Microalbuminuria:

Causes:

- Hematuria
- Fever
- UTI
- Dehydration
- Drugs: sulphonamides, cephalosporins, NSAIDS, penicillin,
tolbutamide

Routine screening test of urinary albumin excreation equivalent to (UAE) >

200 mg/min.

Suggests diabetic nephropathy. This is usually associated with long- standing

diabetes not less than 10 years.

Once diabetic nephropathy occurs, renal function deteriorates rapidly and

renal insufficiency development.

This range of 20 -200 mg/min. or 30 – 300 mg/d, more than this range
defines Microalbuminuria

Investigations

A) Qualitative estimation:

1) Albu. screen and Albu-sure test:

Detect UAE exceeding 20 mg/m. and 30 mg/L respectively. It is a latex

agglutination inhibition test.

2) Microalbumin test: uses bromphenal blue in an alkaline matrix to detect

albumin concentration exceeding 40mg/L only 80% specificity.

3) Microglactoside: is a new product uses a monoclonal antialbumin IgG

conjugated to enzyme: B-galactosidase.
 (60)

The albumin in the urine binds to the antibody enzyme conjugate-Albumin

immunocomplex diffuses to the reaction zone where it reacts with a
buffered substrat (chlorophenol red galactosids) to produce a red dye. The
intensity of the color after 5 min. is proportional to albumin concentration:
yellow = 0, light brown = 10 mg/L and burgwady = 100 mg/L

No interference with drugs, glucose, urea or often protein.

B) Quantitative assay:

All the specific assays for urine albumin immunochemistry with antibodies
to human albumin e.g.:

Radioimmunoassay, ELISA, RID, and immunoturbidity

 (61)

6) Polyuria

*Def.: volume of urine > 2.5 L/d.

1- A. Causes and lab. investigations of a case of polyuria.

The most common cause of polyuria in diabetes mellitus, which causes

osmotic diuresis. Water follows the glucose concentration passively, leading
to abnormally high urine output.

Etiological Classification of Polyuria

• Endocrine: DM, CDI, Cushing's syndrome

• Renal: CRF, relief of urinary tract obstruction, chronic pyelonephritis,

Fanconi syndrome.

• Iatrogenic: Diuretic therapy, alcohol, lithium, tetra cyclines.

• Metabolic: Hypercalcemia, potassium depletion.

• Psychological: psychoiogical diabetes insipidus.

• Other causes: Sickle cell anemia, pulmonary and systemic venous

thromboembolism.

Diagnosis:

Differential Diagnosis of D.I.

Procedure Interpretation
1- Measure plasma & urine * A urine osmolality less than that of
osmolality plasma: consistent with pit or
Nephrogenie D.I
* Psychogenic D.I.: both urine
&plasma are diluted.
 (62)

2- Dehydration: * A rise in urine osmolality above

* If serum osmolality is less than 290
mosm / L do
* water deprivation test
1- Allow no fluids for 12- 18 hr
2- Measure body weight, urine flow,
urine sp. gr., urine & plasma
osmolality every 2HS.
* Terminate study if body weight
falls more than 3%
that of plasma osmolality indicates
psychogenic polydepsia.
* Urine specific gravity less than
1005 (200 mosm/I) Indicates pit or
Nephrogenic DI.
N.B. Normal plasma osmolality
(288- 300) mosmol/Kg
* Normal urine osmolality (600-
1200) mosm/kg.
Test must done in hospital
* Nicotin alcohol & caffeine are
avoided

Vasopressin test:

3- Measure serum vasopressin after dehydration Test.

* Normal or high vasopressin indicates Nephrogenic D.I.

4- Inject 6 units vasopressin S.C. & measure urine flow, urine & plasma
osmolality.

* Failure of urine osmolality to rise indicates nephrogenic D.I.

 (63)

7) Urinary Casts

*Def. & formation:

- Cylindrical, Cigar – shaped structures produced by the kidney & present in

the urine.

- Composed of mucoprotein (Tamm- horsfall).

- DCT

- A significant number of urinary casts usually indicates the presence of

renal disease.

*Types:

A) a cellular

1) hyaline casts:

- The most Common type of Casts which are colorless, homogenous formed
of Tamm- horsfall mucoptn, Secreted by renal tubules.

