Summary procedures for some laboratory tests

‫ ﺑﺴــﺎﻡ ﺍﻟـﺪﺧﻴﻨــﻲ‬/‫ﺍﻟﺪﻛﺘـــﻮﺭ‬

‫ﺃﺧﺼـــﺎﺋﻲ ﲢﺎﻟﻴـــﻞ ﻃﺒﻴـــﻪ‬

Do not accept less than the best

Dr. Bassam Ahmed Aldokhainy

 Manual Procedures of clinical biochemistry tests (by use spectrophotometer)
-
Incubation
Wave-
Class Name of test Reagent Sample Procedures period & Blank Factor Unit
length
condition
Mix well and incubate
B. Glucose RBS/FBS/PPBS 1 ml (spinreact) 10 ul (1ml) Reagent 546 380 Mg/dl
for 10 min. at 37
Mix and start stopwatch. Use Empty
0.5 ml (R1A) + 0.5 ml Transfer to the measuring cuvette
Creatinine (R2B) 100 ul cuvette. Read the ‫اﻟﻜﯿﻮﻓﯿﺖ‬ 510 40 Mg/dl
(QCA) absorbance's at 20 and 80 ‫اﻟﻤﺴﺘﺨﺪﻣﺔ ﻟﻘﯿﺎس‬
K.F.T seconds. ‫اﻟﻌﯿﻨﺔ وھﻲ ﻓﺎرﻏﺔ‬
1ml (R1)+10ul (S).mix & incubate at 37 in W. bath for 5min.
Urea (1ml) Reagent 580 170 Mg/dl
Add 1ml (R2), mix & incubate in w.bath for 5min.
Mix well and incubate
Uric acid (U.A) 1ml (spinreact) 25ul (1ml) Reagent 520 35 Mg/dl
for 10 min. at 37
Mix will and incubate 1.5ml (R1T)
T. bilirubin 1.5ml(R1T)+50ul(R3) 100ul 555 19.1 Mg/dl
for 10 min. at R.T +100ul(S)
Mix will and incubate 1.5ml (R2D)
D. bilirubin 1.5ml(R2D)+50ul(R3) 100ul 555 14 Mg/dl
for 10 min. at R.T +100ul(S)
Use Empty
GPT 1ml (spinreact) 100ul Mix and start stopwatch. 340 1750 U/L
cuvette
Transfer to the measuring
Use Empty
L.F.T GOT 1ml (spinreact) 100ul cuvette. Read the 340 1750 U/L
cuvette
absorbance's at 60 and
Use Empty
ALP 1200ul (spinreact) 20ul 180 seconds. 405 3300 U/L
cuvette
Mix well and incubate
Total protein 1 ml (spinreact) 10ul (1ml) Reagent 630 10 Mg/dl
for 10 min. at R.T
Mix well and incubate
Albumin 1 ml (spinreact) 5ul (1ml) Reagent 630 10 Mg/dl
for 10 min. at R.T
Mix well and incubate
Cholesterol 1ml (spinreact) 10 ul (1ml) Reagent 546 925 Mg/dl
for 10 min. at 37
Mix well and incubate
Triglyceride 1ml (spinreact) 10 ul (1ml) Reagent 505 800 Mg/dl
for 10 min. at 37
and take 10ul of
50ul(R)+500ul(S)
Lipid profile supernatant to 1ml of (1ml)
Mix well and centrifuge for 10
HDL cholesterol reagent, Mix Cholesterol 546 320 Mg/dl
min.
will and incubate Reagent
(spinreact)
for 10 min. at R.T
LDL LDL = Total cholesterol – triglyceride /5 – HDL Mg/dl
Mix well and centrifuge
500ul of precipitate for 3 min. and take 100ul W.R.
Potassium (K) solution 50 ul of supernatant to 1ml of (0.5 ml (R1A) + 578 27 mmol/l
(spinreact) Working reagent (0.5 ml 0.5 ml (R2B)
(R1A) + 0.5 ml (R2B)
0.5 ml (R1A) + 0.5 ml Mix will and incubate
Calcium (ca) 10 ul W.R 570 16.5 Mg/dl
(R2B) for 10 min. at R.T
Electrolyte Magnesium 0.5 ml (R1A) + 0.5 ml Mix will and incubate
10 ul D.W - - -
(Mg) (R2B) for 10 min. at R.T
Mix will and incubate
Sodium (Na) 1ml 10 ul W.R - - -
for 10 min. at R.T
Mix will and incubate
Phosphors (P) 1ml 10 ul D.W. - - -
for 10 min. at R.T
Mix will and incubate
Chloride (Cl) 1ml 10 ul W.R - - -
for 10 min. at R.T
Cardiac Mix and start stopwatch.
enzymes Incubate for 5min. in w.
1ml bath and Transfer to the Use Empty
Ck-MB 40ul 340 1651 U/L
(spinreact) measuring cuvette. Read cuvette
the absorbance's at final
6min. and final 10min.
Use Empty
1.5 ml
LDH 50ul As GOT, GPT & ALP measuring 340 4925 U/L
(spinreact)
cuvette
Calculate the factor= concentration of standard / absorption of standard Dr. Bassam Ahmed Aldokhainy
Calculate the concentration of sample= absorption of sample × factor
 Manual Procedures of Hematological tests

