Clinical Chemistry Full

‫‪Gedair Laboratories‬‬
‫‪Medical Analysis‬‬
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‫ﺗﺣﺎﻟﯾل ﻗﺳم‬
‫ت‪j‬حا‪j‬ليل لا‪rj‬كيمياء‬
‫لاسريرية ولاحيوية‬
‫مقدمة‬
‫الكيمياء الحيوية اإلكلينيكية‪Clinical Biochemistry‬‬

‫مختبر الكيمياء الحيوية السريرية‬
‫)‪(Clinical Biochemistry Laboratory‬‬
‫أھداف القسم‬
‫يھتم ھذا القسم بإجراء التحاليل الخاصة بالكشف عن مدى فاعلية أعضاء الجسم في أداء وظائفھا المختلفة وعن المواد الكيميائية‬
‫الموجودة في سوائل الجسم وخاصة الدم وجميع ھذه المواد تكون بنسب ثابتة وأي اختالف في ھذه النسب يكون له مدلول‬
‫مرضي وسوف يتم توضيح ذلك بالتفصيل‬
‫طريقة العمل في القسم‬

‫ويعتمد العمل في ھذا قسم الكيمياء الحيوية السريرية على أجھزة خاصة في تحليل العينات حيث تعتمد على مواد خاصة‬
‫لالختبارات وعلى ھذا األساس يمكن تقسيم العمل في المختبر من حيث‪:‬‬
‫•فصل العينات ‪ :‬حيث يقوم الشخص المسئول عن ھذا القسم داخل المختبر بالتأكد من الرقم الموجود على العينة ومطابقته مع‬
‫ورقة طلب التحاليل)‪(Request‬‬
‫•ترقيم العينات ‪ :‬يقوم الشخص بترقيم ھذه العينة برقم تسلسلي ويوضع نفس الرقم على ورقة طلب التحاليل ويستمر تسلسل ھذه‬
‫األرقام إلى نھاية اليوم ‪ .‬وينقسم ترقيم ھذه العينات إلى‪:‬‬
‫‪-1‬عينات عاجلة‪(State) .‬‬
‫‪-2‬عينات روتينية‪(Routine) .‬‬
‫تفصل العينات بواسطة جھاز الطرد المركزي وتنقل العينات بعد الفصل إلى كأس صغير )‪ (Cup‬خاص بجھاز التحليل ويكتب‬
‫عليه الرقم التسلسلي للعينة ثم توضع في الجھاز‪.‬‬
‫•أجھزة تحليل العينات‪.‬‬
‫•التحليل اليدوي )‪ (Manual‬الذي يعتمد على خطوات يدوية في أغلب األحيان مثل تحليل الحصوات‪(Stones) .‬‬
‫أنواع العينات القادمة إلى ھذا القسم‪:‬‬
‫‪-1‬عينات الدم‪(Blood Samples) :‬‬
‫يوضع الدم الذي أخذ من المريض في أنابيب تحتوي على مادة مانعة للتجلط وھي )‪ ، (Lithium Heparin‬أو توضع في‬
‫أنابيب تحتوي على مادة مانعة للتجلط وھي )‪ (Floride Oxalate‬في حالة إجراء تحاليل السكر ‪ ،‬أو توضع في أنابيب تحتوي‬
‫على مادة مانعة للتجلط وھي )‪ (K-EDTA‬وذلك عند إجراء اختبار )‪ ، (HbA1c‬أو توضع في أنابيب ال تحتوي على مادة‬
‫مانعة للتجلط مثل تحليل )‪ (Iron‬و ‪ (TIBC) ,‬ثم تؤخذ ھذه العينات وتوضع في جھاز الطرد المركزي)‪ (Centrifuge‬عند‬
‫سرعة تصل إلى ‪ 3500‬لفة ‪ /‬دقيقة لمدة ‪ 5‬دقائق لكي يتم فصل مكونات الدم والحصول على البالزما أو السيرم ‪ ،‬أما عند إجراء‬
‫تحليل )‪ (HbA1c‬فال نضع العينة في جھاز الطرد المركزي ألننا نستخدم الدم الكامل )‪ (Whole Blood‬عند إجراء ھذا‬
‫التحليل‪ ،‬ثم نقوم بإخراج األنابيب حيث نقوم بسحب البالزما أو السيرم من العينة ونضعھا في أنابيب خاصة بالجھاز المستخدم‪.‬‬
‫•السيرم – مصل الدم‪(Serum) :‬‬
‫نحصل عليه بعد وضع عينة الدم في أنابيب ال تحتوي على مادة مانعة للتجلط ثم في جھاز الطرد المركزي )‪ (Centrifuge‬عند‬
‫سرعة تصل إلى ‪ 3500‬لفة ‪ /‬دقيقة لمدة ‪ 5‬دقائق ويكون الجزء العلوي ھو السيرم و يكون اللون الطبيعي له ھو اللون األصفر‪.‬‬
‫‪- 2‬البالزما‪(Plasma) :‬‬
‫نحصل عليھا بوضع عينة الدم في أنابيب تحتوي على مادة مانعة للتجلط مثل )‪ : (Lithium Heparin‬أو ‪(Florid‬‬
‫)‪Oxalate‬أو )‪ (K-EDTA‬ثم تؤخذ ھذه العينات وتوضع في جھاز الطرد المركزي عند سرعة تصل إلى ‪ 3500‬لفة ‪ /‬دقيقة‬
‫لمدة ‪ 5‬دقائق ويكون الجزء العلوي ھو البالزما ويكون اللون الطبيعي له ھو اللون األصفر‪.‬‬
‫•الدم الكلي )‪(Whole Blood‬‬
‫ھذه العينة ال نضعھا في جھاز الطر المركزي وتستعمل ھذه العينة في تحليل الھيموجلوبين السكري • )‪ (HbA1c‬كريات الدم‬
‫الحمراء )‪(Red Blood Cells –RBC‬‬
‫نحصل عليھا بغسيل الدم بمحلول ملح كلوريد الصوديوم )‪ (Na Cl‬تركيزه ‪ % 0.9‬ثم نفصلھا بترسيبھا باستخدام جھاز الطرد‬
‫المركزي والتخلص من الطبقة العليا ويكرر ذلك ‪ 3‬مرات ويكون الراسب بعد الغسيل األخير ھو كريات الدم الحمراء وتستخدم‬
‫الكريات لتقدير نسبة إنزيم نازعة ھيدروجين جلوكوز ‪ 6‬فوسفات – ‪(Glucose 6 Phosphate Dehydrogenase‬‬
‫)‪G6PD‬‬
‫‪ -3‬عينات البول )‪(Urine Samples‬‬
‫يعتبر البول أحد السوائل الحيوية في الجسم حيث يمكن تحليله مباشرة ‪ ،‬حيث يتم وضع جزءاً من عينة البول في األنابيب‬
‫الخاصة بالجھاز المستخدم إلجراء التحاليل المطلوبة‬
‫أما بالنسبة الختبار تحليل البول ‪ 24‬ساعة يكون بتجميع البول لمدة ‪ 24‬ساعة حيث تكون ساعة الصفر من بعد التبول مباشرة ثم‬
‫يجمع البول حتى أخر تبول عند نفس الساعة في اليوم الثاني ثم يتم إجراء بعض التحاليل عليھا لمعرفة مدى كفاءة الكلى في‬
‫القيام بوظائفھا‪.‬‬
‫‪ -4‬عينات سائل النخاع الشوكي ) ‪( C.S.F‬‬
‫يتم إجراء تحاليل السكر والبروتين لھا وذلك للكشف عن مدى فاعلية وكفاءة النخاع الشوكي في القيام بوظائفه ويتم إجراء ھذه‬
‫التحاليل بواسطة جھاز الدايمنشن )‪(Dimension‬‬
‫التحليل الكيميائي للدم‬
‫تحليل السكر‪Glucose‬‬
‫ً‬
‫إن قياس سكر الدم ھو من أكثر االختبارات التي ترد إلى المختبر ‪ ،‬وأھميته ترجع إلى اكتشاف حاالت السكري مبكرا ‪ .‬وأھم‬
‫من ذلك اكتشاف حاالت عدم تحمل السكري وھي الحالة التي تسبق اإلصابة العرضية للسكري‪.‬‬
‫تعود أھمية قياس السكر أيضا ً في متابعة المعالجة لداء السكري ومعرفة ما إذا كانت الحالة مستقرة أو غير مستقرة ‪ .‬كذلك يفيد‬
‫قياس السكر في معرفة حاالت نقص السكر في الدم ويجري أيضا اختبار مساعد في كثير من التجارب الحركية مثل اختبار‬
‫نقص سكر األنسولين وتجارب أخرى كثيرة‪.‬‬
‫أھم الفحوصات الخاصة بالسكر‪:‬‬
‫_________________ __________________________________________________‬
‫الھيموجلوبين السكري‪Glycosylated Haemoglobin – Hb A1c‬‬
‫الھيموجلوبين السكري عبارة عن بروتين ) جلوبيولين ( مرتبط مع الحديد في مجموعة )‪ (Haem‬وھذا البروتين‬
‫)الھيموجلوبين( مرتبط بسكر الجلوكوز وھناك أنوع عديدة من الھيموجلوبين ولكن ما يھمنا ھو نوع ‪ A1c‬ألنه يتميز بارتباطه‬
‫مع الجلوكوز حيث ترتبط نسبة قليلة من الھيموجلوبين ال تتعدى ‪ % 10 – 5‬من الھيموجلوبين بجلوكوز الدم ويطلق على ھذا‬
‫الجزء المرتبط ‪ ( HbA1c ) .‬نسبة ارتباط الجلوكوز بالھيموجلوبين يعتمد على مستواه في الدم فكلما زادت نسبة الجلوكوز‬
‫ازدادت نسبة السكر في المحمولة عليه بالوجبات الغذائية ويعطينا مؤشراً عن نسبة السكر في الدم خالل فترة حياة كريات الدم‬
‫الحمراء وھي حوالي ‪ 120‬يوما ً‪.‬‬
‫المعدل الطبيعي ‪% .8 – 5 :‬‬
‫_________________ __________________________________________________‬
‫اختبار منحنى تحمل السكر)‪Glucose Tolerance Test (GTT‬‬
‫يجري ھذا التحليل عندما يكون ھناك شك في اإلصابة بمرض السكر ويعطينا فكرة عن احتمال اإلصابة بالسكر من عدمه ‪.‬‬
‫وعند إجراء ھذا التحليل البد أن يكون المريض صائما ً من ‪ 12 – 8‬ساعة ثم نأخذ عينة دم وبول ثم يتناول المريض جرعة‬
‫جلوكوز مقدارھا ‪ 75‬جرام ثم نأخذ عينة دم وبول أخرى بعد ساعة ثم بعد ساعتين‪.‬‬
‫المعدل الطبيعي ‪ 110 – 70 :‬ملجم ‪ 100 /‬ملليتر دم‪.‬‬
‫_________________ __________________________________________________‬
‫اختبار تحمل السكر عن طريق الوريد‬
‫يطلب اختبار تحمل السكر عن طريق الوريد في بعض الحاالت التي يتعذر فيھا إعطاء السكر عن طريق الفم كما في بعض‬
‫األمراض المعوية ويجري االختبار بأخذ عينة من دم للصائم ثم يحقن محلول ‪ % 25‬أو أحيانا ً ‪ % 50‬وريديا ً بواقع ‪ 5,0‬غرام‬
‫لكل كلغم من ورزن المريض ويتم الحقن على مدى ‪ 5 – 2‬دقائق ثم تؤخذ عينة دم بعد ساعة واحدة من الحقن ثم بعد ساعتين‪.‬‬
‫وھناك عدة اختبارات للسكر منھا‪:‬‬
‫‪-‬اختبار تحمل السكر بعد إعطاء الكورتيزون‪.‬‬
‫‪-‬اختبار تحمل السكر بعد إعطاء األدرينالين‪.‬‬
‫‪-‬اختبار تحمل السكر بعد إعطاء األنسولين‪.‬‬
‫_________________ __________________________________________________‬
‫قياس السكر الصيامى)‪Fasting Blood Sugar (FBS‬‬
‫يجري ھذا التحليل على المريض بحيث يكون صائما ً من ‪ 12 – 8‬ساعة ‪ ،‬وفي حالة ارتفاع السكر عن الحدود الطبيعية يجب‬
‫إعادة القياس مرتين على األقل بفاصل أسبوعين بين كل قياس‪.‬‬
‫يزداد في‪:‬‬
‫‪-‬عدم تحمل السكر‪.‬‬
‫‪-‬مرض السكري‪.‬‬
‫‪-‬التداوي بمركبات الكورتيزون أو‪ACTH .‬‬
‫‪-‬أورام الغدة النخامية المفرزة لھرمون النمو‪.‬‬
‫‪-‬فرط نشاط الغدة الدرقية‪.‬‬
‫ينخفض في‪:‬‬
‫‪-‬زيادة جرعة األنسولين‪.‬‬
‫‪-‬زيادة جرعة مخفضات السكر‪.‬‬
‫‪-‬قصور الغدة الدرقية‪.‬‬
‫‪-‬قصور الغدة النخامية‪.‬‬
‫‪-‬قصور الغدة الكظرية‪.‬‬
‫‪-‬في الخدج‪.‬‬
‫المعدل الطبيعي ‪ :‬يتراوح ما بين ‪ 110 – 70‬ملجم ‪ 100 /‬ملليتر دم‪.‬‬
‫_________________ __________________________________________________‬
‫تحليل السكر بعد ساعتين من األكل‪Post Prandial Blood Sugar‬‬
‫يجري قياس سكر الدم بعد وجبة غنية بالمواد الكربوھيدراتية وذلك بعد ساعتين من بدأ الوجبة ويفضل إعطاء المريض عن‬
‫طريق الفم محلول من الجلوكوز بواقع ‪ 75‬جم ‪ ،‬ثم قياس سكر الدم بعد ساعتين يجرى ھذا االختبار في الحاالت التي يراد فيھا‬
‫معرفة عدم تحمل السكر أو الحاالت التي يشك فيھا بوجود مرض السكري ومع ذلك فقياس السكر للصائم يكون في المجال‬
‫الطبيعي أو أعلى بقليل من الحدود العليا للمجال الطبيعي‪.‬‬
‫المعدل الطبيعي ‪ :‬أقل من ‪ 140‬ملجم ‪ 100 /‬ملليتر دم‪.‬‬
‫_________________ __________________________________________________‬
‫تحليل السكر العشوائي)‪(Random Blood Sugar‬‬
‫فائدته فقط أنه يعطي فكرة عامة عن مستوى السكر في دم المريض حيث يتم تحليل العينة في أي وقت خالل اليوم وتؤخذ نتائج‬
‫ھذا التحليل إلى الطبيب ليقوم بتقويم حالة المريض‪.‬‬
‫المعدل الطبيعي ‪ 150 – 70 :‬ملجم ‪ 100 /‬ملليتر دم‪.‬‬
Chapter Page
1. Powder preparation 1
2. Acid preparation 2
3. End point method 3
4. Kinetic method 4
5. Quality control 6
6. Electrophoresis 9
7. Semen analysis 13
8. Calculation 15
9. Urine analysis 17
10. Others 24
Chemistry lectures_Clinical
Powder preparation
Steps:
1. Calculation: see calculation chapter
2. Balance
3. preparation
----------------------------------------------------------------------------------------
2. Balance: We use single pan electronic balance.
‫( للتشغيل‬on) ‫ندوس علي زرار‬ -
.‫ النقطه تكون في منتصف الميزان‬.‫ ثم نفردھا علي كفة الميزان‬4 ‫( الي‬filter paper) ‫نطبق ورقة نشاف‬ -
.zero‫ندوس علي ال‬ -
.(‫ و ننظر إلي تدريج القراءه حتي نصل إلي الرقم المطلوب )الوزن‬filter paper‫نضع البدره بالملعقه البالستك بالتدريج فوق ال‬ -
Precautions:
- We click zero after putting filter paper to cancel its weight.
- No centrifuge on the same bench, no fan.
Apparatus principle:
- In null position it is balanced weight → deflection of beam which is α weight
- This need electromagnetic force α weight to return back to null position.
3. Preparation: beaker → funnel → flask
- Choose Beaker near to the volume needed.
- Put small volume of distill water inside the beaker (less than volume needed).
- Put the powder on the beaker and dissolve it.
- We use glass rod to dissolve the powder.
- Transport the beaker continents to flask with funnel.
- Raise the funnel little and make it in touch with flask wall to avoid frothing (air bubbles).
- Wash beaker with distal water (to remove any non-dissolved powder) then empty it in the flask.
- Use the funnel till we reach the neck of the flask.
- Then we finish the volume till we reach the mark using final volume (adapt the meniscus).
- Mix the solution, cover with parafilm.
- Write date, name & concentration over the container and my name.
- Wash all instruments and put them in the shelf.
N.B.
- Beaker volume near to final volume
- Flask volume must be = the final volume exactly.
1
Acid preparation
1. Calculation: See calculation chapter
2. Preparation:
- Never to pipette acid by mouth but with capillarity
- We use 10 mL pipette. We put the pipette (closed by finger) inside the container.
- We can tilt the container carefully to one side if the acid volume is small.
- Then the acid is delivered from the pipette to the center of a beaker containing
distilled water. Never to the side of the wall as the acid that strong to break the wall.
- With gentle swirling of the beaker.
.‫ حركه دائريه خفيفه‬beaker‫ مع تحريك ال‬beaker‫ داخل ال‬distal water ‫ إلي منتصف ال‬pipette ‫ننزل الحمض إللي داخل‬
- Ideally, the beaker should be put in the ice or cold water: due to hear production
- Then adjust the final volume in the measuring flaks by using the funnel till the neck
then use the glass pipette.
- Write date, name & concentration over the container and my name.
- Wash all instruments and put them in the shelf.
2
End point method

 We have 3 tubes: reagent blank – stander – sample.
 Steps:
1. Warm up 15 minutes for stability then VIS
2. Wave length adjustment according to wave length at which the chromophore have maximum
absorbance = W.L complementary to the colour of the chromophore (coloured product).
.λ ‫ ندوس علي الرقم ثم علي زر‬.1