- present in healthy patients (dehydration, exercise, diuretic medications).

- Renal D, CHD.

2) Granular Casts:

- Results from the degeneration of cellular casts or aggregation of plasma

proteins.

- Cigar – shaped appearance mostly.

- Present in after strenuous exercise & chronic renal disease, acute tubular
necrosis, viral infection.
 (64)

3) Fatty Casts:

- Formed by breakdown of lipid –rich epithelial cells, with free droplets

within the protein matrix of the cast.

- present in tubular damage, hypothyroidism, and nephritic syndrome.

4) Waxy Casts: (Renal failure Casts) :

- represents the final stage of degeneration of cellular casts, usually seen in

tubular injury.

- Present in sever CRF, acute & chronic transplant rejection.

- These casts are called renal failure casts

5) Inclusion Casts:

- Contain inclusions within pts (hyaline matrix).

a) Haemosiderin Cast: Stain blue after intravascular hemolytic.

b) Crystal Cast:

Urates, calcium oxalate.

6) Pigmented Casts:

a) Hb Casts: red colour  glomeular D.

b) Myoglobin Casts: red brown; acute muscular damage.

c) Bilirubin Casts: deep yellow brown in obstructive jaundice.

B) Cellular Casts:

1- RBCs Casts: Composition from RBCs, present in Glomerulonephritis,

pyleonephritis, acute interstitial nephritis & sever tubular damage.
 (65)

2- White bl.cell casts: Composition from white blood cells, present in

Glomerulonephritis typical for acute pyelonephrities.

3- Epithelial cells Cast: Composition from renal tubular epithelial cells,

present in: renal tubular necrosis, viral disease, kidney transplant rejection.

4- Bacterial cell Cast: Composition from bacterial cell in protein matrix.

Present in; acute pyleonephritis, renal infection.
 (66)

8) Renal tubular dysfunction tests:

1- Urine concentration test or water deprivation test: it depends on normal

tubular function & normal ADH secretion

2- Pitutrine or pitressin test: AD1H.

3- Ammonium chloride loading test (for urine acidification function):

4- Dye excretion test phenolphthalein (phenol red test)

 (67)

9) Laboratory Tests for Tubular Proteinuria

An increase in the urinary excretion of retinol binding protein indicates

proximal tubule injury and/or impaired proximal tubular function.

(1) Measurement of retinal- binding protein in urine is, therefore,

useful in the monitoring and/or diagnosis of kidney disease.
(2) Elevated excretio n rates can indicate:

a. tubular damage associated with renal tubulointerstitial nephritis or

tubular toxicity from heavy metal or nephrotoxic drug exposure.

b. Glomerulonephropathies and renal vasculopathies

(3) Measurement of urinary excretion of alpha- 1- microglobulin.

N.B.

Urinary excretion of retinol- binding protein can be determined from either

a 24- hour collection or from a random urine collection. The 24- hour
collection is traditionally considered the gold standard.
 (68)

10) Renal threshold

- Renal threshold of a substance = means the conc. of substance in

plasma above which it is excreted in urine, e.g. at normal blood
glucose level, all glucose filtered is reabsorbed
- As the concentration of plasma glucose increase, the amount of
glucose filtered also but still all absorbed.
- At bl. glucose level > 180 mg% glucose begins to appear in urine
& this blood glucose level is called the renal threshold for glucose.
- More in blood glucose, filtered, absorbed & excreted in urine
- Finally the amount of glucose absorbed reaches a maximum level
& remain constant. This amount is called the tubular maximum for
glucose reabsorption & equals mg glucose I minute in filtrate 300 –
350
- Sometimes in elder patient with long standing diabetes, there is
hyperglycemia but no glucose in urine.
- Means renal threshold that may be due to low GFR and glucose
filtered and can be absorbed by the functioning tubules, or due to
over stimulation of glucose receptors
- Urea always presents in urine has a threshold zero called non
threshold substance.
- The threshold for any substance influenced by:
1. Change in renal plasma flow.
2. Glomerular permeability.
3. Tubular reabsorption capacity.
 (69)

11) Renal clearance

- It is the volume of plasma which could be completely cleared of a

substance per minute.
- If each 1ml of plasma contains x mgs of substance.
- Clearance (mls of plasma contains UXV mgs of substance
excreted in urine/min.
𝑈𝑥𝑉
Clearance (vol) = x 1 ml/ min
𝐵

