Thermo Scientific

Small Benchtop Effective and Efficient Application Note Oct 2014

Application Note

4 Pages

Richard Sicard, Thermo Fisher Scientific, Asheville, North Carolina, USA Romana Hakim, Thermo Fisher Scientific, Osterode am Harz, Germany Appli c a ti on N ote Using Thermo Scientific Small Benchtop Centrifuges for the Effective and Efficient Processing of Clinical Samples Key Words Small benchtop centrifuge, Platelet-poor plasma (PPP), Urinalysis, Blood chemistry analysis Introduction Clinical laboratory testing plays an important role in the detection, diagnosis, and treatment of disease. The use of centrifugal force for separation of blood and urine is a very crucial step within the process. It is very important not only to perform this step correctly, but also efficiently so as to derive the best results for the patient sample. Blood is typically separated to yield platelet-poor plasma (PPP), which in turn is used for most coagulation tests. Ideally, PPP should have a platelet count of less than 10 x 103 per µL1. Similarly, the analysis of urine is also very important in the clinical laboratory. Information derived from this testing can predict many important medical concerns, one of which is the function of kidneys or associated infections. This common test measures various compounds such as white blood cells, red blood cells, epithelial cells, bacteria, calcium oxalate crystals, mucous threads, yeast, and hyaline casts that pass through the urine2, 3. capacity than the standard swinging bucket rotor, this rotor’s higher speed does make it a very important rotor in efficiently and effectively processing PPP. These small benchtop centrifuges offer sample protection with the Thermo Scientific™ ClickSeal™ biocontainment lid system certified by Public Health England, Porton Down, UK. They also provide Thermo Scientific™ Auto-Lock™ rotor exchange which allows rotors to be installed and removed from the chamber of the centrifuge with the push of a button – no tools required. Table 1. Thermo Scientific™ Small Benchtop Centrifuges Heraeus™ Megafuge™ 8 small benchtop centrifuge, ventilated Heraeus™ Megafuge™ 8R small benchtop centrifuge, refrigerated Sorvall™ ST 8 small benchtop centrifuge, ventilated Sorvall™ ST 8R small benchtop centrifuge, refrigerated SL 8 small benchtop centrifuge, ventilated SL 8R small benchtop centrifuge, refrigerated Finally, blood chemistry analysis is also a very common test in the clinical laboratory. These results are used to evaluate a variety of components such as potassium, aspartate aminotransferase (AST), phosphorus, and lactate dehydrogenase (LDH)4. The objective of this note is to illustrate the efficiency and effectiveness of Thermo Scientific™ small benchtop centrifuges in critical centrifugation steps of the following clinical testing: (A) platelet-poor plasma (PPP) preparation, (B) urinalysis, and (C) blood chemistry analysis. Thermo Scientific small benchtop centrifuges (see Table 1) can be used with the Thermo Scientific TX-150 swinging bucket rotor for high-speed horizontal processing. The TX-150 rotor accommodates up to 24 x 5/7 mL blood collection tubes or 8 x 50 mL conical tubes at 4500 rpm (3260 x g) but when configured with the Thermo Scientific™ HIGHConic™ rotor, it can achieve up to 8,700 rpm (10,155 x g) with 6 tubes. While a lower Figure 1. Thermo Scientific Small Benchtop Centrifuge.

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Richard Sicard, Thermo Fisher Scientific, Asheville, North Carolina, USA Romana Hakim, Thermo Fisher Scientific, Osterode am Harz, Germany

Appli c a ti on N ote

Using Thermo Scientific Small Benchtop Centrifuges for the Effective and Efficient Processing of Clinical Samples

Key Words Small benchtop centrifuge, Platelet-poor plasma (PPP), Urinalysis, Blood chemistry analysis

Introduction Clinical laboratory testing plays an important role in the detection, diagnosis, and treatment of disease. The use of centrifugal force for separation of blood and urine is a very crucial step within the process. It is very important not only to perform this step correctly, but also efficiently so as to derive the best results for the patient sample. Blood is typically separated to yield platelet-poor plasma (PPP), which in turn is used for most coagulation tests. Ideally, PPP should have a platelet count of less than 10 x 103 per µL1. Similarly, the analysis of urine is also very important in the clinical laboratory. Information derived from this testing can predict many important medical concerns, one of which is the function of kidneys or associated infections. This common test measures various compounds such as white blood cells, red blood cells, epithelial cells, bacteria, calcium oxalate crystals, mucous threads, yeast, and hyaline casts that pass through the urine2, 3.