Incubation
Wave-
Class Name of test Reagent Sample Procedures period & Blank Factor Unit
length
condition
Hb measure 5ml (Drabkin) 20 Mix well and incubate for 5min. at R.T. 540 576 Mg/dl
Use chamber to count cells under
Mix well and incubate for 9
WBC count 380ul (G. acetic acid) 20ul microscope with 10x ,count in 4 large ×10 /l
5min. at R.T.
squares, (No. cells count×50)
Use chamber to count cells under
Hematology Mix well and incubate for microscope with 10x ,count in 5 12
RBC count 400ul (N. saline) 20ul ×10 /l
5min. at R.T. small squares,
(No. cells count×10.000)
Use chamber to count cells under
380ul (ammonium Mix well and incubate for microscope with 10x ,count in 5 9
Platelets count 20ul ×10 /l
oxalate 1%) 5min. at R.T. small squares,
(No. cells count×10.000)

PT measure (Normal Range =12-16 sec.)

1- Take 2 glass tubes and label them from 1 to 2.
Step
2- Pipette into each tube as follow:
No. of tube Tube (1) Tube (2)
Patient plasma 300 – 500 ul -
Reagent one - 100 ul
Step 3 Incubate period For 10 minutes in water bath
Step 4- Add 100 ul of (patient plasma) into tube 2 and start the stop-watch.
5- Hold the tube in water bath and tilt the mixture back and forth looking for clot formation.
6- When a clot form, stop the stop-watch and record the time in seconds.

PTT measure (Normal Range =23.2 36.4 sec.)

1- Take 3 glass tubes and label them from 1 to 3.
Step
2- Pipette into each tube as follow:
No. of tube Tube (1) Tube (2) Tube (3)
Patient plasma 300 – 500 ul - -
Reagent one - 100 ul -
Reagent two (ca2cl) - - 300 ul
Step 3 Incubate period (1) For 3 minutes in water bath
Step 4 Add 100 ul of pa ent plasma into tube 2 and mix.
Step 5 Incubate period (2) For 4 minutes in water bath
7- Add 100 ul of (R3 ca2cl) into tube 2 and start the stop-watch.
Step 8- Hold the tube in water bath and tilt the mixture back and forth looking for clot formation.
9- When a clot form, stop the stop-watch and record the time in seconds.

aPTT measure (Normal Range =35 - 50 sec.)