 Where V = urine volume per minute

 U=concent ration of substance in lml urine
 B= concentration of substance in 1 ml blood or plasma.
𝑈𝑥𝑉
So Clearance = x 1 ml/ min
𝐵

 Conc. of substance in 1 ml urine x volume of urine/minute =

 Concentration of substance in 1 ml blood or plasma
 Clearance as % normal= UXV X 100
 NB:
1. If the substance investigated is not uniformly distributed
between plasma and RBCS.
2. Theoretically a substance may be excreted by
a. Glomerular filtration only
b. Filtration + tubular excretion
c. Filtration + tubular reabsorption
3. As regards substance clearance
 If a substance is completely filtered of the glomeruli and is
completely reabsorbed by the tubules, its clearance will be zero as
glucose and amino acids.
 With the decrease of tubular reabsorption  substance appear
in urine and clearance increase as urea.
 (70)

 With no reabsorption, e.g. inulin & creatinine, clearance will be

equivalent to rate of glomerular Filtration.
 If a substance excreted by tubular epith. Together with
glomerular filtration & if all that is contained in the blood passing
through the kidneys is removed & transferred to the urine. Its
clearance will be complete
 (71)

12) Creatinine Clearance

*Clinical Significance:

1) Creatinine is measured as an indicator of GFR.

2) Creatinine Clearance is less influenced by the rate of urine flow.

3) Creatinine Clearance (Concentration) is independent on Ptn intake.

*procedure:

1) Hydrate the Pt with 2Cups of Water to assure urine flow over 2ml /m.

2) Avoid Caffe, tea or drugs on the day of the test.

3) Empty the bladder & discard Sample.

4) Collect Urine accurately for 2, 4, 12 or 24 hrs & calculate urine vol/min.

(V)

5) Collect bl.Sample for serum Creatinine at any time since its concentration
is constant (B).

6) Estimate urine Creatinine (U)

𝑈𝑥𝑉
- Clearance =
𝐵

- Avoid muscular exercise during test.

- Avoid Loss of urine during collection.

 (72)

13) Water deprivation test:

*It depends on normal tubular function & normal ADH response to detect
the conc. power of the kidney (Sp. Gr: 1020 or more)

*Procedure:

1) The patient is allowed no food or water after 6 PM on night before the

test.

2) A) All urine specimens including at 9 AM on discarded.

B) Collect urine sample 10 AM, another at 11 AM.

3) *Measure Sp. Gr of each specimen if the 1st sample was 1022 or more, so
there is no need for another specimen.

*the test is terminated if less than measure the 2nd specimen.

*Sp. Gr should be higher than 1022.

*the osmolality should be at least 850 mosmol/kg.

*Interpretation:

- Max osmolality < 850 or Sp. Gr < 1022 that indicates: impaired renal
concentration power due to tubular diseases due to Diabetes insipidus.

- The urine to plasma osmolality ratio (U/P) should be >3

Normal 1- 3

*Precautions:

- Water conc. test should be avoided in early dehydrated patients.

- During the test the Patients should be under observation.

 (73)

Chapter 5 - Enzymes & Vitamins

1) Laboratory Tests for acute MI

These include:

1. Troponin- the most commonly ordered and cardiac- specific of

the markers. Blood levels of troponin will be elevated within a few
hours of heart damage and remain elevated for up to, two weeks.
2. The hs- troponin test may also be positive in people with stable
angina and even in people with no, symptoms. When it is elevated in
these individuals, it indicates an increased risk of future heart events.
3. CK- MB- one particular form of the enzyme creatine kinase that
is found mostly in heart muscle and rises when there is damage to the
heart muscle cells.
4. Myoglobin- is a protein released into the blood when heart or
other skeletal muscle is injured; this test is used less, frequently now.
5. Increased levels of BNP- while not diagnostic for a heart attack
indicate an increased risk of cardiac problems, in persons, with acute
coronary syndrome.
6. Other more general screening tests- electrolyte balance, blood
glucose.
 (74)

2) Creatine kinase

1- The CK test may sometimes be used to help detect a second heart attack
that occurs shortly after the first.

2- A CK test may sometimes be used to evaluate and monitor muscle

damage.