3. Blanking (adjust zero absorbance) → 100 % transmission (measure against DW or reagent blank).
- Put the solution inside the cuvette (put in consideration way of light). Then click in Calibrate.
4. Precautions:
- Cuvette
 Clean, dry, not scratched & not stained.
 Should respect minimum volume.
- Other precautions:
 Avoid hemolysed sample: if obligate to use. Do sample blank, if hemolysis interfere
colourimetrically. But if enzyme as LDH. (We must ask for another sample).
 Avoid lipemic sample: fasting of patient, use sample blank & ultra centrifugation (105,000 x
overnight 18 hours).
 Avoid hemolysis or turbid sample to minimize interference of any other colour or it may
interfere as a reaction. If we have to read this sample: use sample blank, read at different wave
length.
 To avoid carry over: we let the darkest sample
5. Reagent kits:
- Check expire date.
- Must be pit in the tube, don’t take from the bottle to avoid contamination.
6. Check the pipette for the proper volume, avoid air bubbles and do proper mixing.
Notes: (Very Very Important Notes) ‫شفوي العملي‬
- Conc. of the sample = Abs of sample x Conc. of standard / abs of standard.
- If they give you quality control sample: we manage it as a sample exactly.
- Be sure that equal volumes of standard, test if not: complete the equation.
Conc. Of test =
(abs of test / abs of standard) x conc. of st. x (amount of st / amount of test) x (dil of st / dil of test) x 100
- Don't forget: (A = 2 – log% trans.) (We can calculate the transmission).
- Conversion from mg/dL to mmol/L: Mmol/L = mg/dL x M.wt
3
Kinetic method
Kinetic assay → measure the rate of change of reaction.
Plateau
Lag phase
phase
Absorbance Linear
phase
Time
 Lag phase: Adaptation between substrate & enzyme = incubation time = equilibrium time.
 Linear phase: Enzyme converts the substrate into product – regular, detect activity every minute
 Plateau phase: Substrate depletion.
NB.
After the lag phase → measure the initial absorbance (absorbance of reagent + sample) (‫)علشان كده مش صفر‬
Then we take reading every 1 minute
STEPS
1.After we take 4 readings of absorbance:
- R1: initial absorbance
- R2: after 1 minute - R3: after 2 minute - R4: after 3 minute
2.Calculate:
Δ 1 = R2 – R1 Δ 2 = R3 – R2 Δ 3 = R4 – R3
Average Δ = Δ 1 + Δ2 + Δ3 / 3
3.Concentration of the enzyme = IU/L or Micromol/min = average Δ x factor.
4.Factor = 1 / micromolar absorptive x 1 / light path x total sample / sample volume.
Important notes:
- Total volume = reagent volume + sample volume.
- Micromolar absorptive = absorbance of the stander (if NADH = 6.3 x 10¯ ³)
- So factor = 1000/6.3 x 1/light path (‫ سم‬1 ‫ )غالبا‬x total volume/sample volume.
5.If reading every 30 seconds → x 2.
6.To ensure linearity of reaction = (each Δ - Average Δ / Average Δ) x 100
- Must be < 10% - Causes of non-linear: air bubbles , substrate depletion
- If not linear → I couldn't use the equation.
4
7.Initial absorption is important to detect:
- Substrate depletion
- Substrate deterioration
8. Enzyme activity is highly sensitive to temperature (should be strict to temperature control).
Important Notes:
- To differentiate between substrate depletion and deterioration:
1. Dilute the sample and repeat the test → If correct = substrate depletion. (Due to ↑ enzyme activity).
2. Use Q.C. (if Q.C ↓ → defect in reagent → substrate deterioration).
3. Use smaller amount of the enzyme.
4. ↓ incubation time.
- If there is substrate depletion: there is ↑↑ in initial absorbance due to high activities of the enzyme.
- If there is substrate deterioration: ↓↓ in initial absorption due to absent of reagent (not containing NAD).
- There is no standard for enzymes → due to rapid deterioration.
- Factor is usually supplied by kits → (‫)أو نطبق المعادله‬
- Micromolar absorptive = absorbance of 1 micromole of the end product of the reaction.
- IU/L = amount of enzyme that catalyze 1 micromole of substrate /min.
- Katal = amount of enzyme that catalyze 1 mole of substrate /sec.
- If the products (NAD, NADH, NADPH) → Maximum absorption at WL = 340 nm.
- In ALP, ACP & GGT → at WL = 405.
- In phosphate (non-enzyme analytes) → at WL = 340 nm. Because its reaction produce NAD , NADP,
NADH, NADPH.
- Reagent:
1.It is powder. 3. Check expire date.
2.No incubation in enzymes. 4. Ask for minimum volume. 5. Checking must be very slowly.
- Micromolar absorbitivity = 6.3 x 10¯ ³
- Molar absorbitivity = 6.3 x 10³

- IU = 16.7 nanokatal: Nanokatal to IU/L x 0.06.
- During reconstitution of the vial:
1. Check expiry date 4. Write the date
2. Should be not turbid 5. Stability and storage
3. Reconstitute the buffer or D.W or saline
- Types of lamp: tungsten, quartz, halogen, mercury vapor arc and laser (intense banc of light).
- Types of cuvettes: circular, square, rectangular, flow through cuvette.
5
Quality control
+3 SD
+2 SD
+1 SD
-1 SD
-2 SD
-3 SD
1st D 2nd D 3rd D 4th D 5th D 6th D 7th D
- QC material:
 Assayed → Kits provides me a range
 Non-assayed → I should calculate my own range under my conditions (pipetting. Temp. …).
- QC should be done in 3 levels
- In clinical exam: (all next notes are very important).
 They give you reading for 20 days
 Then you calculate the SD and X
from 20 days readings. (From calculator)
 Then we calculate the Range = X ± 2SD.
 Some time in exam he gives you directly the range and SD. And you calculate the mean and +ve
1, 2, 3 SD and –ve 1, 2, 3 SD.
 Example:
- Mean = 55 - SD = 3
- So range = 55 ± 2 x3 = 55 ± 6 = (49-61).
 Now the data on the chart is ready to use (mean, SD +ve and –ve)
 Then in the exam he gives you 7 days readings. Draw them on the chart as dots.
Notes:
- SD = Stander of deviation.
- X = Mean
X
- & 3SD (-ve and +ve) = we draw uninterrupted lines.
- While 1SD and 2 SD = we draw interrupted lines.
6
We interpret the results according to Westgrad Multirules (Very important).
(The normal level is that the results falls between ± 2 SD and fluctuate around the mean)
Rules:
1. 12S:
- 1 QC is outside +2SD
- Warning signal but I can release the results.
2. 22S: (‫)يومين ورا بعض‬
- 2 consecutive QC results outside ± 2 SD on the same side.
- Results of the 2nd day is rejected, Systemic error.
3. 13S:
- 1 QC is outside ± 3SD.
- Rejection, Random error.
4. R4S:
- 2 consecutive results: one > +2SD and the other < -2SD.
- Rejection, Random error.
5. 41S:
- 4 successive results outside ± 1SD on the same side.
- Rejection, Systemic error.
6. 10-x:
- 10 successive results on one side of the mean.
- Rejection of last result, Systemic error.
Interpretation of the Results:
- Comment on each day: Accept warning or rejection. (For 12S and 13S Roles)
- Then comment by combination. (22S and R4S Roles)
- Comment on each one and its relation to the next 3 results to it. (For 41S Roles)
- We must write another comment especially if he mentioned it is autoanalyser (ASTRA & Synchron):
In automated machine due high precision and accuracy of the results we consider 41S & 10 X as accepted
results. (Modified Westgrad role)
7
Types of Error:
Cause of:
1. 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.
2. Systemic error:
a. Downward shift:
- Reagent: expired
- Standard: concentrated or improper prepared.
- QC material: deteriorated or not reconstituted by proper volume.
- Change of methodology
b. Upward shift:
- Reagent: indicator lost its sensitivity or prolonged boiling.
- Standard: deteriorated or improperly prepared.
- QC material: No reconstituted by proper volume.
How to correct the error:

1. Random:
1.Check instrument.
2.Calibration of pipettes.
3.Proper steps of the test.
4.Skillful person to do the test.
2. Systemic:
1.Reagent not expired.
2.Standard: avoid evaporation – proper reconstitution.
3.QC: not expired – proper reconstitution.
8
Electrophoresis
- Definition: Migration of charged particles in liquid medium under influence of an electric field.
- Factors affecting migration rate: See Instrument chapter (‫)النظري‬
- Buffer: See Instrument chapter (‫)النظري‬
- Support media: See Instrument chapter (‫)النظري‬
- Types of electrophoresis: See Instrument chapter (‫)النظري‬
- Clinical applications of electrophoresis: See Instrument chapter (‫)النظري‬
- Limitation and errors:
 Buffer:
1. Cold (improve resolution and decrease evaporation).
2. Should be at desired PH, ionic strength.
 Sample application:
1.Should be priming before application.
2.Bent application or overloading of sample of excess drying of strips → distortion of bands.
3.Wet cellulose acetate → irregularities of bands.
 Care of proper storage of stain.
SPE
- Proteins are separated according to their electrical charges.
- Using barbital buffer at PH 8.6
- Cellulose acetate is under 2 powers: electrophoresis and electroendosmosis.
- Albumin: Smallest protein, high –ve charges → fast movable protein.
- λ-globulin → affected by endosmosis, low –ve charge → move just cathodal to the origin.
Values:
- Total protein: 6-8 gm/dL - Albumin: 3.2 – 5 gm/dL
- α-1: 0.1 – 0.4 gm/dL - α-2: 0.6 – 1.0 gm/dL
- β: 0.6 – 1.3 gm/dL - γ: 0.7 – 1.5 gm/dL
Subtypes:
- α-1 proteins: α-1 Antitrypsin – α-1 Acid glycoprotein – α-1 Lipoprotein – α-Fetoprotein – Thyroid
binding globulin (TBG).
- α-2 proteins: α-2 Macroglobulin – Haptoglobin.
- β-protein: transferrin – hemopexin – β-lipoprotein – C3.
- γ proteins: IgA – IgM – IgG.
Sample:
- Fasting: to avoid increase B-lipoprotein in B-region.
- Serum not plasma because fibrinogen make narrow band between β & γ region.
- No hemolysis: false increase in α-2, false increase in β region (Hb free).
9
Abnormal pattern:
a. Specific:
- Liver cirrhosis
- Nephrotic syndrome
- Monoclonal gammopathy
- A gammaglobinemia
b. Non-specific:
- Polyclonal gammopathy
- Hypoalbuminemia
- Hypogammaglobinemia
- Protein loosing enteropathy
- Oligoclonal gammopathy
To write a report:
1. Degree:
2. Band: increase , decrease
3. Specific or non specific
4. Suggesting …………….
5. Further investigations:
Example: SPE showing moderate hypoalbuminemia with moderate increase alpha-2 band suggesting
of nephritic syndrome for further investigations: 24 hours urine protein, urine analysis, kidney
functions and complement assay.
The investigations:
- Liver cirrhosis: viral markers, liver functions, U/S.
- Monoclonal Band in gamma region suggesting MM: BM, IEP, I.F., Bence jones protein in
urine, increase ESR.
NB: Monoclonal Hypergammaglobinemia:
 Suppressed residual Igs: MM
 Present Igs residual: early MM or on treatment.
- Decrease albumin, increase gamma-2 suggesting Nephrotic syndrome. 24 hours urine protein,
urine analysis, kidney functions and complement assay.
10
Important Notes:
1.Monoclonal: narrow base & narrow peak.
2.Oligoclonal: wide base & narrow peak

Kale azar, chronic hepatitis.
3.Polyclonal: wide base & wide peak

Search for bridging
--------------------------------------------------------------------------------------
Lipoprotein Electrophoresis
Patient preparation:
- Mandatory fasting 12-14 hours
- On normal diet, activity
- No recent illness, surgery, MI
- Avoid drugs increase or decrease lipid or thyroid hormones.
Sample: on EDTA blood (on cellulose acetate: cathodal application, Dye = fast red 7B)
Values:
- α = 15 – 40 % = HDL
- pre-β = 5 – 20 % = VLDL
- β = 40 – 55 % = LDL
Diagnosis based on 2 or 3 abnormal samples 2 – 4 weeks apart.
Normal pattern:
α Pre-β β
+ve -ve
11
- Type II:
 Type IIa: Increase β region only
 Type IIb: Increase both β & pre- β (But with space between them).
- Type III: Increase both β & pre- β (But they make broad band). ‫سايحين علي بعض‬
- Type IV: Increase pre- β only.
- Type V: Increase pre- β & Chylomicron.
Note: Type I = Increase β, pre- β & Chylomicron.
 Type IIa: Increase β region only
α Pre-β β
+ve -ve
 Type IIb: Increase both β & pre- β (But with space between them).
α Pre-β β
+ve -ve
 Type III: Increase both β & pre- β (But they make broad band).
‫سايحين علي بعض‬ α Pre-β β
+ve -ve
 Type IV: Increase pre- β only.

α Pre-β β
+ve -ve
 Type V: Increase pre- β & Chylomicron.