3- Take 3 glass tubes and label them from 1 to 3.
Step
4- Pipette into each tube as follow:
No. of tube Tube (1) Tube (2) Tube (3)
Patient plasma 300 – 500 ul - -
Reagent one - 200ul -
Reagent two (ca2cl) - - 300 ul
Step 3 Incubate period (1) For 2 minutes in water bath
Step 4 Add 100 ul of pa ent plasma into tube 2 and mix.
Step 5 Incubate period (2) For 3 minutes in water bath
10- Add 100 ul of (R3 ca2cl) into tube 2 and start the stop-watch.
Step 11- Hold the tube in water bath and tilt the mixture back and forth looking for clot formation.
12- When a clot form, stop the stop-watch and record the time in seconds.

Dr. Bassam Ahmed Aldokhainy

Laws for some hematological tests:
- PT Ratio = PT Patient / PT control
- INR = ISI × Log PT Ratio
- PTT Ratio = PTT Patient / PTT control
- Estimate WBC and platelet from blood film:
1- Rough estimate WBC = Number of WBC in all fields/number of fields × 2000
2- Rough estimate Platelet = Number of Platelet in all fields/number of fields × 10.000
- Retics count (%)= Number of Re cs cells / 10
- (Normal range of re cs count in adult= 0.5 – 2.5% and in infant = 0.5 -5.0%)
- Correction Retics count (%) = patient retics (%) × {pa ent Hct / Normal Hct (45%)}
- Retics production index (RPI)= ½ { patient retics (%) } × pa ent Hct / Normal Hct (45%).
(Normal range of RPI= 1).
(if RPI more than 1:mean Bone marrow effective, while RPI less than 1: bone marrow ineffective)
- Absolute retics count= (No. of retics × No. of RBCs /100) ×1012
- Hb = Hct/3
- To calculate absolute value of each WBC type= (No. of each cell count × Total WBC count)/100
Normal Range for some Coagulation tests:
- PT = (12 – 16) second. (cri cal value more than 60)
- PTT = (23.4 – 36.2) second. (cri cal value more than 300)
- aPTT = (35 – 50) second.
- Target INR in normal person = (0.82 – 1.2).
- Target INR for therapy with warfrine = (2- 3). (critical value more than 6.0)
- PTT Ratio = (0.82 – 1.1) (cri cal value more than 3.0)
Interpretation result of CBC:
- (Hb > 12 g/dl ) = Not anemic.
(> )= more than
- (Hb 10 - 11 g/dl ) = Mild anemia.
(< )= less than
- (Hb 8 – 9 g/dl ) = Moderate anemia.
- (Hb 6 – 7 g/dl ) = Marked anemia.
- (Hb 4 – 5 g/dl ) = Severe anemia.
- Critical value of Hb= (< 4 g/dl ) Or (Hb > 20 g/dl )
- Critical value of WBCs= (< 1.9 ×109 /L) or (30 > ×109 /L).
- Critical value of Neutrophil = less than (20 %)
- Critical value of lymphocyte = less than (4 %)
- Critical value of RBCs= (< 1.0× 1012 /L) or (7.0 > × 1012 /L).
- Critical value of Platelets= (< 30 ×109 /L) or (900 > ×109 /L).
- (Hb = 11-13.5 g/dl) and (WBC= 4-15×109 /L) in pregnant women.

- To convert from SI units to old units multiply by the factor.

- To convert from old units to SI units divide by the factor.
- Old units = (mEq/l + mg/dl) SI units= (ummol/l + mmol/l + g/l)
Specific conversion factors:
1- Creatinine: ummol/l × 0.0011 = mg/dl
2- Uric acid: ummol/l × 0.0017 = mg/dl
3- Urea: mmol/l × 6.01 = mg/dl
4- Triglyceride: mmol/l × 87.5 = mg/dl
5- Glucose: mmol/l × 18 = mg/dl
6- Bilirubin (total + direct)= ummol/l × 0.058 = mg/dl
7- Albumin: g/l × 0.1 = mg/dl
8- Protein: g/l × 0.1 = mg/dl
9- Calcium: mmol/l × 4.0 = mg/dl
10- (Potassium + sodium + chloride): ummol/l × 0.058 =mEq/l

Dr. Bassam Ahmed Aldokhainy