If a CK is elevated and the location of the muscle damage is unclear, order

CK isoenzymes or a CK-MB as follow-up tests, to distinguish between the
three types (isoenzymes) of CK: CK-MB (found primarily in heart muscle),
CK-MM (found primarily in skeletal muscle), and CK-BB (found primarily in
the brain; when present in the blood.

3- It may be ordered when a muscle disease (myopathy) such as muscular

dystrophy is suspected or when someone has experienced physical trauma,
such as crushing injuries or extensive burns. The test may be ordered when
a person has symptoms associated with muscle injury such as:

- Muscle pain or aches

- Muscle weakness
- Dark urine

Increased CK may be seen with, for example:

- Recent crush and compression muscle injuries, trauma, burns

- Inherited myopathies, such as muscular dystrophy
- Hormonal (endocrine) disorders, such as thyroid disorders,
Addison disease or Cushing disease
- Strenuous exercise
- Prolonged surgeries & Seizures
- Infections - viral (such as influenza and HIV), bacterial, fungal,
and parasitic (such as malaria)
- Connective tissue disorders (e.g. lupus, rheumatoid arthritis)
 (75)

- Celiac disease & Renal failure

- High fever accompanied by shivering
- (thrombosis) blocking the flow of blood
- Any drug or toxin that interferes with muscle energy production
or increases energy requirements

Moderately increased CK levels may be seen following:

* strenuous exercise such as in weight lifting, contact sports, or long

exercise sessions.

CK isoenzymes and isoforms:

CK isoenzymes CK isoforms
Diff on genetic base Diff not on genetic base
Due to Hybrid between 2 genes Due to post transcriptional changes
Types Types
CK BB CK MM3 CK MM2
CK MB CK MM1 CK MB2
CK MM CK MB1
 (76)

3) Isoenzyme & lsoform:

Protein isoforms "is a term describing either several different forms of

coded from the same gene, or proteins with and functional similarities, even
when they are products of different genes.

Serum isoforms of Creatine kinase isoenzymes

The CK-2 and CK-3 isoenzymes of human serum creatine kinase (CK) can be
subdivided into five isoforms (subforms derived from the same isoenzyme).
Three are derived from CK-3 and two from CK-2.

Following damage of muscle tissue, the serum isoform distribution changes

as a result of the increased release of CK enzyme. This provides, more
diagnostic information concerning acute myocardial infarction and other
muscle diseases than is available from routine CK isoenzyme analysis.

* The enzyme Lactate Dehydrogenase is made of two (H-form and M-Form)

different sub units.

* Because bone ALP and liver ALP are encoded by the same gene locus, they
are referred to as isoforms of the same isoenzyme.

CK isoenzymes and isoforms:

CK isoenzymes CK isoforms
Diff on genetic base Diff not on genetic base
Due to Hybrid between 2 genes Due to post transcriptional changes
Types Types
CK BB CK MM3 CK MM2
CK MB CK MM1 CK MB2
CK MM CK MB1
 (77)

4) LDH in MI:

Lactate dehydrogenase is composed of four subunits. The two most

common subunits are the LDH-M and LDH-H protein. These two subunits
can form five possible (isoenzymes): 4H, 4M, and the three mixed tetramers
(3H1M, 2H2M, 1H3M). These five isoforms are enzymatically similar but
show different tissue distribution: The major isoenzymes of skeletal muscle
and liver (M4) have four muscle (M) subunits. While LHD1 is the main
isoenzymes for heart muscle in most species, containing four heart (H)
subunits.

• LDH-1 (4H)-in the RBC

• LDH-2 (3H1M)

• LDH-3 (2H2M)

• LDH-4 (1H3M)

• LDH-5 (4M)

 Usually LDH-2 is the predominant form in the LDH-1 level higher than the
LD1 H-2 level (a "flipped pattern") suggests (damage to heart tissues
releases heart LDH which is rich in LDH-1, into the bloodstream).
 (78)

5) Clinical significance of GGT

*Site: in liver, kidney, pancreas, prostate.

Increased in these cases:

1) Obstructive Jaundice.

2) Liver Tumors (1ry, 2ry).

3) Fatty liver, alcoholic cirrhosis.

4) Infective hepatitis.

5) Infectious mononucleosis, CMV.