α Pre-β β Chylomicron
strip‫تظھر كتله كبيره خارج ال‬
+ve -ve
12
Semen analysis
- Composition:
1.Seminal vesicle (60%) → fructose + PG + vesiculase + K
2.Prostatic (20%) → vesiculase, hyalunidase, ACP & Zn.
3.Testicular (5%) → sperms, testosterone, inhinin & transferrin
4.Epididymal duct, efferent ductules → phospholipids & L-carnitine.
- Indications of semen analysis:
1.Assess male infertility 3. Forensic purposes
2.Effectiveness of vasectomy 4. Suitability of semen for IVF
- Collection:
1.After 3-5 days of abstinence period in a clean, sterile wide mouth container.
2.Must be transported within 1 hour to laboratory at T◌۫ 20-40
- Record on the report:
1.Abstinence period
2.Time of collection
3.Complete or incomplete collection
4.Drugs taken
- Universal precautions during handling semen: transmit HIV, Hepatitis & herpes.
- Macroscopic examination:
1.Liquefaction
 Analysis of semen after complete liquefaction
 Normally: 10-20 minutes up to 30 minutes
 If > 60 minutes → specimen is considered abnormal.
2. Appearance
 Normally: turbid, viscous, white grey & seminiferous odour.
 Red or brown (RBCs) = haematospermia
 Dense white turbid (WBCs) = leukocytospermia
3. Volume
 By wide mouth pipette, graduated centrifuge tube or graduated cylinder.
 Not by syringe (because –ve pressure → destruction of sperms).
 Normally: 2-6 mL
4. Viscosity: Aspirate by pipette → allow to drop
 Normally: distinct drops
 If threads: decrease sperm motility due to Abs coating sperms.
5. pH: 7.2-8 → within 60 minutes from collection.
13
- Microscopic examination
1. Motility: Done by wet mount analysis:
→ Rapid progressive → Slowly progressive (Sluggish)
→ Non-progressive (Shaking) → Immotile sperms
 Done immediately after liquefaction: 1st hour & 2nd hour → normally:
→ Class a ≥ 25% (rapidly progressive) → Class a + b ≥ 50% (rapid & slow)
2. Viability:
 By supravital stain (Eosin/Negrosin) → normally: viable > 75% within 1 hour of liquefaction.
 By hypo-osmotic swelling test (HOS) → swollen is alive.
3. Agglutination: For immunological cause of infertility
 Pattern of adhesion → head to head, head to tail or tail to tail.
4. Morphology: By H&E, papanicolour, wrights stain
 Normally: ≥ 30% with normal morphology.
5. Cells & bacteria: Mature sperms, epithelial cells & spermatogenic cells (immature germ cells).
 If bacteria or candida, trichromonas → C/S must be done.
6. Sperm count: On haemocytometer.
 Dilution 1:20. The diluent is: formalin or water. Number x 50,000 = sperm concentration.
 Sperm concentration = ----- million/mL (normally ≥ 20 million/mL).
 Sperm count = ----- millions / ejaculation (normally ≥ 40 million/mL).
- If azospermia:
 Do concentration by centrifugation
 Test must be repeat 3 time with one month interval between one and another.
- Complete associated semen analysis (CASA): ↑ accuracy, reproducibility, measures direction &
speed of sperms.
- Biochemical assay: (Seliwaneffas test for fructose) → Done in cases of azospermia.
 5 mL reagent (recrosinol + conc. HCl) + 0.5mL semen →.boil → red colour (fructose +ve)
within 1/2 minute (qualitative test).
 Sensitivity: 100 mg/dL, normal level of fructose ≥ 150 mg/dL.
 Done with control = fructose of sucrose solution to compare colour.
 Interpretation of fructose test:
→ Azopermia + normal fructose = bilateral epididymis obstruction.
→ Azopermia + -ve fructose = congenital abnormality of vasa deferentia + seminal vesicle or ductal obstruction.
→ Polyspermia + ↓ fructose + ↓ motility = for quantitative.
- Sperm Abs assay: for detection of Antisperm Abs of sperm or on serum. Methods
 Immunobased assay, Mixed antiglobulin reaction (MAR) or ELIZA
 Flow cytometry (if > 20% → +ve antisperm Abs).
14
Calculation
Note: for more detail back to chemistry unit practical book for Prof. Ola Ghanem.
- 1 Liter = 1000 mL - 1 dL = 100 mL - 1 Liter = 10 dL 1 mL = 1000 Microliter
- 1 Liter = 106 microliter = 109 nano = 1012 pico = 1015 femto.
- Dilution factor = total volume needed / sample volume.
- Dibasic Na phosphate = NaH2Po4
- Monobasic Na phosphate = Na2HPo4
- Concentration:
1. W/W:
Example: 5% NaCl = 5 mg of NaCl in 100mg solution total = 5mg Nacl + 95 mg Distal
Water.
2. V/V:
Example: 2% acetic acid = 2 mL acetic acid + 98mL DW
3. Molecular weight (MW) /V
- % solution: grams/100mL or grams/dL
- Molarity: MW in grams/L
- Normality: Equivalent weight in grams/L
Note: Eq weight = MW/valency.
‫أنواع المسائل‬
A. New preparation: usually the question start with (Prepare)
- Amount of powder needed in grams =
1. If % = %needed x volume need (mL) / 100 = RESULT gm/mL
2. If molarity = molarity x MW x volume need / 1000 = RESULT
3. If normality = normality x eq wt x volume needed / 1000 = RESULT
B. ‫حاجه من حاجه‬: Most of the time the question start with (calculate)
- He will ask to change from certain type of concentration to another
- Example: from % to molarity, from normality to molarity ………….. etc.
1. Normality = % x 10 / MW 3. Molarity = % x 10 / Eq W
2. Molarity = Normality / valency 4. gms = molarity x MW or = normality x Eq W
15
Notes
:‫ تحضير محلول بتركيزمعين من محلول اخر بتركيز أكبر‬.1
‫ موجودين‬specific gravity ‫ و ال‬conc.‫ ال‬-
V1 x C1 = V2 x C2 : ‫ تطبق معادلة‬-
V1 & C1 for the stock solution -
V2 & C2 for the diluted solution (the unknown solution ) -
Acids ‫ تحضير‬.2
.‫ تحضير حمض بحجم و تركيز معين من زجاجه بتركيز مختلف‬
.‫ موجودين‬specific gravity ‫ و ال‬Conc. ‫* ال‬
Amount of acid needed = Molarity x MW x V/1000 x 1/Sp. Gravity x 100/conc.% *
Or
Amount of acid needed = Normality x Eq.W x V/1000 x 1/Sp. Gravity x *
100/conc.%
.molarity or normality ‫ إلي‬% ‫ للتحويل من‬
.‫ موجودين‬specific gravity ‫ و ال‬Conc. ‫* ال‬
Molarity = % x 10 x Sp. Gravity / MW *
Normality = % x 10 x Sp. Gravity / Eq.W *
16
Urine analysis
- Urine considers a liquid tissue biopsy of the urinary tract.
- Aim: evaluation of renal function, detection of urinary tract disease & detection of metabolic
or systemic diseases e.g. DM, MM, aminoaciduria.... etc.
Urine report
(‫)مھم جداُ يتحفظ و يتكتب بالكامل في اإلمتحان‬
- Physical examination:
 Volume
 Colour
 Aspect
 Reaction (pH)
 Specific gravity
- Chemical examination:
 Glucose
 Ketone bodies
 Proteins
 Bilirubin
 Urobilinogen
- Microscopic examination:
 WBCs
 RBCs
 Crystals
 Amorphous
 Casts
 Others: epithelial cells, parasitic ova , monilial budding.
Important note: We must known the reaction done when we use strip
17
Physical examination:
 Volume:
 Random:
- For routine urine analysis
- Fresh morning sample because it is more concentrated.
- Collected in a clean dry container.
- Analyzed within one hour of collection or else refrigerated at 2-8 C for not more than 8 hours
because:
1. Ketone bodies volatilized
2. RBCs and casts decomposed with time.
3. Glucose utilized by bacteria
4. Bacterial container: alkaline urine by urease producing organisms (urease converts urea
into ammonia) → alkaline urine.
5. Bilirubin and urobilinogen are affected by light.
 24 hours urine (timed urine specimens):
- e.g. protein measurement / 24 hours.
- Preservative may be needed (Hcl for Calcium).
- Method collection: discard the 1st sample at 8 am and collect until the next day at 8 am also.
- Normal urine volume: 500 – 2000 mL/day (differ with age)
- Measured by cylinder: Polyuria > 2L/day, Oliguria < 400 mL/day, Anuria < 100 mL/day.
Notes
- Causes of polyuria
 Marked polyuria and hypotonic
- Urine after water deprivation - Pituitary diabetes insipidus
- Nephrogenic diabetes insipidus - Chronic lithium toxicity
- Sickle cell nephropathy - Hypokalemia (rarely)
 Moderate polyuria & inability to produce hypertonic urine
- Hypercalcemia - Chronic pyelonephritis
- End stage renal disease - Amyloidosis
- Interstitial nephritis - Hypokalemia
- Causes of oliguria:
- Prerenal causes: dehydration, heart failure …..
- Renal causes: acute GN, acute tubular necrosis …….
- Post renal causes: stones, urethral stricture, prostatic enlargement ……….
18
 Colour
- Normally: yellow colour due to urine pigments (urochrome, uroerythrine and urobilin). The
colour intensity correlated with urine concentration.
- Abnormal colours:
 Greenish brown: due to bilirubin (yellowish green foam on shaking)
 Orange red: due to urobilinogen (colourless) when oxidized to urobilin → orange.
 Reddish brown: due to Hb, RBCs (hematuria) or drugs e.g. rifampicin.
 White (milky urine): due to rupture of lymphatic vessels into urinary bladder or urethra → chyluria
(few drops of ether → clear).
 Pink: due to uroerytherine pigments which is deposited in urate crystals or amorphous urate.
 Dark: due to Homogentisic acid in case of Alkaptonuria or drugs e.g. methyl-DOPA.
 Aspect
- Normally: Clear
- Turbidity is due to phosphates (alkaline pH). Phosphate precipitate in alkaline urine and
redissolved on addition of acetic acid.
- Turbidity increases with heating: Urates, Bactenuria, Mucus, Epithelial cells & Leucocytes
 Reaction (pH)
- Normal pH: 4.5 – 8
- Method of measurement:
 Strip method: containing 2 indicators e.g. Methyl red & Bromthymol.
 Ph meter (glass electrode):
 Titrable method:
- Titrable acidity: 24 hours urine volume → NaOH till pH become (7.4) → calculate amount of
alkaline needed.
- Example: low titrable acidity → in case of RTA because of pH of urine is alkaline.
 Specific gravity
- Definition: It is the ration of the weight of a substance to the weight of an equal volume of
water i.e. the density of urine relative to the density of water.
- N.B. osmolality of urine is more accurate for concentrating power of renal tubules.
- Significance: indicator of the concentrating power of the kidney which is a tubular function.
- Normal range:
 1.025 in 24 hours urine
 1.003 – 1.030 in random sample (according to water intake).
 After 12 hours fluid restriction > 1.025.
19
- Method of measurement:
 Urinometer (hydrometer)
- We must do correction for
1. Temperature: every 3C◌۫ above 15 C◌۫ add 0.001
2. Glucose: every 1 g/dL glucose (100mL) subtracts 0.004.
3. Protein: every 1 g/dL protein substrate 0.003.
- Cause of very low sp. Gravity: D.I. and dilutes sample.
- Isothinuria: low fixed sp. Gravity 1010 in CRF.
- If urine volume is very small → dilute 1:1. Then multiply the reading x 2.
- Glucose and proteins → false increase in Sp. Gravity.
- Temperature → false decrease in sp. Gravity.
- Technique:
1.When we fill the cylinder we put the urine slowly on the wall to avoid froth
2.Put the urinometer in the center of cylinder
3.Slightly twisting of the urinometer then take the reading when it is floating freely
‫ و نلفه زي النحله و ھو جوه و نقرأ بسرعه لما يبطىء بس قبل ما يركن علي الجدار‬Cylinder ‫نلقيه في ال‬
- Notes:
 We use the suitable cylinder without froth or air bubbles
 If there is froth we suck it by pipette or wait it till be ruptured.
 Refractometer
- Measure the refractive index of urine which depends upon number of solutes in urine
and hence the urine concentration.
- Refractive index: It is the ratio of the velocity of light in air to the velocity of light in
solution. This ratio varies directly with the number of dissolved particles in solution.
- Advantage: needs few drops of urine.
 Reagent strips
- The test device for sp. Gravity consists of an absorbent cellulose pad impregnated with
Bromthymol blue, polymethylvinylether, maleic acid & NaOH.
- Increase of electrolytes in urine sample → reagents in the strips release H → lowering of the
pH of reagents and change the colour.
20
Chemical examination:
1. Glucose
a. Benedict's test:
- For detection of reducing substance.
- 5 mL benedict + 8 drops urine → heat → mix → cool 1st then interpret
‫ وبعد كده نسيبھا تبرد نصف دقيقه‬.‫كل لما تفور نبعدھا عن اللھب ثانيه أو إثنين‬
- Interpretation:
1. Green PPT: + 3. Orange PPT: +++
2. Yellow PPT: ++ 4. Red PPT: ++++
- Disadvantages: False positive with other reducing substances e.g. lactose, galactose, fructose
& ascorbic acid (non-specific test).
- Notes:
 During boiling the tube opening must be away from face (toward the bench).
 Principle: In hot alkaline solution (Benedict) the aldehyde group of glucose reduces cupric
ions to cuprous ions (cupper reduction method).
b. Glucose oxidase method (strips):
- Glucose + O2 + glucose oxidase → gluconic acid + H2O2 + peroxidase → O2 + H2O.
- Advantage: It is a specific method.
- Disadvantage: false –ve with reducing substance e.g. ascorbic acid due to O2 consumption.
2. Ketone bodies
Rothera test:
- 5 mL urine + ammonium sulphate (‫ → )بدره‬saturation
- Then small amount of Na nitroprusside (nitoferricyanide) is added
- Then layering by ammonia 2 mints → interpret
 Violet ring: +ve
 No violet: -ve
- We must wait 15 minutes to confirm –ve results (acetone react with Na nitroprusside in
presence of alkali → purple complex).
- Note:
 Ketone bodies are: acetoacetate, acetone, B-hydroxybutyrate.
 They ↑↑↑ with ↓ availability of CHO e.g. fasting, carbohydrate free diet or decrease use of
CHO e.g. DM and glycogen storage diseases.
 ↓ ketonuria in spite of ketonemia in renal failure.
 This test is +ve mainly with acetone & acetoacetate and –ve with B-hydroxybutyrate
acetate which doesn't react with Na nitroprusside.
21
3. Proteins
A. Boiling test:
- turbidity due to protein precipitation (trace, +, ++, +++)
- Then add acetic acid: →Turbidity disappear: phosphates / → Still present: protein (albumin).
B. Strips (Dipstick test):
- Principle: reagent strip is impregnated with tetra bromophenol buffered at pH3. Protein change in pH
& change of color of the dye from yellow to green. They can measure protein in excess of 10 mg/dL.
- Disadvantage:
 +ve with albumin only and not sensitive to globulin. They are excellent screening test for glomerular proteinuria
but unsatisfactory for detection of tubular proteinuria or over load proteinuria of Bence Jones type.
 False +ve in alkaline urine.
Notes: Protein analysis in urine
- Qualitative by boiling test
- Semiqualitative: 1. Latex agglutination inhibition test for albumin: detect albumin > 20mg/dL.
2. Micral: uses a monoclonal Abs: IgG
- Quantitative: by TCAA → turbidimetry or nephelometry
4. Bilirubin
Fouchet test
- 10 mL urine (by glass pipette, by capillarity or Pasteur pipette ‫ )ممنوع الشفط بالفم‬+ 2 mL BaCl2
- → filtration in another tube → Takes the filter paper alone and put on it 2-3 drops of fouchet
reagent → interpret the results:
 Of +ve bilirubin → greenish blue colour on filter paper → due to oxidation of bilirubin into biliverdin.
Notes: - If we use 5 mL urine add 1 mL BaCl2 (barium chloride)
- In dipstick measure of bilirubin → we use diazo reagent.
- Fouchet reagent = FeCl3 + TCAA
5. Urobilinogen
Erlish reagent
- 10 mL urine + 2 mL BaCl2 → filtration in two tubes. One tube with small amount of filtered
urine (just for control). Another one with big amount of urine we add 1 mL of Erlish reagent
- Then wait for 3 minutes and interpret
 Red colour → +ve urobilinogen
 Faint pink colour → normal trace.
Notes: - Erlish reagent = 2gm of para dimetyl amino benzaldhyde + in 100 mL of 20% Hcl.
- Absent or –ve urobilinogen → in obstructive jaundice
- ↑ urobilinogen → in hemolytic anemia
-
22
- Microscopic examination: ‫الزم يكتب بالكلمل‬
Every items should have comment: Nil , few, +, ++ or +++.
 WBCs: normal < 5 /HPF
 RBCs: normal 0 – 2 /HPF
 Crystals:
- Ca oxalate: ‫شبه الظرف أو العظمه‬
- Uric acid: ‫ – أصفر و بيلمع و بأشكال ھندسيه‬must be in acidic urine only.
- Triple phosphate: ‫ شبه التابوت‬. must be in alkaline urine only.
N.B.
- Triple phosphate (phosphorus + ammonium + Mg)
- Dicalcium hydrogen phosphate can be present in acidic or neutral
 Amorphous material: according to pH: acidic → urate, alkaline → phosphate.
 Casts:
- Hyaline: ‫شفافه‬. Disappear by acetic acid
- Granular: ‫لونھا بني غامق‬. Degenerated tubular epithelial cells.
- WBCs: acute pyelonephritis.
- RBCS: acute G.N.
- Waxy: in amyloid kidney.
 Others:
- Epithelial cells
- Parasitic ova
- Monilial budding
- Trichomonas vaginalis
23
Test Principle of the most common used method Comments Reference
- Plasma, no hemolysis
* Glucose + H2O + O2 → Glucuronic acid + H2O2
- Rapid separation
Glucose * H2O2 + phenol + amninoantipyrine → coloured reagent HexoKinase
- In urine: reduction method or by
* It is measured at wave length 520 nm
glucose oxidase in strips
- Serum, no hemolysis
Jandrassek and Grof
- No light or sunlight
* Diazodized reagent + serum → coloured chloroform (direct
- ↑ light or T◌۫ cause ↓ results
Bilirubin bilirubin)
- Δ bilirubin: it is unconjugated bilirubin
* + accelerator (Na benzoate coffein) → total bilirubin.
+ albumin with covalent reaction
* pH 3 – measured at 630 nm
- Act as direct.
- Serum or plasma
Berchelat reaction
- BUN (MW = 28) = urea (MW=60)
Urea * Urea → NH3 + carbonic acid
- BUN / Creatinine ratio.
NH3 + phenol + Na hydrochloride → Blue indophenol complex
Enzymatic end point uricase

* Uric → allantorir + Co + H2O2
- Serum, no hemolysis, no lipaemia
Uric acid * H2O2 + phenol + 4 aminoantipyruvate → coloured reagent
- Urine UA/urine creatinine > 1 = gout.
* Measured at 520
Rate Jaffe method - Serum, no hemolysis (false ↑ due to

Creatinine * Creatinine + picric acid (alkaline solution) → Red colour pseudocreatinine substances as glucose, uric acid) HPLC
* Read after 20 seconds to about pseudo creatinine products - No bilirubin - ↑ T◌۫ → false ↑ results
24
Enzymatic endpoint
- Cholesterol esters → free FA + cholesterol - Serum or plasma
Cholesterol - Cholesterol + O2 → cholesterol + H2O2 - Fasting not required Abell et al
- H2O2 + phenol + antipyrine → coloured reagent at - No prolonged tourniquet or hemolysis
wave length 520.
- Precipitation of LDL & VLDL by heparin
- 3 Types (HDL1 – HDL2 – HDL3
HDL-Cholesterol - Manganese or dextras sulphate then → measure
cholesterol.
- Direct method:
Indirect fridweld formula Used when TG > 400 mg/dL, type III & ↑
LDL-Cholesterol
LDL = cholesterol – HDL – TG/5 chylomicron.
- LDL is 2 types: oxidized & MM
Biuret method
- Serum
- Complexes between cupric acid ions + Nitrogen atom of
Total protein - No: plasma, prolonged tourniquet, icteric or
peptide bonds of protein → reagent (alkaline solution) →
lipidemic or hemolysis
violet colour at WL 540.
Dye binding method
- Serum
- Based on a shift of the absorbance maximum of the dye
- Supine position
Albumin when binds to the albumin.
- No: plasma, prolonged tourniquet, icterus or
- BCG: bronocresal green
lipidemia or hemolysis
- BCP: bronocresal purple
25
Turbidemtric method
Total protein in urine After adding precipitating agent to urine e.g. trichloroacetic acid
"TCAA".
1. Extraction: - Serum or plasma. Fasting 12 – 14 hours
To remove any internal substance (phospholipids & free glycerol). - No: change in diet habits, alcohol or No
2. Hydrolysis of TG: - Free glycerol + F.A. prolonged tourniquet.
TG - By ethanolic acid or by lipase enzyme - Storage reference but not prolonged:
3. Measurement of glycerol: - Glycerol + ATP → G3P + ADP hydrolysis → free glycerol → false decrease
- ADP + phosphate-pyruvate → ATP + pyruvate - Glycerol blanking: when TG > 200 mg/dL
- Pyruvate (with LDH) → lactate  Measure glycerol before and after hydrolysis.
- Fasting due to meal: gastric secretions →
Reduction method
alkaline pH →increase results
- Inorganic P + NH4 malybdate (acidic media) → unconjugated
Phosphorus - No anticoagulants → false ↓.
phosphate malybdate.
- No prolonged tourniquet: false ↑
- Reduction substance: Mobdinium blue colour measured at 680 WL.
No hemolysis: false ↑ (RBCs rich in P)
Spectrophotometeric method
a.O-cresolphthalim complex one: (at WL: 570 – 580 nm)
- O-cresolphthalim complex one + Ca → Red complex.
Total Calcium - Stabilized by → potassium cyanide.
- Inhibit interference of Mg → hydroxy quinoline.
b. Arsenazo III: (at WL: 650 nm.)
- Ca binding reagent at Ph=6 & measure Ca dye complex
26
CHEMISTRY SECTION
I. GENERAL: Specimens for chemistry procedures should be obtained in a fasting state (12-14 hour fast).
If this is not practical, an "order comment" should be made in CHCS to verify this. Accuracy of results on a
lipemic (most commonly caused by a non-fasting specimen) or hemolyzed specimen is questionable. It is
also important to make the Chemistry Section aware of medications so that proper precautions can be taken
to assure the best results. Close adherence to the information and instructions contained herein will insure
more effective laboratory support and services by the Chemistry Section. Our laboratory personnel are as
anxious to provide the highest quality patient support as the physicians who rely on it.
II. REQUEST FORMAT:
1
III. CHEMISTRY TESTS:
A. Blood Chemistry:
1. All blood chemistries are done on samples drawn in the fasting state (12 hours), except in
emergencies. The fasting state means that food and drinks, except for water, are to be withheld from the
patient. Water may be given, except when a gastric analysis, gastric wash or urinary concentrating
ability test is to be done. If at all possible, all drug medications should be
withheld from 24 to 48 hours prior to having blood drawn except for
therapeutic drug monitoring. A minimum of 14 hours fast is necessary for
triglycerides, HDL-cholesterol, and LDL-cholesterol.
2. In the analysis of therapeutic drugs, additional data on the patient will be helpful. When ordering a
therapeutic drug in CHCS, the dose time will be asked and should be answered as accurately as possible in
the Order Comment section.
B. Urine Chemistry:
1. Instructions and appropriate urine containers with required preservative

for 24-hour urine collections are to be obtained at the laboratory front desk. A
24-hour urine test request that requires an acid preservative may be collected in
conjunction with a 24-hour test that does not require any acid or other preservative
if the specimen is refrigerated during collection and is brought to the laboratory
immediately upon completion.
2. If at all possible, instruct patient to withhold all drug medications from 24 to 48 hours prior to
timed-urine collection. For timed specimens, the patient should be instructed to empty the bladder upon
arising in the morning of the starting day and discard that urine. All urine passed throughout the subsequent
timed period is collected in the container provided and refrigerated. Upon arising the next morning, the
patient completely empties the bladder and adds this urine to the container. This last specimen terminates
the 24-hour collection and the urine collection is submitted to the laboratory. If a creatinine clearance test is
requested, a blood creatinine specimen must be collected by the laboratory within the 24-hour time frame
usually after termination of the collection. The patient’s height and weight must be recorded on the
instruction sheet. Complete instructions for collection and diet will be given at the time the collection
container is procured.
3. Collection time for quantitative urine chemistry tests is of utmost importance in order to properly
report urine chemistry results. It is essential to be able to distinguish 24-hour urine collections from those
collections which are less than 24 hours. The volume of urine measured without any written indication of
the collection period cannot be relied upon solely as a means of identifying the time interval of collection.
In order to insure meaningful and accurate reporting, please indicate the time period of urine collection. All
that is required is an indication such as "random", "spot", "2 hour", "12 hour", "24 hour", or other in the
comment section of CHCS. Your attention to the matter will facilitate the initial processing and final
reporting of urine chemistry tests.
2
Tubes used :.
Tubes Additive Sample Tests
Lithium All
Green Tube Heparin Plasma Test
Hormone
Iron
TP
Plain Tube No additive Serum
CSF
Lavender Whole
EDTA HbA1c
Tube Blood
There are 3 technicians including the chief of this department