capacity than the standard swinging bucket rotor, this rotor’s higher speed does make it a very important rotor in efficiently and effectively processing PPP. These small benchtop centrifuges offer sample protection with the Thermo Scientific™ ClickSeal™ biocontainment lid system certified by Public Health England, Porton Down, UK. They also provide Thermo Scientific™ Auto-Lock™ rotor exchange which allows rotors to be installed and removed from the chamber of the centrifuge with the push of a button – no tools required. Table 1. Thermo Scientific™ Small Benchtop Centrifuges Heraeus™ Megafuge™ 8 small benchtop centrifuge, ventilated Heraeus™ Megafuge™ 8R small benchtop centrifuge, refrigerated Sorvall™ ST 8 small benchtop centrifuge, ventilated Sorvall™ ST 8R small benchtop centrifuge, refrigerated SL 8 small benchtop centrifuge, ventilated SL 8R small benchtop centrifuge, refrigerated

Finally, blood chemistry analysis is also a very common test in the clinical laboratory. These results are used to evaluate a variety of components such as potassium, aspartate aminotransferase (AST), phosphorus, and lactate dehydrogenase (LDH)4. The objective of this note is to illustrate the efficiency and effectiveness of Thermo Scientific™ small benchtop centrifuges in critical centrifugation steps of the following clinical testing: (A) platelet-poor plasma (PPP) preparation, (B) urinalysis, and (C) blood chemistry analysis. Thermo Scientific small benchtop centrifuges (see Table 1) can be used with the Thermo Scientific TX-150 swinging bucket rotor for high-speed horizontal processing. The TX-150 rotor accommodates up to 24 x 5/7 mL blood collection tubes or 8 x 50 mL conical tubes at 4500 rpm (3260 x g) but when configured with the Thermo Scientific™ HIGHConic™ rotor, it can achieve up to 8,700 rpm (10,155 x g) with 6 tubes. While a lower

Figure 1. Thermo Scientific Small Benchtop Centrifuge.

Methods5 :

I. METHODS AND MATERIALS

Platelet-poor plasma (PPP) preparation was done by performing centrifugation of whole blood using 2.7 mL BD Vacutainer™ Plus plastic citrate tubes. The initial platelet concentrations from whole blood as well as for each PPP preparation were determined with the aid of a hematology analyzer.

A. Platelet-poor plasma (PPP) Blood was obtained from 10 unknown donors by venipuncture collection at the same day of the testing. 2.7 mL of whole blood was collected into BD Vacutainer Plus plastic citrate tubes and after 30 minutes each sample was tested to get the initial platelet count before processing. The tubes were processed using a small benchtop centrifuge. The rotors and centrifugation conditions are summarized in Table 3. Platelet counts were performed on the whole blood and PPP samples using a hematology analyzer.

Urinalysis is composed of two main steps. In the first step urine was centrifuged using 12 mL KOVA™ tubes in the TX-150 swinging bucket rotor to separate sediment from urine which was then examined in the second step for crystals, casts, cells, yeast, and bacteria under a microscope. Blood chemistry analysis was performed by collecting blood into 7 mL BD Vacutainer SST Plus tubes, followed by centrifugation (TX-150 and HIGHConic rotors). The serum was tested for potassium, aspartate aminotransferase (AST), phosphorus, and lactate dehydrogenase (LDH) using a chemistry analyzer.

Results Clinically acceptable results were derived for all centrifugation steps performed using a Thermo Scientific small benchtop centrifuge with the TX-150 swinging bucket rotor or HIGHConic fixed angle rotor. Acceptable results for PPP were accomplished at 4,500 rpm for 10 minutes using the TX-150 rotor and 1 minute at 8,700 rpm using the HIGHConic rotor. Similarly, it was at 1,700 rpm for 4 minutes using the TX-150 rotor, whereby acceptable results were achieved for the Urinalysis testing. And finally, a 2-minute centrifugation time at 4,500 rpm using the TX-150 rotor for blood samples were the centrifugation parameters suitable for stat clinical blood chemistry testing. Subsequently, centrifugation of peripheral blood was also successfully performed at 7,200 rpm for 1 minute using the HIGHConic rotor. Table 2. Thermo Scientific Rotor Buckets & Adapter Purposea

PPP; Blood chemistry analysis Urinalysis

Rotor

Buckets

TX-150 rotor (PN 75005701)

Round buckets (PN 75005702)

HIGHConic rotor (PN 75005709)

–

TX-150 rotor (PN 75005701)