6) Pancreatic damage ex: acute & chronic pancreatitis (pancreatic tumor).

7) Antiepileptic damage & alcohol.

N.B: GGT Light sensitive & kept in refrigerator (4 - 7 days)

 (79)

6) PSA

1) Prostatic specific antigen

2) It’s organ specific Tr M in cancer prostate
3) It’s aglycoptn enzyme secreted by epithelial cells of prostate
gland.
4) It’s in 2 major form:
a) minor component  free
b) major component  total
 α1 antichromotrypsin
 α2 macroglobulin
5) has 5 isoforms:

PSA –A
Active
PSA –B

PSA – C

PSA – D Not active

PSA – E

 Clinical Significance:
1- Increased in BPH, acute & chronic prostatitis
2- urinary retention, ejaculation,
3- prolonged cycling, prostate biopsy,
4- catheterization, cystoscopy, digital rectal examination.
5- Decrease in hormone therapy in BPH (chloramadinone acetate).
 Interpretation:
1) For Staging > 80% in advanced stage.
a) PSA < 10 g  No L.N, No bone metastasis.
b) The level of PSA, cancer Prostate is divided into:
 (80)

 Low risk if PSA < 10ng/ml

 Intermediate risk if PSA 10 – 20 ng /ml
 High risk ≥ 20 ng/ml.
2) Used for monitoring of ttt

 if decreased below some Limits  assay within 2 – 3 weeks

3) If ≤ 50 µg/L  indicate extracourpusclar tumour extension

4) From 4- 10 µg/L  indicate BPH  if prostatic cancer  confirm
by biopsy
5) Sensitivity of PSA at 4 µg/L is 78% & Specificity is approximately
33%
6) Measuring PSA
a. /3month in the 1st year
b. /4month in the 2nd year
c. /6 months in the after
 (81)

7) α- FP

1- Normal level < 10 ng/ml in adult.

2- Amniotic (fetal) α- FP reaches peak of 2 ng/ml, at 14 weeks then
decreased about 70 µg/ml at term.
3- During pregnancy, maternal, α- FP increased from 12 weeks to
reach a peak of 500 ng/ml in third trimester.
 Diagnostic Significance:
1) Marked increased
 500 ng/ml in advanced HCC
 Used for diagnosis staging, prognosis and monitoring of HCC
2) Moderate increased (100- 200 ng/ml)
 Metastasis from GIT(Cancer of colon, pancreas, stomach)
3) Mild increased up to 100 ng/ml
 Benign liver D (hepatitis & Cirrhosis)
4) Germ cell tumor (testicular cancer, ovarian cancer, malignant
teratoma).
-  10- 20 % for stage I.
-  50 – 80 % for stage II.
-  80 – 100 % for stage III.
5) Neural tube defects, Multiple gestation (16 weak e gestation) ,
GIT Obstruction)
   α- FP
6) Screening of Down syndrome & triosomy 18
 Interpretation:
1) In benign Conditions: Low transient elevation.
2) In Malignancy Conditions: With persistent high levels: poor
prognosis
3) 2ry metastasis from cancer colon:  α- FP + CEA.
4) Follow up 16 months for detection of HCC for patient with 
HCVAb, HBsAg, Cirrhotic.
5) A normal α- FP level doesn’t rule out neural tube defects or
Down syndrome
 (82)

8) Diagnostic value of phosphatase enzymes:

1ry The phosphatase

1- Alkaline phosphatase

The phosphate is most active between pH values of 8 and 9. Alkaline

phosphatase is formed in bone intracellularly by osteoblast and is also
present in liver, kidney, intestine and placenta.

* Alkaline phosphatase in bone disease

Raised serum alkaline phosphatase is found in Paget disease,

Hyperparathyroidism, in healing fractures, and in presence of osteoblastic
carcinoma metastases. Increased serum alkaline phosphate activity
accompanied by raised serum calcium suggests hyperparathyroidism.

* In Liver disorders: to differentiate diagnosis of jaundice.

2- Acid phosphatase

An enzyme that acts to liberate phosphate under acidic conditions and is

made in the liver, spleen, bone marrow, and prostate gland. High serum
levels of acid phosphatase may indicate infection, injury, or cancer of the
prostate.

Acid phosphatase is a lysosomal enzyme that hydrolyses organic phosphates

at an acid pH.

