Overview Of Cell Culture And Harvest

Culture Initiation -> Culture Maintenance-> Cell Harvest

• Sterility • Optimal temperature • Swell cells

• Proper growth medium • Optimal pH • Fix cells

• ± Mitotic stimulant • Optimal humidity • Prepare slide

• Microbial inhibitors • Optimal time interval • Stain/band

The most critical requirement is that living cells capable of cell division be received by the laboratory. The manner in which the sample is collected and subsequently handled will greatly influence whether or not the cells will grow and divide and the quality of the resulting metaphases. Specimen containers must be sterile and must be labeled with the patient's name. The laboratory may reject specimens that are improperly labeled or unlabeled (see Chapter 6).

SPECIMEN COLLECTION AND HANDLING Requirements: Peripheral Blood Specimens

Peripheral blood samples should be collected in sterile syringes or vacuum tubes containing preservative-free sodium heparin. Vacuum tubes should be discarded if outdated. Peripheral blood cultures can be initiated several days after the blood is drawn; however, for best results, blood samples should be set up within 24 hours of collection. Temperature extremes must be avoided if samples are transported or stored. Specimens should be kept at room temperature or refrigerated above 4°C until they

From: The Principles of Clinical Cytogenetics, Second Edition Edited by: S. L. Gersen and M. B. Keagle © Humana Press Inc., Totowa, NJ

can be processed. Culture medium is sometimes added to small blood samples, as these have a tendency to dry up, especially if collected in large containers.

A repeat sample should be requested if these requirements are not met (e.g., the sample is received clotted, on ice, more than 24 hours old, etc.). It is not always practicable or possible to obtain a new sample, and in such cases, the laboratory should attempt to salvage the original specimen. There may be enough viable cells for a cytogenetic analysis, although the number and quality of cells may be compromised.

Requirements: Bone Marrow Aspirates

The collection requirements for bone marrow samples are essentially the same as for peripheral blood. Bone marrow aspirates should be collected in sterile syringes or vacuum tubes containing preservative-free sodium heparin and transported at room temperature. The first few milliliters of the bone marrow tap contain the highest proportion of cells and are the best sample for the cytogenetics laboratory. Blood dilutes the bone marrow sample in later taps and reduces the number of actively dividing cells present in the sample. The success of bone marrow culture is dependent on the number of actively dividing cells. Bone marrow specimens should be processed without delay upon receipt to avoid cell death.

Requirements: Amniotic Fluid Specimens

Amniocentesis can be performed from as early as 10 weeks gestation until term (see Chapter 12). From 15 to 30 mL of amniotic fluid should be obtained under sterile conditions and collected in a sterile container approved for cell culture. For amniocentesis performed earlier than 15 weeks, 1 mL of fluid is generally drawn for each week of gestation. The first few milliliters of an amniotic tap are the most likely to be contaminated with maternal cells and should not be submitted to the cytogenet-ics laboratory. Samples should be transported at room temperature. Temperature extremes and long transport times should be avoided.

The amniocentesis procedure has an inherent, albeit small, risk of miscarriage and should not be repeated unless absolutely necessary. Every effort to salvage samples improperly collected or handled should be made to diminish the need for a repeat tap.

Requirements: Solid Tissue Specimens

Solid tissue sources include skin biopsies, chorionic villi, products of conception, and stillbirth biopsies. Products of conception and stillbirths are one-of-a-kind specimens that cannot be recollected, and repeat collection of chorionic villi increases the risk of abortion, although subsequent amniocentesis is an option here. Microbial contamination is a common problem for many types of solid tissue samples. Unlike amniotic fluid, blood, bone marrow, and chorionic villi, most solid tissue specimens are not sterile prior to collection. In addition, viable cells might be few or even nonexistent. These factors threaten the integrity of the sample and pose problems for the laboratory.

Small samples should be collected and transported in sterile culture vessels containing growth or tissue culture medium (not formalin). Sterile saline is not optimal for this purpose, but should be used if no other option is available. If distance and timing permit the laboratory to receive and process the sample at once, it can be delivered with no liquid added at all. Larger samples can be sent to the laboratory in toto for dissection. Solid tissue samples should be transported and stored on ice until culture is established. Storing tissue specimens on ice slows the action of enzymes that degrade the tissue and slows microbial growth in the event of contamination.

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