Round buckets (PN 75005702)

Adapter 5/7 mL blood collection tube adapter (PN 75005739) Rubber pads (PN 76003266) 5/7 mL adapter (PN 75005804) Rubber pads (PN 005700F) 14 mL urine tube adapter (PN 75005738)

Rotor

RPM

RCF [x g]

Timeb [min]

TX-150

4500

3260

HIGHConic

8700

10155

Urinalysis

TX-150

1700

465

Blood chemistry analysis

TX-150

4500

3260

HIGHConic

7200

6967

PPP

a b

Room temperature Acceleration time/Deceleration time: Standard

C. Blood chemistry analysis Blood was collected into BD Vacutainer SST Plus tubes from 6 random donors (2 tubes per donor). After waiting 30 minutes for samples to clot, the tubes were centrifuged using a small benchtop centrifuge. The rotors and spin conditions are listed in Table 3. The serum was tested for potassium, aspartate aminotransferase (AST), phosphorus, and lactate dehydrogenase (LDH) using a chemistry analyzer.

II. DATA ANALYSIS A. Platelet-poor plasma The values were reported as the mean and the standard deviation (SD) of the Platelet concentrations from whole blood and PPP preparation. B. Urinalysis The white blood cells (WBCs), red blood cells (RBCs), and epithelial cells counts were reported as number of cells/HPF (red blood cells per high power field); the counts of casts (cylindrical particles sometimes found in urine that are formed from coagulated protein secreted by kidney cells) were reported as number of casts/10 LPF (low power field). Bacteria, calcium oxalate crystals, uric acid crystals, mucous threads and yeast were all examined at LPF and estimated as “rare”, “few”, “moderate”, or “many”. C. Blood chemistry analysis The mean and standard deviation were calculated.

Table 3. Centrifugation Conditionsa Clinical Testing

B. Urinalysis Urine specimens were pooled from 10 unknown patients and collected at the same day of the testing into 12 mL KOVA tubes. The tubes were processed using a small benchtop centrifuge until a moderately cohesive button was formed at the bottom of the tubes (see Table 3). The supernatant was decanted and a volume of 1 mL was left inside the tube. After resuspending the sediment a drop was poured onto a glass slide, covered with a coverslip and examined under a microscope at 10x (low power field) and 40x magnifications (high power field).

III. RESULTS A. Platelet-poor plasma (PPP) See Table 4. B. Urinalysis The number of WBCs, RBCs, epithelial cells, casts, crystals and yeast are shown in Table 5 and 6. C. Blood chemistry analysis The mean and SD values of potassium, AST, phosphorus, and LDH in serum are summarized in Table 7.

IV. CONCLUSION With a spin of 10 minutes at 4500 rpm using the TX-150 swinging bucket rotor, a small benchtop centrifuge achieved a clinically acceptable PPP yield per laboratory standards. In addition, when this centrifuge was configured with its HIGHConic fixed angle rotor, acceptable PPP yield were achieved within 1 minute when spun at 8700 rpm. With both rotor options the residual platelet counts in the final PPP sample were definitely < 10000/µL commensurate of an acceptable platelet count the CLSI guidelines for PPP used for coagulation

Table 4. Platelet Count In Whole Blood and PPP Preparation From 10 Unknown Donors WB platelet counta,b [K/µL] (Before spin)

Platelet counta,b [K/µL] (After spin)

TX-150

222.9 ± 94.6

2.5 ± 2.2

HIGHConic

178.7 ± 105.8

4.3 ± 1.3

Rotor

a b

Centrifugation conditions shown in Table 3 Mean ± SD

tests6. Therefore, this study has illustrated the effectiveness and efficiency by which the small benchtop centrifuge generates not only a clinically acceptable PPP separation, but also reduces the time required for routine and prompt results. Similarly, with urinalysis, the sample was also effectively and efficiently processed at 1700 rpm in 4 minutes using the TX-150 swinging bucket rotor and at the end, the acceptable results were comparable with values typically obtained in the lab using other small clinical centrifuges. In addition, this same success was also seen with the results from the blood chemistry samples. With a 2 minute run at 4500 rpm using the TX-150 swinging bucket rotor or a 1 minute run when the HIGHConic fixed angle rotor was spun at 7200 rpm, the results were also commensurate with successful results traditionally seen by clinicians when using other clinical centrifuges. The versatility of Thermo Scientific small benchtop centrifuges enable them to be effective and efficient in performing the multiple centrifugation steps required in the various applications within a clinical lab.