Prostatic: malignancy

Extra-prostatic disease prognosis.

 (83)

* Precaution for the sample:

- lf there is delay then add citrate buffer or acetic acid, there will be rapid
loss of the enzyme activity.

- Avoid hemolysis - Can store at 4 C or -20 C

3- 5' Nucleotidase

5'nucleotidase is a protein produced by the liver and the test can be

measured in the obstructive jaundice, parenchymal liver disease, hepatic
metastases and bone disease.
 (84)

9) Clinical significance of Phosphorus:

Phosphate concentration is characterized by a high physiological variation,

depending on age, gender, physiological state (e.g., pregnancy), and even
season

Interpretation of phosphatase levels

• Serum phosphate 1.5- 2.4 mg/dL - a moderate decrease and no clinical

signs and symptoms

• Serum phosphate lower than 1.5 mg/dl – May lead to muscle weakness,
red cell hemolysis, or coma, bone deformity and impaired bone growth

• Serum phosphate is lower than 1 mg/dL - Considered critical and may be

life- threatening

When rapid elevations of serum phosphate levels , urgent associated

hypocalcemia with tetany, seizures, and hypotension.

Specimen stability

Fasting serum specimens and 24h urine collection (not random) are
preferred

* Phosphate concentration increases by 4- 5 mg/dl per day in hemolyzed

samples stored at 4°C or RT.

* Prolonged storage at RT and delay testing/separation lead to, hydrolysis of

phosphate

Hypophosphatemia in:

• Intracellular shift of phosphate (e.g., post- insulin administration,

respiratory alkalosis, hyperalimentation, glucose administration)
 (85)

• Secondary tubular damage, Fanconi syndrome, familial

hypophosphatemia

• Vomiting, diarrhea, malabsorntion syndrome

• Vitamin D deficiency

• Primary hyperparathyroidism

Hyperphosphatemia in:

Hyperphosphatemia may result from any of the following:

• Decreased renal phosphate excretion (e.g., reduced GFR, renal failure,

acute kidney disease, chronic kidney disease)

• Increased renal tubular resorption

• Hypoparathyroidism and pseudohypoparathyroidism

• Acromegaly

• Increased phosphate intake

• Extracellular shift of phosphate

• Loss of phosphate from intracellular stores

• Cell lysis (e.g., intravascular hemolysis, leukemia, lymphoma, cytotoxic

therapy)
 (86)

10) Clinical significance of Myoglobin

Myoglobin is an intracellular haemoprotein expressed in the heart and

oxidative skeletal myofibres. Mb functions as an oxygen storage & protein
in muscle. Mb facilitates oxygen transport from erythrocytes to
mitochondria to maintain cellular respiration.

 Serum myoglobin levels begin to increase within two to three

hours following a heart attack. These levels reach their highest values
within eight to, 12 hours. Myoglobin levels typically return to, normal
within 24 hours. Troponin levels are more specific to heart damage
than myoglobin levels.
 The normal (or negative) range for the serum myoglobin test is 0
to 85 ng/mL.
 Abnormal results will rule out a heart attack abnormal (above
85ng/mL) results can also be seen in:
- muscular inflammation (myositis)
- muscular dystrophy
- rhabdomyolysis
 (87)

11) Clinical significance of Vitamin E:

Vitamin E is a fat- soluble vitamin, which plays a role as an antioxidant in

the body.

Vitamin E Benefits:

1. Balances Cholesterol 4. Balances Hormones

2. Repairs Damaged Skin 5. Improves Vision

3. Thickens Hair

6. Fights Free Radicals and Prevents Disease Development

7. Helps People with Alzheimer's disease.

8. Improves Physical Insurance and Muscle Strength

Vitamin E benefits include treating and preventing diseases of the heart and
blood vessels and a supplement; such as chest pains, high blood pressure,
and blocked, or hardened arteries.

Sources:

6. Avocado
1. Sunflower seeds
7. Broccoli
2. Almonds
8. Spinach
3. Hazelnuts
9. Kiwi
4. Wheat Germ
10. Tomato
5. Mango
 (88)

Chapter 6 - Qc & acid-base balance and instruments

1) Electrophoresis & its abnormalities

*Clinical Applications:

1) Ptn electrophoresis (serum, CSF, Urine)

2) Lipoptn electrophoresis.