1. Daily, weekly and monthly maintenance of the instruments
2. Doing and checking the calibrations and quality control
3. Checking the results
Equipments:.
4. Rotator (Mix )
5. Centrifuge (separated serum & plasma from blood cell )
6. Dimension( Mex ,Rxl ) ،Bakment : Spectrophotometer
7. Elecsys 2010 : for hormone
8. ABG : Arterial blood gases
Testes performers :.
9. Glucose
10. Lipid Profile
11. Liver Function Test
12. Renal Function Test
13. Routine Urine Analysis
14. Hormone
15. Arterial blood gases
Routine Test :.
16. Glucose
17. Urea
18. Creat.
19. Na/ K
3
SAMPLE RECEIVING AND PROCESSING
1. Upon receiving specimen, see to it that an appropriate amount of blood has been sent,
properly labeled with name, number of patient and the date of collection. Data on the tube
should coincide with the data written on the request slip.
2. Assign laboratory number on the sample and write the number on the tube and on the
request slip.
3. Centrifuge specimen for 5 minutes at 3,500 rpm See if sample is not

hemolyzed. Hemolysis affects certain chemistry determinations (i.e
.serum electrolytes, glucose etc.)
4. Separate serum/plasma from red cells at once and

place inside assay cups labeled with the
corresponding number of the patient.
5. Test for the desired chemistry determination on the Dimension and/or SYNCHRON
autoanalyzer Enter the necessary data in the machine.
6. Record results in the logbook
7. Release results that has been signed and stamped with the name of technician who
performed the test.
TESTS USED IN LABORATORIES :.
1. GLUCOSE:
(1) Fasting blood sugar (FBS)
measures blood glucose after fasting for at least 8 hours. It often is the first test done to
check for diabetes.
(2) 2-hour postprandial blood sugar (2-hour PP)

measures blood glucose exactly 2 hours after eating a meal.
(3) Random blood sugar (RBS)

measures blood glucose regardless of when the person last ate. Several random
measurements may be taken throughout the day. Random testing is useful because glucose
4
levels in healthy people do not vary widely throughout the day. Blood glucose levels that
vary widely may indicate a problem. This test is also called a casual blood glucose test.
(4) Oral glucose tolerance test (OGTT)

measures the body's ability to use glucose. It is used mainly to diagnose prediabetes and
diabetes. An oral glucose tolerance test is a series of blood glucose measurements taken after
you drink a sweet liquid that contains glucose. This test is commonly used to diagnose
diabetes that occurs during pregnancy (gestational diabetes). This test is not commonly
used to diagnose diabetes in a person
(5) Glycosylated hemoglobin HbA1c:
Glycosylated hemoglobin is an indicator of the blood glucose concentration over a longer

period of time than either a single blood glucose measurement (which reflects the glucose
concentration at the time of blood collection)
A glycohemoglobin test indicates how well diabetes has been controlled in the 2 to 3
months before the test. The A1C level is directly related to complications from diabetes:
(The lower the A1C level, the lower the risk for complications)
2. Lipid Profile
CHOLESTEROL
Elevated cholesterol has been seen in artherosclerosis, diabetes, hypothyroidism and

pregnancy. Low levels are seen in depression, malnutrition, liver insufficiency, malignancies,
anemia and infection.
Normal Adult Range: 120 - 240 mg/dl
Optimal Adult Reading: 180
TRIGLYCERIDES
Increased levels may be present in artherosclerosis, hypothyroidism, liver disease,

pancreatitis, myocardial infarction, metabolic disorders, toxemia, and nephrotic syndrome.
Decreased levels may be present in chronic obstructive pulmonary disease, brain infarction,
hyperthyroidism, malnutrition, and malabsorption.
5
LDL (Low Density Lipoprotein)
LDL is the cholesterol rich remnants of the lipid transport vehicle VLDL (very-low density
lipoproteins) there have been many studies to correlate the association between high levels
of LDL and arterial artherosclerosis.
Optimal Adult Reading: 81 mg/dl
HDL (High Density Lipoprotein)
HDL or High-density lipoprotein is the cholesterol carried by the alpha lipoproteins. A high
level of HDL is an indication of a healthy metabolic system if there is no sign of liver disease
or intoxication.
Optimal Adult Reading: +85 mg/dl
3. Liver Function Test ( LFT )

a. Tests of excretion by the liver
Alkaline apahosphatase.
Bilirubin.
b. Evaluation of synthesis in liver.
Total Protein- TP
Albumin-Alb
c. Evaluation of enzyme activity.
Alanine Aminotransferase (ALT)=SGPT

Aspartate Aminotransferase (AST)=SGOT
Gamma Glutamic Transpeptidase (GGT)
Lactate Dehdrogenase (LDH)
6
FIRST: Tests of excretion by the liver
SGOT (Serum Glutamic-Oxalocetic Transaminase - AST)
Serum Glutamic Oxalocetic Transaminase or AST is an enzyme found primarily in the

liver, heart, kidney, pancreas, and muscles. Seen in tissue damage, especially heart and
liver, this enzyme is normally elevated. Vitamin B deficiency and pregnancy are two
instances where the enzyme may be decreased.
Normal Adult Range: 0 - 42 U/L

SGPT (Serum Glutamic-Pyruvic Transaminase - ALT)
Serum Glutamic Pyruvic Transaminase or ALT is an enzyme found primarily in the

liver but also to a lesser degree, the heart and other tissues. It is useful in diagnosing
liver function more so than SGOT levels. Decreased SGPT in combination with
increased cholesterol levels is seen in cases of a congested liver. We also see increased
levels in mononucleosis, alcoholism, liver damage, kidney infection, chemical
pollutants or myocardial infarction.

GGT (Gamma-Glutamyl Transpeptidase)
Believed to be involved in the transport of amino acids and peptides into cells as well
as glutithione metabolism, Gamma-Glutamyl Transpeptidase is mainly found in liver
cells and as such is extremely sensitive to alcohol use. Elevated levels may be found in
liver disease, alcoholism, bile-duct obstruction, cholangitis, drug abuse, and in some
cases excessive magnesium ingestion. Decreased levels can be found in
hypothyroidism, hypothalamic malfunction and low levels of magnesium.
Normal Adult Female Range: 0 - 45 U/L

Optimal Female Reading: 22.5
Normal Adult Male Range: 0 - 65 U/L
Optimal Male Reading: 32.5
7
LDH (Lactic Acid Dehydrogenase)
Lactic acid dehydrogenase is an intracellular enzyme from particularly in the kidney,

heart, skeletal muscle, brain, liver and lungs. Increases are usually found in cellular
death and/or leakage from the cell or in some cases it can be useful in confirming
myocardial or pulmonary infarction (only in relation to other tests). Decreased levels
of the enzyme may be seen in cases of malnutrition, hypoglycemia, adrenal exhaustion
or low tissue or organ activity.

SCEOND : Excretory Function
ALKALINE PHOSPHATASE
Produced in the cells of the bone and liver with some activity in the kidney, intestine,
and placenta, it is mostly found in an alkaline state with a pH of 9. Used extensively
as a tumor marker it is also present in bone injury, pregnancy, or skeletal growth
(elevated readings). Growing children have normally higher levels of this enzyme also.
Low levels are sometimes found in hypoadrenia, protein deficiency, malnutrition and
a number of vitamin deficiencies.

Optimal Adult Reading: 72.5
Normal Children’s Range: 40 - 400 U/L
Optimal Children’s Reading: 220
BILIRUBIN, TOTAL
A by-product of the breakdown of red blood cells in the liver, bilirubin is a good
indication of the liver’s function. Excreted into the bile, bilirubin gives the bile its
pigmentation. Elevated in liver disease, mononucleosis, hemolytic anaemia, low levels
of exposure to the sun, and toxic effects to some drugs, decreased levels are seen in
people with an inefficient liver, excessive fat digestion, and possibly a diet low in
nitrogen bearing foods.
Normal Adult Range 0 - 1.3 mg/dl

Optimal Adult Reading: .65
8
Thirds : Synthetic Function
PROTEIN, TOTAL
Proteins are the most abundant compound in serum. The protein makeup of the
individual is of important diagnostic significance because of proteins involvement in
enzymes, hormones and antibodies as well as osmotic pressure balance, maintaining
acid-base balance and as a reserve source of nutrition for the bodies tissues and
muscles. The major serum proteins measured are Albumin and Globulin (alpha1,
alpha2, beta and gamma). Decreased levels may be due to poor nutrition, liver disease,
malabsorption, diarrhoea, or severe burns. Increased levels are seen in lupus, liver
disease, chronic infections, alcoholism, leukaemia, and tuberculosis amongst many
others.
Normal Adult Range: 6.0 -8.5 g/dl

ALBUMIN
Albumin is the major constituent of serum protein (usually over 50%). It is

manufactured by the liver from the amino acids taken through the diet. It helps in
osmotic pressure regulation, nutrient transport and waste removal. High levels are
seen rarely in liver disease, shock, dehydration, or multiple myeloma. Lower levels are
seen in poor diets, diarrhea, fever, infection, liver disease, inadequate iron intake,
third-degree burns and edemas or hypocalcemia.
Normal Adult Range: 3.2 - 5.0 g/dl

4. Renal Function Test ( RFT , KFT )

B.U.N. (Blood Urea Nitrogen)
The nitrogen component of urea, B.U.N. is the end product of protein metabolism and
its concentration is influenced by the rate of excretion. Increases can be caused by
excessive protein intake, kidney damage, certain drugs, low fluid intake, intestinal
bleeding, exercise or heart failure. Decreased levels may be dur to a poor diet,
malabsorption, liver damage or low nitrogen intake.

9
CREATININE
Creatinine is the waste product of muscle metabolism. Its level is a reflection of the
bodies muscle mass. Low levels are sometimes seen in kidney damage, protein
starvation, liver disease or pregnancy. Elevated levels are sometimes seen in kidney
disease due to the kidneys job of excreting creatinine, muscle degeneration, and some
drugs involved in impairment of kidney function.
Normal Adult Range: .7 - 1.4 mg/dl

URIC ACID
Uric acid is the end product of purine metabolism and is normally excreted through
the urine. High levels are noted in gout, infections, kidney disease, alcoholism, high
protein diets, and with toxaemia in pregnancy. Low levels may be indicative of kidney
disease, malabsorption, poor diet, liver damage or an overly acid kidney.
Normal Adult Female Range: 2.5 - 7.5 mg/dl
Optimal Adult Female Reading: 5.0
Normal Adult Male Range: 3.5 - 7.5 mg/dl
Optimal Adult Male Reading:5.5
10
All test estimation in this Apparatus
BECKMAN
Apparatus photo:
Method
1_Separate blood from serum
2_ Put blood in special cups of the apparatus
3_Put the cups in special rack of the apparatus and ensure the numbers
written on the
4_Put the rack inside the apparatus
5_Go the screen and write patient data (patient ID, name, sample no. )
6_Select type of analysis serum or plasma depending on the tube type normal or
anticoagulant.
7_Select the required investigations (glucose, urea, creatinin) according to what is written
in the request paper.
8_After ending press save
11
Dimension( Mex ,Rxl )
Apparatus photo :
Method
1. Press on button F1 - enter data
2. Write sector number
3. Write patient name
4. Write location, sample ID
5. Write required investigations through keyboard
6. If there is more than one sample press F1 then F3 then F4
7. Press F2 if one sample
8. The system start work automatically
9. After ending the results will be printed automatically
12
13
INTRODUCTION
The clinical chemistry laboratory functions to achieve the accurate investigations
(qualitative and quantitative analyses) on body fluids such as blood, urine, and
spinal fluid, as well as feces, tissue, calculi and other materials.
Application of biochemical investigations

Laboratory tests help in releasing obscurity of disease process so they are used
for:
1- Diagnosis of a disease,
2- Monitor its progress,
3- Response to treatment, and
4- Screen for disease in seemingly healthy individuals.
Supplies (instruments & tools)
Balance Centrifuge
Water bath Oven
Blood gas analysis system Air conditioner
Electrolytes analyzer (ISE) Glasswares
Refrigerator Plastic wares
Electric transistors (shock absorbent) Shakers
Beckman analyzers (for glucose, BUN, creatinine)
Photometers (colorimeter & spectrophotometer)
Record book
A reference book for laboratory results must contains
2
Day: Date:
SN Name Ward Test Results Remarks
1 A CCU CK Rejected
2 B MMW RBG (hemolyzed)
Terms
Specimen
Any material taken from the patient and sent to the laboratory for analysis.
Sample
A given volume or a known concentration of the specimen ready in final form
for analysis.
Standard
A substance whose concentration is exactly known thus is highly purified.
Control
A substance against which experimental results can be evaluated and compared.
Calibrator
A reference material used to standardize or calibrate an instrument or laboratory
procedure.
Blank
A substance used to adjust the photometer at zero reading "no reaction".
3
Bioch lab manual IV yr BLM N. M. ELIAS
LABORATORY SAFETY
Laboratory rules
 Always wear a laboratory white coat, gloves and shoes with closed toes &
heels.
 Don't eat, drink or smoke in the laboratory and never store the food or drink in
the refrigerator.
 Don’t apply cosmetic or contact lenses in the laboratory. Dangling jewelry,
long hair, bread may be risky.
 Don't draw reagents or specimens through a pipettes directly by mouth.
 Put needles & sharps in puncture – resistant containers.
 Don’t throw any solid into the sink. If you have to pour strong acids or alkalis
make sure that you let a lot of tap water rinse it away.
 Don't waste reagents.
 Report to the instructor, if there is any accident of any type.
Chemical safety
o Bottles of chemicals and solutions should be handled carefully, and a cart
should be used to transport a heavy or a multiple number of containers from
one area to another.
o Glass containers with chemicals should be transported in rubber or plastic
containers that protect them from breakage.
o A bottle should never be held by its neck, but instead firmly around its body
with one or both hands.
o When working with acid or alkali solutions, safety goggles should be worn &
acids must be diluted by slowly adding them to water, while mixing; water
should never be added to concentrated acid.
4
o Acids, caustic materials, and strong oxidizing agents should be mixed in the
sink. This provides water for cooling.
o All bottles containing reagents must be properly labeled before adding the
reagent.
o The label should bear the name and concentration of the reagent, the initials
of the person who made up the reagent, the date on which the reagent was
prepared, the expiration date & storage and potential hazards instructions
[corrosive, toxic, irritants, flammable, explosive, reactive].
o Organic solvents represent a potential fire hazard and hazards to health from
inhalation of toxic vapors or skin contact. Their use should be carried out
using a fume hood. Solvents should be stored in a metal storage cabinet.
o Disposal of flammable solvents in sanitary sewers is not allowed.
o Separate safety cans should be used for ether and for chlorinated solvents; all
other solvents may be combined in a third can.
Electrical hazards
o Worn wires on all electrical equipment should be replaced immediately; all
equipment should be grounded using three-prong plugs.
o an extension cord may have to be used temporarily.
o If several outlets are needed in an area, a strip with its own fuse or circuit
breaker may be installed at least 3 in. above bench-top level.
o Electrical equipment and connections should not be handled with wet hands,
nor should electrical equipment be used after liquid has been spilled on it.
o The equipment must be turned off immediately and dried thoroughly; a fan or
hair dryer will speed up the drying process.
o In case of a wet or malfunctioning electrical instrument that is used by several
people, the plug should be pulled.
5
Fire safety
o Fire sources are flammable liquids, electrical and trash fires. Fire
extinguishing by water, CO2, foam, dry chemicals, or by fire extinguishers.
o Gas cylinders must be stored separately away from fire sources.
o Fire blankets for smothering fire on clothing should be available in an easily
accessible wall-mounted case.
o An extinguisher should be provided near every laboratory door & should be
tested by qualified personnel at intervals specified by the manufacturer.
Biological hazards
o Exposure to infectious pathogens can result from:
1- Accidental puncture with hypodermic needles.
2- Spraying of infectious materials by a syringe or spilling and splattering
of these materials on benchtops or floors.
3- Centrifuge accidents.
4- Cuts or scratches from contaminated glassware.
o Never perform mouth pipetting and never blow out pipets that contain
potentially infectious material.
o Barrier protection, such as gloves, masks, and protective eyewear and gowns,
must be used when drawing blood from a patient, when handling all patient
specimens & during removal of stoppers from tubes.
o Phlebotomists should change gloves and dispose of them between patients.
o Wash hands whenever gloves are changed. Encourage frequent hand washing
in the laboratory& whenever leave the laboratory.
o Facial barrier protection should be used if there is a significant potential for
the spattering of blood or body fluids.
o Dispose of all sharps appropriately in rigid containers without handling them.
6
o Wear protective clothing, which serves as an effective barrier against

potentially infective materials. When leaving the laboratory, the protective
clothing should be removed.
o Make a habit of keeping your hands away from your mouth, nose, eyes, and
any other mucous membranes (reduce the possibility of self-inoculation).
o Decontaminate all surfaces and reusable devices after use with disinfectants.
o Before centrifuging tubes, inspect them for cracks. Inspect the inside of the
trunnion cup for signs of erosion or adhering matter.
o Periodically, clean out freezer to remove broken ampules and tubes of
biological specimens using rubber gloves and respiratory protection.
o All samples should be considered as dangerous samples therefore a special
care should be followed during handling or processing of the samples.
Safety equipment
 Two entrances
 Showers
 Fire extinguishers
 Fire blankets
 Fire alarm
 Fume hoods
 First aid kits
 Respirators
 Safety goggles
 Masks
 Gloves
 Fluid resistant coats and plastic or rubber aprons.
7
SERIAL DILUTIONS
A dilution involves two entities, the solute, which is the material being diluted,
and the diluent, the medium making up the rest of the solution. When a solution
is diluted with water, its volume is increased and its concentration is decreased,
but the total amount of solute remains unchanged. A simple formula can be used
only if the concentration of the original solution is known:
C 1 X V 1 = C 2 X V 2, where
C 1: the original concentration of the solution to be diluted
V 1: the unknown volume to be taken from the undiluted solution
C 2: the needed dilution concentration
V 2: the needed volume of diluted solution (total volume)
V 2 = V 1 + volume of diluent
This formula can be used to determine the volume of a concentrated solution that
is required to make a known volume of a solution of a desired lesser
concentration.
The relationship between solute and diluent is expressed as a fraction. For
example, if a 1:20 dilution is called for, this implies 1 part of solute and 19 parts
of diluent. The number on the bottom of the fraction is the total volume, reached
by adding the volumes of the solute and diluent together.
1 Amount of solute

Dilution Total volume
To create a certain volume of a specified dilution, it is helpful to know how to
manipulate this relationship. An algebraic equation can be set up to find either
the total volume, the amount of solute, or the amount of diluent needed to make a
dilution. Consider the following example:
8
2 ml of a 1:20 dilution is needed to run a specific test. How much serum and how
much diluent are needed to make this dilution?
The equation is set up using the fraction for the dilution, indicating the
relationship between the total volume and the solute, or amount of serum needed:
1 

20 2 ml
Note that the 20 represents the total number of parts in the solution, and that 2 ml
is the total volume desired. Solving this equation for x gives 0.1 ml for the
amount of serum needed to make this dilution. The amount of diluent is obtained
by subtracting 0.1 ml from 2.0 ml to give 1.9 ml of diluent. To check the answer,
simply set up a proportion between the amount of solute over the total volume.
This should equal the dilution desired.
0.1 ml 1

2.0 ml 20
Thus the correct answer has been obtained. If, on the other hand, the amount of
serum that is to be used is known, a problem can be set up in the following
manner:
A 1:5 dilution of patient serum is necessary to run a test. There is 0.1 ml of
serum that can be used. What amount of diluent is necessary to make this
dilution using all of the serum? A slightly different formula can be used to solve
this problem.
1 Amount of solute

Dilution - 1 Amount of diluent
1 0.1 ml
 , x = 0.4 ml of diluent
4 
Note that the final volume is obtained by adding 0.1 ml of solute to the 0.4 ml of
diluent. Dividing the volume of the solute by the total volume of 0.5 ml yields
9
the desired 1:5 ratio. Depending on the unknown being solved for, either of these
formulas can he used. To calculate the total volume, the total dilution factor must
be used. If, however, the amount of diluent is to be calculated, the formula using
dilution – 1 can be used. The previous examples represent simple dilutions.
Occasionally in the laboratory it is necessary to make a very large dilution, and it
is more accurate and less costly to do this in several steps rather than all at once.
Such a process is known as a compound dilution. The same approach is used but
the dilution occurs in several stages. For example, if a 1:500 dilution is
necessary, it would take 49.9 ml. of diluent to accomplish this in one step with
0.1 ml of scrum. If only a small amount of solution is needed to run the test, this
is wasteful; furthermore inaccuracy may occur if the solution is not properly
mixed. Therefore, it is helpful to make several smaller dilutions. To use the
example above, a 1:500 dilution can be achieved by making a 1:5 dilution of the
original serum, a 1:10 dilution from the first dilution, and another 1:10 dilution.
This can be shown as follows:
Serum 
1:5 dilution  1:10 dilution  1:10 dilution
0.1 ml serum 0.1 ml of 1:5 dilution 0.1 ml of 1:10 dilution
0.4 ml diluent 0.9 ml diluent 0.9 ml diluent
Multiplying 5 X 10 X 10 equals 500, or the total dilution. Each of the simple

dilutions is calculated individually by doing mental arithmetic, or by using the
formula given for simple dilutions. In this example, the 1:500 dilution was made
using very little diluent in a series of test tubes, rather than having to use a larger
volume in a flask. The volumes were kept small enough so that mixing could
take place easily, and the final volume of 1.0 ml is all that is necessary to
perform a test.
10
If, in each step of the dilution, the dilution factor is exactly the same, this is
known as a serial dilution. Serial dilutions are often used to obtain a titer, or
indicator of the strength of an antibody. A series of test tubes is set up with
exactly the same amount of diluent in each (Fig.). The most common serial
dilution is a doubling dilution, in which the amount of serum is cut in half with
each dilution. For example, six test tubes can be set up with 0.2 ml of diluent in
each. If 0.2 ml of serum is added to the first tube, this becomes a 1:2 dilution:
0.2 ml serum 0.2 ml 1