Table 5. Urinalysis From 10 Unknown Patients

WBCa,b

RBCa,b

Epithelial Cellsa,b

Bacteriaa,c

Calcium oxalate crystalsa, c

Uric acid crystalsa, c

6 - 10

> 50

1-5

rare

none

1-5

> 50

6 - 25

moderate

none

> 50

6 - 10

1-5

moderate

none

0-1

1-5

none

moderate

none

1-5

0-1

6 - 25

moderate

none

rare

1-5

0-1

1-5

none

–

10 - 25

6 - 10

6 - 25

many

none

–

10 - 25

1-5

rare

moderate

–

10 - 25

1-5

moderate

none

–

10 - 25

1-5

6 - 25

moderate

none

–

Centrifugation conditions shown in Table 3 b hpf, high power field c lpf, low power field a

Table 6. Urinalysis From 10 Unknown Patients

a b

Mucous threadsa,b

Yeasta,b

Hyaline castsa,b

Coarse granular castsa,b

WBC castsa, b

none

few

none

–

rare

moderate

few

none

–

none

–

none

moderate

none

1-5

–

rare

few

0-1

–

rare

none

few

none

0-2

none

1-5

none

moderate

few

1-5

none

0-2

none

many

none

Centrifugation conditions shown in Table 3 lpf, low power field

Amorphous sedimenta, b

SPANISH

Table 7. Blood Chemistry Analysis From 6 Unknown Donors

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3.85 ±

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20.50 ±Italia +32 3.9002±95059 552 172.33 ± 4.32 Japón +81 0.383 5826 161627.98 Nueva Zelanda +64 9 980 6700 Paises Nórdicos/Bálticos/CIS +358 9 329 10200

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Reference 1. Shahangian S. et al. (2005), Results of 2001 survey of

ITALIAN hospital coagulation laboratories in the United States, thermoscientific.com Archives of pathology and laboratory medicine, 129,

47 - 60.

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2. Fogazzi GB, Garigali G (2003), The clinical art and

Australia +61 of 39757 4300 microscopy, Curr IndiaOpin +91 22Nephrol 6716 2200 science urine Austria +43 1 801 40 0 Italia +32 02 95059 552 Hypertens, 12, 625 32. Belgio +32 53 73 42 41 Olanda +31 76 579 55 55 Cina +800 810 5118 or Nuova Zelanda +64 9 980 6700 3. Graham ACP practice no. +400 650 5118 JC, Galloway A (2001), Europa delbest Nord/CIS Francia +33 2 2803 2180 9 329 10200 167, the laboratory diagnosis of+358 urinary tract infection. Germania +49 6184 90 6000 Russia +7 812 703 42 15 J Clin+81Pathol., 54, 911 - 9. Giappone 3 5826 1616 Spagna/Portogallo +34 93 223 09 18

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4. A. Van der Laarse (1979), Interference of the

GERMAN measurement of lactate dehydrogenase (LDH) activity thermoscientific.com

in human serum and plasma by from blood cells, Clinica Chimica Acta, 99,vorbehalten. 135 – Alle 142. © 2013 Thermo Fisher Scientific Inc. Alle Rechte (anderen) Warenzeichen sind Eigentum von Thermo Fisher Scientific Inc. bzw. nationalen Niederlassungen. Änderungen von Spezifikationen, Bedingungen und Preisen vorbehalten. Nicht alle Produkte sind in allen Ländern erhältlich. Ausführliche 5. Validation studies for Thermo Scientific small benchtop Informationen erhalten Sie von Ihrem Thermo Fisher Scientific Ansprechpartner.

centrifuges were performed with the help of University

Australien +61 39757 4300 Indien +91 22 6716 2200 Hospital, San Antonio, Rosemarie Martinez MT Belgien +32 53 73 42 41 Italien +32 02 95059 552 China +800 810and 5118Kathleen or +81 3 5826 1616 (ASCP) Born MT Japan (ASCP). +400 650 5118 Niederlande +31 76 579 55 55 Deutschland nationale kostenloseand Hotline Neuseeland +64 9 980 6700 6. Collection, transport processing of blood 0800 1 536 376 Nordeuropa/Baltikum/CIS specimens for testing plasma-based coagulation assays. Deutschland international +358 9 329 10200 +49Approved 6184 90 6000 guidelines, 4th ed. Wayne, Österreich +43 1 801 40 0 Pennsylvania, Frankreich +33 2 2803 2180

Clinical and Laboratory Standards Institute, 2003 (Document H21-A4).

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