3) Isoenzyme separation (CK, LDH, ALP)

*Precautions:

1) Fasting (to avoid  B Lipoprotein in B region.

2) Serum not plasma because fibrinogen  narrow band in B- y region.

3) No hemolysis because:

- False  in α2 (Hb haptoglobin)

- False  in  (Hb free).

*Abnormalities patterns for:

1) Nephrotic syndrome

 albumin due to renal loss

α2 macroglobulin is sufficiently large so that it is not filtered

1) Increased synthesis (from the general hepatic protein synthesis) causes

its accumulation.

2) Multiple myeloma (Monoclonal gammopathy) *Monoclonal band in 

region.
 (89)

An unusually sharp band in the gamma region strongly suggests the

presence of a homogenous, immunoglobulin and thus, the malignant
proliferation of plasma cells from a single cell (multiple myeloma) in
contrast to the broad, heterogeneous or polyclonal, gamma band as in
chronic inflammation from immunoglobulin synthesis by many different
clones of plasma cells are also found in Waldenstrom Jiseme.

3) Hypogammagloinemia ( y = 0.1 – 0.7 g/dl)

* Immunoglobulin Deficiency

Deficient immunoglobulin synthesis is revealed by a markedly diminished

gamma band. Effected individuals are prone to recur rent infection

4) Acute Inflammation:

The alpha- 1 and alpha- 2 bands are increased during the inflammatory
response from increased hepatic synthesis of acute phase reactant proteins

5) A gammaglobinemia ( y < 0.1 g/dl)

6) liver cirrhosis ( alb + polyclonal gammopathy with B- y bridging)

7) Pt Loosing enteropathy ( alb,  y).

*Values:

T.protein = 6 – 8 gm/dl α2=0.6 – 0.1 gm/dl

Alb = 3.2 – 5gm/dl B = 0.6 – 1.3 gm/dl

α1=0.1 – 0.4 gm/dl y=0.7 – 1.5 gm/dl

 (90)

2) Differences between Flame photometer& atomic absorption

Principle of flame photometry

An alkali metal salt drawn into a non-luminous flame will ionize, absorb
energy from the flame and then emit light of a characteristic wavelength as
the excited atoms decay to the unexcited ground state.

A photoelectric flame photometer is a device used in inorganic chemical

analysis to determine the concentration of certain metal ions (sodium,
potassium, lithium, and calcium).

FLAME PHOTOMETER INSTRUMENTATION:

1. Burner
2. Monochromators
3. Detectors
4. Recorder and display

ATOMIC ABSORPTION SPECTROSCOPY THEORY:

The method relies on absorption method of spectroscopy.

The method involves: the spraying of sample in solution state over a burner.
This leads to evaporation of solvent and leave fine dry residue behind which
is nothing but neutral atoms in ground state. To these atoms, a light of
 (91)

specific wave length is passed and the un- absorbed light is recorded over a
detector.

The instrumentation includes:

1. The burner to dry the sample and produce atoms.

2. Sample container.

3. Fuel and oxidant to burn the sample by heat.

4. Hallow cathode lamp to produce light of desired wave length.

5. Detector to detect the absorption intensity.

6. Amplifier and data recorder.

APPLICATIONS:

1. Quantitative analysis of metal elements in any sample.

2. Analyze trace metal elements in plasma and other body fluids.

3. To determine metal elements in food industry.

4. To estimate Lead in petroleum products.

 (92)

3) Types of analytical errors

Random error:

1- Instruments need repair or maintenance.

2- Automatic pipette need calibration. 3- Timing regulation.

4- Lack of stability of temperature bath.

5- Improper mixing of sample & reagent.

Systemic error:

 Reagent: expired & indicator lost its sensitivity or prolonged

boiling
 Standard: concentrated or improper prepared.
 QC material: deteriorated or not reconstituted by proper
volume.
 Change of methodology.
 Standard: deteriorated or improperly prepared.

How to correct the error?

Random:

- Check instrument. - Proper steps of the test.

- Calibration of pipettes. - Skillful person to do the test.

Systemic:

- Reagent not expired. - QC: not expired- proper reconstitution.

- Standard: avoid evaporation- proper reconstitution.