 
0.2 ml serum  0.2 ml diluent 0.4 ml 2
Then when 0.2 ml. of the 1:2 dilution is added to 0.2 ml of diluent, a 1:4 dilution
is obtained. The final dilution is obtained by counting the number of tubes and
setting up a multiplication series in which the original dilution factor is raised to
a power equal to the number of tubes. In this example, if the first tube contains a
1 1 1 1 1 1 1
1:2 dilution, the dilution in tube number six is:      
2 2 2 2 2 2 64
If, in this instance, an endpoint was reached at tube number five, the actual liter
would be 1:32. To avoid confusion this is customarily written as the reciprocal of
the dilution, that is 32. Serial dilutions do not always have to be doubling
dilutions. Consider the following set of test tube dilutions: 1:5  1:25  1:125
 1:625  1:3125. For each successive tube, the dilution is increased by a
factor of five, so this would indeed be considered a serial dilution.
Add Mix Mix Mix Mix Mix Mix

0.2 ml remove remove remove remove remove remove
serum 0.2 ml 0.2 ml 0.2 ml 0.2 ml 0.2 ml 0.2 ml
1:2 1:4 1:8 1:16 1:32 1:64

11
Figure: serial dilution, each tube contains 0.2 ml of diluent. Patient serum (0.2
ml) is added to tube one. This is carefully mixed, and then 0.2 ml is withdrawn
and added to tube two. The process is continued until the last tube is reached.
The sample is mixed, and 0.2 ml is discarded.
Ratio
Refers to part relation, for example ratio between 2 liquids or 2 solids.
Dilution
Refers to part to total volume relation, i.e. relative concentration of a particular
substance or solution.
Examples
1- A solution containing 1 ml of serum + 9 ml of normal saline (NS)
Serum to saline ratio 1:9

Saline to serum ratio 9:1
Serum to total volume ratio 1:10
TV to serum ratio 10:1
Saline to TV ratio 9:10
TV to saline ratio 10:9
2- 5 ml serum is diluted up to 25 ml in saline
Serum dilution 1/5 or 5/25

serum to saline ratio 5:20 or 1:4
3- 3 ml serum is diluted with 25 ml saline
Serum dilution 25/3 or 9.3 (dilution factor)

serum to saline ratio 3:25 or 1:8.33
12
Homework
1- Give the serum to saline ratio of the following dilutions
Dilution Serum to saline ratio

1/15
2/23
7/9
30/45
2.5/5
2- If u add 30 ml saline to 2 ml serum what is serum dilution?

TV = 30 + 2 = 32 ml, serum dilution???
3- 0.2 ml of serum is diluted 1:10 in NS, what's the amount of diluents

added?
TV2 = 10 ml, TV1 =
Note, for 1 part of serum we need 9 parts of diluents for 0.2 ml of serum
we need ? parts of diluents. So amount of diluent added =
4- How much serum is present in 25 ml of 1:5 dilution? TV2 = 5 ml, TV1 =

25 ml. In 5 ml there's 1 ml serum, in 25 ml there's ? ml serum.
So the amount of serum present = 25 /5 = 5 ml
5- What's the amount of serum present in 250 ml of 1 in 10 dilution of serum

in saline? The amount of serum present =
6- Find out the amount of serum in 40 ml of 1/5 dilution of serum in saline?
7- You are provided with 3 ml of urine, you made 1/16 dilution by DW,
what's the TV?
13
Concentration of dilution
Final concentration = original concentration X dilution
Examples:
1. What's the conc of 1/10 dilution of 12 % NaCl solution?
Final conc = original concentration X dilution
= 12 X 1/10 = 1.2 %
2. 4 N solution of HCl diluted by 3/5, what's the final conc of resulting
solution?
Final conc = original concentration X dilution
= 3/5 X 4 = 2.4 N
3. You are provided with glucose solution containing 80 mg/ 100 ml which
has been diluted 50 times, what's the final resulting conc?
Final conc = 80/100 X 1/50 = 1.6 mg/100 ml
The original conc is 80 mg/100 ml = 80 mg %
The final conc = 80 X 1/50 = 1.6 mg %
Dilution (serial dilution)

a) Dependent serial dilution: each tube serves as a primary standard e.g. a
serum sample is diluted with saline 1/15 followed by 1/10 & 1/100.
b) Independent serial dilution: you prepare directly from the stock solution
e.g. a serum sample is diluted with saline 1/5 rediluted by 1/10 & 1/100.
Calculations of unknown conc & volumes

C1XV1=C2XV2
(1st X stock) = (2nd X diluted)
Problems:
1. You are provided with a stock solution of albumin conc 25 %, make a set
of working standards with concs 2, 4, 6, 8, 10 % each in 5 ml.
14
Conc. Amount of mls needed (ml) Total volume

Tubes DW (ml)
(%) C1X V1 = C2 X V2 (ml)
1 2 25 X V1 = 2 X 5  V1 = 0.4 4.6 5
2 4 25 X V1 = 4 X 5  V1 = 0.8 4.2 5
3 6 25 X V1 = 6 X 5  V1 = 1.2 3.8 5
4 8 25 X V1 = 8 X 5  V1 = 5
5 10 25 X V1 = 10 X 5  V1 = 5
2. You have 10 N solution of NaOH, how much water is needed to prepare 2

N solution in 100 ml?
C1X V1 = C2 X V2 or N1X V1 = N2 X V2
10 X V1 = 2 X 100  V1 = 200/10 = 20 ml & Amount of H2O needed =
Homework
You are provided with a stock solution of glucose conc 0.25 %, you have to
make a set of working standards with concs 50, 100, 150, 200, 250 mg % each in
10 ml.
Lab. dilution
Tube dilution, sample to diluents ratio.
Solution dilution, sample to diluting factor ratio.
Example: a serum sample is diluted 1/5 with NS, rediluted 1/10 and again 1/100,
what's solution dilution of each solution?
Dilution type Tube 1 Tube 2 Tube 3

Serum 1 ml 1 ml 1 ml
Tube dilution
Saline 4 ml 9 ml 99 ml
1/5 1/5 X 1/10 1/5 X 1/100

Solution dilution
1:5 1:50 1:5000
15
ASPARTATE AMINOTRANSFERASE; ASPARTATE TRANSAMINASE

ALANINE AMINOTRANSFERASE; ALANINE TRANSAMINASE
Biochemistry
(EC 2.6.1.1; AST, ASAT or GOT) & (EC 2.6.1.2; ALT, ALAT; or GPT)
The aminotransferases or transaminases are a group of enzymes that catalyze the
interconversions of AAs and 2-oxacids (α- keto acids) by transfer of amino
groups.
AST, Pyridoxal 5 P
Aspartate + α KG Oxaloacetate + Glutamate
ALT, Pyridoxal 5 P
Alanine + α KG Pyruvate + Glutamate
Distinct isoenzymes of AST are present in the cytoplasm and the mitochondria
of cells.
Tissue sources
Transaminases are widely distributed in human tissues. Both AST and ALT are
normally present in:
 Human plasma
 Bile
 Cerebrospinal fluid
 Saliva
 None is found in urine unless a kidney lesion is present.
Clinical significance
In viral hepatitis and other forms of liver disease associated with hepatic
necrosis, activities for both enzymes may reach values as 20- to 50-fold UNL.
16
Peak values of are seen between the 7th and 12th days; values return to normal
levels by the third to fifth week if recovery is uneventful.
In toxic or viral hepatitis, ALT/AST ratio, which normally is < 1,
approaches or becomes greater than unity.
In extrahepatic cholestasis, moderately increased levels of AST and ALT.
 In cirrhosis, activity 4 to 5 times normal, with the level of AST activity
higher than that of ALT activity.
Primary or metastatic carcinoma of the liver, 5 to 10 fold elevations with
AST usually being higher than ALT.
Slight or moderate elevations of both AST and ALT activities may be
observed
 After intake of alcohol,
 During delirium tremens, and
 After administration of drugs such as opiates, salicylates, or ampicillin.
Although serum levels of both AST and ALT become elevated whenever
disease processes affect liver cell integrity, ALT is the more liver-specific.
Serum elevations of ALT activity are rarely observed in conditions other than
parenchymal liver disease. Moreover, elevations of ALT activity persist
longer than do those of AST activity.
After myocardial infarction, an increased level of AST activity appears in
serum. ALT is increased in liver damage secondary to heart failure.
In progressive muscular dystrophy and dermatomyositis, AST and
occasionally ALT activity levels are increased.
Pulmonary emboli, acute pancreatitis, crushed muscle injuries, gangrene,
and hemolytic disease can raise AST levels.
17
MEASUREMENT OF AMINOTRANSFERASE ACTIVITY
It can be obtained by coupling the transaminase reactions to specific

dehydrogenase reactions.
The disappearance of NADH is followed by the decrease in absorbance. The
change in absorbance per minute (AA/min) is related directly to micromoles
of NADH oxidized and, in turn, to micromoles of substrate transformed per
minute.
A preliminary incubation period is necessary to ensure that NADH-dependent
reduction of endogenous keto acids in the sample is completed before α KG is
added to start the transaminase reaction.
Determination of AST activity
Principle
AST
Aspartate + α KG Oxaloacetate + Glutamate
MD
+
Oxaloacetate + NADH + H Malate + NAD +
N.B.: Absorbance is decreased.
Specimen
1- Serum is the specimen of choice. Hemolysis should be avoided, since AST
and ALT activities in erythrocytes are some 15 and 7 times higher,
respectively, than those in normal sera.
2- Specimens are best stored frozen if they are to be kept more than 3 to 4 days.
Minimal loss of activity occurs at 0 to 4 °C over 1 to 3 days.
18
Procedure
 Tris is used as a buffer in place of the earlier choice of phosphate because
phosphate appears to:
Increase the rate of NADH decomposition.
Inhibit association of P-5'-P with the transaminase apoenzyme.
 Addition of LD to the coupled enzyme system accelerates the endogenous side
reactions and thus shortens the preincubation period.
 The pH optimum for the coupled enzyme system is between 7.7 & 7.9 and the
stability of NADH is greater at this pH than at pH 7.4.
 Stock preparations of both MD and LD are diluted with glycerol rather than
with (NH4)2SO4 to avoid introducing ammonium ions, thereby eliminating a
possible side reaction catalyzed by GDH in which NADH is consumed.
 Increased concentrations of GLDH may be seen in parenchymal liver disease.
 The ratio of serum volume to total reaction volume (serum dilution) is 1: ….
Wavelength : 340 nm
Cuvette : 1 cm light path
Temperature : 25 ºC/ 30 ºC/ 37 ºC
Measurement : Against air
Method : Semi micro
Pipette into cuvette

Enzyme /coenzyme/ substrate 1.0 ml
Sample 0.1 ml
Mix, read initial absorbance after 1 min, start timer simultaneously.
Read after 1, 2, and 3 min.
Calculation
U/l = Δ A X 1746
19
Normal values in serum

♂ up to 18 U/l
♀ up to 15 U/l
Individuals in the fasting state may show slightly lower values.
Linearity
If the absorbance change per minute exceeds 0.16 at 340 nm dilute 0.1 ml of
sample 0.9 ml of 0.9 % NaCl solution and reassay. Multiply the result by 10.
Determination of ALT activity
Principle
AST
Alanine + α KG Pyruvate + Glutamate
LD
+
Pyruvate + NADH + H Lactate + NAD +
Procedure
 The procedure used is identical to that for measuring AST activity.
 The added LD both speeds up the side reaction and serves as the indicator
enzyme.
 The serum dilution is 1: …...
 Values in men are slightly higher than in women.
 Specimen, method, calculation & linearity are similar to those for AST.

 ♂ up to 22 U/l
 ♀ up to 17 U/l
20
TOTAL & DIRECT BILIRUB1N

Biochemistry
 80 - 85 % of bilirubin originates on degradation of hemoglobin with the other
15-20 % being derived from cytochrome, myoglobin and catalases.
 Unconjugated bilirubin, which binds to plasma albumin, is produced in the
course of degradation in the reticuloendothelial system, liver Kupffer cells,
spleen and bone marrow.
 Unconjugated (primary, indirect, water-insoluble) bilirubin is soluble in lipids
and toxic. With the aid of the glucuronyl transferase, bilirubin is conjugated
primarily by glucuronate in the microsomes of hepatic parenchymal cells to be
secreted in bile to GIT.
 In contrast to unconjugated bilirubin, conjugated (secondary, direct) bilirubin
is soluble in water, and is excreted via the kidneys.
 A direct bilirubin value of < 20 % total bilirubin is an indicator of jaundice of
pre-hepatic origin. This value can increase to > 50 % in hepatic and post-
hepatic jaundice.
Test principle
 In the presence of caffeine accelerator, total bilirubin couples with sulfanilic
acid to form a red azobilirubin dye, the color intensity which is proportional to
the bilirubin concentration.
 Determination of direct bilirubin is performed without caffeine additive. The
addition of alkaline tartrate causes a transformation from the red azobilirubin
dye to a blue dye and the absorbance maximum from 546 nm to 578nm.
Sulfanilic acid+ NaNO2 HCl Diazotized sulfanic acid
Bilirubin + Diazotized sulfanic acid HCl Azobilirubin
21
Specimen
 Fresh serum, heparinized plasma or EDTA plasma.
 Hemolysis interferes with the test. Don't use lipemic sera.
 Keep out of light and protect the sample from the effects of sunlight (false 
level).
 Centrifuge samples containing precipitate before performing the assay.
Procedure
Total bilirubin Direct bilirubin

Wavelength 578 nm 546 nm
Cuvette 1 cm light path
Temperature 20 - 25 ºC
Zero measurement Against sample blank
Total bilirubin Direct bilirubin

Sample blank Sample Sample blank Sample
R1 0.2 ml 0.2 ml 0.2 ml 0.2 ml
R2 - 0.05 ml - 0.05 ml
Normal saline - - 2 ml 2 ml
R3 1 ml 1 ml - -
Sample 0.2 ml 0.2 ml 0.2 ml 0.2 ml
Mix, and incubate at RT for 10 – 60 min, add
Mix, and incubate at RT for 5,
R4 1 ml 1 ml
read OD of sample against
Mix, and incubate at RT for 5 – 30 min, read sample blank
OD of sample against sample blank
22
Calculation
mg /dl = 10.8 X ∆ TB
mg /dl = 14.4 X ∆ DB
Normal value
Total bilirubin Up to 1 mg / dl
Direct bilirubin Up to 0.25 mg / dl
Indirect bilirubin Up to 0.75 mg / dl
Linearity
If the absorbance exceeds 1.5 (TB or DB), dilute sample 1+ 4 with of 0.9 %
NaCl solution and reassay. Multiply the result by 5.
Clinical Significance
Elevated levels of unconjugated bilirubin
1- Hemolytic anemia
2- Rh incompatibility
3-  UDP glucuronate or UDP glucuronyl transferase
4- Viral hepatitis
5- Glibert's or Crigler Najjar syndrome
6- Failure to extract bilirubin from blood
Elevated levels of conjugated bilirubin
1- Obstructive jaundice
2- Infectious hepatitis
3- Liver carcinoma
4- Gallstone
5- Drugs
6- Dubin – Johnson syndrome
23
ALKALINE PHOSPHATASE
Biochemistry (EC 3.1.3.1.; ALP)
Alkaline phosphatases act on a large variety of naturally occurring and synthetic
substrates, but the natural substrates on which they act in the body are not known
but the enzyme is closely associated with the calcification process in bone.
Activators & inhibitors

 Some divalent ions such as Mg2+, Co2+, and Mn2+ are activators of the enzyme,
and Zn2+ is a constituent metal ion.
 The correct ratio of Mg2+/Zn2+ ions is necessary to obtain optimal activity.
 Phosphate, berate, oxalate, and cyanide ions are inhibitors of all forms of the
enzyme.
 The individual multiple forms are inhibited to different degrees by L-
phenylalanine, urea, excess Zn2+, or AsO3-4.
 The type of buffer present may affect the rate of enzyme activity. Buffers can
be classified as inert (carbonate), inhibiting (glycine; propylamine), or
activating. 2-amino-2-methyl-l-propanol (2A2M1P); tris (hydroxymethyl)
aminomethane (tris; and diethanolamine, DEA).
Tissue sources
ALP is present in practically all tissues of the body, especially at or in the cell
membranes of:
 Intestinal epithelium
 Kidney tubules
 Bone (osteoblasts)
 Liver
 Placenta.
24
 Prostate
 WBCs
 Spleen
 In liver, the enzyme is located on sinusoidal & bile canalicular membranes
while in bone, activity is confined to the osteoblast.
 The forms present in the sera of normal adults originate in the liver or the
biliary tract, and up to half the total activity comes from the skeleton.
 The relative contributions of bone and liver isoenzymes to the total activity
are markedly age-dependent.
 A small amount of intestinal ALP may also be present, particularly in the
sera of persons of blood groups B or O who are secretors of blood-group
substances.
 The enzyme found in urine is probably derived from renal tissue.
Multiple forms of ALP (isoenzymes)

o The ALP activity in tissues such as liver, bone, and kidney seems to be due
almost entirely to the presence of a form of the enzyme that is characteristic of
that tissue.
o The tissue-specific characteristics of the ALP isoenzymes are retained when
the enzymes are released into the circulation and can be used to identify the
tissue responsible for the elevation of the serum level of the enzyme.
o When serum specimens are separated by electrophoresis at alkaline pH:
o Liver phosphatase moves most rapidly toward the anode.
o Bone phosphatase, which typically gives a more diffuse zone than the liver
isoenzyme, has a slightly lower anodal mobility.
o Intestinal phosphatase also migrates diffusely but more slowly than the bone
enzyme.
25
o Kidney phosphatase, which occurs very rarely in serum, migrates even more
slowly.
o Placental isoenzymes have mobilities of the same order as those of liver and
bone.
o Additional minor phosphatase zones are also present in tissue extracts and
occasionally in serum. One such zone, named the "fast liver" fraction,
migrates more anodally than the main liver zone has been observed more
frequently in hepatobiliary disease. The second zone is called slow liver
fraction.
+
Migration
Intestine
direction
Bone
Liver
( − ) Anode
 Serum ALP measurements are of particular interest in the investigation of
hepatobiliary disease and bone disease associated with increased osteoblastic
activity.
 The response of the liver to any form of biliary obstruction is to synthesize
more ALP; that is, the effect is one of enzyme induction.
26
I) Causes of raised ALP activity:

 Physiological, e.g. infants, children, pubertal growth & pregnancy (3rd
trimester).
 Pathological
1) Hepatobiliary disease
- Extrahepatic cholestasis (≥ 3 ULN), e.g. stone, tumor & inflammation of
head of pancreas.
- Intrahepatic cholestasis (lesser), e.g. drugs, inflammation of biliary tract,
toxins & primary biliary cirrhosis.
2) Bone diseases
- Transient elevations may be found during healing of bone fractures.
- Paget's disease (10 – 25 ULN)
- Rickets & osteomalacia (moderate)
3) Malignancies
- Bone & liver
4) Liver diseases
- hepatitis & cirrhosis (slight to N)
5) Intestinal
- Duodenal ulcer
II) Low levels of plasma ALP
- Arrested bone growth such as achondroplasia, cretinism & ascorbate
deficiency
- Hypophosphatasia rare
27
DETERMINATION OF ALP ACTIVITY
Principle
APL, Mg2+ 4-Nitrophenoxide +P
4-Nitrophenyl phosphate + H2O
(Colorless) PH 10.3 (Colorless, benzoid form)
Rearranges at
alkaline pH
4-Nitrophenoxide
(Yellow, quinonoid form)
 4-NPP is colorless ester, but 4-NP is yellow at the pH of the reaction, thus the
enzyme reaction can be followed continuously by observing the rate of
formation of yellow color.
 Liberated phosphate group was transferred to water; that is, the reaction was
hydrolytic.
 The rate of phosphatase action is enhanced if certain amino alcohols are used
as buffers (activators) such as 2A2M1P, DEA, and Tris that act by binding
protons at the nitrogen atom and as phosphate acceptors by reacting with
phosphate through the hydroxyl group.
 Increase in absorbance is observed.
Specimen
 Serum or heparinized plasma, free of hemolysis, should be used. Complexing
anticoagulants such as citrate, oxalate, and EDTA must be avoided.
 Freshly collected serum samples should be kept at room temperature and
assayed as soon as possible, but preferably not later than 4 hrs after collection
because slight but real increase in activity occurs, 1% / 6-hrs to 3% to 6% over
a 1 - to 4 day period.
28
 Sera stored at refrigerator temperature, activity  slowly (2 % /day).

 Frozen specimens should be thawed and kept at room temperature for 18 to 24
hours before measurement to achieve full enzyme reactivation.
Procedure
Wavelength : 405 nm
Measurement : Against air or DW
Method : Micro
Substrate start
Sample start
R1 0.5 ml
Enzyme /coenzyme/ substrate 0.5 ml Sample 0.01 ml
Sample 0.01 ml R2 0.1 ml
Mix, (incubate for 30 sec. at 25 ºC for sample start only). Read initial
absorbance and start timer simultaneously. Read after 1, 2, and 3 min.
Calculation
U/l = Δ A X 2757 (for sample start) &
Δ A X 3298 (for substrate start)

60 – 170 U/l
Linearity:
If the absorbance change per minute exceeds 0.250 dilute 0.1 ml of sample 0.9
ml of 0.9 % NaCl solution and reassay. Multiply the result by 10.
29
γ-GLUTAMYLTRANSFERASE
Biochemistry (EC 2.3.2.2; GGT)

 GGT transfers the γ-glutamyl group from peptides and other compounds that
contain it to an acceptor.
 The glutamyl acceptor can be the substrate itself, some amino acid or peptide,
or even water.
 The enzyme acts only on peptides or peptide-like compounds containing a
terminal glutamate residue joined to the remainder of the compound through
the terminal (5- or γ-) carboxyl This is the reaction used in measuring enzyme
activity.
 It may also be involved in some aspects of glutathione metabolism.
Tissue sources
GGT is present in serum and in all cells except those in muscle. It is
predominantly located in the cell membrane.
Clinical Significance
1- Intrahepatic or posthepatic biliary obstruction. More sensitive than AST,
ALT & ALP in detecting obstructive jaundice (5 – 30 ULN)
2- Infectious hepatitis & fatty liver (2 – 5 ULN).
3- Heavy drinkers
4- Alcoholic cirrhosis
5- Drug intoxication: alcohol, phenytoin (transient)
6- Pancreatitis & pancreatic malignancies (marked)
7- Prostatic malignancies
30
DETERMINATION OF GGT
Principle
L-γ-glutamyl-3-carboxy-4-nitroanilide + Glycylglycine
GGT
γ-glutamyl-glycylglycine + 5- amino- 2 nitrobenzoate
L-γ-glutamyl-3-carboxy-4-nitroanilide (as substrate) is readily soluble in water,

and activities in serum are higher with the carboxyl derivative than with the non-
carboxylated substrate. Increase in absorbance is observed.
Specimens
Serum free from hemolysis is the preferred specimen, but EDTA-plasma (up to 1
mg/mL blood) can be used. Heparin produces turbidity in the reaction mixture;
citrate, oxalate, and fluoride depress activity by 10 % to 15%.
Procedure
Wavelength : 405 nm
Method : Micro
31

Sample 0.05 ml
Mix, read initial absorbance, start timer simultaneously.
Calculation
U/l = Δ A X 1158
♂ 6 – 28 U/l
♀ 4 – 18 U/l
Linearity
If the absorbance change per minute exceeds 0.200 dilute 0.1 ml of sample 0.9
32
AMMONIA ESTIMATION
Biochemistry
 Ammonia is constantly produced in the tissues & present at very low levels in
blood due to both rapid removal from the blood by the liver and that many
tissues, particularly muscle, release AA nitrogen in the form of glutamine or
alanine, rather than as free ammonia.
 The amide of glutamate provides a nontoxic storage and transport form of
ammonia so glutamine is found in plasma at concentrations > other AAs.
 Ammonia is produced from the metabolism of a variety of compounds:
From amino acids: many tissues, particularly the liver, form ammonia from AAs
by the aminotransferase and GDH.
From glutamine: the kidneys form ammonia from glutamine by the action of
renal glutaminase. Most of this ammonia is excreted into the urine as NH4+,
which is an important mechanism for maintaining the body's acid-base balance.
Ammonia is also obtained from the hydrolysis of glutamine by intestinal
glutaminase. The mucosal cells obtain glutamine either from the blood or from
digestion of dietary protein.
From bacterial action in the intestine: ammonia is formed by the bacterial
degradation of urea in the lumen of the intestine. Ammonia is absorbed from the
intestine by way of the portal vein and is almost quantitatively removed by the
liver by conversion to urea.
From amines: amines obtained from the diet and monoamines that serve as
hormones or neurotransmitters give rise to ammonia by the action of amine
oxidase.
From purines & pyrimidines: in the catabolism of purines & pyrimidines, amino
groups attached to the rings are released as ammonia.
33
Principle
GDH
α ketoglutarate + NADH + NH3 Glutamat + NAD+
The corresponding decrease in absorbance is proportional to [ammonia].
Sample
Heparinized or EDTA plasma.
Procedure
1- Smoking by the patient or the phlebotomist is a source of ammonia
contamination in the specimen. The patient must not smoke after midnight
before the morning when the fasting blood specimen is to be drawn. Patient
cigarette smoking within 1 hr of venipuncture may produce significant .
2- Muscular exertion can increase venous ammonia. Blood is collected from a
stasis – free vein to prevent IV deamination.
3- The specimen must be put on ice immediately and centrifuged without delay
(within 30 min), and the analysis must be performed immediately to prevent
metabolism of nitrogenous constituents in the specimen which is a source of
contamination.
4- Plasma is preferred to serum since ammonia can be generated during clotting.
The plasma may be stored for 2 hrs at 4 ºC or 3 hrs in ice bath.
5- No detergent or glassware free of detergent.
6- No drugs e.g. asparaginase, barbiturate, ethanol, analgesic, diuretics all lead to
 levels.
7- Kanamycine, neomycine, lactulose all lead to low levels.
8- No exposure to air to prevent loss of CO2.
9- Transient ammonia elevation at 0.5-3 hours and again at 3.5-6 hours after a
meal containing protein.
34
Wavelength : 340 nm
Method : Semimicro
Pipette into cuvette Reagent blank Standard Test

DW 0.1 ml
Standard 0.1 ml
Sample 0.1 ml
R1 1.5 ml 1.5 ml 1.5 ml
Mix, and allow to stand for 5 min. read initial absorbance A1 for
sample and blank.
R3 0.01 ml 0.01 ml 0.01 ml
Mix and incubate for 5 min. read final absorbance A2, for sample and
blank. Read after 5 min A2 & ∆ A = A2 – A1
Calculation
Ablank = blank A1 – blank A2
Asample = Sample A1 – Sample A2
Using a standard
A sample - A blank
g / ml  5
A standard - A blank

0.17 – 0.80 μg/ml
Linearity
If the [ammonia] exceeds 20 μg/ml dilute 0.1 ml of sample 0.2 ml of 0.9 % NaCl
solution and reassay. Multiply the result by 3.
35
Fasting blood NH3 determination is helpful in differential diagnosis of hepatic
encephalopathy.
1) Inherited defects of urea cycle
2) Advanced liver disease
3) Hepatic encephalopathy
4) Liver cirrhosis, cirrhosis of the liver caused by alcoholism, hepatitis (viral or
toxicity), or biliary obstruction may result in elevation of circulating ammonia.
This is due to that portal blood is shunted directly into the systemic circulation
and does not have access to the liver for detoxification.
5) GIT bleeding
6) Excess protein in diet
7) Constipation
8) Infection
9) Organic aciduria
10) In chronic renal disease, levels of AAs that normally metabolized by the
kidney (e.g. glutamine, glycine, proline,citrulline) increase. Nitrogen end
products (urea, uric acid & creatinine) are also accumulating. The
accumulation is worsened by dietary protein intake or accelerated proteolysis
(e.g. starvation).
11) In chronic metabolic acidosis (e.g. DM), the activity of renal glutaminase,
GDH and mitochondrial glutamine transporter increase and correlate with
increased urinary excretion of NH4+ and increased renal GNG from AAs.
Liver participate by synthesizing less urea which makes more glutamine
available for kidney.
12) In alkalosis, urea synthesis increases in liver, GNG and NH4+ excretion by
kidneys decrease.
36
Notes
 [ammonia] in the blood cause the symptoms of ammonia intoxication,
which include tremors, slurring of speech, and blurring of vision. At high
concentrations ammonia can cause coma and death (hyperammonemia is toxic
to the CNS).
 Some hypotheses could explain the biochemical basis of ammonia toxicity:
1. The GDH is present in mitochondria of the brain so that, assuming it catalyses
a near-equilibrium reaction as in liver, in [ammonia] would  [α KG].
H2O + glutamat + NAD+ (P)  α ketoglutarate + NAD (P) H + NH4+ + H+
Since α KG is an important intermediate in the TCA,  in its concentration
could  the flux through the latter half of the cycle leading to a serious depletion
in the [ATP] in the cells of the brain.
2. Through the same equilibrium, in [ammonia] should also lead to an  in
the NAD+/NADH ratio within the mitochondria leading to in the rate of
production of ATP in the ETC.
3. Glutamate is an excitatory neurotransmitter in brain. This action of glutamate
may be arrested by conversion of glutamate lo glutamine via the glutamine
synthetase reaction in the glial cells. This glutamine is eventually returned to
the nerve cell where, by the action of glulaminase. re-synthesis of glutamate
occurs. However, glutaminase can be inhibited by high [ammonia] which
could therefore result in depletion of this excitatory neurotransmitter thus
disturbing brain function.
4. In ammonia toxicity there is membrane permeability to potassium and
chloride ions which could interfere with electrical activity in the brain. The
change in permeability might be brought about by in the [proton], due to
in [ammonia] de-inhibiting 6-phosphofructokinase and thus leading to an 
glycolytic flux with consequent formation of lactic acid.
37
UREA ESTIMATION
Biochemistry
 Urea is produced only in the liver, in a cyclic sequence of reactions (the urea
cycle) that starts in the mitochondria and continues in the cytoplasm.
 Urea is the diamide of carbonic acid. In contrast to ammonia, it is neutral and
therefore relatively non-toxic.
 As a small, uncharged molecule, urea is able to cross biological membranes
easily & it is easily transported in the blood and excreted in the urine.
Principle
In alkaline medium, the ions react with the salicylate and hypochlorite to form a
green color indophenol (2.2 – dicarboxyl – indophenol). The color intensity is
proportional to the conc. of urea in the sample.
O
urease
=
C + H2O + 2 H+ 2 NH3+ CO3-2

2HN NH2 28
M.wt = 60
Urea = BUN X (60 / 28) = BUN X 2.14
Sample: serum, EDTA, citrated or heparinized plasma or urine (diluted with DW

1:10).
Procedure
Wavelength : 580 nm
Measurement : Against reagent blank
Method : Colorimetric
38
Reagent Blank (B) Standard (St) Test (T)

Serum / plasma - - 10 µl
Standard - 10 µl -
Working reagent 1 ml 1 ml 1 ml
Mix well and allow to stand at RT for 10 minutes
R3 200 µl 200 µl 200 µl
or diluted R 3 (1:5) 1 ml 1 ml 1 ml
Mix well and allow to stand at RT for 10 minutes
Calculation
OD for T
mg / dl   conc.of St (50 mg/dl)
OD for St
Normal value
Serum 15 – 45 mg / dl
24 hrs urine 200 – 350 mg / dl
Linearity
If [urea] exceeds 200 mg/dl for R3 or 300 mg/dl for diluted R3 dilute sample 1:2
with of 0.9 % NaCl solution and reassay. Multiply the result by 2.
Increased serum urea level
Physiological
- High & selective protein diet
Pathological
A) Pre – renal causes
1- Mild / sever dehydration
39
2-  protein catabolism e.g. burn, surgery, fever, stress, trauma, sepsis,

malignancies, muscle wasting.
3- Cortisol therapy
4- GIT hemorrhage
5- Impaired renal perfusion e.g. cardiac failure, shock, hypovolemia.
B) Renal causes
1- Glumerulonephritis
2- Nephrotoxicity
C) Post – renal causes (obstruction)
1- Nephrolithiasis
2- Prostatism
3- Tumors of GIT
Decreased serum urea level
I) Physiological
1- Pregnancy
2- In growing age
3- IV fluid infusion
4- Low protein diet
II) Pathological
1- Malnutrition & starvation
2- Malabsorption
3- Urea cycle defects
4- Sever liver damage
40
CREATININE ESTIMATION
Biochemistry
o Creatine and its phosphorylated form creatine phosphate serve as an ATP
buffer in muscle metabolism. Creatine does not derive from the muscles
themselves, but is synthesized in two steps in the kidneys and liver.
o Creatinine is derived from creatine and creatine phosphate in muscle tissue
and may be defined as a nitrogenous waste product.
o Creatinine is not reutilised but is excreted from the body in the urine.
o It is produced and excreted at a constant rate that is proportional to the body
muscle mass. As a consequence of the way in which creatinine is excreted by
the kidney, Cr measurement is used almost exclusively in the assessment of
renal function.
o Creatinine is regarded as the most useful endogenous marker in the diagnosis
and treatment of kidney disease.
o The plasma level of creatinine is relatively independent of protein ingestion,
water intake, rate of urine production and exercise.
Principle
Creatinine in alkaline solution reacts with picric acid to form a colored complex.
The color intensity is proportional to the conc. of creatinine in the sample.
Sample
Serum, heparinized plasma or urine (diluted with DW 1:20).
Procedure
Wavelength : 492 nm
41

Method : Semimicro
Standard (St) Test (T)
Serum / plasma - 100 µl
Standard 100 µl -
Working reagent 1 ml 1 ml
Mix well and after 30 sec read A1. Exactly 2 min
later read A2. ∆A = A2 – A1
Calculation
 A for T
mg / dl (for serum)  2
 A for St
 A for T
mg / dl (for urine)   100
 A for St
mg creatinine /dl urine  ml urine 24 hrs

Creatinine clearance (ml / min) 
mg creatinine /dl serum  1440
Normal value
♂ 0.6 – 1.1 mg / dl
Serum
♀ 0.5 – 0.9 mg / dl
24 hrs urine 1 – 1.5 g / 24 hrs
Linearity
If [creatinine] exceeds 10 mg/dl in serum or 500 mg/dl in urine, dilute sample 1+
4 with of 0.9 % NaCl solution and reassay. Multiply the result by 5.
42
Increased serum creatinine
- Non renal causes
1. Increased muscle bulk
2. High meat intake
3. Vigorous exercise
4. Drugs as salicylates & cimetidine
- Renal causes
1- Pre renal
-  blood pressure
- Fluid depletion
- Renal artery stenosis
- Heart failure
2- Renal: loss of functioning nephrons
- Glumerulonephritis
- Nephrotic syndrome
3- Post renal
- Prostatic enlargement
- Stone
Decreased serum creatinine
- Rare but may indicate atrophy of muscle tissue.
Serum creatinine in prognosis
1- To detect potential renal damage when nephrotoxic drugs are used such as
aminoglycoside antibiotic.
2- Routinely measured for all dialysis clients.
3- For clients who have had renal transplants.
43
RENAL CALCULI
Introduction
Urinary tract calculi can be found in the renal pelvis, ureter, bladder, or urethra.
Calculi in The renal pelvis or ureter are of particular clinical significance
because of their frequent association with serious renal disease or as the etiologic
agent of renal colic. History & examination may suggest an underlying cause of
renal calculi such as inadequate fluid intake. Biochemical tests should be
performed on plasma & urine. However, the single most useful test is to analyse
a stone if available.
Types, frequency & etiology
Composition % Urine PH Etiology/Pathogenesis
Hypercalciuria,
Vit D intoxication
Hyperparathyroidism
Calcium
60  Milk alkali syndrome
oxalate
Osteoporosis
Hypocitraturia Hyperoxaluria
Renal tubular acidosis
Calcium
10 Basic Distal renal tubular acidosis
phosphate
Low urinary pH
Hyperuricosuria
Uric acid < 10 Acidic
Hyperuricemia
Gout
Struvite Infection with urease-producing

25 Basic
(triple phosphate) microorganisms
Cystine <5 Acidic Cystinuria
44
Physical properties of calculi
Main
Color Appearance Consistency Size
constituents
White, gray, Hard, small to medium

Rough Very hard Oxalate
brown size, smooth or spiky
White to Slightly larger more

Chalky Hard Carbonate
reddish brown friable
Gray –white or Yellow, friable, may be
Smooth Crisp Phosphate
gray yellow large (staghorn)
Yellow to
Rough Very hard May be large (staghorn) Uric acid
reddish brown
Brownish or pale yellow

Pale yellow to
Waxy Soft color (maple sugar) may Cystine
brown
be large (staghorn).
Reagents
Reagent (1) Universal Indicator
Reagent (2) Hydrochloric Acid
Reagent (3) Resorcinol (crystal)
Reagent (4) Hydrochloric Acid
Reagent (5) Molybdic Acid
Reagent (G) Stannous Chloride (crystals)
Reagent (7) Sodium Hydride
Reagent (8) Sodium Cyanide
Reagent (9) Sodiurn nitroprusside.- (Crystals)
Reagent (10) Sodium Hydroxide
Reagent (11) Cobalt Chloride
Reagent (12) Potassium hydroxide
Reagent (13) Phosphotungestic Acid
Reagent (14) Na2HPO4
Additional reagent not provided in the kit
Conc. Sulfuric acid & ammonium Hydroxide 25%
45
Procedure
Wash, dry and grind the calculi as a fine powder, then
Test Procedure Observation
Yellow-Red = acidic
PH Sample + 1 drop of R1 Green = neutral
Blue = Alkaline
Carbonate Sample + 2 drops of R2 Effervescence
Sample mixed with equal amount of R3 Dark blue green

Oxalate
+ 1 drop of conc H2SO4 color after 5 min
Sample + few crystals of R6 + 2 drops Dark blue color

Phosphate
of R5 immediately
Sample + 2 drops of H2O + 2 drops of

Calcium White gelatinous ppt
R4 + 3 drops of R7 add gradually.
Sample + 2 drops of R10 + 1 drop 25%

ammonium hydroxide + 2 drops of R8,
Cystine Red color
allow to stand for 5 min, then add few
crystal of R9
Ammonium Sample + 2 drops of R10 + 3 drops of

Blue color
Salts R11
Sample + 2 drops of R12, mix well, then

Uric acid Blue color
add 3 drops of R11
Sample + 2 drops of 25% ammonium

Magnesium White gelatinous ppt
hydroxide + 2 drops of R14
Observations
Specimen A: renal calculi
Specimen B: ve control
46
CREATINE KINASE
Biochemistry (EC 2.7.3.2; CK)

Creatine kinase, also incorrectly referred to as creatine phosphokinase (CPK),
catalyzes the reversible phosphorylation of creatine by ATP, as shown below:
CK, Mg2+ ;PH = 6.7

Creatine + ATP Phosphocreatine + ADP
PH = 9 (creatine phosphate)
 Phosphocreatine (PCr), the major phosphorylated compound in muscle, is

present in about an eight-fold excess over ATP.
 When muscle contracts, ATP is consumed and CK catalyzes the
rephosphorylation of ADP to form ATP, using PCr.
 At neutral pH, PCr has a much higher phosphorylating potential than does
ATP; this higher potential favors the reverse reaction. The reverse reaction
proceeds two to six times faster than the forward reaction, depending on the
reaction conditions.
Inhibitors & stability

As is true for all kinases, Mg2+ is an obligate activating ion, functioning with
ADP and ATP. The optimal concentration range for Mg2+ is quite narrow, and
excess Mg2+ is inhibitory. Many metal ions, such as Mn2+, Ca2+,Zn2+ and Cu2+,
inhibit enzyme activity, as do iodoacetate and other sulfhydryl-binding reagents.
Activity is also inhibited by excess ADP, urate, and cystine.
The enzyme in serum is relatively unstable; activity is lost as a result of
sulfhydryl group oxidation at the active site of the enzyme. Activity can be
partially restored by incubating the enzyme preparation with sulfhydryl
compounds such as N-acetylcysteine (NAC), thioglycerol, dithioerythritol,
47
dithiothreitol (Cleland's reagent), thioglycolic acid, mercaptoethanol, or cysteine.