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4) Validation in clinical chemistry includes:

• Single laboratory method validation 1s appropriate where the method is

used for a specific purpose in a specific laboratory by personnel with the
appropriate training.

• Full method validation includes, in addition to the procedures employed in

single laboratory validation an inter-laboratory study with many
measurement instruments several operators.

• Full diagnostic method validation is establishing the diagnostic properties

of the method eg. in health and disease.

Basic requirements for method validation:

• The method should be fully developed and optimized

• A written standard operating procedure (SOP) for the method should be

available

• The measurement instruments to be used should be regularly technically

controlled and well maintained

• The persons performing: the measurements should have sufficient

training and experience for the task

• Appropriate calibrators should be available and a supply (for at least 1

year) of suitable stable materials (for at least 2 concentration levels) for
internal quality control purposes

• The needs of the method & materials should be known

 (94)

5) Write short notes on:

A. Chemiluminescence (CLIA)

The emission of light by a substance as a result of undergoing a chemical

reaction that does not involve an increase in its temperature.
Chemiluminescence usually occurs when a highly oxidized molecule, such as
a peroxide, reacts with another molecule. The bond between the two
oxygen atoms in a peroxide is relatively weak, and when it breaks the atoms
must reorganize themselves, releasing energy in the form of light.

B. Carryover is defined as ''the contamination of a specimen by the

previous one". Carryover testing is performed to help to, prove or
disprove carryover from the sample probe in clinical laboratory
testing.
C. Monochromator is an optical system that transmits a specific
band of electromagnetic spectrum. Dispersion of different
wavelengths is accomplished with the separating capability of
refraction (prism) or diffraction (diffraction grating).
D. Osmometry

The field of study that deals with the phenomenon of osmosis and the
measurement of osmotic forces.

E. Reference interval

In health- related fields, a reference range or reference interval is the range

of values for a physiologic measurement in healthy persons.

Distribution of test results that are normal for a selected population of

healthy persons.

Reference intervals are interpreted according to age, sex, and race. Also
called reference range, normal range.
 (95)

F. DNA complementarity

In nature, complementarity is the base principle of DNA replication and

transcription as it is a property shared between two DNA or RNA sequences,
the nucleotide bases at each position in the sequences will be
complementary, much like looking in the mirror and seeing the reverse of
things.

This complementary base pairing allows cells to copy information from one
generation to another and even find and repair damage to the information
stored in the sequences.

The degree of complementarity between two nucleic acid strands may vary
from complete complementarity to no complementarity and determines
the stability of the sequences to be together.
 (96)

6) Factors affecting Na balance

1) GFR:

- Decrease in GFR   Na+ excretion in urine.

- Increase in GFR  Na+ excretion in urine.

2) Renin- Angiotensin – aldosterone system:

-  Excretion.

- Aldosterone Stimulate Na+ reabsorption from DCT.

Receptors located in the Juxtaglomerular cells decrease arteriolar pressure

or decrease Na+ in D.C.T and stimulate the production of rennin.

3) (ANP) Atrial Naturic peptide:  excretion

a) Several peptides have been demonstrated in the atrium of the heart  

Na+ execration   GFR

b) ANP antagonize action of aldosterone.

c) It is released into Circulation in response to Changes in atrial filling

pressure   B.P

4) Dopamine release in the kidney:  excretion following an increase intake

of Na+, the PCT cells Can detected,  in Na+ excreted lead to Secretion of
dopamine into tubular Lumen that inhabit reabsorption of Na+ &  its
excretion.
 (97)

5) Kalikrin of kidney

It is secreted into DCT fluid reacts with filtered hepatic kininogen that
changed to kinin that inhibit Na+ &· H20 reabsorption  Bl. Pressure
(antagonism aldosterone)

*Methods of assay:

1) RIA.

2) RID.

3) Elisa.

4) Immunoturbidimetric assay.

5) PCR for Apo E genotyping.

 (98)

7) Causes of hyponatraemia:

1- Hyperosmotic hyponatremia

2- Isosmotic hyponatremia

3- Hypo-smotic Hyponatremia

A) Depletional hyponatremia or increased ECF.

B) Dilutional hyponatremia

Analytical Methods:

Na determined by atomic absorption spectrophotometry, flame emission

spectrophotometry, ion-selective electrodes, or spectrophotometric
method.