NAC is soluble, stable, odorless, and inexpensive and can be lyophilized,
whereas thioglycerol cannot.
Tissue sources
CK activity is greatest in
 Striated muscle (2500 U/g protein),
 Brain (550 U/g protein), and
 Heart tissues (470 U/g protein).
Other tissues, such as
 The Kidney and
 The diaphragm, contain significantly less activity, and
 The liver and erythrocytes are essentially devoid of activity.
Structure
 CK is a dimer composed of two subunits (B, or brain, and M, or muscle),
each with a MW of ~ 40 000, are the products of two distinct structural genes.
 Three different pairs of subunits can exist: BB (or CK-1), MB (or CK-2), and
MM (or CK-3), they are numbered on the basis of their electrophoretic
mobility, with the most anodal form receiving the lowest number.
o CK-1 predominates in brain, prostate, gut, lung, bladder, uterus, placenta,
and thyroid, whereas
o CK-3 predominates in skeletal and cardiac muscle.
o CK-2 is present in heart muscle (25 % - 46 % of CK activity) and also to a
minor degree in skeletal muscle (< 5%).
All three of the isoenzymes are found in the cell cytosol or are associated
with myofibrillar structures.
48
 There exists a fourth, mitochondrial form (differs from the others both
immunologically and in electrophoretic mobility) CK-Mt that is located
between the inner and outer membranes of mitochondria, and in the heart it
constitutes up to 15% of the total CK activity.
 CK activity may be found in 2 macromolecular forms macro CK types 1 & 2:
o Type 1 (CK-1 associated with IgG or CK-3 with IgA) is associated with
GIT diseases, adenoma or carcinoma, myocardial and vascular diseases,
and often occurs in women older than 50 years.
o Type 2 (oligomeric CK-Mt) is found predominantly in adults who are
severely ill with malignancies or liver disease or in children who have
myocardial disease.
 The M subunit undergoes post-translational modification in serum and thus
there exist at least two other M subunits, each of which is capable of
hybridizing with other M or B subunits to form active isoenzyme species,
called isoforms, with electrophoretic mobilities slightly different from the
original, unmodified subunit.
Diseases of skeletal muscle
1. Muscular dystrophy, but values may be normal following a period of
physical inactivity.
2. Malignant hyperthermia.
3. Serum enzyme activity is normal in neurogenic muscle diseases such as
myasthenia gravis, multiple sclerosis, poliomyelitis, and Parkinsonism.
Diseases of the heart

1. Myocardial infarction CK activity in serum is invariably elevated.
49
2. Cardiac trauma following heart surgery including transplantation.

3. Angina pectoris,
4. Cardiogenic shock,
5. Electrical counter shock,
6. Tachycardia,
7. Myocarditis,
8. Congestive cardiac failure,
9. Percutaneous transluminal coronary angioplasty (PTCA) or other
intraarterial procedures.
Notes:
The mere presence of CK-2 activity in serum does not necessarily indicate
myocardial damage. CK-2 (< 6% of tCK activity) can be detected in serum in:
i. Inflammatory diseases
ii. Degenerative muscle diseases,
iii. Traumatic lesions including shock, intoxications, delirium tremens,
hypothyroidism,
iv. Acute psychosis, and
v. In women immediately after obstetric delivery.
Diseases of the CNS

1. After cerebral ischemia,
2. Acute cerebrovascular disease (including subarachnoid hemorrhage),
3. Neurosurgical interventions, and head injury.
4. In Reye's syndrome (a childhood disorder characterized by acute brain
swelling with fatty infiltration and nonicteric dysfunction of the liver),
indicating the severity of the encephalopathy.
Diseases of the thyroid
50
Serum CK activity shows an inverse relationship with thyroid activity. The

major isoenzyme present is CK-3.
Clinical conditions associated with the presence of CK-1 in serum

1. During normal childbirth, elevation in maternal total serum CK activity, CK-
1 from the uterus and possibly the placenta.
2. Surgical intervention during labor will increase the serum activity of CK-1.
3. CK-1 may be elevated in neonates, particularly in brain-damaged or very
low-birth-weight newborns.
4. CK-1, usually at low levels, is reported in multiorgan involvement, following
aortocoronary bypass operations, and in hypothermia.
5. Gastrointestinal infarction or adenocarcinoma, as well as tumors of the lung,
prostate, bladder, testes, kidney, breast, and ovary have been found to be
associated elevations of serum CK-1; these findings suggest that serum CK-1
may be of use as a tumor marker.
Mitochondrial CK activity in serum

Mitochondrial CK activity in serum is rarely observed, and then only in acutely
ill patients with malignancies or severe illnesses with a high associated mortality.
CK activity in CSF
After certain types of neurological injury, CSF-CK-1 elevations can be detected.
Reference intervals
In health, serum CK activity is influenced by age, sex, race, lean body mass and
physical activity, as well as genetic differences.
1. Children have higher CK activities than adults,
2. Men have higher values than women, and
51
3. Blacks have higher values than nonblacks.

The activity in serum appears to be a function of the muscle mass of the
individual. This is presumably the basis for the finding that females have lower
serum activity than males and that slightly built persons often have lower serum
CK activity than more muscular members of the same sex. Ethnic origin is also
important.
 The CK levels in the sera of normal newborns are elevated during the first 24
hours post partum to about three times adult values, and a slight elevation
remains throughout the first year of life.
 In males, serum CK activity remains constant after the first year until 12
years of age; it then increases at about 15 years of age, a reflection of the
increase in muscle mass occurring during puberty. CK activity thereafter
decreases slightly with age.
 In females, activity is stable from the 1st year - 12 years of age and then rises
to the time of menstruation. Thereafter, activity falls, particularly during
pregnancy, so that the mean level during pregnancy is less than half that of
premenarchal girls. After menopause, the serum CK level rises.
 For both sexes CK activities are higher in the summer months (seasonal
variation).
 Exercise and muscle trauma (contact sports, traffic accidents, intramuscular
injections, surgery, convulsions, wasp or bee stings, and burns) can elevate
serum CK values.
 Many drugs cause elevations of serum CK activity because they release
enzyme from cells or exert a direct toxic effect on cell membranes.
52
DETERMINATION OF TOTAL CK ACTIVITY
Principle
PCr + ADP CK Creatine + ATP

PH 6.8
ATP + glucose HK Glucose 6 phosphate + ADP
G6P + NADP+ G6PD 6 phosphogluconate + NADPH + H+
The rate of NADPH formation is a measure of the CK activity, provided that the
concentrations of all other components in the three-enzyme system are present in
suitable excess so that the CK activity is the only limiting factor. Increase in
absorbance is observed.
Sources of interference
The adenylate kinase (AK) effect is caused by an enzyme found in fairly high
concentration in nearly all tissues, including RBCs. AK catalyzes the reaction,
2ADP ATP + AMP. Serum AK activity results in an apparent increase in CK
activity because more ATP is produced than CK activity alone produces. AK
activity can be inhibited by adding to the CK assay AMP (competitive inhibitor)
and diadenosine pentaphosphate (Ap5A) that competitively inhibits the AK of
muscle and erythrocytes but has less effect on the liver and kidney enzymes.
Endogenous inhibitors in serum

Ca2+ is an important competitive inhibitor for Mg2+ which is required by CK for
full catalytic activity. The Ca2+ effect can be nullified by the addition of EDTA,
provided the Mg2+ concentration is increased to compensate for some binding of
Mg2+ by the EDTA.
Note: NAC is included to activate CK, EDTA to bind Ca2+ and to increase the
stability of the reaction mixtures, and Ap5A together with AMP to inhibit AK.
53
Specimen
Serum is the preferred specimen, heparinized or EDTA plasma may be used.
1. CK activity in serum is unstable and is rapidly lost during storage (activity
decreases by < 10 % in 24 hrs at 2-8 °C or 1 hr at 20 – 25 °C). Full activity
may persist at ambient temperatures for 4 hours, at 4 °C for 8 to 12 hours, and
for 2 to 3 days when frozen.
2. CK is inactivated both by bright daylight and by increasing specimen pH
owing to loss of carbon dioxide; accordingly, specimens should be stored in
the dark in tightly closed tubes.
3. The serum specimen should be chilled to 4 °C after collection.
4. The optimized assay formulation, containing EDTA and NAC will reactivate
CK in serum up to 99 % even after it has been stored for 1week at 4 °C.
5. Moderately or severely hemolyzed specimens are unsatisfactory because
enzymes and intermediates (AK, ATP, G-6-P) liberated from the RBCs.
Procedure
Wavelength : 340 nm
Method : Semi micro

Sample 0.02
Mix, incubate for 3 min. at 25 ºC. Read initial absorbance and start
timer simultaneously. Read after 1, 2, and 3 min.
Calculation
U/l = Δ A X 4127
54

♂ 10 – 80 U/l
♀ 10 – 70 U/l
Linearity:
DETERMINATION OF CK – MB ACTIVITY
Principle
Total serum or plasma CK activity represents CK-MM and CK-MB isoenzyme
activity, the contribution of the CK-BB isoenzyme normally being undetectable.
The reagent contains a mixture of monoclonal antibodies directed against the
CK-M subunit, giving complete inhibition of CK-MM and partial inhibition (see
note 1) of CK-MB. The residual activity is determined (NAC activated) and the
CK-MB activity of the sample is calculated.
Procedure
Wavelength : 340 nm
Temperature : 30 ºC/ 37 ºC
Measurement : Against air or distilled water

Sample 0.1 ml
Mix, incubate for 10 min. then read A1
After exactly 5 min, read A2.
55
Calculation
U/l = Δ A (A 2 – A1) X 1200
Normal values in serum:

At 37 ºC: < 25 U/l. CK-MB activity between 6 and 25 % of total activity.
Linearity
If the total CK activity is > 1000 U/l repeat the test using a sample diluted with 9
g /l NaCl to reduce the activity to < 1000 U/l.
Note
A multiplying factor of 1.8 is included in the previous calculation formulae to
take into account the 45 % inhibition of CK-MB activity by the monoclonal
antibodies.
56
LACTATE DEHYDROGENASE
Biochemistry (EC I.I.I .27; LD)

 It is a hydrogen transfer enzyme that catalyzes the oxidation of L-lactate to
pyruvate with the mediation of NAD+ as hydrogen acceptor.
LD, PH 8.8 – 9.8

Lactate + NAD + Pyruvate + NADH + H +
LD, PH 7.4 – 7.8
 The optimal pH varies among the different isoenzymes and depends on the
temperature as well as on substrate and buffer concentrations.
 The specificity of the enzyme extends from L-lactate to a variety of related 2-
hydroxy acids and 2-oxo-acids; for example, catalytic oxidation of 2-
hydroxybutyrate to 2-oxobutyrate is referred to as 2-hydroxybutyrate
dehydrogenase (HBD) activity.
 LD does not act on D-lactate, and only NAD+ will serve as coenzyme.
 The enzyme has an MW of 134 000 and is composed of four peptide chains
(subunits) of two types: M and H, which are determined by loci on human
chromosomes 11 and 12, respectively.
 The subunit compositions of the five isoenzymes, in order of decreasing
anodal mobility in an alkaline medium, are
1- LD-1 (HHHH; H4)
2- LD-2 (HHHM; H3 M)
3- LD-3 (HHMM; H2 M2)
4- LD-4 (HMMM; HM3)
5- LD-5 (MMMM; M4)
6- A different sixth isoenzyme, LD-X composed of four X subunits, is
present in the postpubertal human testis.
57
Inhibitors
 Lactate dehydrogenases are inhibited by reagents such as mercuric ions and
p-chloromercuribenzoate that react with thiol groups; the inhibition is
reversed by the addition of thiol reagents such as cysteine or glutathione (non
competitive inhibition).
 Borate and oxalate inhibit by competing with lactate for its binding site on
the enzyme; similarly, oxamate competes with pyruvate for its binding site.
 Both pyruvate and lactate in excess inhibit enzyme activity, although the
effect of pyruvate is greater (competitive inhibition).
 Inhibition by either substrate is greater for the H form than for the M form,
and substrate inhibition decreases with increase in pH.
 EDTA inhibits the enzyme, perhaps by binding Zn2+.
Distribution
o LD activity is present in all cells of the body and is invariably found only in
the cytoplasm of the cell.
o Enzyme levels in various tissues (in U/g) are very high, compared with those
in serum:
 Liver, 145
 Heart, 124
 Kidney, 106
 Skeletal muscle, 147
 Erythrocytes (U/g hemoglobin),36.
o Thus, tissue levels are about 500-fold higher than those normally found in
serum, and leakage of the enzyme from even a small mass of damaged tissue
can increase the observed serum level of LD to a significant extent.
58
o In addition to their higher enzyme concentration, many of these tissues show

different isoenzyme composition.
Isoenzyme, composition % of total LDH Tissue localization
LDH 1 (HHHH) H4 & 15 – 25 Heart, RBCs & renal cortex

LDH 2 (HHHM) H3M 30 – 40
Lung, lymphocytes, spleen,

LDH 3 (HHMM) H2M2 20 – 25
pancreas, thyroid & adrenals
LDH 4 (HMMM) HM3 10 – 15

Liver & skeletal muscle
LDH 5 (MMMM) M4 5 – 15
Normal electrophoretic pattern
1 + – 5
A B
59
C D
Pattern A, AMI, common pattern showing "flipped" or elevated LD-1 .

Pattern B, lymphatic tissue involvement in infectious mononucleosis.
Pattern C, CHF showing  LD-5 as a result of hepatic anoxia.
Pattern D, acute circulatory shock showing very severe hepatic anoxia.
Raised levels
# Artefactual
o Invitro hemolysis
# Marked increase
1- circulatory failure with shock & hypoxia
2- MI
3- Megaloplastic anemia, leukemia & lymphoma
4- Hemolytic anemia
5- Renal infarction
6- Toxic hepatitis
7- Hepatic coma
# Moderate elevation
1- Viral hepatitis
60
2- Malignancy of any tissue

3- Skeletal muscle disease or injury
4- Myocarditis
5- Glomerulonephritis
6- Pulmonary embolism
7- Infectious mononucleosis
8- SLE
9- Hypothyroidism
10- HTN
11- Head trauma
61
DETERMINATION OF LD
Principle
LD
+
Pyruvate + NADH + H Lactate + NAD +
The L  P assay has the following advantages:

1) substrate inhibition by lactate is less than that produced by pyruvate,
2) NAD+ preparations used in the L P reaction appear to contain fewer
endogenous LD inhibitors than NADH preparations used for P L reaction.
3) The L P reaction linearity is more prolonged than that of the P  L assay.
The advantages of the P  L assay include:
1) A less expensive assay because of the much lower concentration of reactants.
2) Greater change in absorbance with time allowing precise measurements.
3) Greater stability of the working reagents once they are prepared in assay
solutions.
Specimens
 Serum or heparinized plasma specimens are satisfactory. Plasma containing
other anticoagulants, especially oxalate, should not be used.
 Serum or plasma should be separated from the clot as soon as possible after
the specimen has been obtained.
 Hemolyzed serum or plasma must not be used, since erythrocytes contain 150
times more LD activity (particularly LD1 and LD2) than serum.
 The different isoenzymes vary in their sensitivity to cold; LD4 and LD5 are
especially labile.
 Loss of activity may be prevented by addition of NAD+ or GSH. Both H and
M monomers bind a molecule of NAD+ but the binding of NAD+ to the M
form is weaker and exposes the -SH groups to oxidation.
62
 In serum, the sulfhydryl groups in albumin and other proteins retard

inactivation of the M-rich isoenzymes (LD4 & LD5); therefore, serum
specimens should be stored at room temperature, at which no loss of activity
will occur for 2 - 3 days.
 If serum specimens must be stored for longer periods, they should be kept at 4
°C with NAD+ (10 mg/mL) or GSH (3.1 mg/mL) added to retard the
inactivation of LD4 & LD5.
Procedure
Wavelength : 340 nm
Method : Semi micro

Sample 0.04 ml
Mix, read initial absorbance after 0.5 min, start timer simultaneously.
Calculation
U/l = Δ A X 4127

120 – 240 U/l
Linearity
63
ENZYME TESTS IN THE DIAGNOSIS OF MI
 The first symptom of MI is usually chest pain (due to ischemia of the cardiac
muscle), often severe and frequently described as "crushing" or "tightness."
 The patient is sweating markedly, and nausea as well as vomiting occurs
frequently.
 Blood pressure or heart rhythm may be abnormal, and the patient may be in
circulatory shock.
 Chest pain, per se, may be due to causes other than MI such as
1- Aortic dissection.
2- Pericarditis.
3- Pulmonary embolism, pneumothorax, or pneumonia.
4- Esophageal spasm.
5- Rupture or gastric ulcer.
6- Degenerative arthritis of cervical or thoracic vertebrae.
7- Herpes zoster.
8- Emotional origin (depression, anxiety, malingering).
 About a quarter of all MI may be clinically "silent," associated either with
atypical symptoms or with no symptoms at all. Many patients with silent
infarcts are diabetic (autonomic neuropathy).
 In the elderly, a myocardial infarction often presents with the onset of sudden
breathlessness, acute confusion, fainting, or even a stroke.
 In these circumstances the existence of either typical ECG changes or typical
serum enzyme changes may be sufficient for establishing the diagnosis.
 After a myocardial infarction there is an initial period during which all serum
enzyme activities remain within their reference interval.
64
Start to
Enzyme Peak Return to normal Notes
rise
Its transient existence in blood

CK2 4 - 6 hrs 24 - 36 hrs 3rd day
is due to its short half-life
CK3 6 - 8 hrs 18 - 30 hrs 3rd or 4th day
AST 6 - 8 hrs 18 - 24 hrs 4th or 5th day
False-positive results with LD1

or LDl/LD2determinations are
LD1 8 - 12 hrs 24 - 48 hrs 7 – 12 days
caused by hemolysis and renal
infarction.
False positives arise as a result
of skeletal muscle damage and
Myoglobin 2 to 5 hrs ~ 12 hrs 24 hrs are also seen in renal failure
owing to an inability to excrete
this molecule in the urine.
Troponin I
~ 3 hrs
&T
 Cardiospecific (protein) myoglobin is one of the proteins that is found

exclusively in the myocardium and is released into the circulation where it can
be measured after damage to heart muscle, not so easily measured.
 Troponin I and T are structural components of cardiac muscle. Troponin T is a
cardiac protein and part of the myofibril complex, which, like myoglobin, is
released into the circulation during severe ischemia.
 Myosin heavy chains are released into the blood after a myocardial infarct.
65
Enzyme activity (X URR)
Days after the onset of chest pain (infarction)
Typical pattern of changes in serum enzymes activities following an MI.
Test Sequence
A variety of sampling sequences have been suggested (as an aid in the detection
of even small subendocardial infarcts):
 Every 12 hours for the first 48 hours of hospital admission;
 Three samples within the first 36 hours of admission (at admission, 6-12
hours later, and 24 - 36 hours later);
 Sampling every 6 to 12 hours;
 Every 8 hours for the first 48 hours.
66
AMYLASE
Biochemistry (EC 3.2.1.1)

 A group of hydrolases that split complex carbohydrates constituted of α-D-
glucose units linked by α (1 4) located on adjacent glucose residues.
 Both straight-chain polyglucans (e.g. amylose) and branched polyglucans
(e.g. amylopectin & glycogen) are hydrolyzed, although at different rates.
 In the case of amylose, the enzyme splits the chains at alternate α (1 4)
links, forming maltose and some residual glucose; maltose, glucose, and a
residue of limit dextrins are formed if branched-chain polyglucans are used.
 The α 1 6 linkages at the branch points are not attacked by the enzyme.
 β-Amylase (e.g., plant and bacterial exoamylase) acts only at the terminal
reducing end of a polyglucan chain; it splits off two glucose units (maltose) at
a time.
 Human amylases are α-amylases they are also called endoamylases because
they can attack the α (1 4) linkage in a random manner anywhere along the
polyglucan chain. Large polysaccharide molecules are thus rapidly broken
down into small units (e.g., dextrins, maltose, and some glucose units).
 Amylase in human serum has a moderately sharp pH optimum at 6.9 to 7.0.
The enzyme is customarily assayed at 37 °C, although it is active at 50 °C.
 α -Amylases are calcium metalloenzymes, and calcium is required for
functional integrity.
 Full activity is displayed only in the presence of anions such as chloride,
bromide, nitrate, cholate, or HPO2-4. The first two anions are the most
effective activators.
 The amylases normally occurring in human plasma are small molecules with
molecular weights varying from 55 000 to 60 000.
67
 The enzyme is thus small enough to pass through the glomeruli of the kidney,
and amylase is the only plasma enzyme normally found in urine. Serum and
urine amylases migrate electrophoretically with the β- and α -globulins.
1 Isomaltose
Maltose 6
1 4
Starch molecule
 α 1 6 glucosidic bond (not attacked by salivary α amylase).
α 1 4 glucosidic bond serving as branch point(not attacked by amylase).
Terminal reducing end (not attacked by salivary α amylase).
α 1 4 glucosidic linkage (interior bond; target for amylase action).
Amylose
Amylopectin
Tissue sources
Major sources
 Pancreas, where the enzyme is synthesized by the acinar cells and then
secreted into the intestinal tract by way of the pancreatic duct system.
 The salivary glands also secrete a potent amylase to initiate hydrolysis of
starches while the food is still in the mouth and esophagus.
Minor sources
 Amylase activity is found in extracts from semen, testes, ovaries, fallopian
tubes, striated muscle, lung, adipose tissue, colostrum, tears, and milk. There
is little or no amylase activity in the liver.
68
 Tumors of lung and ovary may contain considerable amylase activity.

 The enzyme present in normal serum is predominantly of P-type and S- type
origin, and the enzyme found in urine is derived from the plasma.
Macroamylases.
 These rare forms (sometimes present in sera) are probably complexes between
ordinary amylase, usually S- type, and IgA, IgG, or other normal or abnormal
high-Mwt plasma proteins.
 Cannot be filtered through the glomeruli of the kidney because of their large
size (MW > 200 000) and are retained in the plasma, where their presence
may  amylase activity.
 In macroamylasemia, amylase activity in the urine is < normal, since less
amylase is cleared by the kidneys.
 Assays of amylase activity in serum and urine are largely of use in the
diagnosis of diseases of the pancreas and in the investigation of pancreatic
function.
 In acute pancreatitis, a transient rise in serum amylase activity occurs within 2
- 12 hrs of the onset; peak in 12 - 72 hrs, return to normal by the 3rd or 4th day.
 A significant amount of the serum amylase is excreted in the urine, and
therefore  of serum activity is reflected in the rise of urinary amylase
activity. Urine amylase, as compared with serum amylase, appears to be more
frequently  and persists for longer periods.
 If the pancreatic duct is obstructed by carcinoma of the pancreas, then
elevation of serum amylase activity is likely.
 Although ~ 25% of the serum amylase is normally eliminated in the urine, in
renal insufficiency the serum amylase activity is .
69
Hyper amylasemia & amylasuria

Pancreatic disease (P-type )
Pancreatitis
Acute & chronic
Complications (ascites and pleural effusion & abscess)
Pancreatic trauma, including investigative maneuvers
Pancreatic carcinoma
Disorders of nonpancreatic origin (mechanism unknown)
Renal insufficiency (mixed )
Neoplastic hyperamylasemia-usually bronchogenic or ovarian(S-type )
Salivary gland lesions, e.g., mumps, calculus disease (S-type )
Macroamylasemia (predominantly S-type)
Disorders of complex origin (mechanism unknown or uncertain)
Biliary tract disease
Intra-abdominal disease (other than pancreatic diseases)
Perforated peptic ulcer (P-type )
Intestinal obstruction (P-type )
Mesenteric infarction (P-type )
Peritonitis (mixed ; depends on cause)
Acute appendicitis 
Cerebral trauma (type depends on other organ damage)
Burns and traumatic shock
Postoperative hyperamylasemia (usually S-type )
Diabetic ketoacidosis (mixed )
Renal transplantation (S-type )
Acute alcoholism (mixed )
Drugs
Medicinal opiates (P-type ) & heroin addiction (S-type )
70
Signs, symptoms & lab. Findings in acute pancreatitis

Epigastric pain Vomiting BP /N
Hard abdomen (heavy pain) Nausea Hb /N
TLC  ALP N ALT & AST /N
Amylase  (S&U) Lipase  Ca++  (neurological signs)
Glucose  Bleeding +/-
Amylase Clearance
 Comparison of renal clearance of amylase with clearance of creatinine, the
amylase-creatinine clearance ratio (ACCR), has been found useful in
diagnosis.
urine amaylase (U/L)  serum creatinine (mg/L)
ACCR %   100
serum amaylase (U/L)  urine creatinine (mg/L)
 Timed urine collection is unnecessary and random or short (2–4 hrs)
collections are adequate.
 The reference interval is ~ 2 % to 5 %, but it is affected by the method of
assay.
 In acute pancreatitis tubular reabsorption of amylase and other proteins is 
(due to competition from other low-molecular-weight proteins) and ACCR is
.
 Caution must be exercised in interpreting  ACCR values, because elevations
have been observed also in burns, ketoacidosis, renal insufficiency, myeloma,
light-chain proteinuria, and march hemoglobinuria, and following
extracorporeal circulation, large IV doses of corticosteroids, duodenal
perforations, and extraperitoneal surgical procedures.
 In macroamylasemia ACCR is usually < 2%.
71
DETERMINATION OF AMYLASE ACTIVITY
Principle
α amylase
bl-G7pNP bl-G 2-5 + G 2-5-pNP
bl-G 2-5-pNP α glucosidase

Glucose + pNP- glucoside
pNP- glucoside α glucosidase

Glucose + pNP
bl-G7pNP = blocked 4-Nitrophenyl maltoheptaose
 Substrates (dye-labeled amylase substrates) are synthesized by linking a

defined oligosaccharide to dyes or indicator groups.
 Substrates are oligosaccharides (of 4-7 glucose units) that have the 4-
nitrophenyl (4-NP) group covalently bound to their reducing ends.
 If the oligosaccharide is maltoheptaose (G7), the substrate is then 4-NP - G7.
 Amylase splits this substrate to produce oligosaccharides (G5, G4, G3).
 Problems with the use of the 4-NP glycoside: the poor stability of the
reconstituted assay mixture (due to the slow hydrolysis of the 4-NP glycoside
by α-glucosidase) and the inadequacy of 4-NP as an effective indicator of
amylase activity.
 The poor stability may be reduced by covalently linking a "blocking" group -
either a 4,6-ethylidene (ethylidene-protected substrate) to the nonreducing end
of the molecule.
 4-NP absorbance is affected by the ionic strength, protein, and surfactant
present in the mixture and hemoglobin interference. These disadvantages are
avoided if β- 2 -chloro-4-NP (CNP) is used as the indicator group. This assay
requires the presence of β-glucosidase to hydrolyze the CNP group.
 The increase in absorbance is proportional to the amylase activity of the
specimen.
72
Specimens
Amylase is quite stable in serum. In urine, an acid pH may make the enzyme less
stable, pH should be ~7 before storage. With the exception of heparin, all
common anticoagulants inhibit amylase activity because they chelate Ca 2+.
Procedure
Wavelength : 405 nm
Sample start Substrate start
R1 1.0 ml
Sample 0.02 ml
Mix, incubate for 1 min
Sample 0.02 ml
R2 0.25 ml
Mix, read initial absorbance after 4 min at 25 ºC and start timer
simultaneously. Read after 1, 2, and 3 min.
Calculation
Serum U/l = Δ A X 4554 (for sample start) & Δ A X 5670 (for substrate start)
Urine U/l = Δ A X 9018 (for sample start) & Δ A X 11250 (for substrate start)

Serum < 55 U/l
Urine ♂ < 298 U/l & ♀ < 270 U/l
Linearity:
If the absorbance change per minute exceeds 0.350 dilute 1 part of sample 10
parts of 0.9 % NaCl solution and reassay. Multiply the result by 11.
73
ACID PHOSPHATASE (ACP) AND PROSTATE SPECIFIC ANTIGEN (PSA)
Biochemistry
- The ACP of greatest clinical importance, namely that derived from the
prostate has a pH optimum in the range of pH 5 to 6.
- The optimal pH for the individual ACPs varies, depending on the tissues from
which they originate and the substrate; the more acidic the substrate, the lower
the pH at which maximum activity is obtained.
- The enzymes can hydrolyze a variety of phosphate esters, and indeed every
substrate utilized in measuring ALP activity in serum has also been used to
determine ACP activity.
- The ACPs are unstable, especially above 37 °C and at pH levels above 7.0.
- Some of the enzyme forms in serum (especially the prostatic enzyme) are
particularly labile, and over 50% of the ACP activity may be lost in 1 hr at
room temperature.
- Acidification of the serum specimen with citrate to a pH below 6.5 aids in
stabilizing the enzymes.
- Because of the clinical importance of serum ACP levels in the diagnosis and
monitoring of prostatic cancer, it is desirable to be able to differentiate
specifically between the prostatic and nonprostatic forms.
- The prostatic enzyme is strongly inhibited by dextrorotatory tartrate ions,
whereas the erythrocyte isoenzyme is not.
- Erythrocyte ACP is inhibited by formaldehyde and by cupric ions, to which
prostatic ACP is resistant.
- These inhibitors, particularly tartrate, allow a distinction to be made between
prostatic and erythrocyte ACPs.
74
Tissue sources
- ACP is present in lysosomes, which are organelles present in all cells except
erythrocytes. Extra lysosomal ACPs are also present in many cells.
- The greatest concentrations of ACP activity occur in
1. Liver
2. Spleen
3. Milk
4. Erythrocytes
5. Platelets
6. Bone marrow
7. Prostate gland (richest source, and it contributes about 1/3 to 1/2 of the
enzyme present in sera from healthy males).
- The remainder source of the ACP in sera from healthy males and females is
unknown, but there is evidence that it derives from the osteoclasts of bone.
Prostate-specific antigen
o PSA is a 34 000-MW monomeric protein, related to the kallikrein family of
proteases (serine protease). It is a serine protease produced exclusively by the
epithelial cells lining the acini and ducts of the prostate gland.
o PSA is secreted into the lumen of the ducts to liquefy the seminal coagulum.
o Its half-life in serum is 2 to 3 days. Therefore, a period of 2 to 3 weeks should
elapse after any event causing PSA to rise, to ensure a stable baseline value.
o PSA is produced by both normal prostatic tissue and by hyperplastic and
neoplastic prostatic tissue.
o However, gram for gram, cancerous prostatic tissue produces about 10 times
more PSA than normal tissue.
75
1. Prostatitis
2. Benign prostatic hypertrophy
3. Prostatic carcinoma
4. Operative trauma or instrumentation of prostate gland (cytoscopy)
5. Bone disease: Paget's disease, hyperparathyroidism & malignant invasion of
the bones by cancers such as female breast cancer and is thought to come
from osteoclasts.
6. Lipid storage disease: Niemann – Pick disease & Gaucher disease.
7. Hematological disorders & malignancies
a. Hemolytic anemia
b. Leukemia
c. Polycythemia
d. Thrombocythemia
8. Acute renal impairment & acute retention of urine.
9. Forensic medicine: semen ACP used for investigation of rape & similar
offenses.
10.The osteoclasts are the probable source of the increased tartrate-resistant
ACP activity of growing children.
Prostatic specific antigen (PSA)

1. Produced exclusively by prostatic epithelial cells.
2.  production by cancerous prostatic tissue.
3. Better marker than PACP in detecting & monitoring prostatic carcinoma.
4. PSA levels increase after prostatic massage, perineal biopsy, and transurethral
resection.
76
DETERMINATION OF ACP AND PSA IN SERUM
Principle
ACP
1-Naphthyl phosphate + H2O 1-Naphthol + phosphate
1-Naphthol + FRTR salt Azo dye
FRTR = fast red TR (4-chloro-2-methylphenyl diazonium salt)

Increase in absorbance is observed.
Specimen
 For ACP analyses, serum is separated immediately and stabilized by the
addition of disodium citrate monohydrate at a level of 10 mg/mL of serum.
 Alternatively, 50 μL of acetate buffer /ml of serum may be added to lower the
pH to 5.4, at which the enzyme is stable at room temperature for several hours
and for up to a week if the serum is refrigerated.
 Hemolyzed serum specimens are contaminated with considerable amounts of
RBCs isoenzyme and should be rejected.
 Unclear sera should be avoided because of possible interference with
measurement due to turbidity.
Procedure
Wavelength : 405 nm
Method : Semimicro
77
tartarate
Pipette into cuvette tACP
resistant -ACP
Sample 0.1 ml 0.1 ml
Working reagent 1.0 ml 1.0 ml
Mix, read initial absorbance A1, start timer simultaneously.
Read after 5 min A2 & ∆ A = A2 – A1
Calculation
U/l = Δ A X 149
PACP = tACP – tartarate resistant -ACP

♂ 3.6 U/l
♀ 3 U/l
Linearity
If the absorbance change per minute exceeds 0.5 dilute 0.1 ml of sample with 0.2
78

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‫‪Gedair Laboratories‬‬

‫الكيمياء الحيوية اإلكلينيكية‪Clinical Biochemistry‬‬

‫طريقة العمل في القسم‬

End point method

.λ ‫ ندوس علي الرقم ثم علي زر‬.1

- Micromolar absorbitivity = 6.3 x 10¯ ³

- Molar absorbitivity = 6.3 x 10³

1st D 2nd D 3rd D 4th D 5th D 6th D 7th D

results. (Modified Westgrad role)

How to correct the error:

2.Oligoclonal: wide base & narrow peak

3.Polyclonal: wide base & wide peak

 Type IV: Increase pre- β only.

 Type V: Increase pre- β & Chylomicron.

Enzymatic end point uricase

Rate Jaffe method - Serum, no hemolysis (false ↑ due to

II. REQUEST FORMAT:

1. Instructions and appropriate urine containers with required preservative

Tubes Additive Sample Tests

There are 3 technicians including the chief of this department

3. Centrifuge specimen for 5 minutes at 3,500 rpm See if sample is not

4. Separate serum/plasma from red cells at once and

6. Record results in the logbook

TESTS USED IN LABORATORIES :.

(2) 2-hour postprandial blood sugar (2-hour PP)

(3) Random blood sugar (RBS)

(4) Oral glucose tolerance test (OGTT)

(5) Glycosylated hemoglobin HbA1c:

Glycosylated hemoglobin is an indicator of the blood glucose concentration over a longer

Elevated cholesterol has been seen in artherosclerosis, diabetes, hypothyroidism and

Normal Adult Range: 120 - 240 mg/dl

Optimal Adult Reading: 180

Increased levels may be present in artherosclerosis, hypothyroidism, liver disease,

Normal Adult Range: 0 - 200 mg/dl

Optimal Adult Reading: 100

Normal Adult Range: 62 - 130 mg/dl

Optimal Adult Reading: 81 mg/dl

HDL (High Density Lipoprotein)

Normal Adult Range: 35 - 135 mg/dl

Optimal Adult Reading: +85 mg/dl

3. Liver Function Test ( LFT )

b. Evaluation of synthesis in liver.

c. Evaluation of enzyme activity.

Alanine Aminotransferase (ALT)=SGPT

Serum Glutamic Oxalocetic Transaminase or AST is an enzyme found primarily in the

Normal Adult Range: 0 - 42 U/L

SGPT (Serum Glutamic-Pyruvic Transaminase - ALT)

Serum Glutamic Pyruvic Transaminase or ALT is an enzyme found primarily in the

Normal Adult Range: 0 - 48 U/L

GGT (Gamma-Glutamyl Transpeptidase)

Normal Adult Female Range: 0 - 45 U/L

Lactic acid dehydrogenase is an intracellular enzyme from particularly in the kidney,

Normal Adult Range: 0 - 250 U/L

SCEOND : Excretory Function

Normal Adult Range: 20 - 125 U/L

Normal Adult Range 0 - 1.3 mg/dl

Normal Adult Range: 6.0 -8.5 g/dl

Albumin is the major constituent of serum protein (usually over 50%). It is

Normal Adult Range: 3.2 - 5.0 g/dl

4. Renal Function Test ( RFT , KFT )

Normal Adult Range: 7 - 25 mg/dl

Normal Adult Range: .7 - 1.4 mg/dl

Normal Adult Female Range: 2.5 - 7.5 mg/dl